Kinetics of ligation of fibrin oligomers.
Human fibrinogen was treated with thrombin in the presence of fibrinoligase and calcium ion at pH 8.5, ionic strength 0.45, and the ensuring polymerization was interrupted at various time intervals (t) both before and after the clotting time (tc) by solubilization with a solution of sodium dodecyl sulfate 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 to 10, from the latter. The degree of gamma-gamma ligation was calculated independently from the size distribution with the assumption that every junction between two fibrin monomers remaining intact after solubilization is ligated, and this agreed well with the direct determination. The size distribution at t/tc = 1.3 to 1.6 differed somewhat from that calculated by the classical theory of linear polycondensation on the assumption that all reactive sites react with equal probability and rate. Analysis of the difference suggests that ligation of a fibrin digomer is not a random process; the probability of ligation of a given junction between two monomers increases with the oligomer length. The number-average degree of polymerization, xn, of ligated oligomers increases approximately linearly with time up to a value of 1.6.
D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005176:Factor XIII / Q000494:pharmacology; D005337:Fibrin / Q000032:analysis / Q000096:biosynthesis*; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D011108:Polymers / Q000032:analysis; D011487:Protein Conformation; D013917:Thrombin / Q000378:metabolism
D011108:Polymers; D005337:Fibrin; D005340:Fibrinogen; D005176:Factor XIII; D013917:Thrombin; D002118:Calcium