Knauss Elizabeth A, Guci Johana, Luc Norman, Disharoon Dante, Huang Grace H, Gupta Anirban Sen, Nieman Marvin T
Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio, USA.
Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio, USA.
J Thromb Haemost. 2025 Apr;23(4):1278-1288. doi: 10.1016/j.jtha.2024.12.031. Epub 2025 Jan 9.
Hypercoagulation and thrombin generation are major risk factors for venous thrombosis. Sustained thrombin signaling through protease-activated receptor (PAR) 4 promotes platelet activation, phosphatidylserine exposure, and subsequent thrombin generation. A single nucleotide polymorphism in PAR4 (rs2227376) changes proline to leucine extracellular loop 3, which decreases PAR4 reactivity and is associated with a lower risk for venous thromboembolism (VTE) in a genome wide association studies meta-analysis.
The goal of this study was to determine the mechanism for the association of rs2227376 with a reduced risk of VTE using mice with a homologous mutation (PAR4-P322L).
Venous thrombosis was examined using our recently generated PAR4-P322L mice in the inferior vena cava stasis and stenosis models. Coagulation and clot stability were measured using rotational thromboelastometry. Thrombin-generating potential was measured in platelet-rich plasma. Phosphatidylserine surface expression and platelet-neutrophil aggregates were analyzed using flow cytometry.
Mice heterozygous (PAR4) or homozygous (PAR4) at position 310 had reduced sizes of venous clots at 48 hours. PAR4 and PAR4 platelets had progressively decreased phosphatidylserine in response to thrombin and convulxin, in addition to decreased thrombin generation and decreased PAR4-mediated platelet-neutrophil aggregation.
The leucine allele in extracellular loop 3, PAR4-322L, leads to fewer procoagulant platelets, decreased endogenous thrombin potential, and reduced platelet-neutrophil aggregation. This decreased ability to generate thrombin and bind to neutrophils offers a mechanism for PAR4's role in VTE, highlighting a key role for PAR4 signaling.
高凝状态和凝血酶生成是静脉血栓形成的主要危险因素。通过蛋白酶激活受体（PAR）4的持续凝血酶信号传导促进血小板活化、磷脂酰丝氨酸暴露以及随后的凝血酶生成。PAR4中的一个单核苷酸多态性（rs2227376）将细胞外环3中的脯氨酸变为亮氨酸，这降低了PAR4的反应性，并且在全基因组关联研究的荟萃分析中与较低的静脉血栓栓塞（VTE）风险相关。
本研究的目的是使用具有同源突变（PAR4-P322L）的小鼠来确定rs2227376与VTE风险降低之间关联的机制。
使用我们最近培育的PAR4-P322L小鼠在腔静脉淤滞和狭窄模型中检测静脉血栓形成。使用旋转血栓弹力图测量凝血和血凝块稳定性。在富含血小板的血浆中测量凝血酶生成潜力。使用流式细胞术分析磷脂酰丝氨酸表面表达和血小板-中性粒细胞聚集体。
在第310位杂合（PAR4）或纯合（PAR4）的小鼠在48小时时静脉血栓的大小减小。除了凝血酶生成减少和PAR4介导的血小板-中性粒细胞聚集减少外，PAR4和PAR4血小板对凝血酶和convulxin的反应使磷脂酰丝氨酸逐渐减少。
细胞外环3中的亮氨酸等位基因PAR4-322L导致促凝血小板减少、内源性凝血酶潜力降低以及血小板-中性粒细胞聚集减少。这种凝血酶生成和与中性粒细胞结合能力的降低为PAR4在VTE中的作用提供了一种机制，突出了PAR4信号传导的关键作用。
