63rd Annual SSC meeting, in conjunction with the 2017 ISTH Congress in Berlin, Germany Meeting Minutes

Standing Committees Coagulation Standards Committee ...... 3 Subcommittees Animal, Cellular and Molecular Models ...... 5

Biorheology ...... 7

Control of Anticoagulation ...... 10

Disseminated Intravascular Coagulation ...... 12

Factor VIII, Factor IX and Rare Coagulation Disorders ...... 14

Factor XI and the Contact System ...... 19

Factor XIII and Fibrinogen ...... 21

Fibrinolysis ...... 27

Genomics in Thrombosis and Hemostasis ...... 33

Hemostasis and Malignancy...... 46

Lupus Anticoagulant/Phospholipid Dependent Antibodies ...... 49

Pediatric and Neonatal Hemostasis and Thrombosis ...... 54

Perioperative Thrombosis and Hemostasis ...... 59

Plasma Coagulation Inhibitors ...... 61

Platelet Immunology ...... 66

Platelet Physiology ...... 73

Predictive and Diagnostic Variables in Thrombotic Disease...... 77

Vascular Biology ...... 79

Von Willebrand Factor ...... 83

Women’s Health Issues in Thrombosis and Hemostasis ...... 91

Coagulation Standards Standing Committee Minutes

9 July 2017 12:30 - 13:30 Chairman: Anthony R. Hubbard

Review of ISTH/SSC Secondary Coagulation Standard Lot #4 (A Hubbard)

Dispatch of Lot #4: Between the beginning of June 2016 and the end of May 2017 there were a total of 58 orders from 23 different manufacturers and 2 external quality assurance schemes (College of American Pathologists & UK NEQAS); a total of 13,872 vials were issued. Lot #4 was also included in multi-centre studies for the value assignment of Lot #5. Remaining stock at the end of May 2017 was 31,700 vials. Based on the dispatch of Lot #4 since 2012 it is predicted that stocks will be exhausted around 2019 – 2020. Stability of Lot #4: Stability monitoring by accelerated degradation is now complete for Lot #4 and the predicted degradation rates for the four test analytes (factor V, factor VII, factor VIII, Antithrombin) are all less than 0.1% loss per year at -20°C. This is consistent with previous lots of the standard and confirms that Lot #4 is extremely stable. Real-time stability testing will be carried out in 2018 to compare vials stored at the bulk storage temperature (-20°C) with vials stored at -70°C for the 4 analytes in order to have an objective measure of stability of -20°C. Troubleshooting: It was reported that the use of the standard for troubleshooting purposes, by EQA schemes, has been renewed for a further 4 years from November 2016. Calibration for VWF:GPIbR & VWF:GPIbM methods: Following the VWF sub- committee proposal to adopt the VWF:RCo value on the WHO 6th IS FVIII/VWF Plasma for these new methods it was announced that the calibration of Lots #4 and #5 for VWF:GPIbR & VWF:GPIbM will be carried out by assay against the WHO 6th IS in Q4 2017.

Experience of EQA schemes with Lot #4 UK NEQAS (S Kitchen) The use of Lot #4 for troubleshooting was reviewed. Between 2008 and 2016 there were 49 vials issued for this purpose (covering factors II, V, VIII, IX, XI, VWF, fibrinogen, Antithrombin, Protein C, Protein S). In the last 12 months vials were issued for troubleshooting problems in factor VIII, Protein C, fibrinogen and VWF. There was an update on a survey result for factor II, previously reported in 2016, where results were 15% higher with one commercial reference plasma. Tests indicated this was not related to the use of different thromboplastin reagents but to the reference plasma used. A survey in April 2017 showed this issue was still unresolved. Correspondence with the manufacturer indicated they were addressing the calibration of their product and that the last batches affected by this issue were dispatched in January 2016. A survey of VWF testing in May 2017 returned median results for VWF:Ag, VWF:CB and VWF:RCo in excellent agreement with the labelled values for Lot #4; results for VWF:Ab and VWF:GPIbR were higher than VWF:RCo whereas the median for VWF:GPIbM was close to the VWF:RCo value. These results were very similar to a survey carried out in 2012.

College of American Pathologists (R A Higgins)

Lot #4 was issued in two surveys in 2016; one for thrombophilia testing and a special survey for VWF. Analytes are tested at the discretion of participating laboratories. Results are presented as mean and median for 10 or more participants and only as median for less than 10 participants. Results for Antithrombin activity were close to the assigned value for Lot #4 whereas the median from one antigen method was around 10% higher. Mean estimates for Protein C activity by chromogenic methods were close to the assigned value whereas one clotting end-point method gave much higher results (116 vs 92 IU/dl). Results for Protein S activity and free Protein S antigen were generally close to the assigned value whereas one estimate for total Protein S antigen differed by around 10%. Results for factor VIII activity agreed closely to the assigned for one method (21 labs) but differed by around 10% with another high use method (15 labs). A study of trends for factor VIII indicated an approx. 10% increase in survey results by 3 different methods during 2015; the reason for this increase in unclear and is not linked to any change in Lot #4 or the WHO IS. VWF activity (VWF:RCo) results were close to the assigned for the most popular method; results for the VWF:GPIbR method were slightly higher than the VWF:RCo value. Review of the trends for VWF:RCo estimates indicated that the very large variability between methods seen in 2014 has been reduced in more recent surveys. VWF:Ag results from 3 different methods were in good agreement with the assigned value.

Towards Lot #5 (C Thelwell) The project to develop Lot #5 was initiated following approval by the ISTH Council in November 2015. Procurement of Lot #5 was completed in 2016 after the review of bids from two manufacturers. Lot #5 consists of a batch of 100,000 vials and was delivered to NIBSC on 31 January 2017 where it is now stored at -20°C. Some vials were placed at elevated temperatures on 09 March 2017 for the accelerated degradation studies. The value assignment will be carried out in 3 separate exercises during 2017. The first exercise will cover Protein C, Protein S, Antithrombin and Fibrinogen; the second will cover factors, II, VII, IX, X, XI and XIII; the third exercise will cover factor VIII and VWF. Value assignment for factor V will be carried out as part of the project to replace the WHO IS. The whole calibration exercise will include 77 laboratories recruited from 30 countries. Four independent estimates will be requested for each analyte from each testing laboratory and the assigned values will be calculated as the geometric means of results returned by the participants. The objective is to complete this value assignment in time for endorsement at the SSC meeting in Dublin 2018. It is intended to also value assign Lot #5 for FXII following establishment of the new WHO IS Factor XIII plasma in October 2017; this will be undertaken in a separate study.

There were 22 attendees.

Animal, Cellular and Molecular Models

9 July 2017 8:00 – 12:15

Chairman: Jose A. Diaz

Co-Chairs: Brian Cooley, Alexandra Kopic, Nobuo Nagal, Lesile Parise, Denise E Sabatino

8-10am:

AAA MODEL–Elastase Model

AAA Peri-Adventitial Model

AAA Angiotensin II Model

Cremaster Arteriole Laser-Injury Model of Thrombosis

Summary: Drs. English, Grover and Daugherty covered advantages, disadvantages, limitations and applications of each model. All speakers engaged the audience and this was translated into many questions following the talks (all talks had at least 3 questions)

Break 15 minutes

10:15 – 11:15

Educational session:

Murine IVC thrombosis model: Tips and Tricks

IVC Stenosis Model: Latest modifications

Summary: Drs. Diaz and Saha delivered two educational talks. Diaz highlighted tips and tricks for VT models, how to approach the abdominal cavity, anatomical signature and landmarks, as well as postoperative care of the animals. Saha presented an overview of the stenosis model (one of the most widely used animal models) and delivered, as he was asked, all the modifications introduced during the last 7 years that had caused some confusion in the field.

11:15 – 12:15

Lectures:

Directing Your Research: Unmet Needs from the Clinical Perspective

Dr. Saha (last minute replacement for Dr. Lurie) presented gaps in the field for both AAA and venous thrombosis providing a lecture on what is needed from the clinical perspective.

Bench-Side to Bedside to Community: The Role of Pre-Clinical Models

Dr. Wolberg delivered an excellent lecture on how animal models contribute to current knowledge and how they can be used in the future to increase our knowledge on thrombotic diseases. She emphasized the scientific process in choosing appropriate models (in vitro, in vivo, and computational) through a descriptive example from her own lab.

Publications since last ISTH meeting:

None.

Publications in preparations:

Consensus on animal models of venous thrombosis – Jose A. Diaz. American Venous Forum project that will be submitted for evaluation of potential endorsement from ISTH, based on previous conversation with Dr. Walter Ageno during ISTH in Berlin.

Future publications planned:

Standardization on selection and conduct of murine bleeding models.

Brian Cooley. ISTH SSC grant. Dr. Cooley reported by phone on August 11, 2017: the work has been completed and manuscript is in preparation. Lacey Schmeidler will send an email to Dr. Cooley notifying him about the next steps toward to a publication through our SSC. A progress report has been requested by Jose Diaz. I will keep you posted with the progress in this project.

Topics for consideration in conversation with experts:

Dr. Steven Grover (expert from Harvard) is working on a review of the Cremaster arteriole laser-injury model of thrombosis models and he expressed interest in working with the SSC co-chairs to make this project an SSC project.

Dr. Abhishek Jain (expert from Texas A&M) is working in in-vitro assays realted to thrombosis. He approached me to initiate work toward to standardization of in-vitro models of thrombosis. We are in conversations, nothigh to report yet in this area.

As you can see, since Berlin I was actively working to represent SSC as much as I can.

Looking forward for a very productive year.

Submitted by: Jose Antonio Diaz, MD

Biorheology

8 July 2017 14:00 – 18:15

Chairman: Keith B. Neeves Co-Chairs: Judith Cosemans, Erik Westein, Pierre Mangin, Warwick Nesbitt, Wilbur Lam

Education Session: The biomechanics of thrombus formation

1. John Weisel, United States, “Fibrin biomechanics” Understanding fibrin biomechanics is important for hemostasis and thrombosis because these are basically mechanical processes. Viscoelastic properties of fibrin are unique: small volume fraction, strain stiffening, large volume decrease. Changes in clot structure and mechanical properties with deformation are different for shear, tension, or compression. Rearrangements of fibers in the fibrin network occur with deformation. Mechanical properties of fibrin fibers have been determined, and many molecular unfolding events with fibrin deformation have been defined. Fibrin mechanics can only be described and modeled by incorporating structural changes at all levels from centimeters to nanometers. Clot elasticity is necessary for hemostasis, but stiff clots are associated with heart attacks and strokes, while weak clots are associated with bleeding. Clot plasticity and contraction by platelets may be necessary to prevent obstruction by thrombi. Mechanical properties determine whether or not embolization of thrombi occurs. Finally, fibrin is a promising material for clinical uses, such as tissue engineering and fibrin sealants.

2. Wilbur Lam, United States, “Platelet Biomechanics” Upon vascular injury and the onset of hemostasis, platelets are exposed to a myriad of mechanical microenvironments including the hemodynamic forces of the circulation as well as the mechanical structures of the subendothelial matrix and the nascent clot itself. How platelets physiologically respond to the physical cues of the microenvironment and, in turn, how aspects of platelet function, such as platelet contraction, alters clot mechanics was discussed. The complex interplay of these processes and their implications for bleeding and thrombotic disorders were also discussed.

Novel Applications of Flow-Based Devices. Moderator: Judith Cosemans

1. Hendrik Feys, Belgium, “Flow chambers in transfusion medicine” Few techniques are available to determine platelet quality in platelet concentrates for transfusion. Microfluidic flow chambers were used to determine hemostasis following transfusion in vitro. Using this technique, Feys and colleagues demonstrate that pathogen inactivation significantly decreases platelet function. Changes in platelet membrane packing underlie the reduced respondes of these platelets. For platelets that are cryopreserved, it was found severely reduced platelet adhesion to collagen but despite this, initiation of coagulation was faster. The clinical relevance of both

observations is unknown but a matter of ongoing research.

2. Laura Haynes, United States, “Coagulation kinetics in a flow reactor” Studying coagulation kinetics under flow provides important insight into the function and regulation of the coagulation process under conditions that approach physiology in which key reactions are isolated on the walls of vessels or on platelets that establish an initial plug to halt bleeding. The use of flow reactors permits for the enrichment of platelets that can support prothrombinase complex assembly along collagen fibrils and also allows for the visualization of the components of the prothrombinase complex binding to appropriate membrane surfaces. As a point of comparison, the activation of prothrombin by the prothrombinase complex under flow is dictated by flow mediated convection, while factor X activation by the extrinsic tenase complex is regulated by both flow convection and limited substrate diffusion. Finally, using a flow reactor it is possible to elucidate the dynamics of thrombin “trapped” within a venous clot. Despite the relatively poor reported affinities of thrombin for fibrin, it remains stably incorporated into fibrin clots—presumably a result of its dynamic movement between potential binding sites.

Building Endothelial Cell Function into Flow Chambers. Moderator: Keith Neeves

1. Wilbur Lam, United States, “Endothelial cell culture in microfluidic systems” As microfluidics are gaining traction as research enabling systems to investigate hemostatic and thrombotic processes, interest in culturing endothelial cells in these novel devices has rapidly increased. A thorough discussion of how to culture endothelial cells in microfluidics, the advantages and disadvantages of integrating and culturing endothelial cells in microfluidics, practical tips on how to troubleshoot issues that may arise during this very specific culturing process, as well as novel methods to mitigate the limitations of “endothelialized” microfluidics were presented.

2. Abhishek Jain, United Stated, “Thrombosis-on-a-chip” A clot or a thrombus that forms inside a blood vessel in vivo functions as an organ, collaborating dynamically with blood constituents, flow, endothelium, extracellular matrix and epithelial tissue. Current models do not provide a robust means to analyze the organ-level signaling that occurs during thrombosis or bleeding in a human. Engineering of 3D microfluidic cell culture assays, also known as organs-on- a-chip, offers to meet this technological gap and provide a tool to assess various levels of complexity when hemostasis is pathologically altered. As an example of a thrombosis-on-a-chip model, Dr. Jain presented a microchip that mimics the architecture of a vascular bed and permits real-time analysis of clotting. This device was shown to integrate with blood extracorporeal devices. As a second example, it was shown that a lung alveolus-on-chip containing human primary alveolar epithelium interfaced with an endothelial lumen was used to study pulmonary thrombosis and drug-tissue interactions under whole blood flow. In future, this new methodology may complement animal models in advancing our basic understanding of thrombosis and identifying new drug targets.

New Mechanisms in Blood Flow-Dependent Platelet Function. Moderator: Pierre Mangin

1. Martine Jandrot-Perrus, France, “Modeling in flow the role of GPV in the

initiation of thrombus formation, growth, and lysis” Flow experiments have strongly contributed to demonstrate the role of GPVI in thrombus formation, growth and lysis: these studies demonstrated that when GPVI is deficient (quantity or function), platelets are not activated and do not form aggregates when perfused on collagen surface showing the role of GPVI is the initiation of thrombus formation. Additionally, the role of GPVI in thrombus growth was demonstrated by studies assessing platelet procoagulant activity, fibrin formation and GPVI activation by fibrin in flow. As a consequence, blocking GPVI accelerates fibrinolysis. Interestingly also, the critical role of GPVI in platelet accumulation on atheroma plaques observed in flow experiments has been confirmed in in vivo models of atherothrombosis. Thus, studies performed on GPVI illustrate the usefulness of flow experiments to model platelet functions.

2. Erik Westein, Australia, “Targeting of VWF in shear gradient mediated thrombus formation” Although proven effective, current anti-platelet drugs have dose limitations associated with bleeding complications. We are exploring the functional differences between haemostatic and thrombotic processes with the aim to selectively target the latter. The hemodynamic environment at sites of large, near occlusive, thrombi is fundamentally different to those at sites of haemostatic plug formation and may be a target for improved anti-platelet therapy. VWF is a plasma protein that mediates the adhesion and aggregation of platelets under high shear conditions and is also sensitive to the fluid shear environment due to its large size. Westein and others have identified a hyper active state of VWF that is present only at sites of large gradients of shear stress which occur exclusively at thrombotic sites. The generation of function blocking antibodies against this unique form of VWF may provide a basis for a new and improved class of anti-platelet therapy.

3. Keith Neeves, United States, “Update on SSC Multicenter Study of Flow Chambers” The Biorheology Subcommittee of the SSC has started a project with the objectives of (i) implementing previous Subcommittee recommendations from previous projects, (ii) compare custom and commercial flow chambers to a ‘standard’ flow chamber, and (iii) quantify varaiion in results between centers. An initial prototype of the standard flow chamber has been fabricated and includes a two-step process of micropatterning prothrombotic proteins followed by whole blood flow assay. Initial studies have been conducted on types I and III collagen, fibrinogen, and VWF. A solicitation for interested investigators/centers that would like to participate in the project was made to the audience.

Control of Anticoagulation

9 July 2017 8:15 – 12:15

Chairman: Mark Crowther Co-Chairs: Benilde Cosmi, Ismail Elalamy, Pieter W. Kamphuisen, Jonathan Douxfils, Chatree Chai-Adisaksopha, Adam Cuker

Part 1: The meeting was convened at 14:15

Dr Helin presented an abstract outlining his observation that a genetic dosing algorithm underestimated the vitamin K antagonist dose required in selected patients with thrombosis. This case series suggested that such patients have a vitamin K dosage requirement that exceeds that which would be predicted by their genetic characteristics.

Dr Gibson reported the results of the APEX study of betrixaban for the prevention of thrombosis in patients admitted with medical illnesses who were identified to be at high risk of thrombosis. The study did not meet its primary analysis objective and thus was “negative”. Exploratory secondary analyses suggested benefit. A great deal of discussion entailed.

In the first symposium on laboratory testing:

Dr Cuker provided a comprehensive review of “commonly accessible” tests for ascertaining the presence of DOACS in the acute setting.

Dr Kitchen provided an overview of NEQAS programs to set standards for testing of anticoagulants and outlined plans to establish standards for DOACs

Dr Gouin-Thibault outlined currently available specific tests for Xa inhibitors

Dr Mullier presented his experience with various specific tests for IIa inhibitors

Dr Lindhoff-Last outlined currently available and proposed tests for anticoagulants, including DOACs, with a particular focus on point of care tests. A discussion entailed about various tests, including urine based tests

In the second session outlining committee activities:

Yan Xu from Canada presented a survey on restarting anticoagulation after an intracerebral bleed. There were no standards or widely accepted treatment patterns identified

Walter Ageno presented a proposed prospective registry of cerebral vein thrombosis. The registry is supported by ISTH and will use ISTH database technology to collect information

Dr van den Besselaar presented the current efforts to fine suitable replacement material for the Standards for Thromboplastin. The process is largely complete and has identified two suitable materials; animal derived and recombinant. The process will now be carried forward to the WHO

Dr Tripodi presented a comprehensive review of lupus anticoagulant testing and its performance in patients on anticoagulants. There is variability in sensitivity but the bottom line is such testing should probably not be performed in anticoagulated patients.

In the ensuing discussion a number of potential new projects were outlined.

In the second day, in the first part, we discussed management of bleeding:

Dr Bayer-Westendorf presented his experience, and the experience of others, with rates and consequences of bleeding in patients receiving anticoagulants, including the DOACs.

Dr Verhamme presented his experience, and the published evidence for idarucizumab and non-specific reversal agents for dabigatran associated bleeding over two sessions. Dr Verhamme’s centre has extensive experience with dabigatran reversal.

Dr Crowther presented up-to-date information on the use of andexanet and briefly focussed on non-specific reversal agents. The results of the various studies currently in the public domain were presented

In the second day, in the second session:

Dr Weitz presented a lecture on the future of anticoagulants, and focussed on inhibition of the intrinsic pathway which might offer the opportunity to inhibit pathological thrombosis, without causing bleeding. He also outlined the use of DOACS in settings other than they have been studied for.

Dr Middeldorp outlined the American Society of Hematology guideline efforts, including the development of methodology and evidence tables. The project is estimated to be completed Q1/Q2 of 2017

Dt Tosetto outlined the process for Guidance documents and Guidelines from the International Society of Thrombosis and Haemostasis. The guidance documents have been well received and there are plans for a number of new guidance documents

Dr Kearon presented the recently published ACCP guidelines and outlined ACCP plans for future updates on a “rolling basis.” The recent update has been very well received.

Dr Konstantinides outlined European Society of Cardiology plans for Pulmonary embolism. He reviewed the previously published recommendations that have been well received and have had an influence on care in many centres

Dr Crowther outlined the AC Forum Guidance documents. This series of papers, which provides evidence “guided” recommendations was published earlier this year, has been well received, and heavily downloaded.

Disseminated Intravascular Coagulation

9 July 2017 8:00 – 12:15 Chairman: Jecko Thachil

Co-Chair: Marcello Di Nisio, Toshiaki Iba, Takashi Ito, Shinichiro Kurosawa, Bernd Jilma, Alexandro Squizzato, Sacha Zeerleder

The DIC session for this year commenced with a talk from Dr. Claushuis from the Netherlands on the various ways platelets interact with neutrophil in patients with sepsis and its relevance to those who develop DIC. Understanding more on this interaction between the two crucial blood cells may help reduce the poorer outcome in patients with sepsis. This was followed by a review of the complement system in thromboinflammation by Dr. Nilsson from Sweden. He explained in detail the various interactions along the coagulation pathways with the complement system. The significance of this bidirectional pathway is only starting to be explored. Dr. Nilsson has set precedent to researching more the role of complement in sepsis and coagulation disturbances.

In the next talk, Dr Yamakawa from Japan updated us on the new evidence which supports anticoagulant therapy as effective adjuvant therapy to reduce mortality in sepsis. He presented information from multicenter, retrospective registry which noted favorable associations between anticoagulant therapy and mortality in patients with the highest risk subset as evidenced by higher SOFA score. Dr. Gando, also from Japan then highlighted the controversy which exists in the setting of trauma – whether the entity of trauma-related DIC really exists or this should be completely replaced by the newer term, acute coagulopathy of trauma. He provided adequate evidence to support that DIC is clearly observed in trauma and as such this age-old concept should remain.

Following on, Dr. Ruth Kleinpell from the United States gave a critical care perspective of the management of sepsis and related complications. She stressed the need for a close liaison between critical care specialists and coagulation experts to understand and limit one of the biggest complications of sepsis. The issue of sepsis was highlighted by Mr. Marvin Zick from Germany who is associated with the Global Sepsis Alliance. This consortium has been fundamental in raising awareness worldwide about the very high mortality from sepsis and its effects. He requested membership from the many attendees in helping with the goal of widening his attempts in controlling the burden of sepsis.

The education session started with a very interesting talk from Dr. Toh on the role of histones and neutrophil extracellular traps in DIC. He explained at length, how these ‘new markers’ are pivotal in causing acute lung injury, thrombocytopenia and end organ damage and possibly by interfering with them can improve mortality. The next topic was perturbations in hemostasis in Viral Hemorrhagic Fevers by Dr. Bausch from Switzerland, who related to us his experiences with the recent Ebola outbreak in West Africa and the clinical and laboratory perspectives. He called for action in working together towards understanding these diseases more in the future.

In the second SSC session, Dr. Warkentin from Canada gave us an update on the new entity, ‘Acute DIC, Shock Liver, and Symmetrical Peripheral Gangrene’ with clinical cases and decrease in natural anticoagulants in tandem with liver disease. Next, Dr. Squizzato from Italy highlighted the issues in DIC diagnosis and management across

the world. He presented data from surveys at the ISTH conferences and among DIC experts which revealed a huge amount of variation in both diagnosis and management of DIC. The need for an international registry has been highlighted and is being organized. Dr. Solh from Canada presented his work from the pediatric SSC on the international survey which gave an insight into the issues in management of DIC in children. Similar to the adults, an international registry is being set up to study more and logically approach DIC in pediatrics in the future. The last talk was from Dr. Erez from Israel who gave an obstetricians perspective on DIC. There was discussion about whether DIC really is a problem in obstetrical disorders and why it should be a major concern. He suggested a modified DIC score from his group could form the basis of future diagnostic studies in this area.

Factor VIII, Factor IX & Rare Coagulation Disorders

8 July 2017 9:00 – 13:15 and 9 July 2017 8:00 – 12:15 Chairman: Guy Young Co-Chairs: Manuel Carcao, Peter Collins, Alfonso Iorio, Gili Kenet, Johnny Mahlangu, Maria Elisa Mancuso

Achieving Higher Factor Levels: The “New” Normal

Factor VIII deficiency

Alfonso Iorio discussed how the variability of the pharmacokinetics of the different factor VIII concentrates impacts on factor levels and the choice of product and dosing regimens. He discussed the fact that the extended half-life products which have more variability in their pharmacokinetics lead to more challenges in terms of determining a dosing regimen. He then discussed the cost issues with respect to dosing for prophylaxis and reviewed recent studies evaluating the cost-effectiveness of prophylaxis in general and with some specific patient examples.

Factor IX deficiency

David Keeling discussed the pharmacokinetics of standard half-life versus extended half-life factor IX products demonstrating theoretical modeling showing how various dosing regimens result in different peaks and troughs. He then compared dosing of extended half-life factor IX products between weekly dosing and longer intervals such as every 10 days. He compared the costs of the different regimens showing that extended half-life factors can be cost-effective when aiming for a certain trough levels but at the cost of higher peak levels. He also compared the costs of various regimens of extended half-life factors demonstrating that the costs of extending the regimen from weekly to every 10 days to every 14 days is very costly. Finally, he compared Alprolix and Idelvion demonstrating that the costs are similar in the United Kingdom but much higher troughs are achieved with Idelvion.

Debate on Extended Half-Life Factor IX Concentrates versus Factor IX Gene Therapy

Manuel Carcao and John Pasi took different sides debating the merits of extended half- life factor IX concentrates and the potential for gene therapy. Dr. Carcao argued that the extended half-life factor IX products are a known entity that provide safety and known efficacy with many patients and years of experience. Dr. Pasi argued that gene therapy is the future of treating hemophilia B and that the techniques will be refined to

the point of making them safe and allowing patients to have a one-time therapy to cure their hemophilia.

