Mechanisms Involved in Preventing Unwanted Blood Clots
Summary: Charles Esmon is interested in the mechanisms that control the process of blood clotting and the links between the control of blood clotting and inflammation.
Our laboratory is interested in the mechanisms that control the process of blood clotting and the links between this process and inflammation. Blood clots contribute to many serious human diseases, including heart attacks, strokes, pulmonary emboli, and venous thrombosis (phlebitis). They contribute to the mortality and morbidity of septic shock, acute trauma injury, and some of the complications of diabetes.
To understand why abnormal clots occur, we examine the mechanisms by which the normal blood-clotting system is regulated and compare the regulation under normal circumstances to pathogenic circumstances. To accomplish this, our laboratory seeks to (1) identify new factors that are involved in regulating the blood-clotting process, (2) understand how the proteins function in the control of the process, (3) understand how the genes are regulated, (4) examine the influence of defects in the function of the proteins on human disease processes, (5) examine the influence of inhibition of the function of the proteins in animal models of human disease, (6) use crystallographic and biophysical techniques to determine the molecular structure of the proteins and complexes, and (7) determine how the regulatory proteins of the coagulation system control inflammation, and vice versa.
Work from our laboratory and many others has shown that venous thrombosis is most commonly the result of defects in the proteins that participate in the protein C anticoagulant pathway. These proteins are also involved in protection from the deleterious effects of bacterial infection (a process leading to septic shock, sometimes referred to as blood poisoning) of the bloodstream, where the engagement of the protein C anticoagulant pathway is critical to the survival of the patient. Ongoing studies are aimed at elucidating how the pathway protects the individual from septic shock.
Earlier we demonstrated that activated protein C (APC) could protect animals, including nonhuman primates, from the lethal effects of Escherichia coli infusion. APC was shown to block coagulation induced by E. coli infusion, facilitate clot lysis, limit cytokine elaboration, and minimize vascular leak. As such, it is uniquely poised to serve as a candidate for the treatment of severe sepsis in humans, a disease that has a mortality rate of approximately 30–50 percent. It has now been shown that infusion of APC (this drug is now called Xigris) can decrease mortality approximately 20 percent in severe sepsis patients. These clinical results provide impetus to establish the multiple modes of action by which APC accomplishes this function. To understand better how to use APC clinically, we have developed a variety of assays to understand changes in the function of the pathway as the disease progresses.
The most recently identified member of the protein C pathway, the endothelial protein C receptor (EPCR), was identified in our laboratory. We have now shown that this protein plays an important role in regulating both the blood-clotting and inflammatory responses in septic shock. Indeed, the vast majority of the protein C pathway's biological influences are manifested primarily in a manner dependent on EPCR. Specifically, overexpression of the receptor dampens both the coagulation and inflammatory cytokine responses seen with blood-borne bacterial infiltration and reduces the death rate in experimental animals. The opposite responses are seen when the receptor levels are decreased. At least part of this function is manifested because of an unexpectedly strong contribution of EPCR to protein C activation.
EPCR gene deletion results in early embryonic death in mice. To address the mechanisms involved, we selectively deleted EPCR from the developing embryo while sparing EPCR in the placenta. This strategy resulted in viable offspring. With the offspring we have now begun to address the function of vascular and blood cell–associated EPCR. Vascular EPCR is critical to the regulation of coagulation and an appropriate host response to bacteria. In contrast, blood cell–associated (hematopoetic) EPCR increases lethality, in part by modulating the inflammatory response and altering the clearance of the bacteria. Our current work is directed at understanding which processes are governed by the vascular and hematopoetic EPCR. For example, since EPCR is found at high levels on hematopoetic stem cells and decreases as the cells develop lineage specificity, we wished to determine if it plays an important role in stem cell functions. Using the above system, we found, surprisingly, that EPCR is not important for stem cell function.
We have recently shown that, in addition to its role in controlling blood coagulation and cytokine production in response to inflammation, the protein C pathway plays an important role in limiting complement activation at the blood-vessel interface. Several components of the pathway are down-regulated in a variety of disease processes, including diabetes and atherosclerosis; up-regulation of the pathway can ameliorate disease progression. We are investigating orally active agents that may up-regulate the pathway as a means to limit disease progression.
Bleeding is a significant concern in trauma, a major killer of people younger than 55. Some of the bleeding complications are due to excessive protein C activation. Blocking protein C completely halts the bleeding, but leads to death. APC not only limits blood coagulation but also has cytoprotective effects on the vasculature, improves endothelial barrier function, and decreases inflammation. We have identified monoclonal antibodies that block the anticoagulant activity of APC but allow retention of the above properties in animal models of trauma. These protective antibodies may prove useful in clinical settings.
Recently we have found that histones released from damaged cells or activated neutrophils elicit damage to the blood vessel wall, eliciting severe inflammatory responses, thrombosis, and bleeding into the tissues that ultimately leads to multiorgan failure and death. Blocking this activity of extracellular histones can protect against inflammatory disease progression.
These studies should improve our understanding of the blood-clotting process in health and disease.
Some aspects of this work were supported by grants from the National Institutes of Health and the Leducq Foundation.
As of May 30, 2012