From www.bloodjournal.org by guest on January 5, 2017. For personal use only. Regular Article

THROMBOSIS AND HEMOSTASIS Design and characterization of an APC-specific for the treatment of hemophilia

St´ephanieG. I. Polderdijk,1 Ty E. Adams,1 Lacramioara Ivanciu,2 Rodney M. Camire,2 Trevor P. Baglin,3 and James A. Huntington1

1Department of Haematology, University of Cambridge, Cambridge Institute for Medical Research, Cambridge, United Kingdom; 2Division of Hematology, The Children’s Hospital of Philadelphia, Philadelphia, PA; and 3Department of Haematology, Addenbrooke’s Hospital, Cambridge University Hospitals National Health Service Trust, Cambridge, United Kingdom

Key Points Hemophilia is a bleeding disorder caused by deficiency in factors VIII or IX, the two components of the intrinsic Xase complex. Treatment with replacement factor can lead to • The endogenous inhibitors of the development of inhibitory antibodies, requiring the use of bypassing agents such as APC also inhibit other factor VIIa and factor concentrates. An alternative approach to bypass the Xase complex is to inhibit endogenous activities. Activated (APC) breaks down rendering them unacceptable the complex that produces by proteolytically inactivating factor Va. Defects in for treatment of hemophilia. this mechanism (eg, factor V Leiden) are associated with thrombosis but result in less • Rationally designed APC- severe bleeding when co-inherited with hemophilia. Selective inhibition of APC might therefore be effective for the treatment of hemophilia. The endogenous inhibitors of APC specific rescue are members of the serpin family: protein C inhibitor (PCI) and a1-antitrypsin (a1AT); thrombin generation in vitro however, both exhibit poor reactivity and selectivity for APC. We mutated residues in and and restore hemostasis in around the scissile P1-P19 bond in PCI and a1AT, resulting in serpins with the desired hemophilia mouse models. specificity profile. The lead candidate was shown to promote thrombin generation in vitro and to restore fibrin and platelet deposition in an intravital laser injury model in hemophilia B mice. The power of targeting APC was further demonstrated by the complete normalization of bleeding after a severe tail clip injury in these mice. These results demonstrate that the protein C anticoagulant system can be successfully targeted by engineered serpins and that administration of such agents is effective at restoring hemostasis in vivo. (Blood. 2017;129(1):105-113) Introduction

Hemostasis is a crucial part of the physiological response to tissue Hemophilia A and hemophilia B are X-linked genetic disorders damage. When blood components come into contact with extravascular with rates of 1:5000 and 1:20 000 live male births, respectively.7 The cells and proteins, platelets accumulate and the coagulation cascade is bleeding associated with these disorders is the result of a defect or initiated.1 The sequential activation of zymogens to active serine deficiency in fVIII (hemophilia A) or fIX (hemophilia B), the two proteases culminates in the formation of the effector serine components of the intrinsic Xase complex. The mainstream treatment thrombin.2,3 Thrombin activates platelets and cleaves fibrinogen to of hemophilia consists of replacing the affected factor on demand when fibrin, the two major components of a stable blood clot.4,5 Thrombin bleeds occur or by using prophylaxis.8-13 Prophylactic treatment is also cleaves and activates the critical factors VIII (fVIII) and fV, thereby not completely effective and reduces only the frequency of bleeds. allowing the formation of the highly efficient intrinsic Xase (fVIIIa- Neither treatment regimen prevents hemophilic arthropathy (extrava- fIXa) and prothrombinase (fVa-fXa) complexes, resulting in the burst sation of blood into the joints), a major cause of morbidity associated of thrombin formation necessary to establish and maintain the integrity with hemophilia.14 In addition, because the replacement factor is of the hemostatic clot (supplemental Figure 1, available on the Blood effectively a foreign protein, treatment is often associated with Web site). In addition to these procoagulant activities, thrombin plays formation of inhibitory antibodies,15,16 which necessitates using a a crucial role in downregulating itsownformationbyactivationof different class of therapeutics termed “bypassing agents.”17 Bypassing the protein C anticoagulation pathway.6 When bound to its agents increase thrombin generation through mechanisms independent thrombomodulin (TM), thrombin efficiently cleaves protein C to of the intrinsic Xase complex, the most commonly used of which activated protein C (APC), a powerful anticoagulant that proteolytically are fVIIa (NovoSeven), prothrombin concentrates, and FEIBA. inactivates fVIIIa and fVa, thereby shutting down the intrinsic Xase and However, these agents suffer from short half-lives and result in prothrombinase complexes (supplemental Figure 1). variable responses in patients.18-21 They are also less effective than

Submitted 25 May 2016; accepted 6 August 2016. Prepublished online as The publication costs of this article were defrayed in part by page charge Blood First Edition paper, 27 October 2016; DOI 10.1182/blood-2016-05- payment. Therefore, and solely to indicate this fact, this article is hereby 718635. marked “advertisement” in accordance with 18 USC section 1734.

