Douglas M. Tollefsen, M.D., Ph.D.

DEPARTMENT OF Internal Medicine
Keywords: blood coagulation, extracellular matrix, glycosaminoglycans, thrombin, vascular biology

Thrombin is the major protease generated during blood coagulation at sites of vascular injury. It converts fibrinogen to fibrin monomers, which polymerize to form a clot, and cleaves G-protein–coupled receptors on platelets and endothelial cells, causing platelet aggregation, secretion of chemokines, and recruitment of inflammatory cells. Thrombin appears to induce smooth muscle cell proliferation in developing atherosclerotic plaques. Therefore, thrombin participates not only in normal blood clotting, but also in pathologic processes such as thrombosis and atherogenesis.

We are interested in proteins that regulate the activity of thrombin. One of these is heparin cofactor II (HCII), a plasma protein that inhibits thrombin by formation of a covalent 1:1 complex. Dermatan sulfate and heparan sulfate bind to HCII and increase the rate of thrombin inhibition >1000-fold. Biosynthesis of these glycosaminoglycans by cells in the vessel wall may determine the site of action of HCII in vivo. We find that specific oligosaccharide sequences in dermatan sulfate chains are required to stimulate HCII, and we are determining the structures of these oligosaccharides by enzymatic degradation and mass spectrometry. We are also investigating the protease- and oligosaccharide-binding regions of HCII by site-directed mutagenesis of the cDNA. We have generated HCII-deficient mice by homologous recombination in embryonic stem cells and find that these mice develop thrombi more rapidly than do wild-type animals after injury of the carotid arterial endothelium. Using these mice, we have begun to investigate HCII in a variety of other models in which thrombin may play a role.