Divergent modulation of activated protein C pleiotropic functions by antibodies that differ by a single amino acid

Activated protein C (APC) is a pleiotropic protease generated by the activation of protein C zymogen by the thrombin/thrombomodulin complex. APC exerts anti-inflammatory and cytoprotective functions by the cleavage of endothelial protease-activated receptor (PAR)¹ and extracellular histones.² APC, with protein S as a cofactor, also performs anticoagulant functions by inactivating factor Va (FVa) and FVIIIa. As such, APC has been investigated over the years for its potential in addressing diseases associated with these pathways.³ For example, the role of APC in anticoagulation makes it a potential therapeutic target as a treatment for bleeding. This hypothesis is supported by a murine trauma-induced coagulopathy (TIC) model, in which an APC-resistant ^superFVa variant was able to significantly reduce bleeding.⁴ Furthermore, the blockade of APC with an engineered KRK-α₁-antitrypsin has been shown to reduce annualized bleed rates in hemophilia trials.^5,6 

APC-mediated anti-inflammatory signaling and histone cleavage activities have therapeutic potential in addressing diseases such as sepsis and ischemia, which undergo highly inflammatory conditions and histone cytotoxicity. A recombinant APC (drotrecogin alfa (activated) [DrotAA]) was developed as a treatment for patients with severe sepsis.⁷ However, the efficacy of DrotAA was not replicated in the subsequent PROWESS-SHOCK trial.⁸ Interestingly, a recent analysis of the PROWESS-SHOCK data showed DrotAA was beneficial for patients with a hyperinflammatory phenotype but harmful for hypoinflammatory patients.⁹ Critical to the successful development of an effective therapeutic is the ability to selectively modulate the pleiotropic effects of APC.

Attempts are being made to selectively modulate APC functions. APC-mediated anti-inflammatory signaling and cytoprotective effect are induced by the cleavage of endothelial cell PAR1 at R46^1,10 and extracellular histones.² Potential therapeutics such as TPP-4885 immunoglobulin G2 (IgG2) antibody,¹¹ SR604 IgG4 antibody,¹² and LP11 nanobody¹³ have been developed to target selective inhibition of anticoagulant activity without perturbing its cytoprotective functions. Some of these selective APC-blocking antibodies have shown to be efficacious in ameliorating bleeding in hemophilic monkeys¹¹ and mice.^12,14,15 Oligonucleotide-based aptamers have also been developed in pursuit of this inhibition profile, such as APC-167,¹⁶ HS02,^17,18 and G-NB3.¹⁹ Among them, HS02 had been reported to provide protection against bleeding and coagulopathy in mice.²⁰ APC variants with normal levels of cytoprotection activities but devoid of anticoagulant activity have also been engineered. 3K3A-APC and 5A-APC variants have anticoagulant activity reduced to <10% and <3%, respectively, compared with wild-type APC, while retaining cytoprotective activity. These variants have been shown to be effective in providing anti-inflammatory, antiapoptotic, cytoprotective gene expression and endothelial barrier function.^21-23 Recently, 5A-APC has also been reported to have enhanced activity in cleaving histone H3 and neutralizing its cytotoxicity to endothelial cells.²⁴ Importantly, among the different APC-targeted approaches, antibody-based therapeutics have the potential for a long circulating half-life of 1 to 2 weeks. Although TPP-4885 IgG2 antibody had a long half-life and was efficacious in hemophilic monkeys and mice, the potency of the antibody required a dose of ≥3 mg/kg for efficacy. Therefore, further affinity maturation and optimization are desirable.

The therapeutic potential of APC through selective uncoupling and modulation of its activities by antibodies remain to be fully explored. For example, patients with TIC suffer from acute hemorrhage, endotheliopathy, and organ failure.^25-27 High histone levels in TIC were associated with increased mortality.²⁸ An anti-APC antibody that could specifically inhibit APC anticoagulant activity while enhancing histone cleavage by APC could provide simultaneous prohemostatic and endothelial barrier protection. In this study, we characterized antibodies from the directed evolution of a parental antibody, TPP-26870, a non–active site binder of APC, to understand the role of alterations in the complementarity-determining regions (CDRs) in uncoupling and modulating APC’s pleiotropic activities. Importantly, we found that single amino acid mutations within the CDRs of TPP-26870 were sufficient to elicit diverse changes on APC functions. These represent potential novel antibody therapeutics for the treatment of APC-associated indications including traumatic bleeding, hemophilia, ischemia, and sepsis.