The online version of this article contains a data supplement. There is an Inside Blood Commentary on this article in this issue. © 2017 by The American Society of Hematology

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106 POLDERDIJK et al BLOOD, 5 JANUARY 2017 x VOLUME 129, NUMBER 1

replacement therapy before inhibitor formation and are not point. The apparent (observed) first-order rate constant kobs was calculated from 22 k commonly used prophylactically. the slope of a plot of the natural log of residual protease activity over time. obs Currently all approved bypassing agents improve thrombin was measured for at least 5 different serpin concentrations and plotted against generation by bolstering the levels of coagulation factors. An serpin concentration. The slope of this linear plot gave the second-order rate k alternative approach is to reduce the efficiency of natural anticoagulant constant 2. For each determination, the standard error of the slope is given. mechanisms (eg, using small interfering RNA to knock down Sodium dodecyl sulfate polyacrylamide gel electrophoresis of levels or an antibody to inhibit tissue-factor pathway complex formation inhibitor).23,24 The protein C system is particularly attractive because partial APC resistance reduces the frequency and severity of bleeding in Serpins and human plasma-derived proteases were diluted into 20 mM Tris hemophiliacs, with the common fV Leiden variant providing an early [pH 7.4] and 100 mM NaCl before the experiment. For reactions with APC and proof-of-concept in humans.25-27 ThemodeofactionofanAPC fXa, 2.5 mM CaCl2 was included in the buffer. Serpin and protease were m fi inhibitor is to prolong the life-span of the prothrombinase complex, incubated at equimolar concentrations (1 M nal), and samples were removed at the indicated time points. Sodium dodecyl sulfate (SDS) sample buffer was thereby directly increasing thrombin generation at the site of tissue added with (reducing) or without (nonreducing) 50 mM dithiothreitol, and damage (supplemental Figure 1). samples were place in a boiling water bath to stop the reaction. Samples were then The endogenous inhibitors of APC are members of the serpin run on SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and reaction 28 family. Serpins use a well-characterized mechanism of protease products were visualized by Coomassie staining. inhibition, in which the protease recognizes a stretch of the exposed reactive center loop (RCL) as a (supplemental Figure 2) and is Prothrombin time and activated partial thromboplastin time then trapped in a covalent complex after a large conformational 29 a Fresh frozen human plasma from 3 different donors was mixed to generate change. Several serpins are able to inhibit APC, including 1-antitrypsin pooled plasma. For the prothrombin time (PT) assays, plasma was diluted a 30 31 ( 1AT), inhibitor 1, and protein C inhib- twofold in 20 mM Tris [pH 7.4], 100 mM NaCl, and 2.5 mM CaCl . Then, 50 mL 32 2 itor (PCI). PCI is thought to be the main physiological inhibi- of diluted plasma was mixed with 50 mL of serpin and incubated for 60 seconds, tor of APC; however, its rate-constant for inhibition of APC is and 100 mL PT reagent (TriniClot PT Excel) was added to initiate the clotting – 2 only ;500 M 1s 1, and it is also capable of inhibiting procoagulant reaction. For activated partial thromboplastin time (aPTT) assays, 50 mLplasma proteases such as thrombin, fXa, and fVIIa.32,33 The other circulating was mixed with 50 mL serpin diluted in 20 mM Tris [pH 7.4] and 150 mM NaCl, serpins are also inefficient APC inhibitors that lack specificity. It is 100 mL aPTT reagent (Triniclot automated aPTT reagent) was added and m therefore unlikely that the administration of an endogenous inhibitor incubated for 5 minutes, and 100 L25mMCaCl2 was added to start the clotting of APC would have the desired hemostatic effect. Herein we describe reaction. Time to clot formation for both assays was measured on a Stago STart coagulation analyzer. All reactions were carried out at 37°C and used a final the engineering and characterization of a serpin specific for APC and serpin concentration of 5 mM. show that it is effective in increasing thrombin generation in vitro and normalizing hemostasis in hemophilia B mice. Thrombin generation assays

