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,3
* Institute of Experimental Clinical Research and
Center for Hemophilia & Thrombosis, Department of Clinical Biochemistry, University Hospital of Aarhus/Skejby, Brendstrupgaardsvej, DK-8200 Aarhus N, Denmark
3To whom correspondence should be addressed. E-mail: lse{at}dadlnet.dk.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSAnalytical methodsRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • blood coagulation • folic acid deficiency • homocysteine • thromboelastography • rats
Hyperhomocysteinemia (HH) is epidemiologically associated with an increased risk of myocardial infarction, stroke and peripheral arterial thrombosis (1–6) as well as venous thromboembolism (7). Folic acid is the major dietary determinant of the plasma homocysteine concentration, as shown in a Dutch population study (8). Folate deficiency may be caused by suboptimal folate intake, by increased physiologic demands and through altered metabolism of iatrogenic origin (3). In such deficiency states, folic acid supplementation will quickly and efficiently normalize plasma homocysteine (9,10).
Little is known about the mechanisms underlying the thrombogenic effects of homocysteine or the in vivo mechanisms promoting thrombosis in HH individuals. A substantial number of mechanisms by which homocysteine may act have been investigated with focus on the regulation of single coagulation factors, platelet activity or endothelial markers (11–18); to our knowledge, however, the net effect of hyperhomocysteinemia on the whole blood coagulation profile (WBCP) has not been investigated.
The aim of the present study was to test our hypothesis that elevated homocysteine levels in vivo might promote changes in the WBCP that lead to thrombogenesis. For monitoring changes in whole blood coagulation in our model, a thromboelastographic method was selected. Thromboelastography has been used in various clinical conditions, e.g., during liver transplantation, cardiovascular surgery (19), trauma and obstetric anesthesia (20) and during treatment with recombinant factor VIIa of hemophiliacs with inhibitors to factors VIII or IX (21).
The WBCP expresses quantitative as well as qualitative characteristics of the coagulation process. This represents a major advantage over a simple plasma coagulation assessment by global routine assays based on the activated partial thromboplastin time and prothrombin time plasma methods (22). Furthermore, thromboelastography is more sensitive than standard laboratory tests in detecting hypercoagulable states (23), and this method provides additional information on interactions of all components of the clotting process in whole blood, allowing for study beyond the first stages of clot formation.
Thrombin generation has been evaluated by measuring thrombin-antithrombin (TAT) complexes, the only available method suited for studies in rats, as reported by Ravanat et al. (24).
In the present study a rat model of HH was developed. The rationale for the study was based on the well-characterized and most common cause of HH, folate deficiency. Two series of rats were investigated. The first series consisted of 60 rats, of which 30 suffered severe HH, and a second series consisted of 32 rats, of which 16 suffered intermediate HH. In each experimental series the same number of rats, with a normal homocysteine level, served as controls.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSAnalytical methodsRESULTSDISCUSSIONLITERATURE CITED |
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Wistar Kyoto inbred male rats (M & B, Ry, Denmark) were randomly assigned to consume a purified amino acid-defined pelleted diet (TD00351, Harland, Madison, WI) with a normal amount of folate or an identical diet deficient in folate (TD00350, Harland). The diets were amino acid defined to induce folic acid deficiency (25).
The diets contained 17.23 g protein/100 g diet, 8.03 g fat/100 g diet, 64.9 g carbohydrate/100 g diet. The diets contained the same amount of vitamins and minerals as the AIN-93G diet, except they contained 6.3 g Ca/kg and 4.0 g P/kg (26). Both diets contained succinylsulfathiazole, 10 g/kg, to prevent bacterial proliferation and subsequent intestinal folate neoproduction.
The control and folate-deficient diets were analyzed for the quantity of folate (1.63 and <0.06 mg/kg), L-methionine (4.7 and 4.8 g/kg), vitamin B-12 (0.0172 and 0.0163 mg/kg), and vitamin B-6 (6.27 and 6.63 mg/kg).
The rats were housed in pairs in stainless steel cages with sawdust-covered bottoms and had free access to food and drinking water. The room temperature was 23°C and the humidity was 45–55%. Light cycles consisted of 12 h light/12 h darkness. The rats were weighed when they were assigned to groups and subsequently every 7 d. The rats were killed by exsanguination after blood collection.
