Isolation of size homogeneous preparations of high molecular weight and low molecular weight fibrinogens

Abstract

Molecular sizes of bovine fibrinogen (F) similar to FI, higher MW form, and FII, lower MW form, were found by Lipinska and colleagues. A procedure was developed to isolate for the 1st time each of the FI and FII forms of fibrinogen which are free of each other and of high MW fibrin-fibrinogen complexes. This process involved removing the complexes by A-5 m chromatography. This chromatography also reduced a protein contaminant (X) and removed plasminogen. (NH4)2SO4 subfractionation at pH 5.9 was then done. A subfraction (16-18%) containing 90% FI and another (22-25% or 25-28%) containing 96% FII were obtained. Reprecipitation of the 1st 16-18% subfraction yielded a subfraction containing 97% FI. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of FII revealed that it contains 1 intact A.alpha. chain per (B.beta., .gamma.)2. Clot opacity studies on FII suggested that the carboxyl terminal portion of the .alpha. chain of fibrin plays an important role in the lateral associations in fibrin polymerization. The pattern of (NH4)2SO4 precipitation of the endogenous fibrin-fibrinogen complexes was studied. The complexes precipitated mostly in the least soluble subfractions, but small amounts could be found in all subfractions. Examination of the complexes by SDS-polyacrylamide gel electrophoresis showed that most of the complexes could be dissociated to FI and FII. There were complexes which remained; these were covalently cross-linked forms probably produced by factor XIII.