Skeletal Muscle Myosin Is Procoagulant By Binding Factor XI Via Its A3 Domain and Enhancing Factor XI Activation By Thrombin

Abstract Blood coagulation mechanisms play key roles in health and disease. Pilot studies using selected human plasmas showed the potential associations of plasma skeletal muscle myosin (SkM) isoforms and phenotypes with pulmonary embolism and thrombin generation, suggesting SkM may contribute to blood coagulation reactions in plasma. Here we report that ex vivo studies of the coagulability of fresh flowing human blood over SkM-coated surfaces showed that an anti-factor XI (FXI) mAb, but not an anti-tissue factor mAb, inhibited clot formation, indicating that FXI is an essential contributor for the normal observed procoagulant response of blood during its exposure to immobilized SkM. This raised the question of whether procoagulant SkM's requirement for FXI involves direct or indirect effects on FXI. To assess direct interactions between SkM and FXI, Bio-Layer Interferometry (BLI) (Octet Red system) was used to record kinetics for binding of soluble FXI to immobilized SkM. BLI data showed that FXI bound to SkM with a Kd of 0.2 nM (k on= 2.92x10 6 M -1s -1 and k off=9.25x10 -3 s -1) (Fig. 1A). In contrast, prekallikrein (PK) did not bind to the SkM (Fig.1A), indicating the specificity of SkM for binding FXI. The anti-FXI mAb1A6, which recognizes the Apple (A)3 domain of FXI, potently inhibited binding of FXI to immobilized SkM, implying SkM binds the FXI A3 domain. Studies using purified clotting factors were made to identify which FXI-related activities might be affected by SkM. When FXI activation by thrombin was evaluated under conditions where polyphosphate (PolyP) 100-mer and 700-mer enhance FXI activation, SkM concentration-dependently enhanced FXI activation by thrombin (Fig. 1B). Whereas alkaline phosphatase destroyed PolyP's ability to stimulate FXI activation by thrombin, it did not cause a reduction of SkM's ability to enhance FXI activation, indicating SkM's activity is independent of PolyP-like sequences in SkM. Small unilamellar phospholipid vesicles (20% phosphatidylserine (PS) / 80% phosphatidylcholine) did not affect FXI activation by thrombin; furthermore, reagents that neutralize procoagulant PS, i.e., lactadherin, annexin V, and phospholipase A2, did not affect SkM's enhancement of FXI activation by thrombin, indicating that this activity is not due to anionic phospholipids linked to SkM. The effects of SkM on FXI autoactivation and FXI activation by FXIIa were evaluated. As is well known, PolyP and some other anionic reagents, e.g., nucleic acid polymers, enhance not only FXI activation by thrombin but also FXI autoactivation and FXI activation by FXIIa. However, SkM did not significantly affect FXI autoactivation or FXI activation by factor XIIa, further emphasizing that SkM's enhancement of FXI activation by thrombin is not due to any PolyP-like compounds and that it is a unique property of procoagulant SkM. This also suggests that SkM has a unique mechanism for its procoagulant activity on FXI activation which is limited to the thrombin positive feedback loop. To evaluate further the basis for interactions between FXI and SkM, we employed FXI- PK chimeras because BLI binding studies showed that, in contrast to FXI, PK did not bind to SkM. Recombinant FXI proteins in which each of the four A domains of the heavy chain (designated A1 through A4) were individually replaced with the corresponding A domain from PK and were used to identify the site of factor XI to interact with SkM for FXI activation by thrombin. The FXI chimera with the substitution of the PKA1 domain was not activated by thrombin, which is consistent with the fact that the FXI A1 domain is an interactive site for thrombin. Thrombin activation of the two FXI chimeras (FXI/PKA3 and FXI/PKA4) with substitutions of either the A3 or A4 domains was not enhanced by SkM, whereas substitution of the A2 domain (FXI/PKA2) did not reduce the enhancement of activation by thrombin compared to wild type FXI. Furthermore, mAb1A6, which recognizes the A3 domain and which inhibited the prothrombotic activity of fresh blood flowing over a SkM-coated surface, potently inhibited FXI binding to SkM in BLI studies. These data strongly suggest that functional interaction sites on FXI for SkM involve the A3 and A4 domains of FXI. In summary, we found that SkM's ex vivo procoagulant activity requires FXI, that SkM enhances FXI activation by thrombin and this requires FXI's A3 and A4 domains, and that SkM's high affinity binding of FXI requires the FXI A3 domain (Fig. 1C). Figure 1 Figure 1. Disclosures Gruber: Aronora Inc.: Current Employment, Current equity holder in publicly-traded company; Oregon Health and Science University: Current Employment. Gailani: Anthos Therapeutics: Consultancy; Aronora: Membership on an entity's Board of Directors or advisory committees; Bayer Pharma: Consultancy; Bristol Myer Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees; Ionis: Consultancy; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees.

