Skeletal muscle myosin (SkM) promotes thrombus formation in flowing blood, supports prothrombinase activity in plasma, and promotes prothrombin activation by factors (F) Xa and Va in purified systems.1 SkM binds FXa and FVa and effectively replaces the procoagulant activity of phospholipid vesicles. This discovery challenges a long-standing paradigm for blood coagulation in which coagulation reactions, except for contact activation reactions, are thought to occur only on surfaces containing negatively charged phospholipids, which include phosphatidylserine.62 Thrombin provides both positive feedback upregulation of coagulation by activating various clotting factors and negative feedback downregulation of coagulation by activating protein C to the potent anticoagulant, activated protein C (APC).62 Because APC is potent for negative feedback downregulation of thrombin generation in vivo,7 here we assessed and describe that SkM enhances the anticoagulant activity of APC in purified human clotting factor reaction mixtures and in human plasma. This indicates that SkM can promote both thrombin generation and negative feedback downregulation of thrombin generation.
Because the major mechanism of the anticoagulant activity of APC involves irreversible proteolytic inactivation of FVa, purified human FVa was incubated with APC and varying concentrations of SkM; then residual FVa activity was determined (see the Online Supplementary Appendix for details of the methods and the reagents used). These SkM dose-response studies for FVa inactivation by APC showed a maximum effect at SkM concentrations of 30-40 nM and a half-maximal effect (EC50) at 10 nM SkM (Figure 1A). When the time-course for FVa inactivation by APC in the presence of 40 nM SkM was monitored and compared to that in the presence of 25 μM phospholipids, the rate and extent of FVa inactivation by APC were very similar for SkM and for phospholipids (Figure 1B). To evaluate whether phosphatidylserine contamination of SkM might be responsible for the activity of SkM, the purified SkM reagent was submitted to Avanti Polar Lipids, Inc. (Alabaster, AL, USA) and analyzed using liquid chromatography mass spectrometry. This analysis showed that 40 nM SkM contained only 1.0 nM phosphatidylserine which could not possibly explain why 40 nM SkM is as potent as the optimal level of 25 μM phospholipids. Notably, in separate studies, 40 nM phospholipids provided negligible inactivation of FVa (data not shown).
Because protein S is an anticoagulant cofactor for APC, the ability of protein S to enhance SkM-dependent inactivation of FVa by APC was assessed. When the time course for FVa inactivation by APC in the absence and presence of protein S was determined, protein S significantly enhanced this reaction (Figure 1C). The initial rate of FVa inactivation by APC was maximally enhanced four-fold by the optimal level of 100 nM protein S (Figure 1C). The effects of SkM on specific proteolytic cleavages of FVa by APC in the absence and presence of protein S were examined by immunoblotting using a monoclonal antibody that is directed against an epitope in the 307-506 amino acid region of the FVa heavy chain (Figure 1D). Bands were identified by epitope, by apparent molecular mass, and by comparison to bands previously identified in studies of FVa and of FVa cleavage site mutants.98 As seen in the immunoblot (Figure 1D), the most visually prominent FVa heavy chain fragment due to cleavage by APC in the presence of SkM but absence of protein S was due to cleavage at Arg506, resulting in appearance of the 1-506 amino acid fragment. But strikingly, when protein S was present, the visually evident cleavage by APC in the presence of SkM was at Arg306 because the most prominent fragment was the 307-506 amino acid fragment. Such SkM-supported cleavages are consistent with previous studies which showed that, in the presence of phospholipids, protein S enhances the cleavage of APC at R306.1110 Thus, SkM may function as an anticoagulant APC cofactor.
