59th Annual Scientific and Standardization Committee Meeting

Amsterdam, The Netherlands

Table of Contents Animal, Cellular, and Molecular Models of Thrombosis ...... 3 Biorheology ...... 6 Control of Anticoagulation ...... 13 Disseminated Intravascular Coagulation ...... 16 Registry of Exogenous Hemostatic Factors ...... 19 Factor VIII and IX ...... 21 Factor XI and the Contact System ...... 44 Factor XIII and Fibrinogen ...... 46 Fibrinolysis ...... 52 Hemostasis and Malignancy ...... 58 Lupus Anticoagulant/Phospholipid-Dependent Antibodies ...... 60 Pediatric and Neonatal Hemostasis and Thrombosis ...... 63 Plasma Coagulation Inhibitors ...... 66 Platelet Immunology ...... 72 Platelet Physiology ...... 80 Predictive Variables in Cardiovascular Disease ...... 83 Vascular Biology ...... 86 von Willebrand Factor...... 95 Women’s Health Issues in Thrombosis and Haemostasis ...... 102 Coagulation Standards Standing Committee ...... 105

Animal, Cellular, and Molecular Models of Thrombosis

29 June 2013

Chair: Susan Smyth Co-Chairs: David Motto, Tom Knudsen, Tim Nichols, Cecile Denis, Denise Sabatino, Toshiyuki Miyata, Eva-Maria Muchitsch, Hugo Ten Cate

Educational Session: Humanized Animal Models – their role in drug discovery and validation

8:00 Nigel Key, UNC-Chapel Hill, NC, USA, Need for improved models for drug discovery and antidote testing

This presentation reviewed literature and relevant animal models that have been used for anti- thrombotic and anti-coagulant drug development, gaps in knowledge, and areas where there is need for improved standardization.

8:20 Thomas Diacovo, Columbia University, NY, NY, USA, Modifications in mice for assessment of human therapies

This presentation described the development and validation of a novel mouse model for the study of human platelet function. The small animal model, which supports human but not mouse platelet-mediated thrombosis, was developed by genetically modifying mouse von Willebrand factor (VWF(R1326H)). The model has been validated with the use of the αIIbβ3 inhibitors, abciximab, eptifibatide, and tirofiban, and with platelets from individuals who were treated with aspirin or clopidogrel.

8: 45 Henri Spronk, Maastricht University Medical Center, the Netherlands, Animal models to validate therapies for antidotes for anticoagulant therapy

With increasing use of the new oral anticoagulants has come renewed interest in developing antidotes for their reversal. This presentation reviewed the potential usefulness and limitations of different animal models for predicting efficacy of hemostatic intervention and ability to predict risk of thrombosis.

Committee Sessions

9:15 David Motto, University of Iowa, USA Insights into hemostasis and thrombosis from studying murine models

Ferric chloride has been widely used to initiate arterial thrombosis in small animals, especially in mice. Using scanning electron and brightfield intravital microscopy, the presenter demonstrated that red blood cells that first adhere to the damaged vessel surface in the ferric chloride model and that with time, the red blood cells and derived structures recruit platelets.

Studies demonstrated the key role for GPIba in red blood cell adhesion to the endothelium. The results have important ramifications for the interpretation of results in this widely used experimental model of thrombosis.

9:45 Fumiaki Banno, National Cerebral and Cardiovascular Center, Suita, Osaka, Japan. Genetic mouse models of venous thrombosis for Japanese

This presentation reviewed the development of two novel animal models of genetic causes of thrombosis in the Japanese population and validated that their introduction in mice resulted in a hypercoagulable state.

10:00 Hitomi Yamamoto, National Cerebral and Cardiovascular Center, Suita, Osaka, Japan Temporary cerebral ischemia induced by three-vessel occlusion in mice

The author described the adaption of a three vessel occlusion model to investigate the response to cerebral ischemia in mice. The temporary three-vessel occlusion technique involved a surgical approach for middle cerebral artery and produces consistent cerebral infarction in the neocortex, with reductions in regional cerebral perfusion.

10:20 Tom Knudsen, Haemophilia Pharmacology, Novo Nordisk, Denmark. Optimized bleeding model in mice

This presentation described a method to optimize tail bleeding time in mice. The method resulted in predictable times

10:40. Timothy Nichols , UNC-Chapel Hill, NC, USA. Major advances using genetic approaches in pig

This presentation highlighted the success in developing genetically altered pigs by phenotypic selection of spontaneous mutations and through gene targeting. Examples

Business Meeting of the Animal Models Subcommittee

11:15 to 12:00

The committee discussed publication and distribution of reports of Animal Models Subcommittee. Standardization of bleeding assays has been an ongoing focus of the subcommittee. One communication on the topic has been published (Greene TK, Schiviz A, Hoellriegl W, Poncz M, Muchitsch EM. Towards a standardization of the murine tail bleeding model. J Thromb Haemost. 2010;8:2820-2822. [PMID:21138523]) and another submitted for consideration in J Thromb Haemost (Schiviz A, Resch M, Leidenmühler P, Schuster M, Muchitsch EM, Höllrieg W. Genetic background influences bleeding phenotype in the tail-tip bleeding model). Support for the submission was voiced by committee members. Drs. Smyth, Nichols, and Muchitsch will meet to discuss the manuscript with Dr. Sabine Eichinger-Hasenauer, Editor,

on Tue July 2, 2013. Dr. Smyth will send general comments and recommendations to the subcommittee members about the format for future publications.

The following motions were made, seconded, and approved as manuscript submissions from this subcommittee for consideration for publication in J Thromb Haemost:

1. The work on animal models to validate therapies for antidotes for anticoagulant therapy presented by Dr. Henri Spronk.

2. The "Optimized Tail Bleeding Model in Mice” work presented by Tom Knudsen

The following topics were discussed for inclusion in the Animal Models Education Session during the XXX SSC meeting in Milwaukee WI in 2014:

1. The Pre-analytical conditions in "Point of care assays” for platelet function, platelet inhibitors, anticoagulation for ventricular assist devices, ECMO, and cardiopulmonary bypass. This could include VerifyNOW, activated clotting times (ACT), thromboelastography (TEG), and thrombin generation assays among others.

2. The current role of nonhuman primates in atherosclerosis, thrombosis studies and for safety and efficacy studies during drug development

3. A review and update of the previously published consensus and guidelines for platelet function studies and coagulation assays in mice. 1-3

4. Guidelines for the "basic package” for characterization of animal models of hemophilia

1. Emeis JJ, Jirouskova M, Muchitsch EM, Shet AS, Smyth SS, Johnson GJ. A guide to murine coagulation factor structure, function, assays, and genetic alterations. J Thromb Haemost. 2007;5:670-679. [PMID:17403201]

2. Jirouskova M, Shet AS, Johnson GJ. A guide to murine platelet structure, function, assays, and genetic alterations. J Thromb Haemost. 2007;5:661-669. [PMID:17403200]

3. Matsuo O, Lijnen HR, Ueshima S, Kojima S, Smyth SS. A guide to murine fibrinolytic factor structure, function, assays, and genetic alterations. J Thromb Haemost. 2007;5:680-689. [PMID:17403202]

Biorheology

June 29, 2013

Chairman: Michael R. King (USA) Co-Chairs: Lawrence Brass (USA), Shaun Jackson (Australia), David N. Ku (USA), Owen J. McCarty (USA), Keith B. Neeves (USA), Armin Reininger (Austria), Mitsuhiko Sugimoto (Japan)

2:00-2:20PM

Topic: Flow-dependent thrombin and fibrin generation

Speaker: Keith Neeves, Colorado School of Mines

Educational Aim: The objective of this talk is to provide (i) a brief review of the literature on flow-dependent thrombin and fibrin generation, (ii) methods for measuring these phenomena in vivo and in vitro, and (iii) recommendations on measurements that can be incorporated into flow assays.

 Flow as a regulator of coagulation  Biophysical mechanisms: 1. Flow modulate surface reactions. 2. Microscale perturbations of flow. 3. Hindered transport  Squires et al., Nat Biotech 26 (2008). Scaling analysis of convection/diffusion/reaction. Peclet number. Damkholler number.  Computer simulation of X on TF:FVIIa. Diffusion dominated regime, increase flow to balanced convection/diffusion. Increase flow further, develop boundary layer.  Experiments: Repke et al., PNAS 87 (1990). More Xa generation with inc. flow rate. Da=1000, diffusion limited.  Experiment: Haynes et al., Biophys J 100 (2011). Da=0.1, reaction limited. Increasing flow, dilution.  Fibrin formation under flow. Neeves et al., Biophys J 98 (2010). Thrombin boundary layer gets thinner with higher flow. Low shear, fibrinogen all converted. Experimentally, thinning fibrin fibers, then no fibers at higher flow rate.  Microscale perturbations of flow. Roughness protects surface roughness from flow, then more accumulation of soluble factors. Flamm et al., Blood 112 (2012).  Fibrin formation on TF. TF coated silica (1um) beads. 50 molec TF/um2. Need the roughness of beads or fibrin does not form on TF surface.  Hindered transport. Hathcock and Nemerson, Blood 104 (2004). Leiderman and Fogelson, Bull Math Biol 2012. Assume slower diffusion through porous thrombus. Clot predicted to be smaller due to hindered transport. Wufsus et al., Biophys J 104 (2013). New paper, permeability measured as a function of platelet volume fraction.  Now characterizing pore structure in thrombi by FIB-SEM.

2:20 – 2:40pm

Topic: Assaying thrombus formation under flow: the role of coagulation

Speaker: Johan Heemskerk, Maastricht University

Educational aim: Overview of the latest methods and developments on the role of coagulation in platelet thrombus formation.

 Versteeg Physiol. Rev. 2013; 93:327. Collagen and TF-induced thrombus formation.  Reviews previous Biorheology SSC papers of the ISTH. Zwagina et al., JTH 2006 Parts 1 and 2. Roest et al., JTH 2011. Heemskerk et al., JTH 2011.  Mice. High correlation with arterial thrombosis (93%). Less correlation with tail bleeding (68%). Detect hemostatic insufficiency. Monitoring anti-thrombotic therapy. Predicted test in individual to asses A/V thrombosis.  Conventional flow devices. Platelets 2012; 23:229-242. Protocols for making flow chambers and assays with and without coagulation. Microscopic imaging. Parallel-plate chambers 50 – 100 uM height, 3 – 5mm width. Reduce cross-section to need less blood volume. Coatings: collagen, fibronectin, peptides, VWF, etc. Monitor thrombus formation, spreading etc.  Gas6-free to change thrombus stability. Prevent coagulation: PPACK, heparin, corn trypsin inhibitor. Sodium citrate and recalcify.  Pitfalls: airbubles, signs of coagulation, local platelet-fibrin clots. Berny PLOS ONE 2010: controlled coag with added TF.  Higher shear increases platelet deposition, but not local fibrin formation.  Microfluidic devices. Platelets 2012; 23:501-509. Make your own disposable PDMS. Can have multiple channels, small blood volumes, multiple adhesive surfaces and endothelial cells. Stenotic regions. Stamped microspots of collagen.  Disadvantages: if channel too small, will impair thrombus formation. Variation in design hinders standardization. Platelet function sensitivity varies in different devices.

2:40 – 3:00pm

Topic: Measuring and modeling the thrombus interior microenvironment

Speaker: Lawrence Brass, University of Pennsylvania

Educational Aim: Recent studies show that the hemostatic response to penetrating injuries produces an architecturally complex structure that is shaped in part by the intersection of local conditions with the platelet signaling network. This talk will review work showing how thrombus structure affects not only thrombus growth and stability, but also the local generation of thrombin and the passage of molecules within the growing hemostatic mass. In keeping with the theme of this SSC session, the methods employed for data collection are a

combination of confocal intravital microscopy, microfluidic flow chambers and computational modeling.

 Close collaboration with Scott Diamond and Tim Stalker.  Confocal intravital fluorescence microscopy in mice (w/ T Stalker, Blood 2013). Platelets (red) P-selectin (active platelets, green), vessel (Dextran-blue). Can almost resolve individual platelets. Question: no P-selectin staining of the endothelium? Thrombus only P-selectin positive in "core” 3 – 20 min after injury.  As platelets accumulate over first 2 minutes, albumin leakage goes down. P-selectin expression in activated platelets comes after 3 min.  Diamond flow chamber, can introduce transthrombus pressure gradient. Muthard and Diamond, ATVB 2012.  Thrombus structure: core (P-sel+), shell (P-sel(-)) and boundary. Welsh et al., JTH 2344 (2012). Thrombin biosensor in vivo. Inner core where thrombin is active. Same labeling carried out in microfluidics. Thrombin+/P-sel+ core. ADP involved in shell.  Poor plasma penetration in the core region of thrombus. New computational work with Maurizio Tomaiuolo. Pile of impenetrable platelets: average rate = 1 um/s. Equally spaced platelets, or crowded in core: average rate = 0.1 um/s. Predicted, then measured with albumin with caged fluorescein: can uncage with laser pulse.

Session 2: Mechanical degradation of VWF in medical devices

Moderators: Michael King, Cornell University; Jorge Di Paola, University of Colorado School of Medicine

3:00 – 3:10pm Break and Introduction

3:10 – 3:30pm

Title: VWF Mechanoenzymatic Stability Under Fluid Dynamic Conditions Germane to LVADs

Speakers: Vincent Turitto and Zoe Demou, Illinois Institute of Technology

 Talk given by Demou at 4:10pm. LVAD-induced pathology in heart failure patients. Modeled by closed in vitro flow loop: tubing connected to blood bag, flow created by LVAD. VWF multimer analysis for human blood after shearing. HVAD vs. HeartMate II. Degradation seen over 60 min time frame.  Explore impact of shear stress level, and exposure time. PPP from healthy human donors. Analyzed on SDS agarose gels. At 550 dyn/cm2, degradation occurs over 20 min. Over shorter 1 min expts, see degradation >400 dyn/cm2. Inhibited with EDTA (10 mM), implying enzymatic degradation. Blood in LVAD exposed to non-physiological high shear stress over very short times (CFD provided by HeartWare, Inc.).  Pulsatility promotes VWF degradation in vitro over 10 min, with 550 dyn/cm2 flow pulses with 10 sec. no flow.

3:30 – 3:50pm

Title: Force-induced self-association and disruption of von Willebrand factor strands

Speaker: José López, University of Washington

 Dong JF Blood 2002. ULVWF:GPIb supports attachment of plaetlets to activated EC. Savage et al., PNAS 2002; 99:425-30. VWF self-association in synthetic microvessels.  Ying Zheng and coworkers. PDMS mold with ridges and collagen gels. Can be seeded with endothelial cells, and other vascular cells. Engineered microvessels. PECAM-1 stain shows EC cell junctions. EC cross-section becomes circular over time.  Whole blood perfusion. Some platelet adhesion. Vessel branches show strands of secreted VWF and will grow to occlusion when stimulated. In tortuous vessels, can find continuous VWF strands that connect from different EC that take the shortest path through vessel lumen… up to 5cm long! Fibers form where the shear stress is highest.  Can make large grids of interconnected vessels using microfluidics.  D Chung et al., effect of HDL/apoA-1 ON VWF self-association.

3:50 – 4:10pm

Title: Regulation of VWF structure and function by hydrodynamic shear

Speaker: Sriram Neelamegham, University of Buffalo

 Due to travel delays, gave talk at 5:11pm. Showed example of elevated shear stress in mechanical heart valve. Girdhar and Bluestein, Exp. Rev. Med Dev 2008. Biomaterials vs. design and rheology.  VWF, focused on A1 (platelet binding to GPIb) and A2 (cleavage) domains. What does shear do to VWF? Cone-and-plate viscometer studies. Isolated VWF activates platelets under shear within 10 sec. Big jump in activation at 8000 1/s in PRP, or at lower shear rate in whole blood.  Blood 101:2637, 2003. SLS light scattering to measure VWF size of sheared VWF. For better (structural, high resolution) information, use neutron scattering. Singh et al., JBC 281 2006, Biophys. J. 2009.  Fluoresence dye sensor able to detect conformational change. Biophys J. 2009.  Dayananda et al., Blood 2010 116:3990. Tested VWF vs. VWF lacking A1.  Biophys. J. 2004, 86:576: conformational change causes VWF self association, and can lead to platelet aggregation. 0.1pN predicted to be a relevant force threshold. Actual estimated forces expected to be higher.  VWF-FRET proteins to study A2-domain conformational change. Recombinant proteins made in bacteria. ADAMTS13 cleavage causes change in FRET ratio based on relative motion of neighboring regions. Reagents available for sharing with researchers. Observation is calcium dependent.

 Calcium regulates VWF size, when ADAMTS13 is active. Active ADAMTS13 in endothelial cells responsible for intracellular VWF cleavage.

4:10 – 4:30pm

Title: Fluid shear stress and VWF proteolysis by ADAMTS13

Speaker: X. Long Zheng, University of Pennsylvania

 Length of ULVWF strings (platelet/leukocyte decorated) are modulated by ADAMTS13. Cleaves cell-anchored strings even in the absence of flow. Turner JTH 2009; 229-32.  Cleaved VWF remains ultra large in size.  Cleavage of soluble multimeric VWF by ADAMTS13 on the other hand requires high shear stress. Seyfried Thromb Haemost 2010; 104: 523-30. Heterogeneous molecule shapes. Stretched and globular regions after cone-and-plate shear.  Fluid shear stress applied by stainless steel tubing system: Tsai and Lian, N Engl J Med 1998; 339:1585-94. Not very efficient.  Mini-vortexer induced shear stress: Zhang et al., Blood 2007; 110:1887-94. Dose response with vortex speed. Cao et al., PNAS 2008; 105:7416-21. Factor VIII accelerates the proteolysis of VWF by ADAMTS13.  PCR mixer induced shear stress. 2500rpm, 0-60 min. Skipwith et al., JBC 2010; 285:28596-603. Comparison of methods with cone-and-plate viscometer, to calibrate shear stress magnitude based on generation of cleavage product.  bioFlux microfluidics, 48 chambers/wells. ADAMTS13 inhibits platelet aggregation on collagen at high shear stress (~200 dyn/cm2, but not at 20 dyn/cm2).  Wall shear stress in different vessels: Pries et al., Circ Res 1995; 77:1017-23.  rADAMTS13 as anti-thrombotic.

4:30 – 4:50pm

Title: Thrombosis Differences in Vascular Grafts and ECMO Circuits

Speaker: David Ku, Georgia Institute of Technology

 Clinical problem: vascular grafts. Extracorporeal membrane Oxygenators form clots in ciruits, and can shed clots. Virchos' triad: Stasis, hypercoag, vessel wall injury.  Aortic graft.:EVAR limb thrombosis, Aptus endograft. Surprising thrombosis in the lumen, particularly in smaller lumens. Using CFD, standard grafts <300 dyn/cm2. In thrombosed grafts, ~500 dyn/cm2. Comparison with other commercial grafts. Patient specific predicted clinical outcomes. Problem in how many stitches to use in the "crotch” area in the graft… construction is similar to a pair of pants.  Thrombosis always occurs in highest stress regions, 10,000 1/s shear rate. Predicts multivalent bonds necessary, Almost a 1000 bonds predicted to be needed at highest

shear stresses. So, VWF must be laying down to present enough A1 domain to the platelets. VWF "basket” captures platelet, so the physics works out.  High shear thrombus is a "white” clot, platelet rich.  ECMO circuit: Extracorporeal Membrane Oxygenator. Life support for lungs. Originally for adults, now more children on the device. Clinical data shows difficulties in standardization. Connectors are made of different materials than tubing. Clots in ECMO form at the junction between tubing and connector. Clots in ECMO is mostly fibrin, few platelets.  CFD shows strong concordance with LOW shear rate. Not a biomaterials problem, clots form at very low shear rates. Us fluid mechanics to redesign connectors to reduce clotting by 80%. VWF and platelets not the problem in ECMO clotting.

4:50 – 5:10pm

Title: Acquired von Willebrand Syndrome in Continuous Flow Left Ventricular Assist Device Recipients

Speaker: Rong He, Mayo Clinic

 Abnormality, not a syndrome. Clinical case study: warfarin effect, aspirin effect, but what about VWF? Acquired VW syndrome.  Thoratec HeartMate II (continuous new type), Thoratec HeartMate XVE (pulsatile old type).  Letsou et al., 2005. J. Heart Lung Transplant. Jarvik 2000 axial flow LVAD, GI bleeding, comparing nonpulsatile and pulsatile LVAD. Crow et al. JTCVS 2009. Cannegieter et al., Circulation 1994.  Heyde's Syndrome 1958, similarities with continuous flow LVAD observations. N. Uriel et al., JACC 2010. AvWS in CF-LVAD.  Multi-institution clinical study of AvWS in CF-LVAD: Crow et al., STS 2010. Suarez et al., Circulation: Heart Failure 2011. GI First bleed: Shah et al., STS 2011.  Predicting at risk patients: vWF function tests, vWF:LX assay applied to LVAD patients. Joyce et al., J Heart Lung Transplantation 2012. Trend found, but not predictive enough for clinical use.

Session 3: Emerging Topics in Biorheology

Moderator: Keith Neeves, Colorado School of Mines

5:10 – 5:20pm Break and Introduction

5:20 – 5:40pm

Title: Live cell imaging of coagulation

Speaker: Owen McCarty, Oregon Health Sciences University

 New JOVE video coming out on these methods. Activated platelets on surface, can perfuse and visualize fibrin formation in vitro. How to compute the volume and density? Through focus imaging, DIC + brightfield. Volume measurement: high NA Hilbert Transform HT-DIC, and can calibrate with polystyrene beads. Baker et al., Cell Molec Bioeng. 2012, 5:488. Also validated with RBC and WBC volume. Mass measurement: Low NA NI-QPM. "Transport of Intensity equation”.  Question: can someone download your program to do this analysis? What computing platform?  Validate phase measurement on "noise floor”. Can go from 0.1 to 20 uM beads.  Baker et al., J. Biomed. Opt. 2013; 18:16014: aggregate vs. Thrombi +/- tissue factor.  Cell density measurements of SW480 and SW620 prostate cancer cells, will appear in a publication soon. Look for "heat maps” of mass density in cytoplasm vs. nucleus.  Summary: NI-QPM and HT-DIC can be used with off the shelf microscope to quantify basic physical features of aggregates/thrombi.

5:40 – 6:00pm

Title: Multiscale simulations of blood cell interactions with a microvascular thrombus

Speaker: Michael King, Cornell University

 Contrasted hydrodynamic, vs. biochemical environment in hemostasis.  Previous work: single tethering platelet. Mody and King, 2005; Physics of Fluids. Wang et al., 2012; J. Comp. Physics. Simulation can differentiate between the slip bond and catch/slip bond of GPIb:VWF.  Previous work: two-platelet collisions. Mody and King, 2008; Biophys. J. Wang et al., J. Comp. Phys. 2012.  Humanized mouse developed by Diacovo and colleagues. Computational technique for deformable blood cells developed by Freund and colleagues.  Model results obtained for a range of 3D thrombus shapes, predicting stagnation point upstream and downstream of thrombus.  Increase in velocity with increase area stenosis agrees with mouse and human clinical data. Detailed comparison between simulation and mouse flow trajectory data. Calculation of "trajectory density” and streamline displacement to collapse data.  RBCs shown to enhance platelet:thrombus contact in several ways. Future work: add multiple receptor-ligand binding pairs and increased vessel diameter and hematocrit.  Presenters and attendees are invited to submit manuscripts to Cellular and Molecular Bioengineering journal. King has taken over as Editor-In-Chief.

Control of Anticoagulation

June 30, 2013

Chairman: Walter Ageno (Italy) Co-Chairs: Rebecca Beyth (USA), Benilde Cosmi(Italy), Mark Crowther(Canada), Ismail Elalamy (France), Elaine M. Hylek (USA), Pieter W. Kamphuisen (The Netherlands), Peter Verhamme (Belgium), Henry G. Watson (Scotland)

The program of the meeting was divided into four distinct parts, the first was an educational session, the second provided an update on ongoing SSC registries, the third focused on laboratory issues with the new oral direct inhibitors, and the fourth addressed research issues on the management of anticoagulant drugs.

The meeting started with the educational session entitled: "Predicting and managing bleeding in patients on anticoagulant therapies” and was chaired by Dr. Walter Ageno and Dr. Mark Crowther. The three lectures were presented by Dr. Elaine Hylek (Risk factors and bleeding scores for AF patients), Dr. Benilde Cosmi (Risk factors and bleeding scores for VTE patients) and Dr. Rebecca Beyth (Evidence based management of major bleeding).

Dr. Elaine Hylek reviewed risk factors for major hemorrhage among individuals with atrial fibrillation and provided updates on published scores to assess bleeding risk. Emphasis was clearly placed on the appropriate use of bleeding scores and their methodological strengths and limitations. Similarly, Dr. Benilde Cosmi reviewed available evidences on the incidence and risk factors for bleeding during anticoagulation in patients with venous thromboembolism and discussed the usefulness of bleeding scores in clinical practice and recommendations from the most recent practice guidelines. Issues for future research were also considered. Finally, Dr. Rebecca Beyth provided a comprehensive review on currently available reversal strategies for the management of patients with major bleeding during anticoagulant treatment with the "old” and the "newer” anticoagulant drugs.

In the second session chaired by Dr. Peter Verhamme and by Dr. Henry Watson, Dr. Sam Schulman presented an update of the SSC registry entitled Recurrent venous thromboembolism in anticoagulated patients with cancer. There are currently about 160 patients enrolled and the planned sample size is 200. All those who are interested in contributing to this registry may find all necessary information and the case report form online by accessing the ISTH SSC website. All patients with cancer who experienced recurrent venous thrombosis while on anticoagulant therapy (any treatment) are eligible for this registry aimed to gather information on the current therapeutic management of these patients. Subsequently, Dr. Walter Ageno presented an update of the registry on patients with splanchnic vein thrombosis. This study has completed enrolment with more than 600 patients from 33 centers in 12 countries. The 2-year follow-up is expected to be completed in early 2014. The results of the 6-month follow will be presented during this ISTH meeting. Finally, Dr. Gualtiero Palareti proposed a registry on the management of acute clinical events, including bleeding complications, need for emergency surgery or

thrombotic complications in patients treated with the new oral anticoagulant drugs: the START- SSC-Events study. The electronic CRF could be prepared in the currently active electronic database of the START-SSC registry. A protocol will be developed by an appointed steering committee and proposed to this subcommittee for approval. Meanwhile, all interested centers are always welcome to contribute to the main START-SSC register which is enrolling consecutive patients starting any anticoagulant therapy.

In the third session, chaired by Dr. Pieter Kamphuisen and Dr. Ismail Elalamy, Dr. Job Harenberg has reported the results of the SSC project entitled Evaluation of tests to assess the effect of apixaban. The project is now completed and a paper will be prepared and will be submitted as an SSC communication. Briefly, in this in vitro study all coagulation assays used, including coagulation and chromogenic assays, adequately determined the concentration of apixaban using adequate controls. Adaptation of the protocols for the coagulation platforms is now needed to further improve the quality of the measurement of apixaban in human plasma samples. After Dr. Harenberg presentation, Dr. Elaine Gray has also presented a quick update on the status of her SSC project on the Evaluation of tests to assess the effect of dabigatran. The study is still ongoing, but it should be completed in few months. Dr. Jovan Antovic presented an overview of the tests to assess the effect of the oral direct inhibitors comparing in vitro and in vivo results. Based on the results of studies from his and other groups, Dr. Antovic concluded that for coagulation assays there is a reasonable agreement between in vitro and in vivo data. In clinical practice, HTI and ECA can be considered the assays of choice for measuring dabigatran activity, although neither of those two assays can be used to infer the total absence of the drug. APTT may be used for qualitatively assessment if HTI and ECA are not available, but normal APTT levels may be observed even with therapeutic levels of dabigatran. Dr Elalamy from France addressed the question of which patients to monitor and which tests to use for the new anticoagulant drugs. Dr. Elalamy stressed the need to restrict testing to very selected patients, such as those requiring emergency procedures, but also stressed the need to provide continuing education to clinicians about the correct prescription of these newer agents. Further clinical validation of available and reliable assays is required. Finally, Dr. Kluft reported on the clinical relevance and of methodological issues related to the inhibition of factor XIa with the oral direct inhibitors. Dr. Kluft observed that oral direct inhibitors can influence the formation of molecular markers such as TAT and F 1+2. For example, direct thrombin inhibitors increase F 1+2. The oral direct inhibitors appear to be less specific than natural inhibitors and may act on other target enzymes, such as factor XIa. In particular with dabigatran, the inhibition of factor XIa may result to be relevant.

In the final session, chaired by Dr. Kamphuisen and Dr. Crowther, Dr. Scott Kaatz proposed to standardize the definition of clinically relevant non-major bleeding in clinical investigation of antihemostatic medicinal products in surgical and non surgical patients and Dr. Clive Kearon from Canada proposed a standardization for the definition of unprovoked VTE in clinical studies. Dr. Kaatz reported the results of previous work from this subcommittee on the standardization of the definition of major bleeding in clinical trials, and stressed the need to obtain a similar recommendation on non-major bleeding events. Dr. Kaatz acknowledged the high variability in defining a bleeding event as relevant, and proposed a careful literature review

to summarize all previously used definitions, with the aim to subsequently interview international experts in this field and to obtain a consensus on a standardized definition. Dr. Kearon initially stated that whether VTE is provoked by a reversible factor, unprovoked, or provoked by a continuing risk factor (usually cancer), influences the risk of recurrence. These factors, particularly the categories of "provoked by a reversible risk factor” and "unprovoked” are part of a continuum; criteria to distinguish the two categories and to define "unprovoked”, therefore, will have an arbitrary and subjective component. Despite these limitations, standardized criteria to define "unprovoked VTE” may still facilitate comparisons between studies. It was then proposed that unprovoked VTE can be defined as: "VTE that occurs without a convincing provoking factor”. Convincing provoking factors would include: reversible provoking factors that are divided into major factors (associated with a >10 relative risk of a first VTE) in the past 3 months, and minor factors (associated with a 3-10 relative risk of a first VTE) in the past 2 months; and continuing risk factors (associated with a >2 relative risk of recurrent VTE) which will usually be active cancer. Finally, Dr. Verhamme highlighted the need for independent trials in orphan areas such as in the case of patients with cancer associated thrombosis or patients with atypical site venous thrombosis. The treatment for rare thrombotic disorders is often consensus-based rather than evidence-based in the absence of randomized controlled trials, which are difficult in orphan areas. Vitamin K antagonists may have limitations for patients with rare thrombotic disorders, but clinical development programmes of oral direct inhibitors will not investigate these therapeutic areas. However, patients with thrombotic disorders in orphan areas may also benefit from alternative oral anticoagulation, e.g. patients with cerebral or splanchnic vein thrombosis, or patients with arterial thrombo-embolism.Dr. Buller from the Netherlands discussed how such studies could be designed and financially supported. He proposed as an example the setting of cancer associated venous thrombosis, in which new data on the oral direct inhibitors in comparison with low molecular weight heparin are needed. This type of study should be investigator intiated, multicentre and low budget, but of course with ad adequate steering committee, data safety monitoring board, and adjudication committee. Dr. Buller suggested that finances could be collected by public (e.g. government, foundations, research institutions) or industry sponsors, possibly under SSC auspices.

