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Prepublished online as a Blood First Edition Paper on May 24, 2002; DOI 10.1182/blood-2002-03-0727.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Veterans Affairs Medical Center, Iowa City,
IA; Departments of Internal Medicine and Pharmacology, University of
Iowa College of Medicine, Iowa City; Scripps Research Institute, La
Jolla, CA; Oregon Regional Primate Research Center, Beaverton; and
Baylor Institute of Metabolic Disease, Dallas, TX.
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 µM versus 5.6 ± 1.0 µM; P < .05). After infusion of human thrombin (25 µg/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
Hyperhomocysteinemia is a risk factor for
stroke, myocardial infarction, peripheral arterial disease, and venous
thrombosis.1,2 An association between moderate
hyperhomocysteinemia and clinical cardiovascular events has been
observed in more than 35 case-control and observational epidemiologic
studies, as well as several prospective studies.3 Because
elevated levels of plasma total homocysteine (tHcy) can often be
lowered by oral administration of folic acid or combinations of B
vitamins, treatment of hyperhomocysteinemia has been proposed as a
strategy for preventing cardiovascular disease and its complications.
This approach is currently being evaluated in several prospective
randomized intervention trials.4
Like other cardiovascular risk factors, hyperhomocysteinemia produces
endothelial dysfunction, which can be detected on the basis of impaired
responses to endothelium-dependent vasodilators.5 Hyperhomocysteinemia may also adversely affect the protein C
anticoagulant pathway.6 Incubation of cultured endothelial
cells with exogenous homocysteine decreased the activity of
thrombomodulin, which is the major endothelial cofactor for activation
of protein C by thrombin.7-9 In agreement with these
findings in vitro, we have observed decreased thrombomodulin activity
in the aorta of hyperhomocysteinemic monkeys10 and
mice.11 Clinical evidence for impaired activation of
protein C in hyperhomocysteinemia has not been
convincing,12 however, and the effect of
hyperhomocysteinemia on activation of endogenous protein C has not been
examined in animal models.
In addition to possibly inhibiting the activation of protein C,
hyperhomocysteinemia has been proposed to impair the ability of
activated protein C (APC) to inactivate its major substrate, coagulation factor Va (FVa). Undas et al13 reported that
homocysteine can react rapidly with free cysteine residues of FV in
vitro, resulting in APC resistance similar to that of FV Leiden. This effect of hyperhomocysteinemia could have direct clinical implications because FV Leiden is an established risk factor for venous
thrombosis.14
Thus, hyperhomocysteinemia may interfere with the protein C
anticoagulant system through 2 mechanisms: decreased activation of
protein C by thrombin and thrombomodulin, and resistance to APC caused
by decreased inactivation of FVa. In this study, we used a diet that
produces hyperhomocysteinemia in monkeys to test the hypothesis that
generation of APC by thrombin is impaired during hyperhomocysteinemia
in vivo. We also tested the hypothesis that hyperhomocysteinemia alters
the APC sensitivity of plasma FV in cynomolgus monkeys, mice deficient
in cystathionine- Cynomolgus monkeys
CBS-deficient mice
Human volunteers Ten healthy volunteers (6 men and 4 women) without risk factors or clinical evidence of atherosclerosis were recruited by advertisement. Written informed consent was obtained from each subject, and the study protocol was approved by the University of Iowa Institutional Review Board. In a randomized, double-blind crossover study, each subject received oral L-methionine (100 mg/kg) dissolved in cranberry juice on one study day and cranberry juice alone (placebo) on another study day, as described previously.18 The 2 study days were separated by at least 2 weeks. Blood samples for measurement of plasma tHcy were collected into chilled EDTA tubes immediately before and 6 and 8 hours after oral administration of methionine or placebo. Blood samples for hemostasis assays were obtained 6 hours after oral administration of methionine