Anti-protamine/heparin antibodies: further studies on the pathogenesis and preclinical development of approaches to prevent adverse effects in immunized patients

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Heparin (H) is the anticoagulant of choice for interventional and surgical procedures requiring cardiopulmonary bypass (CPB) such as cardiac surgery. Protamine (PRT) is the standard drug to antagonize heparin after CPB. Recently, we observed that patients undergoing CPB develop antibodies (abs) against PRT/H complexes. Moreover, we found that patients testing positive for anti-PRT/H abs before surgery show delayed recovery of their platelet (PLT) counts and an increased risk for early post-surgery arterial occlusions. We demonstrated that the initiating event is the binding of IgG abs to PRT/H complexes; resulting in immune complexes (PRT-H-IgG) being able to activate PLTs via their FcgRIIa. In the proposed project, the following aims will be investigated:1) assessment of the definite clinical impact of anti-PRT/H abs in patients undergoing CPB2) assessment of the half-life of PRT bound to PLTs3) evaluation of potential therapeutic agents to prevent adverse effects in immunized patients.To assess the association of anti-PRT/H abs with early thrombotic complications after CPB 1,000 patients from a multicenter study will be investigated for the presence of anti-PRT/H abs. In the previous study, we observed a more pronounced post-surgery decrease in PLT counts in patients who tested positive for anti-PRT/H abs. PRT has a plasma half-life of less than 5 minutes. It is therefore unclear, how anti-PRT/H abs could mediate thrombocytopenia several days after the last dose of PRT has been given. One possible scenario is that PRT binds to PLTs and is exposed on the PLT surface for several days. To investigate this hypothesis PLTs will be incubated with PRT or PRT/H complexes and the binding of PRT will be assessed by an anti-PRT antibody using flow cytometry and immunoblotting. Recently, it has been shown that desulfated heparin (ODSH) disrupts complexes of heparin with PF4 and inhibit anti-PF4/H antibody binding and PLT activation. This suggests that ODSH may show similar interactions with PRT. We will investigate the characteristics of ODSH, including a) the capability to prevent/reverse structural modifications in PRT, b) to inhibit the binding of anti-PRT/H IgG abs and PLT activation by these abs in-vitro, and c) the capability of ODSH to prevent anti-PRT/H antibody mediated PLT destruction in the NOD/SCID mouse model.Another potential approach to prevent PRT-induced adverse reactions is the use of low molecular weight PRT (LMW-PRT). LMW-PRT was shown to fully maintain the heparin neutralization capability of PRT. It is, however, likely that a truncated protein does not express the neoepitope to which anti-PRT/H abs bind. We intend to investigate the heparin antagonization effect as well as the cross-reactivity of anti-PRT/H abs with different variants of LMW-PRT/H complexes. In addition, the impact of LMW-PRT on the survival of circulating human PLTs in the presence of anti-PRT/H abs will be analyzed in the NOD/SCID mouse model.