Education Program

Gene Therapy Update

Thierry VandenDriessche presented an update on gene therapy approaches for both hemophilia A and B. He discussed different vector approaches and the advantages and disadvantages of each as well as discussing the approaches to factor IX gene therapy using the factor IX Padua variant Next he described briefly results from the most recent clinical trials of both factor VIII and factor IX gene therapy. He also briefly compared gene therapy to extended half-life factors.

Update on Rare Factor Deficiencies

Flora Peyvandi provided an update on the various rare bleeding disorders noting in particular the increasing incidence in the developed nations largely as a result of the efforts by groups like the SSC in increasing the awareness of these disorders. She reviewed the epidemiology, presentation, and the various treatment options available in Europe and the United States. She described how the severity levels for the different rare bleeding disorders vary and how the severity classifications by factor levels is different than in hemophilia. In addition, she specifically brought to light the issues of women with rare bleeding disorders. Lastly, she discussed how several new drugs in development for hemophilia could work for rare bleeding disorders.

Rare Bleeding Disorders

Factor XI Deficiency

Ophira Salomon discussed in detail new data regarding factor XI deficiency including recent publications demonstrating that it is more common than previously thought and present in different populations throughout the world with higher rates in some groups than others. In addition, she discussed new research aimed at identifying which patients are more likely to bleed based on global hemostasis assays. Lastly she discussed treatment issues and in particular concerns regarding safety of factor XI concentrates and the possibility of using recombinant FVIIa for patients with factor XI deficiency.

Fibrinogen disorders

Alessandro Casini reviewed the epidemiology, laboratory diagnosis, and clinical presentation of several fibrinogen disorders including afibrinogenemia, hypofibrinogenememia, dysfibrinogenemia and hypodysfibrinogenemia. He discussed

diagnostic issues stating that depending on the specific reagents used, the diagnosis of these disorders could be missed or misclassified. He then discussed treatment issues in afibrinogenemia including the efficacy and safety of the fibrinogen concentrates.

Project updates

Koen Mertens provided a final on his project on standardizing inhibitor measurement. His group has not been able to reach a final conclusion due to a number of mostly technical barriers and thus no publication will be forthcoming.

Carmen Escuriola-Ettingshausen provided a final report on her project regarding prophylaxis with bypassing agents in inhibitor patients. Her project is complete and her group is working on a publication to be submitted as an SSC recommendation.

Alfonso Iorio presented a final report on his group’s work on pharmacokinetics and population pharmacokinetics and a publication will be forthcoming as an SSC recommendation.

Flora Peyvandi presented a final report regarding her group’s work on post-registration surveillance for new products. Her group developed a case report form which would be used (conceivably) for all patients who start a new medication in order to capture primarily safety data. This work was recently accepted for publication in the Journal of Thrombosis and Hemostasis as an SSC recommendation.

Old and New Challenges in Immune Tolerance

Definitions of Tolerance—What is/Should be Different with Extended Half-life Products

Charles Hay presented data on current definitions of tolerization following immune tolerance induction therapy. He discussed the limitations of the methods for defining tolerization when standard half-life products are used. He then discussed the limited data available on extended half-life factors and their use for immune tolerance induction. Finally, he proposed a suggestion that even when using an extended half-life factor for tolerization that a standard half-life product should be used to assess whether tolerization was achieved or not.

When to Start and When to Stop Immune Tolerance Induction Therapy

Rolf Ljung discussed the issues of when to start and when to stop immune tolerance stating that after more than 30 years of immune tolerance therapy that we still don’t have good data to support decisions regarding when to start and when to stop immune tolerance. He reviewed published consensus group recommendations regarding these issues and offered his interpretation of those recommendations. Finally, he proposed that we need to use the worldwide registries that are currently active to collect data on clinical practice to help answer these questions since conducting clinical trials in this area are not considered to be feasible.

Debate on Using Plasma-derived FVIII versus recombinant factor VIII

Carmen Escuriola-Ettingshausen and Raina Liesner debated the merits of plasma- derived versus recombinant factor VIII for the first line of immune tolerance therapy. Dr. Escuriola-Ettingshausen argued and used supporting to data from Germany and other European countries to demonstrate that the success of immune tolerance is much higher with plasma-derived products. Dr. Liesner argued in favor of using recombinant products showing meta-analysis data that shows no benefit to using plasma-derived products and that plasma-derived products are inherently less safe. Furthermore, she pointed to data, however limited, that extended half-life products may be more effective in immune tolerance yet there are no clinical trial data available for the use of immune tolerance with these products.

Panel Discussion on the Future of Immune Tolerance Therapy

A panel of experts was convened to discuss the future of immune tolerance therapy given the future availability of non-factor replacement products. The panel consisted of Gili Kenet (Israel), Shannon Meeks (USA), Maria Elisa Mancuso (Italy) and Julie Curtin (Australia). They were presented with 3 patient scenarios which included a new onset inhibitor patient, an inhibitor patient who failed a first attempt of immune tolerance and a previously untreated patient at very high risk for inhibitor development, and asked to discuss how they would approach each scenario with the assumption that the year is 2020 and that several non-factor replacement products are on the market.

Laboratory Assay Issues

Establishment of the 2nd International Standard for factor IXa

Elaine Gray presented the results of the establishment of the 2nd International Standard for factor IXa. She pointed out that it has been ~30 years since the last standard was established and that it was important to update the standard. She reviewed the detailed laboratory work that was involved in establishing and validating the new standard.

U.S. National Inhibitor Surveillance Using a Central Laboratory

Connie Miller of the US Centers for Disease Control reported on a broad-based US surveillance project regarding the incidence of inhibitors to both factor VIII and factor IX. First, she reviewed the methods that the Centers for Disease Control used to assess for inhibitors which included an immunologic assay and a chromogenic Bethesda assay. She then reviewed the results of the national testing and determined how often false positives and false negatives occurred via additional testing. Finally, she reported on the number of subjects in the surveillance scheme with new onset

inhibitors and the Centers for Disease Control plan for assessing those patients with clinical characteristics.

Measuring Extended Half-life Factors in the Laboratory

Guy Young reviewed the available data regarding issues with accurately measuring infused factor for extended half-life factors. He pointed out that there both underestimation and overestimation problems when measuring infused extended half- life factors VIII and IX and commented on the potential for negative clinical outcomes. He discussed how the activators of the aPTT reagents can result in significant inaccuracies in the measurement of infused factors and pointed out the importance for the clinician to be aware of this issue. Dr. Young then mentioned that most of these inaccuracies can be solved by using chromogenic factor assays.

Implementation of Chromogenic Factor Assays

Steve Pipe carried forward from Dr. Young’s presentation and discussed how his lab implemented the chromogenic factor assays. He reviewed the many steps required to develop this assay step by step ensuring that the audience understands that while it is eminently feasible to do this, that it also takes time, effort, and funding.

Is all thrombin generation the same?

Claude Negrier discussed the thrombin generation assay demonstrating that the amount of thrombin generated can vary significantly depending on both the pre-analytic and the analytic variables. He pointed out that it is crucial to understand the methods of thrombin generation test in order to be confident that the way the assay is conducted will yield meaningful results.

Factor XI and the Contact System

9 July 2017 8:00 – 12:15 Chairman: Joost Meijers Co-Chairs: Edward Feener, Heiko Herwald, Coen Maas, Owen McCarty, Stephanie Smith, Evi X. Stavrou

The Factor XI and Contact System session on July 9 drew approximately 400-450 attendees.

The session started with a number of discussions on the effects of polyphosphates on the contact system chaired by Thomas Renné.

First, Jonas Emsley from the United Kingdom presented novel structural models of contact factors among which the interaction between factor XII and HK with gC1qR, which suggests that gC1qR may be more specific agent to the contact system compared to activators like polyphosphates that affect many plasma proteases and more focused on control of the inflammatory PK-HK activation than factor XI.

Second, James Morrissey from the United States highlighted the procoagulant activities of nucleic acids and polyphosphates on the contact system. He emphasized that currently used techniques to purify nucleic acids lead to preparations that also contained silica, which hinders our research since we cannot be sure if the effects that are observed are induced by nucleic acids or silica contamination. Long chain polyphosphates are much more procoagulant than cellular DNA or RNA.

Third, Coen Maas from the Netherlands showed data on physical properties of polyphosphates, and showed that polyphosphates are released from platelets upon activation. The secreted polyphosphates are not as potent as the polyphosphates that remain associated to the membrane as insoluble particles.

Finally, in this polyphosphate session, Thomas Renné provided data on targeting polyphosphates by polyphosphatases in order to reduce activation of factor XII and thereby the contact system. On the other side it is possible to increase polyphosphates by interfering with the phosphate receptor XRP1.

The polyphosphate session was concluded with a lively discussion between the speakers and the audience and also between the speakers highlighting the importance of polyphosphates and made clear that there are a number of unresolved issues.

Elaine Gray from the United Kingdom discussed the functional and antigen levels of the 1st International Standard for factor XII, plasma. This standard for both factor XII function and factor XII antigen has been endorsed by SSC and submitted to the Expert Committee for Biological Standardization.

The use of intravenous immunoglobulins (IVIG) is associated with potential prothrombotic complications because of contamination with factor XIa. Mikhail

Ovanesov from the United States explained the problem and provided potential solutions including specific testing for factor XIa and procoagulant activity.

Edward Feener from the United States discussed the role of the contact system in diabetic retinopathy, in which kallikrein plays a role that can be targeted using specific kallikrein inhibitors. A phase 1 study was performed with an intravitreous kallikrein inhibitor in patients with diabetes. The treatment was well tolerated and patients had improvement of vision, which will be further investigated in follow up phase 2 studies.

Alvin Schmaier from the United States gave an educational lecture on the vascular biology of the factor XII and kallikrein/kinin system. He elaborated on the cell biological aspects of the components of the contact system.

The second educational lecture was given by Scott Diamond from the United States. He explained the role of flow in studying the contact system. By using specific inhibitors, he is able to initiate the coagulation system by either the contact pathway or the TF pathway with all intermediary combinations. In situations where the factor XI feedback loop contributes to fibrin formation (low TF), polyphosphates stimulate feedback activation of factor XI under both venous and arterial flow conditions.

Jeffrey Weitz from Canada gave an update on the use of factor XI inhibitors in the clinic. This is currently a highly interesting topic, because of the potential safer strategy for treatment of thrombosis. Targeting of factor XI with antisense oligonucleotides has been studied in a phase 2 clinical study. Targeting factor XI with antibodies is currently entering phase 2. Small molecules inhibiting factor XIa have completed phase 1 studies.

Sara Martinez de Lizarrondo from France gave an overview on her studies that demonstrate that bradykinin is the mediator of the deleterious effects of tissue-type plasminogen activator in stroke.

The business meeting was concluded with a proposal for nomenclature of the contact factors by Alvin Schmaier from the United States. He suggested to harmonize names and abbreviations. His presentation was followed by an extensive discussion in which arguments for and against different names and abbreviations were mentioned and several alternative options were given. A firm conclusion or consensus could not be reached, and it was decided that a survey will be prepared and sent to interested individuals. The results will be tabulated and surveyed. A report will be made and presented at the SSC meeting in Dublin in 2018.

Factor XIII and Fibrinogen

8 July 2017 14:00 – 18:15

Chairman: Verena Schroeder Co-Chairs: Zsuzsa Bagoly, Matthew Flick, Martin Guthold, Helen Philippou, Marlien Pieters, Alisa Wolberg

Educational session (1h) Martin Guthold (Wake Forest University, US): Current view of fibrin structure and clot structure Martin Guthold gave a presentation entitled "Current View of Fibrin Structure and Clot Structure". The talk had five parts: 1) Overall Structure of Fibrin(ogen), 2) Assembly of Protofibrils; 3) Assembly of Fibrin Fibers, 4) Crosslinking, 5) Mechanical Properties of Single Fibers and Internal Structure of Fibrin Fibers. In part 1, the overall structure of fibrinogen was discussed, in particular the overall shape and dimensions, the key interaction sites for protofibril formation and fibrin fiber formation and missing parts in the crystal structure, including the entire alphaC region (alpha221- 610), regions at the amino termini of the alpha and beta chains (alpha1-26, beta 1-57) and the gamma C-terminus (gamma395-411). In part 2, protofibril formation was discussed, in particular the key interactions (A:a interaction, B:b interaction and D:D interface). Protofibril models from the Doolittle lab, AFM images from Protopopova and protofibril simualations by Zhmurov & Barsegov were shown and discussed. The location of the alphaC regions seems to be on the outside of a protofibril. As an aside, the Y- ladder model of Rocco et al. was shown and discussed. Here, fibrin monomers initially form a Y-ladder polymer, in which only one of the two A:a interactions is engaged. The Y-ladders then from a network and coalesce into a fibrin fiber network. In part 3, fiber assembly from protofibrils and the internal structure of fibrin fibers was discussed. This assembly process is currently not fully understood, though there is good evidence that the B:b interactions and alphaC region interactions play a role. Though, neither one are absolutely required because fibers can form without these interactions. Techniques to study the internal structure of fibrin fibers include transmission electron microscopy, small angle X-ray scattering and neutron scattering and turbidimetry. The obtained data from these techniques suggest that fibrin fibers have a very open, perhaps fractal cross- section with holes in it. In part 4, the alpha-alpha and gamma-gamma crosslinks, and their different locations and roles were discussed. Gamma-gamma crosslinks form between abutting monomers across the D:D interface, and they stabilize that interface. Alpha-alpha crosslinks from between interacting alphaC regions and they stabilize the coupling between protofibrils. Crosslinked fibers lyse slower. Both types of crosslinks contribute to making fibers (and clots) stiffer; alpha-alpha crosslinks also increase the elasticity of fibrin fibers. In part 5, the mechanical properties of single fibrin fibers, and their meaning, were discussed. The atomic force microscopy-based technique to study these properties was shown and two summary slides with the mechanical properties of

fibrin fibers (such as modulus, extensibility, elasticity, energy loss, relaxation times, etc) were shown. Fibrin fibers are about as stiff as an elastic rubber band, alpha-alpha crosslinking increases stiffness by a factor of about 2.5, and full crosslinking by about a factor of 3.5. The diameter-dependence of the stiffness of fibrin fibers was then discussed in more detail. The stiffness of fibrin fibers decreases strongly (~D^-1.5) with increasing diameter. The protein density also decreases with increasing diameter. These data indicate that fibrin fibers have a denser, well-connected core and a less dense, less well-connected periphery. These data seem to agree with the turbidimetry and scattering data mentioned above. However, more work is needed to fully understand protofibril assembly and the internal structure of fibrin fibers.

Alisa Wolberg (University of North Carolina, US): Fibrin structure and crosslinking in disease Desire to understand the biophysical characteristics of fibrin(ogen) has motivated innovative and informative studies for decades. These studies have revealed remarkable properties of fibrin, including substantial elasticity and extensibility. In parallel, studies using plasma, blood, and mouse models have interrogated biological and pathophysiological roles of fibrin(ogen) in thrombosis. These have demonstrated direct, causative mechanisms by which fibrin(ogen) promotes the formation of venous thrombi. Mice with elevated fibrinogen levels demonstrate accelerated thrombus formation. Mice with elevated prothrombin levels and consequently, enhanced fibrin deposition and denser fibrin networks, produce larger thrombi that are enriched in red blood cells. Recently, we discovered that by crosslinking fibrin, factor XIII(a) promotes red blood cell retention in venous thrombi. This mechanism is dependent on the ability of factor XIII to bind and circulate with fibrinogen, and is associated with the formation of fibrin alpha- chain crosslinking. Given findings that demonstrate profound effects of fibrin alpha-chain crosslinking on fibrin fiber stiffness, we propose that the physical characteristics of fibrin are important determinants of venous thrombus formation and composition.

Business session (3h in total) First part: Standardisation topics (1h) Sanj Raut (NIBSC, UK): WHO 1st International Standard Factor XIII Plasma, (02/206): Collaborative Study to additionally assign value for Factor XIII B-subunit Dr Sanj Raut presented a proposal to additionally assign value for Factor XIII-B subunit, to the current WHO 1st IS Factor XIII, Plasma (02/206), as standardisation for the FXIII- B subunit is critically needed. This proposal has been approved by the WHO/ECBS (October 2016) and entails carrying out an international multi-centre collaborative study involving manufacturers, clinical/research laboratories and regulatory authorities. The proposal outlined assay designs including a sandwich ELISA for measurement of Total FXIII-B subunit, using biotinylated A/Total FXIII-B subunit monoclonal antibody (MAb1) as capture-antibody and HRP-labelled A/Total FXIII-B subunit monoclonal antibody (MAb3) as tag-antibody. Both monoclonal antibodies will be kindly provided by Dr Éva Katona & Professor László Muszbek, University of Debrecen, Hungary. A feasibility study involving 2-3 laboratories will be initially carried out to assess ELISA designs to be used, relative to locally collected Normal Plasma Pools (both fresh & frozen). This will

help to finalise protocol for the main collaborative study, which will involve between 8-10 laboratories. The study design will ask each participants to carry out 4 independent estimates (independent assays) for Total FXIII-B subunit on the test material (WHO 1st IS FXIII plasma), relative to locally collected Pooled Normal Plasma (n>10), following strict assay designs and raw data from each participants will be analysed at NIBSC. Primary evaluation will be for Total FXIII-B subunit for value assignment to the WHO 1st IS FXIII plasma vs. Normal Pooled Plasma, in International Units (IU) and the objective will be to submit to ECBS for assignment in October 2019.

Another standardisation topic was originally planned in this first part of the business session, but the presentation by Marlien Pieters (SA), entitled “Standardisation of the plasma clot turbidity and fibrinolysis assays revisited” had to be cancelled. Instead, Muriel Maurer agreed to give the following presentation:

Muriel Maurer (University of Louisville, US): Oligomeric State of Factor XIII Changes Upon Activation Factor XIII (FXIII) introduces covalent crosslinks into substrates including the fibrin clot network. Intracellular FXIII exists as an A2 homodimer whereas plasma FXIII exists as an A2B2 heterotetramer. Both forms of zymogen FXIII can be activated proteolytically by thrombin cleavage of the Activation Peptide segment on the catalytic FXIII A2 subunits or nonproteolytically in the presence of high mM CaCl2. Indirect evidence has been accumulating to suggest that FXIII becomes a monomer upon activation. Analytical ultracentrifugation was now used to directly probe the oligomeric states of FXIII A2. Additional supporting data were obtained from other biochemical methods. Results revealed that the zymogen FXIII A2 did indeed dissociate into monomers following both non-proteolytic and proteolytic activation strategies. Furthermore, thrombin cleavage of a single activation peptide in the zymogen dimer was sufficient to initiate the transition to monomer. The results obtained demonstrate that FXIII undergoes a change in oligomerization state upon activation.

Verena Schroeder (University of Bern, CH): A follow-up on the question of novel guidelines for FXIII deficiency Verena Schroeder gave a presentation entitled “Two important standardisation topics: 1.) Novel guidelines for FXIII deficiency? 2.) Nomenclature of FXIII sequence variations. In the first part, the question raised at last year’s meeting was revisited. The earlier SSC communication on the diagnosis and classification of FXIII deficiency is not well visible and does not contain recommendations on treatment of FXIII deficiency in general and in special clinical situations. It was discussed whether a short communication referring to the latest literature in the field was sufficient. A statement from the audience said that a detailed update and extension of a guideline paper would be preferable. In the second part, an important issue was raised. Until now, numbering of the nucleotides in the FXIII-A gene and of the amino acids in the FXIII-A protein were started at the first amino acid of the mature protein, serine, and not the initiator codon and amino acid methionine (according to the publications of Ichinose et al. Biochemistry 1986, and Ichinose et al. Proc Natl Acad Sci USA 1988). In recent years, HGVS nomenclature

(http://varnomen.hgvs.org) has become the international standard when reporting sequence variations. We cannot deny this development and have to think about how we can best deal with this in the future without causing too much confusion. Marguerite Neerman-Arbez reported on the experience with fibrinogen, where always both nomenclatures are given, numbering starting with the initiator codon and numbering according to the mature protein.

Second part: Scientific topics (2h) László Muszbek (University of Debrecen, HU): Novel methodological approach to the detection and characterization of anti-FXIII antibodies

Clinical symptoms and laboratory findings associated with suspected FXIII autoantibodies were presented. It is important to realise that FXIII antigen determination may not be reliable in the presence of a FXIII antibody. FXIII alloantibodies are extremely rare, only 5 cases have ever been described. An algorithm for the diagnosis of anti-FXIII antibodies was suggested including mixing tests and a modified Bethesda assay and problems with Bethesda-type inhibitor assays were highlighted. This was followed by a summary of present and future techniques for binding studies. An outlook was given regarding therapy of FXIII autoantibodies: the elimination of antibodies is often ineffective and remains associated with a high mortality. In the future, the aim may be to neutralise the antibodies.

Zsuzsanna Bereczky, Éva Katona (University of Debrecen, HU): Effect of factor XIII levels and polymorphisms on the risk of myocardial infarction in young patients Introduction. Factor XIII (FXIII) stabilizes and protects the fibrin network. Its role in myocardial infarction (MI) is still to be clarified. Aims. To evaluate the association of FXIII levels with MI in young patients and to investigate how the FXIII-A p.Val34Leu, FXIII-B p.His95Arg and IVS11, c.1952+144 C>G (Intron K) polymorphisms influence FXIII levels and MI risk. Patients and Methods. Patients with ST elevation MI below 40 years of age (MI, n=119), age-matched clinical controls (CC, n=101) without MI and coronary artery disease and healthy controls (HC, n=120) were investigated for FXIII activity, FXIII-A2B2, FXIII-B concentrations and for the polymorphisms. Results. FXIII activity and FXIII-A2B2 antigen were significantly elevated in MI. FXIII activity and antigen were significantly elevated in Arg95, while decreased in Intron K “G” carriers. Smoking had an independent increasing effect on FXIII activity and FXIII-A2B2 antigen. Intron K C>G polymorphism significantly decreased the risk of MI in patients with elevated fibrinogen. Conclusions. Among the investigated factors Intron K C>G polymorphism and smoking have the most powerful effect on FXIII levels and on the risk of MI in the young. The effect of smoking on coronary thrombus formation may partially be attributed to its FXIII increasing effect.

Zsuzsa Bagoly (University of Debrecen, HU): Factor XIII levels and polymorphisms in patients with systemic lupus erythematosus and antiphospholipid syndrome The aim of the study was to find out whether levels of factor XIII (FXIII) might differ in patients with systemic lupus erythematosus (SLE) and/or antiphosphoslipid syndrome

(APS) as compared to healthy individuals and if levels are associated with thrombotic episodes. 137 patients with SLE and/or APS and 140 age and sex-matched healthy controls were enrolled. FXIII activity and FXIII-A2B2 antigen levels were significantly elevated in APS patients (primary or secondary) as compared to controls. Among SLE patients, FXIII A2B2 levels were signicantly elevated in those with a history of atherothrombosis, while no such association was found for FXIII-B levels. A FXIII A2B2 level in the upper quartile conferred a significant risk for arterial but not venous thrombotic events in SLE patients (OR:4.43; 95% CI:1.48-13.18, p=0.008). The presence of FXIII-B intron K c.1952+144 C>G polymorphism rare allele drastically decreased FXIII levels in patients and in controls as well, and conferred a significant protective effect against venous thrombosis in SLE patients (OR:0.192; 95% CI:0.05- 0.70, p=0.008), while the presence of FXIII-B Arg95 allele showed a significant risk for venous thrombosis (OR:3.66; 95% CI:1.00-13.3, p=0.039) in this patient population.

Veronika Farkas (Semmelweis University Budapest, HU): Structure of fibrin in thrombi extracted with interventional treatment of ischemic coronary, peripheral and cerebral artery disease

Coronary (CAD, n=66), peripheral artery disease (PAD, n=64) or acute ischemic stroke (AIS, n=78) patients were prospectively enrolled between 2014 and 2016, basic clinical characteristics and laboratory findings were registered. Thrombi were extracted with acute therapeutic intervention and processed for scanning electron microscopy. Five images from separate, randomly selected parts of each thrombi were taken. The median of fibrin fiber diameters was determined by morphometric analysis and a significantly higher value was found in CAD thrombi compared to PAD or AIS samples, due to the structural pattern of fibrin in male patients. We found that two- and four-fold more patients received chronic aspirin and clopidogrel treatment prior to intervention in the CAD group, compared to PAD and AIS, respectively. Our findings show that long term anti-platelet treatment caused a significant fiber thickening, but only in thrombi from male patients. Regarding comorbidities, only malignancy affected fibrin fiber thickness; in the presence of such disease clots with thinner fibrin fibers developed in PAD patients. We found a parabolic dependence of fiber thickness on fibrinogen level in CAD thrombi, with a minimum at about 4 g/l. In AIS thrombi, the diameter of fibrin fibers shows an inverse correlation with plasma level of C-reactive protein in the range of 0-8 mg/L, but this trend is reversed in more severe inflammation.