At the end of the meeting, after short conclusive remarks, Dr. Ageno invited Dr. Job Harenberg to present the 2013 SSC Control of Anticoagulation Awards recipients. These awards have been made possible thanks to the SSC Control of Anticoagulation, the XXIV Congress of the ISTH, and the corporate sponsor Sanofi and are provided in the form of travel grants to medical trainees and young scientists under 35 years of age who submitted highly rated abstracts to the ISTH Congress in Amsterdam. The awardees were: Tyler Buckner, USA, Elena Campello, Italy, Pichika Chantrathammachart, Thailand, Xue Chen, China, Shanshan Du, Germany, Maarten Hendrickx, Belgium, Sandra Krämer, Germany, Martin Krejczy, Germany, Abimbola Onasoga, USA, Dianne van der Wal, Australia, Min Xuan, China, Shabnam Zolfaghari, Germany.

Disseminated Intravascular Coagulation

June 30, 2013

Chairman: Hideo Wada (Japan) Co-Chairs: Satoshi Gando (Japan), Hyun Kyung Kim (South Korea), Shinichiro Kurosawa (USA), Jorn D. Nielsen (Denmark), Jecko Thachil (UK)

Educational session

Dr Gary Kinasewitz (USA) presented " Coagulopathy of the critically ill”. Many biomarkers were high in almost ICU patients such as D-dimer (99.7%). The frequency of abnormality in PT was 63% and that in platelet count was 32.7 %. There was no significance difference in hemostatic markers and frequency of DIC between gram positive and negative infection. About 25-35 % of ICU patients were associated with overt DIC. 1) Simple scoring system; Platelet count < 100,000/μl was 1 point, 20 % reduction of platelet count was 1 point, prothrombin time (PT) >. 15 sec was 1 point, and 20% prolongation of PT was 1 point. He examined 163 ICU patients on day 0, 1 and 2 by this score. There were 24 patients with capillary leak syndrome (CLS) and 45 with TOF, 57 MOF with survivor and 37 MOF with non-survivor. The simple score was significantly correlated with organ failure score and mortality. 2) Model of healthy human injected with LPS. After injection of LPS, inflammatory cytokines such as IL 6 and IL 8 and plasma levels of D-dimer and TF were elevated.

3) Improved simple scoring system; Platelet count < 100,000/μl was 1 point, 20 % reduction of platelet count was 1 point, PT-iNR >. 1.3 sec was 1 point, and 20% increase of PT-INR was 1 point. He examined the relationship between previous and new simple scoring system in 393 IC patients. New scoring system was well correlated with previous scoring system, APATH II score and mortality. More than 3 points of simple scoring score had poor outcome..

Dr Wada (Japan) reported a Guidan ce for diagnosis and treatment of DIC to harmonize three different guidelines established by the British Committee for Standards in Haematology (BCSH), the Japanese Society of Thrombosis and Hemostaisis (JSTH) and the Italian Society for Thrombosis and Hemostaisis (SISET). !) Diagnosis of DIC: There is no gold standard for the diagnosis of DIC and no single test by itself that is capable of accurately diagnosing DIC. Use of a scoring system is recommended (Moderate quality).The scoring system for DIC criteria is known to correlate with key clinical observations and outcomes (Moderate quality). It is important to repeat the tests to monitor the dynamic changes based on laboratory results and clinical observations (Moderate quality). 2) Treatment of the underlying disease: The cornerstone of DIC treatment is the treatment of the underlying condition (Moderate quality). 3) Transfusion of platelets and FFP: The transfusion of platelets is recommended in DIC patients with active bleeding and a platelet count of <50 x 103/μl or in those with a high risk of bleeding and a platelet count of <20 x 103/μl (Low quality). The administration of FFP may be useful in active bleeding patients with either prolonged PT/APTT (greater than 1.5 times normal) or decreased fibrinogen (less than 1.5 g/dl). It should be considered in DIC patients requiring an invasive

procedure with similar laboratory abnormalities (Low quality). 4) Antifibrinolytic treatment: Patients with DIC generally should not be treated with antifibrinolytic agents (Low quality). DIC patients that present with severe bleeding, haracterized by a marked hyperfibrinolytic state such as leukemia (Low quality) and trauma (Moderate quality), could be treated with antifibrinolytic agents. 5) Anticoagulants: Therapeutic doses of heparin should be considered in cases of DIC where thrombosis predominates (Low quality). The use of low molecular weight heparin (LMWH) is preferred to unfractionated heparin (UFH) in these cases. Prophylaxis for VTE with prophylactic doses of UFH or LMWH is recommended in critically ill, non-bleeding patients with DIC, (Moderate or High quality, respectively), but there is no direct evidence of the effects of anticoagulants on DIC. 6) Anticoagulant factor concentrates: Further prospective evidence from RCTs confirming a benefit is required. The administration of AT, recombinant TM or activated protein C (APC) may be considered in DIC patients.

Section for Diagnosis and treatments for DIC

Dr. Wada reported " Chairman's report” and introduced the project of DIC subcommittee.

1) The relationship between DIC and Trauma has been discussed, and one SSC communication and one review have been published on JTH (2012 and 2013, respectively). 2) ISTH guidance for diagnosis and treatment of DIC has been published on JTH (2013). 3) Standardization and of D- dimer and establishment of cut off value of D-dimer for diagnosis of DIC, 4) Establishment of modified non-overt DIC diagnostic criteria. 5) Investigation of new pathogenic mechanism of the onset of DIC. 6) Survey for DIC

Dr. Di Nisio (Italy) reported " SISET guidelines for DIC?” as following Scoring system for DIC was recommend (Grade C). Single test analysis for DIC was not recommended (Grade D). Treatment of underlying disease was recommended as cornerstone. Transfusion of platelet concentration, FFP and fibrinogen concentrate, cryoprecipitate were recommended (Grade D). FVIIa was not recommended (Grade D). UFH (treatment ) was not recommended (Grade D). UFH (prophylaxis for VTE) was recommended. LMWH was recommended (grade D). Synthetic protease inhibitor was not recommended (grade D). rhAPC was recommended (Grade D). AT and rhTM were not recommended (Grade D). Plasma exchange was not recommended (Grade D). He introduced the survey for DIC.

Dr Igor Bokarev (Russian Federation) presented " How we understand DIC in Russia”. He introduce the history and his practice in management of DIC. He introduced the concept of constant intravascular coagulation and diagnosis of DIC by the fibrinogen level and platelet count.

Pathogenic mechanisms for onset of DIC

Hyun Kyung Kim (South-Korea) presented " Prognostic value of thrombomodulin expression in peripheral monocytes in DIC”. She measured TM mRNA and CD14 levels in monocytes from 78 patients suspected DIC. Out of them, 54 patients were associated with overt-DIC and 24

patients died. TM mRNA levels decreased and CD14 increased in the non-survivor. TM mRNA level was negatively correlated with DIC score and PT and CD14 was correlated with those. .In vitro study, thrombin increases both TM and CD14. LPS increases CD14 but decrease TM in monocyte.

Dr. Shinichiro Kurosawa (USA) presented " DIC Border Patrol: Complement activation. He talked the possibility of sharing same pathogenic mechanism between DIC and TMA. TTP is caused by markedly decreased ADAMTS 13, and HUS is caused by verotoxin, and aHUS is caused by activation of complement. The activation of complement is considered to be high in gram positive infection but low in gram negative infection. Coagulation system and complement system may have a close talk. The activation of complement was observed in baboon injected with E-coli or verotokin.

The activation of complement was evaluated by C5b-9

Dr. Cheng-Hock Toh (United Kingdom) presented " DIC: Emerging histones- emerging relevance”.

Histone is positive charged protein to bind the negatively charged phospholipid of cell membrane. The histone causes endothelial cell death, thrombocytopenia and releases of inflammatory cytokines such as IL-6 and 8, although histone and elastase from neutrophil kills bacteria. In the histone infusion model, histone causes edema, hemorrhage and microvascular coagulation The histone increased plasma levels of TAT and caused the pathological changes in kidney and liver. More than 50 μ g/ml of histone caused dysfunction of respiratory system. In severe trauma, more than 50 μ g/ml of histone levels were observed. The complex of CRP- histone levels were increased in patients with infections.

Dr. Takashi Ito (Japan) presented " Massive thrombosis evoked by extracellular histones: is it DIC?” In preliminary study for sepsis, histone levels were significantly higher in non-survivor than in survivor. In the animal model injected with 1.5 μ g/ml of LPS, plasma levels of histone (1.1 ~ 10.2 μ g/ml) were increased from 8 hours to 12 hours. More than 60 μ g/ml of histone caused fatal DIC. A poikilocytosis, decrease of ADAMTS13, increase of TF and VWF, and microthrombi with rich VWF were observed in this model. Histone activate platelet aggregation and cause pulmonary thrombosis with platelet rich-thrombi and reduced TM expression. rhTM inhibit the effect of histone in animal model and platelet aggregation.

Registry of Exogenous Hemostatic Factors

June 30, 2013

Chairman: Jan Rosing (Netherlands) Co-Chairs: Kenneth J. Clemetson (Switzerland), Ivo Francischetti (USA), Manjunatha R. Kini (Singapore), Francis S. Markland Jr (USA), Mary Ann McLane (USA), Takashi Morita (Japan)

Jan Rosing chaired the session. Francis Markland Jr (US), Takashi Morita (JP), and Mary Ann McLane (US) were absent with apologies

Six members of the registry were in attendance plus about 100-120 guests.

Welcome: Jan Rosing, Chair

Minutes of the last meeting (Honolulu 2012) was approved.

Publication of the subcommittee

None

Educational programs

Two international workshops on "Thrombosis and Haemostasis: Discovery and Development of Tools and Therapeutics” were successfully conducted. The first one was held at Federal University of Santa Catarina, Florianopolis, Brazil (November 12-15, 2012) and the second one was held at University of Mysore, Mysore, India (December 8-11, 2012). These workshops were sponsored ISTH and respective institutions. The workshops were conducted by three international experts, namely, Ken Clemetson, Ivo Francischetti and Manjunatha Kini along with respective local scientists. We have been re-invited to run the workshops in different parts of Brazil and India. The local scientific institutions have agreed to sponsor all local expenses.

New inventories / activities

1. Nomenclature / classification / activities of L- amino acid oxidase is in progress. Ken Clemetson is working on it and the first draft will be ready in 2014.

2. Classification and nomenclature of exogenous factors from hematophagous animals. The project is too big and complicated. Ivo Francischetti will attempt come up with a strategy in 2014.

3. The National Institute for Biological Standards and Control (NIBSC) is responsible for the distribution and replacement of reference materials including WHO International Standards. Stocks of the WHO 1st IS for Ancrod (74/581) are now depleted and a replacement is needed.

The NISBC will propose the replacement project to WHO this year which also propose an approval route for the calibration exercise.

4. The SSC Registration of Exogenous Haemostatic Factors will provide an expert review of the study report i.e. distribute the report to its co-chairs and other related experts for comment. This expert review is a crucial part of the approval process and is required for the formal SSC approval of the project. All comments are then incorporated into the final report that goes to the WHO expert committee on biological standardisation (ECBS) for establishment.

5. Educational programs: Three international workshops on "Thrombosis and Haemostasis: Discovery and Development of Tools and Therapeutics” will be conducted. The first one will be held at Federal University of Rio de Janiero, Rio de Janiero, Brazil (November 12-15, 2013), the second one will be held at All India Institute of Medical Sciences, New Delhi, India (February 2- 6, 2014) and the third one will be held in Rajshahi University, Rajshahi, Bangladesh (February 8- 11, 2014). These workshops are sponsored ISTH, respective institutions, and local scientific agencies. The workshops will be conducted by four international experts, namely, Ken Clemetson (Switzerland), Jay Fox (US), Ivo Francischetti (US) and Manjunatha Kini (Singapore) along with respective local scientists.

6. Guidelines for treatment: Gerhard Johnson, Guidance Committee, ISTH asked whether we could come with guidelines for treatment of snakebite victims. Geoff Isbister is considering to set-up a committee to come up with the guidelines.

7. Fifth International meeting on Exogenous Factors Affecting Thrombosis and Haemostasis: It will be organized as the satellite meeting after the World Congress of the ISTH in Amsterdam in 2013. Prof. Jan Rosing, Chair of the Organizing Committee, informed that the meeting will be held in Amsterdam (July 5-6, 2013). We are planning for 30-50 participants.

Factor VIII and IX

Chairman: Flora Peyvandi (Italy) Co-Chairs: Jan Astermark (Sweden), Kathelijn Fischer (Netherlands), Michael Makris (UK), Danijela Mikovic (Serbia), Steven W. Pipe (USA), Elena Santagostino (Italy), Midori Shima (Japan) ,Leonard A. Valentino USA)

June 29, 2013

8.00 EDUCATIONAL SESSION

Session Chairpersons: David Lillicrap (Canada) and Flora Peyvandi (Italy)

Dr. Peyvandi and Dr. Lillicrap opened the session welcoming the audience. They apologised for the absence of Dr. Mikovic. She was not able to attend the meeting due to personal reasons, therefore more time will be dedicated to the other two presentations by Dr. Pipe and Dr. Collins.

Inhibitor risk associated with switching products including PUPs and PTPs. Steven Pipe (USA)

The prospect of a wave of new recombinant clotting factors advancing out of the development pipeline has highlighted concerns regarding how to assess the potential immunogenicity of these products. In addition, within the hemophilia community there remains a perceived increased risk of inhibitor formation among previously treated patients when switching between factor products, even in the absence of bioengineered modifications . This presentation reviewed the published experience with product switching, novel preclinical models for testing immunogenicity, as well as currently available inhibitor surveillance practices and based on data available in the literature it seems there is no increase of inhibitor rate after switching in PTO patients. Finally, the Delphi technique was introduced as potential approach for a more comprehensive approach to risk forecast/ assessment.

DISCUSSION:

 The importance of post-registration surveillance was underlined. Since for the new longer acting modified rFVIII and rFIX products it is more difficult to predict the risk of inhibitor development due to their genetic modifications and potentially aggravating environmental factors it would be important to request long term post registration surveillance trials.  It was also discussed to involve basic scientists (immunologists) to develop prospective cohort studies to verify a potential influence of the genetic modification and other environmental risk factors for inhibitor development.

Acquired Hemophilia: Diagnosis and treatment. Peter Collins (UK)

Management of acquired hemophilia A should be undertaken in close collaboration with a hemophilia center with expertise in the field. Treatment which is not required in 30% of patients involves controlling and preventing bleeds and immunosuppression to eradicate the auto-inhibitors. Prompt diagnosis is important to allow early haemostatic treatment and prevent unessential invasive procedures. First line hemostatic treatment should be with a bypassing agent and both recombinant activated factor VII and the activated prothrombin complex concentrate anti-inhibitor complex concentrate (FEIBA) are equally efficacious. Both rFVIIa and AICC are associated with thrombotic events when used in acquired hemophilia. The pivotal trial with porcine rFVIII awaits results and could be a treatment alternative, with the promise of fewer thromboembolic events and well measurable FVIII levels. Immunosuppression should be started as soon as a diagnosis has been confirmed. The combination of steroids and cyclophosphamide may induce remission in more patients than steroids alone. Current data do not suggest that rituximab results in better outcomes, rather slightly inferior. Relapse is common (10-20%) in the first 6 months after immunosuppression is stopped and patients need to be followed up regularly to allow early diagnosis and treatment of relapse.. is currently under development

DISCUSSION

Questions and comments from the audience:

 Effect of the delay of immunosuppression on eradication. Data should be thoroughly analysed, however, no association with mortality has been shown so far.  Should immunosuppression be stopped if there is no clinical response? Numbers too small to reach a final conclusion, but 3-5 weeks should be OK.  Dr. Lillicrap: could VWF be involved in unexplained bleeding after treatment? There are no convincing data supporting this.

Dr Mikovic had apologized for not being present due to personal reasons. Summary of her abstract

Laboratory and clinical phenotype comparison in rare bleeding disorders. Danijela Mikovic (Serbia)

According to clinical experience there is a heterogeneous association between coagulation factor activity level and clinical bleeding severity in patients with rare bleeding disorders (RBDs).

The aim of the report is:

 To give an overview of data on the association between the residual FV levels and clinical bleeding symptoms in patients with different RBDs.  To identify factor levels necessary to reduce major spontaneous bleeding  To point the inadequacy of the current available assays to define a minimum residual level of all coagulation factors and suggest the further research in potential role of global coagulation assays in accurate prediction of the hemorrhagic risk

 To indicate that a more detailed evaluation on each single factor deficiency is required for future planning of optimal diagnosis and management.

9.00 REPORT ON SSC FVIII&FIX ACTIVITY 2012 – 2013. Flora Peyvandi (Italy)

SSC reorganization

SOA FVII&FIX SSC (report on on-going and closing projects)

Dr Peyvandi opened the SSC business session by introducing the new SSC rules and strategy plan.

She thanked all co-chairs of this subcommittee for the work done in the last year and she welcomed Dr. Michael Makris, Dr. Danijela Mikovic and Dr. Elena Santagostino as new co-chairs wishing them a fruitful and successful collaboration. She announced that the SSC executive committee approved the new name of this SSC: Factor VIII, Factor IX and Rare Coagulation Disorders.

She reported about the state of the art of the SSC:

1. Recommendations published as Official SSC Communications on JTH

 Consensus definitions in rare bleeding disorders (Peyvandi et al, JTH 2012:10)  Pharmacokinetics (Bjorkman and Collins, JTH 2013:11)  Potency labelling of clotting factor concentrates (Hubbard et al, JTH 2013. doi: 10.1111/jth.12167)  Standardization of methods for performing the clot wave form analysis (Shima et al, JTH 2013. doi: 10.1111/jth.12287)Reports posted on the ISTH website for further comments and discussion

2. Recommendation submitted as Official SSC Communications to JTH

 Consensus definitions in haemophilia (Chair: V. Blanchette)  Standardization of methods for performing the thromboelastogram (Chair: G. Young and M. Chitlur)

3. Recommendation in preparation based on available data by SSC on Control of Anticoagulation (JTH 2011;9:1859–61)

 Standardization of methods for performing the thrombin generation test

4. Closing project:

 Clinical trial design for haemophilia (Chair: D. DiMichele). This project has been completed and the manuscript is in preparation.

Dr. Peyvandi presented new projects that started in late 2012 or early 2013:

 Evaluation of hemostatic efficacy of novel FVIII / FIX concentrates and FVIII-inhibitor by-passing agents (Chairs: A. Lawrie and A. Tripodi) – 2012/2014  Bleeding score in hemophilia: a prognostic tool for clinical outcome (Chairs: E.M. Mancuso and A. Tosetto) – 2012/2014  The definition of mild Haemophilia A (Chair: M. Makris) – 2012/2014  Factor V deficiency, clinical heterogeneity and treatment(Chair: D. Mikovic) – 2013/2015  Standardization and quality management of genetic assays for diagnosis of hemophilia (Chair: V. Jenkins) – 2013/2015  Prophylaxis in patients with and without inhibitors (Chairs: C. Escuriola and V. Blanchette) – 2013/2015  Consensus definitions and recommendations for immune tolerance induction (ITI) in hemophilia with inhibitors (Chair: E. Santagostino) – 2013/2015  Standardization of anti-FVIII inhibitor assays (Chair: K. Moertens) – 2013/2015

Finally Dr. Peyvandi highlighted the importance to start a new project on post-registration surveillance in patients using novel modified rFVIII and rFIX drugs.

Dr. Lillicrap underlines the importance of SSC now and in future.

Dr. Peyvandi reported the importance of collaboration between different SSC sub-committees in order to answer overlapping issues (e.g. bleeding in women affected with hemophilia, bleeding score ( BS) which could involve either FVIII, FIX and women Subcommittee or the issue of FDA- EMA harmonization on new drug registration. EMA requires the full clinical data base in children as part of the submission dossier for registration while FDA requires only data in adults (>12 years of age) for initial registration . The FVIII, FIX and Rare Coagulation Disorders SSC and the Pediatric SSC subcommittees should discuss and release a common recommendation.

9.20 CLINICAL OUTCOME EVALUATION - METHODS

Session Chairpersons: Michael Makris (UK) and Kathelijn Fischer (The Netherlands),

Dr. Fisher introduced the session.

The 2001 SSC communication defining the different levels of severity of haemophilia was a landmark publication that had a major impact in the field both at clinical and research levels. Two other SSC projects will attempt to refine the definitions above by examining the impact of FVIII:C and other parameters which might affect them. Another project on mild haemophilia will examine the issues of borders, discrepancy and inhibitor development.

SSC project: Bleeding score in hemophilia: a prognostic tool for clinical outcome. Alberto Tosetto (Italy)

In patients with haemophilia, the residual levels of clotting factor (FVIII or FIX) are considered as the most important prognostic factors. Based on that, haemophilia patients are classified into the severe, moderate and mild categories, despite several observation suggesting that this may be actually insufficient. For instance, about 10-20% of patients classified as having severe haemophilia A do not have major bleeding complications. Furthermore, there are very few data supporting a strong relationship between factor VIII level and bleeding symptoms in patients with moderate or mild haemophilia A. Despite this lack of knowledge, treatment strategies are often implemented, sometimes pre-emptively, only on the basis of residual factor level (ie., prophylaxis vs. on demand therapies). Appreciation of the true prognostic value of residual factor level is therefore a major, clinically relevant task to be undertaken by the SSC-ISTH Working Group on Clinical outcome evaluation. With this as background the Working Group will establish consensus-based clinical criteria to define the degree of severity of hemophilia independent from residual FVIII/FIX levels and will validate the predictive ability of such residual levels in a pre-defined cohort of haemophilia patients. Such consensus-based criteria on the degree of clinical severity of hemophilia will be established by means of the Delphi method. If FVIII/FIX predictive ability is not entirely satisfactory, the Working Group will define a protocol for eligible measurements to be tested for prognostic improvement over residual factor in patients with hemophilia.

SSC project: The definition of mild haemophilia A. Michael Makris (UK)

The current ISTH definition for severity of haemophilia defines mild haemophilia as a condition with FVIII:C levels between >0.05-0.40u/ml. This definition does not comment on how patients with FVIII:C between 0.40u/ml and the lower end of the normal range should be classified. This working group (WG) will examine the evidence to decide on whether the 0.40u/ml cut-off is justified or needs to be altered.

A further important issue is that the ISTH definition does not specify the type of FVIII:C assay to be used in the definition of haemophilia. It is now recognised that approximately 30% of patients with mild haemophilia exhibit discrepant FVIII:C levels depending on whether they are measured by the one- stage or chromogenic assay. The WG will define discrepancy when applied to FVIII assays so that different studies can be compared and will investigate the most appropriate assay for use in the definition of mild haemophilia A.

The third area to be addressed by the WG will be the issue of inhibitors in patients with mild haemophilia A to try and capture evidence as to which mutations are associated with inhibitor development, whether it is possible to confidently state which mutations are not associated with inhibitors and provide guidance on genetic testing of mild haemophilic individuals.

DISCUSSION

Questions and comments from the audience:

 SSC project on BS prediction: suggestion on using the software based on Delphi approach. Moreover it was suggested that it would be important to collect data also on patients not receiving any or very few treatments.  Which assay should be used to define mild haemophilia or unexplained mild bleeding disorder? One-stage assay or Chromogenic assay?

10.20 INHIBITOR DEVELOPMENT – METHODS AND RESULTS

Session Chairpersons: Steven Pipe (USA) and Marijke van den Berg (The Netherlands)

Dr. van den Berg introduced the session.

SSC project: Inhibitor assays standardization. Koen Mertens (The Netherlands)

The development of a FVIII inhibitor standard has been initiated at the 2003 Subcommittee Meeting, and subsequently endorsed by WHO and EMEA. The initial study, which included 22 laboratories and 5 candidate materials, has been performed by Dr. Raut from NIBSC. In 2006 he presented an interim analysis at the SSC meeting in Oslo and at the WHO-ECBS meeting in Geneva. Unfortunately, none of the candidate materials was considered suitable for establishment as the 1st International Standard for FVIII Inhibitors. This was due to a variety of concerns, including the origin of some of the materials (rabbit versus human), and the overall poor quality of the data generated. The rabbit Mab was deemed most suitable, though not accepted by the competent authorities. Both inter- and intra-laboratory variability were undesirable high (CVs up to 36%), and recalculating the results relative to any of the 5 candidate preparations did only marginally improve the inter-laboratory CVs. Moreover, information was lacking regarding the statistical validity of the individual inhibitor assays performed.

In 2007, a Working Party was established (Chair: K. Mertens) with the aim to perform a ‘post- hoc’ statistical analysis on the raw data in order to assess the validity of the participants’ inhibitor titre estimates. Remarkably, this revealed that most participants performed inhibitor assays in a format that does not allow any statistical evaluation at all. As proposed at the SSC meetings in 2008 and 2010, the only option to proceed would be a new, more controlled collaborative study. This would require an assay format that complies with statistical requirements, and additional candidate materials, including some of human origin.

As for new materials, Dr. Mertens’ laboratory has produced new recombinant antibodies which are cloned from the immune repertoire from inhibitor patients. Whether these monoclonals are suitable for "like versus like” testing of polyclonal patient samples remains to be investigated. At NIBSC, Dr. Raut has performed studies on a new assay format as an alternative to the regular Bethesda/Nijmegen assay. These recent developments may provide a starting point for bringing this project to some completion. The project group will focus on: (1) recommendations for the assessment of inhibitor titres, and (2) characterization of reference reagents (not necessarily standards) to facilitate inhibitor testing. In absence of these tools, the

establishment of an International Standard for FVIII Inhibitors may remain an unrealistic goal. Any comments or suggestions from the Subcommittee are welcome at [email protected] for discussion in the FVIII inhibitor standard project group.

Membrane composition can alter results of factor VIII inhibitor assays. Gary Gilbert (USA)

The C2 domain of factor VIII binds to phospholipid membranes and is a dominant epitope for inhibitory antibodies. These antibodies alter the interaction with phospholipid membranes but, in many cases, do not entirely block membrane binding. Thus there is a need for basic insights that explain the mechanism through which anti-C2 antibodies inhibit activity of factor VIII. There are qualitative differences in the interaction of factor VIII with membranes of low phosphatidylserine content (<12%) where binding results from stereoselective interaction with phosphatidyl-L-serine vs. membranes with high phosphatidylserine content, where negative net charge is critical. Thus, we have hypothesized that the poor predictive value of factor VIII assays may be related to measurement on membranes of non-physiologic composition. mAb ESH4, directed against the factor VIII C2 domain, interferes with membrane binding and is a prototypic factor VIII inhibitor. ESH4 is a type II inhibitor, with residual factor VIII activity in the presence of saturating antibody concentrations.

ESH4 decreased the apparent affinity 4-fold for membranes of 4% PS (KD = 4.8 ± 0.4 M without and 21 ± 4 M with ESH4) but only 2-fold on 15% PS vesicles (KD = 1.3 ± 0.2 M without and 2.5 ± 0.5 M with ESH4). The apparent affinity for factor X in the presence of saturating phospholipid and ESH4 was higher on 15% PS vesicles (KM = 129 ± 18 nM) than on 4% PS vesicles (KM = 284 ± 30 nM). To determine the mechanism through which ESH4 inhibits membrane-bound factor VIII activity, we measured the fluorescence anisotropy of factor IXa-Fl- EGRck. ESH4 decreased anisotropy of the factor VIIIa-factor IXa-factor X complex from 0.280 ± 0.002 to 0.272 ± 0.002 indicating that ESH4 alters the Vmax of the membrane-bound factor VIIIa-factor IXa complex through a change near the factor IXa active site. ESH4 inhibited 28% and 36% of factor VIII activity in aPTT-based and 2-stage commercial factor VIII assays, respectively. In contrast, factor VIII inhibited 78 ± 12% of factor VIII activity supported by thrombin-stimulated platelets.

The results indicate that anti-C2 domain antibodies can inhibit factor VIII activity through modulation of the membrane-bound factor VIIIa-factor IXa complex. The 2-fold difference between inhibition in commercial factor VIII assays vs. an assay supported by thrombin- stimulated platelets suggests a strategy for improving the predictive value of factor VIII assays in patients with inhibitory antibodies.

DISCUSSION

Questions and comments from the audience:

The difference among all new products coming on the market is a big source of variability: one standardized assay for all of them or specific assays for each one? The community decided to go step by step and first focus on one standard assay, which is sensitive and can be used for all new replacement therapy products .

