The intersection of the availability of genetically altered mice, new technologies for intravital microscopy and high speed computing systems has led to the use of animal models to build on the concepts that have emerged from in vitro studies of the molecular and cellular biology of hemostasis and thrombosis. In an effort to improve the understanding of the etiology and pathogenesis of thrombosis, thrombus formation has been initiated in experimental systems via mechanical disruption, laser-induced, photochemical-induced and ferric chloride-induced injury to the vessel wall, among others. None of these methods are physiologic, and as such, remain models that require extrapolation from a living animal – a mouse – to human biology. We have focused on laser-induced injury of the arteriole vessel wall in the cremasteric muscle of the mouse. Using high speed digital imaging of fluorescently labeled components and real time intravital microscopy, our group has been able to demonstrate that platelet accumulation and fibrin generation during thrombus formation occur simultaneously, that tissue factor and collagen are independent initiators of platelet activation, and that monocyte-derived microparticles deliver tissue factor to the site of thrombus development. Perhaps the most important and unanticipated observation has been that thiol isomerases, thought only to be involved in protein biosynthesis via the formation of disulfide bonds in the endoplasmic reticulum, play a critical role in thrombus formation. Protein disulfide isomerase (PDI), ERp5 and ERp57 are among the vascular thiol isomerases that are known to be important for the initiation of thrombus formation. Laser-induced thrombosis in mice is associated with PDI, ERp5 and ERp57 secretion by platelets and endothelium. Inhibition of these thiol isomerases blocks platelet thrombus formation and fibrin generation. The integrins αIIbβ3 and αVβ3 play a key role in this process, binding directly with the secreted thiol isomerases and capturing them in the vicinity of vessel wall injury. These enzymes are required for the initiation of platelet thrombus formation and fibrin generation, but the mechanism by which they function remains to be elucidated. At present, it would appear that there is an electron transfer pathway involving these enzymes that regulates the initiation of thrombus formation. The mechanism by which PDI participates in thrombus generation is being evaluated by using trapping mutant forms to identify substrates of PDI that participate in the network pathways linking thiol isomerases, platelet receptor activation and fibrin generation. Several proteins, including vitronectin, thrombospondin and Factor V, have been identified as forming covalent disulfide intermediates with PDI. We are currently exploring PDI as an antithrombotic target using isoquercetin and quercetin 3-rutinoside, inhibitors of PDI identified by high throughput screening. Tail bleeding times are equivalent for mice treated with quercetin-3-rutinoside and isoquercetin compared to untreated mice. In an in vivo mouse pulmonary embolism model, PDI inhibitors rescue a high percentage of mice from death. The b-domain of PDI binds to quercetin-3-rutinoside, and infusion of the isolated b’ domain into a mouse treated with quercetin-3-rutinoside restores thrombus formation. This suggests a method for reversal of bleeding if these PDI inhibitors are found to be complicated by bleeding. The antithrombotic properties of quercetin and isoquercetin in humans have been tested. A pharmacokinetic study with quercetin and isoquercetin determined optimal oral delivery with isoquercetin. The effectiveness of these PDI inhibitors in human studies is being evaluated in a clinical trial evaluating prophylaxis of thromboembolic events in patients with cancer-associated thrombosis. PDI is a novel target for antithrombotic therapy and is unique in that its inhibition simultaneously blocks platelet thrombus formation and fibrin generation.
Disclosures
Zwicker: Quercegen Pharma: Research Funding.
Endothelium-derived but not platelet-derived protein disulfide isomerase is required for thrombus formation in vivo