Normal human plasma was purchased from George King Biomedical. To begin, 40 mLplasmawasmixedwith10mL Technothrombin TGA reagent B (tissue- factor low/phospholipids mix, TechnoClone) and 5 mL protein sample diluted Methods in 20 mM N-2-hydroxyethylpiperazine-N9-2-ethanesulfonic acid [pH 7.4], 150 mM NaCl, and 0.1% bovine serum albumin. To initiate the reaction, 50 Protein expression and purification mL of 1 mM Z-Gly-Gly-Arg-AMC (Bachem) in 15 mM CaCl2 was added. Human PCI with its N-terminal 21 residues truncated (D21, first residue is Ala22) Fluorescence was read by using 360 nm excitation and 460 nm emission was expressed from Escherichia coli and purified as described34 with some wavelengths. Fluorescence units were converted to thrombin concentration by using a thrombin generation assay (TGA) calibration kit (TechnoClone). modifications. Full-length human a1AT with M358R and C232S mutations was expressed from a pET SUMO protein expression system (Life Technologies) Data were analyzed by using the Technothrombin TGA evaluation construct in Rosetta2(DE3)pLysS cells. Protein was purified from clarified cell spreadsheet (TechnoClone). All reactions were performed at 37°C. lysate by using a combination of HiTrap high performance immobilized metal ion affinity chromatography and Q Sepharose columns (GE Healthcare). Tail clip assays a fi The SUMO tag was removed and the 1AT was repuri ed. The human TM Tail clip assays were performed essentially as described.35 Briefly, male wild- extracellular domain, containing a TEV protease cleavage site between EGF type (wt) or hemophilia B mice (BALB/c) (8 to 16 weeks of age) were domains 3 and 4 was expressed in HEK-EBNA cells. All mutations described anesthetized by using 2% isoflurane at 2 mL/min and weighed. Mice were were introduced by site-directed mutagenesis using the QuickChange injected through the tail vein with 200 mL phosphate-buffered saline (PBS) or mutagenesis method and verified by sequencing at Source BioScience a1AT variant diluted into PBS. Five minutes after injection, the tail was cut at (Cambridge, United Kingdom). Detailed descriptions of methods for protein 3 mm diameter and placed into a tube filled with prewarmed saline solution production are provided in the supplemental Data. (37°C); blood was collected for 10 minutes. Collection tubes were centrifuged,

and the saline solution was suctioned off. Lysis buffer (150 mM NH4Cl, 10 mM Determination of inhibition constants KHCO3, 1 mM EDTA) was added, and red cells were allowed to lyse for 10 minutes at room temperature. Samples were centrifuged, and absorbance of the Second-order rate constants of protease inhibition were measured by a supernatant at 575 nm was determined to measure hemoglobin content. A fi discontinuous method under pseudo rst-order conditions, using at least a standard curve using known volumes of blood was constructed to allow fi vefold molar excess of serpin over protease. All reagents were diluted into assay calculation of total blood loss volume. buffer (20 mM tris[hydroxymethyl]aminomethane [Tris; pH 7.4], 100 mM NaCl, 0.2% bovine serum albumin, 0.1% PEG8,000). For reactions with APC and fXa, Intravital microscopy 2.5 mM CaCl2 was included in the buffer. Reactions were incubated at room temperature and were stopped for each time point by the addition of the The real-time in vivo imaging experiments were performed essentially as chromogenic substrate appropriate for the protease used (S2238 for thrombin, described.35 Briefly, male hemophilia B mice ;12 weeks of age were

S2366 for APC and fXIa, and S2222 for fXa). The slope of the linear part of A405 anesthetized, and the cremaster muscle was exposed and stretched across an (absorbance at 405 nm) over time gave the residual protease activity at each time intravital microscopy tray. Mice were infused with 10 mg rat anti-CD41 Alexa From www.bloodjournal.org by guest on January 5, 2017. For personal use only.