SIPPET study: power and weakness of the study. Pier Mannuccio Mannucci (Italy)

Clinical data stemming from a systematic review that summarized a few retrospective studies suggest that the source of factor VIII used for replacement therapy (plasma or recombinant DNA technology) might have some influence on the cumulative incidence of inhibitors [1]. A more recent systematic review and meta-analysis found a two-fold higher inhibitor incidence with the use of recombinant factors, but the difference in favor of plasma-derived factor VIII was no longer statistically significant when variables such as study design, study period and frequency of inhibitor testing were included as confounders [2]. To tackle this state of current uncertainty on this issue, the randomized, prospective independent SIPPET trial [3], ongoing now in 19 countries from 5 4 continents, is planned to enroll 300 previously untreated or minimally treated patients with severe hemophilia A at risk of developing factor VIII inhibitors, in order to establish whether or not plasma-derived factor VIII products are less immunogenic than recombinant products. At this time, SIPPET has already enrolled 241 patients with 208 patients having received at least one dose and an interim analysis of the available data is planned within the current year when 50% of the planned patients have achieved at least 20 exposure days. So far 40 inhibitor patients have been identified.

References

1. Paisley S et al. Haemophilia 2003;9:405-17. 2. Iorio A et al. J Thromb Haemost 2010;8:1256-65. 3. Mannucci PM et al. Haemophilia 2007;13 (Suppl 5):65-8.

RODIN study: power and weakness of the study. Marijke van den Berg (The Netherlands)

The PedNet registry was initiated in 2004, included patients born from beginning of 2000 and apart from that aimed to collect prospective data of all children diagnosed with severe hemophilia born in one of the participating Haemophilia treatment centers (HTC’s). The RODIN study was the first satellite study and it was set up to investigate both genetic and treatment related factors for inhibitor development. As of May 2011 the 516 PUPs reached the study end point with major center variability in incidences of inhibitor patients. From September 2012 also the N=8 ex-RODIN centers became part of the PedNet study group. The aim for the 30 centers is also to include newborns from 2010 onwards of both hemophilia A and B with factor VIII/IX levels <25%. The strength of multi center observational studies in rare diseases is the possibility to collect large numbers of patients. This makes it possible to study correlation between different variables that have an impact on disease outcome. Selection bias is an important confounder and could largely be prevented in the PedNet study group.

Inhibitor development in PTP: available registries and harmonization. Alfonso Iorio (Canada)

The development of inhibitors in patients previously exposed to factor concentrates is a rare event, with estimates be in the order of 2 per 1000 patient year (95% CI 1-4)(1). Both the UK and Canadian registry have shown an increase of this rate with the age of the patient (2,3). Historically, the interest in inhibitors in PTPs has been mainly related to the use of this population as the preferred one to assess the immunogenicity of new factor concentrates before their approval for clinical use (4). More recently, the analysis of the development of inhibitors in PTPs has been proposed for the comparison of the immunogenicity of different products and for the assessment of the risk of developing inhibitors as a consequence of switching factor concentrates (1,5). Furthermore, recent publications and ongoing trials have employed or suggested the use of PUPs to answer similar or overlapping questions (6,7). Several challenges make this issue interesting for the ISTH SSC on factor VIII. To the purpose of the assessment of inhibitor rates, the rarity of the event requires large scale data collection to gather relatively stable estimates; diagnostic modalities (upon clinical suspicion versus routine testing, laboratory assay used, frequency and modality of testing) should be recorded if standardization and central testing is unfeasible; size of the exposed population under surveillance has to be reliably assessed to approximate as much as possible an inception cohort; the clinical details of the events occurring before inhibitor development and the clinical course of the inhibitors should be collected in a simple but standardized way. To the purpose of performing surveillance in patients undergoing a concentrate switch, surveillance should be performed in a consistent and identical way in switcher and not switcher over a reasonable time frame. Recent surveys of the information currently collected by existing registries and surveillance schemes outline the need for a harmonization process. For most of these aspects, no evidence exists to guide practice, so that empirical protocols would be better developed after gathering advice from immunologist and epidemiologist, and endorsed by the SSC.

References

1. Iorio A et al. Blood, 2012;120:720-7. 2. Hay CRM et al. Blood, 2011;117:6367–70. 3. Webert KE et al. Haemophilia, 2012;18; e254-e259. 4. White GC et al. Thromb Haemost, 2001;85:560. 5. Aledort LM et al. J Thromb Haemost, 2011;9:2180–92. 6. Gouw SC et al. New Eng J Med , 2013;368:231–9. 7. Iorio A et al. J Thromb Haemost, 2010;8:1256–65.

DISCUSSION

Questions and comments from the audience:

 Dr. Rosendaal states that probably we should not talk anymore of retrospective and prospective studies creating a paradox: data are ALWAYS collected after the event.

14.00 NEW HEMOSTATIC DRUGS: CLASSIFICATION, DOSE STANDARDISATION AND MONITORING

Session Chairpersons: Flora Peyvandi (Italy) and David Lillicrap (Canada)

Dr. Lillicrap introduced the session.

SSC project: Clinical trial design for hemophilia. Donna Di Michele (USA)

The Project Group on Clinical Trials for New Products in Hemophilia (CTPG) was created in February 2011 by the FVIII/IX Subcommittee of the International Society for Thrombosis and Hemostasis (ISTH) to examine current rationale and propose alternative strategies for the design of pre- and post-authorization (licensure) clinical trials and studies for new therapeutics in hemophilia. Its mandate arose from pragmatic concerns about the feasibility of populating and conducting multiple simultaneous new clotting factor trials. These concerns led to the question of whether innovative and evidence-based approaches to trial simplification might increase feasibility without compromising assessment of product safety and efficacy. Moreover, the project group was envisioned as a forum for the participatory discussion of the EMA’s and FDA’s respective regulatory goals for new product clinical trials, and the potential for inter- agency harmonization of the most critical regulatory requirements.

Based on this rationale, the CTPG was tasked with exploring alternative statistical models for the pre- and post- licensure study of so called ‘me-too’ and novel biologics for hemophilia A and B (with /without inhibitors), while giving due consideration to: a) safety and efficacy data required by regulators for marketing authorization; b) number of potential subjects available world-wide for multiple simultaneously- conducted trials; and c) innovative clinical trial design suitable for rare diseases such as hemophilia. The group was constituted according to member expertise in clinical care and investigation, immunology, clinical trial design and statistics methodology, and included regulatory science representatives FDA and the EMA. The group met monthly between February 2011 and June 2013 to 1) review the known clinical data on factor VIII and IX product immunogenicity (and any emerging data for novel biologics) in previously treated (PTPs) and previously untreated (PUPs) patients; 2) explore the potential impact of alternative statistical modelling and innovative trial design on the pre-authorization regulatory requirements for product safety and efficacy determination; 3) examine the current scientific concepts of immunogenicity and neo-antigenicity and their potential influence on clinical trial design and novel approaches to antibody surveillance; 4) develop consensus safety and efficacy clinical endpoint definitions (building on the work of the Definitions Project Group); and 5) formulate recommendations. Progress reports were provided and input from critical stakeholders in the hemophilia community was solicited at international meetings that included the Scientific and Standardization Committee of the ISTH (2011, 2012); World Federation of Hemophilia (WFH) meeting (2012); and the WFH Global Research Forum (2011; 2013). The hemophilia biologics industry was also surveyed in 2012 for its input with respect to perceived regulatory barriers to efficient and timely product registration; alternative paradigms for clinical safety and efficacy monitoring in pre- and post- licensure studies; and potential benefits to be

derived from strategic harmonization of key FDA and EMA regulatory requirements. Finally, comments were solicited from selected experts in clinical trial design methodology as well as the FVIII/IX Subcommittee membership prior to publication.

The CTPG ultimately narrowed the project scope to the following recommendations for an alternative approach to statistical modelling and the clinical trial design of pre-authorization trials for both ‘me-too’ and novel factor VIII (FVIII) biologics in PTPs. This approach was rooted in the combined agency regulatory goals for pre-licensure studies, and incorporates both current immunological theories of neo-antigenicity and consensus clinical efficacy endpoint definitions. The group considered several innovative approaches to the clinical design of new product safety (immunogenicity) trials. All proposed strategies were based on the known epidemiology and immunology of FVIII inhibitor development in congenital haemophilia A. The CTPG explored how epidemiology might best inform the traditional design of the single arm pre-licensure new product study with respect to subject number and duration, and considered alternative clinical designs that incorporate either Bayesian modelling or adaptive design. The group also recognized that the optimal design of pre-and post-authorization clinical trials remains hampered by poor understanding of the precise nature of the interactions between the therapeutic FVIII products (both ‘me too’ and novel biologics), and the recipient’s immune system. This is in part due to the incomplete characterization of the PTP subject’s immune status relative to the therapeutic product(s) received prior to recruitment into a new product trial such that immunological ignorance is not routinely distinguished from active tolerance. Critical information about genetics, cross-reactive material (CRM)-status and treatment intensity history is also frequently lacking. The CTPG therefore advocated for the systematic and harmonized collection of clinical and biological data from subjects entering pre- and post authorization new product studies. It envisioned complementary but integrated data sets may be required to satisfactorily address regulatory, scientific and national/ international surveillance priorities. The ultimate intent of this harmonized data collection would be a more evidence-based rational design of clinical trials for new therapeutics in which a smaller but well characterized and homogenous subject cohort would more precisely define the risk for inhibitor development in specific populations.

The CTPG also recommended that the FVIII/IX Subcommittee establish a new project group dedicated to the feasible implementation of international harmonized post-authorization studies using existing national and international database infrastructure and consensus standardized minimum datasets. Consideration should be given to the implementation of consensus clinical outcomes definitions that, while tailored to the specific period of observation, are themselves harmonized with pre-authorization clinical endpoints in order to maximize the potential for continuous longitudinal data collection on well characterized populations. The CTPG suggests that such a coordinated post- marketing effort could collect strategic data that contribute to a greater understanding of immunogenicity and the potential implementation of novel more sensitive antibody detection assays. Finally, the group advocated for greater EMA/FDA harmonization in critical regulatory areas such as evolving landscape of inhibitor assays and the approach to product authorization in children. A manuscript summarizing this PG’s activities and recommendations is nearing final draft form and will be

sent shortly to the FVIII/IX Subcommittee membership for final comment prior to submission for publication.

Immunogenicity of novel products and its evaluation. Sebastien Lacroix-Desmasez (France)

The Project Group on Clinical Trials for New Products in Hemophilia (CTPG) was created in February 2011 by the FVIII/IX Subcommittee of the International Society for Thrombosis and Hemostasis to examine current rationale and propose alternative strategies for the design of pre- and post-authorization (licensure) clinical trials and studies for new therapeutics in hemophilia, with a focus on FVIII biologics.

The CTPG has recognized that the optimal design of pre-and post-authorization clinical trials for FVIII biologics in previously treated patients (PTPs) with hemophilia A is limited by the poor understanding of the precise nature of the interactions between the therapeutic FVIII products and the recipient’s immune system, and by the imprecisely characterized rate of allo- immunization to FVIII therapeutics.

Optimization of clinical trials therefore requires the systematic collection of information that is potentially available but not routinely registered. The latter information includes i) the immune status of the PTPs relative to the therapeutic FVIII products received prior to recruitment in a new product trial, which should distinguish between immunological ignorance and active tolerance, ii) the cross-reactive material (CRM)-status of the patients, and iii) the genetics and the treatment intensity history. Further, the true rate of inhibitor development in PTPs should be determined including parameters such as age, treatment intensity/bleeding pattern, product use and product switch.

Development of novel clinical trials would then proceed in two phases. Firstly, a mandatory international registry should be established for the systematic and harmonized collection of clinical and biological data from subjects entering pre- and post-authorization product studies. A more rationale design of clinical trials could then be based on registry-derived evidence, and might be expected to include a smaller but well characterized and homogenous subject cohort with a more precisely defined risk for inhibitor development.

Efficient model of safety evaluation of novel products. Frits Rosendaal (The Netherland)

New opportunities in drug design and modification pose a paradoxical challenge of luxury: after a decade in which clotting factor concentrates have remained largely unchanged, we are currently observing a series of innovative drugs in various stages of development that offer a view of major advances in, for instance, extended half-life and ease of administration. In addition, biosimilar compounds will be developed that offer the benefits of currently available products at a far reduced price, which may close the treatment gap between the rich and the poor. New drugs need to be tested for efficacy and safety, and here is where the paradox comes in, i.e., that haemophilia is such a rare disease that stringent criteria for licensing studies may lead to a shortage of patients to be entered into clinical trials. Several solutions are

conceivable: firstly, in revisiting the criteria. Secondly, in making informed choices and only allow products in the phase of clinical testing that indeed have the potential of a major advance. Thirdly, to use the most efficient study design. For the latter, we will discuss the approach to establishing safety with regard to neo-antigenicity (inhibitor development), which is currently the sample-size-limiting aspect of required studies. Several approaches can be applied that all share some characteristics. The first is two apply prior experiences with product-related neo-antigenicity, and two develop study designs that incorporate these patterns. A second is to use an adaptive trial design, in which predefined stages of subsequent trials are set, not unlike predefined interim analysis. The third is to use Bayesian approaches, which is the method that relies most strongly on predefined expectations, and which may be appropriate for biosimilars.

Timelines of approval of new products: patients’ view. Brian O'Mahony (Ireland)

The Haemophilia patient community are positively anticipating the licencing and availability of a new generation of longer acting factor concentrates which will provide an additional therapeutic option and the possibility to transform the current treatment paradigm. The availability of these new products, if introduced on a cost sustainable basis, could also lead to a change in the economics and availability of the current recombinant and plasma derived factor concentrates. There are real possibilities of improving access to treatment for countries at all levels of current treatment availability. We are greatly concerned that these positive developments may be negated by delays in licencing of these products in Europe and the impractical application of orphan drug market exclusivity to these products. The requirement to submit paediatric PTP data before initial market approval may delay access to these products for two to three years in the EU compared to the USA and Canada. Apart from depriving adult patients from more attractive product options, this, may lead to unacceptably high launch prices in Europe for these products based on historically higher prices in the USA and Canada. This could create difficulty in reimbursement of these products in Europe. Many of the new products under development have been granted Orphan Drug designation. However, it is the view of the patient community in Europe that market exclusivity should not be granted to the first of the longer acting FVIII or FIX products to be licenced. The products under development use demonstrably different protein modification or enhancement methodology and in our view should not be classed as similar products for this purpose. If market exclusivity is granted, this will lead to a lack of therapeutic options, a monopoly with consequent higher costs and a very significant reduction in the potential benefit to the community.

Global assays standardization: what is needed? Guy Young (USA)

Over the past decade, through the work of a number of investigators worldwide, there has been substantial progress made regarding the understanding of the use of global hemostatic assays in the field of hemophilia. Important studies have evaluated different methodologies, demonstrated the ability of these assays to distinguish different phenotypes of hemophilia, and have also demonstrated in small pilot studies the potential clinical applications for monitoring of factor therapy including bypassing agent treatment in inhibitor patients. In order to move

the field forward and importantly to develop these assays as a clinical tool, the first step is to have a unified, standardized approach to conducting the testing. Thus far, among a number of global assays that have been studied, two specific assays have emerged as the leading candidates for clinical application: thrombin generation test and thromboelastography. With respect to thrombin generation testing, the calibrated automated thombogram has become the main device used in hemophilia studies and a standardized protocol has been developed by one center (1) although this protocol is yet to be accepted as an international standard (2). The situation for thromboelastography is similar although with 2 key differences. First, there are two devices, the TEG®5000 Analzyer from Haemonetics and the ROTEM® delta from Tem International which are available. While the devices differ slightly in their specifications, their output in terms of quantifiable parameters and graphical representations are identical. The second difference is that the Working Party on Thromboelastography Standardization has developed a standardized protocol which will be published shortly as an SSC Communication in the Journal of Thrombosis and Hemostasis (3) and will allow investigators in this area move forward with a unified approach. For a detailed discussion of the state-of-the-art with respect to these assays, the following reference is recommended (4).

References

1. Dargaud Y et al. Thromb Res 2012; 130:929-934. 2. Loeffen R et al. J Thromb Hemost 2012; 10:2544-54. 3. Chitlur M et al. Recommendations for the methodology to perform thromboelastography/thromboelastometry in patients with hemophilia: A SSC communication from the project on standardization of methods for performing thromboelastography. Submitted to J Thromb Hemost. 4. Young G et al. Blood 2013; 121:1944-1950.

SSC project: Evaluation of hemostatic efficacy of novel FVIII / FIX concentrates and FVIII- inhibitor by-passing agents. Andrew Lawrie (UK)

INTRODUCTION: Assessment of new therapeutic agents that are not directly comparable to a WHO (plasma derived) concentrate standard is a topic that concerns. As Pfizer have already identified that this is an issue and produced a ReFacto AF Laboratory Standard, it was felt that this product specific reference preparation could be used to investigate possible alternative analytical methods such as thrombin generation or thromboelastography that could be used to evaluate the potency of new products for treatment in haemophilia.

METHODS: Initial studies were performed using one-stage clotting and chromogenic factor VIII (FVIII) assay techniques, employing two analytical systems (Reagent / Analyser combinations). The analysers were calibrated using the 8th International Standard Factor for VIII Concentrate (07/350) pre-diluted to ~1 IU/mL in system specific FVIII deficient plasma. The 13th British Standard Factor VIII Concentrate (10/188) and ReFacto AF Laboratory Standard (RAFLS Lot 10/246) were pre-diluting as for 07/350. Thrombin generation was performed using the Calibrated Automated Thrombogram (CAT) with Thrombinoscope PPP reagent low. Rotational thromboelastometry (ROTEM) was performed using Sekisui Diagnostics dilute prothrombin

time reagent (reaction concentration ~3 pmol/L Tissue Factor. For both the CAT and ROTEM 0.1mL of concentrate was added to 0.9 mL factor VIII deficient plasma from: Precision BioLogic Inc, Siemens and IL

RESULTS / DISCUSSION: Both the one-stage FVIII clotting assays and chromogenic FVIII assay the BS FVIII (10/188) and ReFacto AF (10/246) gave linear dose response curves that were parallel to the IS FVIII (07/350) calibration curve. Mean relative potency estimates for the BS FVIII were similar by each of the assay techniques; however the mean relative potencies for ReFacto AF (10/246) were clearly influenced by the assay system. Thrombin generation showed that the three sources of factor deficient plasma had different TGT characteristics in terms of lag time, time to peak, peak and endogenous thrombin potential (ETP). These characteristics shown by the deficient plasmas were mirrored in the thrombin generation curved produced when they were spiked with FVIII concentrates. For each of the substrate plasmas of BS FVIII (10/188) and ReFacto AF (10/246) ETP and Peak thrombin were normalised relative to the values from IS FVIII (07/350), however this failed to produce similar results for each of the deficient plasmas systems. Rotational thromboelastometry performed on factor deficient plasmas and those deficient plasmas spiked with the concentrate exhibited only minor changes in the ROTEM trace, those changes relating only to the alpha angle i.e. the rate at which clot firmness increased.

CONCLUSIONS: Under the study conditions results from the CAT and ROTEM were dependent on the source of factor FVIII deficient plasma used to dilute the FVIII concentrate. Therefore these alternative techniques do not appear to offer any advantages over the conventional assay systems.

DISCUSSION

Questions and comments from the audience:

 On the opportunity to use Bayesian approach or two stage approach as statistical analysis: Prof. Rosendaal answers that this is an issue the scientific community have to decide on.  Is it possible to approach the standardization of pharmacokinetics studies in paediatric patients in the same way Dr. DiMichele approached in the clinical trial design recommendation for the safety and efficacy evaluation? This will help to avoid delay due to regulatory requests?: Dr. DiMichele answers this could be done, but it is necessary to set the method first.  What will be the timeline and do we need to obtain harmonization between two regulatory bodies of FDA and EMA for performing clinical studies with novel drugs?: Dr. O’Mahony answers he is afraid it won’t be possible to reach this goal, while Dr. Srivastava invites to continue to look for such an important agreement. He further re-iterated that the community has not strongly enough stood together to allow the EU guideline regulating the ped data base being a pre- requisite for submission of dossier for product approval in EU.

15.30 ASSAYS STANDARDIZATION

Session Chairpersons: Jan Astermark (Sweden) and Midori Shima (Japan)

Dr. Astermark introduced the session.

Precision in estimation of the FVIII and FIX levels is a prerequisite for accurate correlation between activity levels and clinical symptoms. It is also crucial for true characterization and prediction of phenotypes. Assay discrepancies are a well-known occurrence in the case of FVIII measurement, but less so for FIX. In addition, genetic analyses are now more readily available and used for both characterization of the disease as well as for carrier and prenatal testing. These assays, however, require standardization and quality controls. This session will highlight two important projects in the area of laboratory testing

Relationship between results with different FIX assays in post concentrate infusion samples.

Steve Kitchen (UK NEQAS Blood Coagulation)

Approximately 60 centres participated in a collaborative study assessing FIX assays. Samples were collected from a haemophilia B carrier and 2 subjects with severe haemophilia B after infusion of FIX concentrates with written informed consent. Two subjects were infused with recombinant FIX (BeneFIX, Pfizer) and one with high purity plasma derived concentrate (Replenine, BPL ).

The details were as follows

Sample Baseline FIX (IU/dl) Concentrate sample collection details

01 37 IU/dl BeneFIX 20 mins after 3000 units

02 <1 IU/dl Replenine 20 mins after 3000 units

03 <1 IU/dl BeneFIX 20 mins after 6000 units

All samples were lyophilized protocols in 0.5 ml aliquots using established UK NEQAS BC and dispatched to participating centres through the post. Centres were requested to assay these as thought they were routine samples for monitoring patients during treatment with FIX concentrates. Median results for centres calibrating assays using plasma standards are shown in the table below

Assay n sample 01 sample 02 sample 03 median CV median CV median CV

IU/dl IU/dl IU/dl Chromogenic 3 71 - 37 - 61.5 - (Rossix) Chromogenic 2 77 - 37 - 74.5 - (Hyphen)

One stage 27 89 10.1% 39 7.3% 92 11.1%

IL reagents One stage Siemens 16 87.2 6.3% 32.5 9.6% 82.5 7.5% reagents All chromogenic 5 71 - 37 - 63.5 - All one stage 61 87.5 10.3% 37..7 12.7% 87 13.3%

One stage results with the reagent set from IL (Synthasil APTT reagent, IL deficient plasma IL reference plasma) were significantly greater than those obtained with the Siemens reagent set (AFS APTT reagent, Siemens deficient plasma, Siemens reference plasma) for samples 02 ( p<0.0001) and 03 (p<0.02).

When results obtained by different methods were combined chromogenic assay results were significantly lower than one stage results for samples 01 ( p<0.002) and 03 ( p<0.01).

Our data indicate that FIX results vary according to the assay methods used in some samples from patients treated with recombinant or plasma derived concentrates.

SSC project: Standardization of genetic assays for diagnosis of hemophilia. Vincent Jenkins (Ireland)

Mutation analysis of haemophilia A and haemophilia B is now routinely available in many countries and increasingly available internationally. Carrier and prenatal diagnosis by molecular analysis are now standard techniques. Despite the growth in molecular testing there are relatively few guidelines and external quality assurance schemes for the molecular analysis of the F8 and F9 genes. This project will survey current practices in diagnostic laboratories by means of a questionnaire. The aim is to harmonise diagnostic practice especially the reporting of genetic results, and encourage the development of appropriate quality assurance.

DISCUSSION

Questions and comments from the audience:

 Percentage of non-participating centres to quality exercise is high, does it mean there are lot of inadequate labs?: no, only that they decide not to take part.  It seems there is different specific type of intron 22 inversion mutation .Is there any different method to identify these type of variants for intron 22 inversion mutation? how to solve this problem during an exercise? Dr. Jenkins explains that the test we are using now is very basic one and at the beginning of this exercise we just need to understand the basic information and later one we could include more specific questions.

16.25 ORPHAN DRUGS IN RARE BLEEDING DISORDERS (RBDS)

Session Chairpersons: Elena Santagostino (Italy) and Danijela Mikovic

Dr Santagostino introduced the session, explaining that Dr Mikovic had apologized for not being present due to personal reasons and the SSC Project will be presented by Dr. Roberta Palla.

SSC project: Factor V deficiency, clinical heterogeneity and treatment. Roberta Palla (Italy)

Inherited factor V (FV) deficiency is an autosomal recessive rare bleeding disorder with estimated prevalence of one per million in the general population.

The phenotypic expression is variable. Homozygotes and compound heterozygotes show mild to severe bleeding symptoms while heterozygotes are usually asymptomatic or experience mild symptoms. Main clinical manifestations are mucosal and postoperative haemorrhage but haemarthroses and intracranial haemorrhages can also occur. The reasons for clinical heterogeneity are largely unknown. No clear correlation between genotypes and the clinical phenotypes has been identified.

Clinical severity shows weak association with laboratory phenotype. There is no ready explanation for the differences in bleeding phenotype between patients with equally low or undetectable FV levels. Several observations indicate the crucial role of platelets in FV deficiency. Although most FV is present in plasma, approximately 20% of the circulating FV is found within platelet alpha-granules. Residual platelet FV might be responsible for the vast differences in bleeding phenotype observed among patients with equally undetectable plasma FV levels. Moreover, recent findings show that inter-individual differences in tissue factor pathway inhibitor (TFPI) plasma levels might be contributing factors.

Replacement therapy with fresh frozen plasma (FFP), preferably virus-inactivated, remains the mainstay of treatment in the absence of FV concentrate. In most patients FFP is administered on demand, rarely prophylactically in severely affected patients. Potential risks of treatment with FFP are allergic reaction, infection and volume overload. In refractory cases or in patients with inhibitors platelet transfusions and recombinant activated FVIIa were used, with variable efficacy.

The SSC project will enable collection of prospective information related to FV deficiency patients using web-application PRO-RBDD database. Aims of the project are to determine potential genetic, laboratory and clinical predictors of bleeding severity, to evaluate efficacy and safety of treatment options in order to improve care due to increased use of standardized therapeutic approach. Collection of sufficient data should allow design of a clinical trial of newly produced FV concentrate.

Novel products for treatment of RBDs: clinical trial design and methods for efficacy evaluation (antithrombin RNAi) Amy Simon (Alnylam Pharmaceuticals , USA)

Rare Bleeding Disorders (RBD) impact over 14,000 people worldwide, according to the World Federation of Hemophilia 2010 Global Survey. Recently, there has been increased understanding about the epidemiology, clinical course and treatment of these different RBDs due to the establishment of national and international networks and registries. While treatment options are available for RBD patients, such as fresh frozen plasma, activated prothrombin complex concentrates, or factor concentrates in some cases (e.g. FVII, FXIII), there is still an unmet need for better therapeutic options in RBD patients. In addition, there are a subset of RBD patients with a severe clinical phenotype that could benefit from a more feasible prophylaxis option. To this end, we have initiated a therapeutic program targeting the natural anticoagulant antithrombin (AT) through RNA interference (RNAi) as a mechanism to enhance thrombin generation and clot formation in patients with bleeding disorders. Supportive data for this therapeutic approach can be found in the literature from animal models as well as humans with hemophilia who have coinheritance of thrombophilic mutations, such as AT deficiency. We have developed a small interfering RNA (siRNA), ALN-AT3SC conjugated to a hepatocyte targeted ligand Gal-NAc, that results in the potent AT reduction as seen by liver mRNA as well plasma AT levels (by antigen and activity assays) in multiple species, with an ED50 of 1 mg/kg after a single subcutaneously administered dose. Chronic weekly dosing with 6 or more doses at 0.125, 0.25 or, 0.50 mg/kg was well tolerated in wild type non-human primate (NHP) and resulted in approximately 50%, 65% and 85% AT suppression, respectively. Importantly, , data from both in vitro studies using hemophilia patient plasma and in vivo studies from NHP have demonstrated that 50% or greater reduction in plasma AT resulted in increased thrombin generation as seen by a 2 to 4-fold increase in the ETP. Furthermore, reduction of AT by 50% or more resulted in improved hemostasis after laser injury in hemophilia A mice. These combined preclinical studies provide validation for AT as a target for enhancing thrombin generation and improving hemostasis. Current studies are ongoing in large animal models of hemophilia, and clinical studies in man will commence in 2013. If ALN-AT3SC is found to improve hemostasis in clinical trials, it may offer a novel, attractive prophylaxis option for RBD patients, given its subcutaneous route of administration and long duration of action.

Novel Factor V concentrate: orphan drug and clinical trial. Claudia Nardini (Kedrion, Italy)

Patients affected by coagulation factor V deficiencies, classified as rare diseases, cannot lean on a suitable clinical therapy, since so far no factor V concentrate is available on the market. FFP infusion remains indeed the mainstay of treatment, implying several and established side effects, such as volume overload, risk of allergic reactions and pathogens transmission.

The aim of Kedrion’s project is to bridge the gap between patients’ needs and existing therapies, developing a plasma derived factor V concentrate targeted to on demand/prophylaxis replacement treatment, in order to overcome all the drawbacks deriving from the administration of huge volumes of undefined pathogen safety plasma.

The development of such a product implies the planning and execution of several steps in order to make it available for patients’ treatment. First a purification strategy has been defined, including virus reduction steps and product characterization. Then a pilot production plant has

been set up with the aim of manufacturing GMP clinical batches of orphan drugs. On the regulatory side an application for Orphan Drug Designation is planned to be submitted to the regulatory authorities with the aim to obtain the designation within 2013 and preliminary in vitro preclinical studies are being started.

The purification process set up has been developed, bearing to a stable factor V concentrate, suitable for parenteral infusion in deficient patients. The GMP certification application for the pilot plant has already been submitted with the goal of receiving authorization first and to start with the production of cGMP batches within 2013.

In conclusion a factor V concentrate has been developed for clinical use in deficient patients and first steps have already been taken from the regulatory point of view, in order to make it available on the market as soon as possible.

17.15 OPEN SESSION ON NEW PROPOSALS

Session Chairpersons: Alok Asrivastava (India) and Leonard Valentino (USA)

Dr. Srivastava introduced the session.