The animal experiments were carried out in accordance with the guidelines of the Scientific and Standardization Committee of the International Society on Thrombosis and Hemostasis (27), and the study protocol was approved by the Danish Ethical Committee for Animal Experimentation.
The experiments.
Experiment 1 consisted of 30 rats fed the folate-deficient diet and 30 rats fed the control diet for 63–73 d. Experiment 2 was designed to test the reproducibility of the results from Experiment 1 in rats with intermediate HH only. A power calculation on the data from Experiment 1 was adopted to estimate the optimal number of rats for inclusion in Experiment 2. The standardized difference was calculated on the basis of a two-sided level of significance (
) of 0.05, study power (1-ß) at 80% and the SD for the WBCP measurements in Experiment 1. The sample required was estimated to be between 20 and 70 rats depending on the WBCP derivative (CT, CFT, velocity or MCF). In order to employ the same batch of diet, the second series of rats was selected to consist of 32 rats in total. The feeding period to induce intermediate HH was estimated to be 28 d for all rats in this series.
Blood sampling.
The rats were sedated with a mixture of 0.05 mL/100 g rat Hypnorm (Fentanyl Citrate 0.315 g/L and Fluanisone 10 g/L) administered intramuscularly and 0.15–0.2 mL/100 g rat Dormicum (Midazolam) administered subcutaneously. The heart was exposed by a thoracic incision and blood was collected by heart puncture with a butterfly needle. To minimize activation of the blood, aspiration of blood was gentle and slow and a plastic syringe was used. Whole blood was collected in citrated tubes (0.129 mol/L), EDTA tubes and fluoride-heparinized tubes.
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Analytical methods |
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TOPABSTRACTMATERIALS AND METHODSAnalytical methodsRESULTSDISCUSSIONLITERATURE CITED |
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Flouride-heparinized blood was immediately cooled on ice and centrifuged at 4000 x g for 12 min at 4°C. Plasma was immediately separated from cells to limit the release of homocysteine from the RBC (28) and stored at -80°C until assayed. tHcy was measured using GC-MS stable isotope dilution (29).
Hematology.
Hemoglobin, RBC, platelets, leukocytes and hematocrit were determined using the ADVIA 120 system (Bayer Corporation, Copenhagen, Denmark).
Thrombin-antithrombin (TAT) complexes.
Citrated whole blood was centrifuged at 4000 x g for 12 min. Platelet-free plasma was immediately stored in aliquots at -80°C until assayed within 8 wk. TAT complexes were measured using an ELISA test (Enzygnost Micro, Behring, Marburg, Germany).
Red cell folate.
Red cell folate was determined in EDTA-stabilized whole blood using the ADVIA Centaur folate assay (Bayer Corporation, Dusseldorf, Germany).
Plasma fibrinogen.
Plasma fibrinogen was determined using nephelometry on a BNA (Behring, Nephelometer-Analyzer) Instrument (Dade Behring GmbH, Marburg, Germany), using rabbit antibody directed against human fibrinogen (Dakopatts, Copenhagen, Denmark).
Thromboelastographic analyses.
The thromboelastograph (roTEG, Pentapharm, Munich, Germany) assesses whole blood elasticity as recorded through a plastic pin slowly rotating in a plastic cup containing the blood sample to be assessed. During coagulation, the rotating pin is subjected to the increasing elastic forces of the clot during its alternate oscillation. Pin rotation is monitored optically using a diode, a small mirror fixed at the shaft, and a digital charge couple device array. The resistance against the movement of the pin determines the amplitude (Fig. 1). Both cup and pin are kept at 37°C using a thermostatically controlled heating block.
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Using DyCoDerivAn software (AvordusoL, Risskov, Denmark) (30), the digital raw data (amplitude) were differentiated according to time, leading to the definition of two additional derivatives, i.e., the maximum velocity of clot formation and the time to maximum velocity (Fig. 2).
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In separate pilot experiments, we empirically evaluated the thromboelastograph using varying concentrations of TF (Innovin, Dade Behring) and different dilutions of rat blood to optimize the sensitivity of the thromboelastographic method.
Using the roTEG equipment, we investigated the WBCP by two different assays employing TF-activated WBCP as well as nonactivated WBCP. The TF-activated method was performed to simulate in vivo conditions in the bloodstream in which TF triggers coagulation. Our technique closely mimicked in vivo conditions because the thromboelastographic investigations were carried out immediately after collection of the blood samples, as recommended by Camenzind et al. (31).
