The goal of this proposal remains to test a novel and unique inhibitor of plasmin that has the potential to reduce severe bleeding in major surgery as well as in coagulopathy. Until recently, another inhibitor of plasmin, called aprotinin was used as an antifibrinolytic agent to reduce blood loss in cardiac, organ transplant, orthopedic and vascular surgery. In fact, aprotinin was the only FDA-approved drug to reduce blood transfusion in cardiac surgery with cardiopulmonary bypass. Three meta-analyses of aprotinin reported in 1999 indicated that aprotinin decreased mortality (by two fold), the incidence of surgical reexploration, as well as blood transfusions. However, in a recent multi-institutional study, aprotinin was reported to significantly increase the risk of renal, cardiovascular and neurological injury, based on its ability to inhibit kallikrein and therefore kinin formation, leading to disturbed autoregulation of organ blood flow. Using crystallography, molecular modeling and biochemical approaches, we generated two variants of the first Kunitz domain (KD1) of human tissue factor pathway inhibitor-2 (TFPI-2) that are significantly more potent inhibitors of plasmin as compared to the wild-type KD1 or to the full-length TFPI-2. Preliminary data revealed the unique ability of KD1 variants to strongly inhibit human and mouse plasmin with minimal or no inhibition of kallikrein. Based upon this fundamental property of the KD1 variants, which provides a distinct advantage over aprotinin, we hypothesize that KD1 variants will be effective antifibrinolytic agents without causing significant organ injury related to the inhibition of kallikreins. The proposed studies are designed to determine the efficacy and toxicity of KD1 variants using two specific aims.
In specific aim 1, we will express wild type KD1 and the two variants (one modified at P2'residue and the second at P1 and P2'residues) in E. coli, further characterize their binding to plasmin, tissue and plasma kallikreins and other proteases involved in the coagulation cascade. Further, we will obtain crystal structures to validate the molecular interactions between the KD1 variants and plasmin.
In specific aim 2, we will compare the efficacy of the KD1 variants to aprotinin in reducing bleeding from large and small vessels in animal models. Further, we will study the toxicity of the KD1 variants to the vital organs in the animal models and compare it to aprotinin. We anticipate that KD1 variants will reduce total blood loss without having an adverse effect on organ function. The long term goals of this proposal are to study the efficacy and toxicity of KD1 variants in animal models of cardiac surgery with cardiopulmonary bypass.
In patients with open-heart surgery and organ transplant, there is significant blood loss due to breakdown of fibrin by plasmin. Aprotinin, an antifibrinolytic drug that has been used to inhibit plasmin was discontinued in 2007 as it was found to cause significant organ injury. Here, we are proposing to test a new drug that our laboratory has designed to reduce bleeding in high-risk cardiac surgery and transplant patients.
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| Vadivel, Kanagasabai; Ponnuraj, Sathya-Moorthy; Kumar, Yogesh et al. (2014) Platelets contain tissue factor pathway inhibitor-2 derived from megakaryocytes and inhibits fibrinolysis. J Biol Chem 289:31647-61 |
| Vadivel, Kanagasabai; Agah, Sayeh; Messer, Amanda S et al. (2013) Structural and functional studies of ?-carboxyglutamic acid domains of factor VIIa and activated Protein C: role of magnesium at physiological calcium. J Mol Biol 425:1961-1981 |
| Vadivel, Kanagasabai; Bajaj, S Paul (2012) Structural biology of factor VIIa/tissue factor initiated coagulation. Front Biosci (Landmark Ed) 17:2476-94 |
| Bajaj, Madhu S; Ogueli, Godwin I; Kumar, Yogesh et al. (2011) Engineering kunitz domain 1 (KD1) of human tissue factor pathway inhibitor-2 to selectively inhibit fibrinolysis: properties of KD1-L17R variant. J Biol Chem 286:4329-40 |
