Activation of encrypted tissue factor during thrombus formation in vivo

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As a mechanism of host defense, the initiation of the blood coagulation cascade requires tight regulation both in its timing and location. Major causes of death, including stroke, heart attack and pulmonary embolism, are each linked to pathologic thrombosis. Current teaching of coagulation has evolved from in vitro studies in which isolated components of this complex system are studied individually. My long-term goal is to elucidate the mechanism initiating coagulation in vivo and to determine how this critical step is regulated. Recently developed imaging technologies allow for the study of protein interactions at the site of thrombus formation in a living animal in real time. Tissue factor (TF) is required to initiate bloodcoagulation, thrombin formation and fibrin generation. It has been dogma that tissue factor is shielded from the circulation and is only exposed by vascular injury. However, TF has been identified on circulating microparticles, as well as on leukocytes and endothelial cells. Recent studies have led to the novel proposal that oxidation of TF leads to disulfide bond formation, acting as a molecular switch that changes latent TF to a procoagulant form. We hypothesize that an encrypted form of TF exists within the circulation that is activated during thrombus formation and fibrin generation in vivo. This would be a novel mechanism of TF involvement in thrombosis. We base this hypothesis on the following observations: 1) in vivo evidence of circulating TF accumulating in a growing thrombus, 2) a discrepancy between circulating TF concentration as measured by antigen and function - circulating antigen far exceeds the amount required to initiate coagulation and 3) disruption of the Cys186-Cys209 disulfide bond in tissue factor decreases its procoagulant activity, and this is reversible by forming this same disulfide bond. Based on these observations, we will characterize active and encrypted tissue factor isoforms in vivo using intravital microscopy and isoform-specific antibodies. We will characterize known antibodies for isoform specificity and have developed novel antibodies using recombinant phage display technology. These antibodies will allow visualization of TF isoforms during laser-induced thrombus formation in a living mouse. Furthermore, mouse models deficient in TF in individual tissues of interest, including monocytes and endothelial cells, will be employed to investigate the role of these tissues in TF-mediated thrombus formation in vivo.