Wielders S J H, Bennaghmouch A, Reutelingsperger C P M, Bevers E M, Lindhout T
Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, the Netherlands.
J Thromb Haemost. 2007 Mar;5(3):571-6. doi: 10.1111/j.1538-7836.2007.02364.x. Epub 2006 Dec 13.
Blockade of the thrombin receptors protease-activated receptor (PAR)1 and PAR4 with pepducins, cell-penetrating lipopeptides based on the third intracellular loop of PAR1 and PAR4, effectively inhibits platelet aggregation. We have previously shown that PAR1 pepducin also exerts an anticoagulant activity by partial inhibition of the thrombin plus collagen-induced externalization of phosphatidylserine (PS) at the platelet plasma membrane.
In the present study we examined the effects of PAR1 and PAR4 pepducins on tissue factor (TF)-initiated thrombin generation in platelet-rich plasma (PRP) and the interaction between PAR4 pepducin-loaded mouse platelets and a growing thrombus to confirm the relevance of the in vitro data.
Localization of pepducins at the inner leaflet of the plasma membrane was confirmed with a fluorescence resonance energy transfer assay. Both the PAR1 pepducin, P1pal12, and the PAR4 pepducin, P4pal10, inhibited TF-initiated thrombin generation in PRP. Concentrations of P1pal12 and P4pal10, which blocked the thrombin-induced influx of extracellular calcium ions and inhibited platelet aggregation, reduced the rate of thrombin generation during the propagation phase by 38% and 36%, respectively. Whether this anticoagulant effect is relevant in inhibiting in vivo arterial thrombin growth is uncertain because P4pal10 prevented the incorporation of platelets in a growing thrombus.
Our findings suggest that in spite of their potential anticoagulant activities the in vivo antithrombotic effect of intracellular PAR pepducins is mainly based on inhibiting platelet-platelet interactions.
使用基于蛋白酶激活受体（PAR）1和PAR4第三细胞内环的细胞穿透脂肽（pepducins）阻断凝血酶受体PAR1和PAR4，可有效抑制血小板聚集。我们之前已经表明，PAR1 pepducin还通过部分抑制凝血酶加胶原诱导的血小板质膜上磷脂酰丝氨酸（PS）的外化发挥抗凝活性。
在本研究中，我们检测了PAR1和PAR4 pepducins对富含血小板血浆（PRP）中组织因子（TF）启动的凝血酶生成的影响，以及加载PAR4 pepducin的小鼠血小板与正在形成的血栓之间的相互作用，以证实体外数据的相关性。
通过荧光共振能量转移分析证实了pepducins在质膜内小叶的定位。PAR1 pepducin P1pal12和PAR4 pepducin P4pal10均抑制PRP中TF启动的凝血酶生成。阻断凝血酶诱导的细胞外钙离子内流并抑制血小板聚集的P1pal12和P4pal10浓度，分别使传播阶段的凝血酶生成速率降低了38%和36%。由于P4pal10阻止血小板掺入正在形成的血栓中，这种抗凝作用在抑制体内动脉血栓中凝血酶生长方面是否相关尚不确定。
我们的研究结果表明，尽管细胞内PAR pepducins具有潜在的抗凝活性，但其体内抗血栓作用主要基于抑制血小板 - 血小板相互作用。
