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A study of the specificity and effectiveness of HD1, a thrombin-directed DNA aptamer, as an inhibitor of coagulation / Elucidating the anticoagulant properties of the DNA aptamer HD1Kretz, Colin A. 09 1900 (has links)
<p>The coagulation system is initiated when subendothelial tissue factor is exposed to circulating blood, and culminates in the production in the enzyme thrombin, which catalyzes clot formation. However, the failure of built-in controls to limit thrombin generation to sites of vascular damage, leads to pathological clot formation, termed thrombosis. Anticoagulants, such as heparin and warfarin, are used to prevent thrombosis; however, these can cause bleeding. As a result, there is a need for novel anticoagulants that limit clot formation without the risk of bleeding. DNA aptamers are small oligonucleotides designed to bind a specific target with high affinity. HD1 is a DNA aptamer that was developed by Griffin and Leung in 1993 to bind thrombin. The focus of this work was to better characterize the anticoagulant properties of HD1.</p><p>We demonstrate that HD1 binds a subdomain of exosite 1 that is fully developed in prothrombin, unlike other exosite 1-directed ligands, such as Hir⁵⁴⁻⁶⁵(SO₃⁻) and fibrin, which do not bind prothrombin. As a result, HD1 binds prothrombin with high affinity and inhibits prothrombin activation by prothrombinase with an IC₅₀ value of 115 nM. HD1 competes with tV a for binding to prothrombin. Based on its capacity to inhibit both thrombin and prothrombin, HD1 is more potent than Hir⁵⁴⁻⁶⁵(SO₃⁻) at inhibiting standard plasma clotting assays, but not in the thrombin clotting time. Furthermore, HDl inhibits thrombin generation by prolonging the lag time, reducing peak thrombin, and reducing the ETP to a greater extent than Hir⁵⁴⁻⁶⁵(SO₃⁻). Based on thrombin generation assays conducted in cofactor-deficient plasma, we demonstrate that HD 1 inhibits thrombin feedback activation of fV, but not fVIII, and that inhibition of prothrombinase accounts for the reduction in both peak thrombin and prolongation of the lag time. HDI is neutralized in buffer-based and plasma-based coagulation assays by an antisense oligonucleotide, antiHD1. AntiHD1 preferentially targets free HD1 because HD1 bound to either thrombin or prothrombin is protected from neutralization by antiHD1. Based on our data, HD1 may be a useful anticoagulant due to its capacity to inhibit both prothrombin and thrombin. Positioned at the junction of the intrinsic and extrinsic pathways prothrombin could be a useful target for aptamer development into anticoagulant applications.</p> / Thesis / Doctor of Philosophy (PhD)
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