BLOOD, 5 JANUARY 2017 x VOLUME 129, NUMBER 1 CHARACTERIZATION OF AN APC-SPECIFIC SERPIN 107

interaction with the of thrombin.34 Superposition of APC on A thrombin in this structure shows that the features observed with PPACK are largely preserved, namely that APC is permissive of large side chains at the S2 and S19 sites and would interact favorably with basic residues (Figure 1B). Therefore, we hypothesized that bulky residues at the P2 and P19 positions would be disfavored by thrombin and that basic residues at these positions might additionally contribute to conferring specificity for APC. This is further supported by comparison of the natural substrates of thrombin and APC (supplemental Table 1). Thrombin APC B Specificity of PCI RCL Lys variants P1′ P1′ P2 P2 Individual and combined P2 and P19 lysine variants were constructed on the PCI scaffold as described in “Methods.” The RCL of wt PCI has aP2Phe,P1Arg,andP19 Ser, so we refer to wt as FRS. Separate and combined P2 and P19 Lys variants are referred to as KRS, FRK, and KRK. To evaluate the effect of the mutations on the inhibitory activity P1 P1 of PCI, variants were incubated with either thrombin (Figure 2A) or APC (Figure 2B), and the reaction products were run on SDS-PAGE Figure 1. Active site properties of thrombin and APC. (A) Electrostatic surface (Figure 2; supplemental Figure 3). This method allows simultaneous representations of human thrombin (1PPB42) and human APC (from 1AUT44), with the small molecule inhibitor PPACK bound into the active site cleft (sticks). (B) evaluation of both inhibitory and substrate behavior (supplemental Zoomed-in view of the active sites of human thrombin (3B9F34) and APC (from Figure 2). We noted that wt PCI rapidly formed complex with thrombin 1AUT), with modeled KRK sequence corresponding to the P2, P1, and P19 positions with complete inhibition in about 1 minute. APC inhibition was con- 34 found in the crystal structure of the PCI-thrombin complex. siderably slower, with the reaction completed after about 4 hours. The 555–labeled antibody and 10 mganti-fibrin Alexa 647–labeled antibody in a total P2 Lys (KRS) mutation slowed complex formation with thrombin, 9 volume of 100 mL. This was followed by infusion of 100 mLPBSor100mL requiring 30 minutes to achieve full inhibition. The P1 Lys mutation (FRK) essentially abolished thrombin inhibition, although the PCI was a1AT variant diluted in PBS. Laser injury was induced in cremaster arterioles by using a pulse-nitrogen dye laser through the microscope objective. Both bright- consumed as a substrate. The inhibitory profiles of these variants for field and fluorescence images were collected for 3 to 4 minutes. Data were APC were essentially identical to wt PCI, indicating that the analyzed by using SlideBook 5 software (Intelligent Imaging Innovations). substitutions were tolerated but did not improve APC recognition. The combined P2 and P19 Lys mutations (KRK) resulted in complete loss of activity against thrombin, similar to what was found for the P19 variant, but they appeared to slow inhibition of APC, approximately Results doubling the time require to achieve full inhibition relative to wt. Second-order rate constants for the inhibition of thrombin and APC by Engineering specificity the PCI variants were determined (Table 1), and they agree with the qualitative SDS-PAGE results.Boththe FRK and KRK variants of PCI The main determinant of serpin specificity is the RCL sequence in and exhibited a substantial increase in specificity for APC over thrombin around the scissile bond. In the standard substrate nomenclature, the (1600- and 400-fold, respectively); however, the rates of APC inhibition scissile bond is denoted P1-P19, with N-terminal residues numbered P2, remained low. P3, and so on and C-terminal residues numbered P29,P39,andsoon.36 All clotting proteases, including APC, favor an arginine in the P1 Specificity of a1AT RCL Lys variants position because of Asp189 ( template numbering) at the base of the S1 pocket. We examined the structures of APC and a1AT is the most prevalent serpin in the blood, circulating at a 38,39 thrombin bound to the same covalent inhibitor (D-Phe-Pro-Arg- concentration of ;2g/L. It has a Met at the P1 position and chloromethylketone [PPACK]) to see if differences in shape and therefore targets chymotrypsin-type serine proteases, including properties in other parts of the substrate might be exploited neutrophil and G. The Pittsburgh mutation, P1 to confer specificity for APC (Figure 1A). PPACK binds in an identical Met-to-Arg, has been observed in a few patients who generally fashion in the 2 structures, with the P1 Arg deep in the S1 pocket, the experience severe bleeding as a result of the switching of specificity P2 Pro in the S2 pocket, and the D-Phe in the aryl binding site (S4 toward the -type serine proteases, including the clotting factors 40,41 pocket). However, PPACK isburied inthe active sitecleft of thrombin, thrombin and fXa. However, a1AT Pittsburgh is also a potent whereas the active site of APC is wide open, especially on either side of inhibitor of APC, with a rate constant 100 times that of PCI.30 We the P1 residue (the S2 and S19 sites). This is primarily the result of the therefore hypothesized that a1AT Pittsburgh might serve as a better difference in size of the 60-loop in APC and thrombin; APC has a template for creating an APC-specific serpin using the Lys mutations single insertion relative to chymotrypsin and thrombin has a 9-residue identified from our work with PCI. insert. The 60-loop in thrombin has been well characterized and acts as Lysine mutations were made individually and in combination at P2 a hydrophobic cap that restricts the S2 and S19 pockets, favoring small and P19 in a1AT Pittsburgh (a1AT has a Pro at the P2 position and a Ser hydrophobic amino acids like Gly and Pro at S2 and Ser in S19.37 In at the P19 position; PRS). Rates of inhibition of APC, thrombin, fXa, addition, the S19 site is also more acidic in APC than in thrombin and fXIa were measured, and the results are given in Table 2. As seen because of the side chain of Asp60a in the position of Lys60h in with PCI, each single Lys mutation dramatically reduced inhibition of thrombin. We previously reported a high-resolution crystal structure of thrombin (;5800-fold for KRS a1AT and ;1700-fold for PRK the PCI-thrombin complex (3B9F) that revealed the details of the RCL a1AT), and the double mutation abolished thrombin inhibition. From www.bloodjournal.org by guest on January 5, 2017. For personal use only.

108 POLDERDIJK et al BLOOD, 5 JANUARY 2017 x VOLUME 129, NUMBER 1

Figure 2. Inhibition of thrombin and APC by PCI A variants. Nonreducing SDS-PAGE of the reactions of (A) human plasma thrombin and (B) APC with PCI 9 Time (min) variants (FRS is wt, KRS is P2 Lys, FRK is P1 Lys, and 0 20 40 80 440 5 M ThrombinPCI 1 5 15 30 6 1 2 4 1 M ThrombinPCI 1 5 1 30 60 120 240 480 1440 KRK is the combined P2 and P19 Lys variant). Native 66.3 66.3 serpin is indicated by a solid arrow, cleaved serpin by 55.4 55.4 an open arrow, and the covalent serpin:protease complex by a shaded arrow. In each gel, lane 1 (M) 36.5 36.5 is molecular weight markers, lane 2 is the protease 31.0 31.0 alone, lane 3 is serpin alone, and the other lanes are FRS (WT) PCI KRS PCI the reactions of serpin and protease at the indicated times.

