KINETICS OF FORMATION OF FIBRIN OLIGOMERS .2. SIZE DISTRIBUTIONS OF LIGATED OLIGOMERS

Human fibrinogen was treated with thrombin in the presence of fibrinoligase (factor XIIIa) and Ca ion at pH 8.5, ionic strength 0.45, and the ensuing polymerization was interrupted at various time intervals (t) before and after the clotting time (tc) by solubilization with a solution of sodium dodecylsulfate and urea. Aliquots of the solubilized protein were subjected to gel electrophoresis on polyacrylamide gels after disulfide reduction by dithiothreitol, and on agarose gels without reduction. The degree of .gamma.-.gamma. ligation was determined from the former and the size distribution of ligated oligomers, for degree of polymerization x from 1-10, from the latter. Thrombin was inhibited, after partial polymerization, by p-nitrophenyl-p''-guanidinobenzoate. For thrombin concentration .ltoreq. 0.013 U/ml and fibrinoligase .gtoreq. 30 mg/l, oligomer assembly is rapid as compared with peptide A release and ligation is rapid as compared with assembly. Under these conditions, the theory of the 1st paper of this series describes well the time-dependences of the degree of .gamma.-.gamma. ligation, the weight fractions of monomer and small oligomers and the number- and weight-average degrees of polymerization after solubilization of the staggered overlapped assemblies, each of which splits to give 2 strands of end-to-end ligated oligomers. The theory assumes that the 2nd A peptide is released by thrombin more rapidly than the 1st by a factor q, which, from the experimental data, is determined to be 16. The subsequent assembly into staggered overlapped oligomers follows the statistics of linear polycondensation, taking into account the presence of both difunctional and monofunctional combining units. For higher thrombin or lower fibrinoligase concentrations, ligation fails to keep pace with oligomer assembly, and the size distributions after solubilization show a higher proportion of very small and a lower proportion of larger ligated oligomers, owing to separation of the staggered overlapped assemblies into smaller fragments.