| Antithrombin III (ATIII) is an essential endogenous anticoagulant. Its anticoagulant activity is increased by ∼1000x after ATIII binds to and is activated by a specific pentasaccharide component present in vascular wall heparan sulfate proteoglycans (HSPGs) and pharmaceutical heparin. There are two ATIII isoforms in human plasma, alpha-ATIII and beta-ATIII, which have different heparin binding affinities and diffusion coefficients. Why two ATIII isoforms are required in most vertebrates and which factors affect the partitioning of two isoforms between the flowing blood and vessel surface is not fully understood.;An in vitro heparin surface-containing flow circuit, which can simultaneously simulate venous, arterial and pathological flow conditions, was built to solve the questions above. The results showed that the loading of alpha-ATIII onto a heparin-coated surface is reaction-controlled, while beta-ATIII surface-loading is diffusion-contributive at physiological wall shear rates (WSRs), but becomes reaction-controlled at pathological WSRs. A recombinant ATIII with enhanced heparin binding affinity and diffusion coefficient, DES.N135A ATIII, loaded onto heparin-coated surfaces and inhibited flowing thrombin more efficiently than plasma ATIIIs, especially at arterial and pathological flow conditions.;Based on the above findings, the following hypothesis was made that a genetically engineered ATIII, r.503 ATIII, with increased mass transport properties like DES.N135A ATIII and resistance to elastase cleavage, will be a more effective antithrombotic than endogenous ATIII, especially for treatment of arterial thrombosis. A rabbit carotid artery thrombosis model induced by ferric chloride was modified to compare the antithrombotic effect of r.503 ATIII with that of human plasma-derived ATIII infused from the left carotid artery (proximal infusion) or from the right femoral vein (distal infusion). The results showed that proximally infused r.503 significantly prolonged the occlusion time of the injured carotid artery compared to the saline infusion group, whereas distally infused r.503 did not. Proximally infused r.503 inhibited thrombosis through prolonging the thrombosis plateau phase, and the antithrombotic effect was dose-dependent.;Thus, I conclude that both exterior flow conditions and intrinsic mass transport properties (heparin binding affinity and diffusion coefficient) determine the evolutionary conservation of two ATIII isoforms and govern the anticoagulant distribution between flowing blood and vessel surface. Recombinant r.503 ATIII with enhanced mass transport properties and elastase resistance is an injured arterial wall-targeted antithrombotic. |
