Procoagulant Activity of Skeletal Muscle Myosin Is Not Caused By Contaminating Phosphatidylserine-Vesicles

Skeletal muscle myosin (SkM) can bind factor (F)Xa and FVa, thereby providing a surface that promotes thrombin generation by the prothrombinase complex (FXa:FVa:Ca++) that cleaves prothrombin. A recent BLOOD paper (Novakovic & Gilbert, 2020) asserted that this activity of SkM preparations is entirely due to contaminating phosphatidylserine (PS)-containing phospholipid vesicles in SkM preparations because annexin V and lactadherin neutralized the ability of SkM to enhance prothrombin activation. However, annexin V and lactadherin are certainly not monospecific for binding PS as they are globular proteins that can bind other lipids and many proteins. Without any PS measurements or use of any reagents specific for PS (e.g., monoclonal (mAb) anti-PS antibodies), that report, in a gross overinterpretation of its incomplete data, dismissed any direct role for myosin for SkM's procoagulant activity. When we previously observed that annexin V is inhibitory of SkM's support for prothrombinase activity, we sent a sample of SkM (Cytoskeletal Inc) to Avanti Polar Lipids for quantitation of the PS content based on liquid chromatography-mass spectrometry. That analysis showed that only a small amount of PS was present in the SkM, approximately 0.90 µmol PS per 40. µmol SkM. This amount of PS present in SkM preparations is not enough to explain SkM's procoagulant activity. For example, standardized purified prothrombinase reaction mixture assays show that 10 nM SkM enables formation of 3 nmol thrombin/min while 0.22 nM PS (in 1.1 nM phosphatidylcholine (PC)(80%)/PS(20%) vesicles) enables formation of only 0.4 nmol thrombin/min (Figure 1A). We directly assessed the role of contaminating PS for SkM's prothrombinase support using the well characterized anti-PS mAb 11.31 (aka mAb PGN632). When the ability of mAb 11.31 to inhibit prothrombinase enhancement by SkM or, in controls, by PC/PS vesicles was determined, the data showed that, in controls, mAb 11.31 at 1.0 nM severely inhibited PC/PS vesicle's enhancement of prothrombinase by > 90% (Figure 1B). The dose-response gave an inhibitory IC50 value of 0.2 nM which is near this mAb's reported Kd of 0.17-0.35 nM for PS, establishing the potent ability of this anti-PS mAb to neutralize PS procoagulant activity. However, there was no substantial inhibition of SkM's enhancement of prothrombinase by the anti-PS mAb 11.31 at up to 1 nM mAb 11.31 (Figure 1B). This indicates that contaminating, PS-containing vesicles are not a significant factor for SkM-dependent enhancement of prothrombin activation by the SkM preparation -- in direct contradiction of the assertion of Novakovic & Gilbert (BLOOD 2020). That recent report was correct that annexin V inhibits the ability of SkM to enhance prothrombinase as well as the ability of PC/PS vesicles to do so (Figure 1B). So it was the overinterpretation of the annexin V and lactadherin data as well as the failure to provide any direct measurement of PS that were problematic in that report. The observation that annexin V and lactadherin inhibit SkM's enhancement of prothrombinase merits further studies to understand what may be their mechanistic influences. Another informative test for an essential role for PS in SkM's enhancement of prothrombinase involves the use of FXa that lacks its gamma-carboxyglutamic acid (Gla) domain because this N-terminal domain of FXa is required for FXa binding to PS-containing phospholipid vesicles. Data in Figure 1A show that SkM, but not PS-containing PL vesicles, supports the prothrombinase activity of des-Gla Domain (DG)-FXa which lacks its Gla domain. Dose-response data show that, in prothrombinase assays, DG-FXa has only 1% activity in the presence of PC/PS vesicles but has 25-35% activity in presence of SkM (Figure 1A). These data indicate that SkM's procoagulant activity does not absolutely require the Gla domain of FXa whereas PS-containing vesicles do require the Gla domain of FXa for significant activity. These data prove that the recent assertion that PS vesicle contamination, not myosin, explains SkM's procoagulant activity represents an overinterpretation of annexin V and lactadherin data and is simply wrong. In conclusion, both new data here, i.e., PS content of SkM preparations and data for effects of anti-PS mAb 11.31 (Figure 1B) and previous data (Deguchi et al, Blood 2016 and J Biol Chem, 2019), affirm that the myosin protein is a key factor for SkM's ability to enhance prothrombin activation. Figure 1 Disclosures No relevant conflicts of interest to declare.

