Evidence that catalytically-inactivated thrombin forms non-covalently linked dimers that bridge between fibrin/fibrinogen fibers and enhance fibrin polymerization.
Phe-pro-arg-chloromethyl ketone-inhibited alpha-thrombin [FPR alpha-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' gamma' chain thrombin-binding site on heterodimeric 'fibrin(ogen) 2' molecules (gamma(A), gamma'). 'Fibrin(ogen) 1' (gamma(A), gamma(A)) containing only low-affinity thrombin-binding sites, showed concentration-dependent FPR alpha-thr enhancement of polymerization, thus indicating that low-affinity sites are sufficient for enhancing polymerization. FPR gamma-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 FPR alpha-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 B beta 1-42 fibrin(ogen) 1, containing defective 'low-affinity' binding sites, was subdued in its FPR alpha-thr-mediated reactivity, whereas des B beta 1-42 fibrin(ogen) 2 (gamma(A), gamma') was more reactive. Thus, the gamma' chain thrombin-binding site contributes to enhanced FPR alpha-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 FPR alpha-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.
Mosesson
M W
MW
The Blood Research Institute of the Blood Center of Southeastern Wisconsin, P.O. Box 2178, Milwaukee, WI 53201-2178, USA. mwmosesson@bcsew.edu
Hernandez
I
I
Siebenlist
K R
KR
D000590:Amino Acid Chloromethyl Ketones / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D001704:Biopolymers / Q000737:chemistry*; D002384:Catalysis; D019281:Dimerization; D005337:Fibrin / Q000737:chemistry* / Q000378:metabolism; D005340:Fibrinogen / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D013917:Thrombin / Q000737:chemistry* / Q000378:metabolism; D013997:Time Factors
D000590:Amino Acid Chloromethyl Ketones; D001704:Biopolymers; D005337:Fibrin; D005340:Fibrinogen; D013917:Thrombin; C050815:phenylalanyl-prolyl-arginine chloromethyl ketone-thrombin