For the TF-activated measurements, citrated whole blood was immediately diluted 1:3 with NaCl (9 g/L) to a total volume of 300 µL and incubated for 2 min at 37°C in the reaction cup. TF (Dade Behring) was diluted 1:4,000 in barbital buffer 0.03 mol/L (natrium acetate trihydrate, concentration sodium 5.5-diethylbarbiturate) and 20 µL of this mixture was added to the preincubated blood sample. Coagulation was initiated by addition of 20 µL of 0.2 mol/L CaCl2 solution and the WBCP was recorded on one of the 4 channels of the roTEG.
Nonactivated analyses used the same processes as for the TF-activated analyses but without the activating reagent. All samples were measured in duplicate on the same roTEG instrument. In Experiment 2, the same 2 channels were used for the TF-activated measurements and the 2 other channels were used for the nonactivated measurements.
Statistics.
The data were tested for normality in Q-Q-plots. Normal distribution of the data was achieved after logarithmic transformation. A two-sided P < 0.05 was considered significant. The variation between the duplicate roTEG measurements was calculated using logarithmically transformed data.
Comparisons between groups were by independent sample t tests. Multivariate analysis was performed to adjust for possible confounding by platelets and fibrinogen on the WBCP derivatives. The statistical analyses were performed with SPSS 10.0 (Chicago, IL).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSAnalytical methodsRESULTSDISCUSSIONLITERATURE CITED |
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The changes in WBCP in rats with HH were thrombogenic in nature (Table 1). HH rats had a greater maximum velocity of clot propagation than controls (P < 0.001), as estimated by an increased velocity (P < 0.001) and/or a shortened clot formation time (P = 0.001). Furthermore, the maximum clot firmness was greater in HH rats than in controls (P < 0.001). The initiation phase of the coagulation was protracted in the HH rats, as judged by a prolongation of the CT in the HH rats (P = 0.001). The spontaneous thrombin turnover in HH rats tended to be less than in controls, as judged by the basal TAT levels (P = 0.074).
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The body weight differed between groups (P < 0.001). Control rats gained 73.2 g (95% CI, 64.2–82.2) body weight, whereas the HH rats lost 21.4 g (95% CI -5.4 to -37.4) body weight.
Experiment 2.
Intermediate HH was induced in these rats that had a median plasma tHcy level of 42.0 µmol/L compared with 6.6 µmol/L in controls. The WBCP changed similarly to what was demonstrated in Experiment 1, with a prolongation of the initiation phase, a reduction in the clot formation time and an increase in the maximum clot firmness, although the differences between HH rats and controls in the TF-activated experiments were not significant (Table 1).
Nonactivated WBCP experienced a significant prolongation of the initiation phase, as illustrated by the prolongation of CT in HH rats compared with controls (P = 0.035). The CFT tended to be shorter in the HH rats than in the controls (P = 0.172), but the MCF did not differ between groups.
When the CT’s in the two methods used are compared, it can be seen that in the nonactivated measurements the difference was 38.4 s (227.8 - 189.4 s) while in the activated measurements the difference was 15.2 s (204.1 - 188.9 s) only. This difference was significant (P < 0.001). The increase in velocity was dose dependent, as judged by a comparison of results from Experiments 1 and 2. The thrombin generation evaluated by TAT tended to be lower in HH rats than in controls (P = 0.121), as in Experiment 1.
Depletion in RBC folate was not as severe in the intermediate than in the severely HH rats; values were 84.9% of the level in controls. This was paralleled by less severe changes in the hematological values. For example, platelet and leukocyte counts did not differ between the groups. The hemoglobin concentration and the red cell count differed significantly between groups, although the numerical difference was less pronounced than in Experiment 1.
No rats suffered from loss in body weight, but the weight gain in controls was 53.2 g (95% CI, 47.1–59.3) compared with 42.4 g (95% CI, 36.8–48.0) in the HH rats, (P = 0.009).
We assumed that the differences in the absolute values of CT (TF-activated) in Experiment 1 compared with Experiment 2 were caused by change of TF batch.
The imprecision (CV%) of the roTEG-derived coagulation values, as assessed from double determinations on the same thromboelastograph, was 11% for the CT, 10% for the CFT and 3% for the MCF. There were no significant differences among the four channels of the thromboelastograph.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSAnalytical methodsRESULTSDISCUSSIONLITERATURE CITED |
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Increase in maximum velocity.
