Abstract

This dissertation is based on experiments carried out at the Institute of Experimental Clinical Research, University of Aarhus, Centre for Haemophilia & 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).

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

The aim was to evaluate the influence of HH on the whole blood coagulation profile (WBCP), the genetic regulation of blood cells and on balloon-induced neointima formation.

An in vivo model of HH rats induced by folate deficiency was used. Experiment 1 (n=2×30) and 2 (n=2×16) consisted of control and HH animals, while experiment 3 (n=3×12) consisted of control, HH and treated animals.

WBCP analyses demonstrated that the WBCP is changed in a thrombogenic direction, characterized by increased velocity of the coagulation propagation, increased firmness of the formed clot, while the initiation phase of coagulation was prolonged. Gene expression analysis of blood cells, measuring the expression of about 8800 genes, revealed a plausible explanation for the changes in the WBCP. Up-regulation of integrin β 3, PECAM 1, platelet GP V and rap 1 B contributes to increased platelet activation and thereby contributes to explaining the increased velocity of coagulation and the increased firmness of the formed clot. Down-regulation of renal kallikrein contributes to explain the prolonged initiation phase of coagulation through a diminished resting thrombin potential and reduced activities of coagulation factors FXII:C, FX:C and FII:C. In contrast, FVII:C was unchanged by HH. A reduced capacity of fibrinolysis was found by an up-regulation of PAI-1 and slight down-regulation of t-PA.

Treatment of HH rats with the folate containing diet reversed the coagulation changes, as estimated by WBCP analyses and single coagulation factor functions. Indication of increased numbers of GpIIb/IIIa receptors was found in HH animals due to saturation kinetics as measured by WBCP following blockage of the platelet GpIIb/IIIa receptor. Up-regulation of uPAR, L-, E- and P-selectins was found and may contribute to increased tethering, rolling of leukocytes and migration of these to the vessel wall.

A reduced amount of neointima was found in HH animals following endothelial cell denudation. This may be due to a reduced methylation capacity related to the severeness of folate depletion in experiment 1 animals.

The findings need to be confirmed in a human based study. If positive, folate supplementation must be considered as thromboprophylaxis.