Evidence that Catalytically-Inactivated Thrombin Forms Dimers That Bridge between Fibrin/Fibrinogen Fibers and Enhance Polymerization

Michael W. Mosesson, Blood Research Institute
Irene Hernandez
Kevin R. Siebenlist, Marquette University

Biophysical Chemistry, Volume 110, Issues 1-2, pp 93-100 (July, 2004). DOI: 10.1016/j.bpc.2004.01.007


Phe-pro-arg-chloromethyl ketone-inhibited α-thrombin [FPRα-thr] retains its fibrinogen recognition site (exosite 1), augments fibrin/fibrinogen [fibrin(ogen)] polymerization, and increases the incorporation of fibrin into clots. There are two ‘low-affinity’ thrombin-binding sites in each central E domain of fibrin, plus a non-substrate ‘high affinity’ γ′ chain thrombin-binding site on heterodimeric ‘fibrin(ogen) 2’ molecules (γA,γ′). ‘Fibrin(ogen) 1’ (γA, γA) containing only low-affinity thrombin-binding sites, showed concentration-dependent FPRα-thr enhancement of polymerization, thus indicating that low-affinity sites are sufficient for enhancing polymerization. FPRγ-thr, whose exosite 1 is non-functional, did not enhance polymerization of either fibrin(ogen)s 1 or 2 and DNA aptamer HD-1, which binds specifically to exosite 1, blocked FPRα-thr enhanced polymerization of both types of fibrin(ogen) (1>2). These results showed that exosite 1 is the critical element in thrombin that mediates enhanced fibrin polymerization. Des Bβ1-42 fibrin(ogen) 1, containing defective ‘low-affinity’ binding sites, was subdued in its FPRα-thr-mediated reactivity, whereas des Bβ1-42 fibrin(ogen) 2 (γA,γ′) was more reactive. Thus, the γ′ chain thrombin-binding site contributes to enhanced FPRα-thr mediated polymerization and acts through a site on thrombin that is different from exosite 1, possibly exosite 2. Overall, the results suggest that during fibrin clot formation, catalytically-inactivated FPRα-thr molecules form non-covalently linked thrombin dimers, which serve to enhance fibrin polymerization by bridging between fibrin(ogen) molecules, mainly through their low affinity sites.