Molecular Interaction Site on Procoagulant Skeletal Muscle Myosin for Factor Xa-Dependent Prothrombin Activation

Skeletal muscle myosin (SkM) is a muscle protein consisting of a dimer of heterotrimers, each trimer comprising a regulatory light chain (RLC), an essential light chain (ELC), and a heavy chain (HC). Recently it was discovered that SkM has potent procoagulant and prothrombotic activity (Deguchi H, et al, Blood. 2016;128:1870-1878). Mechanistic studies showed that SkM's potent prothrombotic activity involved enhancing thrombin generation due to SkM's ability to bind coagulation factors Xa and Va which accelerates prothrombin activation. However, detailed molecular mechanisms for SkM's binding of these coagulation factors have not been described. Since a well-known myosin inhibitor, trifluoperazine (TFP), inhibited SkM's procoagulant activity and since this inhibitor binds to the ELC in SkM's "neck" region which connects the HC head region to the HC tail (Figure, panel A), we hypothesized that SkM's TFP binding region on the ELC in the neck region directly contributes to SkM's procoagulant activity. To identify potential binding site(s) on SkM for factors Xa and Va, 22 peptides representing the neck region's RLC, ELC, and HC were screened for inhibition of SkM-supported prothrombin activation by purified factor Xa, factor Va, and calcium ions. These peptides contained 25-40 residues and overlapped by approximately 5-10 amino acids. Peptides ELC109-138 and ELC129-159, corresponding to amino acid residues 109-138 and 129-159 of the ELC, inhibited SkM-supported prothrombin activation at 100 μM, whereas their partially overlapping neighboring peptides, ELC99-122 and ELC149-173, did not. Three HC peptides (peptides HC781-810, HC796-835, HC815-854) and one RLC peptide (RLC133-162) inhibited SkM-supported prothrombin activation at 100 μM, and each was also inhibitory, to varying degrees, when assayed at 5 μM. Dose-dependency inhibition assays gave IC50 values (50% inhibition of activity) for the peptides HC781-810, HC796-835, HC815-854, and RLC133-162 of 64, 1.2, 2.3 and 26 μM. Peptides HC781-810 and HC815-854 also inhibited prothrombin activation in the absence of myosin but in the presence of phospholipid vesicles containing 20 % phosphatidylserine (IC50 = 7.5 and 104 μM, respectively). In contrast, the strong inhibitory effects of peptides HC796-835, RLC133-162, ELC109-138 and ELC129-159 seen in the presence of myosin were not at all apparent in the presence of phospholipid-supported prothrombin activation when myosin was absent. This suggests that peptides HC796-835, RLC133-162, ELC109-138 and ELC129-159 specifically inhibit SkM-supported prothrombin activation. The 19 synthetic peptides representing the SkM neck region were also screened at 25 µM (final) for their inhibition of recalcification-induced thrombin generation in human plasma which contains significant circulating levels of SkM. Among the 19 peptides tested, HC796-835 and HC815-854 significantly inhibited thrombin generation when screened at 25 µM in plasma. Immobilized peptide HC796-835 showed direct binding of purified factor Xa with apparent Kd of 1.4 μM. This very potent inhibitory peptide, HC796-835, exhibited 50% inhibition of SkM-enhanced prothrombin activation at 1.2 μM, indicating that this peptide's sequence provides a factor Xa binding site on SkM which contributes to its inhibitory action. More specifically, an overlapping peptide containing amino acid residues 815-835 inhibited SkM-enhanced prothrombin activation by factors Xa and Va while a peptide comprising residues 796-811 did not. These studies suggest that residues 815-835 of SkM's HC are responsible for directly binding factor Xa and implies that this binding is responsible for SkM's procoagulant activity (Figure, panel B). In summary, we identified human SkM peptides which specifically blocked SkM-enhanced thrombin generation but not phospholipid-stimulated prothrombin activation in purified reaction mixtures and which inhibited blood clotting in plasma. The most potent anticoagulant HC peptide also directly binds purified factor Xa. These findings strongly suggest that the neck region of SkM, as defined by these inhibitory peptides (Figure, panel B), provides a phospholipid-independent procoagulant surface for thrombin generation that, depending on the in vivo physiologic context, may contribute to either hemostasis or thrombosis. Figure Disclosures No relevant conflicts of interest to declare.