Protein C activator (Protac), a fast-acting snake venom that rapidly converts available protein C into APC in plasma,²⁹ was used to evaluate the effects of TPP-26870 variants on APC anticoagulant activity in activated partial thromboplastin time (APTT) clotting assay. Healthy human pooled plasma samples (50 μL) with TPP-26870 variants (10 nM) were mixed with Protac (25 μL; 0.25 U/mL) and Stago STA-PTT reagent (75 μL) at 37°C. After a 240-second incubation, the clotting of plasma was initiated with the addition of 75 μL of 25-mM CaCl₂ solution. A Protac-APTT clotting time standard curve was established using healthy human plasma serially diluted with protein C–depleted plasma. The percentage of inducible APC activity in the plasma of the TPP-26870 variants–treated samples were interpolated based on their clotting time against the standard curve.

HEK293 cells expressing wild-type endothelial protein C receptor and a PAR1 cleavage reporter constructed with secreted embryonic alkaline phosphatase (SEAP-PAR1) at the N-terminus^1,30 were cultured in 96-well plates until confluent. Cells were washed with HMM2 buffer (Hanks’ balanced salt solution supplemented with 1.3-mM CaCl₂, 0.6-mM MgCl₂, and 0.1% endotoxin-free bovine serum albumin). Human APC (50 nM) and TPP-26870 variants were preincubated in HMM2. After 30 minutes, these APC/variant incubation mixtures were added to the SEAP-PAR1 expressing HEK293 cells to initiate PAR1 cleavage. After 60 minutes, the release of SEAP into the medium was determined using p-nitrophenyl phosphate as substrate. After correction for background activity in the absence of APC, SEAP-PAR1 cleavage was expressed as the percentage of the total SEAP activity present on the cells determined in separate wells, as described.^1,30 Values are normalized to the SEAP-PAR1 cleavage by APC only (= 100%).

APC-mediated cleavage of histone H3 by TPP-26870 was performed as previously described.¹³ Briefly, 50-nM APC and 500-nM TPP-26870 variants were incubated in HBS buffer (HEPES buffered saline, 100 μg/mL bovine serum albumin, 2-mM CaCl₂, and pH 7.4) for 30 minutes. These mixtures were added to 100 μg/mL H3. The incubation mixtures were sampled at different time points, and the reactions were quenched with reducing sample buffer. The samples were electrophoresed on 12 % Bis-Tris gels (Bio-Rad, 30 μL per well; 750 ng H3) and stained with Biotium One-Step Blue Protein Stain (50 mL per gel). The 20 kDa Odyssey protein molecular weight marker (10 μL) was used as a reference for normalization between gels. H3 cleavage was monitored by measuring the decrease in the intact H3 band over time using a Licor Odyssey (700 channel, intensity 6, 1-mm offset, and resolution 169 μm) with Image Studio V5.2. The amount of H3 cleaved by APC after 120 minutes is defined as 100%.

APC (70 nM) and TPP-26870 variants (700 nM) were preincubated for 30 minutes, followed by the addition of healthy human pooled plasma (90% volume-to-volume ratio). Incubation mixtures were subsampled at 0, 1, 10, 20, 30, 45, 60, 90, and 120 minutes and quenched in ice-cold Tris-buffered saline. The chromogenic activity of APC was determined using Pefachrome PCa. Background chromogenic activity of plasma (without APC addition) was subtracted, and the APC activity was normalized to the t = 0 time point to account for the effects of TPP-26870 variants on APC’s chromogenic activity. The APC in vitro plasma half-life was determined by 1-phase exponential decay curve fitting.

Additional experimental details can be found in supplemental Materials.