Matthew Flick (Cincinnati Children’s Hospital, US): Role of fibrinogen and FXIII in obesity and metabolic inflammation Obesity is a worldwide epidemic that predisposes individuals to numerous comorbidities including atherosclerosis, hypertension, type II diabetes, and non-alcoholic fatty liver disease. Systemic inflammation has been proposed as a contributing factor in virtually all of the obesity-associated diseases. Notably, obesity is further characterized by enhanced coagulation activity including extravascular fibrin deposition in multiple organ systems. Fibrin(ogen) has been shown to promote pro-inflammatory activity through engagement of the leukocyte integrin receptor M2/Mac-1. Immunohistochemical studies identified extravascular fibrin deposits within white adipose tissue and liver as distinct features of both mice fed a high fat diet (HFD) and obese patients. Fibγ390-396A

mice carrying a mutant form of fibrinogen incapable of binding leukocyte αMβ2-integrin revealed these animals were significantly protected from HFD-induced weight gain and elevated adiposity. HFD-fed Fibγ390-396A mice had markedly diminished systemic, adipose, and hepatic inflammation with significantly reduced macrophage counts within white adipose tissue, as well as near complete protection from development of fatty liver disease and glucose dysmetabolism. In separate experiments, mice were treated with the direct thrombin inhibitor dabigatran etexilate (DE), which was found to limit HFD- induced obesity development. In rescue experiments, DE suppressed progression of sequelae in mice with established obesity. Finally, preliminary analyses of adipose tissue from obese patients indicated that the amount of cross-linked fibrin in visceral adipose positively correlated with fasting glucose blood glucose levels. Collectively, these data provide the proof-of-principal that targeting thrombin or fibrin(ogen) may limit pathologies in obese patients.

Marguerite Neerman-Arbez (University of Geneva, CH): Congenital fibrinogen disorders: an update In the communication by Marguerite Neerman-Arbez (Faculty of Medicine, University of Geneva) entitled "Congenital Fibrinogen Disorders: an update" a state of the art review of causative mutations for CFDs was presented. CFDs are characterised by extensive allelic heterogeneity. Quantitative fibrinogen disorders are mostly caused by null mutations, in homozyosity or compound heterozygosity (afibrinogenemia) or in heterozygosity (hyporfibrinogenemia) while qualitative fibrinogen disorders (dysfibrinogenemia) are mostly due to heterozygous missense mutations. The natural history of congenital dysfibrinogenemia analysed in a long-term clinical follow-up study (mean of 9 years following the initial diagnosis) of 101 genetically characterised patients was described. The problem of the double nomenclature for mutations (numbered from the initiator Met according to HGVS guidelines, or numbered in the mature chains lacking the signal peptides) was briefly discussed. Finally the structure- function insight gained by genetic studies for the three individual fibrinogen polypeptides as well as for the secreted fibrinogen hexamer was presented.

Fibrinolysis

8 July 2017 9:00 – 13:15 Chairman: Paul Kim Co-Chairs: Krasimir Kolev, Colin Longstaff, Victoria Ploplis, Guy Reed, Tetsumei Urano

Standardization of D-dimer; Development of Shiny App Tools to simplify and standardize the analysis of hemostasis assay data Colin Longstaff, United Kingdom

Colin Longstaff presented an update on Fibrinolysis Standardisation projects being carried out at NIBSC, UK. Two projects are currently recruiting participants to collaborative studies to develop WHO International Standards (IS): The 4th IS for Streptokinase; and a new IS for Thrombin Activatable Fibrinolysis Inhibitor/Procarboxypeptidase U (TAFI/proCPU) in plasma. It is hoped the TAFI/proCPU study will include some results from isotope dilution mass spectrometry, which will enable results to be reported gravimetrically (ng per ml plasma) in addition to International Units. CL also reported on the development of several online apps that facilitate analysis of clotting and fibrinolysis assay data. The work was the subject of a SSC Communication from the Fibrinolysis Subcommittee (and was featured in the editorial) in the May edition of the Journal of Thrombosis and Haemostasis: “Development of Shiny app tools to simplify and standardize the analysis of hemostasis assay data: communication from the SSC of the ISTH”, J Thromb Haemost 15(5) 1044- 6, 2017. Recent work on the on-going efforts to produce an IS for D-dimer determination in plasma was also presented, including a commutability study, conducted with the help of Roche Diagnostics, Germany, which included 2 NIBSC candidate reference preparations and 54 patient plasma samples. On the basis of this study and earlier stability studies on fibrin degradation products in freeze dried plasma, CL made a recommendation to develop a new candidate reference preparation of pooled patient plasma containing D-dimer that will be freeze dried in the presence of trehalose stabiliser. If a suitable source of plasma can be found and this candidate preparation is manufactured, results from a subsequent study should settle to question of whether it will be possible to develop a WHO IS for D-dimer.

Coagulopathy of traumatic brain injury: Pathophysiologic origins distinct from disseminated intravascular coagulation Mark Walsh, United States

Mark Walsh provided a presentation entitled: "Clinical and Pathophysiological Differences Between Trauma Induced Coagulopathy (TIC) and Disseminated Intravascular Coagulation (DIC)". In his talk he reviewed the history of the application of the ISTH definition of Disseminated Intravascular Coagulation (DIC) to Traumatic Induced Coagulopathy (TIC) and addressed the controversy surrounding the accuracy and utility of using only Common Coagulation Testing (CCT) (PT, PTT, fibrinogen, FSP, platelet count) based ISTH definition of DIC to TIC. He made specific reference to the inability of the ISTH criteria for DIC to define those patients with TIC and he also noted the lack of pathological findings characteristic of DIC in patients with TIC. He cited remote and recent literature which highlighted the differences between DIC and TIC. He described the increasing reliance on viscoelastic tests (VET)

Thromboelastography (TEG) and Rotational Thromboelastometry (ROTEM) to further refine the definition of TIC in the clinical arena. He presented septic rat and traumatic brain injury rat model based data comparing TEG with platelet mapping for platelet activation and inhibition parameters and CCT markers for coagulation and inflammation. The conclusions of his presentation were the following.

1. Platelet counts were reduced in septic but not Coagulopathic Traumatic Brain Injured (CBTI) rats. 2. CCT markers were more severely reduced in septic rats. 3. There was similar platelet dysfunction in the septic and CBTI rats. 4. There were no inflammatory marker changes and CTBI rats. 5. CBTI changes were immediate but transient in the rat versus the more delayed and sustained responses in the septic rat model.

These data support the hypothesis the coagulopathies associated with sepsis and TBI are of different etiologies and that inclusion of VETs with platelet function in the definition of TIC would lead to a more accurate and pathophysiologic based definition of TIC that reflects the changes in primary and secondary hemostasis associated with trauma. Based on these combined CCTs and the VETs with platelet function analyses, a consensus regarding an operational definition of TIC would allow for a common research and clinical terminology that would guide trauma researchers and clinicians toward a more standardized diagnostic and therapeutic approach to TIC. A post session meeting with the SSC faculty and organizers of the session proposed a jointly co-authored Consensus Statement by the faculty at the SSC meeting that proposes a uniform operational definition of TIC.

Activation of the hemostasis system in Gram- and Gram+ Sepsis Francis J. Castellino and Victoria A. Ploplis, United States

Group A Streptococcus is a major causative pathogenic agent of superficial and invasive throat and skin infection in humans. Approximately 700M cases are reported worldwide yearly resulting in 0.5M deaths. Post-infection sequelae include rheumatic heart disease and glomerulonephritis. The S. pyogenes strain, AP53, is a pattern D Group A streptococcus consisting of an M-like virulence protein, PAM, that binds directly to host plasminogen (Pg). PAM interacts with human Pg through its a1a2 repeat and kringle 2 (K2) of Pg. Pg on the surface of GAS is converted to plasmin by GAS-secreted streptokinase 2b (SK2b). Deletion of bacterial PAM enhances survival of mice infected by GAS. GAS interacts with the human host innate immune system by regulating hemostasis, inflammation, and complement. The S. pyogenes strain, AP53, activates host contact pathway of coagulation and binds to components of the contact system, as measured by aPTT, thromboelastography, and surface plasmon resonance (SPR). AP53 infection results in enhanced expression of inflammatory cytokines and bradykinin release, a peptide that mediates inflammation and vasodilation. Transcriptional regulation of a number of virulence factors is dictated by both one and two component bacterial regulatory systems. In the case of the one component mga regulatory system, PAM expression is regulated during the different growth phases of the bacteria. The two component system, CovRS, regulates expression of the cysteine protease, SpeB, which degrades both streptokinase and PAM, thus altering the proteolytic surface of the bacteria. In order for the bacteria to survive the host immune system, it needs to regulate host complement system and phagocytosis. During infection, complement C3b, an opsonin, is generated and deposited on the bacteria which targets it for phagocytosis by host inflammatory cells. The cofactor FH, involved in complement inhibition, binds to invading AP53 via fbpA, which is regulated by both mga and CovRS, and, in association with FI, degrades C3b which is facilitated by host

plasmin. Results from this study demonstrate that there is crosstalk between bacterial virulence factors and host components of hemostasis, inflammation, and complement.

Disseminated Intravascular Coagulation in trauma and traumatic shock Satoshi Gando, Japan

Trauma-induced coagulopathy is essentially an endogenously induced primary pathology and is classified as DIC and ACOTS. In discussing DIC in trauma-induced coagulopathy, these must be considered: 1) the definition and diagnostic criteria; 2) the differentiation of blood coagulation inside and outside of the vessels; 3) the differences between the physiologic and pathologic hemostatic responses to trauma; and 4) the understanding of the time courses of hemostatic responses. DIC, defined as an acquired syndrome characterized by the intravascular activation of coagulation with loss of localization arising from different causes, can originate from and cause damage to the microvasculature, which if sufficiently severe, can produce organ dysfunction. DIC is thrombotic due to tissue factor-mediated activation of coagulation, insufficient anticoagulation systems and inhibition of fibrinolysis by plasminogen activator inhibitor (PAI-1) (DIC with the thrombotic phenotype). However, in some situations, DIC and pathological systemic fibrin(ogen)olysis coexist following the same insults. This is referred to as DIC with the fibrinolytic phenotype. Both types of DIC are always accompanied by massive thrombin generation and insufficient anticoagulation systems. The blood inside the vessels is hypercoagulable in DIC patients, while it is hypocoagulable outside of the vessels and hard to clot following injury and bleeding. Therefore, the terms hypercoagulable and hypocoagulable should be used after clarifying the places in which they occur in the discussion of trauma-induced coagulopathy. We should always distinguish between local responses and systemic responses, local physiological responses and systemic pathological responses, and local physiological hemostasis, immunothrombosis, and systemic pathological DIC. Under pathologic condition of DIC, low or insufficient levels of tissue factor pathway inhibitor (TFPI), antithrombin, protein C, protein S, or functional thrombomodulin all enhance systemic thrombin generation. In physiologic hemostatic responses, the immediate generation of thrombin and plasmin is followed by secondary fibrinolysis, and then inhibition of fibrinolysis by PAI- 1, which is referred to as “fibrinolytic shutdown.” The complete disappearance of PAI-1 after the completion of hemostasis is followed by the reactivation of fibrinolysis by plasmin, which is called “fibrinolytic reactivation”. The characteristics of DIC are immediate massive thrombin generation, a significant increase in plasmin followed by persistent PAI-1 generation. It is important to know that there is a time delay between immediate tissue-type plasminogen activator (t-PA) release and plasmin generation, and the induction of PAI-1 mRNA. Using the DIC diagnostic criteria, DIC can be distinguished clearly from normal hemostasis. Tissue factor-induced massive and persistent thrombin generation consumes Factors Va, VIIIa, protein C, and antithrombin. Neutrophil elastase degrades TFPI, antithrombin and endothelial thrombomodulin. Degraded soluble thrombomodulin lacks full domains. These impaired anticoagulation systems further enhance thrombin formation. At an early phase of trauma associated with hemorrhagic shock, t-PA released from the endothelium generates plasmin that degrades Factors Va and VIIIa and further increases fibrin(ogen)olysis and bleeding. Recent studies suggest the participation of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) in the formation of immunothrombosis. Immunothrombosis consists of the local activation of coagulation, insufficient anticoagulation and inhibition of fibrinolysis at the site of insult.

Local thrombosis disseminates into the systemic circulation in association with systemic inflammation, leading to DIC (Crit Care 2015; 19:72). Recent study clearly demonstrated the pathophysiology of DIC in trauma. The study showed that circulating DAMPs (histones) released from injured tissues induce cytokine release, neutrophil extracellular traps (NETs) formation, endothelial damage, platelet aggregation, and systemic thrombin generation, leading to coagulation activation. These changes give rise to DIC and followed by multiple organ dysfunction syndrome (Am J Respir Crit Care Med 2013; 187:160-169). These studies suggest that local physiologic responses of immunothrombosis and hemostasis can change to pathological systemic DIC in severely injured trauma patients and traumatic shock. DIC is an old but established concept. To deepen the understanding of DIC in trauma, we must keep in mind an old Chinese saying, “He that would know what shall be must consider what has been.”

Fibrinolysis shutdown in trauma Hunter Moore, United States

Fibrinolysis following trauma can be driven to two pathologic extremes. Excessive fibrinolysis (hyperfibrinolysis; defined by TEG LY30 > 3%) is identified in 15% of severely injured patients. Tissue plasminogen activator (t-PA) activity if found in excess in these patients, which drives plasmin generation and rapid clot degradation. Not only do these patients have high t-PA levels but its inhibitors and back up plasmin inhibitors are markedly depleted. This refutes the pre-existing theory that hyperfibrinolysis is drive by protein C activation. Emerging data now supports that hyperfibrinolysis is a concomitant depletion of inhibitors with high activity levels of plasminogen activators which cannot be explained by a single pathway. Patient with hyperfibrinolysis have a high rate of mortality ranging from 30 to 50% predominantly attributable to hemorrhagic deaths. While it may seem logical to preemptively treat patients with an antifbrinolytic after severe injury, over inhibition of fibrinolysis may also be pathologic. This is evident as endogenous inhibition of the fibrinolytic system, also known as fibrinolysis shutdown, or hypofibrinolysis has been identified to be pathologic following trauma. Fibrinolysis shutdown is associated with a high rate of mortality (20-25%) in severely injured patients attributable to organ failure and traumatic brain injury. TEG defined fibrinolysis shutdown is an LY30 of < 0.9% and is common following severe injury ranging from 40-65% of patients. Between 0.9 and 2.9 % is a physiologic range of fibrinolysis which has the lowest mortality rate following severe injury. There is data emerging that treat patients with physiologic levels of fibrinolysis with tranexamic acid is associated with increased mortality. After risk adjustment it appears that TXA may increase mortality by 6 fold in patients with physiologic fibrinolysis, but this remains retrospectively collected data, and requires ongoing investigation. It is also important to take into consideration that TEG and other viscoelastic assays measure the functional activity of systemic blood. At the local level of injury clot is actively forming and degrading. Therefore measuring degradation products and plasmin anti plasmin complexes to quantify fibrinolysis require careful evaluation as they do not reflect the patients systemic coagulation function, and will be elevated in severely injured patients that may have an intact and functional coagulation system. In summary balancing fibrinolysis after trauma is an appealing concept that requires validation. The major antifibrinolytic trials have demonstrated modest reduction in mortality, but a goal directed therapy may demonstrate a more dramatic reduction in mortality. Moreover it is now know that hemorrhage control and resuscitation will correct hyperfibrinolysis, suggesting that the use of TXA is only warranted in uncontrolled bleeding early after injury. Perhaps an even more exciting therapeutic target in the future is attenuating fibrinolysis shutdown. As this pathologic change in coagulation is present in the

majority of severely injured patient it may provide the missing link to reduce VTE and organ failure related mortality in the future.

Anti-fibrinolytics in trauma: unresolved issues and effects nobody talks about Dominik Draxler, Australia

For fibrinolysis to occur plasminogen and its main endogenous activator t-PA co- localise on the fibrin surface, which acts as cofactor for efficient activation of plasminogen to plasmin by t-PA. In presence of the lysine analogue tranexamic acid (TXA) plasminogen and t-PA cannot bind to the fibrin clot which is hence protected from plasmin-mediated digestion. In 2010 TXA had been tested for the first time in a large randomised controlled trial (RCT) in the setting of severe trauma. The Clinical Randomization of an Antifibrinolytic in Significant Hemorrhage 2 (CRASH-2) trial found that TXA improved survival if it was administered within 3h after injury. However, later administration was associated with increased mortality compared with placebo. On top of the survival benefit with early TXA treatment there was no increase in thromboembolic complications reported in CRASH-2 and TXA was soon implemented in European guidelines as a 1A recommendation. In Australia and the United States, however, trauma physicians and surgeons were more sceptical about the CRASH-2 results. The trial was criticised because of the high baseline mortality and the lack of blood tests to assess presence or absence of coagulopathy. Other studies reported conflicting results with some raising concerns about safety of TXA in certain trauma patient collectives, others confirming efficacy and safety as demonstrated in CRASH-2. It is also possible that TXA may be influencing other processes in vivo given that plasmin is now appreciated to have a number of critical functions unrelated to classical fibrinolysis. Indeed, more recent findings have suggested that plasmin may modulate the immune response and blood brain barrier breakdown, and these processes might be relevant when TXA is administered to trauma patients. In this study, we explored the possible non-fibrinolytic consequences of TXA in two populations: healthy volunteers and in a subgroup of patients recruited to the Aspirin and Tranexamic Acid for Coronary Artery Surgery (ATACAS) trial. We also performed preliminary experiments to evaluate changes in fibrinolytic and immune cell markers following administration of TXA to mice subjected to traumatic brain injury using the controlled cortical impact model. Our preliminary results indicated that TXA does indeed modulate pro-inflammatory cytokine levels and some key innate immune markers under base line conditions, and some of these parameters are also modulated in patients treated with TXA undergoing cardiac surgery. We moreover provided evidence to suggest that TXA also modulated immune cell function in mice subjected to TBI. In summary, TXA does indeed seem to have non-fibrinolytic effects that are likely to be clinically relevant. We are currently evaluating this more carefully in patients recruited to the ATACAS trial.

Tranexamic acid use in Europe and the United States: a trauma surgeon’s view Scott Thomas, United States

Dr. Thomas’ objectives were the following: 1. Define Trauma Induced Coagulopathy (TIC) 2. Define the Spectrum of Fibrinolysis in trauma 3. Discus Post CRASH-2 use of tranexamic acid (TXA) 4. Examine the future definition and treatment of fibrinolysis in TIC 5. Re explore the indications for TXA in trauma.

Dr. Thomas referred to the evolving definitions of TIC which included a concept of a “term of coagulopathy and Acute Coagulopathy of Trauma Syndrome or (ACoTS) as an early phase of trauma which may equal DIC with the hemorrhagic phenotype associated with consumption coagulopathy and excessive fibrinolysis. This type of DIC may change into DIC with the thrombotic phenotype 24 hours to 48 hours after trauma, leading to multiple organ dysfunction syndrome….” Dr. Thomas contrasted this ACoTS with the ISTH definition of DIC which is: “an acquired syndrome characterized by the intravascular activation of coagulation with loss of localization arising from different causes. It can originate from and cause damage to the microvasculature, which if sufficiently severe, can produce organ dysfunction”. Yet timing of the coagulopathy and quantitative analyses of the evolution of TIC lead to a different analytic framework. Because of the significance of the 100,000 TM molecules and up to 20,000,000 annexin II molecules (which bind tPA and plasminogen) located on each of the 10 to the 13th power endothelial cells lining all blood and lymphatic vessels within the body covering a surface area of 3,000 to 7,000 m2, the endothelial TM-thrombin complex and fibrinolytic system can ‘‘switch’’ from a fibrinolytic to antifibrinolytic phenotype, and vice versa, in seconds to minutes. Finding the right balance in restoring homeostasis in vivo remains the ongoing challenge in the diagnosis and treatment of early coagulopathy following trauma. With this pathophysiologic foundation Dr. Thomas proposed a definition of TIC that incorporated the definition of the term coagulopathy and Acute Coagulopathy of Trauma Syndrome (ACoTS) at an early phase of trauma which some feel equals DIC with the hemorrhagic phenotype associated with consumption coagulopathy and excessive fibrinolysis. This type of DIC has been felt to subsequently changes into DIC with the thrombotic phenotype 24 hours to 48 hours after trauma, leading to multiple organ dysfunction syndrome. Yet, a more useful and operational definition of TIC would be based on a coagulopathic spectrum of fibrinolysis which would reserve anti fibrinolytic therapy for only those patients who truly demonstrate fibrinolysis which is clinically significant. Which test would predict with clinical utility significant fibrinolysis yet remains to be seen. VETs TEG and ROTEM seem to have assumed a position of significance in this arena. Therefore, Dr. Thomas proposed that the utilization of TXA in trauma be reserved to those patients in shock or who demonstrate significant fibrinolysis on VETs in order to avoid the well described phenomenon of multi organ failure associated with traumatic shock and “fibrinolytic shutdown”. Dr. Thomas cited recent literature that supports his contention that a fibrinolytic spectrum indicated that TXA should be limited to those patients who demonstrate fibrinolysis on a VET.

Genomics in Thrombosis and Hemostasis

9 July 2017 8:00 – 12:15 Chairman: Willem Ouwehand Co-Chairs: Daniel Bellissimo, Paul F. Bray, Kathleen Freson, Anne Goodeve, Michele P. Lambert, Pieter Reitsma

Welcome Chair: Willem H Ouwehand, Cambridge, UK Willem H Ouwehand opened the meeting and stressed two points:

 Data sharing – encourage our community to be more organised and share data  Call for nominations for a new Chair of the SSC to start after 2018 Dublin meeting

Session 1 - Updates on diagnostic high throughput sequencing tests

Molecular diagnosis of rare BPD using NGS Kate Downes, Cambridge, UK

Kate introduced the ThromboGenomics test – an NGS panel testing 78 tier-1 (known) genes involved in inherited bleeding, thrombotic and platelet disorders (BPDs). Simeoni I, et al. (2016) Blood describes platform and how can test be used in a clinical environment.

Encoding phenotype: HPO. Essential to support data sharing across the Rare Diseases community, widely accepted by all large Rare Diseases sequencing programs.

Standardising interpretation: American College of Medical Genetics and Genomics (ACMG) guidelines for reporting of clinically actionable variants

Advantages of panel testing: very high coverage. Every single variant identified has been confirmed in an independent aliquot of DNA, in clinically accredited laboratory.

98.9% of the target coding region high coverage, including UTRs. Allows for better identification of copy number variants (CNVs); e.g. of CNVs for F9, DIAPH1

Benefits of having the DNA samples from a large number of patients analysed on a single NGS panel test: 1,200 referrals, 900 cases reviewed by a Multi Disciplinary Team (MDT), positive report for ~50% of the referred patients, and half of those variants are novel and therefore labelled as ‘likely pathogenic’.

Example of intronic F8 single nucleotide variants being upgraded from VUS to Likely Pathogenic due to re interpretation of multiple cases.

Q (Alan Nurden, France): Do you report for a little bit more than what’s being asked? For Glanzmann, for e.g., there are extra variants in other genes than the Glanzmann ones that are modifier. Do we report on those?

A: No, we only report on what is being asked by the requester. Moreover to have statistical confidence in modifiers of monogenic conditions 100s so not 1000s of cases will need to be genome-wide typed (which is not feasible because we are in the realms of rare diseases).

Q (David Lillicrap, Canada): How do we screen for regulatory elements?

A: We look in sequence promoter regions. In the future we may be including the regulatory elements. These have been defined in many different types of blood cells by the BLUEPRINT epigenomics project (Stunnenberg et al, Cell 2016). Similar regulatory maps of the genome in liver cells will be required to better comprehend the regulation of the coagulation genes. When a patient presents with a convincingly documented Factor deficiency but no pathogenic variants in the relevant gene then we will proceed with whole genome sequencing.

Simeoni I, et al. (2016) A high-throughput sequencing test for diagnosing inherited bleeding, thrombotic, and platelet disorders. Blood 127(23):2791-2803.

Experiences from the Öresund Region with implementation of WES in patients with IBD Eva Leino, Copenhagen, Denmark

Eva introduced a project of whole exome sequencing (WES) of the DNA from patients with Inheritable Bleeding Disorders (IBD) from the different health regions of Denmark and including referrals from other Scandinavian countries, Leinoe E, et al. (2017).

Aim: to evaluate the sensitivity and specificity of WES at an early stage in the diagnostic algorithm.

Description of Öresund score - scoring mechanism making use of consequence prediction tools to infer the pathogenicity of coding variants.

Conclusion:

 WES at an early phase may save complex and expensive laboratory tests  The Öresund scoring algorithm requires further evaluation  Interpretation of WES results can be aided by the results of other lab tests (e.g. flow cytometry)  Having a MDT review for reporting is essential

Q (Keith Gomez, London): when a variant is reported, do clinicians know the variant has an additional layer of evidence with the Öresund score?

A: Yes, where possible the results from a functional test is included in the report

Q (Willem H Ouwehand, Cambridge): You rely on in silico tools that are inherently unreliable and known to have poor Receiver Operating Curves. Are you not concerned to miss variants with LP or LPV by HGMD?

A: The Öresund score is only part of the interpretation. We also use previous evidence from the literature to interpret variants.

Leinoe E, et al. (2017) Application of whole-exome sequencing to direct the specific functional testing and diagnosis of rare inherited bleeding disorders in patients from the Oresund Region, Scandinavia. Br J Haematol.

Tier-1 genes: New genes and modes of inheritance Kathleen Freson, Leuven, Belgium

Kathleen explained the definition of a Tier-1 gene:

 high level of confidence of harboring variants which are disease-causing  proven in at least three unrelated pedigrees with co-segregation  OR: if less < 3 pedigrees, then supporting results from functional studies, including mouse KO phenotypes are required  Only variants in internationally agreed Tier 1 genes should be used for clinical reporting  This ISTH-SSC provides an ideal forum to ratify the proposed new Tier 1 genes

Seventeen genes were proposed for inclusion on the Bleeding, Thrombotic, Coagulation and Platelet disorders (BPD) clinically reportable Tier 1 gene list (Table 1 in Annex) and an alternative mode of inheritance was proposed for six genes (Table 2 in Annex).

Session 2 - What should I tell my patients?

Genetic counseling for a child with bleeding disorder Shoshana Revel-Vilk, Jerusalem, Israel

Shoshana explained the points to take into consideration when a patient with a possible genetic condition presents to a Consultant Haematologist:

First consideration: is a DNA test indicated?

 medical condition  potential benefits  potential risks

The American Society of Human Genetics (ASHG) recommendations for testing children (Botkin JR, et al. 2015), are:

 targeted gene panels  single gene  WGS only for certain cases

The use of a ‘test information leaflet’ and a ‘informed consent’ is recommended explaining what the value of the test result may be to inform clinical management, family planning. The leaflet should also deal with issues of turn-around time, incidental findings (e.g. non-paternity) and privacy concerns.

