Reaction of Fibrinogen with Homocysteine Thiolactone Mimics the Dysfibrinogenemia Produced by Hyperhomocysteinemia.

Elevated plasma homocysteine (HCys) is associated with atherosclerosis and thrombosis. We have previously found that clots from plasma, whole blood and purified fibrinogen from homocysteinemic rabbits are composed of thin, tightly-packed fibers and are resistant to fibrinolysis. HCys thiolactone is a metabolite of Hcys that can react with lysine residues under physiologic conditions. This reaction introduces a new free sulfhydryl group into the protein. The current study was designed to test the hypothesis that HCys thiolactone modifies key lysines in fibrinogen, thereby causing the alterations in fibrin structure and susceptibility to fibrinolysis that characterize the homocysteine-induced dysfibrinogenemia. We examined the effect of incubation of HCys thiolactone with purified human fibrinogen. The control and homocysteine thiolactone treated fibrinogen (HCys-fibrinogen) samples were added to fibrinogen-deficient plasma, which was then clotted by addition of thrombin and calcium in the presence of tissue plasminogen activator (tPA). Clots from HCys-fibrinogen lysed more slowly than control clots and the magnitude of this effect depended on the concentration of HCys thiolactone used. At 300 μM Hcys thiolactone, the fibrinogen formed clots with characteristics similar to fibrinogen from hyperhomocysteinemic rabbits. These clots 1) had thinner fibers than controls; 2) the reduced fiber thickness could be partially corrected by increasing calcium and 3) were more resistant to fibrinolysis than controls. In addition, the HCys-fibrinogen formed disulfide-stabilized high molecular weight forms, which are also found in the plasma of hyperhomocysteinemic rabbits. Mass spectrometric analysis of Hcys-fibrinogen revealed 12 different HCys-modified lysines - primarily in the D- and alpha-C-domains. Some of these are close to sites of tPA and plasminogen binding and/or plasmin cleavage. Two of them are very close to sites of naturally-occurring prothrombotic mutations that introduce a new cysteine into the alpha-C-domain. A new Cys at these sites mediates mixed disulfide formation with albumin in the plasma - a structural modification that is thought to underlie their prothrombotic effects. Similarly, we find that HCys-fibrinogen forms mixed disulfides with human albumin. Our data suggest that homocysteinylation of fibrinogen could lead to abnormal resistance of fibrin clots to lysis and thus might contribute to the increased risk of cardiovascular disease in hyperhomocysteinemia.