Physicochemical Characteristics of Platelet Factor 4 under Various Conditions are Relevant for Heparin-Induced Thrombocytopenia Testing.
Physicochemical Characteristics of Platelet Factor 4 under Various Conditions are Relevant for Heparin-Induced Thrombocytopenia Testing.
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DOI：
10.1021/acs.jpcb.9b11695
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发表时间：
2020-01
期刊：
影响因子：
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通讯作者：
V. Bui;Patrycja Gebicka;Holger Hippe;R. Raschke;Thuy-Linh Nguyen;A. Greinacher;Thi‐Huong Nguyen
中科院分区：
文献类型：
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作者：
V. Bui;Patrycja Gebicka;Holger Hippe;R. Raschke;Thuy-Linh Nguyen;A. Greinacher;Thi‐Huong Nguyen
肝素诱导的血小板减少症(HIT)是一种药物不良反应，已引起人们的广泛关注。受影响的患者发生新的血栓并发症的风险很高。此外，HIT也是研究免疫介导性疾病机制的模型。血小板第4因子(PF4)是参与其中的关键蛋白。它是许多HIT诊断试验的基础，并用于体外研究和小鼠模型中HIT的发病机制。众所周知，纯化的PF4很容易形成聚集体，这可能会在实验中造成伪影。采用动态光散射法(DLS)和酶免疫分析法(EIA)检测抗PF4/H抗体(aPF4/H Abbs)与PF4/H复合物的结合情况。与磷酸盐缓冲盐水(PBS)相比，在Hank平衡盐溶液(HBSS)中PF4尺寸更稳定，尤其是在储存过程中。冻干进一步促进了PF4聚集体的形成，而重组的冻干PF4在37℃的PBS中孵育则减少了PF4聚集体的形成。与未冻干的PF4和肝素在HBSS和PBS中形成的复合物相比，冻干的PF4与肝素形成的复合物更大，在EIA中对aPF4/H抗体的结合作用更强，可能对体外试验结果有很大影响。我们的结果可能有助于对PF4生物学功能的机制研究和改进检测a PF4/H抗体的方法。
Heparin-induced thrombocytopenia (HIT), an adverse drug effect, has gained much attention. Affected patients have a high risk of new thrombotic complications. In addition, HIT is also a model to study mechanisms of immune-mediated disorders. Platelet factor 4 (PF4) is the key protein involved. It is the basis for many diagnostic tests for HIT and is used for in vitro stud-ies and in mouse models on the pathogenesis of HIT. Purified PF4 is known to easily form aggregates, which can cause artefacts in experiments. The impact of storage buffer, storage period, lyophilization, and temperature on the size of PF4 and PF4/heparin (H) complexes were assessed by dynamic light scattering (DLS), while enzyme immunoassay (EIA) was used to test binding of anti-PF4/H antibodies (aPF4/H Abs) to PF4/H complexes. PF4 size was more stable in Hank's balanced salt solution (HBSS) compared to phosphate-buffered saline (PBS), especially during storage. Lyophilization further facilitated formation of PF4 aggregates, while incubation of reconstituted lyophilized PF4 in PBS at 37 oC reduced PF4 aggregates. Complexes formed between lyophilized PF4 and heparin were bigger and en-hanced binding of aPF4/H Abs in EIA compared to complexes between non-lyophilized PF4 and heparin, both in HBSS and PBS and may strongly influence in vitro test results. Our results may be helpful for mechanistic studies on the biological function of PF4 and for im-provement of assays for detection of aPF4/H Abs.