ALBERT

Description: 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.

Description: Thrombin generation and fibrin formation can cause occlusive thrombosis and myocardial infarction is caused by occlusive thrombi. Exposure and release of cardiac myosin (CM) are linked to myocardial infarction, but CM has not been accorded any thrombotic functional significance. Skeletal muscle myosin (SkM), which is structurally similar to CM, was previously shown to exert procoagulant activities (Deguchi H et al, Blood. 2016;128:1870), leading us to undertake new studies of the in vitro and in vivo procoagulant activities of CM. First, the setting of hemophilia A with its remarkable bleeding risk was used to evaluate the procoagulant properties of CM. In studies of human hemophilia plasma and of murine acquired hemophilia A plasma, CM was added to these plasmas and tissue factor (TF)-induced thrombin generation assays were performed. Plasmas included human hemophilia A plasma and C57BL/6J mouse plasma with anti-FVIII antibody (GMA-8015; 5 microgram/mL final). CM showed strong procoagulant effects in human hemophilia A plasma, which is naturally deficient in factor VIII (

Description: Neural micro-electrode arrays that are transparent over a broad wavelength spectrum from ultraviolet to infrared could allow for simultaneous electrophysiology and optical imaging, as well as optogenetic modulation of the underlying brain tissue. The long-term biocompatibility and reliability of neural micro-electrodes also require their mechanical flexibility and compliance with soft tissues. Here we present a graphene-based, carbon-layered electrode array (CLEAR) device, which can be implanted on the brain surface in rodents for high-resolution neurophysiological recording. We characterize optical transparency of the device at 〉90% transmission over the ultraviolet to infrared spectrum and demonstrate its utility through optical interface experiments that use this broad spectrum transparency. These include optogenetic activation of focal cortical areas directly beneath electrodes, in vivo imaging of the cortical vasculature via fluorescence microscopy and 3D optical coherence tomography. This study demonstrates an array of interfacing abilities of the CLEAR device and its utility for neural applications.