Botkin JR, et al. (2015) Points to Consider: Ethical, Legal, and Psychosocial Implications

of Genetic Testing in Children and Adolescents. Am J Hum Genet 97(1):6-21.

Dealing with Variants of Uncertain Significance Keith Gomez, London, UK

Keith explained how to deal with variants of uncertain significance (VUS) – starting with two uncertainties:

 variant effect  phenotype (late onset, challenge- the phenotype might not have fully shown) Pathogenic/likely pathogenic (classes 4 and 5) variants: there is robust evidence for labelling variants in these classes.

Benign / likely benign (classes 1 and 2): this class of variants is not easy to ascertain and therefore adequate levels of evidence can only be obtained by long period of observation (large population cohort studies like UK Biobank, 1M Veteran study)

VUS (class 3): the vast majority of rare variants and unique variants (never observed variants) are in this class.

ACGS guidelines: reporting of VUS depend on local clinical practices. But aggregation of VUS information across laboratories is essential, so that we can improve their future interpretation - see ACGS Best Practice Guidelines for Variant Classification 2017 (Ellard S. et al., 2017)

There is a risk when reporting VUS for confusion.

Reporting of VUS should only be considered if it concerns a request for further exploratory research studies.

VUS should only be reported if appropriately contextualized and the referring clinician is informed of the reasons why the VUS is reported, e.g. request for co-segregation (ACMG guidelines - Richards S, et al. 2015).

Bring in other sources of evidences:

 control datasets like ExAC, GnomAD  literature,  family history

Necessary to create link with referring clinicians to the team responsible for reporting. Create an MDT involving clinicians, bioinformatician, clinical geneticists and trainees.

Q: We should be careful with trying to explain all observed clinical phenotypes with the genetics

A: I entirely agree that we must be careful not to over-interpret variant data. DNA variant data can only in part explain the geno- to pheno-type relationship in an ‘outbred’ population

Ellard S. et al., 2017: ACGS Best Practice Guidelines for Variant Classification 2017

http://www.acgs.uk.com/media/1059605/uk_practice_guidelines_for_variant_classificati on_2017_24_05_17.pdf

Richards S, et al. (2015) Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med 17(5):405-424.

Session 3 - Challenges of interpretation

New control datasets and updates to variant catalogues Karyn Megy, Cambridge, UK

Karyn introduced the bioinformatics resources behind the main steps of the variant filtering process.  choice of transcripts (LRG)  selecting on frequency in control populations (ExAC, GnomAD)  presence in curated variant databases (ClinVar, HGMD, LSDB and gene(s)- specific ones: EHAED, Mount Sinai Glanzmann thromboastenia)

She explained how, as a community, we can improve them: - standardization (HGVS nomenclature, ACGM guidelines for reporting) - control databases – now linked to Primary Care records and hospital entries (UK BioBank, UK 100,000 Genomes Project, 1M US Veterans Project) - LRG – gene curation - Submission of variants in ClinVar

Challenges in interpreting genetic data in females at-risk for bleeding disorders Jill Johnsen, Washington, USA

Jill presented the challenges in interpreting genetic data in ‘carrier’ females who are at- risk of bleeding – example used Haemophilia A.

Females are often thought of as ‘carriers’ - but they are actually more frequently symptomatic than assumed (high bleeding score).

Therefore, female in a pedigree should not just be considered as “carriers” but also at risk of bleeding.

Disease severity in males is determined by the factor level. Bleeding only weakly associated with factor levels in females.

Moving forward for testing - NGS panel should be used to sequence F8 region, panel also incorporates inversion intron 1 and 22 testing

Jill presented some examples of VUS variants and how they were reported

Global Collaborations on Variant Pathogenicity Assertions through Expert Panels - ClinGen/ClinVar Jonathan Berg, Chapel Hill, USA

Jonathan introduced the ClinGen consortium. Started in 2013. Funded by NHGRI and NCBI.

The aims are to:

 facilitate data sharing  coordinate expert curation  dissemination of data He explained the difference between ClinVar (repository of medically relevant variant maintained by the NCBI) and the ClinGen “ecosystem” of clinical domain working groups, expert panels and gene-disease curation committees.

Plans for haematology:

 benign haematology working group, starts autumn 2017  Nigel Key and Kirsty Lee  Focus - platelet disorders, VWD, thrombophilia, haemoglobinopathies The American Society of Hematology is planning to contribute to 2 panels.

 malignant haematology diseases  germline platelet disorders Looking for collaborators and community experts contact [email protected]

Q: How do you see ClinVar as a tool for larger sequencing projects

A: Ideal repository for aggregation of data after they have been reviewed locally; submission to ClinVar will allow disease-specific variants data to be integrated in the bigger scheme of gene and genome annotation.

Human Phenotype Ontology to support clinical reporting and genomic diagnostics. Peter Robinson, Connecticut, USA

Human Phenotype Ontology (HPO) terms library was initiated in 2007 from Berlin. Now a decade later it has become the most frequently used terms library for the coding of clinical phenotype data from patients with inherited disorders (Robinson PN. & Mundlos S. 2010).

Peter explained the reason behind the HPO ontology: aim to capture any terms that might be used in clinical settings.

Other ontologies do not provide enough coverage of the medical conditions observed in rare diseases patients (other terminologies, such as MESH and SNOMED, together are 30% of the corpus of HPO).

HPO cover:

 sign, symptom, lab finding, imaging results, etc.  detailed clinical phenotyping results  individual components of the disease

HPO also allows for:

 Computable representation of human diseases  Translational research / diagnostics  Interoperable with model organism and basic research standard  Link to other ontologies (e.g. gene ontology) and allows for the linking of HPO data to similar data obtained in e.g. mice and zebrafish  Standard to exchange phenotypic data

It also allows to match phenotypic terms and to perform computational disease models (fuzzy matching) – e.g. Phenomizer.

He mentioned the tool ‘Exomiser’ which assists in prioritising DNA variants based on phenotype (Smedley D, et al. 2015)

Recently developed algorithm: Genomizer.

Future: extending the HPO for common, complex disease

Q (Paul Bray, Utah): how easy is it to enter data in phenomizer, exomizer and genomizer?

A: we develop the algorithms and other groups have developed software for integrating the algorithms in ‘easy to use’ applications, e.g. Phenotips

Q (Willlem H Ouwehand, Cambridge, UK): do you have idea of how many patients have been coded using HPO?

A: HPO do not record who is using their data … but at least 250,000 patients.

Q (Ernest Turro, Cambridge, UK): Second relationships associated to an HPO terms, which we might not be interested in. How can we deal with those?

A: The goal of HPO is to capture as much information as possible, to help with interpretation. We should take this interesting question offline.

Robinson PN & Mundlos S (2010) The human phenotype ontology. Clin Genet 77(6):525-534.

Smedley D, et al. (2015) Next-generation diagnostics and disease-gene discovery with the Exomiser. Nat Protoc 10(12):2004-2015.

Session 4 - What’s new?

Assessing the relevance of non-coding DNA variants

Dominic Seelow, Berlin, Germany

Dominic introduced an algorithm he developed: MutationTaster

Algorithm used to predict the consequence of non-coding variants in protein-coding transcripts (e.g. synonymous, UTR, intronic)

Training of the algorithm is challenging because the number of proven disease-causing mutations in the non-coding space is small, e.g. limited to known variants underlying rare diseases, such as thalassemia, thrombocytopenia and absent radii syndrome.

Other programs:

MutationDistiller:

Algorithm designed to identify potentially causal variants from a VCF input file

RegulationSpotter

Identifies likely regulatory variants affecting candidate genes and outputs information about each variant (e.g. likelihood of being a modifier of histone states, transcription factor binding sites, etc.) ePOSSUM predicts the effect of variants on transcription factor binding sites.

When searching for the consequences of non-coding variants:

 Do not neglect InDels and compound heterozygous mode of inheritance  beware of incomplete penetrance  consider the relationship between genotype and phenotype  share data

New platelet population studies for genomic discovery Andrew Johnson, Boston, USA Population cohort analysis of platelet function.

No large cohort for platelet function studies - largest study to date brought together data from 4,000 individuals with platelet function data.

Uses a variety of techniques: transcriptomics, genome-wide typing, epidemiological approaches

Framingham population study for platelet function using several platelet function tests, including LTA and a plate-based assay (FHS Gen3) – 4,000 participants will be enrolled over a 2 year period.

Aims: to identify a larger number of loci associated with platelet function.

Loci identified may be rare and coding:

Whole exome sequencing of Framingham Heart Study (Eicher JD, et al. 2017)

Common phenotypes – rare and common SNPs – metaanalysis of 157,293 individuals (Eicher JD, et al. 2016)

Introduction to FHS3

Eicher JD, et al. (2017) Whole exome sequencing in the Framingham Heart Study identifies rare variation in HYAL2 that influences platelet aggregation. Thromb Haemost 117(6):1083-1092.

Eicher JD, et al. (2016) Platelet-Related Variants Identified by Exomechip Meta-analysis in 157,293 Individuals. Am J Hum Genet 99(1):40-55.

A fast association test for identifying exonic and non-exonic pathogenic variants involved in rare diseases Ernest Turro, Cambridge, UK

Ernest explained BeviMed, a new Bayesian statistical method for the analysis of whole exome sequencing (WES) or whole genome sequencing (WGS) data to identify possible rare variants underlying inherited diseases.

Desirable properties for such a test:

 Share information across variants to overcome local genetic heterogeneity  Variants need to be classified as pathogenic or benign  Different modes of inheritance  Variable penetrance  Fast and scalable He illustrated the power of the test by analyzing the WGS results from the NIHR BioResource projects in the UK, with a focus on bleeding, thrombotic and platelet disorders (BPD). Four of the 5 Top results are known loci (ANKRD26, RUNX1, MYH9, ACTN1) but it also revealed that variants in GP1BB can cause a dominant inherited macrothrombocytopenia, sometime accompanied by mild bleeding (Sivapalaratnam S, et al. 2017).

BeviMed gives P-value and also tell you which variant will drive the finding. It is faster and scalable, whilst other methods (e.g. SKAT) are not (Greene D. et al, 2017).

Sivapalaratnam S, et al. (2017) Rare variants in GP1BB are responsible for autosomal dominant macrothrombocytopenia. Blood 129(4):520-524.

Greene D, BioResource N, Richardson S, & Turro E (2017) A Fast Association Test for Identifying Pathogenic Variants Involved in Rare Diseases. Am J Hum Genet 101(1):104- 114.

Closing remarks - Michele Lambert, Philadelphia, USA

Michelle summarised the session by highlighting the following three points:

 Sharing of genotype and phenotype data is essential if we wish to resolve the genetic architecture of the BPDs not yet resolved

 The community must adopt standards in the coding of the clinical and laboratory phenotypes by using terms libraries like HPO and in reporting the findings from next generation sequencing (NGS) tests  Collaboration between clinicians at hospitals, experts in genomics, bioinformatics and statistical genomics is essential if we want to bring the benefits of NGS to the bedside.

Annex 1 – List of proposed bleeding, platelet, coagulation and thrombotic disorder Tier 1 reportable genes. MOI: AR – Autosomal Recessive; AD – Autosomal Dominant Gene symbol Gene name Disorder MOI Category Reference (HGNC) (HGNC) ABCG5 ATP binding cassette subfamily Sitosterolemia /macrothrombocytopenia AR platelet Rees DC, et al. 2005: Br J G member 5 (OMIM 210250) Haematol 130(2):297-309. ABCG8 ATP binding cassette subfamily Sitosterolemia /macrothrombocytopenia AR platelet Rees DC, et al. 2005: Br J G member 8 (OMIM 210250) Haematol 130(2):297-309. ADAMTS13 Disintegrin-like metallopeptidase Familial thrombotic thrombocytopenic AR thrombotic Levy GG, et al. 2001: Nature with thrombospondin type 1 purpura, 413(6855):488-494. motif 13 (OMIM 274150) AP3D1 Adaptor- related protein Hermansky-Pudlack syndrome 10 AR platelet Ammann S, et al. 2016: Blood complex 3, Delta-1 subunit (OMIM 617050) 127(8):997-1006. ARPC1B Actin-related protein 2/3 Thrombocytopenia & Immune Deficiency AR platelet Kuijpers TW, et al. 2017: J complex, subunit 1B (no OMIM) Allergy Clin Immunol 140(1):273- 277. COL1A1 Collagen Type I Alpha-1 Ehlers-Danlos syndrome, classic type AD bleeding Pope FM, et al. 1985: J Med (OMIM 130000) Genet 22(6):466-478. COL5A1 Collagen Type V Alpha-1 Ehlers-Danlos syndrome, classic type AD bleeding Nicholls AC, et al. 1996: J Med (OMIM 130000) Genet 33(11):940-946. COL5A2 Collagen Type V Alpha-2 Ehlers-Danlos syndrome, classic type AD bleeding Michalickova K, et al. 1998: (OMIM 130000) Human molecular genetics 7(2):249-255.

Annex 1 – continued

MOI: AR – Autosomal Recessive; AD – Autosomal Dominant

MOI: AR – Gene name Disorder MOI Category Reference Autosomal (HGNC) Recessive; AD – Autosomal DominantGene symbol (HGNC) F12 Coagulation Factor XII Factor XII deficiency AD/AR coagulation Bernardi F, et al. 1987: Blood (OMIM 234000) 69(5):1421-1424. FYB1 (FYB) Fyn Binding Protein Thrombocytopenia 3 AR platelet Levin C, et al. 2015: J Thromb (OMIM 273900) Haemost. 13(7):1285-92. KDSR 3-ketodihydrosphingosine Thrombocytopenia with skin AR platelet Takeichi et al. J. of Investigative reductase hyperkeratosis/ichthyosis Dermatology. 2017 KNG1 Kininogen 1 High Molecular Weight Kininogen AR coagulation Cheung PP, et al. 1993: The Deficiency Journal of biological chemistry (OMIM 228960) 268(31):23361-23365. MPIG6B Megakaryocyte and platelet Thrombocytopenia, anemia and AR platelet Melhem M, et al. 2017: Eur J inhibitory receptor G6B myelofibrosis Haematol 98(3):218-227. (OMIM 617441) TPM4 Tropomyosin 4 Macrothrombocytopenia AD platelet Pleines I, et al. 2017: The Journal of clinical investigation 127(3):814-829. PTPN11 Protein-Tyrosine Noonan Syndrome AD bleeding Tartaglia M, et al. 2002: Am J Phosphatase, Non- (OMIM 163950) Hum Genet 70(6):1555-1563. Receptor-Type 11 RNU4ATAC Small Nuclear RNA U4atac Roifman Syndrome AR platelet Merico D, et al. 2015: Nat (thrombocytopenia / primary immune Commun 6:8718. disorder) (OMIM 616651) SLFN14 Schlafen Family Member Bleeding Disorder, platelet-type, 20 AD platelet Fletcher SJ, et al. 2015: The 14 (OMIM 616913) Journal of clinical investigation 125(9):3600-3605.

Annex 2 – List of Tier 1 genes with multiple modes of inheritance (MOI)

MOI: AR – Autosomal Recessive; AD – Autosomal Dominant

Gene symbol Disorder OMIM MOI Reference for alternative MOI (HGNC) ITGA2B Glanzmann Thrombasthenia 273800 AR ITGA2B Platelet-type bleeding disorder 16 187800 AD Peyruchaud O, et al. 1998: Blood 92(11):4178-4187. ITGB3 Glanzmann Thrombasthenia 273800 AR ITGB3 Platelet-type bleeding disorder 16 187800 AD Ghevaert C, et al. 2008: Blood 111(7):3407-3414. GP1BA Bernard Soulier syndrome type A1 231200 AR GP1BA Bernard Soulier syndrome type A2 153870 AD Savoia A, et al. 2001: Blood 97(5):1330-1335. GP1BA Platelet-type von Willebrand disease 177820 AD Miller JL, et al. 1991: Proc. Nat. Acad. Sci. 88(11):4761-4765. GP1BB Bernard Soulier syndrome type B 231200 AR GP1BB Macrothrombocytopenia none AD Sivapalaratnam S, et al. 2017: Blood 129(4):520-524. 188025 FLI1 Paris-Trousseau and Jacobsen syndrome AD 147791 FLI1 Platelet-type bleeding disorder 21 617433 AD Stockley J, et al. 2013: Blood 122(25):4090-4093. FLI1 Platelet-type bleeding disorder 21 617433 AR Stevenson WS, et al. 2015: Blood 126(17):2027-2030. GFI1B Platelet-type bleeding disorder 17 187900 AD GFI1B Alpha-delta storage pool disease none AR Ferreira CR, et al. 2017: Mol Genet Metab 120(3):288-294.

Hemostasis and Malignancy

8 July 2017 14:00 – 18:15 Chairman: Marc Carrier

Co-Chairs: Cihan Ay, Guy Meyer, Christophe Dubois, Casey O Donnell, Nigel Mackman and Simon Noble Education Session:

1) Intracranial Hemorrhage in Cancer Patients (Jeffrey Zwicker, United State)

Dr. Zicker provided an excellent presentation on the risk of ICH among cancer patients receiving anticoagulant therapy (parenteral or oral). A number of knowledge gaps were identified. Specifically, lack of standardizations in the diagnosis (e.g. diagnostic modality, size, etc.) and definitions of expansion (and how to quantify) are important issues for this clinically important topic.

Action: Dr. Zwicker to explore the elaboration of a SSC Guidance statement on this issue (term July 2018).

2) Mice Model of Thrombosis and Cancer (Christophe Dubois, France)

Dr. Dubois delivered an elegant review of the literature on the pearls and pitfalls of the different mice models of cancer-associated thrombosis (CAT). During the question period, Dr. Mackman brought up the significant heterogeneity around the use of the different mice models. It was discussed that a SSC statement might be desirable to guide scientist on which model to use depending on the setting and planned experiments.

Action: Dr. Dubois to follow-up with Dr. Mackman on the logistics of a potential SSC guidance on this issue

SSC Committee work:

1) New Guidance: Occult Cancer Detection in Patients With VTE (Aurélien Delluc, France)

Dr. Delluc reviewed the statements of the recently accepted SSC guidance document on occult cancer screening for patients with VTE. Other co-authors: Delluc A, Antic D, Lecumberri R, Ay C, Meyer G, Carrier M. Committee used data from recently published trials and an IPDMA (Dr. Nick van Es)

Actions: Review proofs (Now done)

Guidance published in JTH: J Thromb Haemost. 2017 Oct;15(10):2076-2079.

2) Developing a Tool to Assess QoL in Patients With CAT (Simon Noble, United Kingdom)

Dr. Noble provided a presentation of qualitative studies in cancer associated thrombosis as well as an update on his QoL in Patients with CAT too. The second PELICAN Study (co-PI Agnes Lee) will be recently completed and once analyzed will be used to derive the QoL tool and provide guidance to clinicians. The first meeting of the QoL is expected to occur in the end of 2017. New members have shown interest in participating to the project including Dr. Casey O Donnell. Other interested participants to contact Dr. Noble directly

Actions: First meeting of SSC subgroup before the end of the year.

3) D-Dimer Use in Cancer Patients (Speaker: Cihan Ay, Austria)

Dr. Ay presented the final recommendations to be included within the SSC Guidance on the use of D-dimer in Cancer patients (Stratification for risk of CAT’ and use for CAT diagnosis, etc.)

Actions: Manuscript to be submitted by early 2018

Future Committee Projects

1) Update of Guidance: Management of Thrombocytopenia in CAT (Bethany Samuelson, United States)

Dr. Samuelson is a young investigator from Seattle US. She presented and highlighted that a large number of studies assessing the management of thrombocytopenia among cancer patients requiring anticoagulation have been published since the publication of our SSC guidance on challenging cases in CAT patients (2013). Dr. Samuelson proposed to update the Guidance statement on the management of thrombocytopenia in this patient population.

Actions: i) Participants: A lee, A Khorana, Simon Noble, Marc Carrier, Jeff Zwicker, Bethany Samuelson ii) Submit proposal to writing committee in September 2017 iii) First meeting in September 2017

2) Update of Guidance: New Insights on DOACs Use in CAT (Alok Khorana, US)

Dr. Khorana summarized the new DOAC data to manage CAT. Two RCTs comparing LMWH to DOACs are expected to be presented in December 2017. Clinicians will need to have guidance on which agent to use (parenteral or DOAC) depending on different clinical scenario. It was felt that an update of the Guidance Document on the use of DOAC for CAT would be desirable in early 2018 Actions: Form a working group and plan for a first meeting in early 2018

3) New Guidance: Anticoagulation for Atrial Fibrillation While Undergoing Chemotherapy (Simon Noble, United Kingdom)

Dr. Noble presented on the management of anticoagulation among cancer patients with atrial fibrillation. He highlighted the paucity and data and the importance to provide

clinicians with Guidance on the type and dose of anticoagulants (e.g. parenteral vs. oral; dosing; etc.).

Actions: Dr. Delluc and Noble to meet to establish a working group and send a proposal to the writing committee for early 2018

Lupus Anticoagulant/Antiphospholipid Antibodies

8 July 2017 9:00 – 13:15

Chairman: Bas De Laat Co-Chairs: Maria Laura Bertolaccini, Katrien Devreese, Doruk Erkan, Masahiro Ieko, Rolf T. Urbanus, Denis G. Wahl

The session was chaired by Bas de Laat and Katrien Devreese

Dr. Bas de Laat opened the session. Dr. Devreese first presented an update of the multicenter study on solid phase assays. Because the latest systematic review on the added value of IgM aPL in the classification of APS patients did not provide a clear answer, she aimed to determine the added value of IgM antibodies in the prediction of thrombosis and pregnancy morbidity and compared IgG and IgM assays from different providers, both automated and solid phase assays. In total, about 1400 samples of 8 different centers (including APS patients with thrombosis and pregnancy complications, control diseased patients, autoimmune disease patients and healthy controls) were tested in 4 different assays in one central laboratory and according to the latest SSC guidelines. The classification of the patients was based on the original center. Furthermore, the added role of anti-domain I, a new TG-based assay, IgA and aPS/PT antibodies will be investigated in the same population. The choice of the assays depended on which were most frequently used based on ECAT reports, and the willingness of the manufacturers to participate. Manufacturer’s cutoff values for all tested platforms were confirmed in 20 normals. Results will be presented by W. Chayouâ.

Dr. Devreese continued on the importance of the determination of cut-off values. In the multicenter study, clear differences were found if the manufacturer’s cut-off was used (upon verification in 20 normals) versus the determination of the 99th percentile in a population of 120 to 200 normals. Furthermore, cut-off values depended on the method of outlier detection, and the execution of a box-cox transformation. Together with the ISTH, Dr. Devreese sent a questionnaire to all SSC and ECAT members to have an idea what is done regarding cut-off values determination in daily practice. 139 answers from all over the world were obtained. About 41% of the centers calculated in-house cut-off values, of which about 54% uses at least 120 normals. Regarding the statistical method, 42% uses a parametric method (mean + x.stdev) and 58% uses a non-parametric method (95th or 99th percentile). Looking at the exclusion of outliers, 38.5% doesn’t check for outliers, 28% determines them visually and the remaining uses variants of Tukey or the Reed method. About 30% checks the calculated cut-off values by a clinical approach and 72% of these adapt the cut-off value using sensitivity, specificity or odds ratio values. About 90% of the centers agree with the alternative method to determine interlaboratory cut-off values by users of identical devices and with the same population characteristics. In a previous study of Dr. Devreese she demonstrated that the inclusion of larger numbers of normals results in lower cut-offs with more strict confidence intervals. She concluded that most of the labs feel the need for uniformity in the determination of cut-off values. During the questions, Dr. Devreese mentioned that most of the labs do not discriminate between age in cut-off determinations, but some indicated the need for specific cut-offs for children.

W. Chayouâ presented the data of the multicenter study, regarding the agreement between different platforms of aCL and aβ2GPI IgG and IgM antibodies as well as their correlation with

thrombosis and pregnancy morbidity. Using kappa agreement, he demonstrated a good to very good agreement between all corresponding platforms, except for aCL IgM (moderate agreement). Given the high overlap between aCL and aβ2GPI, for some platforms it may be enough to measure only aCL or only aβ2GPI antibodies. Despite the good correlation, a large variability between the platforms was shown in the number of all aPL positive patients in the thrombotic as well as the obstetric population. Data obtained in the study (all tested in Ghent University hospital) would lead to a reclassification of a significant number of patients compared to the classification of the original center. The OR for thrombosis and pregnancy morbidity varied per platform, and the detection of aPL proved to be especially useful for the risk stratification in LAC positive patients. Importantly, despite earlier suggestions in literature, also triple positivity was found to be platform dependent. Analysis of the importance of IgM and IgA aPL, as well as anti-domain I, anti-PS/PT and a thrombin generation based assay is currently ongoing.

Dr. Belizna presented her work on Hydroxychloroquine (HCQ) for secondary prevention of relapses of primary APS. Based on several preclinical and clinical data and among them the results of their own group, the orphan designation of HCQ in APS has been granted to the University Hospital Angers by the European Commission (EC) on January 2017. A national French trial leaded by their team, the PHRC PAPIRUS, on the use of HCQ for prevention of thrombotic relapses in primary APS has been granted and will start in the coming months. Based on PAPIRUS, they have proposed an international trial for the use of HCQ for preventing thrombotic and obstetric relapses. This trial, HIBISCUS, obtained the scientific advice from the EC on May 2017. An international consortium has been created by members of Europhospholipid in order to perform this double blind randomized versus placebo multicenter trial, HIBISCUS: HCQ for the prevention of thrombotic and obstetric relapses in primary APS (incident and prevalent cases). The adhesion is open for other teams willing to join them. If interested, please contact [email protected] and/or [email protected].