or placebo and collected into sodium citrate tubes. All blood samples were placed on ice immediately, and plasma was isolated by centrifugation at 2500g for 30 minutes at 4°C.Hemostasis assays Plasma APC was measured by enzyme-capture assay using the antihuman protein C light- chain monoclonal antibody C319 and chromogenic substrate S-2366 as described previously.15 One unit of APC was defined as the amount present in 1.0 mL pooled monkey plasma. Activated partial thromboplastin time (APTT) was measured with an ACL-300+ coagulometer (Instrumentation Laboratory, Lexington, MA) by using the Platelin L reagent (Organon Teknika, France). To measure the APC sensitivity of FV, plasma was diluted 1:10 into human FV-deficient plasma (George King Bio-Medical, Overland Park, KS).20 APTT assessment was then performed in the presence of 0 to 20 nM human APC (Enzyme Research Laboratories) as described previously.15Plasma tHcy Plasma tHcy, defined as the total concentration of homocysteine after quantitative reductive cleavage of all disulfide bonds,21 was measured by high-performance liquid chromatography. Samples from monkeys were analyzed in Dr Malinow's laboratory,22,23 samples from mice were analyzed in Dr Bottiglieri's laboratory,24 and samples from humans were analyzed in the University of Iowa General Clinical Research Center.25Other assays Serum creatinine, total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, and triglycerides were measured in the clinical laboratories of the University of Iowa Hospitals.Statistical analysis APC-sensitivity results and changes in APTT and APC in response to thrombin were analyzed by using a 2-way repeated-measures analysis of variance with Bonferroni multiple-comparison analysis. Paired 2-tailed Student t tests were used to compare values obtained on separate study days in monkeys or human volunteers. Unpaired Student t tests were used to compare values in CBS+/ or CBS/ mice fed different diets. A
P value of less than .05 was considered to represent
statistical significance. Values are reported as mean ± SE.
Anticoagulant responses to thrombin in monkeys Monkeys were fed a control diet and a hyperhomocysteinemic diet, each for 4 weeks, in a randomized crossover study. The monkeys' body weights and serum levels of creatinine and cholesterol were not affected by the diets (Table 1). Plasma tHcy was approximately 2-fold higher when monkeys were fed the hyperhomocysteinemic diet (P < .05).
Infusion of thrombin produces activation of protein C and APC-dependent
prolongation of APTT in monkeys.15,26 To determine whether
these anticoagulant responses are impaired in hyperhomocysteinemia, we
measured plasma APC and APTT in response to infusion of human thrombin
(25 µg/kg given intravenously over 10 minutes) in monkeys after they
had been on each diet for 4 weeks. Baseline values for APC and APTT
before infusion of thrombin did not differ significantly between the 2 diets (Figure 1). After infusion of
thrombin, the peak level of plasma APC was 136 ± 16 U/mL when
monkeys were fed the control diet and 127 ± 13 U/mL when monkeys
were fed the hyperhomocysteinemic diet (P > .05; Figure
1A). APTT was prolonged to a similar extent by infusion of thrombin
during the 2 diets, although there was a trend toward less prolongation
when monkeys were fed the hyperhomocysteinemic diet (maximal
prolongation, 62 ± 16 seconds versus 82 ± 20 seconds for
the control diet; P = .07; Figure 1B). These findings
indicate that the hyperhomocysteinemic diet did not produce significant impairment of thrombin-induced activation of protein C in vivo, but do
not exclude a possible effect of hyperhomocysteinemia on the ability of
APC to prolong APTT in monkey plasma.
APC sensitivity of monkey FV To determine whether hyperhomocysteinemia in monkeys produces resistance to APC by impairing APC-mediated inactivation of FV, the sensitivity of monkey FV to human APC was measured after monkey plasma was diluted 1:10 into human FV-deficient plasma. The sensitivity to APC when monkeys were fed control diets was identical to that when they were fed hyperhomocysteinemic diets (Figure 2). This finding suggests that FV from hyperhomocysteinemic monkeys is not resistant to inactivation by APC.
