Experimental Thrombosis and Atherosclerosis (ETA)

The ETA program focuses on the biochemistry of blood coagulation and fibrinolysis as it relates to thrombosis. The term thrombosis includes abnormal clotting in arteries, the cause of most heart attacks and strokes, or in the deep veins of the legs. Clots in the leg veins can break off and travel to the lungs (so-called pulmonary embolism), a condition that can be fatal.

Thrombosis in arteries usually complicates atherosclerosis (hardening of the arteries). Disruption of atherosclerotic plaque exposes material that triggers the clotting mechanism. Blood clot formation superimposed on disrupted atherosclerotic plaque occludes the lumen of the artery, thereby blocking blood flow. Stoppage of blood flow in heart arteries leads to a heart attack, whereas obstruction in brain arteries causes a stroke.

Clotting in veins can be triggered by surgery or trauma, conditions that can directly damage the vein walls. Immobilization because of medical or surgical illness also can cause clotting because blood stagnates in the deep veins of the leg when the leg muscles are not contracting. Inflammatory mediators also can contribute to clotting in veins or arteries because these substances activate the endothelial cells that line blood vessels. Activated endothelial cells promote clotting and tether circulating blood cells onto their surface. These tethered cells not only block the lumen of the vessel, but also release substances that cause further damage to endothelial cells and induce more clotting.

The ETA program focuses on (a) mechanisms involved in blood clot formation and dissolution, (b) development and application of blood tests used for diagnosis of abnormal clotting, and (c) treatment of blood clotting disorders. Each of these areas will be briefly described.

(A) Mechanisms of Thrombosis: Clotting occurs on the surface of injured arteries or veins. Understanding how arteries and veins become injured and promote clotting requires study of the interaction between genes and the environment. For example, high blood pressure, diabetes, and obesity promote hardening of the arteries, a condition that predisposes to clotting. Study of the effect of these conditions on the genetic makeup of blood vessel cells permits understanding of how atherosclerosis and clotting develop. Likewise, accumulation of homocysteine (an amino acid) in the blood and inflammatory cells also can trigger blood vessel injury and clotting. Using cultured cells and molecular biology techniques, the effect of these substances on gene expression profiles can be examined. Mouse models of diabetes, obesity and hyperhomocysteinemia provide another avenue for the study of gene-environment interactions. These investigations are being spearheaded by Drs. R. Austin, B. Werstuck, P. Liaw and A. Sharma.

Clotting in injured blood vessels involves a cascade of enzymatic reactions with numerous positive and negative feedback steps. Understanding these reactions requires study of the structure and function of clotting enzymes and their inhibitors. Dr. Weitz’s group used light scattering spectroscopy, fluorescence spectroscopy and surface plasmon resonance to study the complex protein/protein interactions that characterize clotting. The importance of specific domains on clotting enzymes or their inhibitors can be assessed by using site-directed to mutagenesis to generate recombinant analogs that have these domains deleted or altered. Comparison of the biochemical characteristics of the recombinant analogs with those of the native proteins identifies those domains that are important. These techniques have been used to study clotting enzymes (including thrombin and factor Xa) and their inhibitors (including antithrombin, heparin cofactor II and á1-antitrypsin). Studies are ongoing to identify the triggers that initiate clotting, particularly in patients with cancer. These studies are being conducted in conjunction with Drs. J. Rak and A. Lee.

Clotting represents a dynamic balance between the biochemical pathways that promote clot formation and those that trigger clot dissolution. In addition to studying clotting pathways, Dr. Weitz’s group is investigating the biochemical processes involved in clot dissolution. This includes analysis of the structural determinants of plasminogen activators, the molecules that initiate clot dissolution, and assessment of positive and negative feedback pathways that regulate the clot digesting pathway.

(B) Development and Application of Blood Tests to Monitor or Diagnose Clotting: In the last stages of blood clotting, fibrinogen, a soluble protein in the blood, is converted into insoluble fibrin. Molecules of fibrin polymerize to form the matrix of the blood clot within which blood cells are trapped. Although many different enzymes can degrade fibrinogen or fibrin, each leaves a characteristic footprint by cleaving specific bonds. Capitalizing on this fact, Dr. Weitz has developed specific assays for fragments of fibrinogen released by different enzymes. The levels of these fragments in blood provide a direct measure of specific enzyme activity and can be used to study the activity of these enzymes in various disease states. Dr. Weitz has developed assays that monitor the activity of a key clotting enzyme (thrombin), a clot-digesting enzyme (plasmin), and an enzyme released by activated white blood cells (elastase).

Fragments of fibrin are formed when clots in veins or arteries are broken down by the clot digesting system. The levels of the specific fibrin fragments can be used as diagnostic tests for clotting. Working with Drs. J. Ginsberg, S. Bates, and C. Kearon, Dr. Weitz has been studying the utility of these tests in the diagnosis of clots in the veins of the legs or clots in the lungs.

(C) Treatment of Clotting Disorders: Numerous drugs are available to treat clotting disorders. These include antiplatelet drugs, agents that inhibit the activity of platelets, small cells that clump together to form the first part of a blood clot, and anticoagulants, drugs that inhibit various steps in blood clotting. Working with Dr. S. Yusuf, Dr. Weitz has been involved in studies examining the use of new antiplatelet drugs or anticoagulants in patients with arterial thrombosis. In conjunction with Drs. J. Hirsh, J. Ginsberg, M. Levine, and C. Kearon, Dr. Weitz also is involved in studies examining new anticoagulant drugs for prevention and treatment of venous thrombosis.

Dr. Weitz’s group has been involved in the development of new drugs to treat thrombosis. Serving as a Research and Development arm of GlycoDesign, Inc., a Toronto-based biotechnology company, Dr. Weitz’s laboratory has developed three new anticoagulants. One drug, known as Vasoflux, went to Phase II clinical testing and spawned the development of more potent agents. GH9001, a new drug that is just finishing Phase I evaluation in healthy volunteers, will enter Phase II testing later this year.

At a cell-based level, disrupting the processes that tether circulating blood cells to activated endothelium provides another means for attenuating abnormal clotting. Cell/cell interaction is mediated, at least in part, by sugar molecules expressed on the surface of circulating white blood cells. Dr. Weitz’s group is investigating the utility measures that block the synthesis of cell surface sugars or prevent the sugars from binding to their counter receptor. Oral drugs that disrupt this process may be useful as long-term therapy to prevent thrombosis in arteries or veins.

Heparin is a blood thinning medication that is widely used to prevent or treat thrombosis. Osteoporosis is a complication of long-term heparin therapy. Dr. S. Shaughnessy has been exploring the mechanisms responsible for heparin-induced osteoporosis. These studies have revealed a pathway that not only explains heparin-induced osteoporosis, but may also be involved in postmenopausal osteoporosis. Using small molecules that block this pathway, Dr. Shaughnessy has been able to prevent heparin-induced or estrogen deficiency-induced osteoporosis in animals. Studies are underway to explore the potential clinical utility of these new blocking agents.