Plasmin desensitization of the PARI thrombin receptor: Kinetics, sites of truncation, and implications for thrombolytic therapy
Plasmin desensitization of the PARI thrombin receptor: Kinetics, sites of truncation, and implications for thrombolytic therapy
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DOI：
10.1021/bi9824792
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发表时间：
1999-04-06
期刊：
影响因子：
2.9
通讯作者：
Costello, CE
中科院分区：
文献类型：
--
作者：
Kuliopulos, A;Covic, L;Costello, CE
据推测，蛋白酶激活的受体可能被一种以上的蛋白酶激活和减弱。在这里，我们探讨了抗凝酶蛋白酶对PAR1凝血酶受体的脱敏机制，并解释了为什么纤溶酶/组织纤溶酶原激活剂（t-PA）在凝血酶治疗前既能激活血小板又能使血小板失活。通过使用可溶性n端外结构域（TR78）作为全长受体的模型，我们能够明确地比较血清蛋白酶之间的裂解率和特异性，凝血酶在R-41-S-42肽键上裂解TR78， k（cat）为120 s(-1)， k -M为16 μ M，以产生TR62（残基42-103）。我们发现，在抗凝蛋白酶中，只有纤溶酶能在连接配体和受体的连接体区域的R70/K76/K82位点上快速截断可溶性外结构域。TR78外结构域的纤溶酶裂解几乎等同于凝血酶在R41的裂解，具有相似的速率(k（cat) = 30 s(-1)）和亲和力（KM = 18 μ M）。特异性被证明，因为在其他五个潜在的纤溶蛋白切割位点没有观察到切割。我们直接证明，在酵母和COS7成纤维细胞中表达的全长膜嵌入受体中，纤溶酶也能在R41凝血酶裂解位点切割TR78外结构域，从而产生瞬时激活的外结构域。在广泛糖基化的COS7表达受体和非糖基化的酵母产生的受体中，纤溶酶的截断率相似。R70/K76/K82位点突变为A70/A76/A82，消除了纤溶蛋白截断和凝血酶依赖性Ca2+信号的脱敏，并将PAR1转化为纤溶蛋白激活受体，具有纤溶蛋白的完全激动剂活性。纤溶蛋白不会使血小板或COS7细胞对SFLLRN的Ca2+反应脱敏，这与分子间配体结合位点位于K82的c端一致，在c端纤溶蛋白切割位点截断野生型受体可去除n端系链配体或预配体，从而为体内PAR1脱敏提供了有效途径。
It has been hypothesized that protease-activated receptors may be activated and attenuated by more than one protease. Here, we explore a desensitization mechanism of the PAR1 thrombin receptor by anticoagulant proteases and provide an explanation to the enigma of why plasmin/tissue plasminogen activator (t-PA) can both activate and deactivate platelets prior to thrombin treatment. By using a soluble N-terminal exodomain (TR78) as a model for the full-length receptor, we were able to unambiguously compare cleavage rates and specificities among the serum proteases, Thrombin cleaves TR78 at the R-41-S-42 peptide bond with a k(cat) of 120 s(-1) and a K-M of 16 mu M to produce TR62 (residues 42-103). We found that, of the anticoagulant proteases, only plasmin can rapidly truncate the soluble exodomain at the R70/K76/K82 sites located on a linker region that tethers the ligand to the body of the receptor. Plasmin cleavage of the TR78 exodomain is nearly equivalent to that of thrombin cleavage at R41 with similar rates (k(cat) = 30 s(-1)) and affinity (KM = 18 mu M). Specificity was demonstrated since there is no observed cleavage at the five other potential plasmin-cleavage sites. Plasmin also cleaves the TR78 exodomain at the R41 thrombin-cleavage site generating transiently activated exodomain, We directly demonstrated that plasmin cleaves these same sites in full-length membrane-embedded receptor expressed in yeast and COS7 fibroblasts, The rate of plasmin truncation is similar between the extensively glycosylated COS7-expressed receptor and the nonglycosylated yeast-produced receptor. Mutation of the R70/K76/K82 sites to A70/A76/A82 eliminates plasmin truncation and desensitization of thrombin-dependent Ca2+ signaling and converts PAR1 into a plasmin-activated receptor with full agonist activity for plasmin. Plasmin does not desensitize the Ca2+ response of platelets or COS7 cells to SFLLRN consistent with intermolecular ligand-binding sites being located to the C-terminal side of K82, Truncation of the wild-type receptor at the C-terminal plasmin-cleavage sites removes the N-terminal tethered ligand or preligand, thereby providing an effective pathway for PAR1 desensitization in vivo.