440 Time (min) M ThrombinPCI 1 5 15 30 60 120 240 480 1 M ThrombinPCI 1 5 15 30 60 120 240 480 1440 66.3 66.3 55.4 55.4

36.5 36.5 31.0 31.0 FRK PCI KRK PCI B Time (min) 440 M APC PCI 5 15 30 60 120 240 480 1440 M APC PCI 5 15 30 60 120 240 480 1 116.3 116.3 97.4 97.4 66.3 66.3 55.4 55.4

36.5 36.5 FRS (WT) PCI KRS PCI

Time (min) M APC PCI 5 15 30 60 120 240 480 1440 M APC PCI 5 15 30 60 120 240 480 1440 116.3 116.3 97.4 97.4 66.3 66.3 55.4 55.4

36.5 36.5 FRK PCI KRK PCI

Inhibition of fXa, although still detectable, was reduced by 400-fold plasma with PRS (control) or KRK a1AT and conducted PT and compared with only ;10-fold for the single Lys mutants. Inhibition of aPTT assays (Table 3). The PRS a1AT control increased clotting fXIa was reduced by about 1000-fold for the double mutant. Crucially, timesinbothassays;however,additionofKRKa1AT to normal APC inhibition was reduced by only sevenfold. The resultant increase plasma had no effect on PT and only a marginal effect on aPTT, in specificity ratio of KRK a1AT relative to PRS a1AT for APC over consistent with some residual activity against fXIa (Table 2). thrombin, fXa, and fXIa is .8000, 50, and 120 times, respectively. The residual anti-fXa activity of KRK a1AT appears to be too – 2 To determine whether the observed decrease in rate of procoagulant low (116 M 1s 1; Table 2) to affect clotting times in these assays. protease inhibition was the result of increased substrate behavior, We concluded that KRK a1AT does not possess any relevant reactions were visualized by SDS-PAGE (Figure 3). For these anticoagulant activity. experiments, fIXa and thrombin:TM were alsoincluded (supplemental To assess the procoagulant activity of KRK a1AT, we used a TGA Figure 4). KRK a1AT rapidly formed a covalent complex with APC in the absence and presence of TM with PRS a1AT as control (Figure 4; (Figure 3A), but no complex formation or cleavage was observed with supplemental Figure 5). Two concentrations of TM were chosen thrombin, even after 4 hours (Figure 3B). Small amounts of complex were observed with fXa (Figure 3C) and fXIa (supplemental Figure 4F), consistent with the results from the kinetic studies. For Table 1. Second-order rate constants of inhibition of thrombin and fIXa, only a hint of inhibitory complex was observed (supplemental APC by PCI variants k 21× 21 Figure 4E). The addition of TM to thrombin had no effect on the ability 2 (M s ) of thrombin to react with KRK a1AT (supplemental Figure 4D). These Variant Thrombin APC SR a fi results indicate that KRK 1AT has a better inhibitory pro le than wt PCI (2.82 6 0.15) 3 104 (6.80 6 0.32) 3 102 0.024 KRK PCI and justify its selection as the candidate anti-APC serpin for KRS PCI* 1.18 3 103*6.03 102* 0.5* efficacy studies. FRK PCI (2.24 6 0.24) 3 101 (8.84 6 0.74) 3 102 39.5 KRK PCI (2.80 6 0.24) 3 101 (2.80 6 0.13) 3 102 10

Effect of KRK a1AT on clotting times and thrombin generation Values are rate constants plus or minus standard error. in vitro *The rate determination for KRS PCI is a preliminary determination from only 2 a measurements (therefore, no error is shown) and was calculated by dividing kobs by For KRK 1AT to be a useful hemostatic agent, it must be devoid of any the serpin concentration. SR stands for specificity ratio of rate constant for APC over residual anticoagulant activity. We therefore spiked normal pooled thrombin. From www.bloodjournal.org by guest on January 5, 2017. For personal use only.