A Plasma-Based Assay to Measure the Susceptibility of Factor V(a) to Inhibition By TFPIα

Background: Coagulation factor V (FV) is the precursor of activated FV (FVa), which assembles with factor Xa (FXa) on phospholipid surfaces to form the prothrombinase complex, accelerating prothrombin activation >1000-fold. FV is activated by FXa or thrombin via limited proteolysis at Arg709, Arg1018 and Arg1545. These cleavages progressively expose the FXa-binding site, generating activation intermediates with increasing affinities for FXa. Recently, it has been shown that tissue factor pathway inhibitor α (TFPIα) inhibits both the activation of FV (by interfering with cleavage at Arg1545) and the ability of partially activated FV(a) species to enhance prothrombin activation. These effects are mediated by interactions of the C-terminus of TFPIα with an acidic region in the B-domain of FV as well as with the FV(a) heavy chain. The pathophysiological relevance of these novel anticoagulant activities of TFPIα is still unexplored, but evidence has been provided that prothrombinase complexes assembled with FV(a) Leiden are less susceptible to TFPIα inhibition. Moreover, FV splicing variants (FV-short) with increased affinity for TFPIα have recently been discovered in two unrelated families (from East Texas and Amsterdam, respectively) with bleeding tendencies. Rationale and Aim: The FV present in plasma from different individuals may differ in its sensitivity to inhibition by TFPIα, with potential implications for the risk of venous thrombosis or bleeding. Therefore, the aim of this study was to develop a plasma-based assay that measures the susceptibility of FV(a) to TFPIα inhibition. Methods: FV in 1/1000 diluted plasma was activated for 3 minutes with a suboptimal FXa concentration on 20/60/20 DOPS/DOPC/DOPE lipids in the presence or absence of a peptide mimicking the C-terminus of TFPIα (TFPIα C-term). Purified prothrombin and a chromogenic substrate for thrombin were then added, and the activity of the prothrombinase complex was monitored continuously up to 30 minutes. The parabolic absorbance curves were fitted to second-order polynomial equations and the rate of prothrombin activation was calculated from the coefficient of the x2-term. The assay outcome was expressed as residual prothrombinase ratio (RP-ratio), defined as the ratio between the rates of prothrombin activation obtained in the presence and absence of TFPIα C-term. The assay was validated using plasma from 4 FV Leiden homozygotes and 4 normal controls. In addition, we tested plasma from 3 members of the FV Amsterdam family (2 carriers of the mutation up-regulating FV-short Amsterdam and 1 non-carrier). Results: The rate of prothrombin activation in the absence of peptide was a function of plasma FV level and pre-incubation time, and was inhibited by TFPIα C-term in a dose-dependent manner. A pre-incubation time of 3 minutes and a peptide concentration of 100 nM, yielding an RP-ratio of 0.30 in normal pooled plasma, were chosen. The RP-ratio was independent of the plasma FV level in the 75-150% range. Moreover, control experiments indicated that, at this high dilution, the plasma background did not influence the assay outcome. The intra- and inter-assay coefficients of variation of the RP-ratio were 5.4% and 12%, respectively. FV Leiden homozygotes had higher RP-ratios than normal controls (0.45 ± 0.04 vs. 0.30 ± 0.03, p=0.002), indicating resistance to inhibition by TFPIα C-term. Differently, the 2 carriers of the FV Asterdam mutation, who express high levels of FV-short Amsterdam, had markedly reduced RP-ratios (0.18 and 0.16 vs. 0.29 in the non-carrier), as expected from the high affinity of FV-short Amsterdam for TFPIα. Conclusions: We have developed and validated an assay that measures the susceptibility of plasma FV(a) to inhibition by TFPIα. This assay can be used to test whether TFPIα-mediated inhibition of FV activation and prothrombinase activity differs for (genetically) different FV variants and whether it correlates with the risk of thrombosis or bleeding. Supported by grant 2014-1 from the Dutch Thrombosis Foundation. Disclosures No relevant conflicts of interest to declare.

