Dysfibrinogenemias: Probes for Establishing Relationships Between the Structure of Fibrinogen and Its Function in Blood Coagulation.

Abstract

Inherited abnormal fibrinogens that exhibit altered behavior in one or more of the steps in the conversion of fibrinogen to fibrin should be useful for determining structure-function relationships important in the final stages of blood coagulation. This thesis contains the functional characterization of three new fibrinogen variants and the structural characterization of one of these on a molecular level. The fibrinogen variant fibrinogen Ann Arbor was shown to exhibit altered end-to-end aggregation of fibrin monomers, as well as a more rapid rate of (gamma)-dimer formation. Fibrinogen Gr and Rapids was associated with hypofibrinogenemia and was shown to exhibit abnormal aggregation of fibrin monomers. Fibrinogen Petoskey exhibited a decreased initial rate of fibrinopeptide A (FPA) and fibrinopeptide B (FPB) release. Using a high performance liquid chromatography (HPLC) method for the separation of human fibrinopeptides, an abnormal FPA (FPA Petoskey) along with normal FPA and FPB was shown to be released upon incubation of fibrinogen Petoskey with thrombin. The molar ratios of released fibrinopeptides were one FPA Petoskey, one normal FPA, and two FPB. Amino acid analyses and carboxypeptidase Y digestion of purified FPA Petoskey indicated that it was identical to normal FPA, except that Arg-16 in normal FPA was replaced by a histidyl residue in FPA Petoskey. The thrombin-catalyzed cleavage of fibrinogen Petoskey reported here is the first demonstration of thrombin-catalyzed hydrolysis at a peptide bond other than at an arginyl or lysyl residue. Batroxobin, unlike thrombin, was incapable of cleaving the unusual His-A(alpha)16-Gly-A(alpha)17 bond in fibrinogen Petoskey. Less than one-quarter of the fibrinogen from an affected individual aggregated following incubation with batroxobin, suggesting the presence of heterodimers containing one normal and one abnormal chain. The removal of both FPA molecules from a fibrinogen molecule appears necessary for the molecule to aggregate properly. The HPLC separation of human fibrinopeptides developed in this work allows a simple and direct measurement of the action of thrombin on fibrinogen. The application of this method for characterizing the thrombin mediated release of fibrinopeptides from normal fibrinogen and fibrinogen Petoskey and its usefulness for determining the mechanism of thrombin-fibrinogen interaction is discussed.