TPP-26870 is an APC non–active site antibody derived from the germlining of TPP-24727, an IgG4 isotype of the reference antibody TPP-4885.¹¹ The directed evolution of TPP-26870 through single amino acid substitutions identified positions and residues for improving association rate (k_a), dissociation rate (k_d), and affinity constant (K_D) against APC (Figure 1A; supplemental Figures 1 and 2). Eight positions within the heavy chain (V_H) improved K_D (Figure 1B). In the 3 positions within V_H-CDR1, substitutions with amino acids with non–polar side chains (I, F, and P) increased the K_D. In V_H-CDR2, the K_D was improved by substitutions within the polar amino acid stretch (S52-T57), with P, R, and T residues. In V_H-CDR3, the substitution of the aromatic F103 to a non–polar aliphatic methionine improved K_D. Eight positions within the light chain (V_L) improved K_D. In V_L-CDR1, substitutions of Q27 with a negatively charged glutamate and S28 with a positively charged arginine improved the K_D. In V_L-CDR2, the K_D was improved by the substitutions of the uncharged region (A55-N57) with positively charged amino acids, specifically lysine at 55, arginine at 56 and 57, and histidine at 57. The hydrophobic tryptophan also benefited positions 56 and 57. In V_L-CDR3, the replacement of polar Q93 with threonine, a shorter polar residue and the substitution of the polar Y100 with hydrophobic phenylalanine improved the K_D. A number of amino acid positions not included as the paratope of TPP-4885 for APC binding¹¹ were found to influence the K_D of the antibodies. All substitutions improved the K_D of TPP-26870 (K_D, 28 ×10^–9 M; k_a, 0.68 M^–1sec^–1; k_d, 0.027 sec^–1) by reducing its k_d (Figure 1C-D). Besides a reduction in k_d, some of these substitutions improved TPP-26870 by additionally increasing its k_a. The highest improvements in K_D were N31F in V_H-CDR1 (1.2 ×10^–9 M; Figure 1E) and N57W in the V_L-CDR2 (3.1 ×10^–9 M; Figure 1F), which represent a 23-fold and ninefold improvement compared with TPP-26870, respectively.

The APC anticoagulant activity of TPP-26870 variants were evaluated by Protac-APTT plasma clotting assay. Protac is an enzyme derived from snake venom for activating protein C zymogen to APC. The addition of Protac prolonged the APTT clotting time of healthy human plasma from 43 seconds to 143 seconds. A standard curve of plasma clotting time in the presence of different amounts of protein C was established by the mixing of protein C–depleted human plasma into healthy plasma (Figure 2A). When TPP-26870 variants were tested at 10 nM in the Protac-APTT plasma clotting assay, 5 of the 10 V_H amino acid substitutions (M34F, S54R, S53T, F103M, and N31F) shortened the plasma clotting time compared with TPP-26870, and 5 variants (S56R, Y32F, T57P, N31P, and N31I) resulted in a longer plasma clotting time and weaker inhibition of plasma APC activity (Figure 2B). The amino acid substitutions in the V_L also modified TPP-26870 plasma clotting time in different ways. Six of the 11 V_L amino acid substitutions (N57R, S56R, N57W, Y100F, Y100W, and A55K) shortened the clotting time in compared with TPP-26870, 1 variant (N57H) had minimal effect, and 4 variants (S56W, Q27E, Q93T, and S28R) resulted in a longer plasma clotting time (Figure 2C). Interestingly, the gain in the inhibition of APC anticoagulant activity was not correlated with the level of improvement in K_D for both the V_H (Figure 2D; supplemental Figure 3A) and V_L variants (Figure 2E; supplemental Figure 3A).

The cleavage of PAR1 by APC induces anti-inflammatory signaling and cytoprotective effects.^1,10 The cleavage of PAR1 was measured by monitoring the release of a SEAP-PAR1 reporter fragment into the medium from HEK293 cells expressing SEAP-PAR1 and endothelial protein C receptor. The correlation between APC anticoagulant activity inhibition and PAR1 cleavage in the presence of 10-nM TPP-26870 variants was evaluated. At 10 nM, TPP-26870 reduced APC anticoagulant activity down to 24%, whereas APC-mediated SEAP-PAR1 cleavage was only mildly reduced to 80% of the “no antibody” control (Figure 3A). Of the 5 V_H CDR1 variants, 4 variants showed less inhibition than TPP-26870, with 1 variant (N31F) being similar to TPP-26870 (Figure 3B). All 4 CDR2 variants were similar to TPP-26870 (Figure 3C), and the single CDR3 variant (F103M) showed increased inhibition (Figure 3D). Variant M34F in the V_H CDR1 led to a concurrent improvement in APC anticoagulant activity inhibition and the enhancement of PAR1 cleavage compared with TPP-26870. The TPP-26870 V_L variants also possess different effects of APC-mediated SEAP-PAR1 cleavage (Figure 3E). The 2 V_L CDR1 variants showed less inhibition than the parental TPP-26870 (Figure 3F). Of the 6 V_L CDR2 variants, 2 variants (S56W and N57W) showed decreased inhibition, 3 variants (A55K, N57R, and N57H) were similar to TPP-26870, and 1 variant (S56R) showed increased inhibition of SEAP-PAR1 cleavage (Figure 3G). For V_L CDR3 variants, Y100F showed less inhibition, whereas Q93T and Y100W were similar to the parental TPP-26870 (Figure 3H). At 10 nM, N57R and N57W in CDR2 as well as Y100F and Y100W in CDR3 improved APC anticoagulant activity inhibition and enhanced PAR1 cleavage at the same time compared with TPP-26870 (Figure 3E). The 50% inhibitory concentration of TPP-26870 variants on APC-mediated PAR1 cleavage (supplemental Table 1) provides additional quantification of the effects.