The increased maximum velocity of the WBCP as estimated by the reduced CFT and/or the increased maximum velocity cannot be explained by an exaggerated spontaneous thrombin turnover because the TAT levels in the HH rats tended to be lower than in controls in both series. In our laboratory, we tested the influence of different platelet counts on the WBCP. The lower the platelet number, the lower the velocity achieved. The velocity was greater than in controls in severely HH rats despite their reduced platelet number. A probable explanation for the increase in the maximum velocity could be increased platelet activation and/or increased platelet aggregation, as shown previously in both animal (12,32) and human studies (33,34).
Prolongation of the initiation phase.
The prolongation of the initiation phase was mirrored by a reduced turnover of thrombin, as measured by the tendency toward lower TAT levels in the HH rats. This reduction in TAT in HH rats is in agreement with the work of Bos et al. (35), who investigated the endogenous thrombin potential in HH healthy persons compared with controls and showed that the elevated risk for venous thrombosis is not reflected in an increased endogenous thrombin potential.
However, other studies (36–38) have described a positive correlation between HH and TAT levels. Marcucci et al. (37) reported a positive correlation between homocysteine and TAT in patients with atherosclerosis. The rats in our study did not have atherosclerosis. Klerk et al. (36) described a positive correlation between homocysteine and TAT, although the r was small (r = 0.25).
Increased levels of tissue factor pathway inhibitor (TFPI) could be another explanation for the prolongation of the initiation phase. An indication of increased levels of TFPI in HH was demonstrated indirectly by the difference (P < 0.001) between the difference in the nonactivated CT measurements compared with the difference in the TF-activated CT measurements. This possible increase in TFPI in the HH rats could prolong the CT because the inclusion of TF (TF-activated measurements) "neutralized" the circulating TFPI, as estimated by the difference in CT (HH and controls) in the TF-activated compared with nonactivated measurements. In patients with homocystinuria, the activity of TFPI after heparin-induced release was greater than that in healthy controls (39).
The multivariate analysis did not confirm a significant relationship between homocysteine and CT in Experiment 1 when adjusted for platelet number. However, in Experiment 2, the CT (nonactivated) was prolonged, and these rats were identical in platelet number; we believe that in HH a significant prolongation of the initiation phase of the WBCP occurs.
Increase in maximum clot firmness.
In Experiment 1, the MCF was significantly greater in the HH rats than in controls. A similar nonsignificant trend was seen in Experiment 2. Platelets, as well as fibrinogen, are important determinants of development of clot elasticity, and the MCF indicates the combined function of both. However, platelets seem to play a more important role in determining clot elasticity than does fibrin (40), and a linear relationship between MCF and log10 platelet count has been described (41). Mild qualitative platelet defects diminish the MCF, whereas thrombocytopenia has to be relatively marked to modify the MCF (22).
The multivariate analysis adjusting for platelet and fibrinogen, did not change the level of significance (P < 0.05). This was despite the discrepant platelet counts as well as levels of fibrinogen in Experiment 1. The MCF was greater in the HH rats.
The changes in the WBCP were dose dependent and for that reason, the assumptions of the power calculation were changed. Under these conditions, the number of rats was too small to obtain significant results in Experiment 2 except for the CT in the nonactivated measurements. If we instead had ordered a new batch of diet for Experiment 2 animals, the basic conditions would not have been comparable. The limitation in this study is related to the severity of folate depletion in Experiment 1, which was reflected by loss in body weight and reduction in red cell, white cell and platelet counts. This might have been the result of hypomethylation (42) and impaired DNA synthesis influencing normal cell proliferation and growth. The implications for our understanding of the pathogenesis of thrombosis in hyperhomocysteinemia, however, are related to determining the nature of the whole blood coagulation in HH.
Future questions relate to the primary causes of these changes in the WBCP. Future studies involving other ways of inducing HH must be conducted to verify that homocysteine itself produces these changes in the whole blood coagulation.
In summary, WBCP changes caused by HH were characterized in this model by a prolongation of the initiation phase of coagulation, an increased velocity of clot formation, and an increased firmness of the formed clot. If the coagulation profiles in humans are altered similarly, a more rapid development of a clot with greater firmness might well be responsible for the increased risk of thrombosis in individuals with HH.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by grants from the Danish Heart Foundation, J.nr.00–1-2–15-22788 and J.nr.99–1-2–16-22676; The Aarhus University Research Foundation; The Institute of Experimental Clinical Research, Aarhus University; Kirsten Anthonius’ Foundation; "Bønnelykke" Foundation; and "Direktør Jacob Madsen and Hustru Olga Madsen" Foundation.
