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

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.

Skeletal Muscle progenitors are sensitive to collagen architectural features of fibril size and crosslinking

Muscle stem cells (MuSCs) are essential for the robust regenerative capacity of skeletal muscle. However, in fibrotic environments marked by abundant collagen and altered collagen organization, the regenerative capability of MuSCs is diminished. MuSCs are sensitive to their extracellular matrix environment, but their response to collagen architecture is largely unknown. The present study aimed to systematically test the effect of underlying collagen structures on MuSC functions. Collagen hydrogels were engineered with varied architectures: collagen concentration, crosslinking, fibril size, and fibril alignment, and the changes were validated with second harmonic generation imaging and rheology. Proliferation and differentiation responses of primary mouse MuSCs and immortal myoblasts (C2C12s) were assessed using EdU assays and immunolabeling skeletal muscle myosin expression, respectively. Changing collagen concentration and the corresponding hydrogel stiffness did not have a significant influence on MuSC proliferation or differentiation. However, MuSC differentiation on atelocollagen gels, which do not form mature pyridinoline crosslinks, was increased compared to the crosslinked control. In addition, MuSCs and C2C12 myoblasts showed greater differentiation on gels with smaller collagen fibrils. Proliferation rates of C2C12 myoblasts were also higher on gels with smaller collagen fibrils, while MuSCs did not show a significant difference. Surprisingly, collagen alignment did not have significant effects on muscle progenitor function. This study demonstrates that MuSCs are capable of sensing their underlying ECM structures and enhancing differentiation on substrates with less collagen crosslinking or smaller collagen fibrils. Thus, in fibrotic muscle, targeting crosslinking and fibril size rather than collagen expression may more effectively support MuSC-based regeneration.

Filamentous tangles with nemaline rods in MYH2 myopathy: a novel phenotype

The MYH2 gene encodes the skeletal muscle myosin heavy chain IIA (MyHC-IIA) isoform, which is expressed in the fast twitch type 2A fibers. Autosomal dominant or recessive pathogenic variants in MYH2 lead to congenital myopathy clinically featured by ophthalmoparesis and predominantly proximal weakness. MYH2-myopathy is pathologically characterized by loss and atrophy of type 2A fibers. Additional myopathological abnormalities have included rimmed vacuoles containing small p62 positive inclusions, 15–20 nm tubulofilaments, minicores and dystrophic changes. We report an adult patient with late-pediatric onset MYH2-myopathy caused by two heterozygous pathogenic variants: c.3331C>T, p.Gln1111* predicted to result in truncation of the proximal tail region of MyHC-IIA, and c.1546T>G, p.Phe516Val, affecting a highly conserved amino acid within the highly conserved catalytic motor head relay loop. This missense variant is predicted to result in a less compact loop domain and in turn could affect the protein affinity state. The patient’s genotype is accompanied by a novel myopathological phenotype characterized by centralized large myofilamentous tangles associated with clusters of nemaline rods, and ring fibers, in addition to the previously reported rimmed vacuoles, paucity and atrophy of type 2A fibers. Electron microscopy demonstrated wide areas of disorganized myofibrils which were oriented in various planes of direction and entrapped multiple nemaline rods, as corresponding to the large tangles with rods seen on light microscopy. Nemaline rods were rarely observed also in nuclei. We speculate that the mutated MyHC-IIA may influence myofibril disorganization. While nemaline rods have been described in myopathies caused by pathogenic variants in genes encoding several sarcomeric proteins, to our knowledge, nemaline rods have not been previously described in MYH2-myopathy.

The Formin Inhibitor, SMIFH2, Inhibits Members of the Myosin Superfamily

The small molecular inhibitor of formin FH2 domains, SMIFH2, is widely used in cell biological studies. It inhibits formin-driven actin polymerization in vitro, but not polymerization of pure actin. It is active against several types of formins from different species (Rizvi et al., 2009). Here, we found that SMIFH2 inhibits retrograde flow of myosin 2 filaments and contraction of stress fibers. We further checked the effect of SMIFH2 on non-muscle myosin 2A and skeletal muscle myosin 2 in vitro and found that SMIFH2 inhibits myosin ATPase activity and ability to translocate actin filaments in the in vitro motility assay. The inhibition of non-muscle myosin 2A in vitro required a higher concentration of SMIFH2 than for the inhibition of retrograde flow and stress fiber contraction in cells. We also found that SMIFH2 inhibits several other non-muscle myosin types, e.g. mammalian myosin 10, Drosophila myosin 7a and Drosophila myosin 5, more efficient than inhibition of formins. These off-target inhibitions demand additional careful analysis in each case when solely SMIFH2 is used to probe formin functions.

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.