APC ameliorates the cytotoxicity mediated by extracellular histones by proteolytic cleavage, including the degradation of histone H3.² In this study, histone H3 (100 μg/mL) was incubated with APC (50 nM) and TPP-26870 variants (500 nM). Over a period of 2 hours, the incubation mixtures were subsampled at 15, 30, 60, and 120 minutes for sodium dodecyl sulfate–polyacrylamide gel electrophoresis analyses of APC-mediated histone H3 cleavage. TPP-26870 parental antibody did not alter H3 cleavage compared with control (Figure 4A). Of the 10 TPP-26870 V_H variants, only the variant with the S56R substitution led to a modestly lower level of H3 cleavage (92% of APC only). The APC-mediated H3 cleavage activity in the presence of other V_H variants was either similar or higher than the presence of TPP-26870 parental antibody. Among them, N31F substitution increased H3 cleavage by 33% (Figure 4B). The amino acid substitutions in the V_L generated a wider variety of effects (Figure 4C). Of the 11 substitutions, higher H3 cleavage was observed with 5 substitutions (Q27E, N57W, S56W, Q93T, and Y100F), minimal effect on cleavage was observed with 2 substitutions (S28R and N57H), and lower activity was observed for 4 substitutions (N57R, A55K, Y100W, and S56R). V_L S56R reduced H3 cleavage to 81% of APC only control. In contrast, Q27E increased the H3 cleavage level to 179% of APC only control over a period of 2 hours (Figure 4D). The correlation between APC anticoagulant activity inhibition and histone H3 cleavage was evaluated. Under the experimental conditions used for the 2 assays, TPP-26870 reduced APC anticoagulant activity down to 24%, whereas APC-mediated H3 cleavage was unchanged compared with APC alone with “no antibody.” Five substitutions within the V_H (N31F and M34F of CDR1, S53T and S54R of CDR2, and F103M of CDR3; Figure 4E) and 2 substitutions within the V_L (N57W of CDR1 and Y100F of CDR3; Figure 4F) simultaneously increased the inhibition of APC anticoagulant activity and enhanced H3 cleavage compared with the TPP-26870 parental antibody, a combination of properties desirable for ameliorating bleeding without perturbing anti-inflammatory signaling and cytoprotection.

The APC in vitro half-life is regulated by plasma serine protease inhibitors (SERPINs). The effect of TPP-26870 V_H variants on the decay of APC activity in plasma was determined by chromogenic activity using Pefachrome PCa as substrate (Figure 5A). TPP-26870 parental antibody increased APC plasma half-life 3.3-fold compared with the APC only control. Five amino acid substitutions at 3 different positions within CDR1 (N31F, N31P, N31I, M34F, and F103M) led to an increase in APC plasma half-life by 3.7-fold to 9.5-fold (Figure 5B). These 5 substitutions also achieved this prolongation of APC half-life along with an enhancement in APC-mediated histone H3 cleavage activity (Figure 5C), a combination of properties desirable for achieving the prolongation of the enhanced histone H3 cleavage activity. For the V_L variants, the amino acid substitutions generated antibodies with a wider variety of effects on APC half-life (Figure 5D). Three variants (S28R, S56R, and Q93T) led to a short APC-half-life compared with the presence of TPP-26870, whereas N57H had minimal impact on the effect of TPP-26870 on APC half-life (Figure 5E). Seven variants (N57W, S56W, Y100W, A55K, N57R, Q27E, and Y100F) led to an increase in APC plasma half-life by 3.9- to 5.6-fold. Four of these variants (Q27E in CDR1; S56W and N57W in CDR2; and Y100F in CDR3) achieved simultaneous prolongation of APC half-life and improved H3 cleavage compared with the TPP-26870 parental antibody.

