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 in all higher animals. Deficient function of this system can lead to fatal bleeding following even a minor injury, whereas overactivity can produce unwanted blood clots, resulting in blockages to critical blood vessels, as occurs in heart attacks and stroke.
von Willebrand Factor
The blood-clotting protein von Willebrand factor (VWF) functions as the initial bridge connecting blood platelets to injured blood vessels and 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 inherited bleeding disorder. Our lab played a key role in defining the genetic defects responsible for VWD and identifying the VWF-processing protein, ADAMTS13, as the cause of the often catastrophic blood-clotting disease, thrombotic thrombocytopenic purpura.
VWF levels differ by as much as fivefold in normal individuals. This variation is determined primarily by genetic differences between people. The genes responsible for these differences contribute to wide variation in bleeding severity among individuals with VWD, whereas elevated VWF levels caused by these same "modifier" genes result in increased risk for blood-clotting diseases. To understand how such modifier genes might work, we studied the genetic basis for different VWF levels among inbred mice. The first mouse VWF modifier gene that we identified produces a subtle alteration that results in accelerated removal of VWF from the blood. We found this same gene variant in mice from many parts of the world, suggesting an evolutionary advantage to low VWF levels, at least in some situations. Using a similar approach, we have located six additional VWF modifier genes in the mouse.
Advances in genomic technology have now made it possible to also approach this problem directly in humans. We analyzed the genetic makeup and VWF levels in two large groups of healthy college students, confirming a major effect of ABO blood type on VWF levels. Taking advantage of sibling relationships in our study, we identified a novel gene on chromosome 2, which accounts for close to 20 percent of VWF variation not previously detected by standard genetic approaches. Further characterization of this and other genes controlling VWF levels may lead to improved VWD diagnosis and prediction of bleeding and blood-clotting risk.
Genetic Susceptibility to Thrombosis
Venous thromboembolic disease causes 300,000 deaths in the United States each year. Though approximately 60 percent of the overall risk for thrombosis is determined by genetic factors, most of this genetic risk remains unexplained. About 20 percent of this risk is due to elevated VWF-FVIII levels, with another 25 percent due to a common human variant, factor V Leiden (FVL), present in 5 percent of Europeans. We engineered mice producing FVL in place of normal factor V. By combining FVL with mutations in other genes, we identified several blood proteins that, when only mildly reduced, cause markedly increased clotting in FVL mice. We are now screening mice carrying mutations randomly induced throughout the genome, to identify other genes that are capable of acting as modifiers for FVL. Characterizing these genes may help us distinguish the 10 percent of humans with FVL who will develop a serious blood clot during their lifetime from the 90 percent who will remain asymptomatic.
Blood Clotting and Bacterial Infection
Plasminogen is the protein that breaks down blood clots after they have formed. 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 a type of severe skin infection ("flesh-eating" bacteria). Introducing a human plasminogen transgene rendered 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. Our results also suggest that blocking streptokinase function might provide a novel approach to treatment of GAS infections. We thus screened more than 300,000 chemicals to identify a small set that specifically turn off streptokinase in the bacteria. Several of these compounds proved effective in protecting mice from fatal GAS infection. These compounds also inhibit production of disease-causing factors by another important human pathogen, Staphylococcus aureus, suggesting that this class of drugs could also be effective for treating S. aureus infections.
The Role of Endoplasmic Reticulum (ER)–to–Golgi Transport in Blood-Clotting and Vascular Diseases
We showed that the inherited bleeding disease, combined deficiency of coagulation factors V and VIII, is due to mutation in one of two genes, LMAN1 or MCFD2. The MCFD2-LMAN1 complex serves as a carrier from the ER to the Golgi 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 to identify other proteins that also depend on this 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.
Our discovery of the LMAN1-MCFD2 cargo receptor led us to study the effects of mutations in other components of the cell's protein export machinery. Humans with mutations in one of these genes, SEC23B, exhibit a unique abnormality of their red blood cells. Surprisingly, we found that SEC23B-deficient mice have normal red blood cells and instead develop a severe degeneration of the pancreas. This evolutionary difference provides important clues about the balance between SEC23A and SEC23B. We also found that mice lacking SEC24A are remarkably normal, except for an ~50 percent reduction in blood cholesterol. We subsequently showed that PCSK9, a key regulator of cholesterol, is uniquely dependent on SEC24A for export from the cell, accounting for the reduced blood cholesterol in these animals. These findings suggest that this pathway may represent a novel target for the treatment of elevated cholesterol.
This research is supported in part by grants from the National Institutes of Health.
As of February 07, 2013