4 Abbreviation used: CFT, clot formation time; CT, clotting time; HH, hyperhomocysteinemia or hyperhomocysteinemic; MCF, maximum clot firmness; roTEG, thromboelastography; TAT, thrombin-antithrombin; TF, tissue factor; TFPI, tissue factor pathway inhibitor; tHcy, total plasma homocysteine; WBCP, whole blood coagulation profile.
5 g/kg diet: L-alanine, 3.5; L-arginine HCl, 12.1;L-asparagine, 6.0; L-aspartic acid, 3.5; L-cysteine, 3.5; L-glutamic acid, 43.2; glycine, 23.3; L-histidine, HCl H2O, 4.5; L-isoleucine, 8.2; L-leucine, 11.1; L-lysine HCl, 18.0; L-methionine, 5.0; L-phenylalanine, 7.5; L-proline, 3.5; L-serine, 3.5; L-threonine, 8.2; L-tryptophan, 1.8; L-tyrosine, 5.0; L-valine, 8.2; sucrose, 354.4; cornstarch, 150.0; maltodextrin, 150.0; soybean oil, 80.0; cellulose, 30.0; mineral mix, AIN-93M-MX 35.0; calcium phosphate, monobasic Ca(H2PO4)2 · H2O, 8.2; nicotinic acid, 0.03; Ca pantothenate, 0.016; pyridoxine · HCl, 0.007; thiamin · HCl, 0.006; riboflavin, 0.006; D-biotin, 0.0002; vitamin B-12 (0.1% in mannitol), 0.025; dl--tocopheryl acetate (500 IU/g), 0.15; vitamin A palmitate (500000 IU/g), 0.008; cholecalciferol (500000 IU/g), 0.002; phylloquinone, 0.0008; choline bitartrate, 2.5; tert-butylhydroquinone (antioxidant), 0.016.
Manuscript received 13 January 2003. Initial review completed 17 February 2003. Revision accepted 19 March 2003.
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LITERATURE CITED |
|---|
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1. Whincup, P. H., Refsum, H., Perry, I. J., Morris, R., Walker, M., Lennon, L., Thomson, A., Ueland, P. M. & Ebrahim, S. B. (1999) Serum total homocysteine and coronary heart disease: prospective study in middle aged men. Heart 82:448-454.
2. Nygard, O., Nordrehaug, J. E., Refsum, H., Ueland, P. M., Farstad, M. & Vollset, S. E. (1997) Plasma homocysteine levels and mortality in patients with coronary artery disease. N. Engl. J. Med. 337:230-236.
3. Boushey, C. J., Beresford, S. A., Omenn, G. S. & Motulsky, A. G. (1995) A quantitative assessment of plasma homocysteine as a risk factor for vascular disease. Probable benefits of increasing folic acid intakes. J. Am. Med. Assoc. 274:1049-1057.[Abstract]
4. Perry, I. J., Refsum, H., Morris, R. W., Ebrahim, S. B., Ueland, P. M. & Shaper, A. G. (1995) Prospective study of serum total homocysteine concentration and risk of stroke in middle-aged British men. Lancet 346:1395-1398.[Medline]
5. Malinow, M. R., Kang, S. S., Taylor, L. M., Wong, P. W., Coull, B., Inahara, T., Mukerjee, D., Sexton, G. & Upson, B. (1989) Prevalence of hyperhomocyst(e)inemia in patients with peripheral arterial occlusive disease. Circulation 79:1180-1188.
6. Danesh, J. & Lewington, S. (1998) Plasma homocysteine and coronary heart disease: systematic review of published epidemiological studies. J. Cardiovasc. Risk 5:229-232.[Medline]
7. den Heijer, M., Rosendaal, F. R., Blom, H. J., Gerrits, W. B. & Bos, G. M. (1998) Hyperhomocysteinemia and venous thrombosis: a meta-analysis. Thromb. Haemost. 80:874-877.[Medline]
8. de Bree, A., Verschuren, W. M., Blom, H. J. & Kromhout, D. (2001) Association between B vitamin intake and plasma homocysteine concentration in the general Dutch population aged 20–65 y. Am. J. Clin. Nutr. 73:1027-1033.
