Structure and Function of Heparin Cofactor II

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DESCRIPTION (provided by applicant): Heparin cofactor II (HCII) is an inhibitor of thrombin in plasma that can be activated by dermatan sulfate (DS) proteoglycans on vascular smooth muscle cells and fibroblasts. In a murine model of HCII deficiency, we showed that HCII modulates the response to arterial injury. In comparison with wild-type mice, HCHII-/- mice require less time to form a thrombus in the carotid artery following endothelial injury and have a greater degree of neointimal smooth muscle cell proliferation. HC-/- mice also develop more atherosclerotic lesions in the aorta when hypercholesterolemic. To build upon these observations, we propose the following specific aims: (1) Investigate the cause of neonatal mortality in HCII-deficient 129/SvJ mice and the phenotype of mice with combinations of HCII deficiency and other thrombophilic mutations. Although HCII-/- mice in the C57BL/6 background produce litters of normal size and viability, HCII-/- 129/SvJ mice do not produce viable offspring. Breeding studies will be done to determine whether neonatal mortality is related to maternal, paternal, or fetal HCII deficiency, and pathologic abnormalities will be identified. C57BL/6 mice with HCII deficiency in combination with factor V Leiden or protein Z deficiency will be observed for evidence of spontaneous thrombosis or other abnormalities. (2) Obtain evidence for activation of HCII upon binding to DS in the vessel wall, and identify HCII-binding sites in other tissues. HCII-/- mice will be reconstituted with recombinant HCII variants having specific defects in binding to heparin or DS to determine the importance of HCII-glycosaminoglycan interactions in models of arterial and venous thrombosis. Immunohistochemical methods will be used to localize HCII and glycosaminoglycans in the vessel wall after injury, and HCII-binding sites in other tissues will be identified. (3) Determine the location and amount of thrombin activity and the time course of smooth muscle cell and macrophage accumulation in neointimal and atherosclerotic lesions of HCII+/+ and HCII-/- mice. Hirudin-binding and chromogenic substrate assays will be used to examine arteries of HCII-/- mice for evidence of increased thrombin activity during neointima formation or development of atherosclerotic lesions. Rates of accumulation of fibrin(ogen), platelets, smooth muscle cells, and macrophages in these lesions will be determined in HCII-/- and wild-type mice. (4) Compare the structures of HCII-binding DS oligosaccharides derived from porcine skin and mucosa with those of vascular smooth muscle cells and fibroblasts. Size-fractionated DS oligosaccharides will be subjected to HCII affinity chromatography, and the structures of selected high- and low-affinity oligosaccharides will be determined. Metabolically-labeled vascular smooth muscle cells and fibroblasts will be assayed for the presence of HCII-binding oligosaccharides. This work will provide insight into the mechanism of activation of HCII by vascular glycosaminoglycans and the pathogenesis of human diseases such as thrombosis and atherosclerosis.