Biological Significance of Protatmine/Heparin Antibodies

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DESCRIPTION (provided by applicant): Biological Significance of Protamine/Heparin Antibodies Protamine (PRT) is currently the only FDA approved therapy for reversing heparin (H) anticoagulation. Several adverse reactions have been described with protamine use including anaphylaxis, cardiovascular collapse, and death. Populations at risk for developing a protamine reaction include: patients receiving protamine-containing insulin preparations (such as Neutral Protamine Hagedorn, NPH insulin), patients with fish allergies, and vasectomized men. My mentor's laboratory has shown that PRT interacts with heparin to form immunogenic multi- molecular complexes in a murine model. Based on these findings, I undertook studies during my research fellowship (starting July 2011) to demonstrate antibodies to PRT/H complexes in humans. To do so, I investigated 500 patients from a recent multicenter, prospective study investigating the incidence of platelet factor 4 (PF4)/H antibodies after cardiac surgery (HIT 5801). With IRB approval, I determined that PRT/H antibodies occur commonly after cardiac surgery (29% incidence 30 days post-CPB) and share several essential properties with HIT antibodies including: heparin-dependence, antigen-specificity, and platelet activation. Based on these observations, I hypothesize that PRT/H antibodies are pathogenic in the presence of circulating antigen (cell bound PRT or PRT/H complexes). I will test this hypothesis in the following two Aims: Aim 1: Demonstrate the cellular pathogenicity of PRT/H antibodies. In preliminary studies, I demonstrate that PRT/H antibodies activate monocytes to express procoagulant activity and antibodies cross-react with PRT in NPH insulin. In this aim, I will test the hypothesis that PRT/H antibodies exert diverse biological effects through antigen and antibody-mediated effects. Specifically, I will examine the functional effects of PRT/H antibodies on glucose uptake, insulin receptor activation and cellular procoagulant activity. Additionally, I will investigate the cross-reactivity of PRT/H antibodies with other protamine like substances (histones). Aim 2: Generate murine monoclonal antibodies to PRT/H. Due to limited availability of human specimens, I will generate a murine monoclonal PRT/H antibody. This monoclonal antibody will be an important tool for future in vitro and in vivo studies. If successful, these studies will inform future investigations on the clinical impact and pathogenic potential of PRT/H antibodies in susceptible patient populations requiring protamine exposure, such as patients with insulin dependent diabetes, peripheral arterial disease or repeat cardiac surgery.