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Hyperhomocysteinemia due to folate deficiency. Impact on haemostasis and vascular biology

Liselotte M. Sabroe Ebbesen  

Summary

This thesis is based on experiments conducted at the Institute of Experimental Clincial Reseach, University of Aarhus, Centre for Hemophilia & Thrombosis, and the Molecular Diagnostic Laboratory at the Department of Clinical Biochemistry, University Hospital of Aarhus (Skejby Sygehus) and at the Institute of Pathology, University Hospital of Aarhus (Kommunehospitalet) in the period between 1999 and 2003.

Hyperhomocysteinemia (HH) has been identified as a risk factor for thrombosis both in arterial thrombosis and venous thromboembolism in epidemiologic studies, although the pathogenesis is still largely unresolved.

An in vivo model of HH rats induced by folate deficiency was used in three series of experiments. Experiment 1 (n=2x30) and experiment 2 (n=2x16) consisted of control and HH animals, while experiment 3 (n=3x12) consisted of control, HH and treated animals (treated animals were HH animals which were subsequently fed a folate containing diet). Whole blood coagulation analyses have demonstrated that the whole blood coagulation is changed in a thrombogenic direction, characterized by increased velocity, increased firmness of the formed clot, while the initiation phase was prolonged. Gene expression analysis of blood cells (buffy coat cells), measuring the expression of about 8,800 genes, revealed a plausible explanation for the changes in the whole blood coagulation. Up-regulation of integrin beta 3, PECAM 1, platelet glycoprotein V and rap 1 B contribute to increased platelet activation and thereby contribute to explaining the increased velocity of whole blood coagulation and the increased firmness of the formed clot, as detected. Down-regulation of renal kallikrein contribute to explain the prolonged initiation phase of coagulation through a diminished resting thrombin potential and reduced activities of coagulation factors contributing in the contact activation pathway of coagulation. FXII:C, FX:C and FII:C were reduced in HH animals. In contrast, the tissue factor dependent pathway of coagulation, FVII:C, was unchanged. The down-regulation of the contact activating pathway contributes to an impaired fibrinolysis, which may also contribute to increased risk of thrombosis. The gene expression analysis also demonstrated an up-regulation of PAI-1 and slight down-regulation of t-PA, both contributing in reduced capacity of fibrinolysis. Treatment of HH rats with the folate containing diet reversed the coagulation changes, as estimated by whole blood coagulation analysis and single coagulation factor functions. Addition of increasing dosages of Integrilin (inhibitor of the platelet GpIIb/IIIa receptor) did not change the HH induced increased velocity and the HH induced increased firmness. Indication of increased numbers of GpIIb/IIIa receptors was found in HH animals due to saturation kinetics. Up-regulation of uPAR, L-, E- and P-selectins was found by gene expression analysis and may contribute to increased tethering, rolling of leukocytes and migration of these to the vessel wall. The influence of HH on atherosclerosis was demonstrated by a model of balloon induced neointima formation in the left common carotid artery. A reduced amount of neointima was found in HH animlals, indicating HH does not contribute to the development of atherosclerosis. These findings may be explained by a reduced methylation capacity related to the severeness of folate depletion in experiment 1 animals.