BLOOD, 5 JANUARY 2017 x VOLUME 129, NUMBER 1 CHARACTERIZATION OF AN APC-SPECIFIC SERPIN 109

Table 2. Second-order rate constants of inhibition of thrombin, APC, fXa, and fXIa by a1AT variants 21 21 k2 (M ×s ) Variant Thrombin APC fXa fXIa

5 5 4 5 PRS a1AT (2.93 6 0.18) 3 10 (1.08 6 0.071) 3 10 (4.13 6 0.24) 3 10 (4.00 6 0.13) 3 10 1 4 3 KRS a1AT (5.10 6 0.28) 3 10 (6.48 6 0.71) 3 10 (3.93 6 0.31) 3 10 ND 2 4 3 PRK a1AT (1.74 6 0.17) 3 10 (9.57 6 1.37) 3 10 (4.89 6 0.16) 3 10 ND 4 2 2 KRK a1AT No detectable inhibition (1.51 6 0.17) 3 10 (1.16 6 0.10) 3 10 (4.71 6 0.37) 3 10

Values are rate constants plus or minus standard error. ND, not determined.

empirically: one that resulted in an ;50% reduction in thrombin Effect of KRK a1AT on clotting in vivo generation and one that essentially abolished thrombin generation. The control anticoagulant PRS a AT completely inhibited thrombin The high sequence identities between human and mouse APC and 1 thrombin (72% and 87%, respectively) suggest that the relevant generation in normal plasma when added at a concentration of m features of the active sites are preserved. This was confirmed by 0.5 M (supplemental Figure 5A). Consistent with results from the PT 42 43 assay, KRK a AThadnoeffectonthrombingenerationwhenaddedto comparing structures of human and mouse thrombin (supplemental 1 Figure 8A) and by comparing a mouse APC model with the crystal normal pooled plasma (Figure 4A). However, when TM was added, 44 KRK a AT dose-dependently increased thrombin generation structure of human APC (supplemental Figure 8B). Mouse thrombin 1 a (Figure 4B-C). Similar results were obtained in a TGA using plasma did not react with KRK 1AT (supplemental Figure 9), and inhibition from patients with hemophilia A or hemophilia B (Figure 4D-E; of mouse APC was preserved (twofold reduction in rate constant; a supplemental Figures 6 and 7). supplemental Table 2). In addition, KRK 1AT dose-dependently increased thrombin generation in mouse hemophilia B plasma spiked with TM (supplemental Figure 10). We concluded that mouse models of hemostasis could be used to test the in vivo efficacy of KRK a1AT. Two assays were used to assess the ability of KRK a1AT to promote hemostasis in a hemophilia B mouse: tail clip and intravital laser injury. A In the tail clip assay, the tail was transected, and blood was collected for AT a 1 Time (min) 10 minutes after administration of either KRK AT or PBS, and total MMAPC α 1 5 10 15 30 60 120 240 1 97.4 blood loss volume was calculated from a standard hemoglobin curve 66.3 after red cell lysis.35 In this model, KRK a AT dose-dependently 55.4 1 decreased the blood loss (Figure 5A), with the highest dose (15 mg/kg) reducing bleeding to the same level as for wt mice. The effect of KRK 36.5 a1AT was also assessed by using the cremaster arteriole laser injury 31.0 model.45 Clot formation was visualized by fluorescence microscopy APC following deposition of platelets and fibrin with time. KRK a1AT dose- B dependently increased in the amount of platelet (Figure 5B) and fibrin deposition (Figure 5C). Example images taken as snapshots from

AT representative recorded videos are provided in supplemental Figure 11. 1 Time (min) MMThrombinα 1 5 10 15 30 60 120 240 116.3 At the 7.5 mg/kg dose of KRK a1AT, stable clots (platelets and fibrin) 97.4 66.3 occurred in 55% of injuries compared with 0% when only PBS was 55.4 given. For the higher dose (15 mg/kg), stable clots were observed in 85% of injuries (Table 4). Fluorescence quantitation showed a shorter 36.5 lag time for fibrin deposition at the higher dose (Figure 5C). 31.0 Thrombin C

AT Discussion MM1 Time (min) 97.4 fXa α 1 5 10 15 30 60 120 240 66.3 This study was motivated by the observation that bleeding frequency 55.4 and severity are reduced in patients with hemophilia A who co-inherit the fV Leiden mutation.25-27 These observations were supported by

36.5 31.0 Table 3. Clotting times for PT and aPTT assays with PRS and KRK a m fXa 1AT (5 M) Clotting time (s) a Figure 3. Inhibition of proteases by KRK 1AT. SDS-PAGE of the reactions of (A) Variant PT aPTT APC, (B) thrombin, and (C) fXa with KRK a1AT. Native serpin is indicated by a solid 6 6 arrow, cleaved serpin by an open arrow, and the covalent serpin:protease complex Buffer 16.6 0.22 55.0 3.8 by a shaded arrow. (A) and (B) are run under nonreducing conditions, and (C) is run PRS a1AT 50.5 6 5.6 .300 under reducing conditions. In each gel, lane M shows molecular weight markers, lane KRK a1AT 16.5 6 0.3 62.1 6 4.2 2 is the protease alone, lane 3 is KRK a1AT alone, and the other lanes are the reactions at the indicated times. The values shown are an average of 3 measurements with standard deviation. From www.bloodjournal.org by guest on January 5, 2017. For personal use only.