A statistically significant correlation was observed for the variants between the enhancement in K_D and a reduction in the inhibition of PAR1 cleavage (r = −0.44; P < .042), with V_L variants being the most significant contributor to this correlation (supplemental Figure 3A-C). A statistically significant correlation between enhancement in K_D values and increased H3 cleavage was observed for the V_H variants (r = −0.60; P < .049) but not for the V_L variants (supplemental Figure 3A,D-E). Additionally, statistically significant correlations between K_D and prolonged APC half-life were observed for the variants (r = −0.70; P < .0003; supplemental Figure 3A,F-G).

APC is a pleiotropic protease with anticoagulant, anti-inflammatory, and cytoprotective activities. In this study, we characterized antibody variants from the directed evolution of a parental antibody, TPP-26870, a non–active site binder of APC, to understand the role of its CDRs in the modulation of APC’s pleiotropic activities. TPP-26870 is an antibody derived from the germlining of TPP-24727, an IgG4 isotype of TPP-4885 reported to target the APC autolysis loop and also potentially amino acids L429-N431.¹¹ TPP-26870 shared some of the same residue positions identified as the paratope of TPP-4885 within 5 Ǻ to APC. Interestingly, there were also substitutions, not included as the paratope, found capable of influencing the K_D and the functional activities of the antibodies. We observed that affinity maturation of TPP-26870 by single amino acid substitution can have significant effects on the various functions associated with APC. Depending on the position of the substitutions and the identity of the newly introduced amino acids used to improve K_D, affinity maturation resulted in variants that exerted diverse functional activity profiles on APC’s pleiotropic activities compared with the parental TPP-26870. These observations illustrate that the pleiotropic functions of APC were very sensitive to epitope-CDR interactions. Because the variants only have single amino acid substitutions, the variants were expected to bind to the same APC epitope as the parental TPP-26870. Therefore, the activity changes observed were likely mediated by the changes in the angle of binding, resulting in differential steric effects on APC interaction with substrates, and cofactors and/or allosteric conformational changes in APC induced by antibody binding to the autolysis loop.

Twenty-one different substitutions in 15 different amino acid positions over all 6 CDRs were identified to improve the K_D of TPP-26870. Interestingly, these K_D improvements led to divergent effects on APC anticoagulant activity inhibition for the different TPP-26870 variants. For example, N57W substitution in the V_L-CDR2, which had the highest improvement in K_D in the V_L, led to a parallel improvement in inhibition of APC anticoagulant activity compared with TPP-26870. In contrast, N31P substitution in V_H-CDR1, which had the second highest improvement in K_D in the V_H, resulted in a significant decrease in the inhibition of APC anticoagulant activity. This decrease in inhibition with N31P is contrary to the general approach of optimizing an antibody for higher affinity to achieve higher potency. This difference may lie in the fact that this inhibition of anticoagulant activity was mediated via the binding of TPP-26870 to a nonactive site on APC. Although K_D against the nonactive site was improved, different amino acid substitutions affected the epitope-CDR interactions differently. As a result, the spatial constraint exerted by the TPP-26870 variants on the interaction of APC with FVa and FVIIIa may differ in a manner that could either increase or decrease the inhibition of APC anticoagulant activity.

These results have significant ramifications to the development of APC-mediated treatments to disease. APC-mediated cleavage of endothelial cell PAR1 at R46 induces anti-inflammatory signaling and cytoprotective effects. The development of an anti-APC antibody for the purpose of promoting hemostasis in bleeding situations should, ideally, inhibit APC anticoagulant activity with minimal impact on PAR1 signaling. In the Protac-APTT clotting assay, 5 V_H substitutions and 6 V_L substitutions had increased inhibition of APC anticoagulant activity compared with TPP-26870. Besides being more potent in inhibiting anticoagulation, M34F in the V_H, as well as N57R, N57W, Y100F, and Y100W in the V_L, also gained enhancement in PAR1 cleavage compared with TPP-25870, a combination of properties desirable for ameliorating bleeding without perturbing anti-inflammatory signaling and cytoprotection.

