Carboxypeptidases in Thrombosis and Inflammation

描述（由申请人提供）： 炎症和促凝血反应通常具有保护作用，但也可能导致败血症和心脏病发作等疾病。本研究旨在阐明两种羧肽酶（CP）在调节纤溶和炎症反应中的作用。两种CP，活化的TAFI（TAFla）和羧肽酶N（CPN）都从蛋白质底物去除C-末端赖氨酸和精氨酸残基。 然而，虽然TAFla优选Arg，但CPN优选Lys底物。此外，TAFI需要激活，并可能在“按需”的情况下发挥作用，而CPN是天生活跃，并可能发挥“管家”的作用。TAFla抑制纤维蛋白溶解并可能加剧血栓形成，而CPN可能不会。相反，CPN被认为通过灭活补体激活产生的过敏毒素在调节炎症中起主要作用，而TAFI在该过程中的作用尚不清楚。这两种酶是如何独立调节的，其中一种酶的存在如何影响总的或整体的活性？我们认为TAFla和CPN在不同程度上介导纤维蛋白溶解和炎症的调节，但不是相互排斥的。它们协同作用，以保持对止血和炎症反应的精确控制。我们的具体目标是：1）确定人血浆中TAFI的功能浓度和抗原浓度之间的相关性，2）评估TAFla和CPN在纯化系统中的抗纤维蛋白溶解作用，3）确定细胞表面上的糖胺聚糖参与纤溶酶介导的TAFI活化的作用，4）确定CPN和TAFla对抑制C3 a介导的钙离子流出的作用。活性测定将在2 × 2基质中进行，改变TAFla和CPN的两种浓度，以评估协同效应。从长远来看，这种理解将导致新的治疗干预措施，这些干预措施可能特异性地和显著地影响血栓形成，而不会对炎症反应产生负面影响，反之亦然。

DESCRIPTION (provided by applicant): Inflammatory and procoagulant responses are often protective and yet may contribute to maladies such as sepsis and heart attacks. This study focuses on elucidating the role(s) of two carboxypeptidases (CP) in regulating the fibrinolytic and inflammatory responses. The two CP, activated TAFI (TAFla) and carboxypeptidase N (CPN) both remove C-terminal lysine and arginine residues from protein substrates. However, while TAFla prefers Arg, CPN prefers Lys substrates. Also, TAFI requires activation and may play a role in "on demand" situations whereas CPN is constitutively active and may play a "house-keeping" role. TAFla inhibits fibrinolysis and could exacerbate thrombosis whereas CPN may not. In contrast CPN is considered to play a predominant role in regulating inflammation by inactivating anaphylatoxins generated by complement activation whereas the role of TAFI in this process is unknown. How are these two enzymes independently regulated and how does the presence of one affect the total or overall activity? We believe that regulation of fibrinolysis and inflammation are mediated to different extents by TAFla and CPN but are not mutually exclusive. They act synergistically in order to maintain exquisite control of both hemostatic and inflammatory responses. Our specific aims are to; 1) determine the correlation between functional and antigenic concentrations of TAFI in human plasma, 2) to assess the antifibrinolytic effects of TAFla and CPN in a purified system, 3) to determine the role of glycosaminoglycans on cell surfaces to participate in plasmin-mediated TAFI activation, 4) to determine the effect of CPN and TAFla on inhibition of C3a-mediated calcium ion efflux. Activity assays will be performed in a 2 x 2 matrix varying both concentrations of TAFla and CPN in order to assess synergistic effects. In the long-term this understanding this will lead to new therapeutic interventions which may specifically and significantly affect thrombosis without negatively affecting inflammatory responses or vice versa.