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Clinical Institute and Center for Hemophilia and Thrombosis, Department of Clinical Biochemistry, University Hospital of Aarhus (Skejby Sygehus), 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 METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • homocysteine • folic acid deficiency • blood coagulation factors • thrombelastography
Epidemiologically, hyperhomocysteinemia (HH)4 has been associated with an increased risk of arterial thrombosis (1–5) as well as venous thromboembolism (6,7); a large meta-analysis recently suggested a causal relation (8). However, the pathophysiological linkage of elevated levels of circulating homocysteine and the increased risk of thrombosis is still largely unknown. In an attempt to elucidate the pathogenesis of the associated increased risk with HH, we demonstrated previously that the whole-blood coagulation profiles (WBCP) in HH rats were changed in a thrombogenic direction, as assessed by an increased velocity of the coagulation propagation and an increased firmness of the formed clot, whereas the initiation phase of the coagulation was prolonged (9). A prolongation of the activated partial thromboplastin time (APTT) was described previously in HH rabbits, whereas the prothrombin time (PT) was unaffected (10). In severe and intermediate HH rats, we suspected a diminished spontaneous thrombin turnover (9). We hypothesized that there is a linkage among the prolongation of the initiation phase of the WBCP (9), the suspected diminished thrombin turnover, and the prolongation of the APTT (10) through a downregulation or impaired function of the coagulation factors participating in the contact activation pathway.
The aim of the present study was to test our new hypothesis by investigating the functional activities of single coagulation factors in FD-HH rats. Furthermore, the influence of platelet glycoproteine IIb/IIIa receptor on the WBCP was investigated to determine whether platelets might be involved in the changes in WBCP we found previously in HH rats (9). The present study included a subgroup of FD-HH rats that were subsequently fed a folic acid–containing control diet.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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This series of 36 rats comprised 12 FD-HH rats fed the folic acid–deficient diet for 28 d to induce HH, 12 control rats fed the control diet for 28 d, and 12 rats who were fed the folic acid–deficient diet for 28 d to induce HH and then fed the control diet for 28 d to normalize the homocysteine level. All rats were weighed at randomization and subsequently every 7 d. 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 (11), and the study protocol was approved by the Danish Ethical Committee for Animal Experimentation.
Analytical methods. The blood sampling procedure and determination of total homocysteine (tHcy), hematological variables, red cell folate, and thrombin antithrombin (TAT) complexes were described previously (9).
Single coagulation factors (F) II, VII, VIII, X, and XII were recorded on a Thrombolyzer (Behnk Elektronik) coagulation instrument using routine 1-stage coagulometric methods, in each case utilizing a commercial test base depleted (human deficiency plasma) in the specific coagulation factor to be determined. Measurements of FII, FVII, and FX were carried out using recombinant human tissue factor (TF) Innovin (Dade Behring) as the activating agent. For determination of FXII and FVIII, the activator was Platelin Excel (Organon Teknika). Recorded values of single coagulation factors in rat plasma were based on an in-house human normal plasma pool from 30 healthy volunteers. Hence, rat plasma factors are expressed here relative to their human counterpart, with 1 unit of respective single coagulation factor corresponding to the amount present in 1 mL of normal human plasma.
The principle of the thrombelastographic analysis and the set-up for the TF-activated measurements were described previously (9). All WBCP were recorded on 1 of the 4 channels of the roTEG instrument (Pentapharm). The Integrilin-TF–activated measurements were obtained using the same procedure as for the TF-activated measurements, but with the prior addition of 0.025, 0.1, or 0.4 mg Integrilin (Schering-Plough A/S). Integrilin (Eptifibatid) is a specific and reversible inhibitor of the platelet glycoprotein IIb/IIIa receptor.
Statistics. The data were tested for normality in Q-Q-plots showing skewness. A normal distribution was achieved after logarithmic transformation. Data were analyzed by 1-way ANOVA and a modified t test with Bonferroni correction. A two-sided P < 0.05 was considered significant. Data are presented as means with 95% CI. The statistical analyses were performed using SPSS Ver. 11.0.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Thrombelastographic analyses. The velocity of coagulation and the clotting time (CT) were greater in FD-HH rats than in controls and were normalized in the treated rats (Table 2, P < 0.05). The maximum clot firmness (MCF) was greater in FD-HH rats and in controls than in the treated rats (P < 0.05).
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When 0.025 mg Integrilin was added, the CT pattern was changed from the pattern that occurred when no Integrilin was added; it was shorter in the FD-HH rats compared with controls or treated rats, P < 0.05. At a dose of 0.1 mg Integrilin, the groups did not differ, and at 0.4 mg Integrilin, the CT was shorter in controls than in the 2 other groups.