Dr. Oku elaborated on a new subset for antiphospholipid antibodies for the diagnosis of APS. Most of the aβ2GPI antibodies are also detected by aCL assays. Single aCL are often considered not specific nor pathogenic. Dr. Oku described results from a human derived IgG antibody EV35102, produced by immortalizing B lymphocytes from APS patients with an EBV infection. Interestingly, this antibody proved to specifically bind to β2GPI, but only when coupled to CL. As a consequence, none of the ELISAs detecting aβ2GPI IgG antibodies (exposing either β2GPI in the open or closed conformation), recognized the antibody. Furthermore, the antibody tested positive in an anti-domain I assay but did not display LAC activity. EV35102 induced TF expression in human monocytes, suggesting the antibody may be pathogenic. Interestingly, the antibody profile aCL positive and aβ2GPI negative was also found in 20 out of 182 APS patients, of which 4 patients were also positive for anti-domain I. These data suggest the presence of a new pathogenic subset of antibodies in APS patients, with reactivity towards domain I of β2GPI. As these antibodies are not recognized by aβ2GPI assays, aCL assays remain useful for the screening of this subset of antibodies.

Dr. Kumano discussed about the usefulness of the APTT mixing test for the diagnosis of LAC. So far, the ISTH suggests the mixing test is useful to distinguish an inhibitor pattern from a factor deficiency pattern. His study aimed to differentiate LAC from other coagulation inhibitors and factor deficiencies by creating new formulas for the mixing test. The index of circulating

anticoagulant (ICA) values were calculated from the clotting times without incubation (ICAi) and after 2h incubation (ICAd) in LAC positive samples, samples with factor deficiency or with unfractionated heparin. Interestingly, both the ratio and difference between ICAi and ICAd resulted in a good Area under the curve in a ROC analysis, and a good sensitivity (93.8%) and specificity (98%). The new formula can therefore differentiate well between the presence of LAC, factor deficiency and other coagulation inhibitors, and may be useful to add to the guidelines. So far, they did not measure samples in which both a LAC and a factor deficiency are present, but this is interesting for the future.

Dr. Moore continued with the debate on the mixing test. He questioned in which conditions mixing tests are necessary and investigated the reliability of the test. Although it is possible to detect LA without mixing, in some cases mixing studies are required. In the presence of a strong antibody, a strong LAC is visible in the screening, which also remains positive in the confirm, because of which the mixing test is necessary to reduce false positives. In the mixing test, we have to be aware of the dilution effect, which may lead to false negative results. We also have to keep in mind that not all normal pooled plasmas are the same, which may lead to false results if higher/lower than normal. Importantly, even if the same reagents and devices are used, a large variability is found for cut-off values, illustrating the need to determine local cut-off values. The test can be improved by using a reference interval of the undiluted plasma, resulting in lower cut-off values. A mixing test specific cut-off proved to be more sensitive than an ICA. If ratios are reported, make sure to use the reference interval mean to exclude false positive or negative results.

Due to technical problems, the remaining two presentations were delayed to the second part of the SSC program.

Dr. de Laat opened the educational session and discussed about antibody-antigen structures to diagnose APS patients. He went back in time to explain the discovery of the pathogenic epitope located on domain I of β2GPI. This epitope, highly conserved in different species, only becomes available after a conformation change of the protein induced by binding of domain V to a negative surface such as phospholipids. In both a single and multicenter study, the presence of these anti-domain I antibodies proved to be associated with thrombosis, illustrating their pathogenicity. 2 patient-derived antibodies (P1-117 and P2-6) proved useful in the discrimination of β2GPI exposing the pathogenic domain I epitope and its native closed counterpart. A huge variation in the exposure of the domain I epitope in commercially available anti-β2GPI assays was shown using these antibodies, with clinical implications (considerable number of APS patients is missed in assays with a low exposure.) Using small angle X-ray scattering, β2GPI in open and closed conformation was visualized, as well as the complexes of β2GPI with the two patient-derived antibodies. By looking at the conformation of β2GPI and the interaction sites with pathogenic antibodies, we may be able to develop more specific assays for APS, resulting in a better treatment.

Dr. Erkan discussed the clinical challenges an APS patient is faced with. A first challenge lies within the name: antiphospho what? Also, the definition of aPL is confusing for the patient, given the different aPL profiles and problems of over and underdiagnosis. Thirdly, the definition of APS is confusing, with the contradiction between classification and diagnostic criteria. They are working on updated APS classification criteria, and need the help of the SSC. Increased

awareness and education of patients and doctors are necessary; any congress on APS should include a session for patients. Additionally, controversies exist on the management of APS (INR; PAPS versus CAPS, no medication versus LMWH/HCQ, elimination of other risk factors). Also, what is found on the internet is confusing. The highest scoring quality websites do not rank accordingly on search engines. Lastly, patient’s perspectives are not clear. The APS foundation of America asked patients for their story. Main conclusions were that their hematologists don’t know APS and that APS is difficult to manage (at first stable INR but after a while all medication stops to work). Fear and hope is present among patients. Both physicians and patients should be educated as much as possible, by improving collaboration.

Subsequently, the two presentations of the morning session were given. Dr. Cohen shortly introduced the RISAPS study, a trial on Rivaroxaban versus Warfarin for stroke patients with APS, with or without SLE. This study was initiated after the RASP trial, showing that Rivaroxaban is at least as good as warfarin in APS patients with stroke. RISAPS is a randomized controlled phase 2/3 noninferiority trial, composed of 140 patients. Rivaroxaban (15 mg, twice daily) is compared to a standard care warfarin (INR 3.5). Primary outcome is the ischemic damage after 2 years assessed by MRI. Other neurological markers, clinical efficacy and safety are considered as secondary outcome.

Dr. Pengo provided us with an update on the treatment of APS patients with DOACs. DOACs are an option to treat APS patients as they are at least as efficient as warfarin to prevent venous and arterial thrombosis and reduce cerebral bleeding. The usage of DOACs in APS patients is not encouraged today as there is no clear evidence of the efficacy nor safety. Additionally, the single randomized trial RAPS used a laboratory surrogate endpoint (ETP) and case reports as well as case series are conflicting. In 4 of 6 case reports, a recurrence of thrombosis was observed. In two case series, characterized by the same number of patients, the same DOAC and usage, completely different results were obtained: 100% versus 0% recurrence of thrombosis. Different trials are ongoing today, such as the ASTRO-APS (prospective randomized warfarin – apixaban; results expected December 2019); a trial comparing Rivaroxaban versus Acenocoumarol in APS patients, the RISAPS and the TRAPS (113 triple positive patients randomized warfarin – Rivaroxaban).

Dr. Kremers discussed about the standardization of thrombin generation (TG) assays and APS. TG assays may be useful in APS, as previously a prolongation of the lag time (LT) and time to peak was demonstrated (in line with a prolonged aPTT), as well as a resistance to activated protein C. Studies in her laboratory demonstrated a prolonged LT and increased peak in APS patients compared to healthy controls. APS patients with thrombosis were characterized by an increased peak and ETP compared to those without thrombosis. By fine tuning the TG assay (thrombin dynamics), she additionally demonstrated an increased prothrombin conversion in APS patients compared to healthy controls, while the thrombin decay capacity was comparable. Taken together, a TG assay may be beneficial in a clinical setting but so far TG has only been used in research. Standardization of the method may help to improve its use in diagnosis. The usage of different TG methods, different sources and concentrations of reagents and temperature issues within the assay remain important problems to tackle. Dr. Kremers suggests to start a working party on TG, that should inventarize the current TG methods used, as well as source/concentration of reagents by a questionnaire. This should enable us to tailor the methodology to the patient group, fine tune the reagents and investigate which of the TG parameters are most interesting. The questionnaire will be send to the SSC members in 1 to 2 months.

Dr. Wahl continued on the TG characteristics in APS according to antiphospholipid antibody profile. Their study aimed to find a relationship between the APC resistance determined by TG and the antiphospholipid profile. In a prospective multicenter cohort (N=137 women), APC resistance was measured using TG by adding APC. APC resistance resulted in an increased risk of thrombosis (HR=6.07) and the thrombotic risk was correlated with the strength of the resistance. Additionally, aCL, aβ2GPI and anti-domain I IgG were significant predictors of thrombosis. Interestingly, the higher the IgG concentration of these antibodies, the higher the APC resistance. No correlation was found for IgM or IgA which were also no significant predictors of thrombosis. In conclusion, new functional tests (APC resistance by TG) and specific antibodies (eg anti-domain I) seem promising for the risk stratification in APS patients.

Dr. de Groot received the difficult task to elaborate on what we don’t know about APS. Many unknowns exist in the clinic (is APS a unique pathology; thrombotic versus obstetric APS; when does disease start; is there a second hit; long term prognosis for recurrent thrombosis/ for asymptomatic patients) and the biology of the disease (one or more epitopes; target = cells or proteins?; APS in the absence of antibodies?; need of better animal models). Importantly, we don’t have a single assay that helps to understand the increased thrombotic risk: LA (prolongation of clotting time normally indicates bleeding risk); aCL (marker for infections?); aβ2GPI (a protein that you can miss); anti-PT (inhibition of prothrombin should protect). Why do we develop the antibodies so often? Is it infection-related? What determines the transition from transient to persistent? What is the relation between the titer of the antibody and the risk? Disappearance of antibody equals disappearance of the risk? So far there is no solid evidence that transient antibodies are not pathogenic, or that we need two positive measurements of aPL. Dr. de Groot ends that he personally thinks that our knowledge did not evolve significantly the last 17 years and encourages us all to do better.

Dr. Rand closed the session by showing results on APS-mediated complement activation. His group visualized the exposure of APS patient plasma to phospholipid vesicles by SEM and significant differences were found compared to healthy plasma. The question remains if the observed immune complexes are able to activate the complement sytem. Indirect evidence for the importance of complement activation in APS comes from animal models and CAPS patients, in which complement inhibition proved to work. They now developed a two-stage approach in which APS plasma pre-incubated with phospholipid vesicles generated significantly higher levels of C5a than patients with cancer, SLE, or VTE. In conclusion, the exposure of phospholipid vesicles results in complex formation with aPL, that consequently can activate complement. The two-stage assay can be used to monitor complement activation in APS patients.

Pediatric and Neonatal Hemostasis and Thrombosis

8 July 2017 14:00 – 18:15

Chairman: Christoph Male Co-Chair: Anthony K. Chan, Fiona Newall, Shoshana Revel-Vilk, Heleen Van Ommen, Manuela Albisetti, Sarah O’Brian

The focus of the Subcommittee is to address issues 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 reports and recommendations relating to patient care for the pediatric population. Work within the SSC is organized through project-related working groups lead by one of the Co-chairs.

1. Administrative issues: Two co-chairs (Leonardo Brandao, Neil Goldenberg) stepped down end of 2016. Christoph Male was appointed chairman in July 2016. Dr. Sarah O’Brian (US) and Dr. Manuela Albisetti (CH) have been appointed for new co-chair positions in early 2017. The new co-chair team has met by TC and, apart from the public SSC meeting, had a face-to- face meeting on 08-July-2017 to discuss ongoing and future projects. Anthony Chan will step down as co-chair in 2017.

2. Educational session:

Post-Thrombotic Syndrome in Children Speaker: Maria Laura Avila, Canada

Therapeutic Options for Post-Thrombotic Syndrome Speaker: Oliver Schlager, Austria

Primary Prevention of CVC-Related VTE Speaker: Sarah O'Brien, United States

Diagnostic Imaging of VTE in Vascular Abnormalities Speaker: Leonardo Brandão, Canada

3. Communications/reports:

SSC position paper: Recommendations for risk factor definitions in pediatric hospital-acquired venous thromboembolism (HA-VTE) to inform future prevention trials: Communication from the SSC of the ISTH. Brian R. Branchford, Arash Mahajerin, Leslie Raffini, Elizabeth Chalmers, C. Heleen van Ommen, A.K.C. Chan, and Neil A. Goldenberg. The manuscript has currently been resubmitted to JTH after revisions.

SSC position paper: Recommendations for future research and standardization of definitions in relation to pediatric pulmonary embolism: Communication from the SSC of the ISTH Tina.T. Biss, Suzan William, C. Heleen Van Ommen, Anthony K.C. Chan, Neil A. Goldenberg and Madvi Rajpurkar. The manuscript has passed SSC co-chair review and has recently been submitted to JTH.

4. Ongoing Projects: a. VTE Risk Factors/Models and Prevention Responsible co-chair: Neil Goldenberg Working group: B. Branchford, A. Mahajerin, L. Raffini, E. Chalmers, H. van Ommen, A. Chan

Publications:  Hospital-associated venous thromboembolism in pediatrics: a systematic review and meta-analysis of risk factors and risk-assessment models Arash Mahajerin, Brian R. Branchford, Ernest K. Amankwah, Leslie Raffini, Elizabeth Chalmers, C. Heleen van Ommen, Neil A. Goldenberg Haematologica August 2015 100: 1045-1050  SSC position paper (Branchford et al.; see above).

b. Management of pulmonary embolism Responsible co-chair: Neil Goldenberg, Working group: Madhvi Rajpurkar, Tina Biss, Heleen van Ommen Progress report at current SSC meeting presented by Tina Biss.

Publications:  Pulmonary embolism and in situ pulmonary artery thrombosis in Paediatrics - a systematic review Madhvi Rajpurkar; Tina T. Biss; Ernest K. Amankwah; Denise Martinez; Suzan Williams; C. Heleen van Ommen; Neil A. Goldenberg. Thromb Haemost 2017; 117: 1199–1207  SSC position paper (Biss et al; see above).

c. Catheter-related arterial thrombosis Responsible co-chair: Neil Goldenberg, Manuela Albisetti Working group: Mariana Bonduel, Shoshana Revel-Vilk, Mattia Rizzi Progress report was presented at current SSC meeting by M. Albisetti.  Systematic review (Mattia Rizzi et al.): manuscript anticipated for summer 2017  SSC position paper (Manuela Albisetti et al.): analysis completed; manuscript anticipated for Q3 2017

d. Diagnostic criteria for venous thrombosis in children Responsible Co-chair: L. Brandao Working group: J. Journeycake, C. Male, A. Chan, R. Krishnamurthy, C. Loewe

The project targets four areas: a. Central vein thrombosis b. Pulmonary embolisms c. Cerebral venous thrombosis d. Imaging of VTE in Vascular Abnormalities

The fourth topic was covered by an educational presentation at the current SSC meeting (see above). All topics are planned to be covered by separate but related clinical guidance papers, currently being drafted under the lead of L. Brandao.

e. DIC Survey Responsible co-chair: A. Chan Working group: Revathi Rajagopal (lead), Paul Monagle, Ziad Solh Collaboration between pediatric SSC and DIC SSC (chair: Jecko Thachil) Survey has been completed (2016-09) with about approximately 250 respondents. The results were presented at the current SSC meeting. A manuscript is in preparation.

f. Registry of congenital antithrombin deficiency with a focus on genotype and phenotype correlation Responsible co-chair: A. Chan Working group: Riten Kumar (lead), Suzan Williams, Mike Tarantino, Vicky Price  Completed developing the RedCap database for the study; received IRB approval at Nationwide Children’s Hospital  received a grant from ISTH for the pilot validation  next steps are to get IRB approval at participating centers and additional grant support

g. Registry of Congenital Severe Purpura Fulminans Responsible co-chair: L. Brandao Working group: Vicky Price, Adrian Minford, Paul Monagle, Progress Presented at SSC meeting in 2016 Received ISTH grant ~ 6,000 CAD Red cap database established; website open since early 2017 [cases already identified in Saudi Arabia, China, Brazil, US (Indiana), and Canada (Calgary)]

h. Guidance paper on Catheter-related thrombosis prevention and treatment in the pediatric population Responsible co-chair: A. Chan Working group: Ketan Kulkarni (lead), Leonardo Brandao, Paul Monagle, Vince Faustino, Christoph Male Activities: systematic review in progress

i. Development of Standards for Bleeding Disorder Clinics for Adolescent Females

Responsible co-chair: S. Revel-Vilk Working group: Ayesha Zia (lead), Meera Chitlur, Susan Halimeh Collaboration between pediatric SSC and women health SSC. Activities: Protocol for international panel study on "Setting standards for appropriate and necessary care for young women with heavy menstrual bleeding and potential bleeding disorder" completed. Currently seeking research support Guidance communication on "Development of bleeding disorder clinics in adolescent females" is planned. Progress report at current SSC meeting by Meera Chitlur.

j. Anticoagulation and monitoring in extracorporal circulation (ECMO, VAD) Responsible co-chair: Heleen van Ommen

Working group: Meera Chitlur, P. Monagle, P. Massicotte, C. Neunert, T. Nguyen, Lisa Bomgaars.

Progress report at current SSC meeting by Meera Chitlur. Collaboration with ECMO/VAD interest group (chair: Jun Teruya) of the Perioperative Hemostasis SSC. The pediatric ECMO-VAD Anticoagulation working group will be separate because neonatal ECMO and VAD’s are a distinct entities. A meeting of the overall group took place on July 10 at the ISTH conference in Berlin. Planned activities: - survey among participating centres on ECMO/VAD and anticoagulation protocols - define key issues essential to comparing anticoagulation protocols - literature review - white paper

k. International Paediatric Thrombosis Network Responsible co-chair: H. van Ommen Working group: Manuela Albisetti, Mihir Batt, Suzanne Holzhauer, Christoph Male, Paul Monagle, Heleen van Ommen, Shoshana Revel-Vilk, Ulrike Nowak-Göttl, Elizabeth Chalmers Goal: Collaborative research on epidemiology, diagnosis, management of thrombosis, including interventional studies on new treatments. Activities: charta in preparation, invitation of other SSC members to participate, develop registry of paediatric thrombosis cases using REDcap database; definition of core data set; obtain funding.

l. Management of VTE detected incidentally Responsible co-chair: Shoshana Revel-Vilk Working group: Anjali Sharathkumar (lead), Sanjay Ahuja, Tina Biss, Victoria Price, Matt Regan Goal: Define epidemiology, significance, outcome of incidental VTE; define optimal management Activities: systematic review in progress; clinical guidance paper planned

m. Definition, Promotion, and Measurement of Adherence to Pediatric Anticoagulant Therapies Responsible co-chair: Fiona Newall A pilot study will be undertaken investigating self-reported adherence to anticoagulant therapy in one large paediatric anticoagulation service. Data will be collected not only on the self- (or parent-) reported rates of adherence to anticoagulant therapy, but also the feasibility and validity of self-report adherence tools in this cohort. Data will be presented at the next ISTH SSC meeting in Dublin.

n. Task force for pediatric anticoagulant drug development Responsible chair: Christoph Male Working group: P. Monagle, A. Chan, H. van Ommen, T. Biss; more SSC members to be invited Goal: to ensure that pediatric developments of various new anticoagulant drugs target paediatric needs and are feasible; liase with all concerned stakeholders (health care professionals, patient groups, regulatory agencies, pharmaceutical companies) Activities: position paper; develop platform for communication with stakeholders.

Perioperative Thrombosis and Hemostasis

8 July 2017 14:00 – 18:15

Chairman: C. Marc Samama

Co-Chair: James Douketis, Pierre Albaladejo, Andreas Greinacher, Jerrold Levy, Alex Spyropoulos, Beverley Hunt

The session took place on Saturday, July 8th, 2017, in room A8, Berlin City Cube. The room was packed with around 800 attendees during the two first sessions and, at least, 500 people during the last part of the programme.

The education session (1 hour) was dedicated to venous thromboembolism (VTE) prevention Two lectures were proposed: In-patients. Beverley Hunt (London, UK) made a global overview of the VTE risk. She emphasized the need for risk assessment protocols and she demonstrated the benefit of the implementation of such protocols in the UK

Day surgery and fast-track surgery patients: Marc Samama (Paris, France) showed the recorded decrease of the VTE risk in fast-track and ambulatory procedures. He presented the new ESA driven European Guidelines on VTE prophylaxis and especially he focused on fast- track and day surgery, aspirin and mechanical prophylaxis.

Jim Douketis (Hamilton, Canada) chaired the first part of the business session (1 hour) dedicated to Hemostasis and Thrombosis in the Perioperative Setting Pharmacokinetic" vs "laboratory" based strategy on management of OACs in elective procedures. Alex Spyropoulos, (Northwell, USA) using large published studies and evidence- based data advocated the supremacy of pharmacokinetics over biology for the management of oral anticoagulants in elective procedures.

Bridging for VKA and DOACs : new data. Jim Douketis presented the last available data on the lack of benefit of bridging oral anticoagulants (especially for non-valvular atrial fibrillation patients, and the future directions of research in the field

Use of antidotes for surgical patients Marc Samama took over Prof. Albaladejo’s lecture because he wasn’t able to attend the meeting. He first questioned the need for antidotes in patients treated with oral anticoagulants. Then he reviewed the evidence with non-specific agents and he critically focused on the specific antidotes and their limits.

After the break, the second part of the business session was chaired by Marc Samama

Platelet transfusion in patients with acquired platelet defects. Andreas Greinacher (Greifswald, Germany) proposed different ways of using platelet transfusion in patients treated with antiplatelet agents. He especially reported the results of a study transfusing platelets preemptively.

Use of factor concentrates for managing bleeding. Jerrold Levy (Durham, USA) reviewed the evidence on the use of fibrinogen concentrates, prothrombin complex concentrates and factor VIIa in massively bleeding patients.

Utility of TEG/ROTEM for monitoring? Sophie Susen (Lille, France) demonstrated how an haematologist may comply with viscoelastic methods. She underlined the benefits of these methods while reporting the data from recent large studies and she questioned several limits.

As a global evaluation of this session (education + business) the feedback from the audience was very positive, leading the SSC to re-schedule several topics next year in Dublin and to keep one part of the business section to be dedicated to a special topic, namely ECMO/VAD

Plasma Coagulation Inhibitors

8 July 2017 14:00 – 18:15

Chairman: Richard A. Marlar

Co-Chair: Herm-Jan Brinkman, Cecilia Guillermo, Ian Jennings, Jun Teruya, Hiroko Tsuda

Educational Session:

Unique non-anticoagulant functions of protein C Laurent O. Mosnier, PhD (USA) Activated protein C (APC) is a multifunctional protease and provides natural anticoagulant functions by inactivating the coagulation cofactors, factor Va and factor VIIIa. In addition to its natural anticoagulant functions, APC conveys direct effects on cells that involve multiple receptors including protease-activated receptor (PAR) 1 and the endothelial protein C receptor (EPCR). Depending on the cell type and cell stress involved, these unique non-anticoagulant functions of APC, collectively referred to as “APC’s cytoprotective activities,” can be characterized as anti-apoptotic and anti-inflammatory activity, beneficial alterations of gene expression profiles, protection of endothelial barrier function, and regenerative activities.

Numerous studies have shown beneficial effects of activated protein C (APC) in rodent injury and disease models. The use of molecular engineered APC variants with altered selectivity profiles to rodent stroke models, such as the 3K3A-APC and 5A-APC variants with minimal anticoagulant activity but normal cytoprotective activities, demonstrated that the beneficial effects of APC primarily require its cytoprotective activities but not its anticoagulant activities. In addition to applications of APC and APC variants as a therapeutic strategy, there is increasing evidence that the also the endogenous cytoprotective protein C pathway provides important protective effects in normal physiology and pathophysiology.

Recent insights into the molecular mechanisms for APC’s cytoprotective activities identified non-canonical PAR1 activation by APC that give rise to novel tethered-ligands capable of inducing biased cytoprotective signaling as a key distinction from the traditional canonical thrombin-mediated PAR1 signaling that generally results in prohemostatic and proinflammatory effects. Based on the current available data, the consensus model for APC’s cytoprotective activities involves the binding of APC to the endothelial protein C receptor (EPCR) which permits APC-mediated non-canonical activation of PAR1 at Arg46 resulting in the initiation of biased signaling and the corresponding cytoprotective effects. In addition, it has become clear that additional receptors including ApoER2, MAC1, PAR2, PAR3, Tie2, factor V, and protein S

are involved to provide cell-type specific variations to this model, but their integration into the overall precipitation of APC’s cytoprotective activities remains incompletely understood.

Tissue factor pathway inhibitor: New insights in an old inhibitor

E. Castoldi (The Netherlands)

Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type protease inhibitor present on the endothelium, in plasma and in platelets. The plasma pool accounts for ~10% of all TFPI and consists entirely of the TFPIá isoform, which is found in the circulation with various degrees of C-terminal truncation. Only the full-length form, which circulates in complex with factor V (FV) and/or protein S, expresses full anticoagulant activity.

TFPIá is best known for its ability to inhibit tissue factor/factor VIIa and factor Xa (FXa) via its Kunitz domains 1 and 2, respectively. However, recent work has uncovered a role for full-length TFPIá in the regulation of FV activation and prothrombinase activity as well, which makes TFPIá an all-round regulator of the initiation of coagulation. In addition, protein S and FV have been shown to act as (synergistic) cofactors of TFPIá in the inhibition of FXa.

Variations in the levels of plasma TFPIá have been associated with the risk of venous thrombosis and bleeding. In particular, an ~10-fold increase in plasma TFPIá levels has been recently shown to underlie the previously unexplained East Texas and Amsterdam bleeding disorders. Interestingly, the mutations responsible for these disorders reside in the F5 gene (rather than in the TFPI gene) and act by up-reguating FV splicing isoforms (FV-short) that bind TFPIá with high affinity.

This lecture will review these novel findings with an emphasis on the functional significance of the FV-TFPIá interaction.