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Prothrombotic skeletal muscle myosin directly enhances prothrombin activation by binding factors Xa and Va

Blood ◽

10.1182/blood-2016-03-707679 ◽

2016 ◽

Vol 128 (14) ◽

pp. 1870-1878 ◽

Cited By ~ 21

Author(s):

Hiroshi Deguchi ◽

Ranjeet K. Sinha ◽

Patrizia Marchese ◽

Zaverio M. Ruggeri ◽

Jevgenia Zilberman-Rudenko ◽

...

Keyword(s):

Skeletal Muscle ◽

Thrombus Formation ◽

Procoagulant Activity ◽

Trauma Patients ◽

Skeletal Muscle Myosin ◽

Acute Trauma ◽

Key Points ◽

Muscle Myosin ◽

Prothrombin Activation

Key Points Skeletal muscle myosin promotes thrombus formation and enhances prothrombin activation by binding factors Xa and Va. The procoagulant activity of skeletal muscle myosin might contribute to the hypercoagulability in plasmas of acute trauma patients.

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

Blood ◽

10.1182/blood-2019-129161 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3622-3622

Author(s):

Hiroshi Deguchi ◽

Zihan Guo ◽

Mohammed Hayat ◽

Elsa Pflimlin ◽

Weijun Shen ◽

...

Keyword(s):

Skeletal Muscle ◽

Thrombin Generation ◽

Factor Xa ◽

Neck Region ◽

Coagulation Factors ◽

Procoagulant Activity ◽

Amino Acid Residues ◽

Skeletal Muscle Myosin ◽

Muscle Myosin ◽

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.

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

Blood ◽

10.1182/blood-2020-139574 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 20-21

Author(s):

Shravan Morla ◽

Hiroshi Deguchi ◽

Rolf Brekken ◽

John H. Griffin

Keyword(s):

Skeletal Muscle ◽

Dose Response ◽

Annexin V ◽

Procoagulant Activity ◽

Phospholipid Vesicles ◽

Skeletal Muscle Myosin ◽

Gla Domain ◽

Muscle Myosin ◽

Prothrombinase Activity ◽

Prothrombin Activation

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.

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Novel blood coagulation molecules: Skeletal muscle myosin and cardiac myosin

Journal of Thrombosis and Haemostasis ◽

10.1111/jth.15097 ◽

2020 ◽

Vol 19 (1) ◽

pp. 7-19 ◽

Cited By ~ 1

Author(s):

Hiroshi Deguchi ◽

Shravan Morla ◽

John H. Griffin

Keyword(s):

Skeletal Muscle ◽

Blood Coagulation ◽

Cardiac Myosin ◽

Skeletal Muscle Myosin ◽

Muscle Myosin

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Skeletal muscle myosin promotes coagulation by binding factor XI via its A3 domain and enhancing thrombin-induced factor XI activation

Journal of Biological Chemistry ◽

10.1016/j.jbc.2022.101567 ◽

2022 ◽

pp. 101567

Author(s):

Shravan Morla ◽

Hiroshi Deguchi ◽

Jevgenia Zilberman-Rudenko ◽

András Gruber ◽

Owen J.T. McCarty ◽

...