APC sensitivity of FV in hyperhomocysteinemic mice Beginning at weaning, CBS+/ and CBS+/+
mice were fed either a control diet, a high-methionine diet, or a
high-methionine/low-folate diet. At 5 weeks of age, all mice appeared
to be healthy, and body weight did not differ between
CBS+/+ and CBS+/ mice or between mice fed
control and experimental diets (data not shown). Compared with the
control diet, the high-methionine and high-methionine/low-folate diets
produced graded elevations of plasma tHcy in both CBS+/+
and CBS+/ mice, and plasma tHcy was higher in
CBS+/ mice than in CBS+/+ mice for each diet
(Table 2).
The sensitivity of murine FV to human APC was measured after plasma
from CBS+/+ or CBS+/
Effect of methionine loading on APC sensitivity in human volunteers To determine whether acute hyperhomocysteinemia impairs the sensitivity of human FV to APC, 10 healthy human volunteers received an oral L-methionine (100 mg/kg) load in a placebo-controlled, randomized, double-blind crossover study. Plasma tHcy levels increased from 11.5 ± 1.6 µM to 45.4 ± 5.5 µM (P < .001) 6 hours after administration of L-methionine (Figure 4A). Plasma tHcy levels did not change significantly (10.2 ± 1.2 µM to 10.5 ± 1.3 µM; P > .05) after administration of placebo. The sensitivity of plasma FV to APC was essentially identical in samples collected 6 hours after administration of L-methionine and in samples collected from the same volunteers 6 hours after administration of placebo (Figure 4B). These observations show that marked elevation of plasma tHcy in humans does not acutely alter the susceptibility of FV to APC.
A great deal of epidemiologic evidence indicates that hyperhomocysteinemia is a risk factor for thrombotic vascular disease,1-3 but the mechanisms responsible for vascular pathology in hyperhomocysteinemia are not completely understood. Homocysteine may create a predisposition to thrombosis and other adverse cardiovascular events through several different mechanisms, including impairment of the protein C anticoagulant system.5,6,13 Because much of the evidence for homocysteine-induced impairment of the protein C system has been obtained from experiments performed in vitro, we tested the hypotheses that activation of protein C and susceptibility of FV to APC are altered during hyperhomocysteinemia in vivo. Our major findings were that thrombin-induced activation of endogenous protein C is not significantly impaired in hyperhomocysteinemic monkeys and that plasma FV does not become resistant to APC during hyperhomocysteinemia in monkeys, mice, or humans. The hyperhomocysteinemic diet that we fed to monkeys produced about a 2-fold increase in plasma tHcy. Although moderate, this degree of hyperhomocysteinemia is likely to be pathophysiologically relevant because it is very similar to that obtained in a previous study in which we observed significant impairment of endothelium-dependent vasomotor function in hyperhomocysteinemic monkeys.10 Abnormal endothelial function has also been observed in CBS-deficient mice with mild elevations of plasma tHcy (about 10 µM),27 and relatively small increases in plasma tHcy are associated with adverse cardiovascular events in humans.1 Our finding that normal APC responses to thrombin were preserved during hyperhomocysteinemia in monkeys suggests that impairment of protein C activation is unlikely to be a major mechanism of vascular pathologic conditions in mild hyperhomocysteinemia. The thrombin-infusion protocol is a reliable method for detecting abnormalities of endogenous protein C activation in monkeys: we previously used this protocol to demonstrate impairment of the protein C system in hypercholesterolemic monkeys.15,16 Although there was no difference in activation of protein C when monkeys were fed the control diet compared with when they were fed the hyperhomocysteinemic diet, we did observe a trend toward less prolongation of APTT in response to thrombin during the hyperhomocysteinemic diet (Figure 1B). This effect was probably not caused by resistance of FV to APC because in vitro assays of the sensitivity of plasma FV to human APC demonstrated equivalent dose-response relations in control and hyperhomocysteinemic monkeys (see below). It is also unlikely that this effect was caused by resistance of FVIII or other plasma factors to APC because prolongation of APTT in response to infusion of APC is not impaired in monkeys with combined hyperhomocysteinemia and hypercholesterolemia.15 It remains possible that hyperhomocysteinemia may alter the change in APTT in response to thrombin through APC-independent mechanisms. Exploration of such mechanisms will require additional studies with larger numbers of animals. The activated form of coagulation FV (FVa) is a major substrate for