Working Session #1

Investigation into discrepancies in Protein S activity assay results

Ian Jennings

Aim of Project: To elucidate the cause of marked differences between PS activity results obtained with different manufacturers kits, and observed in multiple EQA program distributions. The protein S (PS) activity assay has been shown in External Quality Control studies to have significant variation among laboratories and these issues extend to patient results as well. This study will attempt to elucidate the causes of the variation observed in the clinical PS activity assays. Up to 40 laboratories will participate in this study. The design of the study has been completed. The difficulty was obtaining the appropriate samples for distribution to the participating laboratories. However, normal and protein S deficient individuals have been identified and obtaining sufficient plasma for the proposed studies is in progress. Both

lyophilized and frozen plasma will be distributed in the fall of 2017. The results will be analyzed during the spring of 2017. It is anticipated to have the data by the next meeting in Ireland.

Investigation into racial differences in genetic risk factor (AT, PC, PS) for venous thrombosis

Hiroko Tsuda (Japan).

Factor V Leiden and prothrombin G20210A, well-known hereditary thrombophilia in Caucasians, are not found in Asians, Africans and Australoid. Protein S (PS) Tokushima (PS p.Lys196Glu) and two protein C (PC) gene variants (PC p.Arg189Trp, PC p.Lys193del), all representing type II deficiency, are hereditary thrombophilia in Japanese and Chinese, respectively. However, the prevalence of these variants has not been determined in the races other than East Asians. Because the PS activity assay using clotting-time method is not sensitive to identify PS Tokushima, we have developed total PS assay system that accurately determines both the activity and antigen levels of total PS, enabling to calculate PS specific activity. From 2013, we started the project entitled “Investigation into racial differences in genetic risk factor for venous thromboembolism (VTE)”.

Update: 1) PS Tokushima was found in Japanese VTE patients and healthy controls, but not in South Korean, Singaporean, and Hungarian. Analysis of Japanese healthy controls revealed the cutoff value of PS specific activity for diagnosis of PS Tokushima to be 0.78, with both accuracy and specificity being almost 100%. 2) PC p.Arg189Trp was found in Japanese and Singaporean VTE patients, being most prevalent in patients of Chinese origin. 3) PC p.Lys192del was found in Japanese, South Korean, and Singaporean VTE patients. Homozygotes were found both in control and VTE patients in South Korea. 4) FV Leiden was prevalent in Hungarian. Analysis of Hungarian healthy controls revealed that PS specific activity of the heterozygotes of FV Leiden was decreased, but the decrease was significantly smaller than PS Tokushima. 5) According to the1000 Genomes Project Final Reports, the three variants were found only in the East Asian population, being consistent with our findings.

Working Session #2:

Summary of the Status of Thrombophilia Testing in the Clinical Lab.

Richard Marlar (USA)

Clinical laboratory assessment of plasma levels of AT, PC, PS and APC-

Resistance is difficult and associated with many problems and pitfalls. The working groups are producing four manuscripts on the state-of-the-art clinical assessment of plasma levels of the

common thrombophilic risk factors. The manuscript progress for these four thrombophilic factors was presented:

Protein C- Manuscript is prepared and will be submitted this summer

Protein S- Currently being written and will be submitted in November, 2017

Antithrombin- Development was reassigned and will be submitted in

January 2018

APC-R- Manuscript is finalized and will be submitted in summer of 2017

(The basic aspects of the testing requirements were reviewed by

Gary Moore (UK). See section below

Review of APC-Resistance Testing recommendations.

Gary Moore (UK)

The concept of APC Resistance was presented. The various modifications of the APC-R assay were reviewed. Discussions about the relationship with the genetic mutations and the assay results was also presented. Other causes (non-genetic) of APC-Resistance were reviewed.

Clinical Testing Issues associated with Protein S Activity Assays.

Richard Marlar (USA)

The artefactual decrease of Protein S activity seen in normal patients in the clinical assays were reviewed with pre-analytical variables as the cause was presented. It appears that the amount of time between collection and separating of the blood cell and platelets from the plasma may be the cause of this intermittent false abnormal result. Further studies of the mechanism was described in the next presentation.

The cleavage of Protein S as the cause of the loss of Protein S activity

Herm-Jan Brinkman (The Netherlands)

Protein S is cleaved and inactivated in a region known as the thrombin sensitive loop. Thrombin and other enzymes can cleave protein S in this region and subsequently inactivate the PS activity. There at least 3 specific sites that can be cleaved in this specific loop by different enzymes. Work was presented showing the mechanism and parameters of this cleavage by the various enzymes. This work may have clinical significance for assay performance.

Working Session #3

Clinical relevance of other potential plasma inhibitors.

Jun Teruya (USA)

While antithrombin, protein C, and protein S are widely monitored natural plasma inhibitors, there are more inhibitors that have clinical significance.

He listed plasma protein inhibitors that may have clinical relevance. Hereditary deficiency of a1 antitrypsin may cause chronic obstructive lung disease, liver cirrhosis, panniculitis, and vasculitis. The abnormal a1 antitrypsin that had antithrombin activity caused bleeding has been reported. C1 esterase inhibitor has multiple actions affecting complement system, coagulation system, fibrinolytic system, and kallikrein-kinin system. It also interacts with cells and infectious agents. Deficiency of C1 esterase inhibitor is known to cause angioedema. Clinical trials of the use of C1-esterase inhibitor include sepsis, ischemia reperfusion injury, and transplantation. Tissue factor pathway inhibitor (TFPI) is not clinically monitored routinely. However, it is a potent anticoagulant and there are numerous papers about its clinical significance. Increased level of TFPI causes bleeding such as East Texas bleeding disorder. Antibody against TFPI is a very promising treatment modality for hemophilia. Thrombomodulin has been used to treat DIC in Japan. Abnormal thrombomodulin is known to cause atypical hemolytic uremic syndrome. SERPIN5 (aka protein C inhibitor) has multi-function such as inactivating activated protein C, urokinase, and others. Protein Z is not clinically utilized, but its deficiency is linked to ischemic stroke and recurrent miscarriages. Fibrinolysis inhibitors such as a2 antiplasmin, plasminogen activator inhibitor 1 and thrombin activatable fibrinolysis inhibitor were also discussed. Lastly heparin-like inhibitors are not well recognized and thus underdiagnosed, but it may be associated with liver disease, liver transplant and lymphoma. Although it may be transient, it may cause significant bleeding.

Conclusions:

Richard Marlar (USA)

Conclusions of the Session were presented

Platelet Immunology

8 July 2017 9:00 – 13:15 Chairman: Donald Arnold Co-Chairmen: Tamam Bakchoul, Brian Curtis, Shigeki Miyata, Francois Mullier, Rachel Peterman, Chris Ward

Welcome (9:00 – 9:20)

D. Arnold welcomed everyone to the SSC meeting in Berlin. He reviewed the membership of the SSC, its focus and its mandate.

Chair: Donald M. Arnold; Co-Chairs: Tamam Bakchoul, Brian Curtis, Shigeki Miyata, Francois Mullier, Rachel Petermann, and Christopher M. Ward

Focused on immune disorders of platelets: Fetal/neonatal alloimmune thrombocytopenia; Primary immune thrombocytopenia; Drug-induced thrombocytopenia; Heparin-induced thrombocytopenia. The main topics relate to: Development and evaluation of biological assays detecting and assaying allo, auto, or drug-dependent antiplatelet antibodies; Translational focus.

Mandate is to: Develop international laboratory standards, methods, and nomenclatures; Address issues of practical importance to the research community in the field of platelet immunology; Evaluate existing data, determine issues for which data are missing, identify areas of controversy or pressing clinical need and discuss methodological approaches to answer questions; Create international collaborations for planning and executing projects related to the defined needs in the field; Suggest and organize collaborative studies as a result of these activities; Generate, publish and distribute reports, recommendations and other documents concerning the above.

Papers: 2 publications from SSC projects in 2016:

Morel-Kopp MC, Mullier F, Gkalea V, Bakchoul T, Minet V, Elalamy I, Ward CM; subcommittee on platelet immunology. Heparin-induced multi-electrode aggregometry method for heparin-induced thrombocytopenia testing: communication from the SSC of the ISTH. J Thromb Haemost. 2016 Dec;14(12):2548-2552.

Fuhrmann J, Jouni R, Alex J, Zöllner H, Wesche J, Greinacher A, Bakchoul T. Assessment of human platelet survival in the NOD/SCID mouse model: technical considerations. Transfusion. 2016 Jun;56(6):1370-6.

Projects: 3 ongoing projects are:

1) NAIT standardization of laboratory testing;

2) International validation of HIMEA assay for HIT;

3) new study (Bakchoul, Mullier, Michael Nagler) on standardized algorithm for HIT testing. Dr. Nagler is the recipient of an ISTH project grant in support of this latter project which was briefly discussed.

DA challenged each co-chair to lead one project in the coming year; to consider applying for ISTH funding; encouraged to use ISTH resources (staff, database platform), and to plan on presenting those project in Dublin 2018.

Section 1: Immune Thrombocytopenia (ITP)

Arnold- Laboratory investigations for ITP, with a focus on H. Pylori testing

DA presented on laboratory investigations for patients with suspected ITP and highlighted some gaps in current published guidelines. Specifically whether H. Pylori testing should be included in this work up is inconsistent in guideline recommendations. He discussed H. Pylori associated ITP in more depth showing data from McMaster which suggested that only a very low proportion of patients screened ultimately had a platelet count response after successful HP eradiation. He proposed an idea for a new SSC project: To survey practice patterns across a broad geographic representation on HP investigations and treatments for patients with ITP. This could be a survey type study plus retrospective data from 4 – 6 representative centres.

Discussion:

 The experience from Japan is that all patient are screened and HP eradication is given as ‘first line’ therapy. In Japan, the rate of platelet count response is very good, but mostly for patients with mild-moderate TCP, not for severe disease.  Lack of data in pediatric population  Suggestion to store samples from patients with anti-HP antibodies to assess the interactions with platelets, and determine what type of antibodies these are – anti-HPA-1a like (persistent for many years), HIT-like (transient after 30 days) or other.

Mullier- Method of counting platelets

FM presented on the need for ISTH guidance (on each type of analyser) to select the most accurate method for counting platelets. He showed some illustrative examples of when the platelet count can be falsely low due to inaccuracy of impedance or optical readings in certain clinical situations: e.g. very low platelet counts, fragmented red blood cells, leukemia/lymphoma cells, cryoglobulins, bacteria, lipids, haemolysis, etc. PLT-F (flow cytometry) is more accurate than PLT-I (impedance) and PLT-O in low platelet counts and in presence of fragmented red blood cells or cryoglobulins. Flow cytometry can be considered the gold standard (more accurate than haematological analysers in case of chemotherapy or to measure transfusional efficiency post acute leukemia). There is no reference technique for calibration of platelet counting, as this is not provided by the manufacturers and calibration methods will be different for the impedance and the fluorescence mode. In addition, there is a lack of adapted controls (i.e only one acceptable level for PLT-F on XN instruments as the other levels are not fluorescent)

Discussion

 This inaccuracy of platelet counting might explain why some patients with the same low platelet count bleed while others do not; maybe the results are inaccurate.  MPV is not a reliable indicator, flow may be better.  To get the accurate platelet count we need additional, specific platelet markers.

Bakchoul – new destruction pathway in ITP

TB presented on the effects of ITP autoantibodies on glycan patterns on PLTs. Autoantibodies can induce changes in the glycan pattern on the PLT surface detectable with different lectins. These glycan changes occur irrespective of the antibody’s glycoprotein specificity. Antibody- mediated desialylation seems to contribute to PLT destruction in vivo presumably via neuraminidase activation. To address this question more fully, a standardized SSC-biobank for ITP sera should be established.

Discussion:

 Could the SSC support an ITP biobank? This would have to be from well characterized patients so that the diagnosis is as accurate as possible. Serostatus (antibody-positive or antibody-negative) should be confirmed by 2 different laboratories.  This could be a novel tool for standardization purposes. Logistics would have to be figured out.

Section 2: Fetal and Neonatal Alloimmune Thrombocytopenia (NAIT)

Boehlen- Standard practice for FNAIT in France and Switzerland

A Working Group on « Foetomaternal Platelet Alloimmunisation » was created in 2015 under the auspice of the GFHT (French Group of Haemostasis and Thrombosis. This multidisciplinary group is co-chaired by F. Boehlen (haematologist, University Hospitals of Geneva) and G. Bertrand (biologist, Blood Bank of Brittany EFS) and has of 47 members (obstetricians, haematologists, neonatologists, biologists) from France, Switzerland and Belgium. The aims of this working group is to establish a statement of French practices in terms of management, to design French guidelines for the management of neonatal thrombocytopenia (index case) and next pregnancies in case of foetomaternal alloimmunisation, to create a discussion forum to present difficult clinical cases and to share scientific knowledge. Eighteen months after its formation, we had 7 meetings, discussed 24 difficult cases, proposed an algorithm for the management of neonatal thrombocytopenia and work actually on a French consensus for the management of next pregnancy in case of confirmed foetomaternal alloimmunisation based on literature review and local experience. Indeed foetal and neonatal alloimmune thrombocytopenia is not always well known (often underdiagnosed) and there is no clear consensus concerning the management of the next pregnancy. A multidisciplinary approach is necessary.

Discussion:

 Work on clinical management guidelines is occurring in parallel e,g, at least one other international group (ICTMG) is working on this. There would be opportunities to harmonize these initiatives.  Suggestion to support a national advisory board for difficult NAIT cases hosted by the platelet immunology SSC.  Important to emphasize the need for paternal crossmatch in complete investigations for FNAIT.

Petermann – Improving test methods for NAIT

To determine the status of the fetus in FNAIT case, fetal platelet genotyping is done routinely by invasive procedure. A new non invasive method allowing early and reliable diagnosis on maternal plasma is developed by droplet digital PCR on 4 main HPA systems, HPA-1,-3,-5 and -15. This new method which includes a fetal DNA marker (RASSF1A promoter gene) has many advantages : screening of many systems in the same time, early determination of fetal platelet genotyping (6WG) and results available for physicians in 48 hours after blood sample management.

By contrast, even if MAIPA test was a turning point in platelet serology investigation, French users of Complete MAIPA Kit (apDia, Belgium), the only EC marked in vitro medical device for MAIPA, are confronted in crossmatch with paternal platelets to extrareactions directed mainly to GPIbIX, but others too, in HLA immunized patients. Despite the introduction of chloroquine treatment, many questions have been araised in users and all these questions need further evaluation and guidelines.

Discussion:

 Question about ethnicity in the patients who have co-precipitation of GPIbIX antibodies, which would be unusual in a European population  Consider a workshop to deal with issues related to platelet cross matching with paternal platelets.

Educational Sessions

Sachs – New Diagnostic Tools for NAIT

In this presentation, Dr. Sachs presented novel predictors of bleeding in babys with NAIT. Specifically, he highlighted the importance of anti- αvβ3 antibodies, which are associated with ICH. The target antigen of this alloantibody is on platelets and endothelial cells which might explain why babies with this antibody are more prone to bleeding. This marker may represent an important prognostic tool for patients and help direct therapy to those mothers at risk.

Warkentin – State-of-the-art: HIT diagnostic strategies.

In more than 99% of cases of HIT, platelet-activating antibodies directed against PF4/heparin complexes are implicated. The talk began by discussing the concept of "autoimmune HIT", in which HIT antibodies activate platelets in both heparin-dependent and heparin-independent fashion. Autoimmune HIT disorders include: delayed-onset HIT, persisting HIT, "flush" heparin-

induced HIT, fondaparinux-induced HIT, and spontaneous HIT syndrome. A current controversy in HIT diagnosis was also discussed, namely the sensitivity of the serotonin-release assay (SRA) for diagnosing HIT: some labs have found the SRA to be highly sensitive for HIT (>95% sensitivity), whereas another lab (BloodCenter of Wisconsin) has suggested that the sensitivity of the SRA (in their hands) may be as low as 54%. In the second half of the presentation, the topic of rapid immunoassays for HIT was discussed: recent studies suggest that certain rapid assays have diagnostic sensitivity for HIT as high as PF4-dependent EIAs, but with greater diagnostic specificity than the EIAs; this offers both the potential for rapid assays to speed up diagnostic evaluation of HIT while at the same time minimizing "overdiagnosis"). Finally, a Bayesian approach to HIT diagnosis was described in which (by way of example) the use of a particular rapid assay−the latex immunoturbidimetric assay (LIA)−was highlighted, whereby likelihood ratios associated with a positive and a negative test result were used to increase and decrease, respectively, the post-test probability of HIT. Used in real time, this approach can dramatically help the clinician in making appropriate treatment decisions.

Section 3: Heparin induced thrombocytopenia

Shigeki Miyata - Nationwide heparin-induced thrombocytopenia (HIT) registry: towards appropriate diagnosis and treatment of HIT in Japan

SK spoke about his reference centre for HIT testing, which uses antigen immunoassays and a washed platelet activation assay, the platelet microparticle assay (PMA). The PMA detects platelet-derived microparticles activated by HIT antibodies at therapeutic concentrations of heparin (or by heparin-independent strong HIT antibodies). We made modifications to the assay, such as donor selection for washed platelets, which increased sensitivity for detecting HIT antibodies with weak platelet-activating properties. (Thromb Haemost. 2017; 117, 127-138) We have been conducting a nationwide HIT registry by asking each treating physician who sent blood samples of a HIT-suspected patient to fill out a case report. This nationwide HIT registry clarified that the incidence of HIT-associated thromboembolic events was very high (61%) in patients who tested strongly positive in the PMA.

A post-marketing survey on the use of argatroban in HIT-suspected patients was conducted, since most of HIT patients were treated with argatroban, a direct thrombin inhibitor only approved for HIT treatment in Japan. These studies suggests that the Japanese treatment strategy with argatroban, in which we recommend less than half of the initial dosage recommended in Western countries due to the ethnic difference for the appropriate dosage, was effective in preventing HIT-associated thromboembolic events, when argatroban was initiated immediately after HIT suspicion. However, a delay in the initiation of argatroban was significantly associated with HIT-associated thromboembolic events, which occurred after HIT suspicion, in patients who tested strongly positive in the PMA.

These results provide further evidence that HIT patients should be treated with an alternative anticoagulant immediately after HIT suspicion. In addition, these results suggest that we may need different treatment strategies for patients with strong HIT antibodies and those with weak antibodies. To identify appropriate treatment strategies for each patient population, a washed platelet activation assay that can distinguish strong HIT antibodies clearly from weak antibodies could be useful in the clinical management of HIT patients.

SK proposed the establishment of a worldwide HIT registry for more appropriate diagnosis and treatment of HIT patients. Such strategies may also clarify ethnic differences in the pathogenesis, diagnosis, and treatment of HIT.

Discussion:

 HIT samples from japan might be available to share as a resource

Nazy- cellular immune cells

IN presented the cellular immune response in patients with HIT. HIT is caused by pathogenic antibodies that bind to complexes of platelet factor 4 and heparin (PF4/heparin) causing platelet activation. Studies have shown cellular sensitization to PF4/heparin occurs early in life, before heparin exposure; however, the immunogenesis of HIT is not well characterized. The objective of this study was to investigate the cellular immune response to PF4/heparin complexes in patients diagnosed with HIT. Peripheral blood mononuclear cells (PBMCs) from healthy controls (n=28), patients post-cardiac surgery requiring cardiopulmonary bypass (CPB, n=14) and patients with confirmed HIT (n=36) were cultured in the presence of PF4 and PF4/heparin. Cellular proliferation was assessed by the 3H-thymidine uptake assay. In all groups, PBMCs proliferated in the presence of PF4 and this reaction was enhanced by the addition of heparin. Patients with HIT and patients post-CPB had higher proliferative responses compared to healthy controls. PBMC proliferation was antigen-specific and depended on the presence of platelets, T cells, B cells and monocytes. Culture supernatants were tested for the levels of regulatory cytokines IL-10 and TGF- β1. PBMCs from both HIT and CPB patients produced significantly lower levels of IL-10 and TGF-β1 compared to healthy controls and these regulatory cytokines had an inverse correlation with PF4/heparin-dependent proliferation. This study documents that, cellular immune sensitization to PF4/heparin complexes is present before heparin exposure and suggests that immune dysregulation can contribute to the immunogenesis of HIT.

Discussion:

 One direction for the SSC eventually would be to apply these findings to identify patients at increased risk for HIT before cardiac surgery.  Consider testing a different protein, besides PF4, to see if cellular proliferation was the same or different.  Consider checking the immunophenotype of the immune cells that are responsible for this break in immune tolerance.

Ward- Multicenter study of Heparin-Induced Multi-Electrode Aggregometry for HIT testing

CW provided an update on the whole-blood impedance assay for functional HIT testing (HIMEA). A consensus method for this assay has been published in JTH late last year. The working group is currently planning a multicentre study to compare donor responses to a common set of patient samples, but collecting a sufficient number of high-volume samples has proved challenging. We have accumulated around half the target (12) and several attendees at the meeting indicated that they may have suitable patient material. We propose to test these

samples in 4 expert laboratories (using at least 2 donors per site) and compare results with a centralised SRA. In discussion, Prof Gruel recommended that a humanised antibody like 5B9 be used to compare donor reactivity and the group is happy to include this if the antibody can be made available. The group expects to have results from this multicentre study for presentation at the Dublin SSC.

Discussion:

 Need for additional samples to complete this study  Consider the variability in FcR activation between donors that might impact on the test results. Consider using the humanized HIT antibody 5B9 rather than a mouse monoclonal.

Closing Remarks

D. Arnold thanked the committee and all members for their hard work as part of the SSC. Co- chairs should begin planning what they will present in Dublin 2018.

Platelet Physiology

9 July 2017 8:00 – 12:15

Chairman: Paolo Gresele Co-Chairmen: Hans Deckmyn, Andrew L. Frelinger III, Martine Jandrot-Perrus, Shinji Kunishima, Marie Lordkipanidzé, José Rivera Pozo

The Platelet Physiology SSC organized a 2+2 hour session on Sunday July 9th 2017. The first 2 hr session, from 8:00 to 10:00, A3 Room, was a Business Session reporting on the ongoing projects of the SSC. It was chaired by Paolo Gresele (Italy) and Marie Lordkipanidzé (Canada). The business session started with an introduction by Paolo Gresele (Italy) who gave an overview of the mission of the ISTH-SSCs and of the specific recently updated mandate of the Platelet Physiology SSC. The Platelet Physiology subcommittee web page within the ISTH site was illustrated, including the options for interaction by registered members, and the audience was encouraged to register. A list of the concluded projects and relative publications was given. Ongoing projects and projects about to be started were listed: • Evaluation of the Bleeding Assessment Tool (BAT) for the bleeding history of inherited platelet disorders (P. Gresele, P. Harrison): cross-sectional phase: completed; first year follow-up: completed; expected final completion: April 2018 • Assessment of the correlation between the ISTH/SSC bleeding assessment tool score and result of laboratory tests employed for the diagnosis of inherited platelet function disorders (BAT-LAB substudy) (P. Gresele): enrollment closed. • Consensus/guidance on the methods for the study of platelet secretion (D. Mezzano): second round survey concluded. • Laboratory monitoring of P2Y12 inhibitors: a position statement (A. Frelinger): ready for submission. • Generation of guidance on the measurement of platelet dimensions: methods and clinical use (P. Noris): second round survey concluded. • Position statement on the use of platelets in regenerative medicine (P. Harrison): ready for submission. • Prospective evaluation of the bleeding phenotype in PT-VWD to support evidence-based diagnosis and management (M. Othman): ongoing. • Standardization of flow cytometry for the assessment of platelets disorders (A. Frelinger, J. Rivera Pozo) (to be started). • Measurement of soluble markers of platelet activation: methods and use. A project for generation of guidelines (H. Deckmyn, M. Jandrot-Perrus) (to be started). • Diagnosis of inherited platelet disorders on a blood smear: survey and inter-laboratory validation exercise (S. Kunishima) (to be started). • Methods for the conservation and shipment of platelets for platelet studies (M. Lordkipanidzé) (to be started). Other topics of interest as possible new projects were listed: • Utility of the ISTH BAT for acquired platelet function disorders • Standardization of methods for the measurement of platelet procoagulant activity • Methods for the study of platelet hyperreactivity in ischemic cardiovascular disorders • Guidelines for the analysis of the platelet transcriptome (perhaps in collaboration with the Working Group on Genomics in Hemostasis?) • Ageing and platelets

• Standardization of methods for the study of the immature platelet fraction • Standardization of transmission electron microscopy in the study of platelet disorders The audience was invited to interact with the SSC Platelet Physiology about the ongoing and new projects and to send expressions of interest to participate in the new projects to be started.

