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

抗鱼精蛋白/肝素抗体：对免疫患者的发病机制和预防不良反应的临床前开发方法的进一步研究

• 批准号: 253407448
• 负责人: Professor Dr. Tamam Bakchoul
• 金额: --
• 依托单位: Institut für Klinische Experimentelle Transfusionsmedizin
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/253407448?language=en
• 关键词: Anti; protamine; heparin; antibodies; further

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.

肝素(H)是需要体外循环（CPB）的介入性和外科手术（如心脏手术）的首选抗凝剂。鱼精蛋白（PRT）是CPB后抗肝素的标准药物。最近，我们观察到接受CPB的患者产生针对PRT/H复合物的抗体（abs）。此外，我们发现术前抗prt /H抗体检测阳性的患者，其血小板（PLT）计数恢复延迟，术后早期动脉闭塞的风险增加。我们证明了启动事件是IgG抗体与PRT/H复合物的结合；导致免疫复合物（PRT-H-IgG）能够通过它们的FcgRIIa激活plt。本项目拟开展以下研究目的：1)评估抗PRT/H抗体在cpp患者中的确切临床影响；2)评估PRT与PLTs3结合的半衰期；3)评估免疫患者可能使用的治疗药物以预防不良反应。为了评估抗PRT/H抗体与CPB术后早期血栓并发症的关系，一项多中心研究将调查1000例患者的抗PRT/H抗体的存在。在之前的研究中，我们观察到抗PRT/H抗体阳性的患者术后PLT计数下降更为明显。PRT的血浆半衰期小于5分钟。因此，目前尚不清楚，抗PRT/H抗体如何在最后一次PRT给药后几天介导血小板减少。一种可能的情况是PRT与PLT结合并暴露在PLT表面数天。为了研究这一假设，plt将与PRT或PRT/H复合物一起孵育，并使用流式细胞术和免疫印迹技术通过抗PRT抗体评估PRT的结合情况。最近，有研究表明，去硫肝素（ODSH）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有类似的相互作用。我们将研究ODSH的特性，包括a)阻止/逆转PRT结构修饰的能力，b)抑制抗PRT/H IgG抗体的结合和这些抗体在体外激活PLT的能力，以及c) ODSH在NOD/SCID小鼠模型中阻止抗PRT/H抗体介导的PLT破坏的能力。另一种预防PRT诱导不良反应的潜在方法是使用低分子量PRT （LMW-PRT）。LMW-PRT被证明可以完全维持PRT的肝素中和能力。然而，截断的蛋白很可能不表达抗prt /H抗体结合的新表位。我们打算研究肝素的拮抗作用以及抗prt /H抗体与不同LMW-PRT/H复合物的交叉反应性。此外，将在NOD/SCID小鼠模型中分析LMW-PRT对存在抗prt /H抗体的循环人plt存活的影响。

项目成果

Partially desulfated heparin modulates the interaction between anti-protamine/heparin antibodies and platelets

部分脱硫肝素调节抗鱼精蛋白/肝素抗体与血小板之间的相互作用

• DOI: 10.1160/th15-07-0539
• 发表时间: 2015
• 期刊: Thrombosis and Haemostasis
• 影响因子: 6.7
• 作者: Jouni R;Zollner H;Khadour A;Wesche J;Delcea M;Krauel K;Schwertz H;Sachs UJ;Greinacher A;Bakchoul T
• 通讯作者: Bakchoul T

Platelet activation in the presence of neutral protamine Hagedorn insulin: a new feature of antibodies against protamine/heparin complexes

中性鱼精蛋白哈格多恩胰岛素存在下的血小板激活：鱼精蛋白/肝素复合物抗体的新特征

• DOI: 10.1111/jth.13547
• 发表时间: 2017
• 期刊: Journal of Thrombosis and Haemostasis
• 影响因子: 10.4
• 作者: Zollner H;Jouni R;Panzer S;Khadour A;Ten Berg M;Schellong S;Heinken A;Greinacher A;Bakchoul T
• 通讯作者: Bakchoul T

Flucloxacillin‐induced immune thrombocytopenia

• DOI: 10.1111/trf.13284
• 发表时间: 2016-01
• 期刊: Transfusion
• 影响因子: 2.9
• 作者: G. Jatzlauk;K. Althaus;U. Strobel;V. Kiefel;J. Wesche;K. Muehlenberg;A. Greinacher;T. Bakchoul

Assessment of human platelet survival in the NOD/SCID mouse model: technical considerations

• DOI: 10.1111/trf.13602
• 发表时间: 2016-06-01
• 期刊: TRANSFUSION
• 影响因子: 2.9
• 作者: Fuhrmann, Julia;Jouni, Rabie;Bakchoul, Tamam
• 通讯作者: Bakchoul, Tamam