APC, and impaired inactivation of FVa by APC in patients with FV Leiden is associated with an increased risk of venous thrombosis.14 Homocysteine can react rapidly with free cysteine residues of FV in vitro, resulting in "homocysteinylation" of FV.13 Homocysteinylation of FV does not alter its rate of activation by thrombin, but homocysteinylated FVa is resistant to inactivation by APC.13 Using a direct assay for APC resistance in which human APC was added to undiluted monkey plasma, we did not detect resistance to APC in a previous study of monkeys with combined hyperhomocysteinemia and hypercholesterolemia.15 In the current study, we used a modified assay for APC resistance in which monkey plasma was diluted 1:10 into human FV-deficient plasma to measure the APC sensitivity of FV specifically.20 We found that the sensitivity of monkey FV to APC when monkeys were fed a control diet was identical to that when monkeys were fed a hyperhomocysteinemic diet (Figure 2). This finding shows that FVa from hyperhomocysteinemic monkeys is not resistant to inactivation by APC. To determine the effect of higher levels of plasma tHcy on the
sensitivity of FVa to APC, we performed APC-sensitivity assays with
plasma from CBS+/+ or CBS+/ To test the hypothesis that acute hyperhomocysteinemia produces resistance to APC in humans, we conducted APC-sensitivity assays of plasma obtained 6 hours after administration of an oral methionine load. A limitation of this experimental approach is that the hemostatic effects of acute hyperhomocysteinemia induced by oral methionine loading in healthy volunteers may differ from those in patients with chronic hyperhomocysteinemia. Nevertheless, oral methionine loading does produce impairment in endothelium-dependent vasomotor responses in the brachial artery of healthy humans.18 Moreover, because homocysteinylation of purified FV occurs within minutes in vitro,13 we anticipated that resistance of plasma FV to APC should be apparent within minutes to hours after oral methionine loading. However, despite a large increase in plasma tHcy (45 ± 6 µM versus 11 ± 1 µM), the sensitivity of plasma FV to APC was essentially identical in samples collected 6 hours after oral administration of methionine and in those obtained 6 hours after oral administration of placebo. Our observations that hyperhomocysteinemia in vivo in monkeys, mice, and humans does not induce resistance to APC in circulating FV is in contradiction to the hypothesis generated from the in vitro studies of Undas et al.13 This discrepancy may be explained by variations in the reactivity of FV with homocysteine in an in vitro purified system compared with an in vivo environment. Undas et al13 found that purified FV was homocysteinylated in vitro by the reduced thiol DL-homocysteine (30 µM-1.0 mM), presumably through a copper-dependent oxidation reaction. Homocysteinylation of FV was also detected when human plasma was exposed to a high concentration (10 mM) of DL-homocysteine that far exceeded clinical pathologic levels. Disulfide forms of homocysteine account for more than 95% of plasma tHcy, even in patients with moderate hyperhomocysteinemia.21 Therefore, the plasma concentration of the reduced thiol form of homocysteine that is available to react with FV through oxidative reactions in vivo is at least 2 to 3 orders of magnitude lower than that used in the in vitro plasma experiments. Furthermore, homocysteinylation of FV in vivo may not depend on direct oxidation of homocysteine because conversion of homocysteine to its disulfide forms in plasma is mediated mainly by thiol-disulfide exchange reactions rather than by copper-dependent oxidation.30 Thiol-disulfide exchange appears to be a major mechanism for homocysteinylation of albumin,30 but it is not known whether FV is susceptible to homocysteinylation through this mechanism. In summary, our findings in monkeys, mice, and human volunteers constitute evidence against a major effect of hyperhomocysteinemia on activation of protein C or on inactivation of FVa by APC. Therefore, these mechanisms are unlikely to be clinically important in most patients with thrombosis or other adverse cardiovascular events associated with mild to moderate hyperhomocysteinemia. Our data do not, however, exclude the possibility that impairment of the protein C anticoagulant pathway may occur in some patients with more severe hyperhomocysteinemia. This possibility could perhaps be addressed in studies of patients with end-stage renal disease or hereditary homocystinuria due to homozygous CBS deficiency.21
We thank Lorie Leo for technical assistance. Genotyping was performed by Lucinda Robbins, Norma Sinclair, and Patricia Lovell in the University of Iowa Transgenic Facility under the direction of Curt D. Sigmund.