Paolo Gresele (Italy) then presented the final results of the cross-sectional phase of the evaluation of the Bleeding Assessment Tool (BAT) for the assessment of inherited platelet disorders study and the preliminary results of one year follow-up. The initial enrolment in the study was successfully completed with a total of 1,103 subjects enrolled, in the three subpopulations of inherited platelet disorders (IPD), von Willebrand disease type 1 (VWD-1) and healthy controls (HC), with contribution from 43 investigators from 17 countries worldwide. Results showed that inherited platelet function disorders (IPFD) have a bleeding score (BS) almost the double that of VWD-1, while inherited platelet number disorders (IPND) show an only mildly increased BS as compared with HC and significantly lower than VWD-1. Data of the one-year follow-up showed a significantly higher incidence of clinically significant bleeding events in the IPFD as compared with the VWD-1 group. Patrizia Noris (Italy) presented an update on the 2nd round questionnaire on "Platelet guidance on the measurement of platelet dimensions. Methods and clinical use". The experts involved in the project reached an agreement on several methodological issues for the standardized measurement of the mean platelet volume: in particular on type of anticoagulant, the time to analysis, and the blood storage temperature. Most of the experts agree that the normal range of the mean platelet volume must be generated by each laboratory and that optical counters may offer advantages over impedance counters in case of abnormal CBC parameters, such as fragmented or small erythrocytes, platelet anysocytosis. Jeremy Magalon (France) presented an update on the use of PRP in Regenerative Medicine setting the stage for the overview presented by Paul Harrison for the generation of guidance on the use of platelets in regenerative medicine. His conclusions were that there is an urgent need for guidelines regarding the use and the quality control of PRP in regenerative medicine. Paul Harrison (UK) presented an update on the PATH 2 trial (Platelets in Achilles Tendon Healing) and reported that 201 out of 214 patients have been recruited and that the trial should complete this summer. He then presented the overview of the SSC guideline on the use of platelets in regenerative medicine. In 2016 a working party of 9 experts was formed and undertook a rand survey comprising 45 statements. 12 statements were scored as appropriate with complete agreement by the panel, 14 were scored as appropriate after removal of 2 extreme scores and 19 statements were scored as uncertain. He also presented a proposed new classification system for platelet preparations that will be included in the guidelines. The guideline is now being drafted and will be submitted as soon as possible. Diego Mezzano (Chile) presented the results and analysis of the second round survey (RAND Methodology), on the Platelet Secretion Assays for the diagnosis of Inherited Platelet Function Disorders (IPFD). This project will end in the generation of Recommendations for the measurement of platelet secretion. 14 experts answered the new round survey. An analysis of the replies was presented and a detailed analysis will be circulated among the Sub-Committee members to decide if there are areas of uncertainty that need to be dealt with before publishing the final Recommendations. Shinji Kunishima (Japan) presented the new project “Diagnosis of inherited platelet disorders on a blood smear: survey and workshop”. Several well-characterized inherited platelet disorders can be diagnosed on a routine peripheral blood smear with immunofluorescence staining. An expert

agreement on the use of immunofluorescence for platelet diagnostics and an interlaboratory cytochemistry exercise will be carried out. Marie Lordkipanidzé (Canada) presented the project “Methods for the conservation and shipment of platelets for platelet studies”. This project was well received by the community. Several members have commented that they have over the years documented their data on shipped platelets and their sensitivity to aggregation in comparison with a fresh sample. Many have offered to share their data with us, which opens the possibility of a collaborative sub-project in the upcoming years. The second part of the session, from 10:15 to 16:15, A3 Room, was opened by an education session on the platelet cytoskeleton in platelet function chaired by Hans Deckmyn (Belgium) and Andrew L. Frelinger III (United States). The education session was opened by Robert Flaumenhaft (Boston) presenting “The granule secretion mechanisms and actin dynamics”. Platelet actin dynamics and granule exocytosis have significant interactions. Actin acts as a barrier to platelet exocytosis, but also participates in the expulsion of alpha-granule contents. Conversely, platelet granules provide membrane to cover and matrix proteins to support growing actin-rich pseudopodia and lamellipodia.

Markus Bender (Würzburg) presented “Cytoskeletal dynamics in megakaryocytes and platelets”. Reorganization of the cytoskeleton is critical to platelet production and function. It has been shown that actin dynamics is important for the initiation of proplatelet formation (Schulze H et al. Blood 2006, Antkowiak J et al. J Thromb Haemost 2016, Bender M. et al. Blood 2010), whereas microtubule sliding powers proplatelet elongation (Patel SR et al. Blood 2005, Bender M et al. Blood 2015). Staining of the platelet cytoskeleton on standard peripheral blood smears can be useful to narrow down or to confirm the diagnosis of several well-characterized hereditary platelet disorders (Greinacher A et al. J Thromb Haemost 2017). Loredana Bury (Italy) presented “Abnormal αIIbβ3 and cytoskeletal perturbation causing platelet dysfunction”. This communication focused on variants in ITGA2B and ITGB3 that cause Glanzmann Thrombasthenia by interfering with ouside-in signalling and thus cytoskeletal reorganization. Variants that impair triggering of outside-in signalling cause impaired platelet function despite normal expression of alphaIIbbeta3 and normal fibrinogen binding. Gain of function variants that cause constitutive triggering of outside in signalling cause platelet dysfunction by interfering with the cytoskeletal remodelling required for platelet aggregation. The session continued with business activities of the Platelet Physiology Subcommittee, chaired by Martine Jandrot-Perrus (France) and José Rivera Pozo (Spain). Yvonne M.C. Henskens (The Netherlands) presented “How to reach consensus on using the SSC LTA guidelines: the Netherlands experience”. The Dutch society for Hematological laboratories succeeded to translate the SSC guideline on light transmission aggregometry to a practical and accepted procedure. More than 90 % of the recommendations were taken over and were implemented by a pilot group of 12 laboratories. Results of this standardisation process will be published after thorough analysis later this year.

Andrew L. Frelinger III (United States) and José Rivera Pozo (Spain) presented the preliminary results of the “Standardization of flow cytometry for the assessment of platelets disorders” project. The goal of this project is to provide guidelines for the use of flow cytometry for the evaluation of platelet function in patients with possible inherited or acquired disorders of platelet number and function. The results of the initial RAND survey of experts showed that there are large areas of agreement on the appropriateness of statements with respect to clinical utility, pre-analytical variables, instrument and reagent standardization, methods, reporting, and quality control.

However, a significant number of statements on the clinical utility and pre-analytical variables of flow cytometry of platelets were judged uncertain or inappropriate. Feedback from experts and the ISTH community will be used to reformulate ambiguous statements to generate a second round of survey questions. Hans Deckmyn (Belgium) and Martine Jandrot-Perrus (France) presented the project ”Measurement of soluble markers of platelet activation: methods and use. A project for generation of guidelines”. Robust platelet activation markers may be useful to assess the contribution of platelets in prothrombotic states and also in a growing number of situations in which platelets have a pathogenic role, including inflammation, infection and cancer. The purpose of the project is to come to a consensus on which biomarkers are considered as most useful/promising to serve as an incentive to whoever to start building an easy robust test system to finally allow standardized large studies by multiple research/clinical teams to proof or disproof the usefulness of particular markers.

Attendance to the session was excellent, ranging from approximately 300 to possibly more than about 400 attendees in the different phases of the SSC meeting. Discussion from the audience was active and lively. The room session was reasonably good, but for the rest organization and facilities were bad, because microphones did not work at the beginning of the session, and technical assistance was slow, delayed and unprepared. This lead to a serious delay in the actual starting time of the session, creating problems for the coffee break and ending time. Moreover, some of the uploaded presentations did not open and here too it took time to have them working by the room technicians. Finally, the screens on the moderators desk did not work properly and often went black during the presentations.

Predictive and Diagnostic Variables in Thrombotic Disease

8 July 2017 9:00 – 13:15 Chairman: Paul A. Kyrle Co-Chairs: Cecilia Becattini, Suzanne Cannegieter, Geert-Jan Geersing, John-Bjarne Hansen, Gregoire Le Gal, Marc Righini

Welcome, introduction of co-chairpersons and update on activities in the recent year (P Kyrle)

Diagnosis of VTE

Chairmen: G Le Gal, M Righini

Disease prevalence and dependent failure rates (F Klok): a safe threshold for DVT exclusion is defined, which would also facilitate patient number calculation for future studies.

Adjudication of death from PE (P Girard): a better definition of PE, in particular of fatal PE is needed and an SSC project on this topic is urgently required.

Common data elements projects (G Le Gal): the need for easier access to patient data by the use of large databases is discussed.

Ongoing Clinical Studies

Chairpersons: S Cannegieter, GJ Geersing

Updates on the following studies were presented:

VISTA (GJ Geersing): Validation of the Vienna Prediction Model

VALID (L Eischer): Validation of the Vienna Prediction Model

L-TRIP (S Cannegieter): Construction of recurrence prediction rules for various VTE states

Apidulcis (P Prandoni): Algorithm consisting of repeated D-Dimer measurement and apixaban therapy to optimize secondary thromboprophylaxis in VTE patients.

Hypercan (M Marchetti): VTE incidence in cancer patients, prediction tool for cancer patients

HOT-PE (S Konstantinidis): Home treatment of patients with low-risk pulmonary embolism with the oral factor Xa inhibitor rivaroxaban

HOME-PE (PM Roy): randomized controlled trial comparing hospitalization and out-of hospital treatment in patients with pulmonary embolism

THEIA (F Klok): Diagnostic Management of suspected recurrent ipsilateral deep vein thrombosis of the leg with magnetic resonance direct thrombus imaging

CLOT-3 (S Stevens): the role of CUS VTE detection in pregnancy

All studies are ongoing and final results have not been published yet.

Education session

Chairmen: PA Kyrle, JB Hansen

Big data and IPD-analysis: novel concepts for future VTE research (GJ Geersing):

The advantage of using big data bases and independent data-analysis (PD meta-analysis is a type of systematic review. Rather than extracting aggregate data from publications or from investigators, the research data are sought directly from the researchers responsible for each study) was presented.

Management of pulmonary embolism in pregnancy (C Becattini):

Pros and cons of different approaches to exclude or prove PE in pregnancy were extensively described.

Prediction of VTE

Chairperson: C Becattini

D-Dimer measured at diagnosis of first VTE and future risk of cancer and recurrence (E Bjori)

Elevated D-Dimer levels at the time of VTE were associated with a higher risk of VTE recurrence and cancer occurrence.

Impact of prothrombotic genotypes on the risk of VTE in cancer patients (S. Braekken)

Prediction of VTE in cancer patients is facilitated by genetics.

Discovery of novel biomarkers of VTE by plasma proteomics (J Odeberg):

End of meeting: 1:15 p.m.

Vascular Biology

8 July 2017 9:00 – 13:15

Chairman: Rienk Nieuwland Co-Chairs: Alexander Brill, Chris Gardiner, Elizabeth E. Gardiner, Anna Randi, Florence Sabatier, Pia R. Siljander

Complex relationship between low density lipoprotein and extracellular vesicles. Pia Siljander; EV group, Department of Biosciences, University of Helsinki, Finland Extracellular vesicles (EVs) are released by cells, and EVs are widely distributed in (human) body fluids. Although high hopes are directed towards EVs in clinical applications, analysis of EVs from human blood or fractions thereof is yet impaired by the concurrent presence of e.g. lipoprotein particles. Consequently, studying EVs from variable sources requires knowledge of not only EVs, but also of common cofounders, which is why we set up the first “EV core facility” in the world (https://www.helsinki.fi/en/news/new-extracellular-vesicle-core-facility-launched-at- the-university-of-helsinki). For example, both EVs and lipoproteins are considered to carry circulating RNA, and it may be relevant to be able to accurately pinpoint the RNA source in a sample. Unfortunately, due to their overlapping physical parameters -the extent of which has previously not been sufficiently appreciated, separation and analysis of EVs from lipoprotein containing samples is difficult, which may make drawing appropriate conclusions impossible. In our laboratory, we are developing a quick method to allow the detection of lipoprotein presence in the EV samples based on flow cytometry.

Because also complexing of EVs with lipoprotein particles has been reported, and typical apolipoprotein proteins such as apo E are present in proteomes of highly purified EVs derived from e.g. cultured cancer cells and urine, the crosstalk between EVs and may be physiologically relevant For example, in our on-going studies of platelet-derived EVs, we can see that native and modified lipoproteins induce a time and dose-dependent release of EVs (Neuvonen et al.).

Taken together, the relationship of lipoproteins with EVs is more complex than previously appreciated. To fully understand the contribution of both EVs and lipoproteins in physiological functions, particle enumeration (EV quantification) or circulating biomarkers, we first have to be fully aware of sample confounders.

Standardization of vesicle detection by flow cytometry. Rienk Nieuwland, Academic Medical Centre of the University of Amsterdam, Amsterdam, The Netherlands To establish extracellular vesicles (EVs) as clinical biomarkers, standardization of concentration measurements is essential. During the last decade alone, a 10-fold increase in the concentration of platelet-derived EVs has been observed (Gascecka A et al.; Platelets, 2017). This increase is explained by improved protocols for collection and handling of blood, improved protocols to label EVs, knowledge of artefacts, and improved sensitivity of flow cytometers. Regarding standardization, apart from the pre-analytical variables, flow rate, scatter and fluorescence need to be measured and compared between flow cytometers. The SSC on Vascular Biology has a track record of standardization studies of EV concentrations measurements by flow cytometry based on measuring the light scatter of polystyrene particles of known diameter. Based on the light scatter of these beads, EV size gates are set, provided

samples of EVs are measured, and measured concentrations of EVs are reported and compared.

In a new standardization study, two problems were overcome. Firstly, the refractive index (RI) of EVs was unknown in the earlier SSC studies. In 2014, the RI of EVs was found to be much lower than that of polystyrene particles of similar diameter. Secondly, the sensitivity of detectors for light scatter differs between instruments. Both problems were solved by developing a software model that takes the physical properties and differences between EVs and reference materials into account as well as the flow rate, and which corrects for the optical differences between instruments. A comparison study, financially supported by the ISTH, was performed and a manuscript is expected to be submitted coming month.

Because in parallel not only the ISTH but also International Society of Extracellular vesicles (ISEV) and International Society on the Advancement of Cytometry (ISAC; were working on the standardization of EV measurements by flow cytometry, in 2015 a collaboration was initiated (www.evflowcytometry.org). Recently, a review was written by members of before mentioned societies on the methodological guidelines regarding blood collection, isolation and measuring single EVs by flow cytometry and published in Circulation Research earlier this year (Circ Res. 2017;120:1632-1648. DOI: 10.1161/CIRCRESAHA.117.309417). Taken together, major progress is being made in standardization of EV detection.

New strategies for detection of leucocyte-derived microvesicles by fluo-sensitive flow cytometry. Romaric Lacroix, Vascular Reseach Center of Marseille, Marseille, France Romaric Lacroix presented new strategies, based on a broad panel of antigen specificities combined with size exclusion chromatography (SEC), to reduce the flow cytometry (FCM) background noise for detection of leukocyte-derived microvesicles (LMV) using a fluorescent- sensitive flow cytometer (CytoFLEX*). The screening of 52 myeloid-specific antibodies on MV identified the best specificities for the detection of LMV (monocytes MV: HLA DR, CD11c; granulocytes MV : CD15, CD66c; Myeloid MV: CD18, CD157, CD45). The LMV detection improved significantly by fluorescence background noise reduction with SEC and by using a fluorescence-sensitive instrument. Combining high-sensitivity cytometry, broad antibody screening and pre-analytical optimization will help to set up optimized reagents and specific strategies to improve the detection of MV at the a level required for relevant biomarker detection in clinical practice.

Measuring extracellular vesicle concentrations: standardize the unknown. Edwin van der Pol, Academic Medical Centre of the University of Amsterdam, Amsterdam, Netherlands Blood plasma contains heterogeneous populations of extracellular vesicles (EVs). In many diseases, including thrombosis, the concentration and composition of EVs is elevated. Accurate determination of the concentration of EV populations therefore provides a biomarker for diseases. An accurate concentration measurement requires the detection and characterization of all EVs in a known sample volume at a clinically relevant rate. At present, no technologies exist that meet these requirements. For example, modern flow cytometers can detect EVs at a clinically relevant speed of 10,000 EVs per second, but they lack the sensitivity to detect the smallest EVs, leading to erroneous EV concentrations. Nano flow cytometers can in principle detect the smallest EVs, but the maximum count rate is only several hundreds of EVs per second. Microfluidic resistive pulse sensing can count 50 nm EVs at a rate of 10,000 EVs per second, but lacks the possibility to differentiate between types of EVs. Nanoparticle tracking analysis provides an order of magnitude estimate of the particle concentration.

Since hitherto flow cytometers are the most clinically applicable instruments to characterize single EVs in body fluids, laboratories should focus on standardization of flow cytometry measurements. To measure EV concentrations by flow cytometry, researcher should calibrate

(1) the flow rate (µL/min), the fluorescence intensity (MESF) and the scatter intensity (nm*nm) and determine and report the detection limits of their instrument.

Measuring NETs. Thea Tilley, Zychlinsky Lab, Cellular Microbiology, Max Planck Institute for Infection Biology, Berlin, Germany

Neutrophils are a major player in host defence. One of the most dramatic of their activities is neutrophil extracellular trap (NET) formation. NETs consist of large extracellular DNA structures decorated in histones and neutrophil granule proteins which are extruded into the extracellular space in response to a growing list of microbial and host derived stimuli, in infectious and non- infectious disease contexts. This rapidly growing field lacks a "gold standard" assay for their identification and measurement. Many published NET related studies only examine surrogate markers of NETs (extracellular DNA or nucleosomes) in parallel with neutrophil activation (e.g. neutrophil elastase [NE] and myeloperoxidase [MPO] release), neither of which are unique to NET formation. Presented here were two microscopy methods developed in the Zychlinsky Lab that utilise colocalisation of DNA, chromatin and azurophilic granule proteins (NE and MPO), in line with the original definition of NETs, for their accurate identification. These methods rely on the differential binding of anti-chromatin antibodies to decondensed (NETs) versus condensed (resting cells) chromatin, in parallel with DNA and granule protein colocalisation, for the measurement of in vitro generated NETs and identification of NETs in formalin fixed paraffin sections from humans and mice. Future directions to improve the measurement of NETs from complex samples include the development of monoclonal antibodies to de novo NET specific antigens which will improve their detection and measurement and our overall understanding of this novel activity and its role in human health and disease.

 NETs consist of large extracellular DNA structures decorated in histones and neutrophil granule proteins which are extruded into the extracellular space in response to a growing list of microbial and host derived stimuli, in infectious and non-infectious disease contexts.  This rapidly growing field lacks a "gold standard" assay for their identification and measurement. Many published papers on NETs only examine surrogate markers of NETs (extracellular DNA or nucleosomes) in parallel with neutrophil activation (e.g. neutrophil elastase [NE] and myeloperoxidase [MPO] release), neither of which are unique to NET formation.  Presented here were two microscopy methods developed in the Zychlinsky Lab that utilise colocalisation of DNA, chromatin and azurophilic granule proteins (NE and MPO), in line with the original definition of NETs, for their accurate identification.  Methods rely on the differential binding of anti-chromatin antibodies to decondensed (NETs) versus condensed (resting cells) chromatin, in parallel with DNA and granule protein colocalisation, for the measurement of in vitro generated NETs and identification of NETs in more complex formalin fixed paraffin sections from humans and mice.  Future directions to improve the measurement of NETs from complex samples include the development of monoclonal antibodies to de novo NET specific antigens which will improve their detection and measurement and our overall understanding of this novel activity and its role in human health and disease. Histone citrullination as a marker of NETosis in mice. Kim Martinod, Kortrijk, Belgium

NETs provide a scaffold for platelets and RBCs, thus promoting thrombosis.

The mechanisms by which NETs form in different host response or pathological conditions are being actively investigated by many groups. This talk focused on one such pathway, describing what has been published regarding peptidylarginine deiminase 4 (PAD4). PAD4 is a protein deiminating enzyme, converting arginine to citrulline, and is unique among PAD family members as it has a nuclear localization signal. Elevated PAD4 activity can result in histone hypercitrullination and eventually NETosis. In mouse peripheral blood neutrophils, H3Cit can be associated with NETosis under sterile inflammatory conditions. This can be examined in vitro by immunofluorescence microscopy, comparing H3Cit-positive cells to DNA staining for spread “NET” morphology. Of note, it is important that these be analyzed separately, as NETs are defined as extracellular chromatin originating from neutrophils, while H3Cit+ cells can also include neutrophils that are at an earlier stage of NETosis. Neutrophils from PAD4-deficient mice, or wild-type mouse neutrophils treated with specific PAD4 inhibitors, have a significant defect in NET formation. In human neutrophils, the effect of PAD4 inhibition is less clear and may depend on the stimulus. In vivo, PAD4-/- mice have favorable outcomes in disease models where histone citrullination occurs (thrombosis, wound healing, ischemia/reperfusion injury, aging). Lastly, citrullinated histones are seen in human thrombi (both venous and arterial), and it remains of interest to investigate what factors drive NETosis during thromboinflammation, and in which pathologies PAD4 contributes.

Challenges when studying neutrophil extracellular traps (NETs) by Prof. Dr. Maren von Köckritz-Blickwede, University of Veterinary Medicine Hannover, Germany

 Serum nucleases in fetal calf serum degrade NETs and may limit the formation of NETs during in vitro experiments.  Cationic host antimicrobial peptides as the cathelicidin LL-37 may block visualization by interfering with DNA intercalating-dyes.  Neutrophil density and medium impacts formation of NETs during in vitro experiments.  Heparin versus EDTA used as anticoagulant during isolation of blood-derived neutrophils exhibit different effects on functionality of neutrophils.

von Willebrand Factor

8 July 2017 14:00 – 18:15 Chairman: Sandra Haberichter Co-Chairs: Simon De Meyer, Jorge Di Paola Veronica Flood, Kochi Kokame, Frank Leebeek, James O’Donnell

1. Education session: Advances in the biology and treatment of TTP

Moderators: Sandra Haberichter (USA), Jorge Di Paola (USA)

Simon De Meyer (Belgium)

ADAMTS13 mediated thrombolysis of t-PA-resistant occlusions in ischemic stroke in mice

Dr. De Meyer presented data regarding rapid vascular recanalization as the basis for successful treatment of cerebral ischemia. Currently, tissue plasminogen activator (t-PA) is the only approved thrombolytic drug for ischemic stroke. However, t-PA does not always result in efficient thrombus dissolution and subsequent blood vessel recanalization. To better understand thrombus composition, Dr. De Meyer and colleagues analyzed thrombi retrieved from ischemic stroke patients and found a distinct presence of von Willebrand factor (VWF) in various samples. Thrombi contained on average 20.3% VWF, and was inversely correlated with thrombus red blood cell content. They hypothesized that ADAMTS13 can exert a thrombolytic effect in VWF-containing thrombi in the setting of stroke. To test this, they generated occlusive VWF-rich thrombi in the middle cerebral artery (MCA) of mice. Infusion of t-PA did not dissolve these MCA occlusions. Interestingly, administration of ADAMTS13 5 minutes after occlusion dose-dependently dissolved these t-PA–resistant thrombi resulting in fast restoration of MCA patency and consequently reduced cerebral infarct sizes (P < .005). Delayed ADAMTS13 administration 60 minutes after occlusion was still effective but to a lesser extent (P < .05). The data presented show for the first time a potent thrombolytic activity of ADAMTS13 in the setting of stroke, which might become useful in treatment of acute ischemic stroke. (Blood. 2016;127(19):2337-2345)

Marie Scully (United Kingdom)

Advances in the treatment of TTP

Dr. Scully discussed thrombotic thrombocytopenic purpura (TTP), which has undergone some major changes in the last decade, resulting in improved diagnosis and treatment pathways of the disorder. The mainstay of treatment remains plasma based therapy, replacing the metalloprotease, ADAMTS13. Steroids still have an impact in the initial disease, but therapy to

remove ADAMTS13 autoantibody has primarily been replaced by anti CD 20 monoclonal therapy. The timing of this therapy remains debated, but early use is more likely associated with improved outcomes. Furthermore, use prophylactically can prevent TTP relapse. However, diagnosis and follow up relies on the availability of ADAMTS13 assays. The current role of ADAMTS13 parameters and other prognostic factors were discussed. There are 2 significant therapies that are undergoing clinical trials for the treatment of TTP. First is the nanobody Caplacizumab. Its use at Initial presentation provides an important adjunct therapy, bridging the period for immunomodulatory treatment to be effective, but also preventing further microthrombi formation and potentially faster normalisation of platelet count. Secondly, the role of recombinant ADAMTS 13. Initial studies which are planned in congenital TTP as a replacement for plasma infusion, but this provides an exciting alterative in immune TTP.

2. Main Session Topic: ADAMTS13 and TTP

Moderators: Simon De Meyer (Belgium), Koichi Kokame (Japan)

Karen Vanhoorelbeke (Belgium)

Immunoprofiling of ADAMTS13 Autoantibodies in TTP

Dr. Vanhoorelbeke presented data on autoantibodies in targeting ADAMTS13 to cause the life threatening disease acquired TTP. Recently it was shown that ADAMTS13 can adopt a ‘closed’ conformation by interaction between its spacer and CUB domains. Disruption of this spacer-CUB interaction by addition of the VWF D4CK fragment or an activating anti-CUB antibody (Ab) changes the ADAMTS13 conformation, resulting in exposure of cryptic epitopes. Dr. Vanhoorelbeke and colleagues showed that ADAMTS13 has an altered conformation during acute phase in acquired TTP and identified one anti-cysteine/spacer domain Ab (1C4) that recognized a cryptic ADAMTS13 epitope. Ab 1C4 captured full length ADAMTS13 only when its conformation was altered by addition of the activating Ab 17G2. ADAMTS13 in healthy individuals (n=33) adopts a closed conformation where the spacer and CUB domains interact and the epitope of 1C4 is inaccessible. The conformation of ADAMTS13 is altered in acquired TTP patients (n=6) as the cryptic epitope of 1C4 was readily available. The conformation of acquired TTP patients is altered compared to healthy people and sepsis patients. Exposure of cryptic epitopes in the cysteine/spacer domain might induce immune responses and explain autoAb development in acquired TTP. Dr. Vanhoorelbeke’s group also cloned anti-ADAMTS13 autoAbs from aTTP patients and showed that these autoAbs recognize cryptic eptiopes. Epitope mapping revealed that Ab TTP73-1 is directed against the cysteine/spacer domain, while the Ab ELH2-1 recognizes the TSP1-2/3 domain of ADAMTS13. The Abs did not capture ADAMTS13, MDTCS or T2C2, implying that their epitopes are cryptic. They cloned two anti-ADAMTS13 autoAbs from aTTP patients which recognize cryptic epitopes in ADAMTS13. Identifying which factors induce conformational changes in ADAMTS13 in aTTP patients will allow to elucidate autoantibody formation in these patients.