Body weight. All rats gained weight during the 4 wk of study. The rats fed the control diet gained 57.0 (range 51.7–62.4) g body weight, whereas the rats fed the folate deficient diet (the FD-HH rats and the treated rats) gained 49.9 (range 46.1–53.7) g body weight during the 4 wk of consuming the folate-deficient diet (P < 0.05).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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This study demonstrated that FD-HH diminishes the functional activities of FXII:C, FX:C, and FII:C, whereas FVII:C was unperturbed by FD-HH, and FVIII:C increased in FD-HH rats. The thrombelastographic measurements confirmed our previous findings of increased velocity of coagulation, increased clot firmness, and a prolonged initiation phase in FD-HH rats. The novel finding of the present study is the demonstration that most of the FD-HH–induced changes in WBCP and single coagulation factor functions were reversed by treatment with a folic acid–containing diet. Furthermore, blockage of the platelet GpIIb/IIIa receptor by Integrilin did not abolish the FD-HH–induced increase in coagulation velocity.
Moderate HH resulted from feeding the rats a folate-deficient diet. The treated rats had a plasma tHcy concentration that did not differ from that of the controls. The RBC folate level was lower in FD-HH rats than in controls. The RBC folate in treated rats was not normalized during the short observation period because RBC folate concentrations are a better reflection of long-term distribution than actual serum levels due to the half-life of RBC (120 d) (12).
The contact activation pathway of coagulation was influenced by FD-HH. Coagulation FXII activity was significantly reduced after induction of FD-HH, and FXII:C increased in FD-HH rats that were subsequently treated with the normal folic acid–containing diet. FX:C, the factor that forms the intersection between the coagulation pathways of contact activation and TF dependent activation, was lower in FD-HH rats than in controls and treated rats. FII:C, a key element in fibrin formation, also was reduced in the FD-HH rats compared with controls and the folate treated rats.
Our finding of reduced functional activities of FXII, FX and FII is consistent with the finding of a prolongation of the APTT in rabbits suffering HH (10). This diminished function of coagulation factors involved in the contact activation pathway of coagulation could be responsible for the longer initiation phase in WBCP in the FD-HH rats in this study as well as in our previous study (9). The prolongation of the initiation phase of coagulation may seem paradoxical because HH is associated with thrombotic diseases (13–15). A similar paradox occurs in humans with FXII deficiency, which is also reflected by a prolongation of the APTT and/or a prolongation of the initiation phase of WBCP (16). An in vivo model of human FXIIa deficiency demonstrated a prolonged initiation of thrombin formation, whereas the relative thrombin generation was unaffected by the decrease in FXIIa (17). A reduction in thrombin during HH was also described in a human study of HH patients with venous thrombosis (18). In this study, we report data on the spontaneous thrombin status as measured by FII:C; however, the total thrombin generation during the coagulation process warrants evaluation.
The initiation of the TF–dependent coagulation pathway may not be affected by FD-HH because the levels of FVII:C were unchanged in FD-HH compared with controls and treated rats. This is in accordance with an unaffected PT in HH rabbits (10). In contrast, a positive correlation between homocysteine and FVII:C was found in a human cross-sectional study comprising middle-aged people suffering from atherosclerosis (19). However, the FD-HH rats in this study did not have atherosclerosis.
FVIII, which participates in activating FX, was increased in FD-HH rats, whereas the interpretation of the results derived from the treated rats is limited due to the limited numbers of measurements. Increased levels of FVIII, as in the FD-HH rats, represent a risk factor for venous thromboembolism, independent of the acute phase reaction (20,21), and FVIII is correlated with von Willebrand factor (vWF) in patients with thromboembolic diseases (22).
The methods employed for measuring the functional activities of the single coagulation factors utilized a human plasma-based set-up in which only differences in factor activities among the groups could be estimated and not the actual coagulation factor antigen level. Therefore, the detected downregulation of the factor activities could be the result of unknown regulating factors inhibiting or changing the activity of these coagulation factors. Testing the actual antigen levels would have been desirable; unfortunately, rat-specific antibodies directed against the respective coagulation factors were not available.
Consistent with our previous findings (9), an increase in the velocity of clot propagation and a prolongation of the CT occurred in FD-HH rats. The novelty is the finding that the FD-HH–induced changes in the WBCP were reversed by treatment with the folate-containing diet. This finding of increased velocity of whole-blood coagulation in FD-HH may be of clinical importance because homocysteine is a proven risk factor for a secondary event in patients suffering ischemic heart disease (3,23–25). In atherosclerotic patients who develop a plaque rupture, the velocity of the coagulation must be decisive, irrespective of whether the lumen in the affected vessel eventually occludes, resulting in ischemia in the heart, brain, leg, or elsewhere.