Assessment of CFC- The end points towards long term outcomes. Alok Srivastava (India)

Dr. Srivastava introduced this session describing the current situation of the evaluation of clotting factor concentrates (CFC). He mentioned that in the lifecycle of the a new CFC, there is a reasonably well defined process of assessment with the pivotal clinical study that evaluated short term safety particularly with regard to incidence of inhibitors in PUPs and hemostastic efficacy in prevention and treatment of bleeds as well as surgical hemostasis. The work of this subcommittee through several project groups has helped in this process by providing definitions of clinical events and treatment responses that should help standardize some of the short term endpoints. However, there is currently no well defined and implemented process for the systematic assessment of long term safety and efficacy of CFCs. This can lead to delays or non detection of potential adverse events in the post marketing phase. There are examples of this happening in the past. The question therefore is whether such processes should be put in place and if so, what should they be and finally what should the additional responsibilities therefore of the manufacturers, regulators, treating physicians and very significantly the patients who need to participate in many of these additional assessments.

New European pharmacovigilance legislation: first experiences. Anneliese Hilger (EMEA)

New pharmacovigilance legislation (Regulation EU 1235/2012 and Directive 2010/84 was adopted by the European Parliament and European Council in December 2010 and amended by Regulation 520/2012, 1027/2012 and Directive 2012/26. The legislation was the biggest change to the regulation of human medicines in the European Union since 1995. It has significant

implications for applicants and holders of EU marketing authorizations including products for haemophilia treatment.

Impacts of the new legislation comprise adverse drug reactions reporting only into EudraVigilance data base. Safety monitoring as laid down in periodic safety update reports has been simplified e.g. electronic reporting, single assessment for same active substance instead of individual routine PSUR reporting. There is a strengthened legal basis for the requirement of post-authorization safety and efficacy studies.

In view of the limited availability of patients suffering from haemophilia, data from pre- authorisation studies only are considered to provide basic knowledge at the time of marketing authorisation, especially with respect to immunogenicity. Therefore, to collect additional clinical data and to ensure consistency in the long-term between the outcome from pre- authorisation clinical studies and from routine use, post-marketing investigation should be performedfor Factor VIII and IX products. This post-authorisation investigation concept is included in the Risk-Management-Plan and thus, will be approved and evaluated by the recently established Pharmacovigilance Risk Assessment Committee (PRAC).

New FDA regulation for post-marketing surveillance. Nisha Jain (FDA)

In 2007, the provisions made under the FDA Amendments act gave the FDA authority to mandate post-marketting surveillance. This could include studies or clinical trials if there were credible information to suggest that there were potential issues with safety or efficacy of the licensed products. For approval of a product, if a serious risk is identified in the clinical trial or there is potential for serious mandate a post-marketting surveillance. For an approved product, if the FDA becomes aware of of a new safety information, then a post-marketting study / clinical trial can be also be mandated. Prior to mandating a post-marketting study, the FDA has to establish that routine pharmacovigilance will not be adequate to identify these risks. Some examples of such post-marketting studies include pharamaco epidemiologic studies and clinical trials with specific safety end-points.

DISCUSSION

Questions and comments from the audience:

 Have been post-marketing surveillance methods for new products already set? Too premature.  Dr. Mannucci asked why do not make easier rules for the pre-marketing registration and then applying demanding rules for the post-marketing surveillance? Dr. Hilger reported that both regulatories are evaluating this point but strongly believe that the requested number of patients to be enrolled is an essential requisite to licence treatment products to the market.

NEW SSC PROJECTS

Consensus definitions and recommendations for immune tolerance induction (ITI) in hemophilia with inhibitors. Elena Santagostino (Italy)

In recent years some randomized clinical trials have provided high levels of evidence in the field of hemophilia including immune tolerance induction (ITI) treatment. In fact, the International ITI Study (Hay and DiMichele, Blood 2012) provided milestone results on treatment of children with good prognosis and pointed out the safety issue of the bleeding risk during ITI. Nonetheless, heterogeneous dosing regimens are used in routine practice and additional efforts are needed to optimize and standardize ITI therapy. Solid evidences concerning ITI treatment in other patient groups (i.e., patients with bad prognostic factors at first ITI course, adults with long-standing inhibitors or patients candidate to a rescue ITI) are scant and the premature closure of the randomized Resist study (Gringeri A, Haemophilia 2007) betrays the challenge of conducting rigorous studies in this setting.

At this stage, consensus definitions of clinical and laboratory endpoints in the different subsets of patients undergoing ITI are required because these would allow more reliable data analyses and comparisons between studies, furthermore, prompting the initiation of international prospective surveys able to collect more homogeneous data during a prolonged follow-up period.

Based on this background, the aim of this project proposal is to provide consensus definitions of ITI outcome standardizing the methodologies for the assessment of treatment endpoints. An additional aim will be to establish harmonized strategies for ITI therapy in inhibitor patients belonging to different and well defined prognostic groups.

The rationale, the methodology and the workflow of the project was presented and is posted on the ISTH SSC website.

Prophylaxis in hemophilic patients with inhibitor. Carmen Escuriola (Germany)

Increasing more long term prophylaxis is applied in inhibitor patients using Novo 7 or FEIBA alike. Novo 7 is only licensed for prophylaxis very few countries. Both treatment options lead to a reduction of ABR by approximately 60% independent of the status of the inhibitor patients, i.e. early secondary, secondary/tertiary. The down-side, though is that there is an unpredictable response, the long term outcomes as to protection from joint degeneration, not measurable factor levels and the incidence of thromboembolic events. It is strongly requested to optimize prophylaxis regimen/dose levels and establish a clear analysis of cost/ benefit ratio. The details of the composition and mandate of this project group is posted on the ISTH SSC website.

Prophylaxis in hemophilic patients without inhibitor. Victor Blanchette (Canada)

Dr. Blanchette described the current status of prophylaxis in patients with hemophilia. He presented data to drive home the point that despite decades of observational data and more recent data from prospective clinical trials, randomized or otherwise, there is no clear directions on how prophyalxis should be initiated. To address this situation a project group has been created. This group will look at the following issues that are relevant to long-term prophylaxis in persons with hemophilia and will focus on the following key areas: definitions of

prophylaxis; when and how to start prophylaxis; dose regimens for use in prophylaxis; strategies for adjusting prophylaxis regimens in different patient cohorts e.g. children, adolescents, adults; outcome measures relevant to long-term prophylaxis aimed to prevent/delay progression of bleed related arthropathy; potential impact of prophylaxis on adverse outcomes other than joint damage e.g. intracranial hemorrhage, inhibitor formation; and adherence to prophylaxis regimens and factors that influence adherence. The potential impact of long-acting factor VIII/IX concentrates on prophylaxis in the future will also be reviewed and discussed. The details of the composition and mandate of this project group is posted on the ISTH SSC website.

Factor XI and the Contact System

Chairman: Thomas Renne (Sweden) Co-Chairs: Jonas Emsley (UK), David Gailani (USA), Jose W. Govers-Riemslag (Netherlands), Christine Mannhalter (Austria), Joost Meijers (Netherlands), James Morrissey (USA), Ophira Salomon (Israel)

June 30, 2013

Summary of SSC meeting Factor XI and the contact system The factor XI and contact system was extremely well attended with 250 for the educational session and up to 400 in the main session. The educational session had three talks from jonas emsley, edward feener and james morrisey covering topics on the structure of contact factors, the role BK receptors in hereditary angioedema (fact or fiction), and polyphosphates in coagulation respectively. The subcommittee session contained seven talks on a variety of topics including the involvement of coagulation factor XII in fibrinolysis, the link to NETs, and studying FXI variants using next generation sequencing. The most well attended talks were the last two on new inhibitor developments targeting FXII and FXI for safer anticoagulation.

Structures of contact factors. Jonas Emsley. In the first part of the talk an overview of the domain structure for contact factors and FXI was presented and then data presented on a new structure of the FXI complex with a peptide from HK. Later information on the structure of the FXII protease domain and a review of old and new electron microscopy data on contact factors was also presented.

The contact system in diabetes. Dr Edward Feener . In the first part of the talk he showed the role of kallikrien in controlling intercerebral hemorrage in hyperglycaemic states. In the second part he was alluding to the activation of the contacts system and BK formation in diabetic retinopathy.

Polyphosphates in coagulation. Dr James morrisey presented an elegant overview of the polyphosphatees in coagulaton inlcuding the role of platelet polyphates in activating FXII, role in factor XI feedback activation by thrombin and function of the polymer in fibrinolysis.

Hereditary angioedema - facts and fiction. Eric Hack. Highlighted the role of the B1R receptor for increased vascular permeability in edema. He summarised studies on the relative importance of B2R versus B1R for targeting edema. The last part of his talk was on the role of inflammatory stimuli for local and systemic activation of the contact system.

Increased factor XIIa activity in patients on extracorporeal membrane oxygenation - a link to NETs. Simon Davidson. He gave a background of the clincial use of extracorproal circulation and summarised classical and new data showing activation of the contact system in ECMO. He discussed potential activators of the contact system in ECMO including NET formation and artifial surfaces.

The involvement of coagulation factor XII in fibrinolysis. Joke Konings. This talk summarised the recent blood paper on the crosstalk of the intrinsic pathway and fibrinolytic systems with implications for changes in the density of clot structure in the presence of FXII or FXIIa.

Understanding the role of sequence variation in the factor XI gene in the next- generation sequencing era. Stephano Duga. This talk outlined the new of new technologies in next generation sequence to pinpoint novel sequence variants and identify phenotypes.

FXI inhibitors. Eric Tucker. Summarised new data on different antibodies targeting FXIIa mediated FXI acitivation or FXIa. The FXI binding antibodies recognising FXI do not affect the thrombin feedback loop and selectively block the FXIIa activation of the intrinsic pathway. The FXI binding antibody that blocks FXIa cleavage of FIX has a more potent antithrombotic effects in a baboon model of thrombosis.

Factor XIIa inhibitors. Marc Nolte. A novel phage display based antibody interferes with FXIIa activity and FXIIa driven thrombus formation in mouse models of thrombosis and anterial venous shunt systems in rabbits. This recombinant monoclonal is in a stage 4 clinical trial in humans.

Factor XIII and Fibrinogen

June 29, 2013

Chairman: Helen Philippou (UK) Co-Chairs: Moniek P. de Maat (Netherlands), Aida Inbal (Israel), Vytautas Ivaskevicius (Germany), Hans P. Kohler (Switzerland), Marguerite Neerman-Arbez (Switzerland), Sanj Raut (UK), Verena Schroeder (Switzerland)

14.00 EDUCATIONAL SESSION

Session Chairpersons: Moniek de Maat (The Netherlands) and Helen Philippou (United Kingdom)

Dr Philippou opened the session welcoming the audience.

14.00 In vivo imaging of simultaneous FXIII activity, fibrin formation and fibrinolysis in a murine model. Helen Philippou (United Kingdom)

The effects of Factor (F)XIII activity on fibrin structure and the resistance of a fibrin clot were described using in vitro purified systems (with fibrinogen free of FXIII activity), independent of FXIII crosslinking of a2-antiplasmin. In essence FXIII causes more compact less porous structures that are more resistant to fibrinolysis. Subsequently, an in vivo model of thrombosis, induced by ferric chloride was described where both fibrin formation and FXIII activity can be monitored in real time. The main conclusion of the presented results was that FXIII determines the rate of fibrin growth determined using FXIII deficient mice compared to their wildtype counterparts. Infusion of recombinant FXIII A subunit partially corrected the rate of fibrin formation. The methods described for quantitative assessment of the fibrin formed over time with or without simultaneous assessment of FXIII activity can be applied to determine novel therapeutics such as novel anticoagulants and fibrinolytic agents (an example of the analyses employed was shown).

14.30 Structural molecular transitions underlying deformation of fibrin clots and thrombi. John Weisel (USA)

Fibrin clots become stiffer as they are being stretched which reduces the likelihood of breakage of fibres. The stretching of the fibres leads to a dramatic decrease in volume in part due to expulsion of water and fibre densification. The aC domains and unfolding of g-nodules of fibrin were shown to play an important role in the response to mechanical deformations. In fibrin a- helical coiled coils acts as highly elastic springs. In highly stretched and compressed fibrin clots a-helix to b-sheet transitions occur. The structure and plasticity of fibrin clots is uniquely suited to responding to external forces. Fibrin polymerisation is a reversible process before cross- linking, which can contribute to irreversible plastic deformations. In vivo, fibrin rather than fibrinogen is likely to play a role in mediating platelet aggregation. In clots taking place in vivo the ratio of fibrin to platelet incorporation increases as the clot ages.

15:00 -18:00 Subcommittee session

Session Chairpersons: Moniek de Maat (The Netherlands) and Helen Philippou (United Kingdom)

Dr Philippou opened the SSC subcommittee session by introducing the slides emailed by Cary Clark encouraging participation and use of the website.

15.00-15:10 Update on FXIII-B subunit standardisation. Verena Schroeder and Hans Kohler (Switzerland)

Dr Schroeder gave an update of the status of where we are with trying to introduce a FXIII-B subunit standard and why such a standard is required. It was stated that in order to initiate the introduction of such a standard, two specific ELISAs need to be developed (i) to specifically measure free FXIII-B subunit and (ii) to measure total FXIII-B with that bound to FXIII-A subunit. The general process of the subsequent work required to achieve a FXIII-B subunit standard was described.

15:10-15:25 Free and total FXIII-B subunit assays. Eva Katona and Laszlo Muszbek (Hungary)

Professor Muszbek gave an overview of what the status of the development of the B-subunit ELISAs was at our last meeting. Monoclonal antibodies to the FXIII-B subunit have been identified; one of these antibodies recognises only free B-subunit whilst a second recognises FXIII-B subunit in complex with FXIII-A subunit. Essentially whilst the ELISA for free FXIII-B subunit was working in a purified system, the recovery of FXIII-B subunit was underestimated by 50%. It is proposed that fibrinogen binding to the B-subunit of FXIII may be playing a role in the diminished recovery of FXIII-B subunit from plasma. Dr Muszbek then described an alternative FXIII-B subunit ELISA conformation that yields a good recovery of added FXIII-B subunit in both purified and plasma based systems. Recovery of added FXIII-B subunit has also been confirmed in various clinical samples and is very close to the predicted recovery. Therefore, this new ELISA looks very encouraging to initiate the first phase of developing a FXIII- B subunit standard. The ELISA will need a small amount of further development work and further characterisation.

In addition, Professor Muszbek showed the site on FXIII-B subunit responsible for binding to FXIII-A subunit.

Discussion: The ELISAs look very encouraging for the assessment of both total and free FXIII-B subunit so help to progress this project forward. The Chair asked the audience if anyone has any samples of patients that are receiving rFXIII-A therapy to further characterise the ELISAs. At the end of the session someone offered their samples of such studies.

15:25-15:35 Update on the proposed WHO 2nd International Standard (IS) on fibrinogen concentrates. Sanj Raut (United Kingdom)

Characterisation of the fibrinogen concentrates showed that total protein level determinations across centres are acceptable but there are discrepancies between clottable protein potency. There is an approximate discrepancy of 20% between the CLOTr and Clauss assay methods. This therefore suggests that there is a case for assigning potency using the CLOTr method.

15:35-15:50 Clottable protein assays for fibrinogen concentrates: the effect of fibrinogen and thrombin concentrations on clot formation, structure and clot opacity/turbidity. Sanj Raut (United Kingdom)

The aims of the study were to investigate the influence of fibrinogen and thrombin concentrations in fibrinogen concentrates. Three commercial fibrinogen concentrates were prepared using identical buffer formulations and lyophilised at the NIBSC. The current guidelines for assessment of fibrinogen suggest a 1 in 10 dilution of fibrinogen but do not give any guidelines on the concentration of fibrinogen that should be aimed for in the clottable assay. One of the three concentrates did not clot unlike the other two concentrate samples. Dr Raut explained how changes in concentrations of thrombin and fibrinogen affect polymerisation and the maximum absorbance using the turbidity assay. The conclusion of the study was that an optimal concentration of fibrinogen that should be used is in the range of 1-4 mg/ml with a 1-5IU/ml range of thrombin. In addition a mechanical end-point assay rather than a photo-optical instrument is preferred.

Session Chairpersons: Verena Schroeder (Switzerland) and Helen Philippou (United Kingdom)

15:50-16:05 Misconceptions regarding turbidity and clot density. Robert Ariens (United Kingdom)

Dr Ariëns presented data on the turbidity assay for fibrin polymerisation and listed a number of points which could lead to confusion in the literature and which we should keep in mind when performing these tests. First he showed data that an increase in turbidity or optical density associates with an increase of fibre thickness at constant fibrinogen concentration. When reporting turbidity results he pointed out that we should clearly state the parameter we measure. So for example density could be interpreted as optical density or fibre density (number of fibres per area) which are not the same and in fact are opposite at constant fibrinogen (higher optical density means lower fibre density). Dr Ariëns also explained that the maximal optical density or turbidity is related to both fibre thickness and fibrinogen level. A higher fibrinogen level (concentration) causes an increase in optical density. Fibrinogen levels are therefore important and need to be taken into account when analysing these results. Finally, Dr Ariëns suggested that it is always best, if possible, to visualise the changes in fibrin structure using microscopy (electron or confocal) to determine what causes the changes in turbidity observed.

16.05-16.15 Coffee break

16.15-16.30 Stable expression of FXIII Variants in flp-in CHO cells. Helena Handrkova (Switzerland)

Although there are many published methods for protein expression of FXIII, an alternative method to enable comparison of stability of mRNAs encoding mutated FXIII with quantification of differences in protein expression and stability, alongside the ability to purify and directly compare FXIIIA-subunits is still warranted. Dr Handrkova described a protein expression system available from Invitrogen that will enable targeted integration of genes into the host chromosome using a flp-in system with CHO cells. Early data is encouraging showing that FXIII-A subunit can be expressed using this system using a variety of tags with the ability to yield high levels of protein expression.

16.30-16.45 Modelling fibrinogen disorders in zebrafish. Marguerite Neerman-Arbez (Switzerland)

The haemostatic system of zebrafish is highly conserved to those of humans. Therefore zebrafish are a good model for characterisation of haemostasis. Elevated fibrinogen is an independent risk factor of cardiovascular disease therefore understanding the regulation of fibrinogen levels in plasma is important. A laser-induced thrombosis model has been established to monitor clot formation in the zebrafish. If the a-chain of fibrinogen is knocked in the zebrafish there is a significant decrease in thrombus formation, furthermore spontaneous bleeding occurred in the tail of fga-/- zebrafish.

16.45-17.00 FXIII in Hemophilia A. Catherine Rea, Benny Sorenson (United Kingdom)

Hemophilia A ultimately results in decreased thrombin generation thereby forming clots with decreased stability. In vitro studies have shown that administration of FVIII or FXIII to plasma from haemophilia A patients improves the clot stability. This has also been confirmed in whole blood samples from these patients. Unfortunately, FXIII administration to haemophilia A mice did not correct the phenotype of these mice.

17.00-17.15 Measurement of the knob-hole interactions of fibrin at the single-molecule level. Rustem Litvinou (USA)

Dr Litvinou gave an overview of studying nano-mechanics of the knob-hole interaction at the single-molecule level. The knob-hole interactions in fibrin should be studied in physiologically relevant dynamic conditions as opposed to bulk equilibrium and static measurements. Single- molecule studies provide insights into intrinsic mechanisms of binding and unbinding apart from effects of cooperativity and the methodology of single-molecule forced unbinding provide unique mechanistic information, not available with other techniques. The optical trap serves as a mechanical spring. The force ramp is when external mechanical perturbation increases linearly in time during bond dissociation and here the force at which the bonds break or the rupture force is measured. The force clamp is when a tensile force remains constant during unbinding and bond lifetimes are measured . Both the rupture forces and bond lifetimes allow

one to estimate the thermodynamic and kinetic parameters of the forced unbinding. Measuring the strength of individual knob-into-hole interactions provide unique information about the mechanisms underlying fibrin polymerization. The main findings of the work presented were

 Knobs ‘A’ in the central part of fibrin and holes ‘a' in D-region of fibrin(ogen) produce a binding strength of ~125-130 pN and zero-force off-rate of the order of 10-4 s-1, comparable with avidin-biotin complexes.  The B:b bonds are found to be about 6-fold weaker that the A:a bonds with a binding strength of ~20 pN.  The difference in the binding strength between the A:a and B:b knob-hole complexes is due to extended binding interface between the g-nodules and the central nodules of fibrin(ogen) molecules.  The strength of knob-hole interactions follows a dual catch-slip behavior: it first increased with forces up to 40 pN and then decreased with larger forces.

17.15-17.30 An update on Japanese criterion 2012 for the diagnosis and treatment of autoimmune/acquired hemorrhaphilia XIII/13; a proposal of algorithm of laboratory tests and differential diagnosis. Akitada Ichinose (Japan)

Acquired FXIII deficiency is a severe haemostatic problem that is often not diagnosed correctly. In Japan there are 38 cases with 4 recently diagnosed cases in 2013. Autoimmune/acquired hemorrhaphilia XIII/13 (AH13) is caused by an autoimmune antibody directed against FXIII which impairs FXIII cross-linking and patients present with a bleeding phenotype. Professor Ichinose with colleagues has developed a rapid immuno-chromatography method that is a very good method for the detection of AH13 (by detecting the presence of anti-FXIII antibodies). If diagnosis is able to be made early upon presentation of symptoms this can be life-saving as appropriate therapy (treatment with FXIII supplementation) can be administered.

17.30-17.45 Algorithm to determine inherited FXIII deficiency. Vytautas Iaskevicius (Germany)

Dr Iaskevicius presented an algorithm that highlight methods for the diagnosis of the various forms of FXIII deficiency, showing reference to the formal SSC communication on this topic that was published by Professor Kohler and colleagues published in JTH. The frequencies of each type of FXIII deficiency were discussed, alongside treatment guidelines for FXIII deficiency. The overall implications of Dr Iaskevicius and Prof Ichinose’s presentations were that a formal algorithm of FXIII deficiency diagnosis needs to be described to aid the diagnosis of FXIII deficient patients by clinicians that have little experience of diagnosing FXIII deficiency.

Discussion

Unfortunately we ran out of time for formal discussion in the session. The take home messages as far as SSC projects is concerned though are that

1. The development of a Value assignment for FXIII B-subunit (total & free) to the WHO 1st International Standard Factor XIII Plasma (2/206) is set to continue as the appropriate ELISAs are now established and just require further characterisation. 2. The pilot study led by Dr Marlien Pieters on a permeation study to fibrin structure was published as an official communication in JTH and initiates the path for a larger study to be performed. 3. The introduction of an algorithm to determine inherited and acquired FXIII deficiency will be led by Professor Akitada Ichinose and Dr Vytautas Iaskevicius with a view to also potentially include an algorithm for how these patients are to be treated. The aim would be to publish this study as an official SSC communication in JTH.

Fibrinolysis

Chairman: Ann Gils (Belgium) Co-Chairs: Jonathan H. Foley (Canada), Paul Y. Kim (Canada), Nicola J. Mutch (UK), Craig Thelwell (UK), Shirley Uitte de Willige (Netherlands), Tetsumei Urano (Japan)

Educational session: Unexpected/unconventional factors that partake in upregulation of fibrinolysis

8-8.20h "Effect of cells and flow on fibrinolysis” by Krasimir Kolev

Red blood cells and leukocytes as well as shear forces profoundly affect the mechanical and chemical stability of thrombi through modification of fibrin structure. RBC bind fibrinogen and facilitate thrombin generation resulting in a decrease in fiber diameter and pore size of fibrin with two ambivalent consequences: plasminogen activation is enhanced in fine-mesh fibrin, but the action of plasmin is retarded. Shear forces cause longitudinal alignment of fibrin fibres with smaller diameter and pore size. The resulting fibrin is a poor matrix for plasminogen activation by tPA and a poor substrate of plasmin. Leukocyte proteases directly degrade fibrin in thrombi and release extracellular matrix components from the vascular wall, which compromise the chemical and mechanical stability of thrombi at the interface with vascular lesions. In contrast, NET (neutrophil extracellular trap) components render fibrin resistant to mechanical and enzymatic destruction. Selected examples prove that interference with these effects can be used in prevention and treatment of thrombotic vascular diseases.

8.20-8.40h: "Roles of factor Xa cleavage products in upregulation PA by t-PA” by Ed Pryzdial

Coagulation factor Xa (FXa) is cleaved by plasmin to first produce FXa-beta and then Xa33/13. These enhance tPA-mediated fibrinolysis because C-terminal lysines are exposed approximately 50-fold faster than on fibrin. In plasma, the likely source of the FXa-derived tPA cofactors is the Xa-antithrombin complex, which is cleaved by plasmin an additional 10-fold more efficiently than FXa. Xa33/13 rapidly loses fibrinolytic activity in plasma. By chemically modifying FXa, we have stopped the cleavage at FXa-beta, which also contains a C-terminal lysine tethered to the active site. This compound, Xa-K, functions as a novel cofactor-based thrombolytic agent in murine models of thrombolysis.

8.40h-9.00h: "The role of microparticles in initiating and regulation clot lysis” by Eduardo Angles-Cano

It was recently discovered that circulating cell-derived microvesicles might behave as a template for plasmin formation. The plasminogen activator expressed by the cell of origin: tPA from endothelial cells and uPA from leukocytes are localised at the vesicular membrane that also have the capacity to bind plasminogen. The assembly of plasminogen and its activator results thereby in plasmin formation at the surface of microvesicles.

Furthermore, it was recently discovered that these microvesicles might participate in a new mechanism of plasmin formation requiring a cross-talk between two different surfaces. In this fibrinolytic cross-talk one of the surfaces bear plasminogen (fibrin, extracellular matrix or platelets) whereas the other surface carry the plasminogen activator, typically leukocyte- derived microvesicles bearing uPA. These new actors and concepts in plasminogen activation represent hitherto unknown pathways in our comprehension of the physiopathology of fibrinolysis and thrombosis.

Dr. Angles-Cano suggested that the fibrinolytic activity of endothelial and leukocyte microvesicles compensates locally the activity of procoagulant microvesicles. This phenomenon may explain the spontaneous re-canalization observed in 10-20% of patients with acute occlusion of the coronary arteries. Accordingly, the functional balance between these two types of microvesicles would result in a physiological haemostatic response, while the lack of fibrinolytic microvesicles may promote the formation of a thrombus.

SSC-session:

9.00h-9.15h: "Quantifying the added value of a diagnostic test” by Carl Moons

Traditionally, diagnostic tests and (bio)markers are evaluated on their accuracy in isolation, i.e. neglecting diagnostic results or information that will standardly be available in clinical practice before that test or marker would be applied. However, a single test's diagnostic accuracy measure often provides no guidance on the true clinical value of a test. Researchers should more focus on estimating the added diagnostic value of new tests or markers. Perhaps guidelines for market access may need to be updated in a conform way.

9.15h-9.30h: "Improvement of fibrinclot structure after the treatment in hemophilia A. Do TAFI and microparticles play any role?” By Antovic A,Mikovic D, Fariborz Mobarrez, Zabczyk M, He S, Elezovic I,Hutenby K,Woodhams B,Antovic JP.

Fibrin clot structure is altered in patients with hemophilia, revealing porous fibrin composed of thick and short fibers. Treatment with FVIII decreased fibrin clot permeability and improved fibrin clot structure. It seems that thrombin generation is the main determinant of fibrin structure in hemophilic plasma while TAFI and MP may, albeit to a lesser extent, contribute to fibrin clot formation on the site of injury. A larger mechanistical study to evaluate the influence of TAFI and MP on fibrin clot in the patients with hemophilia is desirable.

9.30-9.45h: "The antifibrinolytic function of factor XIII” by Nicola Mutch

Factor XIIIa has been postulated to play a role in regulating fibrinolysis, but the exact mechanism was not clearly defined. Using a flow model we have show that factor XIIIa exerts its antifibrinolytic function via cross-linking of a2antiplasmin to fibrin. Platelet factor XIII also has the capacity to regulate fibrinolysis via cross-linking of functional plasma a2antiplasmin to fibrin during thrombus formation.

9.45h-9.55h: "Update on Standard Development for D-dimer Assays” by Colin Longstaff and coworkers

D-dimer is widely used to test for fibrin breakdown products (FDP) present during fibrinolysis of blood clots that cause deep vein thrombosis, pulmonary embolism and a number of other clinical conditions. However, measurement and interpretation of results is well known to be hampered by a lack of standardisation. Several attempts to generate a common D-dimer standard have been unsuccessful in the past but our latest initiative has focussed on generating a pool of patient plasma from many donors with elevated D-dimer values that could be freeze dried and used as a common calibrator to harmonise standards used by manufacturers of D- dimer kits. A batch of plasma was collected and freeze dried successfully but investigations on ampoules stored at elevated temperatures, which is a method used to predict long term stability, showed there was a loss of D-dimer antigen over time, which was unexpected. Further studies on a preparation of FDP made from time courses of lysing fibrin have shown that stability may be markedly improved by adding the sugar trehalose.This and other observations suggest that FDP and D-dimer mixtures may form aggregates containing cross-b structures that are less reactive to antibodies against D-dimer. Further work is need to investigate the possibility that trehalose can stabilise D-dimer in a plasma matrix and to explore the utility of complex but controlled mixtures of FDP as standards for D-dimer assays.

9.55h-10.05h: "Program to replace the WHO 3rd IS for Plasmin” by Craig Thelwell

An update on the collaborative study to calibrate a replacement for the WHO 3rd International Standard for Plasmin (97/536) was presented. The plasmin standard was originally developed to standardise plasmin measurements in fibrinolysis assays, rather than for potency assigning therapeutic preparations. This replacement provides the opportunity to ensure the 4th IS is appropriate for potency assignment for therapeutic plasmin, currently being investigated in clinical trials, as concerns were raised over the suitability of the 3rd IS for this purpose at meetings of the Fibrinolysis Subcommittee of the SSC (Geneva, Switzerland 2007 and Vienna, Austria 2008). Therapeutic grade plasmin has now been sourced as the candidate IS, and stability studies on trial fills are underway. The collaborative study is scheduled for Q1 2014 to assign a potency to the candidate relative to the 3rd IS using chromogenic and fibrinolytic methods. It is also planned to propose a molar concentration as a dual label alongside the IU, in line with therapeutic product labelling. Recruitment for the study is still open and willing participants are encouraged to complete the questionnaire on the SSC website. It is planned to submit the study for SSC and then ECBS of WHO approval in 2014.

