Abstract
The Aα R16C mutation is a common cause of dysfibrinogenemia, but the complete implications of this mutation on the process of hemostasis have not been fully characterized. Because of its critical position at the fibrinopeptide A cleavage site, this mutation leads to delayed fibrinopeptide release and subsequent delayed fibrin polymerization. The point mutation responsible for this dysfibrinogen leads to a clinical paradox, however, with both hemorrhage and thrombosis as reported complications. Of previously identified patients with this dysfibrinogen, approximately 30% have experienced bleeding and 15% thrombosis, with the remainder asymptomatic. In this report, the biochemical properties of Aα R16C dysfibrinogens that contribute to either hemorrhage or thrombosis are characterized. Blood samples were obtained from two young siblings who presented with excessive trauma-induced bleeding. Functional fibrinogen levels were 46–55 mg/dL and fibrinogen antigen levels were 427–429 mg/dL, consistent with the diagnosis of dysfibrinogenemia (Fibrinogen Hershey III). DNA sequencing demonstrated both siblings to be heterozygous for the Aα R16C mutation. Fibrinogen was then purified from plasma by classical glycine precipitation. In order to determine if this dysfibrinogen has altered rates of factor XIIIa cross-linking, cross-linking kinetics were assessed by incubating normal or mutant fibrinogen with factor XIII and thrombin and quantifying band intensity at successive timepoints for the resultant γ-γ dimers and α multimers by SDS-PAGE. Analysis of factor XIIIa cross-linking showed a significant decrease in the amount of γ-γ dimer formation when compared to normal fibrinogen (p<0.05 for both siblings) but no significant difference in the rate or quantity of α multimer formation. After an initial lag, the rate of γ-γ dimer formation was not appreciably different from that of the control. This decreased amount of cross-linking, which may also reflect the delay in fibrin polymerization, likely contributes to the hemorrhagic phenotype sometimes seen with this dysfibrinogen. Fibrinolysis kinetics were next measured by monitoring the optical density of purified Fibrinogen Hershey III clotted with thrombin in the presence of factor XIII, tissue plasminogen activator, and Glu-plasminogen. For the propositus, fibrinolysis was significantly delayed, with t1/2 of 51 ± 3 minutes (mean ± SEM) compared to 38 ± 0.2 minutes for normal fibrinogen. Similar results were obtained for the second sibling. The decreased rate of fibrinolysis could explain the paradoxical thrombotic phenotype sometimes seen with this dysfibrinogen. Thus the dual nature of the Aα R16C mutation is demonstrated by the simultaneous presence of deficient fibrinolysis and deficient fibrin cross-linking. Slower clot formation results from the delays in fibrinopeptide cleavage and fibrin polymerization. The delay in fibrinolysis, however, represents a hypercoagulable state leading to potential thrombosis. For this particular dysfibrinogen, the balance of procoagulant versus fibrinolytic factors may be most important in determining its clinical phenotype.
A gamma Gly-268 to Glu substitution is responsible for impaired fibrin assembly in a homozygous dysfibrinogen Kurashiki I