Formation of soluble fibrin oligomers in purified systems and in plasma

Abstract

The kinetic parameters for release of fibrinopeptide A (FPA) from human fibrinogen by thrombin are: Km = 2.3 X 10(-6)M and Vmax. = 1.1 X 10(-10)mol of FPA/s per unit of thrombin; for fibrin formation, Km is similar to that for FPA release, but, 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 in purified fibrinogen incubated with thrombin proceeded as a 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 two fibrinogen molecules, each of the latter lacking one FPA molecule, i.e. each oligomer, regardless of molecular size, retains two 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 calcium 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, 30s or less, with thrombin ranging in concentration up to 1 N.I.H. unit/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-Mr variety (tetramer and greater); these high-Mr polymers contained less than 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.

Evaluation of a Soluble Fibrin Assay in Patients with Suspected Deep Vein Thrombosis

“…Soluble fibrin is produced when thrombin cleaves librinopeptides A and B from fibrinogen. The resultant soluble fibrin can polymerize to form an insoluble fibrin clot (35)(36)(37). There is good evidence that patients with venous thromboembolism have inereased thrombin for mation (19) and therefore, it is reasonable to hypothesize that patients with DVT have elevated plasma levels of soluble fibrin.…”

Platelet binding sites for factor VIII in relation to fibrin and phosphatidylserine

“…10 We asked whether SF bound to a platelet might serve as a binding site (Figure 3). Accordingly, we prepared SF 38 in the presence of antifibrinogen antibodies coupled to Superose. Immobilization of fibrin was verified with a fluorescein-labeled antifibrinogen/antifibrin antibody.…”

Nanoscale Probing Reveals that Reduced Stiffness of Clots from Fibrinogen Lacking 42 N-Terminal Bβ-Chain Residues Is Due to the Formation of Abnormal Oligomers

“…We investigated the initial interactions involved in fibrin oligomer formation using NC-AFM experiments at high resolution. Previously, oligomer formation has been investigated by transmission electron microscopy, gel filtration, and light scattering techniques (41)(42)(43)(44). Marchant et al (33) used AFM to investigate early protofibril formation, however, these images did not reach the same resolution or clarity as those presented in this study.…”