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

肝素(H)是需要体外循环(CPB)的介入和外科手术(如心脏手术)的首选抗凝剂。鱼精蛋白(PRT)是CPB术后拮抗肝素的标准药物。最近，我们观察到接受体外循环的患者会产生针对PRT/H复合体的抗体(Abs)。此外，我们发现，术前抗PRT/H abs检测呈阳性的患者表现出他们的血小板(PLT)计数恢复延迟，术后早期动脉闭塞的风险增加。我们证明，起始事件是免疫球蛋白抗体与PRT/H复合体结合，导致免疫复合体(PRT-H-IgG)能够通过其FcgRIIa激活PLT。在拟议的项目中，将调查以下目标：1)评估抗PRT/H abs在接受CPB的患者中的确切临床影响2)评估与PLT结合的PRT的半衰期3)评估潜在的治疗药物以防止免疫患者的不良反应。评估抗PRT/H abs与体外循环后早期血栓并发症的相关性将从一项多中心研究中调查1000名患者中是否存在抗PRT/H abs。在先前的研究中，我们观察到抗PRT/H抗体检测呈阳性的患者术后PLT计数下降更为明显。PRT的血浆半衰期不到5分钟。因此，目前尚不清楚在最后一剂PRT给药几天后，抗PRT/H abs如何介导血小板减少症。一种可能的情况是，PRT与PLT结合，并在PLT表面暴露数天。为了研究这一假设，将PLT与PRT或PRT/H复合体孵育，并用抗PRT抗体用流式细胞术和免疫印迹法评估PRT的结合。近年来，有研究表明，脱硫肝素(ODSH)可破坏肝素与PF4的复合物，抑制抗PF4/H抗体结合和PLT活化。这表明ODSH可能与PRT表现出类似的相互作用。我们将研究ODSH的特性，包括a)阻止/逆转PRT的结构改变的能力，b)体外抑制这些抗体结合抗PRT/H抗体和激活PLT的能力，以及c)在NOD/SCID小鼠模型中ODSH防止抗PRT/H抗体介导的PLT破坏的能力。另一种预防PRT引起的不良反应的潜在方法是使用低分子PRT(LMW-PRT)。LMW-PRT完全维持了PRT的肝素中和能力。然而，截短的蛋白很可能不表达抗PRT/H抗体结合的新表位。我们打算研究不同变体的LMW-PRT/H复合体对肝素的抗凝作用以及抗PRT/H抗体的交叉反应性。此外，在NOD/SCID小鼠模型中，将分析在抗PRT/H抗体存在的情况下，LMW-PRT对循环中人PLT存活的影响。

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.