Hisanori Horiuchi (Japan)

A proposal of VWF large multimer index for standardization of the quantitative description of VWF multimers among laboratories

Dr. Horiuchi and co-workers proposed the VWF large multimer index as a quantitative value to evaluate acquired von Willebrand syndrome (AVWS) caused by excessive high shear stress (Tamura et al, J Atherosclerosis Thrombosis 22, 1115-1123, 2015). Patients with extraordinary high shear stress by some diseases such as aortic stenosis and under mechanical circulatory support often develop AVWS characterized by loss of the VWF large multimers and is diagnosed by VWF multimer analysis. The data are qualitatively evaluated with exceptions where the 'large multimer ratios' are evaluated in some previous papers. However, the values are so various among papers since the VWF multimer analysis is based on a western blot analysis and the band intensities are intentionally determined. In analysis of healthy subjects these ratios are 20-35%, and severe aortic stenosis patients are 15-30% (Tamura et al, 2015) while the another group has shown that those with severe aortic stenosis are 5-14% (Vincentelli et al, NEJM, 349:343-349, 2003). Further, the ratios of patients treated with left ventricular assist device (LVAD) have been reported 15-60% (Meyer et al, JACC HF, 2, 141-145, 2014). Dr. Horiuchi and colleagues have started the acquired von Willebrand syndrome co-existing with cardiovascular diseases (AVeC) Study and the LVAD-AVWS Study. Both studies are multi- institutional prospective studies where AVWS is quantitatively evaluated using the VWF large multimer index, defined as the large multimer ratios of a patients divided by that of a standard plasma. The index expresses the VWF large multimer amount of a patient as a ratio of standard plasma. They showed that the index is inversely-correlated with severity of aortic stenosis (Tamura et al, 2015) and that the severity of AVWS in patients treated with LVAD is far severer than those with severe aortic stenosis (Sakatsume et al, J Artificial Organs 9, 289, 2016).

3. Main Session Topic: Comparison and Standardization of VWF-platelet binding assays

Moderators: James O’Donnell (Ireland), Sandra Haberichter (USA)

Johan Boender (The Netherlands)

VWF:GPIbM, VWF:Ab, and VWF:RCo in the Willebrand in the Netherlands (WiN) cohort

Dr. Boender reported on measuring platelet-dependent von Willebrand factor (VWF) activity that is crucial for the diagnosis and classification of von Willebrand disease (VWD). There are now several widely used VWF activity assays, but it is not known if differences in assay characteristics translate to clinically relevant differences. Dr. Boender and colleagues compared three widely used VWF activity assays: VWF:RCo (BC von Willebrand reagent, Siemens), VWF:GPIbM (INNOVANCE VWF Ac, Siemens), and VWF:Ab (HemosIL® VWF Activity, Intrumentation Laboratory) in 661 VWD patients included in the WiN Study. A high correlation

was observed between the three assays (Pearson r>0.95) with only a small bias. However, assays had an absolute difference of 10 IU/dL or more in 16-24% of cases. Differences in assay result led to a different VWD classification in up to 26% of patients, depending on the assay used. The choice of platelet-dependent VWF activity therefore has a significant impact on the classification of VWD. Future studies incorporating VWF genetics will help determine which assay most accurately classifies VWD.

Sandra Haberichter (USA)

VWF:GPIbM, VWF:GPIbR, VWF:RCo in the Zimmerman Program

Dr. Haberichter reported on the comparison of platelet-dependent VWF activity assays. VWF platelet-binding activity was assayed for healthy controls, type 1 VWD, and types 2A, 2B, 2M VWD subjects recruited through the Zimmerman Program (USA) using 1) in-lab developed VWF:GPIbM ELISA, 2) in-lab developed VWF:GPIbR ELISA, and 3) automated VWF:RCo assay (Siemans). VWF:GPIbM and VWF:GPIbR assay results were compared to VWF:RCo that has been considered the gold standard. Good correlation was observed for healthy controls and type 1 VWD subjects for both VWF:GPIbM and VWF:GPIbR. VWF:GPIbM assay results were not affected by the common VWF variant p.D1472H which results in artifactually reduced VWF:RCo activity. The in-lab developed VWF:GPIbR ELISA did not detect reduced platelet-binding activity in Type 2M VWD subjects which would lead to misdiagnosis of these subjects. VWF:GPIbM activity was increased in type 2B VWD patients resulting in a normal or increased VWF:GPIbM/VWF:Ag ratio. These patients could be identified based upon reduced VWF:CB/VWF:Ag and abnormal multimer structure. The VWF:GPIbM assay demonstrated reduced CV and, as well as a reduced LLOD (2.0 U/dL) compared to VWF:RCo. VWF:GPIbM is an excellent alternative to the long-standing VWF:RCo activity assay.

Anthony Hubbard (United Kingdom)

Assignment of standard for VWF:GPIbM and VWF:GPIbR

Establishment of an agreed unitage for VWF:GPIbR and VWF:GPIbM methods is required to support harmonisation and long-term continuity of test results. The objective is to assign IU values for these methods to the WHO 6th International Standard Factor VIII / VWF Plasma (07/316). Two different approaches were presented, 1) the assignment of a new IU by assay of the WHO 6th IS relative to local normal plasma pools, or, 2) adoption of the VWF:RCo value assigned to the WHO 6th IS. The latter approach relies on consensus that VWF:RCo, VWF:GPIbR and VWF:GPIbM methods measure the same analyte and this is consistent with the SSC recommendations on nomenclature which group all three methods as platelet- dependent VWF activity / GPIb binding activity. A survey of ISTH members with a registered interest in the VWF sub-committee indicated a clear majority (28:3) for adopting the VWF:RCo value for all three methods. This proposal will be circulated to the SSC sub-committee chairs and experts for endorsement before submission to WHO. A poll of the VWF SSC subcommittee

audience demonstrated acceptance of the proposal of adopting the VWF:RCo value for all three methods.

4. Main Session Topic: Delineation of “Low VWF” and type 1 VWD – Results from large population studies

Moderators: Frank Leebeek (Netherlands), Veronica Flood (USA)

Paula James (Canada)

Results from the Canadian study

The ISTH bleeding assessment tool (ISTH-BAT) bleeding score (BS) includes all bleeding episodes in a patient’s history. Dr. James presented interim BS collected in 489 subjects from the US Zimmerman Program, Canada and Irish LoVIC cohort to determine its potential utility in patient management by evaluating bleeding symptoms over time in relation to VWD type, levels, age and gender. Original ISTH-BAT scores (BS) included entire history at time of enrollment and interim bleeding scores (1BS) represented bleeding that had occurred since the BS was obtained. The original BS/year (BS/yr) and interim BS/year (1BS/yr) were calculated as a means to standardize the scores over time. Index case (IC) BS correlated with VWF levels in subjects with VWF:RCo<30 IU/dl (8) followed by 30-50 (7) and >50 (5) (p<0.05). There was a significant difference in 1BS and 1BS/yr in VWD subjects with levels <30 (5, 1.2) and 30-50 (4, 0.8) (p<0.05), but no difference between levels 30-50 and >50 (4, 0.8). 1BS in type 3 IC (13) was greater than type 2 (7) and type 1 (4) (p<0.01). Type 1 IC females had higher BS (7) than males (5) (p<0.01), however the 1BS and 1BS/yr were similar between males (4, 0.8) and females (4, 0.9). Original BS were lower in pediatric patients (5) than adults (8) (p<0.0001), however there was no significant difference in 1BS or 1BS/yr. When type 1 IC (VWF<50) were separated by abnormal or normal BS, there was a difference in 1BS (4, 3.5) (p<0.05), but not 1BS/yr (1, 0.7) nor differences in VWF levels. Higher interim 1BS in subjects with VWF:RCo <30 show correlation between low VWF levels and increased bleeding. There was no difference in 1BS between males and females nor pediatric vs. adults. Having a positive or negative original BS did not predict a difference in 1BS/yr in type 1 patients. The interim 1BS may be a valuable tool to help monitor the success of treatment and bleeding symptoms over time in VWD patients.

Frank Leebeek (The Netherlands)

Results from the WiN study

There is lack of consensus regarding the cut-off values for the diagnosis of von Willebrand disease (VWD), and criteria differ between countries and guidelines. Although the clinical relevance of von Willebrand factor (VWF) levels ≤30 IU/dL is well-established, the relevance of “low VWF” (VWF levels 30-50 IU/dL) is less clear. Dr. Leebeek reported on the examination of phenotypic and genotypic data from patients with type 1 VWD included in the WiN Study. All patients had historically lowest VWF levels ≤30 IU/dL. Type 1 VWD was defined as centrally measured VWF activity/antigen ratio ≥ 0.6. The 404 patients with type 1 VWD were then divided into four groups based on centrally measured VWF:Ag at inclusion in the WiN study (<30 IU/dL,

30-39 IU/dL, 40-49IU/dL, and ≥50 IU/dL).Age increased per group (median age 41 years in the VWF:Ag 30 IU/dL group, median age 51 years in the VWF:Ag 50 IU/dL group). All groups had a female predominance of 64-71%. There was no difference in bleeding score between groups, nor was there a difference in bleeding incidence in the year prior to inclusion in the WiN Study. The prevalence of a variant in the VWF gene decreased with higher VWF:Ag: 79% of patients with VWF:Ag <30 IU/dL, compared with only 32% of patients with VWF:Ag ≥50 IU/dL, had a VWF variant. In conclusion, bleeding phenotype in previously diagnosed type 1 VWD patients with current VWF:Ag >30 IU/dL was as severe as of patients with VWF:Ag <30.

Pamela Christopherson (USA)

Results from the Zimmerman study

Dr. Christopherson showed results from the type 1 cohort (n=310) of subjects enrolled in the US Zimmerman Study. Data was separated by VWF:Ag (<30, 30-39, 40-49, and >50) and VWF:Ag, VWF:RCo, VWF:GPIbM, bleeding score and sequence variant data summarized for each of the VWF:Ag groups. There were 133 subjects with VWF:Ag<30 who had mean VWF:Ag, VWF:RCo and VWF:GPIbM of 12, ISTH bleeding score (ISTH BS) of 5 and in this group, 86% of the subjects had sequence variants (SV) identified by full-length VWF sequencing. In the VWF:Ag 30-39 group, there were 56 subjects with mean VWF:Ag 36, VWF:RCo 35, GPIbM 40, ISTH BS of 5 and 59% with SV. 79 subjects had VWF:Ag between 40-49 with a mean VWF:Ag 45, VWF:RCo 43, GPIbM 55, ISTH BS of 4 and 41% with SV. There were 42 subjects who had VWF:Ag>50 who had been diagnosed on a low VWF:RCo result. The mean VWF:Ag of this group was 56, VWF:RCo 47, VWF:GPIbM 60, ISTH BS 5 and only 41% SV. The results showed that between the <30 and 30-50 group there is a difference in mean levels and percent sequence variants identified, but no difference in bleeding scores, even when scores were separated by positive or negative by age and gender. In the group of VWF:Ag >50 where the diagnosis was made on a sole low VWF:RCo level, that the majority of those cases were found to be in the normal range when VWF:GPIbM was performed, confirming the utility of VWF:GPIbM over VWF:RCo.

Michelle Lavin (Ireland)

Results from the Irish (LoVIC) study

Dr. Lavin presented data on the prospective, longitudinal Low VWF in Ireland Cohort (LoVIC) study that recruited only adults registered on the national bleeding disorder registry with Low VWF levels (plasma VWF 30-50 IU/dL on two occasions, three months apart). For the 144 individuals enrolled to date they determined bleeding phenotype using the ISTH BAT and Condensed MCMDM-1 VWD bleeding scores (BS), including only symptoms prior to their diagnosis. They observed significant bleeding histories in the majority of LoVIC patients, with positive BS in 82.5% using the ISTH BAT and 68.5% using the Condensed MCMDM-1 score. Although a trend was seen towards higher ISTH BAT score with lowest plasma VWF levels within the 30-50 IU/dL range, this did not reach significance (p=0.06). To further investigate this bleeding phenotype, additional hemostatic studies (including platelet aggregation, nucleotides and factor assays) were performed. Concomitant mild coagulation defects were identified in only 13 subjects and did not

impact on bleeding phenotype. Although platelet VWF:Ag and VWF:CB levels were reduced in the subgroup of LoVIC patients studied (n=54) in comparison to controls (n=22), this was not observed to result in higher ISTH BAT scores.The pathophysiology underlying Low VWF levels was examined, however, deleterious VWF mutations were detected only 42% of patients. Plasma FVIII:C/VWF:Ag ratios were significantly increased in LoVIC patients compared to controls (mean 1.29 versus 1.07; p<0.0001). In contrast, increased plasma VWF:pp/VWF:Ag ratios > 3 were observed in only 6% of the total cohort. Taken together, these platelet and plasma VWF data suggest that reduced plasma VWF levels in LoVIC patients are predominantly attributable to decreased VWF synthesis rather than enhanced VWF clearance. In those LoVIC patients with timed fall-offs post DDAVP available, plasma VWF:Ag and VWF:RCo levels were >50 IU/dL in all patients at 1 and 4 hours. Indeed, peak 1 hour VWF:Ag and VWF:RCo levels exceeded 100 IU/dL in 88% of patients (n=42). Importantly, the response to DDAVP was also sustained with both VWF:Ag and VWF:RCo >100 IU/dL after 4 hours in 72% subjects. When utilized, DDAVP was observed to be an effective prophylaxis for procedures in LoVIC subjects.

Augusto B. Federici (Italy)

Results from the 3Winters-Ips Study and MAGICWIA-ops Project

Dr. Federici gave an update on the type 3 von Willebrand disease international registries and inhibitor prospective study (3Winters-Ips). This study includes assessment of bleeding score, assessment of VWF levels and function, VWF molecular defect identification, and detection of anti-VWF inhibitors. The clinical prospective observational study on the management of confirmed type 3 VWD cases includes follow-up studies such as repeated BAT, efficacy and safety of VWF concentrates and inhibitor formation. Dr. Federici also introduced the MAGICWIA-OPS study. The primary objective of this study is to identify patients with GI bleeds in a large cohort of cases with inherited or acquired VWD. Clinical bleeding information and diagnosis of patients will be determined and efficacy and safety of VWF treatment products evaluated.

Veronica Flood (USA)

Discussion of definitions and guidelines

Questions raised include whether the VWD community should consider “Low VWF” as a “disease state” or more as a “risk factor” for bleeding. This question brought up the issue of cause and effect – If 2% of the general population has VWF levels consistent with “Low VWF”, then how does one establish causality? In addition, few VWF mutations are identified in these subjects to date, and the causative nature of most mutations is unknown. VWF mutation analysis is not clinically available for many physicians. It was also stated that it is unclear whether sequence variants, including variants of unknown significance, are identified in people who do not bleed. Much discussion revolved around bleeding scores in addition to determination of VWF levels. The reliability of bleeding score and potential bias when obtaining

the bleeding score was also considered. The utility of various assays in the diagnosis of VWD was also discussed. Many patients are diagnosed on the basis of one low assay value, such as VWF:RCo which has a notoriously high CV. It was proposed that median levels may be used, rather than VWF levels from one patient sample. In addition, interpretation of VWF levels in children may be problematic, partilcularly in patients who have yet to be exposed to major hemostatic challenges. A prospective study was proposed to examine low VWF level individuals in whom no bleeding is found. These individuals potentially could have another hypercoaguble variable (such as FV Leiden, PAI-1 level, fibrinolytic activity) that may offset bleeding. It may be that current individuals diagnosed with VWD due to low VWF are diagnosed because of bleeding, but likely many others with similar levels remain undiagnosed.

Women's Health Issues in Thrombosis and Hemostasis

8 July 2017 9:00 – 13:15 Chairman: Rezan A. Abdul-Kadir Co-Chair: Hannah Cohen, Ian Greer, Susan Halimeh, Ida Martinelli, Predrag Miljic, Maha Othman Rezan A Kadir (UK) welcomed all participants on behalf of the co-chairmen. She thanked the co-chairs for their contribution to the programme .She provided a brief history of women subcommittee and how the membership has expanded from 26 members 2014 to over 300 in 2017. She also gave an overview of current SSC collaborative work and encouraged attendees and SSC members to become involved with future projects. Report on the “Women’s Health Issues in Thrombosis and Haemostasis Symposium 2017” was provided by professor Benjamin Brenner

Subcommittee session 1 chaired by Hannah Cohen, UK and Susan Halimeh, Germany - Use of IVC filter in pregnancy by Dr Maeve P Crowley (UK) DR Crowley presented a literature review on the topic, Cochrane review in 2010 revealed inadequate evidence, thus unable to make recommendations. PREPIC study – excluded pregnant women. Showed decrease in risk of P.E. but increased DVT. No effect on mortality. PREPIC2 study – Data did not support use of IVC filter. An American observational study by Sarosiek et al 2013, looked at suprarenal IVCs (esp. relevant to pregnant women). Kalva et al 2008 – American study, did include pregnant women. Both studies highlighted high number of complications. Review of current guidelines - Overall poor evidence base, mostly using case series. Australia/NZ 2011 – consider for pregnant women with recurrent DVT. SIR 2011 – use suprarenal IVC for pregnant women BLSH 2006 – pregnant women with contraindications to anticoagulation with extensive VTE before delivery. Based on 3 case series. RCOG 2015 – consider in the peri-partum period SOGC 2014 – pregnant women with VTE and contraindications to anticoagulation ACCP 2012 – restrict to proven DVT with recurrent PE despite anticoagulation as increased risk of complications Conclusions: Poor evidence base, Often extrapolated from studies using non-pregnant population, Importance of multidisciplinary working

-Thrombolysis (systemic or catheter directed) in pregnancy by Prof James Douketis Canada PEITHO-2 trial – right ventricular dysfunction cases. Looked at lytic vs conventional treatment. Better outcomes for lytic but risk of stroke and bleeding. ACCP 2016 – Recommendations : no thrombolysis if no hypotension. If PE and hypotension or clinical deterioration then thrombolysis to be considerd. CaVent trial 2012 (non-pregnant population). Significant reduction in post-thrombotic syndrome (PTS) and risk of major bleed <3%. ATTRACT trial 2017 - no difference overall seen in PTS. 25% reduction in PTS for moderate –to-severe iliofemoral DVTs (relevant to pregnant population). <2% rate of bleeds for catheter-directed thrombolysis (CDTL). This may influence recommended management of pregnant women soon.

Thrombolysis in pregnancy in the literature is mostly case reports. Guideline by Kearon 2016 - CDTL if increased risk of bleeding/failed systemic lysis/adequate personnel. Recommendations – consider CDTL/surgical thrombectomy in immediate postpartum for women after Caesarean section

- Paroxysmal Nocturnal Haemoglobinuria (PNH) in pregnancy - Morag Griffin, UK PNH – rare and life-threatening condition with 35% mortality within 5 years before introduction of eculizumab treatment. High maternal and fetal mortality with PNH in pregnancy. Case report of woman with PNH established on eculizumab who got pregnant and decided to continue. No complications and no drug found in umbilical cord or breast milk (overall studies show small amounts found in cord). Pregnancy in PNH patient is now an indication to start eculizumab treatment. Thrombosis is leading cause of death for these patients. Recommend monitor LDH during pregnancy to titrate eculizumab dose. Within presenter’s own unit – use eculizumab and treatment dose LMWH in pregnancy unless granulocyte clone <20% and not already on eculizumab.

- Anti-thrombin deficiency in pregnancy - Hannelore Rott, Germany This is a rare condition with increases risk VTE. >200 mutations reported. Type I – quantitative, type II qualitative. More risk with Type I and missense mutation. There is possibly increased risk of arterial thrombosis with null and type II mutations. 50% risk VTE if past history VTE. Risks for women with AT while pregnant – VTE despite LMWH ACCP 2012 recommendations – criticised for basing guidelines on study using composite of Protein C, S and anti-thrombin deficiency (AT) outcomes. Presenter’s own centre has established reference ranges for d-dimer levels per trimester. Used to monitor and titrate LMWH for AT patients when pregnant. Recommends genetic testing ideally pre-conceptually to identify Type I patients.

- Ligneous conjunctivitis in women - Pratima Chowdary, UK Case presentation and review of reported cases in literature was provided for this rare condition. Gynaecological presentation includes ligneous cervicitis and endometritis. Option for management discussed. Trial is in progress for plasma derived plasminogen concentrate therapy – results are awaited

- Pelvic vein thrombosis - Tejal Amin, UK Ovarian vein thrombosis – increased risk in pregnancy especially post-partum/post Caesarean. Prevalence is higher than previously thought. Dilemma and controversy on diagnosis and management discussed

-Risks of thromboembolism associated with hormonal contraceptives in Japanese women compared to Western women - Takao Kobayashi Dr Takao Kobayashi presented VTE risk with different types of hormonal contraceptives in Japanese women in comparison to western women.

Educational session (chair: Maha Othman, Canada and Ian Greer, UK - Harnessing the power of next generation sequencing for the diagnosis of bleeding disorders in women - Dr Keith Gomez

Excellent presentation provided up-to-date and progress in the field. Generally the clinical uses of genetic diagnosis are; predict phenotype, confirm severity, identify carriers and Pre- natal and PGD to prevent transmission Gouw 2012 – Evidence that factor levels for carriers is inversely proportional to ISTH bleed score but not completely predictive. Many with normal factor level and increased score. Next generation sequencing also referred to as high throughput sequencing. Different methods used: exome sequencing, whole genome sequencing Targeted gene pool – all genes in physiological network, increases pick up of variants of unknown significance. Next generation sequencing very effective for when candidate gene already suspected, less if no candidate Flowchart for suggested role of different genetic tests in diagnosis

- DOACs and women – overview - Jan Westendorf- Beyer Germnay An excellent overview was given in relation to DOACs in women. Common indications are – management of VTE and atrial fibrillation (AF). Gender-related safety and efficacy – no difference in efficacy between men and women but women are at an increased risk of bleeding complications with DOAC (with subgroup analysis) – vaginal bleeding a major factor. Study by Martinelli 2016 highlighted significant risk of HMB with DOACs as well as study by Kline 2016 et al. Data from Jan Westendorf – Beyer 2016 – Women with underlying abnormalities more likely to develop and get progressive HMB. May be some confounders in these findings – recall/selection bias, need to stop hormonal contraception due to VTE Contraception in young VTE patients – no data supporting cessation of combined oral contraceptive pill (COC) – risk is theoretical. But should only continue COC while on anticoagulation, would need to discontinue after treatment stopped. DOAC in pregnancy- animal studies showed potential harm. Lab models with human placenta showed can transfer across placenta. Case series collated (233 cases) – 137 with known outcomes; 39 TOPS, 31 miscarriages, 67 live births. 64 births with no abnormalities. ISTH registry is discussed and call for data entry to the registry

Subcommittee session II - Registries and projects (chair: Ida Martinelli, Italy Predrag Miljic, Serbia)

-The use of thrombo-elastography in pregnancy: registry - Maha Othman, Canada Report on findings of this SSC project presented. Challenges for the use of these tests include cost, availability and training. Overall use is poor; most commonly used in PPH, DIC, to guide transfusion. Data gathered not sufficient to inform guidance/position statement, therefore literature review performed. PRISMA systematic review – 48 abstracts examined, no RCTs. TEG/ROTEM show pregnancy is a hypercoagulable state, reference ranges now reported. Strong correlation with conventional testing has been shown especially fibrinogen. Transfusion algorithms based on these tests now available.

-PPH core outcome set - Beverley Hunt, UK PPH RCTs – 14 different outcomes used, difficult to compare and variation in definitions of outcomes. Surrogate outcomes used that are often less relevant Core outcomes sets = minimum set of critically important outcomes to be used in future trials. Aim to develop outcomes for prevention and treatment of PPH.

-PPH and tranexamic acid TA– WOMAN trial - Beverley Hunt, UK International multicentre trial of TA in PPH. Women > 16 years and who had a clinical diagnosis PPH were randomly to 1 g of intravenous TA or a matching placebo, in addition to usual care.

Excluded those needing tranexamic acid (TXA) clinically of who had contraindications to TXA. Treatment was 1g IV, repeated if ongoing bleeding after 30 mins or later if further bleed episode End point changed to death and sample size increased to adequately power (found too many doctors recruiting patients who already had a plan for hysterectomy e.g. women with placenta praevia). Found significant reduction in death due to bleeding with TXA use. No increase in rate VTE mortality TA was Eequally effective for vaginal delivery and Caesarean section and with different causes for bleeding. No difference in rates of hysterectomy, organ failure or amount of blood products used. Significant reduction in rates of laparotomy for bleeding with TA. Similar use of uterotonics in both groups. Similar outcomes for quality of life and neonatal outcome in both groups Conclusion of the study - TA reduces death due to bleeding in women with post-partum haemorrhage with no adverse effects. When used as a treatment for post-partum haemorrhage, tranexamic acid should be given as soon as possible after bleeding onset

- Artemis – the YEARS diagnostic study in pregnant patients with symptomatic PE- Menno Huisman- Netherland Looking at diagnosis and management of PE in pregnancy. Controversies in role of D dimers, v/q vs CTPA discussed as well as risks of radiation. Study method presented - Ongoing recruitment

- On-line interventional survey on management of hormonal contraceptive use after diagnosis of an acute VTE- Frederikus Klok, Netherland Literature review. Expert opinions sought. The project aims to evaluate practice with survey. Discrepancies found between all three sources of information, e.g.: Management of abnormal uterine bleeding (AUB) in women with diagnosis VTE; literature – LNG-IUD, experts – tranexamic acid, do not stop anticoagulation, survey – 20% reduce dose anticoagulation; 10% stop anticoagulation and insert IVC filter; 50% TXA Is COC associated with risk of recurrent VTE?: Literature – no, Experts in anticoagulation – no increase in risk - AUB – 50% stop COC, Survey – 84% stop COC

-Assessment of the thrombogenic potential of ovarian stimulation/in vitro fertilization - Angela Pereira, Maria Efthymiou UK This Project aims are to determine : 1. Whether raised basal endogenous thrombin potential (ETP), as assessed by thrombin generation (TG) testing, has predictive value for the success of IVF (embryo transfer); 2. Whether there is an association between ex vivo TG and oestrogen levels during ovarian stimulation / IVF treatment. 49 patients recruited. Interim results presented. Final results to be presented next meeting

Rezan A Kadir – thanked the presenter for their valuable contribution and attendee for their attendance and interest- the meeting closed.