10.15h-10.30h:"Evaluation of the profibrinolytic effect of TAFI and PAI-1 inhibitors” by Tine Wyseure, Ann Gils, Paul Declerck

An overview on how profibrinolytic properties of inhibitors against TAFI and PAI-1 could be evaluated in vitro en in vivo was given. The talk particularly focused on models to which both proteins contribute in order to study dual TAFI/PAI-1 inhibition. Methods for whole blood analysis, such as in vitro thrombus lysis assay and thromboelastometry, were presented in

which TAFI and PAI-1 inhibitors were compared in human and mouse blood. Because in mice baseline levels of PAI-1 are extremely low, endotoxemia was induced to obtain upregulated PAI-1 levels which allowed the evaluation of PAI-1 inhibition, alone or combined with TAFI inhibition. Also, dual TAFI/PAI-1 inhibition was tested in an in vivothromboembolic model induced by tissue factor using mice with or without pretreatment of endotoxins. In conclusion, we have created an ex vivoand in vivo model in mice in which we can evaluate dual TAFI/PAI-1 inhibition as a robust fibrinolytic enhancer.

10.30-10.45h: "Interindividual CPU (TAFIa) generation profiles during a standardised clot lysis assay” by Dorien Leenaerts, Dirk Hendriks

Research on CPU/TAFIa generation during in vitro clot lysis was presented. To gain insight into CPU formation between individuals, the time course of CPU generation was determined for 10 healthy volunteers. A computer algorithm was developed to plot and analyze the raw data. Based on the algorithm, clot lysis and CPU generation related parameters were quantified. It was demonstrated that there can be marked differences between individuals, especially in height and area under the curve of the first and second peak of CPU activity.

Our method offers a technique to determine someone’s endogenous CPU potential. In a later stage, we will set up a reference range that can be used to investigate whether the calculated parameters differ significantly in patients with thromboembolic disease.

10.45-11.00h: "Extrahepatic TAFI expression and regulation of TAFI gene expression: What does it all mean?” by Michael Boffa

While substantial evidence has accumulated for the existence of TAFI expression outside the liver as well as for the ability of a variety of stimuli to modulate TAFI gene expression, there is scant evidence directly linking these phenomena to physiology or pathophysiology. My presentation will summarize the current state of knowledge related to TAFI gene expression, and will attempt to chart a course whereby these insights can be translated into an enhanced appreciation of the role of TAFI in vivo.

11.00-11.10h: "Nomenclature of TAFI” by Jonathan Foley

At the SSC in Kyoto (2011), a project was initiated to evaluate TAFI/CPU nomenclature to see if previous recommendations (SSC 2000) were being followed and if new recommendations were warranted. Previously, it was recommended that both TAFI and CPU and the accession number be mentioned in the text of publications. The large majority of publications do not follow these recommendations and as a result, performing a comprehensive literature review is becoming more difficult. To address this growing problem, the following nomenclature options were considered:

1. Reiterate our support for TAFI and/or carboxypeptidase U 2. Redefine the TAFI acronym to incorporate fibrinolytic and inflammatory roles

3. Recommend that carboxypeptidase B2 be adopted 4. Identify and recommend a novel name

These options were sent to SSC fibrinolysis committee members and ISFP Council members for feedback and feedback was gathered at the SSC fibrinolysis meeting in Liverpool 2012. A consensus on the nomenclature could not be reached and therefore, no recommendation regarding the use of TAFI/CPU is being made at this time.

Since the gene name CPB2 is universal, we suggest that CPB2 be used as a keyword in publications describing work related to TAFI/CPU. If this suggestion is followed, performing comprehensive literature searches will become more straightforward regardless of the nomenclature used. A call for feedback will be posted to the SSC Fibrinolysis website in order to gather feedback before making a formal recommendation via a JTH communication.

11.10h-11.25h: "Global Haemostasis assays, from bench to bedside” by Waander Van Heerde

Detection of aberrations in the hemostatic balance require a large range of both screening and confirmation assays. To better reflect the hemostatic potential of the patient efforts have been undertaken to develop "global assays” that cover not only the behavior of one single compartment of the hemostatic balance (coagulation, fibrinolysis or platelets/vessel wall interaction) but combines multiple compartments. These global assays may be of great benefit for "Personalized Medicine”. To date, several global assays exists and the technical "in and outs” including the detection system (turbidimetry, enzymatic activity or visco-elasticity) were discussed. Furthermore, the "pro and cons” of the different global assays in clinical practice were also reviewed. These include fixed time point determination, continuous measurements and real time measurements, correction for alpha-2-macroglobulin and sensitivity and specificity. Finally, the applicability of these assays as point of care devices using whole blood was discussed. The final remark was that still a lot of standardization hurdles need to be overcome before these assays could be used in daily clinical practice. Nevertheless "Personalized Medicine” requires proper "Personalized Diagnostics” and as such sensitive and specific global assays may be of great potential.

11.25h-11.40h: "Detection of fibrinolytic microvesicles using a chemosensor capture assay” by Laurent Plawinski, Eduardo Angles-Cano

There is an urgent need for non-invasive detection of cell-derived microvesicles in a variety of frequent pathologies including atherosclerotic vascular disease, diabetes, cancer, and autoimmune systemic and inflammatory diseases. There is currently no available test in the routine laboratory to accurately evaluate in biological fluids the properties and role of microvesicles as potential biomarkers of specific cell or tissue activation processes in a number of pathologies. To circumvent this problem, we propose an assay where microparticles are directly captured by a chemosensor onto a solid surface and then revealed with specific labelled probes. The method is based on the affinity of phosphatidylserine for immobilized dinuclear metallic complexes.

Upon capture of microvesicles, it is possible to identify the phenotype (cell origin) and different active biomolecules with the use of specific antibodies directed against molecular motifs present at the membrane. It will be also possible to detect procoagulant and fibrinolytic activities by functional assays; potential identification of specific RNA (miRNA and mRNA) content of microvesicles by qPCR is a promising development of the assay.

The advantage of the proposed method is that the ligand is a synthetic compound that does not present potential complications encountered with the use of biological molecules. The test could be easily automatized and performed in a routine laboratory setting using a capture procedure and single step detection. It will therefore be both clinically useful and economically advantageous.

11.40h-11.55h: "Clinical utility of D-dimer in the diagnosis and prediction of VTE” by Shannon Bates

The use of D-dimer testing for suspected venous thromboembolism and the use of D-dimer in determining the risk of recurrent venous thrombosis was presented. During the first part of the presentation, the currently accepted uses for D-dimer testing in the evaluation of patients with suspected deep vein thrombosis and pulmonary embolism were discussed. Subsequently, new research that is attempting to improve the efficiency of D-dimer utilization by varying the D- dimer threshold values (for example, increasing the threshold value in patients with a low pre- test probability of disease, in the elderly, and in pregnant women) was presented. The presentation was concluded with a review of recent studies in which D-dimer testing (both alone [for example, the Prolong study] and as part of a clinical prediction rule [for example, the "Men Continue and HERDOO2” rule and the "DASH” rule ) has been used to stratify patients risk of recurrent venous thromboembolism post anticoagulant discontinuation.

11.55h-12.00h: closing remarks by Ann Gils

Everyone is invited to visit the SSC-fibrinolysis website and to take part in the discussion concerning standardization of fibrinolysis parameters and the nomenclature of the protein encoded by the CPB2 gene.

Hemostasis and Malignancy

June 29, 2013

Chairman: Alok A. Khorana (USA) Co-Chairs: Marc Carrier (Canada), Agnes Y. Lee (Canada), Howard A. Liebman (USA), Marina Marchetti (Italy), Ingrid Pabinger(Austria), Joseph S. Palumbo (USA), Wolfram Ruf (USA), Jeffrey Zwicker (USA)

Additional Invited Speakers: Chris Holmes, Thomas Ortel, Cihan Ay, Henri Versteeg, P. W. Kamphuisen, M. Di Nisio, Guy Meyer

There were 12 presentations, including 3 educational talks, updating subcommittee activity and discussing new proposals.

The 3 educational talks comprised the first session. Dr. Lee reviewed state-of-the-art treatment of cancer-associated thrombosis, given the arrival of novel oral anticoagulants (NOACs) as an option. She provided a summary of the evidence base (or lack thereof) supporting the use of low-molecular-weight heparins, warfarin and NOACs in this setting. Dr. Holmes provided a comprehensive overview of anti-platelet therapy and its role in the setting of malignancy, given multiple recent epidemiologic and trials data suggesting benefit in terms of patient outcomes in this setting. Dr. Ortel (who spoke later in session owing to late arrival) reviewed incidence and prevalence of bleeding complications in malignancy, both in the presence and absence of anticoagulation.

The first SSC non-educational session focused on activity by the Subcommittee in developing guidance statements. Dr. Carrier provided an update on a recently developed guidance statement evaluating the appropriate treatment of recurrent VTE in the setting of malignancy, keeping in mind bleeding concerns. This paper was recently accepted for publication by JTH. Dr. Zwicker provided an overview of a guidance statement on the treatment of catheter-associated thrombosis. Dr. Khorana discussed emerging and recent data on outpatient thromboprophylaxis in malignancy including results of SAVE ONCO, PROTECHT and Microtec; validation of a risk score; and recently published subgroup analyses of SAVE ONCO and PROTECHT utilizing the risk score. A summary of these data has led to the development of new guidance from the SSC on outpatient thromboprophylaxis in cancer patients and a draft version of these statements was reviewed as well. Dr. Lee provided an update on the SSC work on developing a guidance statement on incidental VTE including aspects related to diagnosis and management in an era where a high proportion of VTE are diagnosed incidentally.

The second SSC session provided updates of SSC activity on clinical and translational projects. Dr. Palumbo was scheduled to speak in this session but spoke earlier in lieu of Dr. Ortel and provided a comprehensive overview of the literature around the role of hemostatic factors in malignancy including their role in non-hemostatic processes such as angiogenesis. Dr. Pabinger provided an update on results from their registry which will soon be celebrating its 10th

anniversary. This registry, the Vienna Cancer and Thrombosis Study (CATS) is an ongoing prospective non-interventional study for definition of risk factors for venous thromboembolism. Recently novel clinical risk factors were identified, including high-grade histological phenotype and regional (lymph node positive) stage. In addition the group has found that presence of varicose veins increases the risk of cancer-associated VTE. Dr. Carrier provided an update of a pooled analysis of cancer patients from clinical trials of prophylaxis in acutely ill medical inpatients. Unfortunately, the results of this analysis do not confirm a clear benefit to cancer patients from prophylaxis, likely due to under-powering of sample size. Clearly, more data are necessary in this regard and Dr Carrier outlined potential proposals. Dr. Versteeg outlined a call for new translational studies in the setting of malignancy and VTE, focusing on tumoral coagulation factors, and immunohistochemistry. Finally, multiple investigators provided brief updates on the larger ongoing clinical trials in the field of cancer-associated VTE including the CATCH trial of treatment of VTE with tinzaparin (Lee), the PHACS trial evaluating the use of dalteparin as prophylaxis in cancer outpatients at high risk for VTE (Khorana), the SOME and PERIOP trials (Carrier), long-term treatment of VTE after 6 months the LONGHEVA trial (Kamphuisen), Dr. DiNisio presented enrollment data from a registry of incidental VTE. In this multicenter, prospective study, patients with unsuspected pulmonary embolism are followed up for to one year to assess the incidence of recurrent symptomatic VTE, bleeding, and mortality. The status of the study was presented with an update of the number of centers involved and patients included Dr. Zwicker presented study design of a new proposed trial of prophylaxis (CAT IQ).

The session chairs thanked the audience for their participation. The meeting was adjourned at 18:00.

Lupus Anticoagulant/Phospholipid-Dependent Antibodies

Chairman: Thomas L. Ortel (USA) Co-Chairs: Tatsuya Atsumi (Japan), Bas de Laat (Netherlands), Katrien M. Devreese (Belgium), Silvia S. Pierangeli (USA), Armando Tripodi (Italy)

Educational session

Dr. Devreese presented an overview on the latest developments on solid phase assays. In aCL and aβ2GPI testing, several factors contribute to variability in pre-, post- and analytical conditions. Despite consensus guidelines and proposals published in the past, standardization is not yet achieved. The standardization of the solid phase assays is an on going process and new consensus guidelines are in preparation. Meanwhile, it is important to be aware of the methodological shortcomings of the assays and interpret the results accordingly.

Dr. Kelchtermans continued on solid phase assays emphasizing on factors that explain differences between solid phase assays of different manufacturers. It is known that antibodies against domain I, specifically G40-R43, are associated with thrombosis. Using two patient derived monoclonal antibodies Dr. Kelchtermans showed that it was able to detect differences in solid phase assays in exposure of epitope G40-R43 on beta2GPI. These antibodies were proposed to be used as standards to control for the conformation of beta2GPI is commercially available assays.

Dr. McCrae gave a complete overview of pregnancy related antiphospholipid syndrome. It was shown that lupus anticoagulant proved to be best related to APS-defined pregnancy morbidity. Antibodies against domain I were likely to be associated with pregnancy morbidity, although this shown by only one group. Dr. McCrae emphasized that there is less data on pregnancy morbidity than on thrombosis making it difficult to advise which assays to be used in cases of suspected APS.

SSC meeting

Dr. Wahl presented data on antiphospholipid testing in the "real world”. This presentation reported the incidence of antiphospholipid antibodies in a large database comprising all tests performed in a single haematology laboratory of a tertiary medical care hospital (Lorraine University Hospital, Nancy, France). The impact of Sydney criteria and ISTH guidelines have been illustrated for definition of cut-offs and confirmation of positivity. It was shown that that even after confirmation of positivity at 12 weeks antibodies could disappear. The audience asked for the clinical significance of these antibodies. At his moment no clinical data was present.

Dr. Gray showed results of the first international reference panel for lupus anticoagulant. The panel consisted of patient plasmas representing a negative, medium positive and strongly positive sample for lupus anticoagulant. Every sample was the result of a combination of

several patient plasmas. The sample was send to several international recognized expert centers for testing. The results showed acceptable inter-laboratory variation and will be discussed during the SSC business meeting for acceptance.

The updated guidelines for measurement of lupus anticoagulant state that mixing studies should be performed to exclude coagulation factor deficiencies. Dr. Urbanus investigated whether it is necessary to exclude a coagulation factor deficiency with a mixing test. In fact, lupus anticoagulant ratios determined with both the dRVVT and the silica clotting time in coagulation factor deficient plasma indicate that the phospholipid confirm readily discriminates between lupus anticoagulant and a coagulation factor deficiency. Whether mixing tests are necessary to accurately assess lupus anticoagulant in plasma with a high INR of the new oral anticoagulants remains to be determined.

Dr. Tripodi proposed to determine cut-off values for detection of lupus anticoagulant. This proposal was embraced by the members of the SSC subcommittee. Dr. Tripodi was asked to write a proposal to the SSC subcommittee.

Dr. Devreese presented new recommendations for aCL and aβ2GPI measured by solid phase assays. Recommendations for patient selection, blood collection, choice of assays, performance characteristics, interferences, duplicate versus single testing, calibration, expression of results, cut-off values and interpretation of results were included. A manuscript approved by all SSC subcommittee members is currently under review at The Journal of Thrombosis and Haemostasis.

Besides dr. Gray, dr. Pierangeli also presented new standards including both monoclonal and polyclonal antibodies. These standards are aimed to be used in solid phase assays, anticardiolipin antibodies assays and anti-beta2GPI antibody assays. After questions from the audience dr. Piernageli stated that the polyclonal standards included domain I antibodies.

Dr. Wahl proposed possible issues that could be studied within the SSC. Proposal 1 included how to report the diagnostic value of a new test for antiphospholipid antibodies : recommendations for analysis and reports. Reports of diagnostic value of antiphospholipid antibodies are very heterogeneous and there are no current guidelines to propose a methodology according to the type of study needed and the statistical tests to be used. This is due in part to the lack of a gold standard in the clinical and laboratory criteria of antiphospholipid syndrome. We propose to establish a consensus for guidelines and report of studies using the most adequate statistical/methodological standards to evaluate new tests. Proposal 2 included thrombin generation and the diagnosis of antiphospholipid syndrome: review of available data and choice of methods. Thrombin generation studies have been used in antiphospholipid syndrome to assess patient characteristics, in particular activated protein C resistance. However results are reported in a heterogeneous manner and there is a need for standardization. We propose a systematic review of available data and establish a consensus for methods and reporting of these studies. This should lead to multicenter standardization studies in a second step. Both items can be studied within the SSC but it was requested from

out the SSC to send in detailed proposals so that these can be discussed within the SSC subcommittee.

A prospective, randomized clinical trial comparing rivaroxaban vs warfarin in high risk patients with antiphospholipid syndrome was proposed by dr. Pengo. The primary objective is to determine the efficacy and safety of rivaroxaban as compared to warfarin in the primary thrombosis prevention of persistently triple aPL-positive APS patients. The primary end point is cumulative of incident thrombosis (arterial or venous), major bleeding and death.

Dr. Cohen presented a study investigating rivaroxaban in patients suffering from APS. Thrombin generation was used to study the anticoagulative effect compared to VKA.

Pediatric and Neonatal Hemostasis and Thrombosis

June 30, 2013

Chairman: Anthony K. Chan (Canada) Co-Chairs: Mariana Bonduel (Argentina), Leonardo R. Brandao (Canada), Elizabeth A. Chalmers (UK), Neil Goldenberg (USA), Christoph Male (Austria), Paul Monagle (Australia), Paolo Simioni (Italy), Guy Young (USA)

The name of the subcommittee has been formally changed to Pediatric/Neonatal Thrombosis and Hemostasis Subcommittee. The focus of the Subcommittee is to address issues in the area of thrombosis and hemostasis in children and neonates by developing clinical standards for evaluation, foster international collaboration in research and clinical trials, establish/maintain registries and to generate, publish and distribute reports and recommendations relating to patient care for the pediatric population.

Overall, work within the Subcommittee is done through the Working Groups lead by one of the Co-chairs.

Education

The Education Session was well-received with the room at 90 percent filled. Many of the participants requested for the presentations to be posted online for approximately one month in order for them to have an opportunity to view it. I request the SSC to investigate the possibility of posting the slides on-line for a period of time.

Position Papers

Three position papers were published:

1. Revel-Vilk S, Brandao LR, Journeycake J, Goldenberg A, Monagle P, Sharathkumar A, Chan AKC, on behalf of the Perinatal and Paediatric Haemostasis Subcommittee of the Scientific and Standardization Committee of the International Society of Thrombosis and Haemostasis. Standardization of the post-thrombotic syndrome definition and outcome assessment following upper venous system thrombosis in pediatric practice. J Thromb Haemost 2012; 10(10):2182-5. 2. Bruce AK, Bauman ME, Jones S, Massicotte MP, Monagle P. Recommendations for measuring health-related quality of life in children on anticoagulation. J Thromb Haemost 2012; 10(12):2596-8. 3. Bauman ME, Bruce A, Jones S, Newall F, Massicotte MP, Monagle P. Recommendations for point of care home INR testing in children on vitamin k antagonist therapy. J Thromb Haemost 2012; 11:366-8.

Three position papers are at advance stage of preparation and will be submitted within a next few months:

1. Recommendations for the introduction of new anticoagulant drugs into paediatric clinical use. ( lead by Christoph Male and Guy Young ) 2. Recommendations for the development of a dedicated paediatric anticoagulation service ( ( lead by Paul Monagle working with Fiona Newall) 3. Recommendations for the assessment of non-extremity venous thromboembolism outcomes ( lead by Anthony Chan working with Madhvi Rajpurka)

Two guidance papers have been submitted and are under revision. Unfortunately, one of the two guidance papers may need to be withdrawn due to authorship limitations. A third guidance paper has been prepared and waiting for instructions from the Guidance Paper Committee to ensure the paper is prepared in the proper format.

The guidance papers are:

1. Venous thromboembolism in children: Considerations for thrombophilia testing. [Update 2012] ( lead by Ulrike Nowak-Gottl ) 2. The use of inferior vena cava filters (IVCF) in children. ( lead by Anthony Chan working with Suzan Williams ) 3. Management of post-thrombotic syndrome (lead by by Anthony Chan working with Shoshanna Revel-Vilk)

Ongoing Projects

Below is the list of ongoing projects with the intention of developing a position statement or a guidance paper (to be determined by the Working Group lead by the Co-chair):

1. Diagnostic criteria for thrombosis in children: (Responsible Co-chair: Leonardo Brandao) 2. Investigation of severe bleeding child. (Responsible Co-chair: Paolo Simioni) 3. Antiplatelet therapy in children. (Responsible Co-chair: to be determined) 4. HIT in children. (Responsible Co-chair: Guy Young) 5. Antithrombin replacement in heparin therapy. (Responsible Co-chair: Elizabeth Chalmers) 6. APLA in children. (Responsible Co-chair: Mariana Boundel working with Shoshanna Revel-Vilk ) 7. Coagulopathy in liver disease. (Responsible co-chair: Paul Monagle working with Maria Magnusson) 8. Liver and renal biopsies in coagulopathic patients. (Responsible co-chair: Paul Monagle working with Maria Magnusson) 9. VTE prophylaxis ( Responsible co-chair : Neil Goldenberg ) 10. Interpretations of coagulation inhibitors in the context of developmental Hemostasis ( Responsible co-chair : Anthony Chan )

11. Congenital Severe Purpura Fulminan Registry 12. Management of Pulmonary Embolism ( Responsible co-chair : Neil Goldenberg ) 13. Management of Arterial Thrombosis ( Responsible co-chair : Neil Goldenberg working with Manuela Albisetti )

We plan to make use of the ISTH SSC website for communicating with ISTH members beyond the Co-chairs such that ISTH members can be more engaged with the work of SSC.

Anthony K C Chan

Chair, Pediatric/Neonatal Thrombosis and Hemosasis Subcommittee

Plasma Coagulation Inhibitors

June 29, 2013

Chairman: Steven Kitchen (UK) Co-Chairs: Elisabetta Castoldi (Netherlands), Tilman M. Hackeng (Netherlands), Richard A. Marlar (USA), P Meijer (Netherlands), Laurent O. Mosnier (USA), Jun Teruya (USA)

Educational programme _ Chairs S Kitchen, L Mosnier

14:00-14:30 Laboratory Diagnosis and Characterisation of AT deficiency. Peter Cooper (UK)

14.30- 15:00 Structure-Function of activated protein C: deficiencies and endogenous anticoagulant activities. Laurent Burnier (USA)

Minutes/Summaries

Laboratory Diagnosis and Characterisation of AT deficiency. Peter Cooper (UK)

Antithrombin (AT) deficiency may increase the risk of VTE by 5-50 folds, but risk depends upon AT level and type of deficiency. Most patients with inherited AT deficiency have type II defects where the AT activity level is significantly lower than the level of AT antigen. In functional AT assays, AT is activated by heparin, and an enzyme (thrombin or FXa) cleaves the arginine 425 serine 426 bond in AT (numbering according to the Human Genome Variation Society guidelines) and AT is inhibited, the residual enzyme is detected using a chromogenic substrate and optical density (405nm) is inversely proportional to AT level. On identifying a low or borderline AT activity level measurement of AT antigen establishes whether the defect is type I or type II, and the ratio of activity/antigen facilitates this, and can identify type II defects even when AT activity level is not reduced. AT antigen can be accurately measured by techniques such as latex-agglutination and the more labour-intensive ELISA assay, the radial immunodiffusion assay is widely available but can have poorer precision.

Samples for AT level should be citrated plasma, and clotted samples have reduced AT activity. Testing the prothrombin time (PT) confirms that the sample is not clotted and can identify liver disease which can reduce AT level. PT, thrombin time and APTT can help identify presence of anticoagulation, especially important, as direct thrombin/FXa inhibitors cause overestimation of AT by thrombin and FXa-based assays, respectively. Antithrombin from healthy individuals is relatively robust, but sample storage in a self-defrosting freezer can greatly reduce functional AT level.

Within run precision of AT activity when a pooled normal sample is tested can display a coefficient of variation of <2%, but instrumentation and reagent problems can cause drift and imprecision.

AT activity assays should be sensitive to all AT defects but there is no guarantee that one assay will be sensitive to all type II defects. AT assay in the absence of heparin will not pick up type IIHBS (heparin binding site) defects, FXa-based assays do not detect AT Cambridge II (p.Ala416Ser) and we show a type IIRS (reactive site) defect, AT Denver (p.Ser426Leu) that is clearly detected by a thrombin-based assay but undetected by a FXa-based AT assay.

We measured AT in a patient with AT Budapest 3 (p.Leu131Phe) AT levels were much lower in a human-FXa based AT assay (Innovance, Siemens) than three other assays and the very low results appeared more in keeping with heparin-resistance in two patients. Similarly, patients with AT Basel (p.Pro73Leu) had clearly low level by the Innovance AT assay but borderline levels with Berichrom assay even with 30s incubation and had normal AT levels using the Berichrom assay with the manufacturer’s recommended 180s incubation time. Interestingly, with AT Basel, the Coamatic AT assay (bovine FXa-based) had poor sensitivity to the deficiency, and it has been reported that the Liquid AT assay (Instrumentation Laboratory) is also insensitive to this defect. Shortened incubation time of enzyme with sample can increase sensitivity to type IIHBS defects and to at least one type IIRS defect (AT Glasgow, p.Arg425His).

When investigating type II AT deficiency, more than one activity assay is required but there is little data on the sensitivities of different manufacturers’ kits to type II AT defects, however, EQA schemes have helped highlight differences between kit sensitivities as have some published studies.

Structure-Function of activated protein C: deficiencies and endogenous anticoagulant activities. Laurent Burnier (USA)

Activated protein C (APC) is a serine protease in blood that helps maintain a regulated balance between hemostasis and host defense systems in response to vascular injury. APC interacts with numerous cofactors and receptors on various cells. These interactions help APC express multiple anticoagulant and cytoprotective activities that contribute to maintaining the health and integrity of the vasculature. These different activities of APC will be reviewed in light of available structure-function information for APC and supported by experimental and clinical data. Recent advances and in vivo proof-of-principle data for activity selective APC mutants, targeting of APC activities by monoclonal antibodies, and studies of APC cofactors and receptors combined with insights from human congenital or acquired deficiencies and genetically modified murine models support an intricate system safeguarding vascular homeostasis. Although traditionally known as "anticoagulant APC” these novel activities put APC in a different spotlight with important implications for normal physiology, pathophysiology, and therapeutic applications in ischemic stroke and potentially other vascular diseases.

Subcommittee Business section

Laboratory testing Chairs : Jun Teruya (USA) and Richard Marlar (USA)

Project proposal : Proposed Guideline for assays of AT, PC and PS. Piet Meijer (The Netherlands)

Guideline for laboratory testing of natural coagulation inhibitors

Person responsible (Chair / Principal Investigator): Piet Meijer, NL (other principal investigators: Steve Kitchen, UK; Richard Marlar, US)

Current guideline for testing of heritable thrombophilia focuses mainly on clinical aspects and which tests are relevant to perform. No detailed information is provided how to measure thrombophilia parameters. Detailed information is on the technical aspects of the tests, test characteristics, interferences etc. is limited or lacking. Within the framework of the Scientific Standardization Committee on Plasma Coagulation Inhibitors a laboratory guideline for the measurement of coagulation inhibitors will be developed including the fore-mentioned analytical aspects. This projects starts in 2013 and will be finalized in 2014/201

Pre analytical issues for AT, PC and PS testing. Richard Marlar (USA)

Pre-analytical variables (PAV) affect coagulation testing more often than tests in other areas of the clinical lab. These variables can cause an erroneous diagnosis if PAV issues are not recognized or taken into account when a patient’s sample is analyzed. This is especially true of the natural regulatory factors (AT, PC, PS). In its broadest sense, PAV issues include both patient status (food, general health and medications) and sample preparation (collection, transport, processing and storage of the blood specimen and plasma sample). The discussions covered some of the more common PAV affecting clinical testing of AT, PC and PS.

Diagnosis of AT Budapest variants. Ingrid Hrachovinova ( Czech republic)

Deficiency of AT is the most clinically severe defect of plasma coagulation inhibitors. In the last fifteen years, we documented 126 patients from 62 families with a suspected hereditary AT deficiency due to low or borderline AT activity of plasma levels. Mutational analysis of the AT gene had been performed. Mutations were found in all of patients – 54 patients had subtype I, 10 subtype II RS (reaction side), 58 subtype IIHBS (heparin binding side) and 4 had subtype IIPE mutations. Most common mutations in our group were Budapest variants (Leu99Phe and Arg47His). They represented 0.28 of all cases and about half patients with II subtype (35pts).

Screening of AT activity in family members together with the genetic screening of the family mutation revealed that some FXa based assays may fail to detect these mutations and in some cases, even FIIa based assays measured AT activity results which were in the normal range.

AT activity in plasma from 20 patients with AT Budapest variants (no significant difference was found between two mutations) was measured in parallel by reagents DG-Chrom AT(FXa), Biophen Antithrombin (FXa) and Stachrom ATIII (FIIa) with the results of 62±14(IU/dl), 80±32(IU/dl) and 73±20(IU/dl)(x±2SD).

However, not only type of reagent can influence the result of AT activity. We have observed considerable variations in activity in one patient during several years measured with the same method. This could lead to additional factors which might affect the activity of antithrombin.

Molecular genetics Chairs : Elisabetta Castoldi ( The Netherlands), Tilman Hackeng (The Netherlands)

Subcommittee Project update : Update and maintenance of the antithrombin, protein C and protein S mutation databases. Elisabetta Castoldi (The Netherlands)

Despite the availability of several general open-access genetic databases that are maintained by major institutions and regularly updated (OMIM, HGMD, LOVD, etc.), recent years have witnessed a revival of locus-specific mutation databases. These databases focus on single genes and diseases and report not only the details of each identified mutation, but also a wealth of additional information specifically targeted to researchers and clinicians studying that particular gene/protein or disease.

