Our Division has a rich history of pioneering research in the science of thrombosis and hemostatis
Platelet granule exocytosis – As recently as the late 1990s, the mechanisms by which platelets release their granule contents was a black box. It was understood that platelet agonists stimulate release of bioactive compounds and proteins, and that this ‘platelet release reaction’ was important in the pathophysiology of myocardial infarction and stroke. Yet the machinery responsible for membrane fusion events required for granule exocytosis had not been described. The Flaumenhaft laboratory was the first to demonstrate a functional role for SNARE proteins in platelet granule exocytosis. We and other laboratories subsequently identified the SNARE isoforms required for platelet secretion. We described the signaling cascades that mediate granule secretion and determined how the exocytotic machinery interacts with the cytoskeleton in order coordinate cargo release with the dramatic morphological transformation that occurs during platelet activation. We have worked with mice deficient in specific SNARE isoforms to dissect the role of platelet granule release in thrombus formation, demonstrating roles for VAMP-8 and VAMP-7 in platelet function during thrombus formation.
Thrombosis is a leading cause of morbidity and mortality in cancer patients. While the clinical association between cancer and thrombosis was established over 100 years ago, the underlying mechanisms and corresponding biomarkers are poorly understood. Work in the Division has focused on defining novel biomarkers contributing to thrombosis in cancer patients and optimizing the administration of anticoagulants in high-risk populations through targeting new thrombotic mechanisms.
Coagulation in Sepsis
Sepsis affects more than 750,000 Americans yearly, and mortality is estimated between 17-50%. Aberrant coagulation occurs throughout the circulation in sepsis and is characterized by both spontaneous bleeding and clotting, with fibrin formation in the vasculature of vital organs. Known as disseminated intravascular coagulation (DIC), this phenomenon is associated with increased mortality in sepsis, but is poorly understood and difficult to treat. We have found that the endothelium serves an essential and early role in mediating clot formation in the setting of sepsis. This insight has led to the identification of endothelial-derived proteins as biomarkers to facilitate early identification of DIC in human sepsis (JCI, 2018). Strikingly, we have also found that targeting the endothelial Tie2 system can reverse the prothrombotic transformation of endothelium that occurs with sepsis and reduce fibrin deposition in vital organs in mice.
A separate project within the Division is aimed at studying the functional genomics of purpura fulminans (PF), a rare extreme phenotype of sepsis characterized by a highly thrombotic subtype of DIC. We have found that rare defects in genes of the complement system are associated with PF and a maladaptive proinflammatory phenotype. Ongoing studies in the Division will explore the genetic underpinnings of DIC and the molecular mechanisms behind the prothrombotic transformation of the endothelium in systemic inflammation. Our goal is to develop novel strategies for targeting the endothelium, clotting system, and innate immunity to prevent the coagulopathy of sepsis.
Video from an intravital microscope showing platelet aggregation (red) and fibrin deposition (red) after laser injury to a cremaster arteriole of a septic mouse
The Contact Pathway in Thrombosis and Hemostasis
Current treatments for thrombotic diseases such as myocardial infarction and stroke remain limited because available anticoagulant medications carry an inherent risk of major hemorrhage. A therapy that could prevent intravascular thrombosis while maintaining physiologic hemostasis would thus be a vital breakthrough for millions of patients worldwide. Recent studies of the contact pathway of coagulation, comprised of the coagulation factors XII (FXII), XI (FXI), kallikrein, and kininogen, have provided new and exciting insights as to how such a drug may be developed. Strikingly, mice lacking FXII are protected from thrombosis even though severe congenital FXII deficiency in humans does not cause bleeding. Consistent with this observation, the Bendapudi lab has shown that a novel therapeutic antibody targeting FXIIa prevents arterial thrombus formation in mice without impairing hemostasis, a proof of concept that it may be possible to “decouple” antithrombotic efficacy from risk of bleeding. Dr. Bendapudi currently studies the mechanisms of FXII recruitment, activation, and propagation at sites of arterial thrombosis with a view towards the development of novel therapeutics and understanding the key differences between thrombosis and hemostasis.
Thrombus formation in vivo– The study of thrombus formation is complicated by the fact that thrombi form rapidly, are highly variable, and do not respond in a predictable manner to injury. Quantitation of thrombus formation in live animals is therefore challenging. We have worked with murine models to image and measure thrombus formation and hemorrhage in live animals. These approaches have employed high-speed, intravital microscopy to assess the platelet accumulation, fibrin formation, and multiple other elements of thrombus formation (e.g., tissue factor, calcium flux, phosphatidylserine exposure, nitric oxide, etc.) in real-time during thrombus formation. We use this technology to assess thrombus formation in disease states such as sepsis, antiphospholipid syndrome,and sickle cell disease. Experimental interventions to reduce thrombus formation in these disease models are tested.
A real-time 4-D view of a mouse cremaster arteriole after laser injury
Protein Disulfide Isomerase (PDI) in thrombus formation
Although much has been learned about the molecular interactions of the coagulation proteins and signaling pathways in platelets, the events that initiate thrombus formation in vivo remain poorly characterized. Following the discovery in our division that thiol isomerases serve a critical function in the initiation of thrombus formation in vivo, we performed high throughput screening to identify inhibitors of PDI that could be used in our in vivo assays. This screen identified glycosylated quercetin flavonoids as inhibitors of PDI, but not other thiol isomerases. Based on these studies, we performed a much larger screen and identified several other PDI inhibitors. We are currently using these inhibitors to identify characteristics of PDI inhibitors that are optimal for inhibiting thrombus formation. Our evaluation of the molecular pharmacology of these small molecules has lead us to the discovery that S-nitrosylation is an important post-translational modification for controlling PDI activity and that the nitrosylase and denitrosylase activities of PDI serve an essential role its prothrombotic activities. In collaboration with other members of the Division, we are currently testing one of our PDI inhibitors, isoquercetin, in phase II/III clinical trials.
Diagnosis and Management of Thrombotic Microangiopathies (TMA)
Thrombotic microangiopathies are a group of rare but life-threatening hematologic disorders that involve the formation of platelet-rich thrombi in the microvasculature, leading to end organ damage and death. Included under the umbrella of TMA are conditions like thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), and disseminated intravascular coagulation, as well as transplant- and drug-associated TMA. Dr. Bendapudi serves as co-principal investigator of the Harvard TMA Research Collaborative, an interdisciplinary, multi-institutional project that maintains a large registry of patients with TMA. The consortium has produced a number of novel insights, including development of the PLASMIC clinical prediction tool to rapidly identify patients who are likely to have TTP (http://harvardtma.partners.org/plasmic). Dr. Bendapudi currently oversees several ongoing projects in this area, including the creation of a specimen bank to aid in future translational research studies
Genomic and Proteomic Studies of Hemostatic and Thrombotic Disorders
Large multiomic datasets increasingly represent an exciting and productive area of translational research. Members of the Division are currently working in collaboration with the Broad Institute of MIT and Harvard to leverage the UK Biobank and NIH All of Us biorepository programs to address fundamental questions in hemostasis and thrombosis. Projects in this area include studying the clinical impacts of rare genetic variation in HSP47 (SERPINH1), factor XII (F12), and protein Z (PROZ), as well as the first large-scale, biorepository-based analysis of double heterozygosity for the prothrombin gene mutation G20210A and factor V Leiden alleles.