9. Ubbink, J. B., Vermaak, W. J., van der, M. A., Becker, P. J., Delport, R. & Potgieter, H. C. (1994) Vitamin requirements for the treatment of hyperhomocysteinemia in humans. J. Nutr. 124:1927-1933.
10. Brattstrom, L. (1996) Vitamins as homocysteine-lowering agents. J. Nutr. 126:1276S-1280S.
11. Khajuria, A. & Houston, D. S. (2000) Induction of monocyte tissue factor expression by homocysteine: a possible mechanism for thrombosis. Blood 96:966-972.
12. Durand, P., Lussier-Cacan, S. & Blache, D. (1997) Acute methionine load-induced hyperhomocysteinemia enhances platelet aggregation, thromboxane biosynthesis, and macrophage-derived tissue factor activity in rats. FASEB J. 11:1157-1168.[Abstract]
13. Fryer, R. H., Wilson, B. D., Gubler, D. B., Fitzgerald, L. A. & Rodgers, G. M. (1993) Homocysteine, a risk factor for premature vascular disease and thrombosis, induces tissue factor activity in endothelial cells. Arterioscler. Thromb. 13:1327-1333.
14. Harpel, P. C., Chang, V. T. & Borth, W. (1992) Homocysteine and other sulfhydryl compounds enhance the binding of lipoprotein(a) to fibrin: a potential biochemical link between thrombosis, atherogenesis, and sulfhydryl compound metabolism. Proc. Natl. Acad. Sci. U.S.A. 89:10193-10197.
15. Undas, A., Williams, E. B., Butenas, S., Orfeo, T. & Mann, K. G. (2001) Homocysteine inhibits inactivation of factor Va by activated protein C. J. Biol. Chem. 276:4389-4397.
16. Lentz, S. R., Erger, R. A., Dayal, S., Maeda, N., Malinow, M. R., Heistad, D. D. & Faraci, F. M. (2000) Folate dependence of hyperhomocysteinemia and vascular dysfunction in cystathionine beta-synthase-deficient mice. Am. J. Physiol. 279:H970-H975.
17. Graeber, J. E., Slott, J. H., Ulane, R. E., Schulman, J. D. & Stuart, M. J. (1982) Effect of homocysteine and homocystine on platelet and vascular arachidonic acid metabolism. Pediatr. Res. 16:490-493.[Medline]
18. Hajjar, K. A. (1993) Homocysteine-induced modulation of tissue plasminogen activator binding to its endothelial cell membrane receptor. J. Clin. Investig. 91:2873-2879.
19. Calatzis, A., Haas, S., Gödje, O., Calatzis, A., Hipp, R. & Walenga, J. M. (2001) Thromboelastographic coagulation monitoring during cardiovascular surgery with the roTEG coagulation analyzer. Pifarré, R. eds. Management of Bleeding in Cardiovascular Surgery 2001:215-226 Hanley & Belfus Philadelphia, PA. .
20. Whitten, C. W. & Greilich, P. E. (2000) Thromboelastography: past, present, and future. Anesthesiology 92:1223-1225.[Medline]
21. Ingerslev, J., Christiansen, K., Calatzis, A., Holm, M. & Sabroe, E. L. (2000) Management and monitoring of recombinant activated factor VII. Blood Coagul. Fibrinolysis 11(suppl. 1):S25-S30.
22. Traverso, C. I., Caprini, J. A. & Arcelus, J. I. (1995) The normal thromboelastogram and its interpretation. Semin. Thromb. Hemost. 21(suppl. 4):7-13.
23. Zuckerman, L., Cohen, E., Vagher, J. P., Woodward, E. & Caprini, J. A. (1981) Comparison of thromboelastography with common coagulation tests. Thromb. Haemost. 46:752-756.[Medline]
24. Ravanat, C., Freund, M., Dol, F., Cadroy, Y., Roussi, J., Incardona, F., Maffrand, J. P., Boneu, B., Drouet, L. & Legrand, C. (1995) Cross-reactivity of human molecular markers for detection of prethrombotic states in various animal species. Blood Coagul. Fibrinolysis 6:446-455.[Medline]
25. Walzem, R. L. & Clifford, A. J. (1988) Folate deficiency in rats fed diets containing free amino acids or intact proteins. J. Nutr. 118:1089-1096.