To determine whether SkM enhances APC anticoagulant activity in the milieu of plasma, clotting assays were employed using protocols that avoided adding exogenous phospholipid vesicles which themselves would promote the anticoagulant effects of APC. When the effects of varying levels of SkM on FXa one-stage assays were studied, there was a procoagulant effect of increasing SkM concentrations in the absence of addition of APC, i.e., there was a shortening of the clotting times from 87 to 52 s (Figure 1E), consistent with the known procoagulant effects of SkM in plasma.1 Strikingly, in the presence of APC, SkM addition increased rather than decreased the clotting times, e.g., from 129 to 207 s (Figure 1E), showing that SkM enhanced the anticoagulant actions of APC in plasma. The APC sensitivity ratio, defined here as the ratio of clotting times in the presence of APC compared to in its absence, rose significantly with increasing SkM concentrations from 1.5 in the absence of SkM to 3.9 at 200 nM SkM (Figure 1E). Kaolin-induced clotting time assays were also used with plasma conditions chosen to emphasize that changes were due to FVa inactivation in plasma. For this purpose, when FV-deficient plasma was reconstituted with 2 nM of FVa, addition of increasing concentrations of SkM produced significant procoagulant effects as clotting times were reduced from 126 to 71 s (Figure 1F). However, when APC was present, addition of SkM produced significant anticoagulant effects as clotting times were prolonged from 187 to 306 s (Figure 1F). The calculated APC sensitivity ratio rose significantly, as a function of increasing SkM concentrations, from 1.4 to 4.2 (Figure 1F). Thus, data here from two different coagulation assays unambiguously demonstrate that SkM can enhance the anticoagulant actions of APC, not only in purified reaction mixtures but also in the plasma environment.
SkM can provide a surface on which both procoagulant and anticoagulant reactions, namely prothrombin activation1 and FVa inactivation, respectively, are enhanced. So here we tested the ability of SkM to provide a surface for enhancement of protein C activation by thrombin or thrombin:thrombomodulin, and we found no influence of SkM on protein C activation. When SkM was tested at concentrations ranging from 5 to 100 nM, it did not change the rate of protein C activation by thrombin in purified reaction mixtures with or without calcium ions (Figure 2A), by thrombin and soluble thrombomodulin in the presence of calcium ions (Figure 2B), or by thrombin in the presence of endothelial cells (Figure 2C). Thus, SkM had no effect on protein C activation. The fact that SkM does not support the activation of protein C by thrombin ± thrombomodulin in purified reaction mixtures or on endothelial cells suggests that SkM enhancement of protein C activation is unlikely to occur in vivo, although this idea needs in vivo experimentation in order that a firm conclusion can be reached.
Since procoagulant and anticoagulant mechanisms must normally be balanced overall in vivo and since phospholipids enhance both thrombin generation and APC anticoagulant activity, here we investigated whether SkM could support not only procoagulant prothrombinase reactions but also the balancing anticoagulant activities by APC and protein S. In purified clotting factor mixtures, SkM enhanced APC/protein S proteolytic inactivation of FVa. In two distinct types of plasma clotting assays, kaolin-induced clotting time and FXa one-stage assays, SkM dose-dependently shortened plasma clotting times in the absence of APC by enhancing prothrombinase activity; however, in the presence of APC, SkM strikingly prolonged clotting times, implying that SkM is an anticoagulant APC cofactor. Thus, these studies extend the potential role of SkM in supporting procoagulant reactions1 to that of supporting the anticoagulant actions of APC and protein S, indicating that SkM may contribute to negative feedback downregulation of thrombin generation (Figure 3).
The possibility that SkM might contribute to trauma-induced coagulopathy was suggested by the observation that SkM is increased in plasma of trauma patients1 and that anti-SkM antibodies can significantly reduce thrombin generation in plasma from patients with trauma-induced coagulopathy.1 The novel APC cofactor activity of SkM discovered here may have direct relevance for trauma-induced coagulopathy. One major mechanism thought to contribute to this form of coagulopathy involves hyperactivation of the protein C system, with consumption of FV and FVIII, and hyperfibrinolysis.1512 It has been noted that 25-33% of severely injured patients present with elevated APC plasma levels, which are associated with increased blood transfusion requirements and mortality.15 This so-called “activated protein C trauma-induced coagulopathy mechanism” is linked to suppression of coagulation and enhanced fibrinolysis.15 Our discovery that SkM has APC cofactor activity raises a number of new questions about the mechanisms of trauma-induced coagulopathy, such as whether trauma-induced exposure of SkM can causally contribute to excessive suppression of coagulation and hemostasis in subsets of trauma patients with increased bleeding risk. Further studies of SkM levels and activities in patients with trauma-induced coagulopathy and in preclinical trauma models are needed. Coagulation studies in the future will need to take these newly revealed mechanisms for diverse activities of SkM (Figure 3) into account and determine the potential physiological relevance for the procoagulant and anticoagulant properties of SkM, especially in the context of trauma-induced coagulopathy.
