Interaction of fibrinogen-binding tetrapeptides with fibrin oligomers and fine fibrin clots.

Abstract

AbstractThe polymerization of fibrin, at pH 8.5 and ionic strength 0.45, and under conditions where the action of thrombin on fibrinogen was the rate‐determining step, was interrupted by inactivating thrombin with p‐nitrophenyl‐p′‐guanidinobenzoate (NPGB). Addition of the tetrapeptide Gly‐Pro‐Arg‐Pro (GPRP) partially dissociated the fibrin oligomers as shown by subsequent ligation with Factor XIIIa and calcium ion followed by denaturation and gel electrophoresis; polyacrylamide gel electrophoresis with reduction showed a decrease in the proportion of γ‐γ ligation compared with controls untreated by GPRP, and agarose gel electrophoresis showed a shift in the distribution of oligomer sizes. The dissociation was accomplished within 15 min and its extent was consistent with establishment of an equilibrium in which two molecules of GPRP react to sever an oligomer. When GPRP was introduced into fine unligated fibrin clots by diffusion, there was some dissociation as shown by differences in the degree of γ‐γ ligation after treatment by Factor XIIIa; but the action of GPRP was much slower and less complete than on soluble oligomers. However, even a small amount of dissociation affected the mechanical properties of fine clots profoundly. The shear modulus (measured 25 s after application of stress) decreased progressively with increasing concentration of GPRP introduced by diffusion. The rate of shear creep under constant stress and the proportion of irrecoverable deformation also increased enormously. If the steadystate creep rate is interpreted in terms of an effective viscosity, the latter is decreased by up to three orders of magnitude by the presence of GPRP. In terms of transient network theories of viscoelasticity, the average lifetime of a network strand is greatly diminished. However, the total density of strands remains constant during creep and creep recovery as shown by constancy of the differential modulus or compliance. Removal of GPRP by diffusion only partially restores the original shear modulus and creep behavior of the original clot. Some limited data on the effect of the tetrapeptide Gly‐His‐Arg‐Pro are also reported.