Within the SSC Subcommittee on Plasma Coagulation Inhibitors, locus-specific mutation databases for antithrombin (AT, encoded by the SERPINC1 gene), protein C (PC, encoded by the PROC gene) and protein S (PS, encoded by the PROS1 gene) have been established and maintained until the Nineties, but have since been abandoned and become outdated. During the SSC Subcommittee Meeting in Kyoto, Japan (2011), a plenary discussion on the usefulness of these locus-specific databases triggered an SSC project proposal for the update and indefinite maintenance of the AT, PC and PS mutation databases, which was approved shortly before the SSC Subcommittee Meeting in Liverpool, UK (2012).

This presentation discussed the challenges (recruitment of an expert committee for each gene, financial support, ethical issues, etc.) and opportunities (desirable information to be included in the database, relationships with other locus-specific databases, etc.) of this enterprise. The bottom line is that updating and maintaining the locus-specific AT, PC and PS mutation databases is going to be a major effort which requires the long-term commitment of all researchers in the plasma coagulation inhibitors field for the shared benefit of the whole scientific community.

Subcommittee Project proposal : Racial differences in genetic risk factors for venous thromboembolism. Hiroko Tsuda ( Japan)

Aim/Mandate: Factor V Leiden and prothrombin G20210A are well-known hereditary thrombophilias in Caucasians, in contrast, protein S (PS) and protein C (PC) deficiencies are much more prevalent among Asians than non-Asians. PS Tokushima (K155E), a PS gene mutation with a phenotype of type II PS deficiency, is a genetic risk factor for venous thromboembolism (VTE) in Japanese. Two PC gene mutations with phenotypes of type II PC deficiency, PROC c.565C>T and PROC c.574_576del, are identified as genetic risk factors for VTE

in Chinese. In order to elucidate the racial differences in genetic risk factor for VTE, we build up a global network and investigate worldwide distributions of PS and PC type II deficiencies.

Methodology: 1) Build up a network of professional contacts for researching racial differences in genetic risk factors for VTE, which includes Asian and European countries, and USA. The latter two geographic regions consist of different racial groups. 2) Collaborative study to determine the racial differences in the frequencies of PS and PC type II deficiencies and their causal gene mutations, in both general populations and the VTE patients. Plasma PS is analyzed using a new quantitative total PS assay system for high performance screening for type II PS deficiency [1] as well as conventional PS assays. Plasma PC is also analyzed, using chromogenic and clotting assays for PS activity. Gene analysis is performed to find out race-specific genetic risk factors for VTE.

This project starts in 2013 and will be finalized in 2015.

Reference: 1. Tsuda T. et al. Blood Coagul Fibrinolysis. 23: 56-63, 2012

Can plasma levels of PS, PC and AT predict the presence of an underlying genetic defect? Anna Pavlova ( Germany)

Interplay of inherited and environmental factors and life events contributes the pathogenesis of thromboembolism. Of all inherited factors, deficiencies of natural anticoagulant proteins, including antithrombin (AT), protein C (PC) and protein S (PS) are the most important causes for thrombophilia. Thus, the ultimate purpose of thrombophilia investigation in a clinical setting is to enable accurate prediction of the thrombotic risk in a particular individual. First-line diagnosis depends entirely on performing a plethora of functional and antigenic assays to measure the factors that predispose to thrombosis. Despite current standardisation procedures, all these assays show significant inter-laboratory variability and inconsistency in phenotype between individuals with apparently similar levels of functional protein. Therefore, plasma activity levels of natural anticoagulant proteins have not always been found to be a good prognostic indicator of clinical severity of the disorder. This observation raises the question if plasma levels of AT, PC and PS and can predict a causative genetic defect.

The molecular bases of AT, PC and PS deficiencies are highly heterogeneous. The most frequent genetic defects are missense mutations, but other types of gene defects, such as nonsense mutations, splice-site mutations, deletions and insertions have also been reported. A review of literature reveals a significant discordance between laboratory diagnostic phenotype data and genotype data in the ranked order PS > PC>AT, the later with the highest concordance of phenotype and genotype. Our data have shown that the highest mutation detection rate (MDR) was found for the SERPINC1 gene (83.5%), followed by the PROC (69%) and PROS1 (43%) genes. Even at AT activities close to the normal range (75%), the MDR was 70%. In contrast, for PC and PS deficiencies, the MDR dropped significantly at mildly lowered to subnormal values. Additionally, evidence has been provided that genetic analysis of patients with PC levels above 70% and PS levels above 55% may not be indicated because of the very low likelihood of detecting a mutation associated with inherited PC or PS deficiencies. DNA-based testing has the

advantage of detecting a mutation independent of phenotypic characterization and therefore, represents a useful diagnostic tool for confirming inherited AT, PC or PS deficiencies, especially for patients with borderline activities or patients on an anticoagulation therapy. Thus, genetic testing in thrombophilia patients can be helpful to address specific issues of patient management, to guide the duration of anticoagulant therapy, to quantify the need for primary or secondary thrombosis prophylaxis, or determine whether family members also need evaluation.

Standardisation of Protein S activity assays

The problem : Lack of agreement in PS activity results by different methods. Steve Kitchen (UK)

Recent data form UK National External Quality Assessment Scheme (NEQAS) exercises related to protein S activity assays were presented. These show statistically different and clinically relevant differences between results obtained with different reagents. This occurs in normal samples and in the presence of protein S deficiency. Not all PS activity kits were well represented amongst cebtres who participated in the calibration of the SSC plasma standard which also complicates potency assignment to commercial standards.

This short presentation formed the introduiction to the final session in the subcommittee programme.

Moderator : Piet Meijer (The Netherlands) Panel : Steve Kitchen (UK), Ian Jennings (UK), Richard Marlar (USA).

Manufacturers of Protein S activity assays that are used in external quality assessment programes or are otherwise known to the panel members had been invited to make a 5 min presentation describing p. Manufacturers were given some guidance and were invited to address interferences and the effects of test sample lyophilisation on assay results. This was considered important to facilitate interpretation of data form EQA programs where this a regular cross assessment of assays but which is almost always done using lyophilised samples. Not all manufacturers accepted the invitation. Those who did were allocated a slot in alphabetical order. The following assays were described

 Hyphen _ Jean Amiral  Instrumentation laboratory – Ralph Bottenus  R2 diagnostics – Marc Goldford  Siemens – Jorgen Patze  Stago – Francois Depasse

There is a subcommittee project investigating the discrepancies betwee results with different assays which will involve a collaborative exercise led by Ian Jennings and including Pioet Meijer

and Ricahrd Marlar. The panel felt it would be important to inbvitre manufacturers to aprticpate in this in addition to expert laborarories.

Platelet Immunology

June 29, 2013

Chairman: Yves Gruel (France) Co-Chairs: Donald M. Arnold (Canada), Tamam Bakchoul (Germany), Sentot Santoso (Germany), Yoshiaki Tomiyama (Japan), Christopher M. Ward (Australia)

Part 1. Educational session

A Greinacher (Germany): New insights on the pathophysiology of heparin-induced thrombocytopenia.

The HIT immune response is atypical. A Greinacher presented the most recent studies supporting that prevalent infections are playing a role in the pathophysiology of HIT, which is presumably a misdirected host defense response. A recent study (Krauel et al, Blood 2012) defined that PF4 binds to the lipid A core of LPS (lipopolysaccharid of bacteria), and this binding mainly depends on the presence of phosphate groups, which are negatively charged. PF4 may also bind to DNA and expose the epitopes to which HIT antibodies binds, as recently showed by Jaax et al (Blood 2013, in press). In addition, PF4/aptamer complexes can also induce an immune response in mice resembling to those of HIT. Recent data also support the hypothesis that the HIT immune response is an ancient (innate) immune response: PF4 "knock-out” mice may produce PF4-specific antibodies when challenged in a polmicrobial sepsis model; Using the ELISPOT technique, the team of A Greinacher also demonstrated the presence of IgM B cells specific for PF4/heparin in the cord blood, and in some adults not exposed to heparin. On the other hand, they also found that higher levels of fibronectin, which binds to PF4/hearin complexes, may contribute to the decrease response of platelets to HIT antibodies in their plasma milieu.

A Cuker (USA): Laboratory testing for secondary forms of ITP: What is the evidence?

Routine laboratory testing for predisposing conditions in unselected patients with ITP is commonly requested, though the basis for this practice is uncertain. Recommendations from clinical practice guidelines and international consensus reports differ with respect to which tests should be routinely ordered and are based largely on expert opinion rather than evidence. Dr. Cuker and his colleagues conducted a systematic review of existing evidence on the utility of laboratory testing for predisposing conditions in unselected patients with ITP. They defined 4 key questions to guide their review: (1) What is the prevalence of predisposing condition x in unselected patients with ITP? (2) What is the diagnostic accuracy of test y for predisposing condition x in unselected patients with ITP? (3) Does identification of predisposing condition x

inform management of ITP? (4) Does identification of predisposing condition x inform management in other respects. This approach was applied to and presented for ANA testing. The prevalence of ANA positivity in adults and children with ITP was 17.0% and 15.2%, respectively. Concerns regarding the diagnostic accuracy of ANA testing due to the high frequency of abnormal results in healthy controls and the effect of ITP treatment on test results was raised. While ANA was found to have a high negative predictive value for the subsequent development of SLE in patients with ITP, the positive predictive value was only 9.3% in adults and 16.3% in children. There is insufficient evidence regarding the benefits of identifying ANA status in unselected patients with ITP to recommend routine testing. Moreover, the poor positive predictive value of ANA testing is likely to yield frequent false-positive results, which may in turn lead to additional evaluations of unproven benefit and undue patient anxiety.

Part 2. Drug-induced and autoimmune thrombocytopenia (Chairs: D Arnold & Y Tomiyama)

Update on the DITP standardization project (D. Arnold, Canada). Dr. Arnold presented a clinical case demonstrating how DITP can be a challenging diagnosis to make based on clinical grounds alone. Laboratory testing is needed to confirm the diagnosis. This may be especially important for patients exposed to multiple drugs or drugs that are used frequently (penicillin, acetaminophen). Proper testing can successfully demonstrate drug-dependent antibodies either by flow cytometry (platelet-associated IgG) or with a GP-specific assay which often shows differences in drug-dependent antibody specific for GPIb vs. GPIIbIIIa. Current testing for DITP is limited for 3 main reasons: 1. There are many drugs potentially involved in DITP reactions and each one would require proper validation as a reagent in the test; 2. there is a lack of standardization; and 3. limited accessibility. Limited accessibility might improve if we concentrated efforts on standardizing procedures for at least the most commonly implicated drugs. Several investigators have examined the frequency of DITP reactions, and among 5 systematic reviews that were published in this area, the most commonly implicated drugs were: quinine, vancomycin and piperacillin. We could start this project with the standardization of those 3 drugs. Since the development of this proposal, Dr. Arnold has identified the following 6 key standardization issues that need to be addressed: 1. Determine the test method; 2. Determine the method of sample collection; 3. Selection of donor platelets for the test; 4. Determine the proper drug concentration for the test; 5. Establish positive and negative controls; and 6. Provide guidance on the interpretation of positive and negative results.

The next steps in this standardization project are: 1. Identify participating laboratories; 2. Formally survey DITP labs for their method of dealing with each of these 6 key issues; 3. Hold discussion groups either by teleconference or email exchange to reach consensus on these items; 4. Prepare a working document. Additional work may be required to establish optimal drug concentrations through sample sharing.

Anti-GP IIb/IIIa drug-dependent antibodies induced by ampicillin and methylprednisolone in patients with sudden-onset thrombocytopenia (V Kiefel, Germany). A great variety of therapeutically used chemical compounds may cause drug induced thrombocytopenia (DIT).

Among the most common were quinine and quinidine (at least in the past). The diagnosis is often suspected using clinical criteria, but detection of drug-dependent antibodies (ddab) contributes significantly to the diagnosis. DIT is one of the differential diagnoses under consideration in patients with acute, unexpected severe thrombocytopenia. In the first patient of this presentation, DIT followed administration of ampicillin after cardiac surgery. In the second patient DIT followed high dose therapy with methyprednisolone (MP) for immunotherapy of multiple sclerosis. In both cases ddab were identified in an ELISA with intact platelets used as screening test (with the necessity of adding the drug in buffer) and GIIb/IIIa specificity was confirmed in the ddab-modification of MAIPA. To the knowledge of the author this is the second patient with MP-induced DIT.

Autoantigenic epitopes on GPIIb-IIIa in ITP (Y Tomiyama, K Kiyomizu, H Kashiwagi, Japan). GPIIb-IIIa and/or GPIb-IX are major autoantigens in primary ITP, and 40-60% patients possess platelet-associated (PA) anti-GPIIb-IIIa, whereas 10-20% patients possess PA anti-GPIb-IX antibodies. Dr Tomiyama’s team and others demonstrated a significant difference in the specificities between platelet-associated and plasma antibodies. Plasma appears to contain secondary antibodies produced by platelet destruction such as anti-vinculin and anti- cytoplasmic domain of GPIIIa antibodies. In addition, PA antibodies are more frequently detected than the plasma antibodies, and PA antibodies, but not plasma antibodies, correlated with the changes in platelet counts in ITP. Thus, characterization of PA antibodies would provide a further insight into the pathophysiology of primary ITP. Dr Tomiyama’s team eluted PA antibodies by diethyl ether from ITP platelets as platelet eluates. Platelet eluates were kept into -80 ºC until use. The PA antibodies kept their reactivity even after elution and clearly re- bound to intact platelets. They detected PA anti-GPIIb-IIIa Abs in 26 out of 76 (34%) patients with ITP in a flow cytometry using HEK293 cells expressing GPIIb-IIIa. They further examined 15 patients having PA anti-GPIIb-IIIa antibodies. PA anti-GPIIb-IIIa bound poorly to mouse GPIIb- IIIa, mainly because of the difference in GPIIb sequence between human and mouse. Employing various human-mouse chimera GPIIb complexed with human GPIIIa, Dr Tomiyama’s team revealed that their epitopes were mainly localized in the N-terminal half of the β-propellar domain in GPIIb. In addition, in three patients they succeeded to further determine the autoantigenic epitopes with a restricted usage of κ or λ chain in anti-GPIIb-IIIa antibodies, which suggests that only limited B cell clones are activated in a certain ITP patients.

Auto-antibodies to GPVI (M Jandrot-Perrus, France). Platelet glycoprotein VI (GPVI), is an important collagen receptor for platelet activation, but is also the target of autoantibodies in some patients. These autoantibodies induce an acquired GPVI deficiency. Autoantibodies to GPVI are rare but are not often searched since adequate tools are not widely available, and above all, they are not associated to severe clinical manifestations. Most frequently, affected patients present with a mild bleeding disorder that may result from GPVI deficiency and another environmental or genetic factor. Anti-GPVI antibodies are most often discovered in the context of immune thrombocytopenia in which abnormal platelet responses to collagen are

detected. The biological effects of anti GPVI antibodies are heterogeneous: the GPVI deficiency is associated with a FcR deficiency either (i) of similar intensity suggesting that the GPVI-FcRg complex is processed possibly by an internalisation/degradation mechanism, or (ii) of lower intensity suggesting that GPVI is separately processed, possibly via the shedding of its extracellular domain by metalloproteases. Most often, autoantibodies are detected in the plasma. In vitro, these antibodies are capable, or not, to activate platelets and rarely to induce the shedding of GPVI. However, in many cases, purified antibodies do not recapitulate the GPVI deficiency in vitro suggesting that additional factors are involved in vivo. Many points remain to be clarified: if anti-GPVI autoantibodies are usually associated to mild bleeding, when and why bleeding could occur has to be determined. The mechanisms leading to the GPVI deficiency have clearly to be better defined and tools are now available. M Jandrot-Perrus outlined the potential interest to identify predisposing factors and the role for polymorphisms. She also proposed an algorithm to identify autoantibodies to GPVI in patients with an acquired thrombocytopenia or mild bleeding disorder. Such systematic studies would allow determining the frequency of autoantibodies to GPVI, to progress in the diagnosis of acquired mild bleeding disorders and to improve the knowledge on the mechanisms regulating GPVI expression and function.

Part 3. Alloimmune and isoimmune thrombocytopenia (Chairs: T Bakchoul & S Santoso)

Treatment

Development of a prophylactic treatment for the prevention of NAIT (Skogen B, Norway)

After an introduction on the Norwegian FNAIT study (Kjeldsen Blood 2007), B Skogen showed data suggesting that anti-HPA-1a immunization most commonly occur after delivery. Therefore, it would be presumably possible to prevent this immunization by administration of anti- Pantibodies as a prophylaxis after delivery. Experiments performed using a mouse model support tthis hypothesis (Tiller et al, Transfusion 2012). In this model, a lower incidence of miscarriages and dead pups was observed in mice receiving prophylaxis. B Skogen presented a project of a Phase III trial, which aims to evaluate this prophylaxis in women.

Postnatal treatment of NAIT and new approach (Bakchoul T & Santoso S, Germany).

NAIT often occurs in the first pregnancy. Random platelets were suggested to be a therapeutic option (Kiefel, Blood 2006) but with a reduced survival than HPA1a negative platelets (D Allen 2007). Tamam Bakchoul presented a study performed with 3 groups of patients: the first were newborns with NAITP who were treated with random platelets only. In the second group, IvIg were associated with random platelets, and 2 cases were treated with HPA1bb platelets (3rd group). The platelet increment after 1st transfusion was not different in the 3 groups. In addition, the platelet count was also equally maintained after transfusion.

Conclusion: Random platelets can be used as a first treatment in infants with NAITP. The benefit if IvIg is questionable. The use of HPA1bb /5a platelets should be considered in case of refractoriness to random platelets.

Diagnosis

Impact of divalent cation on the detection of HPA-1a antibodies (Allen DL, UK).

In this presentation, Dave Allen (scientist of the University of Oxford and NHS Blood and Transplant), outlined how important are divalent cations for the sensitive detection of anti- HPA1 antibodies. He showed by flow cytometry and by treating platelet-rich plasma by TBS- EDTA that epitopes recognized by different MoAb were variably affected by chelation of calcium from integrin IIb3. Dave Allen also demonstrated that the presence of EDTA either in the sample tested or in assay buffers reduced the MAIPA sensitivity. This finding was confirmed by performing a NIBSC international Quality Assurance workshop. Serum and EDTA plasma samples (from same venepuncture) from a mother with NAIT-affected children were distributed to 39 laboratories. These samples contained a potent anti-HPA-1a (15 i.u./mL) antibody, which was not detected by 2 of the 39 labs when the plasma sample was analyzed. This reduced sensitivity was not related to the capture MoAb used in the MAIPA. In conclusion, this study strongly supports: 1. To avoid testing EDTA plasma in MAIPA (and likely in PIFT) for the detection of anti-HPA1 antibodies; 2. To ensure a careful selection of capture mabs.

A Greinacher suggested that a specific work could be conducted on this practical and important question.

Tamam Bakchoul also suggested a workshop comparing the sensitivity of MAIPA using EDTA vs. Hirudin or Citrate platelets.

Detection of platelet alloantibodies using a multiplex bead assay (Boldt B & Visentin GP, USA)

Current technologies for the detection of platelet alloantibodies are at times constrained by a limited number of available platelet donors with desirable HPA types, a requirement for relatively large sample volumes, and long testing times. With the commercial availability of color-coded microspheres and corresponding detection system, it has become possible to build panels of glycoprotein-coated beads, which can be simultaneously used to detect antibodies reactive with HPA 1-5 targets as well as GPIV and Class I HLA (Pak Lx). Results from a multi- center clinical study (Sanquin Diagnostics, BloodCenter of Wisconsin, and Puget Sound Blood Center) comparing the multiplex bead immunoassay with MAIPA showed the co-positivity to be nearly 80% - 100% (depending on the HPA system), the co-negativity to be 98% or higher, and the overall agreement to be greater than 97%. However, Some concerns were raised about the test sensitivity in detecting anti-HPA-3a and anti-HPA-15a antibodies.

Basic Research

Molecular structure of HPA-1a and -HPA-1b and its consequence on immune response (Jallu V, France). The human platelet antigen (HPA)-1 system is characterized by a leucine (allele 1a)- to-proline (allele 1b) substitution in position 33 of the mature β3 subunit. The HPA-1 system also presents a third variant defined by a valine in position 33, not described as immunogenic (Santoso et al, Transfusion 46:790-799, 2006). Sera containing alloantibodies to the HPA-1a antigen react variably with the V33 form of β3. This suggests structural alterations of the V33 β3 although leucine and valine have similar physical and chemical properties; V Jallu et al studied the structural modifications related to the L33V substitution, and compared the structures of the three β3 variants. He showed i/ that the high flexibility of the PSI, I-EGF-1, and I-EGF-2 domains is conserved in the three variants; ii/ that the L33V substitution mainly displaces structural equilibriums toward a loop tightly maintained by hydrogen bonds for residues 27-31 of the PSI domain, and toward a β-turn with a start of a helical motif for residues 435-338 of the I-EGF-1 domain; iii/ these structures are in dynamical equilibriums with extended conformations (more than 40 % of occurrences) in L33 and P33 β3 isoforms. Despite common features between leucine and valine amino acids, he also showed that the L33V substitution displace a dynamic equilibrium of β3. Common structures between L33 and V33 β3 models underlie the reactivity of some anti-HPA-1a alloantibodies with V33 β3. Nonetheless the variability of this reactivity suggests that molecular dynamic play a key role in the antigenicity of the HPA-1a alloantigen.

Part 4. Heparin-induced thrombocytopenia (Chairs: C Ward & Y Gruel)

Genomic Approaches to Variation in Platelet Activation via FcgRIIa (S McKenzie, USA). Steven McKenzie (USA) addressed the clinical variability in platelet activation that is observed in HIT but still largely unexplained. He noted similarities between HIT and other platelet activation syndromes (TTP, sepsis, antiphospholipid antibodies), which also feature intravascular activation of platelets, endothelium and the immune system, leading to microvascular thrombosis and organ failure. Although much work has been done characterizing downstream signaling for FcgRIIa, such as the Syk pathway, it is unclear why only a subset of antibodies trigger platelet activation. He outlined two ongoing projects which seek to answer this question – firstly, collaborative studies of the platelet transcriptome, including microRNA, and secondly a genomic analysis of two strains of mice with differing susceptibility to FcgRIIa-dependent platelet activation, triggered by anti-CD9 antibodies. Differentially expressed genes in humans (154 with platelet reactivity phenotype, mRNA and miRNA profile, and 5 million Genomic SNPs) and mice appear to come from poorly defined pathways such as ubiquitination, protein-protein interactions and protein phosphatases, rather than from expected candidates in the Syk pathway.

Novel laboratory assays for heparin-induced thrombocytopenia (A. Cuker, USA). Laboratory testing for heparin-induced thrombocytopenia (HIT) has important shortcomings. Immunoassays fail to discriminate platelet-activating from non-pathogenic antibodies. Specific functional assays are often impracticable due to the need for platelets and radioisotope. A Cuker described two assays that may overcome these limitations. The KKO-inhibition test (KKO- I) measures the effect of plasma on binding of the HIT-like monoclonal antibody KKO to

PF4/heparin. DT40-luciferase (DT40-luc) is a functional test comprised of a B-cell line expressing FcgRIIa coupled to a luciferase reporter. A Cuker et al (Blood, 2013) compared these assays to polyspecific and IgG-specific PF4/heparin ELISAs in samples from 58 patients with suspected HIT and circulating anti-PF4/heparin antibodies. HIT was defined as a 4Ts score ≥4 and positive 14C- serotonin release assay. HIT-positive plasma demonstrated greater mean inhibition of KKO binding than HIT-negative plasma (78.9% vs. 26.0%, p<0.0001) and induced greater luciferase activity (3.14-fold basal vs. 0.96-fold basal, p<0.0001). The area under the ROC curve (AUC) was greater for KKO-I (0.93) than for the polyspecific (0.82, p=0.020) and IgG-specific ELISA (0.76, p=0.0044) and for DT40-luc (0.89) than for the IgG-specific ELISA (p=0.046). KKO-I and DT40-luc showed better discrimination than two commercially available immunoassays, are simple to perform, and hold promise for improving the specificity and feasibility of HIT laboratory testing.

The "Rapid Test PF4", a new assay for the diagnosis of HIT (Boldt B & Visentin GP, USA) The rapid assessment of anti-PF4-heparin associated antibodies may assist in the diagnosis of the HIT syndrome. However, current assays, which are configured for "batch” testing are not easily performed as a stat assay. The lateral flow technology lends itself well to screening single samples in a short period of time. Recent results from an internal method comparison study comparing the lateral flow assay with ELISA showed the estimate of co-positivity/co-negativity/ overall agreement to be 97%, 80%, and 84% for the combined detection of IgG, IgA, and IgM; and the estimate of co-positivity/co-negativity/ overall agreement to be 98%, 77%, and 81% for the detection of IgG.

Proposal for a standardized method for the whole blood impedance assay (C Ward, Australia). Christopher Ward (Australia) presented a report on a project of the SSC initiated at Liverpool, and aiming to develop a standardized method for whole blood impedance aggregometry as a function HIT assay. Five groups from Australia, Belgium, France, Germany and the UK have trialed different methods and compared results at a recent teleconference. The contributors noted that this assay should be optimized for high specificity and positive predictive value, and used as a confirmatory assay in samples shown to contain heparin-PF4 antibodies. Platelet donors must be known to be sensitive to a range of HIT antibodies, and this cannot be predicted by FcgRIIa genotype or response to activating monoclonal antibodies. It was suggested that these donors should be tested against both strong and weak HIT positive samples available to each laboratory, in the absence of an established standard for a positive control. Aggregation responses were improved by use of hirudin tubes for donor blood, but similar results could be obtained using citrated samples. A provisional method was outlined, including testing against 200U/ml heparin if equivocal inhibition was noted in the 100U/mL sample. The optimal low-dose heparin concentration was still under discussion, as was the definition of a "positive” aggregation response.

Anti-PF4/heparin serology in the PROTECT-ICU trial (T Warkentin, Canada). Dr. Warkentin summarized the serological HIT studies of the 3746-patient randomized controlled trial, PROTECT, which compared antithrombotic prophylaxis with unfractionated heparin (UFH) vs low-molecular-weight heparin (LMWH), dalteparin, in intensive care unit (ICU) patients (N Engl J Med 2011;364:1305-11). There were 17 patients identified in the PROTECT trial that were

classified as having had HIT, based upon positive testing in the serotonin-release assay (SRA): 12 in the UFH arm of the trial, and 5 in the LMWH arm of the trial. In a prespecified "per-protocol” analysis (where patients with deep-vein thrombosis [DVT] on their initial study ultrasound were excluded), the HIT breakdown was 12 UFH, 3 LMWH, which was statistically significant (12/1561 [0.8%] vs 3/1566 [0.2%], hazard ratio, 0.27; p=0.046; Fisher’s exact test, p=0.021). In the presentation, it was shown how a detailed analysis of these 17 cases—examining both "seroconversion” (i.e., study drug-attributable HIT antibody formation) and "breakthrough” (study drug-attributable development of thrombocytopenia and/or thrombosis at time of HIT antibody presence)—further supports the difference in HIT frequency between UFH and LMWH as being real, since the analysis supported a reduction in HIT frequency with LMWH even when taking into account various "confounding” exposures to open-label heparin that occurred quite commonly in this ICU trial. Furthermore, among the 409 patients who underwent testing for HIT antibodies in the central lab, either because of thrombocytopenia or thrombosis (or both), there was a much higher frequency of anti-PF4/heparin IgG antibodies by ELISA in the UFH (vs LMWH) study arm: 59/216 (27.3%) vs 26/193 (13.5%); p<0.001. Finally, the study provided evidence for markedly different outcomes among patients with unrecognized HIT antibodies (SRA+ status) who subsequently received UFH to treat DVT or pulmonary embolism: whereas some patients demonstrated initial exacerbation of thrombocytopenia followed by platelet count recovery (consistent with the previously-reported phenomenon of HIT antibody transience despite continuation of heparin), one patient developed a fatal anaphylactoid reaction (cardiac arrest post-intravenous heparin bolus). In conclusion, the PROTECT trial substudy supports a ~75% reduction of HIT with LMWH, dalteparin, compared with UFH, in ICU patients, and also demonstrates variable consequences of further UFH treatment among SRA+ patients in the ICU setting.

Conclusion and perspectives (Y Gruel, France).

Based on the presentations and the discussion of this SSC meeting on "Platelet Immunology”, it was concluded that 2 projects would be continued for the next year. The first is related to drug- induced thrombocytopenia (DITP) and aims to define practical recommendations for the laboratory testing in patients with suspicion of DITP. This group is led by Donald Arnold (Hamilton, Canada). The second ongoing project is coordinated by Chris Ward (Sydney, Australia) and to aims to define a standardized procedure for using the whole blood impedance aggregometry (using the Multiplate analyzer) to diagnose HIT. Another project has also to be considered: it is related to the effect of EDTA on the detection of anti-HPA1 antibodies, but its design has to be defined taking into account the previous studies performed by the ISBT.

Platelet Physiology

June 30, 2013

Chairman: Paul Harrison (UK) Co-Chairs: Christian Gachet (France), Paolo Gresele (Italy), Diego Mezzano (Chile), Andrew D. Mumford (UK), Patrizia Noris (Italy), Alan Nurden (France)

The meeting began with a 1 hour educational session. The first presentation entitled "Platelets and their progeny” was by Dr. Hansjorg Schwertz (Germany) followed by a presentation entitled "Circulating pre-platelets and their role in platelet maturation and size determination” by Dr. Jonathan Thon (USA). The final educational talk was by Dr.Meinrad Gawaz (Germany) and was entitled "platelets and inflammation”. The three educational talks were all of excellent standard and created much discussion amongst the audience in a full lecture theatre.

