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Molecular Genetics of Blood Clotting
Summary: David Ginsburg is interested in understanding the components of the blood-clotting system and how disturbances in their function lead to human bleeding and blood-clotting disorders.
Precise control of the blood-clotting system is essential for maintenance of the circulation in all higher animals. Deficient function of this system can lead to fatal bleeding following even a minor injury, whereas overactivity of this system can produce unwanted blood clots, resulting in blockages to critical blood vessels, as occurs in such diseases as heart attack and stroke.
von Willebrand Factor
The blood-clotting protein von Willebrand factor (VWF) functions as the critical initial bridge connecting blood platelets to the wall of injured blood vessels, thereby helping to control bleeding. VWF also serves as the carrier for factor VIII, the substance missing in patients with hemophilia. Abnormalities in VWF result in von Willebrand disease (VWD), the most common human inherited bleeding disorder.
It is now clear that wide variations in the severity of bleeding among individuals with the same or similar defects in the VWF gene are due to the action of one or more additional "modifier" genes. Such modifier genes are also likely to contribute to the wide variation in VWF levels observed among normal individuals. Elevated levels of VWF due to these same modifier genes may also result in an increased risk of blood clotting, in contrast to the bleeding tendency associated with low VWF.
To understand how such modifier genes might work, we have been studying the genetic factors responsible for variation in VWF level among inbred mouse strains. The first such VWF modifier gene that we identified (Mvwf1) is a novel variant of the B4galnt2 gene. This variant produces a subtle alteration in the VWF protein that results in accelerated VWF removal from the blood. This unique mechanism could explain the action of VWD modifier genes in humans, as well as modifier genes for other common diseases. We have found this same unique Mvwf1 variant as the cause of low VWF levels in several other inbred mouse strains, and also among wild mice in many parts of the world. These observations suggest that there may be an evolutionary advantage to low VWF levels, at least in some situations. Using a similar genetic approach in other inbred mouse strains, we have now found the location of at least five additional VWF modifier genes. Mvwf2 and Mvwf5 turned out to be mild mutations within the VWF gene itself. Mvwf3, Mvwf4, and Mvwf6 are due to mutations in other genes on other chromosomes. Understanding how these modifier genes work in the mouse could help us explain how VWF levels are controlled in humans and lead to improved diagnosis and treatment for patients with VWD.
In another series of studies, we showed that mutations in the gene for ADAMTS13, a protein that normally breaks down VWF in the blood, are responsible for the inherited form of thrombotic thrombocytopenic purpura (TTP), an often catastrophic blood-clotting disease. Our subsequent studies in mice suggest that bacterial toxins, together with additional genetic susceptibility factors, are required to trigger TTP in the setting of ADAMTS13 deficiency. Identification of these factors should lead to improved diagnosis and treatment for patients with this disease. (This work is supported in part by a grant from the National Institutes of Health.)
Coagulation Factor V
Coagulation factor V is a central regulator in the early phases of blood clot formation. Genetic deficiency of factor V results in a rare bleeding disorder called parahemophilia. Conversely, a subtle change in the factor V gene that increases the function of this protein, called factor V Leiden, results in an abnormal increase in blood clot formation. Factor V Leiden is remarkably common (present in ~5 percent of the population) and contributes to up to 50 percent of hospital admissions for blood clot–related illnesses.
Our lab has developed genetically engineered mice completely deficient in factor V as well as mice producing factor V Leiden in place of normal factor V. By combining factor V Leiden with mutations in other mouse clotting factor genes, we have identified several blood proteins that, when only mildly reduced, can cause markedly increased clotting in factor V Leiden mice. In related experiments we are screening mice carrying mutations randomly induced throughout the genome, in an effort to identify many of the genes that are capable of acting as modifiers for factor V Leiden in mice, as well as in humans. Characterizing these genes may provide valuable new information to help us distinguish the 10 percent of humans with factor V Leiden who will develop a serious blood clot during their lifetime from the 90 percent who will remain asymptomatic.
We have recently begun to study the blood-clotting system in zebrafish. Through the analysis of a fish with a mutation that causes bleeding in the brain, we identified a gene that plays an important role in the regulation of the endothelial cells that line blood vessels. We have now engineered fish with mutations in several blood-clotting factors that lead to increased blood clot formation. We plan to use these animals to screen for additional genes that might modify bleeding and clotting risk in humans, as well as to test large collections of chemicals to identify potential new blood-thinning drugs.
Combined deficiency of coagulation factor V and coagulation factor VIII is another inherited bleeding disease under study in our lab, in collaboration with Randal Kaufman (HHMI, University of Michigan Medical School). We originally identified deficiency of the cellular protein LMAN1 (also known as ERGIC53) as the molecular basis of this disorder in 70 percent of patients. We subsequently identified mutations in the MCFD2 gene as the cause of this disorder in the remaining 30 percent. The complex of MCFD2 and LMAN1 serves as a carrier for a subset of proteins, including factors V and VIII, that are destined for export from the cell. These findings provide the first example of such a specific transport pathway within the cells of higher organisms. We are studying genetically engineered mice deficient in LMAN1 and MCFD2, in an effort to identify other proteins that, along with factors V and VIII, also depend on this unique intracellular pathway for their production. In addition to providing insight into the processes by which proteins are synthesized and exported from the cell, this work identifies a novel target for the development of new anticoagulant (blood-thinning) drugs. (This work is supported in part by a grant from the National Institutes of Health.)
Plasminogen Activation
The fibrinolytic system, the body's mechanism for breaking down blood clots, is delicately balanced with the system that forms blood clots. Plasminogen activators are the proteins that turn on the fibrinolytic system. Their activity is controlled by several regulator proteins, including plasminogen activator inhibitor-1 (PAI1) and PAI2. We are using transgenic approaches to study the function of the PAIs and other components of the fibrinolytic system in the whole animal. This work has unmasked previously unappreciated roles for these proteins in the blood-clotting system and in the development of several important diseases, including atherosclerosis (the underlying cause of heart attack and stroke) and bacterial infection.
We showed that the bacterial protein streptokinase, an activator of plasminogen, is a key factor for the invasiveness of group A streptococci (GAS). GAS are the cause of strep throat, as well as an unusually severe form of skin infection in humans ("flesh-eating" bacteria). Introducing a human plasminogen transgene rendered the resulting mice remarkably sensitive to GAS, partially explaining why these bacteria generally only infect humans and suggesting a major role for the blood-clotting system in our body's defense against infection. We are now exploring chemicals that interfere with streptokinase as a potential new treatment for GAS-related diseases.
Last updated March 09, 2009
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