Abstract

SY49 Understanding thrombin Hall 2 09:30 17th July, 2003 Session Type: Symposium Subject area: Invited Speaker Session title: From thrombin to fibrin: structure function relationships Abstract: SY49 Authors: L. Leung Stanford University, USA Thrombin interacts with multiple substrates including clotting factors and cofactors, cellular thrombin receptors, and protein C. Access to thrombin's active site is limited by the 50-insertion loop and the autolysis loop. There are two anion-binding exosites (ABE) that mediate the docking of thrombin to its many substrates, thereby overcoming restricted access to the active site. Using a collection of thrombin mutants generated by systematic alanine scanning mutagenesis, we have studied the interactions of thrombin with its various substrates and identified three key residues in three separate structural domains: Trp50, Tyr71, and Glu229. W50A mutation disrupts thrombin's enzymatic activity in every substrate tested so far, but does not affect the function of ABE-I since it retains normal binding to fibrin and the thrombin DNA aptamer. Thus, Trp50 shows the importance of the 50-insertion loop in maintaining the topology of the active site. Y71A mutation in ABE-I disrupts all ABE-I mediated functions but does not affect interactions within the active site. Y71A is inhibited by ATIII similar to wild type and retains fairly normal FXI activation. Therefore the active site and ABE-I are functionally independent. The Na +-binding site E229A mutation has effects similar to W50A, but with more preservation of its activation of protein C and TAFI when bound to thrombomodulin (TM). However ABE-I function in E229A is intact. Therefore the integrity of Na+binding site is important for catalysis but independent from interactions with ABE-I. ABE-I and -II are functionally independent and not allosterically linked. Recently TAFI, a latent plasma carboxypeptidase, is found to be a second physiologic substrate for the thrombin/TM complex on endothelial cells. TAFI removes the Cterminal lysines from partially digested fibrin clots thereby dampening fibrinolysis. Our preliminary studies showed that TAFIa is efficient in inactivating complement C5a, bradykinin (BK), and thrombin-cleaved osteopontin. Activation of TAFI by the E229K thrombin, a thrombin mutant with minimal pro-coagulant activities while retaining significant anticoagulant function, blocked BK-induced hypotension in mice. Our data suggest that TAFI activation by the endothelial thrombin/TM complex is a pivotal step in vascular biology. TAFIa is an important antiinflammatory molecule and along with aPC, serves to counterbalance the prothrombotic and pro-inflammatory effects of thrombin generation. file:////ddn-453/d/ML3G-Data/JTH-Abstract-2003/HTML/Thursday/Abstract%20SY49.html (1 of 2) [1/27/2014 4:32:06 PM]