The SSC business session then began at 9:00 am. Paul Harrison (UK) briefly outlined the new mission statements of the ISTH SSC and the platelet physiology SSC and followed this with an update of current ongoing and future projects. The LTA guideline that was finished and agreed upon in Cairo (available on the SSC website) in 2010 is now available as a JTH paper in the June 2013 issue. Cattaneo et al, Recommendations for the Standardization of Light Transmission Aggregometry: A Consensus of the Working Party from the Platelet Physiology Subcommittee of SSC/ISTH.J Thromb Haemost. 2013 Apr 10. doi: 10.1111/jth.12231. The detailed survey (from 202 laboratories) of laboratory practice for the diagnosis of inherited platelet disorders coordinated by Paolo Gresele and presented at the 2012 Liverpool SSC will be submitted for publication to JTH as soon as possible. The final presentation of the guidelines for the diagnosis of inherited platelet disorders will be presented later in this session by Paulo Gresele for discussion and will again be submitted for publication to JTH later this year. Other projects include the Diagnostic Utility of the ISTH BAT for platelet function disorders (in collaboration with the ISTH BAT working party) and a new Guideline/position statement on Platelet Release Assays that will be discussed later in this session. The SSC is also working on a Guideline/position statement on monitoring P2Y12 inhibition and a presentation on this is planned be given at the SSC in 2014. We also monitoring developments on the Evaluation of methods for measuring the platelet transcriptome (in collaboration with the thrombogenomics working party) for a possible future project.

Paolo Gresele (Italy) then followed this with the final presentation of the working party for the diagnosis of inherited platelet disorders including a detailed diagnostic algorithm. The proposed diagnostic algorithm was generated taking into account the responses to the world wide survey on current practice for the diagnosis of platelet disorders and based on intense discussion among working party members and includes a step-wise approach which allows even to not specialized laboratories to obtain a clear characterization of their patients and, when required, to refer to more specialized laboratories for definitive diagnosis of the most complex cases. After discussions with the audience this will be further refined and edited for submission and publication as a new guideline in JTH this year. Paolo also announced a new SSC retrospective

study on the evaluation of this new diagnostic algorithm. Invitations to participate will be announced to members and on the website and newsletter.

Yvonne Heskens (Netherlands) then presented an overview of LTA standardization in the Netherlands using a National based protocol based upon the recently published SSC LTA guidelines. 54 participants also took part in a national survey on the laboratory practice in the Netherlands. The inter-laboratory studies demonstrated some interesting differences in samples from healthy volunteers between manufacturers and concentrations of agonists, aggregometer instruments and analytical procedures. The variation tended to be more pronounced with lower concentrations of agonists. Yvonne concluded that adherence to the SSC LTA guideline will require a lot of changes in the laboratory practice in many different laboratories in the Netherlands as each laboratory has developed its own individual practice. Future studies will try to implement a national protocol across all laboratories, establish reference ranges for different equipment and reagents and develop a national quality control scheme.

After the coffee break the secretary of Bleeding Assessment Tool Committee Alberto Tosetti (Italy) gave an overview of the history and evolution of the ISTH Bleeding Assessment Tool (BAT) and its potential role for platelet function disorders. The ISTH Bleeding Assessment Tool (ISTH-BAT) is a consensus-based, publicly available tool designed to evaluate the severity of bleeding symptoms, primarily in patients with congenital disorders of haemostasis (see Rodeghiero et al, JTH 2010:8: 2063–2065). The tool includes a standardized questionnaire and a 0-4 bleeding score for each symptom that is used to compute an overall bleeding score. Based on the ISTH-BAT, a web-based instrument has been developed by Barry Coller at the Rockefeller University, NY, USA, named Bleeding Assessment Tool Repository (BAT-R). The database is designed to collect and store data regarding bleeding symptoms in humans, providing a user- friendly, web-accessible platform, encouraging uniformity in the standardization and collection of bleeding histories. Individual patient data from each centre can be entered, the BAT calculated in a standardized format and data downloaded from the website in excel format. Data can also be shared and compared with the existing database. Preliminary data on inherited platelet disorders was presented although few studies have been performed.

This was followed by Marie Lordkipanidze (UK) who presented new data on the assessment and validation of the BAT in the UK GAPP study (Genotyping and Phenotyping of Platelets) – this data was also presented during the congress and has just been published- J Thromb Haemost. 2013 June 29. doi: 10.1111/jth.12332. The ISTH BAT is no doubt a useful tool in documenting lifelong bleeding history and mild bleeding symptoms. However, in this study the score obtained is not predictive of the presence of a platelet defect defined by lumiaggregometry (aggregation and secretion) in patients with suspected inherited platelet function disorders.

Paul Harrison (UK) then presented a proposal for the Platelet Physiology SSC evaluation of the BAT for platelet function disorders. Participants will be invited to submit both retrospective and prospective data on patients presenting with mucocutaneous bleeding symptoms and already defined inherited platelet defects and normal controls and a possible positive control group

(e.g. Type I VWD). Platelet Physiology SSC data will be anonymised and available for analysis and then sharable within the entire database and will be presented at future SSC’s. A future announcement for participation will be made via the ISTH website etc.

Diego Mezzano (Chile) presented an overview of the measurement and problems of platelet release assays with the aim of the SSC writing a future review, guideline/position statement. ATP secretion by lumiaggregometry is the most popular test.

Elaine Uhr (Australia) then gave an overview of the development of a standardized and optimized clinically diagnostic mepacrine assay for diagnosing storage and release disorders. Buffer selection is important for the assay – Hepes buffer with no magnesium is recommended. Platelet count and size and incubation times with mepacrine and mepacrine concentration are all important variables that need to be taken into account during assay development and interpretation of results. Platelet counts should be adjusted when necessary. Larger platelets take up more mepacrine but the results are predictable in terms of uptake and release. The recommended standardized protocol will eventually be submitted for publication by Elaine.

Predictive Variables in Cardiovascular Disease

June 29, 2013

Chairman: James Douketis (Canada) Co-Chairs: Shinya Goto (Japan), Paul A. Kyrle (Austria), Karel Moons (Netherlands), Marc C. Samama (France), Alex Spyropoulos (USA), Richard H. White (USA)

Listed below are the activities of this Subcommittee for the 2013 ISTH Meeting.

1.0 Predictive Variables in Cardiovascular Disease Subcommittee Session

Educational Session: CHADS2, CHA2DS2VASc and New Approaches to Predicting Thrombosis

8:05-8:25 CHADS2 and CHA2DS2VASc to determine stroke risk in atrial fibrillation: One or both? (M. Coppens, Neth)

8:25-8:45 Platelet function assay: Is there a role to predict adverse cardiovascular events in the PCI setting? (P. Kyrle, Aus)

8:45-9:05 Patients receiving new oral anticoagulants who need surgery: How to manage anticoagulation and use coagulation tests to minimize thrombosis and bleeding risk? (C.M. Samama, Fr)

Update of SSC Research and Related Activities

9:20-9:40 D-dimer in atrial fibrillation: Is there a role to combine with CHADS2 to stratify patients according to stroke risk? (J. Eikelboom, Can)

9:40-10:00 D-dimer and risk for stroke in atrial fibrillation (C. Tait, UK).

10:00-10:20 SSC Communication: Standardization of perioperative outcomes: Why is this clinically important and implications for clinical trials. (A. Spyropoulos, USA)

10:20-10:40 D-dimer to predict recurrent VTE: The MORGAGNI study (P. Prandoni, It)

10:55-11:15 Prognosis after unprovoked VTE: Lessons from the REVERSE study (G. Le Gal, Can)

11:15-11:35 SSC Subcommittee Report: Predictive value of hemostatic variables for first and recurrent arterial events (G. Lowe, UK)

11:35-11:55 Using the CHA2DS2VASc score in clinical practice: A clinical trial (CAFÉ) assessing different approaches (K. Moons, J. Geersing, Neth)

Summary: The meeting was very well attended with excellent engagement by audience

2.0 SCC Working Project on D-dimer and other Predictors of Recurrent VTE

A meeting was convened of a collaborative research groups studying pooled data from several studies assessing patients with VTE and risk for recurrence. To date, there have been 5 publications from this Working Party: Ann Intern Med, BMJ, J Thrombo Haemost (2), J Clin Epidemiol.

The attendees of this meeting included: J. Douketis (Can - Chair), P. Kyrle (Aut), M. Marcucci (It), K. Moons (Neth), J. Geersing (Neth), C. Tait (UK), A. Tosetto (It), G. Lowe (UK), D. Fitzmaurice (UK), A. Iorio (Can), M. Rodger (Can), J. Hendriksson (Neth), Gregoire Le Gal (Can). Regrets: C. Kearon (Can), P. Prandoni (It), G. Palareti (It), Daniela Poli (It), B. Cosmi (It).

This is the 5th meeting of this group, which was formed after the 2008 SSC meeting in Vienna. During this meeting, ongoing and future collaborative initiatives relating to determinants of recurrent VTE were discussed, including:

 A manuscript finalizing a study to validate the Vienna prediction guide (CPG) for recurrent VTE was discussed. It will be submitted to a peer-reviwed journal.  The Working Project members welcomed Dr. David Fitmaurice, University of Birmingham, who discussed the STOP-IT and ExAct studies, which will develop a CPG for recurrent VTE using 3 large databases: RIETE, MEGA, and the pooled IPD. The Subcommittee has facilitated this collaborative research study. The committee eagerly awaits the findings of Dr. Fitzmaurice and his colleagues.  The Working Project members heard a presentation from Drs. Karel Moons, Jan Geersing, and Janika Hendriksson regarding the VISTA Study, a randomized trial assessing a standardized management strategy to determine the duration of anticoagulation for patients with unprovoked VTE. Given some challenges with meeting patient recruitment timelines, there was agreement to share data from concurrent studies, such as STOP-IT, to enhance the study's statistical power.

3.0 SSC Working Project on Perioperative Determinants of Thromboembolism and Bleeding

A collaborative group with an overall aim to study patients who are receiving dual antiplatelet therapy, conventional new oral anticoagulants and who require and urgent or elective surgery/procedure.

To date, there have been two SSC Communications from this Working Party, published in J Thromb Haemost relating to standardization of perioperative outcomes in patients who are receiving anticoagulant or antiplatelet therapy.

The attendees of this meeting included: J. Douketis (Can - Chair), M. Samama (Fr), J. Levy (USA), A. Greinacher (Ger), A. Spyropoulos (USA), G. Hron (Ger), N. Rosencher (Fr), A. Godier (Fr). Regrets: P. Sie (Fr), Beverly Hunt (UK), P. Albaladejo (Fr).

This is the 2nd meeting of this group, following its inception after the Kyoto SSC. During this meeting, ongoing and future collaborative initiatives relating to determinants of recurrent VTE were discussed, including the following projects:

 Dr. J. Douketis presented for discussion a multi-centre observational study assessing a standardized method to manage patients with coronary stents on dual antiplatelet therapy who require a surgery/procedure. This study has commenced patient recruitment in Hamilton, Canada, and the aim is to add other clinical centres in North America and the European Union.  Dr. A. Greinacher presented for discussion a German randomized trial assessing the use of pathogen inactivation of platelets in patients who are receiving dual antiplatelet therapy and require urgent surgery. The Working Party agreed to share the protocol from the above-mentioned study for the development of this study in Germany.  Dr. A. Spyropoulos presented for discussion a multi-national registry relating to patients who are receiving new oral anticoagulant drugs (NOACs) and need an elective surgery/procedure. There was collaboration from colleagues, including M. Samama and P. Albaladejo, to share a template and case-report forms from a similar study that is ongoing in France.  Dr. C. M. Samama discussed the need to have standardization of laboratory-based methods used for intra-operative monitoring of coagulation function (TEG, ROTEM) and platelet function, and the use of procoagulant agents (antifibrinolytics, fibrinogen) for patients with massive bleeding.

The members of this Working Project submitted a proposal to the SSC Executive to formally recognize the activities of this collaborative group as a Working Group. An application was submitted by Dr. J. Douketis, on behalf of the Working Project colleagues, to develop educational programs for the 2014 and 2015 SSC Meetings. The aims of this Working Group would be to (a) showcase the activies and research of the Working Group, and (b) promote education and knowledge translation relating to the burgeoning field of perioperative medicine as it pertains to patients who are receiving anticoagulant and/or antiplatelet drugs. A decision as to the status of this Working Group will be forthcoming by August 2013.

Vascular Biology

June 29, 2013

Chairman: Francoise Dignat-George (France) Co-Chairs: Elizabeth E. Gardiner (Australia), Nigel Key (USA) , R Nieuwland (Netherlands), Florence Toti-Orfanoudakis (France)

The session was divided into two sections: the Educational session and the business session, This business session was divided into four parts addressing respectively: Part A, "Shedded protein and receptors". Part B "Circulating endothelial cells and progenitors", Part C "Microparticles" and Part D "Standardization studies" The success of this SSC VB was attested by the presence of at least 200 participants during the whole session

The Educational session, chaired by Françoise Dignat-George, covered the emerging role of microparticles in cellular therapy and regenerative medicine, the regulation of membrane protein shedding by tetraspanins and the interest of endothelial progenitors in cardiovascular diseases studies with blodd outgrowth endothelial cells.

Florence TOTI, France Microparticles : emerging role in cellular therapy and regenerative medecine

"In her presentation "Membrane Microparticles: emerging role in cellular therapy and regenerative medicine" Florence Toti reviewed the recent data of the literature concerning microparticles as pathogenic markers of tissue remodeling. She focused on the particular settings of cellular therapy and transplantation, namely ischemia reperfusion and allograft rejection and graft function. She proposed that microparticles are not only indicators of vascular and graft tissue remodeling but contributors to the dynamic exchanges between graft and host tissue. She proposed that the pharmacological control of microparticle release during ischemia could improve engraftment at the early stage and emphasized on the need for a better understanding of the mode of action of immunosuppressive drugs on the long term."

Mike Tomlinson, United Kingdom Tetraspanins as new regulators of membrane protein shedding

The tetraspanins, are emerging as important regulators of the trafficking, lateral mobility and clustering of specific so-called "partner proteins” with which they interact. Tetraspanins further self-associate to form membrane microdomains that function as platforms for optimal cell adhesion and signal transduction. A major tetraspanin-associated protein is A Disintegrin and Metalloprotease 10 (ADAM10), a ubiquitous transmembrane protein that acts as a "molecular scissor” by shedding the extracellular regions from at least 40 different transmembrane protein targets. The consequences of ADAM10-mediated shedding include the removal of activatory receptors from the cell surface (e.g. platelet GPVI), the removal of adhesion molecules to increase vascular permeability (e.g. vascular endothelial (VE)-cadherin), the release of

chemokines to promote leukocyte transmigration during inflammation (e.g. endothelial CX3CL1 and CXCL16), or the initiation of intracellular signalling to direct vascular development (e.g. Notch). ADAM10 is a major component of tetraspanin microdomains, Last year the group of M Tomlinson identified a subgroup of six related ADAM10-interacting tetraspanins termed the TspanC8s. ADAM10 interaction with a TspanC8 promotes its exit from the endoplasmic reticulum and maturation into a mature protease at the cell surface. Indeed, endothelial cells and red blood cells rendered deficient for their major TspanC8 (Tspan14 and Tspan33, respectively), had substantially reduced surface ADAM10 expression. Different TspanC8s could therefore localise ADAM10 to distinct substrates and allow ADAM10 therapeutic targeting in a cell type and/or substrate-specific manner, so minimising the likely severe side-effects of targeting ADAM10 on every cell in the body.

Anna. M. Randi, UK Endothelial progenitors in cardiovascular disease: studies with blood outgrowth endothelial cells (BOEC)

Over the last 15 years, many claims have been made regarding the origin of Endothelial progenitor cells (EPC) and their potential as therapeutic as well as biomarkers. Unfortunately, because of differences in the methods employed to isolate or quantify EPC, the field has become rather confusing and the value of EPC as research as well as translational tool risks being undermined. One of the most important properties of EPC is that they allow us to investigate endothelial biology in patients. In this presentation, A Randi reviewed the key steps in the identification and characterisation of circulating EPC and highlighted some of the controversial aspects. She then focussed on a type of circulating endothelial progenitor, named blood outgrowth endothelial cells (BOEC), which more than others have been shown to display the characteristics that a "true” EPC should display: clonal proliferative potential, differentiation to the endothelial lineage, ability to form stable blood vessels when implanted into tissues. She showed that BOEC represent a valid tool to investigate endothelial dysfunction in patients with primary endothelial defects, such as von Willebrand Disease (VWD), as well as patients with acquired endothelial dysfunction, such as Chronic Obstructive Pulmonary Disease (COPD). By studying BOEC from VWD patients she identified complex cellular phenotypes in patients previously thought to carry only a synthesis defect (Type 1 VWD). This approach also allowed to validate a novel function for von Willebrand Factor, namely the regulation of angiogenesis. In COPD, BOEC are senescent due to epigenetic modifications which involve the histone deacetylase SIRT1. Ex vivo treatment with drugs such as resveratrol can improve the function of BOEC. A. Randi concluded with future potential applications of BOEC in translational medicine.

The Business Session was divided into 4 parts: Part A, "Shedded protein and receptors". Part B "Circulating endothelial cells and progenitors", Part C "Microparticles" and Part D "Standardization studies"

PART A: Shedded proteins and receptors, chaired by Elizabeth E. Gardiner

Robert Andrews , Australia Measurement of shed vascular proteins in clinical settings

Robert Andrews gave an excellent introduction to methodology currently available to measure soluble ectodomains and levels of intact and cleaved receptor fragments in biological samples by flow cytometry, western blot and ELISA. He highlighted the need to develop standard protocols for the preparation of samples for measurement including the collection tubes, the processing steps and the timing of collection and processing. He discussed the mechanisms responsible for the activation-dependent shedding of platelet adhesion receptors and in the case of GPIb and GPVI, linked the regulated shedding of these receptors respectively to platelet ageing and to accelerated platelet clearance in diseases with immunodestruction such as ITP and clinical situations where blood is chronically and transiently exposed to elevated levels of shear stress such as in acute coronary syndrome and in individuals who have Left Ventricular Assist Devices (LVADS). He highlighted variability in healthy donors and indicated the potential of soluble receptor ectodomains to be linked to clinical outcomes – for bleeding or thrombotic events. He recommended analysis in combination with other bio-markers.

Giovanni Davi, Italy Shed proteins as biomarkers in ACS

G. Davi gave an update on circulating platelet-derived protein from platelet sheddome. He reviewed several shed proteins that have been proposed as biomarkers of acute coronary syndromes (ACS). He reminded the definition of a biomarker, that should be objectively measured, unaltered by pre-analytical artifacts and reproducibly assayed by easily performed methods.Activated platelets shed surface proteins, potentially modifying cell function as well as providing a source of bioactive fragments.

Recently, about seventy shed proteins have been identified in the human platelet sheddome. Among them, sP-selectin, sCD40L, sGPV, and sGPVI have been found elevated in patients with stable angina. The shedding of membrane-associated CD40L produces sCD40L that in the Women’s Health Study, in the CAPTURE study, and in the MIRACLE study has been identified as an independent risk factor for recurrent cardiovascular (CV) events in ACS, particularly in combination with troponin-T levels. Reduced levels of sGPVI in ACS, at difference with stable angina, may indicate that this shedded protein binds to sites of vascular injury where collagen is exposed, thus being removed from circulation.

Several proteins may be shedded from endothelial cell membrane. In particular, thrombomodulin, E-selectin, ICAM-1, and VCAM-1 have been found elevated within 1-hour of chest pain onset in ACS, and ICAM-1, and VCAM-1 remain elevated for months. High levels of sVCAM-1 on ACS presentation are associated with more in-hospital adverse coronary events and major CV event at 6-months.

Soluble CD14, CD163, LR11 have been identified as protein shedded from macrophage surface membrane and found elevated in the ACS setting.

Emerging shedded (?) proteins form uncertain cell origin (platelets and/or monocytes/macrophage and/or endothelial sheddome) have been identified in the context of ACS. MRP8/14 predicts CV events in both apparently healthy population and survivors of ACS, and may add prognostic information to that conveyed by standard risk factors and CRP evaluation. ROS-modified compounds induce a prothormbotic phenotype via interaction with platelet CD36, and sCD36 is well related to platelet activation markers in type 2 diabetes mellitus. Highly up-regulated expression of CD36 in circulating monocytes have been demonstrated in ACS patients.

Finally, the soluble receptor for advanced glycation end-products (RAGE), an inflammation propagating factor contributing to accelerated atherosclerosis, has been found inversely associated with cardiac troponin in patients with NSTEMI-ACS.

Despite increasing data on the relevance of these shed proteins in the ACS setting, the lack of standardized methodological approaches and the complexity of pathophysiology of these proteins renders not easy the use of these shed molecules as biomarkers in clinical settings.

Lawrence Brass, USA Mining the platelet sheddome for biomarkers and insight into mechanism

Professor Brass discussed data describing proteins released from PMA-treated platelets using mass spectrometry techniques and highlighted the need to treat proteins identified through this process as candidate shed proteins and to specifically analyse and validate individual protein shedding mechanisms. Surface expression, cleavage, and shedding of semaphorin 7A were demonstrated, as was its association with α-granules. Semaphorins are of particular interest because of their role in communication between cells. Cleavage of semaphorin 7A and 12 other proteins was substantially reduced by an inhibitor of ADAM17, a known sheddase. Cleavage of 16 of the proteins, including GPVI, was essentially unaffected. These results define a subset of membrane proteins as sheddome candidates, forming the basis for further studies examining the impact of ectodomain shedding on platelet function. Ectodomain shedding of platelet cell surface proteins may regulate platelet function by modifying adhesive interactions, removing ligand-receptor signaling pairs and liberating soluble fragments that harbour biological activity.

PART B: Circulating endothelial cells and progenitors, chaired by Florence Toti-Orfanoudakis

David M. Smadja, France Circulating endothelial cells and progenitors in pulmonary hypertension: potential in diagnosis and treatment monitoring

The respective abundance of circulating endothelial cells and endothelial progenitor cells may reflect the balance between vascular injury and repair. Pulmonary arterial hypertension (PAH) is a heterogeneous pulmonary vascular disease associated with a strong pulmonary vessel remodelling. The group of D. Smadja aimed to identify non-invasive biomarkers of endothelial

turnover that could be used to identify subgroups of PAH, responses to vasodilatator therapy or clinical deterioration. They quantified Circulating endothelial cells (CECs) isolated with CD146- coated beads, circulating CD34+CD133+ progenitor cells and endothelial progenitor cells in patients with congenital heart disease (CHD) associated with PAH, idiopathic PAH, or post thrombotic PAH. He alos présented the follow up of these cells after PAH-specific vasodilator therapy. A proangiogenic effect of treprostinil (a prostacyclin analog) administration targeting endothelial progenitor cells was described that helps to understand the beneficial effect of this drug in kids with refractory PAH. D. Smadja also presented results allowing to propose CECs as a valuable tool to define reversibility of PAH during CHD and as a biomarker that could help optimization of therapeutic strategies. Because of numerous technical challenges to quantify CECs by the reference method, D. Smadja also presented results of CECs detection using the Attune® Acoustic Focusing Cytometer and in particular results of application of this new technique as a biomarker in PAH.

Michael Hristov, Germany Flow cytometry detection of circulating endothelial progenitor cells

Accumulating evidence intensively advises circulating endothelial progenitor cells (EPCs) as surrogate cellular biomarkers in cardiovascular and cancer disease. However, a general standard on their quantification is still elusive, thus precluding a routine monitoring and comparative interpretation of clinical studies. M. Hristov intended to develop an advanced flow cytometric protocol for proper ex vivo quantification of EPCs in human blood. This protocol employs lyse/no-wash procedure and bead-based determination of absolute cell counts and uses three-color antibody panels at appropriate compensation. Analysis of rare events and low antigen expression in the EPC experiment is strengthening by 1/ sequential gating with strict exclusion of dead cells, as well as by matching high-intensity fluorochromes to low-density markers and 2/ by implementing the fluorescence-minus-one control. This current protocol provides quantitative information under a simple gating logic while using commonly accepted fluorochromes. This assay is therefore highly adapted for routine use.

PART C : Microparticles, chaired by Rienk Nieuwland

Edwin van der Pol, The Netherlands Determination of the refractive index of vesicles using nanoparticle tracking analysis

Microparticles and exosomes are often studied by optical detection methods based on light scattering, such as flow cytometry. Light scattering is a complex process involving the size, shape, and refractive index of these vesicles. Consequently, the refractive index affects the detection limit of optical techniques and is essential to relate the detected light scatter signal to the diameter of vesicles. The refractive index may also provide a new label-free parameter. However, knowledge on the refractive index of vesicles is limited, since no methods are available to determine the refractive index. The group of Van der Pol has applied nanoparticle tracking analysis (NS500, Nanosight Ltd) to detect light scattering from single beads of known size and refractive index. He analytically described the relation between the diameter,

refractive index, and light scattering of beads by Mie theory to determine the refractive index of vesicles from cell-free urine and platelet poor plasma. He obtained a median refractive index of urine vesicles of 1.36, with 95% of the vesicles having a refractive index between 1.35 and 1.45. The refractive index distribution of particles from plasma was much broader and had a median refractive index of 1.49. Thus Nanoparticle tracking analysis can be used to asses the refractive index of vesicles. Urine vesicles had a different median refractive index (ñ=1.36) than particles from plasma (ñ=1.49). This group hypothesizes that the relatively high refractive index of plasma particles is due to the presence of lipoproteins (1.45≤n≤1.50). Without considering light scattering theory, the low refractive index of urine vesicles implies that neither polystyrene beads (n=1.61) nor silica beads (n=1.45) are ideal to calibrate optical instruments for vesicle detection.

Chris Gardiner, United Kingdom An update on light scattering techniques, especially identification of cellular sources of MPs using fluorescent markers

The analysis of extracellular vesicles (EV) presents unique challenges due to their small size, low refractive index, polydispersity, interference from lipoproteins in plasma, and low numbers of antigens. Chris Gardiner recently published a protocol for EV enumeration by Nanoparticle Tracking Analysis using silica microspheres to establish camera settings and calibrate concentration measurements. Specific fluorescent labelling of EV is hampered by low numbers of antigens, steric hindrance, protein-label aggregates and limited availability of bright stable fluorophores. Work of Gardiner’s group, with GFP expressing proteins, membrane specific dyes and DNA has shown that the limitations lie within the labelling techniques rather than detection methodology. Elimination of unbound fluorophore to increase the signal to noise ratio improves sensitivity but there remains a need for suitably conjugated antibodies specific for antigens commonly expressed on EV.

Esther N.M. Nolte-‘t Hoen, The Netherlands Intercellular communication by RNA containing extracellular vesicles

Immune cells can communicate with each other via regulated secretion of nano-sized membrane vesicles, the majority of vesicles being in the nano-sized range (< 200nm). To unravel the molecular composition of individual vesicles, single particle-based, high- throughput, multi-parameter techniques are needed. This group developed a high resolution flow cytometry-based method that allows simultaneous quantification and characterization of individual extracellular vesicles. This method yields integrated information on the light scattering, buoyant density and the presence of specific proteins on extracellular vesicles. In addition to proteins and lipids, extracellular vesicles contain RNA. Horizontal vesicle-mediated transfer of RNA between cells uniquely allows the dissemination of genetically encoded messages, which may modify the function of target cells. Whereas the presence of miRNAs in these vesicles is frequently reported, they applied deep sequencing for an unbiased screen of small (<70 nt) RNAs in vesicles released during interaction of dendritic cells and T cells. They found that miRNAs formed only a minority of vesicle-enclosed small RNA species. In contrast,

several other classes of small noncoding RNAs were highly abundant in extracellular vesicles. The detected small noncoding RNAs overlapped with protein coding regions, repeat sequences, or structural RNAs. Many of these RNAs were enriched relative to cellular RNA, indicating that cells destine specific RNAs for extracellular release. Several of the highly abundant small noncoding transcripts in vesicles have previously been associated to gene regulatory functions. Topical questions in the field are whether all vesicle subsets contain RNA, whether subsets of vesicles contain different types of RNA, and how the RNA content of vesicles changes upon cellular activation. This group recently obtained evidence that subpopulations of vesicles released during dendritic cell-T cell interactions differed in their RNA content. These data present leads for unraveling how immune cells modify the function of other cells via horizontal transfer of specific noncoding RNA species.

PART D: Standardization studies, chaired by Nigel Key

Rienk Nieuwland, The Netherlands First results of the European Metrology Research Program on traceable measurements of vesicles

Rienk Nieuwland presented the advancement of the European Metrology Research Program (EMRP) project METVES (Metrological characterization of microvesicles (MV) from body fluids as non-invasive diagnostic biomarkers) whose goal is "to develop reliable, comparable and quantitative analysis of MV in biological fluids”. A "Questionnaire on particle reference materials and methods” has been prepared and distributed to laboratories working on detection of MV. A total of 20 questionnaires have been returned from 19 laboratories in 7 countries. The results of the questionnaire have been summarized in a "Report on the needs, specification and commercial sources of microvesicle reference materials”, prepared by Anaïs Nicolet and Felix Meli of the Federal Office of Metrology (METAS; Wabern, Switzerland). The report can be downloaded from the METVES website (www.metves.eu), and includes a summary of (i) contributors, (ii) methods comparison, and (iii) requested needs for MV reference materials. Reference materials for size and concentration measurements have been selected and are being measured by 3D metrology atomic force microscopy at METAS. First results have presented showing that not all commercially available reference materials are useful for standardization of measurements on MV. For those willing to participate in future standardization measurements of METVES selected reference materials, please register by providing your name and contact details at www.metves.eu.

