In vitro fibrin clot formation and fibrinolysis using heterozygous plasma fibrinogen from γAsn319, Asp320 deletion dysfibrinogen, Otsu I

Abstract

Introduction: We have reported a heterozygous dysfibrinogenemia, fibrinogen Otsu I, caused by the deletion of γAsn319 and γAsp320, which was originally identified in the dysfibrinogen Vlissingen/Frankfurt IV (V/FIV) associated with thrombosis. Unlike the V/FIV family, the Otsu propositus showed no thrombotic tendencies. To analyze the relationship between thrombosis and the heterozygous plasma variant fibrinogen, we used purified plasma fibrinogen from the Otsu patient and compared it with a normal control. Materials and Methods:Thrombin-induced fibrin clot formation and clot structure were observed by fibrin polymerization and scanning electron microscopy, respectively. For in vitro observation of fibrinolysis, plasmin generation and clot lysis assays were performed by the addition of tissue type plasminogen activation (tPA) and plasminogen. Results and Conclusions:Polymerization of Otsu was markedly impaired, while fibrin fibers were much thicker and the density of the bundles of fibrin fibers was less and porous compared with normal. Lysis of the Otsu clot was not significantly different from normal when a tPA and plasminogen mixture was overlaid onto the clots. For Otsu, the penetration of the tPA/plasminogen mixture into the clot was much faster than normal and the protection against plasmin cleavage was impaired, however, tPA-induced plasmin activation of the Otsu fibrin was slower than that of normal fibrin, resulting in a clot lysis of Otsu similar to normal.

Manipulating the quality control pathway in transfected cells: low temperature allows rescue of secretion-defective fibrinogen mutants

“…[25][26][27] Studies with purified patient plasma comprising a mixture of normal and mutant fibrinogen showed impaired calcium binding, fibrin polymerization, factor XIII (FXIII)-catalyzed cross-linking and platelet adhesion. 25,26,28,29 Other studies with pure recombinant γ p.N345_D346del (p.N319_D320del) fibrinogen expressed from CHO cells confirmed these observations, suggesting that the tworesidue deletion affects the structural integrity of the C-terminal domain of γ chains.…”

Dysfibrinogenemia-associated novel heterozygous mutation, Shanghai (FGAc.169_180+2 del), leads to N-terminal truncation of fibrinogen Aα chain and impairs fibrin polymerization

“…Plasminogen (0.2 unit/mL) and tPA (0.05 ng/mL) were incubated along with thrombin (1.0 unit/mL) and fibrinogen (0.23 mg/mL) to evaluate the effect of mutation fibrinogen Shanghai on fibrin-enhanced plasminogen activation induced by tPA 12. The quantity of plasmin generated in the reaction was estimated using plasmin-specific chromogenic substrate S-2251.…”

Fibrin‐chitosan composite substrate for in vitro culture of chondrocytes

“…In vitro degradation studies of crosslinked Fbn with genipin at different concentrations (0, 1, 2.5, and 5 m M ) were performed at 37°C in presence of plasmin using a modification of protocol described by Terasawa et al29 Briefly, after fibrin polymerization, 100 μL of plasmin (Sigma, Madrid, Spain) with a final concentration of 0.13 μ M was added to each sample and incubated at 37°C for 30 days. In absence of genipin the degradation kinetic was monitored by the decrease of absorbance at 350 nm.…”