26. Reeves, P. G., Nielsen, F. H. & Fahey, G. C., Jr (1993) AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J. Nutr. 123:1939-1951.
27. Giles, A. R. (1987) Guidelines for the use of animals in biomedical research. Thromb. Haemost. 58:1078-1084.[Medline]
28. Rasmussen, K. & Moller, J. (2000) Total homocysteine measurement in clinical practice. Ann. Clin. Biochem. 37:627-648.
29. Stabler, S. P., Lindenbaum, J., Savage, D. G. & Allen, R. H. (1993) Elevation of serum cystathionine levels in patients with cobalamin and folate deficiency. Blood 81:3404-3413.
30. Sorensen, B., Johansen, P., Christiansen, K., Woelke, M. & Ingerslev, J. (2003) Whole blood coagulation thromboelastographic profiles employing minimal tissue factor activation. J. Thromb. Hemost. 1:551-558.
31. Camenzind, V., Bombeli, T., Seifert, B., Jamnicki, M., Popovic, D., Pasch, T. & Spahn, D. R. (2000) Citrate storage affects thromboelastograph analysis. Anesthesiology 92:1242-1249.[Medline]
32. Durand, P., Prost, M. & Blache, D. (1996) Pro-thrombotic effects of a folic acid deficient diet in rat platelets and macrophages related to elevated homocysteine and decreased n-3 polyunsaturated fatty acids. Atherosclerosis 121:231-243.[Medline]
33. Davi, G., Di Minno, G., Coppola, A., Andria, G., Cerbone, A. M., Madonna, P., Tufano, A., Falco, A., Marchesani, P., Ciabattoni, G. & Patrono, C. (2001) Oxidative stress and platelet activation in homozygous homocystinuria. Circulation 104:1124-1128.
34. Di Minno, G., Davi, G., Margaglione, M., Cirillo, F., Grandone, E., Ciabattoni, G., Catalano, I., Strisciuglio, P., Andria, G. & Patrono, C. (1993) Abnormally high thromboxane biosynthesis in homozygous homocystinuria. Evidence for platelet involvement and probucol-sensitive mechanism. J. Clin. Investig. 92:1400-1406.
35. Bos, G. M., Rijkers, D. T., Willems, H. P., den Heijer, M., Beguin, S., Gerrits, W. B. & Hemker, H. C. (1999) The elevated risk for venous thrombosis in persons with hyperhomocysteinemia is not reflected by the endogenous thrombin potential. Thromb. Haemost. 81:467-468.[Medline]
36. Klerk, M., Verhoef, P., Verbruggen, B., Schouten, E. G., Blom, H. J., Bos, G. M. & den Heijer, M. (2002) Effect of homocysteine reduction by B-vitamin supplementation on markers of clotting activation. Thromb. Haemost. 88:230-235.[Medline]
37. Marcucci, R., Prisco, D., Brunelli, T., Pepe, G., Gori, A. M., Fedi, S., Capanni, M., Simonetti, I., Federici, G., Pastore, A., Abbate, R. & Gensini, G. F. (2000) Tissue factor and homocysteine levels in ischemic heart disease are associated with angiographically documented clinical recurrences after coronary angioplasty. Thromb. Haemost. 83:826-832.[Medline]
38. Undas, A., Domagala, T. B., Jankowski, M. & Szczeklik, A. (1999) Treatment of hyperhomocysteinemia with folic acid and vitamins B12 and B6 attenuates thrombin generation. Thromb. Res. 95:281-288.[Medline]
39. Cella, G., Burlina, A., Sbarai, A., Motta, G., Girolami, A., Berrettini, M. & Strauss, W. (2000) Tissue factor pathway inhibitor levels in patients with homocystinuria. Thromb. Res. 98:375-381.[Medline]
40. Chandler, W. L. (1995) The thromboelastography and the thromboelastograph technique. Semin. Thromb. Hemost. 21(suppl. 4):1-6.[Medline]
41. Oshita, K., Az-ma, T., Osawa, Y. & Yuge, O. (1999) Quantitative measurement of thromboelastography as a function of platelet count. Anesth. Analg. 89:296-299.
42. James, S. J., Melnyk, S., Pogribna, M., Pogribny, I. P. & Caudill, M. A. (2002) Elevation in S-adenosylhomocysteine and DNA hypomethylation: potential epigenetic mechanism for homocysteine-related pathology. J. Nutr. 132:2361S-2366S.
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