Formation of soluble fibrin oligomers in purified systems and in plasma

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Abstract
The kinetic parameters for release of fibrinopeptide A (FPA) from human fibrinogen by thrombin are: Km = 2.3 .times. 106 M and Vmax = 1.1 .times. 10-10 mol of FPA/s per unit of thrombin; for fibrin formation, Km is similar to that for FPA release, but, under the conditions of the present study, Vmax was approximately half of that for FPA release. The formation of fibrin polymer before the sol-gel transition was studied by gel-permeation chromatography combined with effluent analysis for fibrinogen antigen and residual FPA. Polymer formation is purified fibrinogen incubated with thrombin proceeded as bimolecular association of exposed sites in a manner predicted by probability calculations and assuming random FPA cleavage. Each oligomer consisted of n molecules of fibrin monomer and 2 fibrinogen molecules, each of the latter lacking 1 FPA molecule, i.e., each oligomer, regardless of molecular size, retains 2 FPA molecules. The addition of 5 mM-CaCl2 to the reaction mixture changed the rate of polymer formation, so that dimer was no longer the prevalent oligomer; in the presence of Ca2+, the trimer was the oligomer in highest concentration. The polymers formed in the presence of Ca were similar in composition to those without, i.e., 2 mol of FPA/mol of oligomer. EDTA-treated plasma samples incubated for short periods of time .ltoreq., 30 s, with thrombin ranging in concentration up to 1 NIH [National Institute of Health] U/ml did not form clots during the 10-15 min period of observation until they were applied to the column, though a large proportion of the available FPA was cleaved (maximum 45%). The soluble polymers in plasma were mostly of the high-MW variety (tetramer and greater); these high-MW polymers contained < 2 mol of FPA/mol of polymer, whereas dimer and trimer in plasma were similar to those in the purified systems, i.e., 2 mol of FPA/mol.