To evaluate the contribution of GpIIb/IIIa receptors to the WBCP changes detected, increasing concentrations of Integrilin, a RGD-mimetic (Eptifibatid) were added to the samples. We detected an increase in the WBCP velocity in FD-HH compared with control and treated rats, despite the blockage of the GpIIb/IIIa, irrespective of the dosage of Integrilin. This suggests that mechanisms other than platelet activities mediated through the GpIIb/IIIa receptor are responsible for the increased velocity of clot formation in FD-HH.
The rapid FD-HH–induced changes in the WBCP and single coagulation factor functions were reversed by folate repletion of the FD-HH rats. Folate supplementation and/or a homocysteine-lowering treatment decreased fibrinogen and increased plasminogen and antithrombin III (26), decreased soluble thrombomodulin and vWF (27,28), attenuated thrombin generation, as estimated by TAT and prothrombin fragment 1 + 2 (29), and reduced levels of asymmetric dimethylarginine, an endogenous inhibitor of NO synthase (30).
The effects of folic acid treatment were evaluated in this study with special attention to whole-blood coagulation and single coagulation factor functions. However, the effects of folic acid fortification on clinical outcomes in patients suffering HH are still unknown because the results of large on-going randomized trials are awaited.
In the present study, we found reduced functional activities of coagulation factors XII, X, and II in FD-HH, whereas the functional FVII activity was unchanged. Furthermore, FD-HH altered the pattern of WBCP by increasing the velocity of coagulation and by slowing down the initiation phase of coagulation. Most of the observed changes were reversible as demonstrated in a subset of rats initially depleted in folic acid, but subsequently fed the folic acid–containing diet. Inhibition of the platelet GpIIb/IIIa receptor diminished the velocity in a dose-dependent manner, but in spite of blockage of this receptor, the velocity was still higher in FD-HH rats compared with control and treated rats.
Overall, our results illustrate that several coagulation mechanistic factors may be implicated in FD-HH–induced thrombosis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported by the Danish Heart Foundation, J.nr. 00–1-2–15-22788 and J.nr. 01–1-3–16A-22891; the Aarhus University Research Foundation; Clinical Institute, Aarhus University; Kirsten Anthonius’ Foundation; Bønnelykke Foundation; Frænkels Foundation; and Direktør Jacob Madsen & Hustru Olga Madsen Foundation.
4 Abbreviations used: APTT, activated partial thromboplastin time; CT, clotting time; F, factor; FD-HH, folate deficient-hyperhomocysteinemia; HH, hyperhomocysteinemia or hyperhomocysteinemic; MCF, maximum clot firmness; PT, prothrombin time; TAT, thrombin antithrombin; TF, tissue factor; tHcy, total homocysteine; vWF, von Willebrand factor; WBCP, whole-blood coagulation profile.
Manuscript received 22 October 2004. Initial review completed 30 December 2004. Revision accepted 6 May 2005.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. 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.
2. Danesh, J. & Lewington, S. (1998) Plasma homocysteine and coronary heart disease: systematic review of published epidemiological studies. J. Cardiovasc. Risk 5:229-232.[Medline]
3. 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.
4. Wald, N. J., Watt, H. C., Law, M. R., Weir, D. G., McPartlin, J. & Scott, J. M. (1998) Homocysteine and ischemic heart disease: results of a prospective study with implications regarding prevention. Arch. Intern. Med. 158:862-867.
5. 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.
6. Martinelli, I., Battaglioli, T., Pedotti, P., Cattaneo, M. & Mannucci, P. M. (2003) Hyperhomocysteinemia in cerebral vein thrombosis. Blood 102:1363-1366.
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. Wald, D. S., Law, M. & Morris, J. K. (2002) Homocysteine and cardiovascular disease: evidence on causality from a meta-analysis. Br. Med. J. 325:1202.
9. Ebbesen, L. S., Christiansen, K. & Ingerslev, J. (2003) Hyperhomocysteinemia due to folate deficiency is thrombogenic in rats. J. Nutr. 133:2250-2255.
10. Sauls, D. L., Wolberg, A. S. & Hoffman, M. (2003) Elevated plasma homocysteine leads to alterations in fibrin clot structure and stability: implications for the mechanism of thrombosis in hyperhomocysteinemia. J. Thromb. Hemost. 1:300-306.
11. Giles, A. R. (1987) Guidelines for the use of animals in biomedical research. Thromb. Haemost. 58:1078-1084.[Medline]
12. Stanger, O. (2002) Physiology of folic acid in health and disease. Curr. Drug Metab. 3:211-223.[Medline]
13. Miller, G. J., Esnouf, M. P., Burgess, A. I., Cooper, J. A. & Mitchell, J. P. (1997) Risk of coronary heart disease and activation of factor XII in middle-aged men. Arterioscler. Thromb. Vasc. Biol. 17:2103-2106.