Keyword(s):

Skeletal Muscle ◽

Factor Xi ◽

Skeletal Muscle Myosin ◽

Binding Factor ◽

Muscle Myosin

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Exome Genotyping Links Venous Thrombosis Risk with the Skeletal Muscle Myosin Gene Cluster and Leads to Discovery of New Family of Procoagulant Factors

Blood ◽

10.1182/blood.v126.23.763.763 ◽

2015 ◽

Vol 126 (23) ◽

pp. 763-763 ◽

Cited By ~ 2

Author(s):

Hiroshi Deguchi ◽

Ranjeet Kumar Sinha ◽

Darlene J. Elias ◽

John H. Griffin

Keyword(s):

Skeletal Muscle ◽

Venous Thrombosis ◽

Myosin Heavy Chain ◽

Heavy Chain ◽

Thrombin Generation ◽

Procoagulant Activity ◽

Skeletal Muscle Myosin ◽

New Family ◽

Functional Variants ◽

Muscle Myosin

Abstract Venous thromboembolism (VTE) exacts a severe toll, and its toll persists even after the acute thrombotic event is resolved. Although several low frequency, racially-specific, genetic variants have been linked to VTE (e.g., factor V Leiden, prothrombin nt20210A, protein S Tokushima, Protein C R147W and del-Lys150), these polymorphisms fail to explain the majority of VTE risk. These variants are causally linked to increased VTE risk and, based on their racial-specific occurrence, they likely provided evolutionary benefits. Thus, to discover new rare missense functional variants linked to VTE, we used the new Affymetrix Axiom array to determine ~ 300,000 mostly exomic rare variant polymorphisms in the Scripps venous thrombosis registry (N=214 controls and 107 VTE cases) and gained multiple novel insights. When recurrent VTE cases were analyzed, 34 SNPs, including two F5 SNPs (rs6025 and rs6687813), were significant (p<0.05) after FDR correction. Analysis of clustering for these SNPs revealed associations of VTE recurrence risk with skeletal muscle myosin (MYH) rare SNPs (chr 17p13.1) in a highly conserved region with clustered myosin heavy chain genes (MYH1, 2, 4, 8). Among 11 rare MYH SNPs, rs111567318 was significantly linked to VTE (p=8.55x10-6, FDR p<0.05). Notably, 16 of 107 (15. %) VTE subjects had ≥ one SNP in the MYH chr17 cluster compared to only 1 of 212 (0.5 %) controls. Studies were initiated to define procoagulant or anticoagulant properties of skeletal muscle myosins which heretofore had no known function in blood clotting and which are broadly found in the body, even in plasma (e.g., at 20 nM). Remarkably, purified rabbit skeletal muscle myosin enhanced TF-induced thrombin generation in plasma. In neat plasma, anti-myosin antibodies reduced TF-induced thrombin generation, suggesting the contribution of endogenous plasma myosin to plasma thrombin generation. Skeletal muscle myosin also enhanced recalcification-induced thrombin generation in plasma. Studies to tests if prothrombinase activity is promoted by skeletal muscle myosin in purified prothrombinase (IIase) assays (factor Xa, factor Va, and II plus Ca++, with or without phospholipid added) showed that skeletal muscle myosin greatly enhanced IIase activity. When factor (F) Va was absent, myosin did not detectably enhance IIase activity, showing FVa was required. The myosin heavy chain can be split into 1 light meromyosin (LMM) and 1 heavy meromyosin (HMM) domains which can be further split into 2 globular subfragments (S1) and 1 rod-shaped subfragment (S2). HMM enhanced IIase activity whereas the myosin S1 subfragment did not, suggesting the S2 subfragment and LMM were required for the procoagulant activity. When prothrombin cleavage kinetics in the presence of skeletal muscle myosin were studied using PAGE, negligible amounts of meizothrombin were observed while fragment 1.2 and prothrombin 2 (or a-thrombin) bands were observed. This implies that skeletal muscle myosin promotes the prothrombin cleavage pathway similar to that enhanced by platelets, but not similar to that enhanced by phospholipid vesicles. The IIase activity of plasma-derived, purified extra cellular vesicles (ECVs) was markedly inhibited (> 60 %) by anti-skeletal muscle myosin antibodies, suggesting that a major fraction of procoagulant extracellular vesicle procoagulant activity involves skeletal muscle myosin. In purified systems, the apparent Kd of skeletal myosin in the presence of FVa for FXa was 9 nM. In summary, we found that MYH SNPs may be linked to VTE risk which led to the discovery of a new family of procoagulant proteins, skeletal myosins, that bind FXa, stimulate thrombin generation in plasma and in purified systems, thus paving the way for future genetic and functional VTE research. Disclosures No relevant conflicts of interest to declare.

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