Innovative Therapies in Hemostasis, INSERM U1140, Université Paris Cité, Paris, France.
Department of Physiology and Pharmacology, Universidad de Salamanca, Salamanca, Spain.
J Thromb Haemost. 2023 Jul;21(7):1943-1956. doi: 10.1016/j.jtha.2023.03.023. Epub 2023 Mar 28.
The circulating form of human endoglin (sEng) is a cleavage product of membrane-bound endoglin present on endothelial cells. Because sEng encompasses an RGD motif involved in integrin binding, we hypothesized that sEng would be able to bind integrin αIIbβ3, thereby compromising platelet binding to fibrinogen and thrombus stability.
In vitro human platelet aggregation, thrombus retraction, and secretion-competition assays were performed in the presence of sEng. Surface plasmon resonance (SPR) binding and computational (docking) analyses were carried out to evaluate protein-protein interactions. A transgenic mouse overexpressing human sEng (hsEng) was used to measure bleeding/rebleeding, prothrombin time (PT), blood stream, and embolus formation after FeCl-induced injury of the carotid artery.
Under flow conditions, supplementation of human whole blood with sEng led to a smaller thrombus size. sEng inhibited platelet aggregation and thrombus retraction, interfering with fibrinogen binding, but did not affect platelet activation. SPR binding studies demonstrated that the specific interaction between αIIbβ3 and sEng and molecular modeling showed a good fitting between αIIbβ3 and sEng structures involving the endoglin RGD motif, suggesting the possible formation of a highly stable αIIbβ3/sEng. hsEng mice showed increased bleeding time and number of rebleedings compared to wild-type mice. No differences in PT were denoted between genotypes. After FeCl injury, the number of released emboli in hsEng mice was higher and the occlusion was slower compared to controls.
Our results demonstrate that sEng interferes with thrombus formation and stabilization, likely via its binding to platelet αIIbβ3, suggesting its involvement in primary hemostasis control.
人内皮糖蛋白（sEng）的循环形式是内皮细胞上的膜结合内皮糖蛋白的裂解产物。因为 sEng 包含一个涉及整合素结合的 RGD 基序，我们假设 sEng 将能够结合整合素 αIIbβ3，从而破坏血小板与纤维蛋白原的结合和血栓稳定性。
在存在 sEng 的情况下进行体外人血小板聚集、血栓收缩和分泌竞争测定。进行表面等离子体共振（SPR）结合和计算（对接）分析，以评估蛋白质-蛋白质相互作用。使用过表达人 sEng（hsEng）的转基因小鼠来测量 FeCl 诱导颈动脉损伤后的出血/再出血、凝血酶原时间（PT）、血流和栓子形成。
在流动条件下，向人全血中补充 sEng 会导致血栓体积减小。sEng 抑制血小板聚集和血栓收缩，干扰纤维蛋白原结合，但不影响血小板激活。SPR 结合研究表明 αIIbβ3 和 sEng 之间的特异性相互作用，分子建模显示 αIIbβ3 和 sEng 结构之间具有良好的拟合，涉及内皮糖蛋白的 RGD 基序，表明可能形成高度稳定的 αIIbβ3/sEng。hsEng 小鼠与野生型小鼠相比，出血时间延长，再出血次数增加。两种基因型之间的 PT 无差异。FeCl 损伤后，hsEng 小鼠释放的栓子数量较高，闭塞较慢。
我们的结果表明，sEng 通过与血小板 αIIbβ3 结合来干扰血栓形成和稳定，这可能与其参与初级止血控制有关。
