Department of Molecular Medicine, The Scripps Research Institute, La Jolla, California, USA.
Department of Molecular Medicine, The Scripps Research Institute, La Jolla, California, USA; Departments of Biomedical Engineering and Medicine, School of Medicine, Oregon Health & Science University, Portland, Oregon, USA.
J Biol Chem. 2022 Feb;298(2):101567. doi: 10.1016/j.jbc.2022.101567. Epub 2022 Jan 7.
Skeletal muscle myosin (SkM) has been shown to possess procoagulant activity; however, the mechanisms of this coagulation-enhancing activity involving plasma coagulation pathways and factors are incompletely understood. Here, we discovered direct interactions between immobilized SkM and coagulation factor XI (FXI) using biolayer interferometry (K = 0.2 nM). In contrast, we show that prekallikrein, a FXI homolog, did not bind to SkM, reflecting the specificity of SkM for FXI binding. We also found that the anti-FXI monoclonal antibody, mAb 1A6, which recognizes the Apple (A) 3 domain of FXI, potently inhibited binding of FXI to immobilized SkM, implying that SkM binds FXI A3 domain. In addition, we show that SkM enhanced FXI activation by thrombin in a concentration-dependent manner. We further used recombinant FXI chimeric proteins in which each of the four A domains of the heavy chain (designated A1 through A4) was individually replaced with the corresponding A domain from prekallikrein to investigate SkM-mediated enhancement of thrombin-induced FXI activation. These results indicated that activation of two FXI chimeras with substitutions of either the A3 domains or A4 domains was not enhanced by SkM, whereas substitution of the A2 domain did not reduce the thrombin-induced activation compared with wildtype FXI. These data strongly suggest that functional interaction sites on FXI for SkM involve the A3 and A4 domains. Thus, this study is the first to reveal and support the novel intrinsic blood coagulation pathway concept that the procoagulant mechanisms of SkM include FXI binding and enhancement of FXI activation by thrombin.
骨骼肌肌球蛋白 (SkM) 已被证明具有促凝活性；然而，涉及血浆凝血途径和因子的这种凝血增强活性的机制尚未完全阐明。在这里，我们使用生物层干涉法 (K=0.2 nM) 发现了固定化 SkM 与凝血因子 XI (FXI) 之间的直接相互作用。相比之下，我们表明，FXI 同源物前激肽原不会与 SkM 结合，这反映了 SkM 对 FXI 结合的特异性。我们还发现，识别 FXI Apple (A) 3 结构域的抗 FXI 单克隆抗体 mAb 1A6 强烈抑制了 FXI 与固定化 SkM 的结合，这表明 SkM 结合 FXI A3 结构域。此外，我们表明 SkM 以浓度依赖的方式增强了凝血酶对 FXI 的激活。我们进一步使用重组 FXI 嵌合蛋白，其中重链的四个 A 结构域（分别命名为 A1 至 A4）中的每一个都被前激肽原的相应 A 结构域取代，以研究 SkM 介导的对凝血酶诱导的 FXI 激活的增强作用。这些结果表明，SkM 不会增强两个嵌合 FXI 蛋白的激活，这些嵌合 FXI 蛋白的 A3 或 A4 结构域被取代，而 A2 结构域的取代与野生型 FXI 相比并没有降低凝血酶诱导的激活。这些数据强烈表明，FXI 上与 SkM 相互作用的功能位点涉及 A3 和 A4 结构域。因此，本研究首次揭示并支持了 SkM 的促凝机制包括 FXI 结合和增强凝血酶对 FXI 的激活的新内在血液凝固途径概念。
