Molecular basis of fibrinogen naples associated with defective thrombin binding and thrombophilia: Homozygous substitution of Bβ 68 Ala → Thr

Abstract

In an abnormal fibrinogen (fibrinogen Naples) associated with congenital thrombophilia we have identified a single base substitution (G → A) in the Bβ chain gene that results in an amino acid substitution of alanine by threonine at position 68 in the Bβ chain of fibrinogen. The propositus and two siblings were found to be homozygous for the mutation, whereas the parents and another sibling were found to be heterozygous. Individuals homozygous for the defect had a severe history of both arterial and venous thrombosis; heterozygous individuals had no clinical symptoms. The three homozygotes had a prolonged thrombin clotting time in plasma, whereas the heterozygotes had a normal thrombin clotting time. Fibrinopeptide A and B (FpA and FpB) release from purified fibrinogen by human α-thrombin was delayed in both the homozygous propositus and a heterozygous family member. Release of FpA from the normal and abnormal amino-terminal disulfide knot (NDSK) corresponded to that found with the intact fibrinogens, indicating a decreased interaction of thrombin with the NDSK part of fibrinogen Naples. Binding studies showed that fibrin from homozygous abnormal fibrinogen bound < 10% of active site inhibited α-thrombin as compared with normal fibrin, while fibrin formed from heterozygous abnormal fibrinogen bound ∼ 50% of α-thrombin. These results suggest that the mutation of Bβ Ala 68 → Thr affects the binding of α-thrombin to fibrin, and that defective binding results in a decreased release of FpA and FpB in both homozygous and heterozygous abnormal fibrinogens. amino-termi.