...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Biochemistry >Plasmin desensitization of the PAR1 thrombin receptor: kinetics, sites of truncation, and implications for thrombolytic therapy.
【24h】

Plasmin desensitization of the PAR1 thrombin receptor: kinetics, sites of truncation, and implications for thrombolytic therapy.

机译：PAR1凝血酶受体的纤溶酶脱敏：动力学，截断部位和溶栓治疗的影响。

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

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 R41-S42 peptide bond with a kcat of 120 s-1 and a KM of 16 microM 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 (kcat = 30 s-1) and affinity (KM = 18 microM). 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.
机译：已经假设蛋白酶激活的受体可以被一种以上的蛋白酶激活和减弱。在这里，我们探索抗凝蛋白酶对PAR1凝血酶受体的脱敏机理，并为为什么纤溶酶/组织纤溶酶原激活物（t-PA）既可以激活凝血酶又可以使血小板失活的谜团提供解释。通过使用可溶性N末端外域（TR78）作为全长受体的模型，我们能够明确比较血清蛋白酶之间的切割速率和特异性。凝血酶以120 s-1的kcat和16 microM的KM切割R41-S42肽键处的TR78，以产生TR62（残基42-103）。我们发现，在抗凝蛋白酶中，只有纤溶酶可以在位于将配体束缚到受体体内的连接子区域的R70 / K76 / K82位点上快速截断可溶性外域结构域。 TR78外域的血浆蛋白裂解几乎与R41处的凝血酶裂解等价（kcat = 30 s-1）和亲和力（KM = 18 microM）。证实了特异性，因为在其他五个潜在的纤溶酶裂解位点没有观察到裂解。纤溶酶还在R41凝血酶切割位点切割TR78外域，从而产生瞬时活化的外域。我们直接证明纤溶酶在酵母和COS7成纤维细胞中表达的全长膜嵌入受体中裂解了这些相同的位点。在广泛糖基化的COS7表达受体与未糖基化的酵母生产受体之间，纤溶酶截短率相似。 R70 / K76 / K82位点突变为A70 / A76 / A82可消除纤溶酶的截断和凝血酶依赖性Ca2 +信号转导的脱敏作用，并将PAR1转化为对纤溶酶具有完全激动活性的纤溶酶活化受体。纤溶酶不会使血小板或COS7细胞的Ca2 +反应对SFLLRN脱敏，这与分子间配体结合位点位于K82的C端侧一致。在C末端纤溶酶裂解位点截断野生型受体可除去N末端拴系的配体或预配体，从而为体内PAR1脱敏提供有效途径。

著录项

相似文献

  • 外文文献
  • 中文文献
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号