- Deguchi H, Sinha RK, Marchese P. Prothrombotic skeletal muscle myosin directly enhances prothrombin activation by binding factors Xa and Va. Blood. 2016; 128(14):1870-1878. PubMedhttps://doi.org/10.1182/blood-2016-03-707679Google Scholar
- Furie B, Furie BC. Mechanisms of thrombus formation. New Eng J Med. 2008; 359(9):938-949. PubMedhttps://doi.org/10.1056/NEJMra0801082Google Scholar
- Versteeg HH, Heemskerk JW, Levi M, Reitsma PH. New fundamentals in hemostasis. Physiol Rev. 2013; 93(1):327-358. PubMedhttps://doi.org/10.1152/physrev.00016.2011Google Scholar
- Bevers EM, Williamson PL. Getting to the outer leaflet: physiology of phosphatidylserine exposure at the plasma membrane. Physiol Rev. 2016; 96(2):605-645. PubMedhttps://doi.org/10.1152/physrev.00020.2015Google Scholar
- Flaumenhaft R. Thrombus formation reimagined. Blood. 2014; 124(11):1697-1698. PubMedhttps://doi.org/10.1182/blood-2014-06-579656Google Scholar
- Ivanciu L, Krishnaswamy S, Camire RM. New insights into the spatiotemporal localization of prothrombinase in vivo. Blood. 2014; 124(11):1705-1714. PubMedhttps://doi.org/10.1182/blood-2014-03-565010Google Scholar
- Gresele P, Momi S, Berrettini M. Activated human protein C prevents thrombin-induced thromboembolism in mice. Evidence that activated protein C reduces intravascular fibrin accumulation through the inhibition of additional thrombin generation. J Clin Invest. 1998; 101(3):667-676. PubMedhttps://doi.org/10.1172/JCI575Google Scholar
- Heeb MJ, Rehemtulla A, Moussalli M, Kojima Y, Kaufman RJ. Importance of individual activated protein C cleavage site regions in coagulation factor V for factor Va inactivation and for factor Xa activation. Eur J Biochem. 1999; 260(1):64-75. PubMedGoogle Scholar
- Hockin MF, Cawthern KM, Kalafatis M, Mann KG. A model describing the inactivation of factor Va by APC: bond cleavage, fragment dissociation, and product inhibition. Biochemistry. 1999; 38(21):6918-6934. PubMedhttps://doi.org/10.1021/bi981966eGoogle Scholar
- Rosing J, Hoekema L, Nicolaes GA. Effects of protein S and factor Xa on peptide bond cleavages during inactivation of factor Va and factor VaR506Q by activated protein C. J Biol Chem. 1995; 270(46):27852-27858. PubMedhttps://doi.org/10.1074/jbc.270.46.27852Google Scholar
- Kalafatis M, Mann KG. Role of the membrane in the inactivation of factor Va by activated protein C. J Biol Chem. 1993; 268(36):27246-27257. PubMedGoogle Scholar
- Brohi K, Cohen MJ, Ganter MT, Matthay MA, Mackersie RC, Pittet JF. Acute traumatic coagulopathy: initiated by hypoperfusion: modulated through the protein C pathway?. Ann Surg. 2007; 245(5):812-818. PubMedhttps://doi.org/10.1097/01.sla.0000256862.79374.31Google Scholar
- Cohen MJ, Brohi K, Ganter MT, Manley GT, Mackersie RC, Pittet JF. Early coagulopathy after traumatic brain injury: the role of hypoperfusion and the protein C pathway. J Trauma. 2007; 63(6):1254-1261. PubMedhttps://doi.org/10.1097/TA.0b013e318156ee4cGoogle Scholar
- Cohen MJ, Call M, Nelson M. Critical role of activated protein C in early coagulopathy and later organ failure, infection and death in trauma patients. Ann Surg. 2012; 255(2):379-385. PubMedhttps://doi.org/10.1097/SLA.0b013e318235d9e6Google Scholar
- Kornblith LZ, Moore HB, Cohen MJ. Trauma-induced coagulopathy: the past, present, and future. J Thromb Haemost. 2019; 17(6):852-862. Google Scholar