Department of Cardiology and Angiology, University Hospital Tübingen, Eberhard Karls University Tübingen, Tübingen, Germany.
Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmaceutical Sciences, University of Tübingen, Tübingen, Germany.
Thromb Haemost. 2023 Jul;123(7):679-691. doi: 10.1055/s-0043-1768057. Epub 2023 Apr 10.
Hemolysis results in release of free hemoglobin and hemin liberation from erythrocytes. Hemin has been described to induce platelet activation and to trigger thrombosis.
We evaluated the effect of hemin on platelet function and surface expression of the platelet collagen receptor glycoprotein VI (GPVI). Isolated platelets were stimulated with increasing concentrations of hemin.
We found that hemin strongly enhanced platelet activation, aggregation, and aggregate formation on immobilized collagen under flow. In contrast, we found that surface expression of GPVI was significantly reduced upon hemin stimulation with high hemin concentrations indicating that hemin-induced loss of surface GPVI does not hinder platelet aggregation. Loss of hemin-induced surface expression of GPVI was caused by shedding of the ectodomain of GPVI as verified by immunoblotting and is independent of the GPVI or CLEC-2 mediated ITAM (immunoreceptor-tyrosine-based-activation-motif) signaling pathway as inhibitor studies revealed. Hemin-induced GPVI shedding was independent of metalloproteinases such as ADAM10 or ADAM17, which were previously described to regulate GPVI degradation. Similarly, concentration-dependent shedding of CD62P was also induced by hemin. Unexpectedly, we found that the subtilisin-like proprotein convertase furin controls hemin-dependent GPVI shedding as shown by inhibitor studies using the specific furin inhibitors SSM3 and Hexa-D-arginine. In the presence of SSM3 and Hexa-D-arginine, hemin-associated GPVI degradation was substantially reduced. Further, SSM3 inhibited hemin-induced but not CRP-XL-induced platelet aggregation and thrombus formation, indicating that furin controls specifically hemin-associated platelet functions.
In summary, we describe a novel mechanism of hemin-dependent GPVI shedding and platelet function mediated by furin.
溶血会导致游离血红蛋白和血红素从红细胞中释放出来。血红素已被描述为诱导血小板活化，并引发血栓形成。
我们评估了血红素对血小板功能和血小板胶原受体糖蛋白 VI（GPVI）表面表达的影响。用递增浓度的血红素来刺激分离的血小板。
我们发现血红素强烈增强了血小板在流动条件下对固定化胶原的激活、聚集和聚集体形成。相比之下，我们发现高血红素浓度刺激下，GPVI 的表面表达显著降低，表明血红素诱导的表面 GPVI 缺失并不妨碍血小板聚集。血红素诱导的 GPVI 表面表达缺失是由 GPVI 的胞外结构域脱落引起的，这通过免疫印迹得到证实，并且独立于 GPVI 或 CLEC-2 介导的 ITAM（免疫受体酪氨酸基激活基序）信号通路，因为抑制剂研究表明。血红素诱导的 GPVI 脱落独立于先前描述的调节 GPVI 降解的金属蛋白酶，如 ADAM10 或 ADAM17。同样，血红素也能诱导 CD62P 的浓度依赖性脱落。出乎意料的是，我们发现枯草溶菌素样前蛋白转化酶 furin 控制血红素依赖性 GPVI 脱落，如使用特异性 furin 抑制剂 SSM3 和六聚精氨酸进行的抑制剂研究所示。在 SSM3 和六聚精氨酸存在的情况下，血红素相关的 GPVI 降解显著减少。此外，SSM3 抑制了血红素诱导但不抑制 CRP-XL 诱导的血小板聚集和血栓形成，表明 furin 特异性控制血红素相关的血小板功能。
总之，我们描述了血红素依赖性 GPVI 脱落和由 furin 介导的血小板功能的新机制。
