Effect of covalent antithrombin-heparin on activated protein C inactivation by protein C inhibitor
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Protein C in its activated form (APC) limits thrombin generation. Protein C inhibitor (PCI) readily neutralizes APC. Heparin accelerates this reaction, which may complicate anticoagulant treatment in patients with varying APC generation potential. A potent anticoagulant conjugate of antithrombin and heparin (ATH) was prepared, and its effect on APC+PCI reactions was tested. Second order rate constants for APC+PCI reactions were measured by discontinuous rate experiments in the presence of heparin or ATH. Similarly, low molecular weight fractions of heparin (LMWH) and ATH (LMWATH) were tested, as was high molecular weight ATH (HMWATH). Mechanisms of heparin or ATH binding to APC or PCI were assessed using electrophoresis. While heparin gave a higher maximal APC inhibition rate compared to ATH, peak inhibition rate was achieved at comparatively lower ATH concentrations. Since LMWH was ineffective at enhancing APC inhibition by PCI, unfractionated heparin likely acts by bridging APC and PCI. Unlike heparin, ATH may conformationally activate either APC or PCI since LMWATH significantly catalyses APC inhibition. Binding studies showed that ATH readily associates with APC. Thus, although a small fraction of ATH efficiently catalyses APC inhibition by PCI, complete ATH preparations induce a decreased maximal rate of APC-PCI formation compared to unfractionated heparin.
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