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

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.