The long-term goal is to define the molecular mechanisms of thrombin (T) inhibition by the serpins, heparin cofactor II (HCII) and plasminogen activator inhibitor-1 (PAI-1), implicated in arterial thrombosis. Thrombin localized on fibrin (Fbn) and the glycosaminoglycans (GAGs) dermatan sulfate (DS)and heparin, reacts with HCII and platelet PAI-1, in GAG-accelerated mechanisms different from thrombin inhibition by antithrombin (AT), accelerated by high affinity heparin, present only in trace amounts. Unlike AT, HCII is a unique inhibitor of arterial thrombosis, as only HCII in the presence of DS is capable of inhibiting Fbn-bound thrombin. Thrombin exosites I and II are hypothesized to play different roles in these processes. Exosite I binds HCII and PAI-1 directly, whereas exosite II - heparin binding may modulate HCII and PAI-1 turnover. These steps are absent in the T - AT reaction. DS bound outside exosite II is hypothesized to act as template for inhibition by HCII of exosite ll-blocked thrombin, meizothrombin (MzT), and MzT(desFI). The identity of this site;the HCII and PAI-1 substrate pathways;the mechanisms of DS- selective inhibition of Fbn-bound thrombin by HCII, and of fibrinogen (Fbg) and Fbn regulation of thrombin inhibition by PAI-1 are all unknown. The studies will resolve these significant gaps, by using fluorescence equilibrium binding, steady-state and rapid kinetics with native thrombin, HCII and PAI-1, and specific loss- of-function mutants. They will test the hypotheses: that the exosite roles in the GAG-catalyzed thrombin inactivation mechanisms by HCII, PAI-1 and AT are distinctly different;that GAG binding outside exosite II on thrombin mediates inhibition by HCII;and that Fbg and Fbn regulate thrombin inhibition by these serpins differentially.
Specific aims are: (1) To quantitate binding and chemical steps in the sequence of molecular events in the GAG-catalyzed thrombin inactivation and substrate pathways of HCII and PAI-1, compared to AT;(2) Tocharacterize the DS-binding site outside exosite II in thrombin and MzT, and its role in thrombin and MzT inhibition;and (3) To determine the contributions of Fbg and Fbn binding to exosite I and GAGs in thrombin protection from HCII, PAI-1, and AT. These mechanism-based studies are relevant to understanding the selective, localized regulation of thrombin activity by HCII and PAI-1, and serpin turnover, in arterial clots. They may facilitate development of novel anticoagulants based on HCII and DS specifically targeted to arterial thrombosis.
|Matafonov, A; Cheng, Q; Geng, Y et al. (2013) Evidence for factor IX-independent roles for factor XIa in blood coagulation. J Thromb Haemost 11:2118-27|
|Geng, Yipeng; Verhamme, Ingrid M; Smith, Stephen B et al. (2013) The dimeric structure of factor XI and zymogen activation. Blood 121:3962-9|
|Geng, Y; Verhamme, I M; Sun, M F et al. (2013) Analysis of the factor XI variant Arg184Gly suggests a structural basis for factor IX binding to factor XIa. J Thromb Haemost 11:1374-84|
|Geng, Yipeng; Verhamme, Ingrid M; Messer, Amanda et al. (2012) A sequential mechanism for exosite-mediated factor IX activation by factor XIa. J Biol Chem 287:38200-9|
|Verhamme, Ingrid M (2012) Fluorescent reporters of thrombin, heparin cofactor II, and heparin binding in a ternary complex. Anal Biochem 421:489-98|
|Thompson, Lawrence C; Goswami, Sumit; Ginsberg, David S et al. (2011) Metals affect the structure and activity of human plasminogen activator inhibitor-1. I. Modulation of stability and protease inhibition. Protein Sci 20:353-65|
|Matafonov, Anton; Sarilla, Suryakala; Sun, Mao-fu et al. (2011) Activation of factor XI by products of prothrombin activation. Blood 118:437-45|
|Sarilla, Suryakala; Habib, Sally Y; Kravtsov, Dmitri V et al. (2010) Sucrose octasulfate selectively accelerates thrombin inactivation by heparin cofactor II. J Biol Chem 285:8278-89|
|Sarilla, Suryakala; Habib, Sally Y; Tollefsen, Douglas M et al. (2010) Glycosaminoglycan-binding properties and kinetic characterization of human heparin cofactor II expressed in Escherichia coli. Anal Biochem 406:166-75|
|Smith, Stephen B; Verhamme, Ingrid M; Sun, Mao-fu et al. (2008) Characterization of Novel Forms of Coagulation Factor XIa: independence of factor XIa subunits in factor IX activation. J Biol Chem 283:6696-705|
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