Discovery and Translational Science Department, Leeds Institute of Cardiovascular and Metabolic Medicine, University of Leeds, Leeds, United Kingdom.
School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom.
J Thromb Haemost. 2023 Mar;21(3):667-681. doi: 10.1016/j.jtha.2022.09.004. Epub 2022 Dec 22.
The glycoprotein VI (GPVI) signaling pathway was previously reported to direct procoagulant platelet activity through collagen binding. However, the impact of GPVI-fibrin interaction on procoagulant platelet development and how it modulates the clot structure are unknown.
To determine the effect of GPVI-fibrin interaction on the platelet phenotype and its impact on the clot structure.
Procoagulant platelets in platelet-rich plasma clots were determined by scanning electron microscopy (wild-type and GPVI-deficient murine samples) and confocal microscopy. Procoagulant platelet number, clot density, clot porosity, and clot retraction were determined in platelet-rich plasma or whole blood clots of healthy volunteers in the presence of tyrosine kinase inhibitors (PRT-060318, ibrutinib, and dasatinib) and eptifibatide.
GPVI-deficient clots showed a higher nonprocoagulant vs procoagulant platelet ratio than wild-type clots. The fiber density and the procoagulant platelet number decreased in the presence of Affimer proteins, inhibiting GPVI-fibrin(ogen) interaction and the tyrosine kinase inhibitors. The effect of GPVI signaling inhibitors on the procoagulant platelet number was exacerbated by eptifibatide. The tyrosine kinase inhibitors led to an increase in clot porosity; however, no differences were observed in the final clot weight, following clot retraction with the tyrosine kinase inhibitors, except for ibrutinib. In the presence of eptifibatide, clot retraction was impaired.
Our findings showed that GPVI-fibrin interaction significantly contributes to the development of procoagulant platelets and that inhibition of GPVI signaling increases clot porosity. Clot contractibility was impaired by the integrin αIIbβ3 and Btk pathway inhibition. Thus, inhibition of GPVI-fibrin interactions can alleviate structural characteristics that contribute to a prothrombotic clot phenotype, having potential important implications for novel antithrombotic interventions.
先前有研究报道，糖蛋白 VI（GPVI）信号通路通过与胶原蛋白结合，指导促凝血血小板的活性。然而，GPVI 与纤维蛋白相互作用对促凝血血小板形成的影响以及它如何调节血栓结构尚不清楚。
确定 GPVI 与纤维蛋白相互作用对血小板表型的影响及其对血栓结构的影响。
通过扫描电子显微镜（野生型和 GPVI 缺陷型鼠样本）和共聚焦显微镜来确定富含血小板的血浆凝块中的促凝血血小板。在存在酪氨酸激酶抑制剂（PRT-060318、伊布替尼和达沙替尼）和依替巴肽的情况下，测定健康志愿者富含血小板的血浆或全血凝块中的促凝血血小板数量、凝块密度、凝块孔隙率和凝块回缩。
GPVI 缺陷型凝块中的非促凝血血小板与促凝血血小板的比例高于野生型凝块。在 Affimer 蛋白存在的情况下，纤维密度和促凝血血小板数量减少，抑制了 GPVI 与纤维蛋白（原）的相互作用和酪氨酸激酶抑制剂。GPVI 信号抑制剂对促凝血血小板数量的影响因依替巴肽的存在而加剧。酪氨酸激酶抑制剂导致血栓孔隙率增加；然而，除了伊布替尼外，在使用酪氨酸激酶抑制剂进行凝块回缩后，最终凝块重量没有差异。依替巴肽存在时，凝块回缩受损。
我们的研究结果表明，GPVI 与纤维蛋白的相互作用显著促进了促凝血血小板的形成，抑制 GPVI 信号通路会增加血栓的孔隙率。整合素 αIIbβ3 和 Btk 通路的抑制会损害凝块的收缩性。因此，抑制 GPVI 与纤维蛋白的相互作用可以减轻导致促血栓形成血栓表型的结构特征，这可能对新型抗血栓干预具有重要意义。
