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性，但可能会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(S)。两个CP，活化的TAFI(TAF1a)和羧基肽酶N(CPn)都能去除蛋白质底物上的C-末端赖氨酸和精氨酸残基。然而，虽然TAFla更喜欢Arg，但CPN更喜欢Lys底物。此外，TAFI需要激活，并可能在“随需应变”的情况下发挥作用，而CPN在构成上是活跃的，可能发挥“管家”作用。TAF1a可抑制纤溶，并可加重血栓形成，而CPN则不能。相反，Cpn被认为通过灭活补体激活产生的过敏性毒素来调节炎症，而TAFI在这一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TAF1a和Cpn在不同程度上介导了纤溶和炎症的调节，但不是相互排斥的。它们协同作用，以维持对止血和炎症反应的精细控制。我们的具体目的是：1)确定TAFI在人血浆中的功能浓度和抗原浓度之间的相关性；2)在纯化系统中评价TAF1a和Cpn的抗纤溶作用；3)确定细胞表面糖胺聚糖参与纤溶酶介导的Tafi活化的作用；4)确定Cpn和TAF1a对C3a介导的钙离子外流的抑制作用。活性分析将在2×2的基质中进行，改变TAF1a和CPN的浓度，以评估协同效应。从长远来看，这将导致新的治疗干预措施，可能会特异性和显着地影响血栓形成，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.