Hyperhomocysteinemia is a risk factor for stroke, myocardial infarction, and venous thrombosis. Moderate hyperhomocysteinemia is associated with impaired endothelial function, but the mechanisms responsible for endothelial dysfunction in hyperhomocysteinemia are poorly understood. We have used genetic and dietary approaches to produce hyperhomocysteinemia in mice. Heterozygous cystathionine beta-synthase-deficient mice (CBS +/-), which have a selective defect in homocysteine transsulfuration, and wild-type (CBS +/+) littermates were fed either a control diet or a diet that is relatively deficient in folic acid for 6 wk. Plasma total homocysteine was 5.3 +/- 0.7 microM in CBS +/+ mice and 6.4 +/- 0.6 microM in CBS +/- mice (P = 0.3) given the control diet. Plasma total homocysteine was 11.6 +/- 4.5 microM in CBS +/+ mice and 25.1 +/- 3.2 microM in CBS +/- mice (P = 0.004) given a low-folate diet. In mice fed the control diet, relaxation of aortic rings in response to the endothelium-dependent vasodilator acetylcholine did not differ significantly between CBS +/+ mice and CBS +/- mice. In contrast, in mice fed a low-folate diet, maximal relaxation to acetylcholine was markedly impaired in CBS +/- mice (58 +/- 9%) compared with CBS +/+ mice (84 +/- 4%) (P = 0.01). No differences in relaxation to the endothelium-independent vasodilator sodium nitroprusside were observed among the four groups of mice. These data indicate that CBS-deficient mice are predisposed to hyperhomocysteinemia during dietary folate deficiency, and moderate hyperhomocysteinemia is associated with marked impairment of endothelial function in mice.

Abstract-To examine the effects of atherosclerosis on the protein C anticoagulant pathway in vivo, we measured anticoagulant responses to intravenous administration of human ␣-thrombin or activated protein C (APC) in cynomolgus monkeys. Two groups of monkeys were fed either a control diet (nϭ18) or an atherogenic diet (nϭ12) that produces both hypercholesterolemia and moderate hyperhomocyst(e)inemia. A third group (nϭ8) was fed an atherogenic diet for 15 months, and then fed the atherogenic diet supplemented with B vitamins for 6 months to correct the hyperhomocyst(e)inemia. The plasma homocyst(e)ine level was higher in monkeys fed the atherogenic diet (9.6Ϯ1.0 mol/L) than in monkeys fed the control diet (3.7Ϯ0.2 mol/L) or the atherogenic diet with B vitamins (3.6Ϯ0.2 mol/L) (PϽ0.001). Infusion of thrombin produced a much greater prolongation of the activated partial thromboplastin time in monkeys fed the control diet (52Ϯ10 seconds) than in monkeys fed the atherogenic diet either with (24Ϯ4 seconds) or without (27Ϯ5 seconds) supplemental B vitamins (PϽ0.02). Thrombin-dependent generation of circulating APC was higher in control (294Ϯ17 U/mL) than in atherosclerotic (240Ϯ14 U/mL) monkeys (PϽ0.05), although levels of fibrinogen, plasminogen, D-dimer, and thrombin-antithrombin complexes were similar in each group. Injection of human APC produced a similar prolongation of the activated partial thromboplastin time in control (31Ϯ3 seconds) and atherosclerotic (29Ϯ2 seconds) monkeys. These findings provide evidence for impaired anticoagulation, due partly to decreased formation of APC, in atherosclerosis. Key Words: atherosclerosis Ⅲ cholesterol Ⅲ homocysteine Ⅲ protein C Ⅲ thrombomodulin T hrombin, a major regulator of hemostasis, has both procoagulant and anticoagulant properties. 1 Procoagulant effects of thrombin include proteolytic activation of factors V, VIII, and XI; cleavage of fibrinogen to form a fibrin clot; and stimulation of platelet aggregation. A major anticoagulant effect of thrombin involves activation of protein C, a vitamin K-dependent plasma anticoagulant. Thrombin's procoagulant and anticoagulant activities are regulated by thrombomodulin, a cofactor that is expressed on the luminal surface of vascular endothelium. 2 When bound to thrombomodulin, thrombin's ability to catalyze procoagulant reactions is inhibited, but its ability to activate protein C is enhanced Ͼ1000-fold. 3 The protein C anticoagulant pathway is impaired in patients with inherited deficiencies of protein C or protein S and also in patients with a mutation in factor V (factor V Leiden) that renders it resistant to activated protein C (APC). 4,5 The physiological importance of the protein C anticoagulant pathway is underscored by the observation that resistance to APC is found in 20% to 50% of patients with venous thromboembolism. 6 Resistance to APC that is independent of factor V Leiden may be associated with increased risk of stroke. 7,8 In a previous study, we observed impaired endotheliumdependent vasodilatation and decreas...

Hyperhomocysteinemia has been proposed to inhibit the protein C anticoagulant system through 2 mechanisms: decreased generation of activated protein C (APC) by thrombin, and resistance to APC caused by decreased inactivation of factor Va (FVa). We tested the hypotheses that generation of APC by thrombin is impaired in hyperhomocysteinemia in monkeys and that hyperhomocysteinemia produces resistance to APC in monkeys, mice, and humans. In a randomized crossover study, cynomolgus monkeys were fed either a control diet or a hyperhomocysteinemic diet for 4 weeks. Plasma total homocysteine (tHcy) was approximately 2-fold higher when monkeys were on the hyperhomocysteinemic diet than when they were on the control diet (9.8 ؎ 2.0 M versus 5.6 ؎ 1.0 M; P < .05). After infusion of human thrombin (25 g/kg of body weight), the peak level of plasma APC was 136 ؎ 16 U/mL in monkeys fed the control diet and 127 ؎ 13 U/mL in monkeys fed the hyperhomocysteinemic diet (P > .05). The activated partial thromboplastin time was prolonged to a similar extent by infusion of thrombin in monkeys fed the control diet and in those fed the hyperhomocysteinemic diet. The sensitivity of plasma FV to human APC was identical in monkeys on control diet and those on hyperhomocysteinemic diet. We also did not detect resistance of plasma FV to APC in hyperhomocysteinemic mice deficient in cystathionine ␤-synthase (plasma tHcy, 93 ؎ 16 M) or in human volunteers with acute hyperhomocysteinemia (plasma tHcy, 45 ؎ 6 M).