Identification, Cloning, and Characterization ofStaphylococcus pseudintermediusCoagulase

ABSTRACTCoagulase activation of prothrombin by staphylococcus induces the formation of fibrin deposition that facilitates the establishment of infection byStaphylococcusspecies. Coagulase activity is a key characteristic ofStaphylococcus pseudintermedius; however, no coagulase gene or associated protein has been studied to characterize this activity. We report a recombinant protein sharing 40% similarity toStaphylococcus aureuscoagulase produced from a putativeS. pseudintermediuscoagulase gene. Prothrombin activation by the protein was measured with a chromogenic assay using thrombin tripeptide substrate. Stronger interaction with bovine prothrombin than with human prothrombin was observed. TheS. pseudintermediuscoagulase protein also bound complement C3 and immunoglobulin. Recombinant coagulase facilitated the escape ofS. pseudintermediusfrom phagocytosis, presumably by forming a bridge between opsonizing antibody, complement, and fibrinogen. Evidence from this work suggests thatS. pseudintermediuscoagulase has multifunctional properties that contribute to immune evasion that likely plays an important role in virulence.

Study of Optimum Condition for Rapid Preparation of Thrombin using Russell’s Viper Venom Factor X Activator

Background: The purpose of this study is to investigate a simple method with the optimum condition for rapid thrombin preparation from Cryoprecipitate-depleted Plasma (CDP) using RVV-X in the process. Methods: Thrombin preparation from human CDP was studied with the presence of different factors in batch condition including: 1) RVV-X; 2) volume of calcium chloride solution; 3) volume of sodium chloride solution for final extraction; and 4) incubation time. The properties of the prepared sample were analyzed for fibrin clot formation, total protein by Kjeldahl method, thrombin time, molecular weight and protein patterns by SDS-PAGE, and thrombin concentration by coagulation analyzer. The method and process of preparing thrombin and the study of optimum condition for rapidly preparing the highest yield of thrombin from starting CDP 100 ml were introduced. Results: The best four conditions were concluded: 1) RVV-X 50 mcg should be present in the process; 2) volume of 0.25 M calcium chloride should be 3 ml; 3) volume of 0.85% sodium chloride for the final protein precipitate extraction should be 10 ml and; 4) no incubation time needed for prothrombin activation process. A solution prepared from the optimum condition showed an obvious band on SDS-PAGE at a molecular weight about 36,000 Da which is our target protein thrombin. The prepared solution had a total protein content of 0.065 g/dl and gave satisfactory results of thrombin time (9 seconds) and fibrin clot formation. The test results of thrombin concentration between the method with and without incubation time were 269.4 and 295.2 IU/ml, respectively. Conclusion: This result showed that the method with RVV-X but without incubation time for prothrombin activation (optimum condition) gave the highest yield of thrombin.