IDENTIFYING NOVEL HEMOSTATIC PROTEINS

Duke University, Durham NC

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Abstract

Arterial thrombosis is associated with a large array of human pathology such as acute myocardial infarction and stroke. It is clear that most thrombotic events require a dynamic interplay between plasma phase blood coagulation proteins, circulating platelets, and local vascular endothelial cells. Detailed information has been developed regarding the regulation of fluid phase blood coagulation proteins and some associated platelet interactions. In spite of this information, it is clear that the regulation of arterial thrombosis in specific vascular beds requires a more detailed understanding of vascular specific interactions with plasma phase proteins and cellular elements. To this end we have developed a two-tiered collaborative research program between the laboratories of Dr. Sullenger and Dr. Lawson. The goal of the first tier is the translational development and evaluation in relevant pre-clinical models of novel RNA-based anticoagulant and antithrombotic compounds (aptamers) to test the efficacy versus bleeding risk associated with inhibitors that target different porcine procoagulant factors in the blood coagulation pathway. The goal of the second tier is the identification of novel plasma phase, platelet and vascular bed specific proteins and their genes by using RNA- based combinatorial chemistry methods to isolate specific ligands to these previously uncharacterized proteins, and to start to unravel the function of these proteins by employing these RNA ligands in functional studies. The successful completion of this collaborative research program will require the joint expertise and personnel of Dr. Sullenger's laboratory in RNA-based combinatorial chemistry and of Dr. Lawson's laboratory in conventional protein biochemistry, blood coagulation assays and animal models of arterial thrombosis. Thus, our specific aims are: l. To isolate high affinity nuclease-resistant RNA aptamers that bind to and inhibit the function of porcine clotting factors Va, VIIa, VIIIa, IXa, Xa and tissue factor, and test these compounds in relevant pre-clinical models of bleeding and anticoagulation. 2. To identify yet unknown regulators of thrombosis in the plasma, on the surface of platelets and on endothelial cells of diseased and vascular bed specific vessel segments by employing multi-target subtractive in vitro selection techniques to isolate ligands capable of specific and high affinity binding to these previously uncharacterized proteins. 3. To assess the anti- or pro-thrombotic effects of individual or combinations of lead RNA ligands and their target proteins in bioassays, including in vitro biochemical assays, plasma based assays and in vivo swine models of bleeding, anticoagulation and arterial injury.