Project Summary/Abstract
Tissue factor (TF), the primary initiator of the blood coagulation cascade, is carefully regulated to prevent
aberrant coagulation activation. However, pathological conditions including bacterial and viral infection induce
intravascular TF expression and lead to thrombosis. As TF-induced thrombosis is a major cause of acute
myocardial infarction, ischemic stroke, and pulmonary embolism, improved understanding of the mechanisms of
pathological TF activation may lead to new therapeutic targets. Pyroptosis, a form of inflammatory cell death,
drives TF-mediated intravascular coagulation activation in bacterial sepsis. Emerging studies also implicate
inflammasome activation in SARS-CoV-2 infection, but its role in TF activation is unknown. Our preliminary
studies demonstrate that SARS-CoV-2 and specifically its accessory protein ORF3A induce TF activation in a
phosphatidylserine (PS)-dependent mechanism that requires TMEM16F, similar to PS-dependent TF activation
in pyroptosis. In Aim 1, we will investigate whether ORF3a-induced TF activation is driven by inflammasome-
mediated pyroptosis. Lipid peroxidation and its highly reactive end products such as 4-hydroxy-2-nonenal (HNE)
are involved in various forms of programmed cell death including pyroptosis. However, the role of HNE, the most
stable and toxic reactive aldehyde produced during lipid peroxidation, in pyroptosis-associated TF and
coagulation activation is not known. Our preliminary data showed that HNE induces PS-dependent TF activation
in LPS-primed macrophages and causes intravascular coagulation activation in mice. However, the complete
mechanism by which HNE induces PS externalization and TF activation is not known. In Aim 2, we will use
chemical genetic approaches to dissect the mechanism of HNE-induced TF activation via pyroptosis in vitro and
in vivo. Although lipid peroxidation plays a central role in cell death and coagulation activation in bacterial sepsis,
a therapeutically targetable enzyme responsible for the unbridled lipid peroxidation and generation of
pathological levels of reactive radicals such as HNE is not known. In Aim 3, we will use genetically modified mice
deficient in lipid peroxidation and HNE formation to investigate TF-dependent pathologic coagulation activation
and thrombosis during sepsis. A successful completion of these studies will help delineate a common pathway
involved in pathologic TF activation across varied pathogenic infections and will also help identify a specific
therapeutically targetable enzyme to attenuate TF activation in disease.