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The effect of homocysteine-lowering treatment on thrombin generation was investigated in 17 subjects with hyperhomocysteinemia (aged 22-60 years), 11 of whom had symptomatic atherosclerotic vascular disease. All subjects had fasting total homocysteine levels above 16 micromol/L. The formation of thrombin was assessed by measuring thrombin-antithrombin III complexes and prothrombin fragment 1+2 in peripheral venous blood and in the bleeding time blood collected at 30-second intervals from skin incisions on a forearm. All the tests were performed before and after an 8-week treatment with folic acid p.o. 5 mg/day, vitamin B6 p.o. 300 mg/day, and vitamin B12 i.m. 1000 microg given on a weekly basis. Following the 8-week therapy, the median plasma homocysteine concentration became significantly reduced from 20 to 10 micromol/L, while plasma levels of fibrinogen, prothrombin, and antithrombin III as well as activity of protein C, S, and factor VII showed no changes. Vitamin treatment was associated with a significant fall in thrombin-antithrombin III complexes and prothrombin fragment 1+2 concentrations in peripheral venous blood. Bleeding time became prolonged by about 60 seconds. At sites of hemostatic plug formation, plasma concentrations of both thrombin markers significantly decreased. Compared with pretreatment values, significantly less thrombin was produced during the first 3 minutes of bleeding after homocysteine-lowering therapy. In subjects with hyperhomocysteinemia a reduction of plasma fasting homocysteine concentration by folic acid and vitamins B12 and B6 administration is associated with attenuation of thrombin generation both in peripheral blood and at sites of hemostatic plug formation.

Interactions of nitric oxide (NO) with various cobalamin species have been examined, apparently for the first time, with both absorption and electron paramagnetic resonance spectroscopy. Only slight shifts in the absorption spectrum of hydroxocobalamin, B12a [Cb(III)], were produced by NO, but dramatic changes in the spectrum of B12r [Cb(III)] were found on addition of NO. The addition of NO shifted the spectrum of Cb(II) to one very similar to that of Cb(III), indicating the oxidation of Cb(II). The addition of NO to Cb(III) resulted in a novel, weak and previously undescribed electron paramagnetic resonance signal. Although it has not been fully characterized, this appears to represent a reversible complex in which NO is liganded to the Cb(III). When NO was added to Cb(II), its strong electron paramagnetic resonance spectrum was replaced by that of this novel species, consistent with oxidation of Cb(II) by NO and then binding of additional NO by the resulting Cb(III). Porcine, aortic endothelial cells were able to partially reduce Cb(III), and release to the supernatant a previously characterized superoxide cobalt(III) complex, but some Cb(II) remained with the cell fraction. These reactions of Cb species could play a role in altering intracellular and intratissue levels of NO.