Effects of Fibrinogen and Thrombin Concentrations on Clot Structure and Internal Clot Lysis Time

Background: In the final steps of the coagulation cascade, thrombin converts fibrinogen to fibrin, which polymerizes into a fibrin network that stabilizes blood clots. The concentration of these two proteins can affect network structure. The fibrin network is dissolved by plasmin, by cleaving fibrin peptide chains. In internal fibrinolysis, tissue plasminogen activator (tPA) activates endogenous plasminogen to plasmin, with activation efficiency much enhanced when tPA and plasminogen are bound to fibrin. The effect of varying fibrinogen and thrombin concentrations and the effect of the resulting varying network structure on fibrinolysis are convoluted, with contradictory trends reported in the literature. Objectives: Investigate how fibrinogen and thrombin concentration quantitatively affect fibrin network structure, and how fibrinogen, thrombin concentrations and the resulting, altered network structure affect fibrinolysis, at a given, rate-limiting tPA concentration. Methods: Fibrin clots were generated from diluted, platelet-poor plasma and purified PEAK 1 fibrinogen, at varying fibrinogen and exogenous thrombin concentrations. The effects of varying fibrinogen and thrombin concentrations on network structure were quantified using non-linear power-law equations derived from confocal images. Internal clot lysis time (CLT) was determined using turbidity-based assays as a function of fibrinogen and thrombin concentrations and network structure at fixed, rate-limiting tPA, and therefore fixed plasmin concentration. Results and Conclusions: Network structural features (fiber density, pore size) and CLT fit well to empirical equations of the form k([Fgn]α× [Thr]β). Generally, increasing fibrinogen resulted in higher fiber density and thicker fibers, whereas increasing thrombin resulted in higher fiber density and thinner fibers. Internal CLT showed strong dependence on fibrinogen, and negligible dependence on thrombin. This implies that fibrinogen is the main determinant of internal CLT in these systems, while thrombin and, thus, thrombin-dependent formation kinetics and network structure, per se, have a near-negligible effect.