110 POLDERDIJK et al BLOOD, 5 JANUARY 2017 x VOLUME 129, NUMBER 1

Figure 4. KRK a1AT rescues the effect of soluble A B TM in normal and factor-deficient human plasma. (A-B) Representative thrombin generation curves 450 NP 450 NP NP + 0.1 M 1AT NP + 1.25 nM TM showing the effect of KRK a1AT (concentrations 400 NP + 0.5 M 1AT 400 NP + 1.25 nM TM + 0.1 M 1AT 350 NP + 1 M 1AT 350 NP + 1.25 nM TM + 0.5 M 1AT indicated) in normal human plasma (NP) in the (A) NP + 2 M 1AT NP + 1.25 nM TM + 1 M 1AT 300 300 NP + 1.25 nM TM + 2 M 1AT absence and (B) presence of 1.25 nM soluble TM. 250 250 Each curve is an average of duplicates. In (B) the no 200 200 TM control from (A) is shown for reference. (C) Bar 150 150 graph of total thrombin generation in normal plasma 100 100 (given as thrombin concentrations) at 0, 1.25, and [Thrombin] (nM) 50 [Thrombin] (nM) 50 10 nM TM, with increasing concentrations of KRK a1AT 0 0 (up to 2 mM). Each bar is the average of 3 experiments 0 153045607590 0 153045607590 of duplicates, with standard deviation shown by vertical Time (min) Time (min) lines. (D) Bar graph of total thrombin generation in fVIII- deficient (hemophilia A) plasma (given as thrombin concentrations) at 0, 1.25, and 5 nM TM, with increas- a m C ing concentrations of KRK 1AT (up to 2 M). Each bar no TM + 1.25 nM TM + 10 nM TM is the average of 2 experiments of duplicates with standard deviation shown. (E) Bar graph of total 7,000 thrombin generation in fIX-deficient (hemophilia B) 6,000 plasma (given as thrombin concentrations) at 0, 1.25, and 5 nM TM, with increasing concentrations of KRK 5,000 a1AT(upto2mM). Each bar is the average of at least 4,000 2 experiments of duplicates, with standard deviation shown. 3,000

2,000 [Thrombin] (nM) 1,000

0 - 0.1 0.5 1 2 - 0.1 0.5 1 2-0.1 0.5 1 2 [1AT] (M)

D no TM + 1.25 nM TM + 5 nM TM 6,000

5,000

4,000

3,000

2,000 [Thrombin] (nM) 1,000

0 - 0.25 0.5 1 2-0.25 0.5 1 2 - 0.250.5 1 2 [1AT] (M)

E no TM + 1.25 nM TM + 5 nM TM 6,000

5,000

4,000

3,000

2,000 [Thrombin] (nM) 1,000

0 - 0.25 0.5 1 2 - 0.25 0.5 1 2 - 0.50.251 2 [1AT] (M)

crossing hemophilia A and hemophilia B mice with fV Leiden mice and The mechanism of all hemophilia treatments is to increase the conducting tail clip and intravital microscopy studies.46 We thus amount of thrombin generated at sites of vascular damage. This is hypothesized that a serpin with specificity for APC might be an generally achieved by adding missing factors or by boosting the effective hemostatic agent. concentration of either the or substrates that lead to the From www.bloodjournal.org by guest on January 5, 2017. For personal use only.

BLOOD, 5 JANUARY 2017 x VOLUME 129, NUMBER 1 CHARACTERIZATION OF AN APC-SPECIFIC SERPIN 111

Figure 5. KRK a1AT reduces bleeding and increases platelet and fibrin deposition in hemophilia B mice. (A) A p = 0.0238 Total blood loss of either wt (gray) or hemophilia B (black) mice p = 0.0648 injected with either PBS (circles), or KRK a1AT (squares) after tail clip. Each data point is from a single mouse. Lines show the 400 average per group. P values were calculated by using an unpaired Student t test. (B-C) Median accumulation of (B) platelets and (C) fibrin over time after laser-induced arteriole injury after administration of PBS (black line; 8 total thrombi, 5 300 mice) or KRK a1AT (gray-shaded dotted and solid lines; 18 total thrombi, 4 mice at 7.5 mg/kg; 20 total thrombi, 3 mice at 15 mg/kg).

200

Total blood loss ( μ l) Total 100

0 PBS PBS KRK KRK 7.5 mg/kg 15 mg/kg B 500,000 PBS KRK α1AT (7.5 mg/kg) 400,000 KRK α1AT (15 mg/kg)

300,000

200,000

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Median fluorescence intensity 0 0 50 100 150 200 250 Time (s) C 140,000 PBS 120,000 KRK α1AT (7.5 mg/kg) α 100,000 KRK 1AT (15 mg/kg)