Submitted March 7, 2002; accepted May 10, 2002.
Prepublished online as Blood First Edition Paper, May 24, 2002; DOI 10.1182/blood-2002-03-0727.
Supported by the Office of Research and Development, Department of Veterans Affairs, and National Institutes of Health grants HL63943, DK25295, HL58972, HL52246, and HL62984.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Steven R. Lentz, Department of Internal Medicine, C303 GH, University of Iowa, Iowa City, IA 52242; e-mail: steven-lentz{at}uiowa.edu.
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© 2002 by The American Society of Hematology.
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  S. Dayal and S. R. Lentz Murine Models of Hyperhomocysteinemia and Their Vascular Phenotypes Arterioscler Thromb Vasc Biol, September 1, 2008; 28(9): 1596 - 1605. [Abstract] [Full Text] [PDF]
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 S. Dayal, K. M. Wilson, L. Leo, E. Arning, T. Bottiglieri, and S. R. Lentz Enhanced susceptibility to arterial thrombosis in a murine model of hyperhomocysteinemia Blood, October 1, 2006; 108(7): 2237 - 2243. [Abstract] [Full Text] [PDF]
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 Y. Aso, K.-i. Okumura, K. Takebayashi, S. Wakabayashi, and T. Inukai Relationships of Plasma Interleukin-18 Concentrations to Hyperhomocysteinemia and Carotid Intimal-Media Wall Thickness in Patients With Type 2 Diabetes Diabetes Care, September 1, 2003; 26(9): 2622 - 2627. [Abstract] [Full Text] [PDF]
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H. Wang, X. Jiang, F. Yang, J. W. Gaubatz, L. Ma, M. J. Magera, X. Yang, P. B. Berger, W. Durante, H. J. Pownall, et al. Hyperhomocysteinemia accelerates atherosclerosis in cystathionine beta -synthase and apolipoprotein E double knock-out mice with and without dietary perturbation Blood, May 15, 2003; 101(10): 3901 - 3907. [Abstract] [Full Text] [PDF]
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G. Podda, E. M. Faioni, M. L. Zighetti, and M. Cattaneo No effect of fasting plasma total homocysteine on protein C activity in vitro Blood, March 15, 2003; 101(6): 2446 - 2446. [Full Text] [PDF]
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Top
Abstract
Introduction
-synthase (plasma tHcy, 93 ± 16 µM) or in human volunteers
with acute hyperhomocysteinemia (plasma tHcy, 45 ± 6 µM). Our
findings indicate that activation of protein C by thrombin and
inactivation of plasma FVa by APC are not impaired during moderate
hyperhomocysteinemia in vivo.
(Blood. 2002;100:2108-2112)
-thrombin (Enzyme Research
Laboratories, South Bend, IN; 25 µg/kg over 10 minutes) in 10 mL
saline was infused through the axillary vein catheter as described
previously.
