In Vivo Regulation of Factor IXa by Protein S in Hemophilia and Systemic Hypercoagulability

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Protein S (PS) is a critically important natural anticoagulant as demonstrated by fatal consumptive coagulopathies in homozygous deficiency for mouse and man. Originally discovered as a cofactor for activated protein C (APC), subsequent studies have demonstrated a plethora of APC-independent anticoagulant mechanisms for PS without clear demonstration of their respective physiologic relevance. Recently, genetic “rebalancing” studies in the mouse and direct biochemical evidence suggest an important interaction between PS and factor IXa (FIXa) The objective of this proposal is to define the role of PS in the in vivo regulation of FIXa activity. The central hypothesis is that PS is an important physiologic regulator of FIXa in both hemostasis and systemic hypercoagulable states. The central hypothesis will be tested by pursuing three specific aims: 1) Identify the predominant mechanism(s) for regulation of FIXa activity in human plasma, 2) Determine the contribution of protein S affinity to the in vivo activity of recombinant FIX(a), and 3) Determine the contribution of protein S deficiency to in vivo regulation of FIX(a) activity. To pursue these aims, we have developed a panel of recombinant human FIX variants that possess reduced affinity for antithrombin (AT), heparin and PS. In the first aim, inhibition of FIXa activity in the intrinsic Xase complex by PS, PS-TFPI and PS-APC will be examined using purified components and the FIXa variants. Likewise, the contribution of AT- and PS-dependent mechanisms to the regulation of plasma thrombin generation will be examined with the FIXa variants and inhibitory antibodies in immunodepleted plasma. In the second aim, the human FIXa variants will first be employed to evaluate the impact of AT, heparin and PS affinity on zymogen and protease recovery and clearance. Secondly, dose-dependent in vivo hemostatic and thrombotic activity will be examined using established models in the hemophilia B mouse to evaluate the contribution of AT and PS-dependent mechanisms. In the third aim, Pros1 specific antisense oligonucleotides (ASO) will be employed to create PS deficiency in wild type and hemophilia B mice. The impact of the ASO-mediated PS deficiency on bleeding and thrombosis phenotypes will be evaluated using established models, including the ability to ameliorate the baseline bleeding phenotype in hemophilia B mice. Further, the effect of ASO-mediated PS deficiency on the in vivo hemostatic and thrombotic activity of injected FIXa variants will be examined in hemophilia B mice, including the ability of human PS to “rescue” the effects of PS deficiency. Completion of these aims will elucidate the critical role of the PS-FIXa interaction in the physiologic regulation of FIXa procoagulant activity within the intrinsic Xase complex. The proposed research is innovative because it addresses the in vivo significance of a novel mechanism for regulating the rate-limiting step in the coagulation response. This contribution will be significant because it will provide a conceptual basis for manipulating the FIXa-PS interaction in hemophilia and systemic hypercoagulable states associated with elevated levels of plasma FIXa activity.