APC functions to dampen the cytotoxicity induced by the pathological release of extracellular histones from immune or necrotic cells by proteolytic degradation.² The incubation of the parental TPP-26870 with APC did not inhibit histone H3 fragmentation. Interestingly, there were multiple single amino acid substitutions distributed over the 6 CDRs that conferred TPP-26870 with agonistic activity for APC-mediated histone H3 cleavage by up to 179%. These agonistic antibody variants likely promoted cleavage through allosteric interaction and stabilization of APC-H3 interaction by shielding positively charged residues in the autolysis loop or by inducing conformational changes in the proximal region. The autolysis loop has also been reported to play a role in the regulation of APC activity by plasma SERPINs.^31,32 In in vitro APC plasma half-life assay, the prolonged APC activity half-life observed with the 5 V_H substitutions and 7 V_L substitutions compared with TPP-26870 was likely mediated by the blockade of SERPINs from binding to APC. The prolonged APC activity half-life could result in prolonged APC duration of action in anticoagulation, PAR1 cleavage, and H3 cleavage in the circulation; and an antibody with combined properties of APC activity half-life prolongation, inhibition of anticoagulant activity, and enhanced H3 cleavage could potentially provide unique therapeutic benefits.

The activity profiling of TPP-26870 variants with enhanced K_D identified 12 categories of variants (supplemental Table 2). Additional categories could potentially be discovered by profiling variants with reduced K_D. N57W V_L substitution exhibited improvement over TPP-26870 uniquely suitable for TIC. Patients with TIC suffer from acute hemorrhage, inflammation, endotheliopathy, and organ failure.^25-27 High histone levels were associated with increased probability of mortality.²⁸ N57W substitution prolonged the APC plasma half-life, increased APC anticoagulant activity inhibition, and provided agonistic effects on histone H3 cleavage, while having less impact on PAR1 cleavage compared with TPP-26870. This unique collection of properties of N57W V_L substitution could be well suited for addressing TIC. The functionality of this variant could potentially be further enhanced by combining with other variants of similar activity profile, for example, M34F V_H substitution, to achieve increased efficacy in vivo.

The conventional approach of optimizing antibodies for higher potencies is based on affinity maturation of an antibody by identifying amino acid substitutions that could improve the K_D against their targets. Although this approach works well for many antibodies, especially for those that target enzyme active sites, it cannot be universally applied, for example, to the optimization of an antibody targeting a nonactive site of the pleiotropic protein as demonstrated in this study. Data from different assays illustrated that the pleiotropic functions of APC are very sensitive to epitope-CDR interactions. Mutations of single amino acid within the CDRs of TPP-26870 were sufficient to elicit diverse functional effects on APC in an unpredictable manner. Therefore, the optimization of antibodies against nonactive site of pleiotropic protein requires subsequent judicious evaluation of the consequence of optimization on the functional activities of the target protein to ensure the optimized antibody continues to exert its desired activity profile. In summary, the single amino acid substitutions of TPP-26870 yielded a panel of antibody variants capable of modulating and uncoupling APC pleiotropic functions. These variants could offer an opportunity to develop novel therapeutics for the treatment of APC-associated indications, including traumatic bleeding, hemophilia, ischemia, and sepsis.

The authors thank Bayer HealthCare for their technical support.

This study was supported by research funding from Coagulant Therapeutics. L.O.M. was supported by research funding from Coagulant Therapeutics and National Institutes of Health, National Heart, Lung, and Blood Institute grants R01HL142975 and R01HL104165.

Contribution: D.S.S., M.S., C.R.M., J.A.F., X.X., D.S., M.B., T.W.H., and L.O.M. designed and performed experiments and analyzed data; D.S.S., C.R.M., T.W.H., and L.O.M. contributed to writing of the original draft of the manuscript; and D.S.S., M.S., C.R.M., J.A.F., X.X., D.S., M.B., T.W.H., and L.O.M. contributed to reviewing and editing of the final manuscript.

Conflict-of-interest disclosure: D.S.S., C.R.M., and T.W.H. are employees of Coagulant Therapeutics. D.S. and M.B. are consultants of Coagulant Therapeutics. D.S.S., C.R.M., M.B., and T.W.H. have stock ownership in Coagulant Therapeutics. L.O.M. received research funding from Coagulant Therapeutics. D.S.S., D.S., M.B., and T.W.H. are listed as a coinventor on patents filed by Coagulant Therapeutics related to this study. The remaining authors declare no competing financial interests.

Correspondence: Derek S. Sim, Coagulant Therapeutics Corporation, 2630 Bancroft Way, Berkeley, CA 94720; email: dsim@coagulanttherapeutics.com.