Romaric Lacroix, France Standardization of pre-analytical variables

Romaric Lacroix presented an update about the SSC workshop on standardization of pre- analytical variables in plasma MP determination. The main objective of this collaborative study was to compare inter-laboratory variability of platelet-MP count by flow cytometry or MP- dependent procoagulant activity, using a common pre-analytical protocol versus a non- standardized protocol. 14 laboratories participated. As a main results, the standardized

protocol resulted in a reduction of the inter-laboratory variability in MP count by flow cytometry although a significant variability did remain. These data have been published this year as a SSC report in Journal of Thrombosis and Haemostasis (Lacroix et al. J. Thromb. Haemost. 2013). Additional information were reported about the impact of pre-analytics on the different MP subpopulations measured by flow cytometry. Large platelet-MP and erythrocyte- MP are the MP subpopulations showing the highest interlaboratory variability compared to leukocytes and endothelial MP. This variability was reduced when preanalytics was standardized. However, for all MP subpopulations significant variations remained between laboratories stressing the need to identify others sources of variability.

Françoise Dignat-George, France Standardization of microparticle enumeration across different flow cytometry platforms : An update

Françoise Dignat-George reported the first data about the SSC workshop on the standardization of MP enumeration across different flow cytometry platforms. This study evaluate the inter- instrument reproducibility of platelet-MP counts among different platforms using a new standardization strategy based on different sets of beads experimentally adapted to each FCM platform. 44 laboratories registered accounting for 59 cytometers. Françoise Dignat-George presented the results of the first stage whose goal was to qualify the instruments according to performance in term of scatter resolution and background noise. The main conclusion was that the standardization strategy was compatible with a large proportion of instruments (78%) including instruments of old generation. The next step will be to evaluate the inter-instrument reproducibility of different PMP levels, using common reagents and the standardized FCM protocol.

Summary of the ongoing collaborative studies and publications

2012-2013

Ongoing collaborative studies on the standardization of microparticles in ISTH SSC Vascular Biology.

1) Standardization of pre-analytical variables in plasma MP determination. It has involved 14 laboratories. Results of on platelet MP and functional procoagulant tests have been published as SSC report at the beginning of this year. And results on others MP subpopulations have been presented at this meeting.

2) Standardization of MP enumeration across different flow cytometry platforms. This study started this year. 44 laboratories registered accounting for 59 cytometers. Results of the first step related to instrument qualification have been presented and final results are expected for Milwaukee.

3) A third project, the European Metrology Research Program (EMRP) project METVES coordinated by Rienk Nieuwland, aims to develop standard for reliable, comparable and quantitative analysis of extracellular vesicles in biological fluids. Reference materials have been selected with the collaboration of 19 laboratories that answered a questionnaire. These materials are being evaluated by 3D metrology atomic force microscopy. A report on the needs, specification and commercial sources of microvesicle reference materials is available online (www.metves.eu).

Publications related to SSC work (2012-2013)

- Lacroix R, Judicone C, Poncelet P, Robert S, Arnaud L, Sampol J and Dignat-George F. Impact of pre-analytical parameters on the measurement of circulating microparticles: towards standardization of protocol. J. Thromb. Haemost. 2012; 10:437-46.

- Robert S, Lacroix R, Poncelet P, Harhouri K, Bouriche T, Judicone C, Wischhusen J, Arnaud L, Dignat-George F. High-Sensitivity Flow Cytometry Provides Access to Standardized Measurement of Small-Size Microparticles. Arterioscler Thromb Vasc Biol. 2012 ; 32 :1054-8.

- Lacroix R., Judicone C., Mooberry M., Boucekine M., Key N.S. and Dignat-George F, on behalf of the ISH SSC WorkshopStandardization of the pre-analytical variables in plasma microparticles determination: results of the ISTH SSC Collaborative Workshop. J. Thromb. Haemost. 2013

von Willebrand Factor

June 30, 2013

Chairman: Jorge A. DiPaola (USA) Co-Chairs: Imre Bodo (Hungary), Jeroen C. Eikenboom (Netherlands), Yoshihiro Fujimura (Japan), Sandra Haberichter (USA), Daniel J. Hampshire (UK), Paula D. James (Canada), Johanna Kremer Hovinga (Switzerland), Alberto Tosetto (Italy)

Education Session

Chairs: Jorge Di Paola and Paula James

Genesis and intracellular processing of VWF Jeroen Eikenboom, Leiden, The Netherlands

VWF Molecular Interactions and their effect on disease Evan Sadler, St. Louis, USA

New developments in VWD diagnosis and treatment Imre Bodo, Budapest, Hungary

Registries VWD/TTP

Chair: Daniel Hampshire

Hereditary TTP registry. Magnus Mansouri, Bern, Switzerland

The hereditary TTP-Registry (www.ttpregistry.net, clinicaltrials.gov NCT01257269) is an orphan disease registry to gain more insight in this disease and eventually establish guidelines to improve diagnostics, treatment and prognosis. Detailed information about patients and their family members are recorded retrospectively up to enrollment and then prospectively. As of today 82 subjects from 9 countries have been enrolled and a first detailed analysis is foreseen when 100 participants have been registered – hopefully by the end of 2013. The registry is open to interested patients and their family members.

VWF database. Daniel Hampshire, Sheffield, UK

A brief overview was presented concerning improvements made to VWFdb over the past year, specifically additional information added to the Leiden Open Variation Database (LOVD) variant registry and the inclusion of VWFdb in the European Association for Haemophilia and Allied Disorders (EAHAD) Coagulation Database. There has been improvement on the variant registry with a new template that includes several additional features. Dr. Lillicrap asked about blasting

these variants against existing genomic databases. There is a plan to be more proactive on matching sequences variants with these databases such as the 1,000 genomes.

Assays for VWF and ADAMTS 13 Chairs: Yoshihiro Fujimura and Johanna Kremer Hovinga

VWF/VWD

Flow cytometry for the diagnosis of VWD. Mark Roest, Utrecht, The Netherlands

The current diagnostic process of Von Willebrand disease is laborious, time consuming, requires a fair amount of blood and can only be performed by specialized technicians in equally specialized laboratories. The authors developed a simple flow cytometry based VWF function micro assay, which requires only 40 µl of unprocessed whole blood. Blood can be collected either by venipuncture or by capillary blood collection from the fingertips. The test is validated in 10 patients of each VWD subtype and compared with 60 controls. The validation shows that the micro assay accurately distinguishes healthy people from VWD patients, but it also identifies various VWD subtypes. Its simplicity, fast results, low blood volume and low costs could significantly improve patient care.

Collagen binding types IV/VI. Veronica Flood, Milwaukee, USA

Von Willebrand factor binds to type VI collagen through the VWF A1 domain. Sequence variations in the VWF A1 domain reduce or eliminate this interaction. The R1399H sequence variation previously reported in 2% of Caucasians, results in diminished (50% of normal) or absent binding for heterozygotes and homozygotes respectively. Additional type 2M mutations located in that same region of the VWF A1 domain also result in decreased or absent binding of VWF to type VI collagen. Patients with these sequence variations experience bleeding, but the clinical significance requires further investigation. Questions were asked about the true clinical significance of this finding. The conclusion is that at this point it is somewhat premature to determine its clinical relevance but further studies will be performed.

Multimer analysis quantification. Sandy Haberichter, Milwaukee, USA

VWF multimer analysis is qualitative, and therefore a subjective assessment open to interpretation. To address this shortcoming a quantitative method for multimer analysis was developed that incorporates performing densitometry, determining area-under-the-curve, and calculating the percentage of low-molecular weight (LMW), intermediate-molecular weight (IMW) and high molecular weight (HMW) multimers. Quantitative analysis revealed subtle loss of HMW multimers in a subset of type 1 patients. Type 2B patients had a loss of HMW and IMW multimers. Type 2A subjects were divided into those with loss of HMW and decreased IMW (similar to type 2B subjects) and those with a virtually complete loss of HMW and IMW. Quantitative analysis of VWF multimer patterns more clearly distinguishes patients with various subtypes of VWD than subjective analysis and provides an objective measure of VWF structure.

Discussion was started and some attendees brought up the issue of gel concentrations and how if not standardized may bring variability to the assay. There will be effort on trying to unify gel concentrations so triplets can be also observed. This would be useful for the diagnosis of Type 1C VWD.

Comparison of VWF activity assays. Imre Bodo, Budapest, Hungary

One of the problems in VWD diagnosis relates to the Ristocetin cofactor assay, which is insensitive (sensitivity > 10-20 IU/dl) and imprecise (inter- and intra-assay coefficient of variation up to 20-30 %). Over the past several years, a number of new methods appeared with higher sensitivity and precision. However, it is currently not known how they exactly relate to each other and to the original VWF:RCo assay in measuring VWF function.

Therefore, at the 2011 SSC von Willebrand Factor Subcommittee meeting in Kyoto, it was decided to design a small collaborative study to compare all the available tests with each other and with the Ristocetin Cofactor assay. The project was to be coordinated by Imre Bodó on behalf of the SSC VWF Subcommittee, and Jürgen Patzke from Siemens. Eight laboratories joined the project, and eight new methods were selected for the comparison (the table in Appendix 1 lists the labs and corresponding tests) along with traditional VWF:Ag and VWF:RCo analysis. Each study site selected its own set of techniques.

The most difficult problem turned out to be the identification of well characterized samples. In the end, we succeeded in including frozen plasma samples from:

 50 healthy blood donors  32 well characterized VWD patients from Budapest and Vicenza  8 well characterized samples (6 patients; 2 normals) from the SSC bank in London (Tony Hubbard) + SSC standard was also used as a sample  4 samples of recombinant VWF protein (Including the D1472H and P1764S polymorphisms) dissolved in type 3 patient plasma (Bob Montgomery)  SSC standards

Altogether there were 95 samples.

The samples were frozen, coded randomly and were distributed to the participating labs in a blinded fashion, on dry ice. Data are just now being submitted (with 2 of the 8 labs having completed the measurements), and analysis has not started yet.

Finally, last year, we had a discussion about the possible nomenclature for the different new tests. We should continue the discussion this year, and – in case consensus can be reached – come up with a recommendation.

The project had support of two companies (Siemens, IL), who provided reagents. There will be some additional financial support for the study from the SSC to cover shipping expenses. The

nomenclature was discussed and comments from Dr. Montgomery were made regarding the use of R in the nomenclature since the GP1b binding assay does not have ristocetin. Dr. Federici made a comment regarding terminology that emphasizes the binding and not the use of the trigger for that binding. It was decided that email conversation will be started and hopefully a nomenclature will be determined.

Multimer methodology and minimal diagnostic requirements, Imre Bodo, Budapest, Hungary

The goal of this project is to standardize the high quality multimer technique developed by Ulrich Budde’s lab, so that similar results can be obtained elsewhere.

As presented last year, the collaboration of Imre Bodo’s lab in Budapest with the Budde lab led to a remarkable improvement of quality of the multimer assay in Budapest, and the two techniques became quite comparable. The goal of the project for this last year was to a) provide a detailed description of the assay, with as much detail as possible to help interested laboratories to reproduce identical gels and b) to develop a database of the most common multimer patterns in an effort to help with the interpretation of the multimer gels. Both aspects – technical details and pattern recognition – seem necessary for a standardized multimer assay.

At the meeting, examples of solutions to technical problems were presented, as well as the current status of the multimer pattern database.

We aim at the following criteria for high quality multimers:

1. Sensitive (detecting ≤0,1% VWF [0,001 IU/ml] VWF in plasma) 2. Should show a clear triplet structure with easily recognizable patterns 3. There should be some clear space between oligomers (obliteration of which is the hallmark of the "smeary” pattern) 4. HMW multimers should be well represented. This could be judged from the following: a. For NP, a low resolution gel should have ≥20% of HMW >10-mers AND b. Some material should be visible above 15 bands counted. 5. Finally, the multimer structure should be highly reproducible.

Dr. Mannucci made a comment about not using the high-resolution gels for clinical purposes since they may not add much to the diagnosis. Dr. Bodo agrees that it may not be important for most patients but for acquired VWS patients may be useful. Dr. Lillicrap discussed the smeary pattern and how to definitely identify those patients. Dr. Bodo does not think that pre analytical variables will cause the smeary patter.

ADAMTS 13/TTP

Standardization of ADAMTS13 Assays. Johanna Kremer Hovinga, Bern, Switzerland and Anthony Hubbard, UK

This presentation was given by Dr. Hubbard from the UK. Development of the WHO 1st IS ADAMTS13, Plasma, has continued with the evaluation of small-scale trial fills of lyophilized pooled normal plasma to establish a suitable formulation for the definitive preparation. Four different formulations (HEPES, PIPES, TRIS, No Buffer) were evaluated by a panel of expert laboratories measuring both ADAMTS13 function and antigen. There were no obvious differences between methods for any of the 3 buffers, and the results from accelerated degradation studies indicated similar stability. It was decided to use a HEPES formulation for the definitive fill and this was undertaken in February 2013. The definitive fill comprises 10,000 sealed glass ampoules each containing 1 ml of pooled normal plasma, lyophilized. The collaborative study for the value assignment of the WHO 1st IS is scheduled for Q4 2013 and over 20 participating laboratories have been recruited. Two patient plasma samples (acquired ADAMTS13 deficiency) have been lyophilized for inclusion in the study. The project is on schedule for submission to WHO for formal establishment of the International Standard in October 2014. There are so far 25 confirmed participants and enrollment continues.

New ADAMTS13 assay, novel substrate and improved sensitivity. Joshua Muia, St. Louis, USA

This presentation was given by Dr. Evan Sadler. Dr. Sadler presented a new assay that has the following advantages over the current assay. A) Increased fluorescence b) reduced interference with hemoglobin c) optimized for inhibitors and d) performed in physiological conditions (normal Ph). FRETS-rVWF71 optimization is ongoing. Citrate and heparinized plasma were similar. Although, citrate required recalcification for optimal resulsts. This new assay gave similar results and no racial differences were observed. Correlation with FRETS-rVWF73 was performed and the new assay appears to have better sensitivity for inhibitors. Dr. Kokame brought up the issue of stability and Dr. Sadler explained that the fluorescence yield is similar even with one-year difference between measurements.

Quantitative PCR-based analysis of ADAMTS13 genetic defects. Koichi Kokame, Osaka, Japan

In most cases of Upshaw-Schulman syndrome (USS), homozygous or compound heterozygous mutations are identified in the ADAMTS13 gene by direct sequencing, while in a small fraction of cases such mutations cannot be identified. We previously analyzed ADAMTS13 in 47 USS patients, and 44 of them had either homozygous or compound heterozygous mutations. Then, we sought to reveal more extensive defects of ADAMTS13 in the remaining three patients. We quantified copy numbers of each ADAMTS13 exon in the patients by using genomic qPCR. Each primer pair was designed to contain at least one of two primers used in direct sequencing, to avoid missing any exonic deletions. The qPCR demonstrated heterozygous loss of exons 7 and 8 in one patient and exon 27 in the other. Genomic qPCR provides an effective method for identifying extensive defects of ADAMTS13. Since a genetic defect could not be found in one patient questions were raised about a potential translocation, or eventually a misdiagnosis.

3) Updates on Multicenter Studies Chairs: Alberto Tosetto and Sandra Haberichter

French Cohort Study. Agnes Veyradier. Clamart Cedex, France Agnès Veyradier on behalf of the French Reference Center for von Willebrand disease (CRMW)

Since the creation of the French Reference Center for von Willebrand disease (CRMW) in 2006, 1175 patients (out of 1900 French patients included) from 650 families have been studied (phenotype and genotype) and found with at least one mutation of VWF gene. Three hundred thirty (330) distinct mutations were found including 58% of new mutations. Globally, missense mutations represent about 70% of all mutations whereas other mutations consisting in deletion, insertion, duplication, nonsense, splice mutations represent 30% of all mutations. The CRMW cohort includes 3% of patients with still undetermined VWD, 5% of patients with type 3, 69% of patients with type 2 and 23% of patients with type 1. The authors emphasized their strict inclusion criteria that eventually allowed finding genetic defects in almost all patients.

EU study. Anne Goodeve, Sheffield, UK

Aims of the group are to continue European Union type 1 VWD collaboration and to facilitate further VWD collaboration across Europe. Current work of the group includes the further mutation analysis on the MCMDM-1VWD cohort, the development of national VWD registries and recent publication on Principles of care for VWD in Europe (Castaman G et al. Principles of care for the diagnosis and treatment of VWD. Haematologica 2013;98:667-74). Additionally, further work in the MCMDM-1VWD study by Eikenboom et al has shown that VWFpp/VWF:Ag >2.2 indicates enhanced VWF clearance whilst the mutations with VWFpp/VWF:Ag substantially above 2.2 are predominantly located in the VWF D3 & A1 domains (VWF propeptide and ratios between VWF, VWF propeptide, and FVIII in the characterization of type 1 VWD. Blood 2013; 121: 2336-9).

Canadian study. Paula James, Kingston, Canada

I will be presenting a Canadian study involving Queen's University in Kingston, the University of Calgary and the University of British Columbia focused on the genetics of excessive mucocutaneous bleeding. We are just coming to the end of recruiting and currently have 444 patients, 348 of whom are female with a median age of 39 (range 18 - 90). Our target is 500, which we should reach in the next few months. All subjects were being seen for bleeding and/or bruising in a tertiary care Hematology clinic, and all were administered the Condensed MCMDM-1VWD Bleeding Questionnaire as well as tested for evidence of a collagen disorder (Beighton and Brighton clinical scoring). Investigations for inherited bleeding disorders were performed as deemed appropriate by the local investigator and DNA samples collected on everyone. The study was originally conceived of as a GWAS, although we will likely take the approach we discussed at last year's Zimmerman meeting (initially to do SNPs with association testing, followed by targeted exomic sequencing).

Zimmerman project. Robert Montgomery, Milwaukee, USA

A brief summary of the Zimmerman Program for the Molecular and Clinical Biology of VWD was provided. This study recruited VWD subjects who had been historically diagnosed at the clinical centers and carried on their registry as having VWD. Recruitment for the type 1 VWD index cases has been completed and the final data being analyzed. 682 total index cases have been recruited and have been phenotypically assigned to VWD, orlow VWF, 1H, which means current studies in the normal range but historically low, and normal. 2117 family members were studied and similarly assigned to these categories. The final group of sequencing results is currently being carried out. Recruiting of type 2 and type 3 families and cases continues and should be completed by this fall. Manuscripts on these cohorts will then be developed.

Dutch WIN Study. Frank Leebeek, Rotterdam, The Netherlands

The Willebrand in the Netherlands (WiN) study is a nation-wide cross-sectional study in patients with moderate or severe VWD, defined as VWF:Ag or VWF:Act <30%. A total of 804 VWD patients have been included of whom clinical characteristics, including bleeding phenotype, plasma and DNA has been collected. In the last year, we have focused on the effect of genetic variations in STXBP5, SCARA5, ABO, STAB2, STX2, TC2N and CLEC4M genes on VWF levels and the bleeding phenotype. We are currently investigating the age-related changes in bleeding phenotype and VWF levels in elderly (aged>65 years) VWD patient and the specific bleeding problems in children with VWD, using the recently developed age-adjusted bleeding scores. In the future we would like to genotype these individuals using next generation sequencing, especially those patients in whom no mutation in the VWF gene has been found. Dr. Leebek also presented the decreased incidence of cardiovascular disease by 39% in patients with VWD type 1 when compared to controls.

Brno-VWD (Belgium-Czech Cooperative Study). Alain Gadisseur, Antwerp, Belgium

The BRNO-VWD Study is a family-based analysis of VWD in the Moravia area of the Czech Republic performed by the University Hospital Brno (Czech Republic) and the Antwerp University Hospital (Belgium). Blood samples were collected from VWD patients (proband) together with at least one affected sibling or parent. Blood was collected from 205 patients from 95 families with suspected VWD. Updated results of the laboratory work were given.

The distribution of different subtypes of VWD was as follows: VWD type 1 in 60/95 families (incl. 1 type 1 Vicenza), and type 2 in 29/95 families (type 2A in 17/95, type 2B in 4/95, and type 2M in 8/95), with 6/95 still unconfirmed/unclassified. Molecular analysis is still ongoing with currently 45/95 families fully analyzed and the remainder partially with mostly exons 14,51, 15 and 51 to do. Thus far, 27 Mutations in the VWF gene have been found in 65/95 families, of which 4 are new to the ISTH VWD database and awaiting gene expression studies. MLPA will follow in all patients. Clinical and laboratory data will be merged when VWF gene sequencing has been completed in all patients.

Women’s Health Issues in Thrombosis and Haemostasis

June 29, 2013

Chairman: Sabine Eichinger (Austria) Co-Chairs: Rezan A. Abdul-Kadir (UK), Takao Kobayashi (Japan), Ida Martinelli (Italy), Claire McLintock (New Zealand), Saskia Middeldorp (Netherlands), Claire S. Phillip (USA), Rochelle Winikoff (Canada)

Attendants: ~300

Educational Lecture

The general format of this year’s SSC meeting was a one hour educational lecture at the beginning of each subcommittee meeting. In line with this we had three lectures:

1. A user's guide to management of ITP in pregnancy - Claire McLintock, New Zealand

2. Risk of thrombosis in postmenopausal women - Astrid van Hylckama Vlieg, the Netherlands

3. Thrombosis and haemostasis issues in preeclampsia - Benjamin Brenner, Israel

Welcome and Introduction of co-chairpersons

Sabine Eichinger welcomed all participants also on behalf of the co-chairpersons. She presented the new SSC Statement of Purpose which was developed during the strategic meeting in December 2012. Also, the mandate of the SSC on Women’s Health Issues and the new webpage features were presented. Attendees were encouraged to participate in the activities of the SSC.

Other educational activities

Benjamin Brenner reported on the 5th International Symposium on Women’s Health

Issues in Thrombosis and Haemostasis, which was held with much success in

February 2013 in Vienna.

Women’s Health Issues in Thrombosis

One of the major ongoing activities of the subcommittee is the standardization of the diagnosis of VTE during pregnancy. Melanie Tan gave an update on the studies on VTE diagnosis during pregnancy also on behalf of Menno Huisman.

Lisbeth Eischer discussed the conundrum of the difference in risk of recurrent VTE between men and women. She presented data from the Austrian Study on recurrent VTE (AUREC) and showed that thus far with the exception of high FVIII no risk factor is predictive of the recurrence risk in both sexes.

Thrombophilia and Pregnancy

Maria T. DeSancho presented own data on a prospective observational study to assess parameters of hypercoagulability in women younger than 45 years of age who undergo in vitro fertilization (IVF). They will assess the hypercoagulable states of women undergoing IVF and will correlate the hypercoagulable state with IVF outcomes defined as: oocyte number, oocyte quality, embryo quality, implantation rate, and pregnancy rate. Secondary objectives will be to evaluate complications from the IVF procedure including ovarian hyperstimulation syndrome, and bleeding.

Elvira Grandone gave an update on a prospective multicenter oberservation study on antithrombotic prevention in thrombophilia and pregnancy loss (OTTILIA study). Pregnancy outcomes in carriers of factor V Leiden or of the prothrombin mutation will be evaluated.

Reiko Neki talked about specific mutations in the protein S gene (K196E) which is frequently found in Japanese population. She presented data on 330 women with an adverse pregnancy outcome. The protein S K196E mutation was found in only 4 womenand was not more frequent than among the general Japanese population.

Claire McLintock gave an update on the EPPI study (enoxaparin in preeclampsia/ intrauterine growth restriction) study and the Australasian Maternity Outcomes Surveillance Systems (AMOSS)-maternal morbidity studies. Of 160 patients required for the EPPI trial, 80 have already been recruited.

She also addressed the aspect of peripartal hysterectomies. Frequencies vary between countries and continents. It is a main aim of AMOSS to assess the reasons fro high frequencies in Australia. Other outcomes of interest are frequencies, causes and management of amniotic fluid embolisation, uterine rupture, ecclampsia, and massive obstetric hemorrhage.

Saskia Middeldorp reported on the current status of the ALIFE 2 in which women with confirmed thrombophilia and recurrent pregnancy loss will be randomized into either LMWH or standard care surveillance to evaluate pregnancy outcomes. The HIGHLOW study will evaluate two different doses of LMWH for preventing VTE recurrence during pregnancy. She called for participation for both studies.

Haemostasis Issues

Claire McLintock described the design of a prospective observational study on massive transfusion in postpartum hemorrhage. The study will be conducted in Australia and the UK. In

this study the effect of 4 g fibrinogen upfront compared to placebo on bleeding will be evaluated.

Rochelle Winikoff gave an overview and update on several Canadian initiatives in women and girls with bleeding disorders. The Code Rouge program is one of these initiatives and to standardize and provide service for women and girls with bleeding disorders.

She addressed the aspect that von Willebrand’s disease is still underdiagnosed and proposed the use of bleeding assessment tools to improve diagnosis.

Initiatives are also ongoing to evaluate the optimal dose of tranexamic acid for menorrhagia.

Also of interest is to study the expulsion rate of the Mirena intrauterine device.

Studies are ongoing to compare hemostatic changes in healthy women during pregnancy compared to women with bleeding disorders, and to evluated obstetric bleeding complications in women with factor XI deficiency.

Report from the Bleeding Assessment Tool Standing Committee

Sabine Eichinger provided an overview of how the project developed and how it works. The chairman of the SSC on Women’s Health Issues is a member of the standing committee. The online bleeding assessment tool provides a user-friendly, web-accessible platform, encouraging uniformity in the standardization and collection of bleeding histories. It creates and maintains a web-accessible database of bleeding symptoms collected by investigators throughout the world that can be used by individual investigators for their own or for collaborative studies. The bleeding assessment tool is available online and a cooperation between ISTH and the Rockefeller University.

The Principal Investigator (single-site study) or the coordinator of a collaborative study shall submit to the Chairman of the committee ([email protected]) the following:

1. A brief outline of the project, including the names, sites and e-mail contacts of all sub- investigators (if any) designed to enter/access data of the BAT-R 2. A statement that an IRB or equivalent ethics committee has approved the study, including an explicit consent by the enrolled patients to use their anonymized data in collaborative studies approved by the ISTH-BAT Standing Committee 3. An Agreement letter with the Rockefeller University

All necessary informations are provided on the ISTH website (www.isth.org).

Coagulation Standards Standing Committee

30 June 2013

Chairman: Anthony Hubbard

Review of Lot #4 (A Hubbard)

Lot #4 of the SSC/ISTH Secondary Coagulation Standard commenced dispatch in April 2012 at a cost of £4.00 (GBP) per vial. Between April 2012 and end May 2013 a total of 17,470 vials were issued. Manufacturers in Europe received a total of 11,170 vials and External Quality Assurance schemes (UK NEQAS, CAP, GEHT) received a total of 3,090 vials. Lot #4 was also made available for inclusion in two collaborative studies – the SSC VWF Activity Assays Study and a Novo Nordisk study on the potency of a new therapeutic product.

The third time-point for the stability testing of Lot #4 was completed in January 2013. Estimates of the residual relative potency for the four analytes (FV, FVII, FVIII, antithrombin) from the two testing laboratories (NIBSC, Royal Hallamshire Hospital, Sheffield) were similar. Mean predicted degradation rates were equal to or below 0.1% per year for vials stored at -20°C for all analytes except for the estimate of FVII based on the NIBSC data (0.14% loss per year). These results indicate that Lot #4 is extremely stable and support the assigned expiry date of "end December 2020”.

Experience of EQA schemes with Lot #4

UK NEQAS (S Kitchen)

Five vials of SSC Lot #4 were used for "trouble-shooting” purposes between July 2012 and June 2013 relating to assay issues for FVIII, FIX and FXI. SSC Lot #4 has also been included as an anonymous sample in various surveys. Median estimates for FVIII, FIX, FVII, FX and antithrombin differed from the assigned values by 5% or less. A larger difference of 8% was found for Protein C activity by clotting assay. Median estimates for free and total Protein S antigen agreed closely with the assigned values whereas the estimates for Protein S activity differed widely between different methods. However the small number of laboratories undertaking some PS activity methods limited the significance of these results.

Estimates for FXII were also obtained in one survey; these indicated an overall inter-laboratory variability (CV) of 10%. Considering the utility of FXII estimation in the clinical laboratory and the good agreement between laboratories it was agreed that development of an International Standard for FXII in plasma was not a high priority. Surveys of VWF have also included results from laboratories employing the new generation (platelet-free) methods for VWF activity. There was discussion over the use of the IU for these methods since the WHO International Standard and Lot #4 were calibrated only using conventional methods for VWF:RCo. It was accepted that the use of the new methods will continue to increase and manufacturers need to

refer to an appropriate definition of unitage. This could be achieved through the assignment of new values to the WHO IS Plasma (and Lot #4) pending evaluation of these methods by the SSC VWF Sub-committee.

College of American Pathologists (D Adcock Funk)

SSC Lot #4 has been issued in two surveys for VWF and one survey for thrombophilia testing in the past year. Mean estimates for FVIII:C were less than 10% different from the assigned value except for one method where the bias was 11.4%. Estimates for VWF:RCo activity also agreed closely with the assigned value except for one method which showed a large bias of 20.2%; this was a surprising result considering that this method returned a 1.2% bias in a study undertaken last year. Mean estimates for VWF:Ag and Antithrombin (activity and antigen) differed by less than 10% from the assigned values. Mean estimates for Protein C activity and antigen differed by less than 10% from the assigned values except for one method for Protein C activity (clotting) where the difference was 21.7%. This is a consistent finding with similar differences reported for this method in surveys from 2009 – 2011. Mean estimates for methods of Protein S total antigen differed from the assigned values by 12.9% lower to 8.6% higher than the assigned value.

Use of Lot #4 for assay validation

During the past year there have been requests from clinical laboratories to use Lot #4 for the validation of new assays or analytical instruments. The Executive Board did not agree to these requests since they were not consistent with the primary objective to promote harmonisation in the calibration of commercial plasma calibrants and diagnostic kits. The discussion supported this decision and furthermore it was thought that the uptake of Lot #4 for this purpose would be unpredictable and there could be a serious impact on the lifetime of the standard.

Lot #4 and the JCTLM database (E Gray)

Previous lots of the Secondary Standard have been accepted on to the JCTLM database as internationally certified reference materials. The application for Lot #4 has been submitted to JCTLM and the decision should be available in approximately 6 months.

There were 30 attendees.