Vincent Laurence, Lapointe Catherine, McDonald Patrick P, Rammos Christos, Rassaf Tienush, Köllnberger Maria, Tinel Hanna, Heitmeier Stefan, D'Orléans-Juste Pedro
Department of Pharmacology and Physiology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada.
UNC Blood Research Center, University of North Carolina at Chapel Hill, NC, United States.
Front Immunol. 2025 Apr 17;16:1511990. doi: 10.3389/fimmu.2025.1511990. eCollection 2025.
Our group recently reported that chymase, a serine protease synthetized and released by mast cells, plays a pivotal role in thrombi stabilization in murine deep vein thrombosis (DVT) models, by interfering with the fibrinolytic properties of endogenous plasmin within thrombi. However, the impact of mast cell-derived chymase on endogenous plasmin activity in human blood clots has yet to be explored.
The antifibrinolytic properties of human recombinant chymase (rCMA-1) were investigated using an thrombolysis assay based on halo-shaped human blood clots. In addition, a fluorogenic assay was used to detect chymase activity in human thromboembolic biopsies. In both assays, the activity of human chymase was validated using a specific chymase inhibitor, fulacimstat (BAY 1142524).
Although rapidly neutralized in plasma, rCMA-1 remains active within the local microenvironment of a blood clot, inducing resistance to endogenous plasmin-mediated fibrinolysis in the presence of recombinant tissue plasminogen activator (tPA). This leads to a concentration-dependent decrease in clot lysis by rCMA-1. The plasmin-inactivating properties of rCMA-1 were inhibited by fulacimstat, resulting in an accelerated clot dissolution. The pro-fibrinolytic effects of fulacimstat in human blood clots were reversed by a plasminogen/plasmin inhibitor, BAY 1214237. Finally, fulacimstat-sensitive chymase activity was identified in thrombi collected from thrombectomy patients, supporting the potential role of the mast cell-derived serine protease in human blood clot stabilization under pathological conditions.
These experiments with human whole blood suggest that mast cell-derived chymase impairs blood clot resolution by interfering with the fibrinolytic activity of endogenous intra-clot plasmin. Our findings provide evidence that recombinant mast cell chymase interferes with endogenous plasmin activity in human whole blood clots and support the potential of chymase inhibitors, such as fulacimstat, as fibrinolytic agents for thrombotic and thromboembolic disorders.
我们的研究小组最近报告称，肥大细胞合成并释放的丝氨酸蛋白酶糜酶，通过干扰血栓内源性纤溶酶的纤维蛋白溶解特性，在小鼠深静脉血栓形成（DVT）模型的血栓稳定中起关键作用。然而，肥大细胞源性糜酶对人血凝块内源性纤溶酶活性的影响尚未得到探索。
使用基于晕圈状人血凝块的溶栓试验研究人重组糜酶（rCMA-1）的抗纤维蛋白溶解特性。此外，使用荧光测定法检测人血栓栓塞活检中的糜酶活性。在这两种试验中，使用特异性糜酶抑制剂富拉西司他（BAY 1142524）验证人糜酶的活性。
尽管rCMA-1在血浆中迅速被中和，但它在血凝块的局部微环境中仍保持活性，在重组组织型纤溶酶原激活剂（tPA）存在的情况下，诱导对内源性纤溶酶介导的纤维蛋白溶解的抗性。这导致rCMA-1引起的血凝块溶解呈浓度依赖性降低。富拉西司他抑制了rCMA-1的纤溶酶失活特性，导致血凝块溶解加速。纤溶酶原/纤溶酶抑制剂BAY 1214237逆转了富拉西司他在人血凝块中的促纤维蛋白溶解作用。最后，在从血栓切除术患者收集的血栓中鉴定出对富拉西司他敏感的糜酶活性，支持肥大细胞源性丝氨酸蛋白酶在病理条件下人血凝块稳定中的潜在作用。
这些用人全血进行的实验表明，肥大细胞源性糜酶通过干扰血凝块内源性纤溶酶的纤维蛋白溶解活性来损害血凝块溶解。我们的研究结果提供了证据，即重组肥大细胞糜酶干扰人全血凝块中的内源性纤溶酶活性，并支持糜酶抑制剂如富拉西司他作为血栓形成和血栓栓塞性疾病的纤维蛋白溶解剂的潜力。
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