80,000

60,000

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Median fluorescence intensity 0 0 50 100 150 200 250 Time (s)

production of thrombin. The fV Leiden mutation and, to a potentially was unacceptably low. Although the rate of inhibition was increased by greater extent, direct APC inhibition increases thrombin generation by adding (data not shown), the relevance of glycosaminoglycans allowing already-formed prothrombinase more time to function. in mediating APC inhibition by PCI in vivo is unclear. Our goal was to engineer a serpin that would inhibit APC with a rate We therefore decided to change the serpin scaffold to a1AT constant of greater than 1 3 104 M–1s21, but without appreciable Pittsburgh, which is known to rapidly inhibit APC and procoagulant inhibition of any relevant procoagulant protease. On the basis of proteases. As with the PCI template, the KRK mutations in the RCL of differences in features of the active sites of thrombin and APC, we a1AT abrogated inhibition of the tested coagulation proteases while chose to alter the 2 residues flanking the primary specificity- maintaining an ability to rapidly inhibit APC. Importantly, coagulation determiningP1residue(P2andP19). Mutating these to Lys in PCI was not inhibited by the addition of KRK a1AT to normal or had the desired effect on selectivity, but the rate of inhibition of APC hemophilia plasma, even at concentrations of 5 mM. KRK a1AT did, From www.bloodjournal.org by guest on January 5, 2017. For personal use only.

112 POLDERDIJK et al BLOOD, 5 JANUARY 2017 x VOLUME 129, NUMBER 1

Table 4. Platelet and fibrin deposition in a cremaster arteriole laser- in plasma,38,39 and immune tolerance to a protein is strongly related to induced injury model 51,52 its endogenous level. Therefore, choosing a1AT as our scaffold for Clot formation developing an APC-specific serpin confers several possible attractive Platelets Platelets 1 features: fast and specific APC inhibition, long half-life, possible No clot only fibrin No. of Total subcutaneous route of administration, and low immunogenic potential. Sample mice injuries No. % No. % No. % If the half-life and subcutaneous availability of KRK a1AT is similar to PBS 5 8 8 100 0 0 0 0 what has been reported for wt a1AT,itispossiblethatweeklyor a KRK 1AT 4 18 2 11.1 6 33.3 10 55.6 fortnightly subcutaneous injections could provide effective prophylaxis (7.5 mg/kg) for either form of hemophilia, even when inhibitors are present. KRK a1AT 3 20 0 0 3 15 17 85 (15 mg/kg) however, retain some residual inhibitory activity against fXIa reflected Acknowledgments in slightly prolonged aPTTs. However, the main function of fXIa in coagulation is the activation of fIX to support formation of the intrinsic This work was supported by a Wellcome Trust studentship (S.G.I.P.). Xase complex. Deficiency in either fVIII or fIX, the basis of hemophilia, therefore renders fXIa redundant. It is important to note that the studies described here used protein purified from an Ecoliexpression system, perhaps explaining the Authorship relatively high doses required for efficacy in the mouse models. 47 Although the activity of a1AT is not affected by glycosylation, its Contribution: J.A.H., T.E.A., T.P.B., and S.G.I.P. conceived of and absence will reduce the half-life of the protein.48 Therefore, the actual designed the study; S.G.I.P. conducted all in vitro experiments; L.I., exposure of KRK a1AT in the efficacy experiments is likely to be R.M.C., and S.G.I.P. designed and L.I. conducted the in vivo substantially lower than what would be expected on the basis of dose. experiments; and J.A.H. and S.G.I.P. wrote the manuscript. PK and efficacy studies using glycosylated KRK a1AT will need to be Conflict-of-interest disclosure: J.A.H., T.P.B., and S.G.I.P. have conducted in the future to determine the actual relationship between shares in ApcinteX Ltd (a company founded to develop KRK a1AT). dose and efficacy. J.A.H., T.P.B., S.G.I.P., and T.E.A. participate in revenue sharing One major advantage of choosing a1AT as the scaffold anti-APC through the commercialization arm of the University of Cambridge. serpin is its 5- to 7-day half-life in the circulation.49 In addition, in The results discussed in this manuscript form part of patent application, contrast to current bypassing agents, which are administered in- Modified Serpins for the Treatment of Bleeding Disorders (WO travenously, the subcutaneous route of administration for KRK a1AT 2015086854 A1). The remaining authors declare no competing might also be available. Studies in rabbits showed that the pharmaco- financial interests. dynamics of subcutaneously administered plasma human a1AT were Correspondence: James A. Huntington, University of Cam- comparable to intravenously administrated protein.50 The risk for cross- bridge, Cambridge Institute for Medical Research, Wellcome Trust/ reacting antibodies developing in response to KRK a1AT is also likely MRC Building, Hills Rd, Cambridge CB2 0XY, United Kingdom; to be low because wt a1AT is present at a high concentration (;2g/L) e-mail: [email protected].

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2017 129: 105-113 doi:10.1182/blood-2016-05-718635 originally published online October 27, 2016

Design and characterization of an APC-specific serpin for the treatment of hemophilia

Stéphanie G. I. Polderdijk, Ty E. Adams, Lacramioara Ivanciu, Rodney M. Camire, Trevor P. Baglin and James A. Huntington

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