Hyperhomocysteenemia induces alterations in fffirinogen function and fibrin clot structure in a rabbit model
Elevated plasma homocysteine is associated with an increased risk of atherosclerosis and thrombosis. However, the mechanisms by which homocysteine might cause these events are not understood. Most studies have focused on the effects of homocysteine on endothelial cell function, with few studies examining effects on coagulation proteins. Jakubowski (FASEB J 13:2277,1999) showed that homocysteine thiolactone, a metabolite of homocysteine, can covalently modify fibrinogen and other proteins in plasma. We hypothesized that homocysteinylation of fibrinogen in vivo could lead to altered fibrin clot structure. We used a rabbit model of hyperhomocysteinemia to test this hypothesis. Three New Zealand White rabbits were injected every 12 hr with 15 mL of 6 mg/mL homocysteine solution through indwelling injection ports. This treatment raised the plasma homocysteine levels in the treated animals to about 30 jiM compared to 15 jiM in three buffer-treated control rabbits. Blood samples were collected into sodium citrate at the end of 9 weeks by cardiac puncture. The plasma was stored at -80°C until assay. Clotting times were determined in a Fibrometer and by monitoring turbidity in a plate-reading specotrophotometer. The turbidity of fibrin gels was measured at wavelengths from 400SOOnm and fiber mass per unit length ratios were calculated by the method of Carr and Gabriel (Macromolecules 13:1473, 1980). The fibrinogen levels (by ELISA) were 222 ± 49 mg% in homocysteine-treated and 197 ±51 mg% in control rabbits. However, the reptilase (atroxin) time was 159 ±9.5 seconds for plasma from controls, but 300 seconds for each sample from homocysteine-treated rabbits. When clotted with thrombin, the control and experimental plasmas had similar clotting times, but the turbidity of the clots was reproducibly different. We calculated that the mass per unit length was smaller for the clots in plasma from homocysteine-treated rabbits compared to clots formed from control plasma. This means that the clots formed from plasma of homocysteinc-trcated rabbits were composed of thinner fibers than control clots. Several congenital dysfibrinogenemias have been reported in patients that led to prolonged reptilase times, formation of tightly-packed clots composed of thin fibers, and recurrent thrombosis. The clots in these patients are abnormally resistant to fibrinolysis. Our results suggest that elevated homocysteine could lead to an acquired dysfibrinogenemia. Alterations in clot structure could render the clots resistant to fibrinolysis and, thereby, contribute to the increased risk of cardiovascular events.
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