14. Grundt, H., Nilsen, D. W., Hetland, O., Valente, E. & Fagertun, H. E. (2004) Activated factor 12 (FXIIa) predicts recurrent coronary events after an acute myocardial infarction. Am. Heart J. 147:260-266.[Medline]
15. Kuhli, C., Scharrer, I., Koch, F., Ohrloff, C. & Hattenbach, L. O. (2004) Factor XII deficiency: a thrombophilic risk factor for retinal vein occlusion. Am. J. Ophthalmol. 137:459-464.[Medline]
16. Pivalizza, E. G. (2003) Perioperative use of the Thrombelastograph in patients with inherited bleeding disorders. J. Clin. Anesth. 15:366-370.[Medline]
17. Obraztsov, I. F., Kuz’min, V. M. & Khanin, M. A. (2002) Blood coagulation dynamics under the conditions of Hageman factor deficiency: a mathematical model. Dokl. Biochem. Biophys. 386:248-250.[Medline]
18. 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]
19. Schreiner, P. J., Wu, K. K., Malinow, M. R., Stinson, V. L., Szklo, M., Nieto, F. J. & Heiss, G. (2002) Hyperhomocyst(e)inemia and hemostatic factors: The Atherosclerosis Risk in Communities Study. Ann. Epidemiol. 12:228-236.[Medline]
20. Koster, T., Blann, A. D., Briet, E., Vandenbroucke, J. P. & Rosendaal, F. R. (1995) Role of clotting factor VIII in effect of von Willebrand factor on occurrence of deep-vein thrombosis. Lancet 345:152-155.[Medline]
21. O’Donnell, J., Mumford, A. D., Manning, R. A. & Laffan, M. (2000) Elevation of FVIII:C in venous thromboembolism is persistent and independent of the acute phase response. Thromb. Haemost. 83:10-13.[Medline]
22. Bombeli, T., Jutzi, M., De Conno, E., Seifert, B. & Fehr, J. (2002) In patients with deep-vein thrombosis elevated levels of factor VIII correlate only with von Willebrand factor but not other endothelial cell-derived coagulation and fibrinolysis proteins. Blood Coagul. Fibrinolysis 13:577-581.[Medline]
23. Acevedo, M., Pearce, G. L., Kottke-Marchant, K. & Sprecher, D. L. (2002) Elevated fibrinogen and homocysteine levels enhance the risk of mortality in patients from a high-risk preventive cardiology clinic. Arterioscler. Thromb. Vasc. Biol. 22:1042-1045.
24. Michalis, L. K., Pappas, K., Tweddel, A., Kolaitis, N., Katsouras, C. S., Loukas, S., Goudevenos, J., Bourantas, K. & Sideris, D. A. (2001) Relatively low red cell folate levels and acute coronary syndromes. Coron. Artery Dis. 12:665-668.[Medline]
25. De Bree, A., Verschuren, W.M.M., Kromhout, D., Kluijtmans, L.A.J. & Blom, H. J. (2002) Homocysteine determinants and the evidence to what extent homocysteine determines the risk of coronary heart disease. Pharmacol. Rev. 54:599.
26. Mayer, O., Filipovsky, J., Hromadka, M., Svobodova, V., Racek, J., Mayer, O., Jr, Stehlik, P., Trefil, L. & Zarybnicka, M. (2002) Treatment of hyperhomocysteinemia with folic acid: effects on homocysteine levels, coagulation status, and oxidative stress markers. J. Cardiovasc. Pharmacol. 39:851-857.[Medline]
27. van den Berg, B. M., Boers, G. H., Franken, D. G., Blom, H. J., Van Kamp, G. J., Jakobs, C., Rauwerda, J. A., Kluft, C. & Stehouwert, C. D. (1995) Hyperhomocysteinaemia and endothelial dysfunction in young patients with peripheral arterial occlusive disease. Eur. J. Clin. Investig. 25:176-181.[Medline]
28. Constans, J., Blann, A. D., Resplandy, F., Parrot, F., Renard, M., Seigneur, M., Guerin, V., Boisseau, M. & Conri, C. (1999) Three months supplementation of hyperhomocysteinaemic patients with folic acid and vitamin B6 improves biological markers of endothelial dysfunction. Br. J. Haematol. 107:776-778.[Medline]
29. 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]
30. Holven, K. B., Haugstad, T. S., Holm, T., Aukrust, P., Ose, L. & Nenseter, M. S. (2003) Folic acid treatment reduces elevated plasma levels of asymmetric dimethylarginine in hyperhomocysteinaemic subjects. Br. J. Nutr. 89:359-363.[Medline]
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