DataSheet_1_In vitro analysis of anti-HPA-1a dependent platelet phagocytosis and its inhibition using a new whole blood phagocytosis assay (WHOPPA).pdf

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a serious bleeding condition mostly caused by the reaction between maternal anti-HPA-1a antibodies and fetal platelets. This reaction leads to Fc-dependent platelet phagocytosis. Although several serological methods have been developed to identify maternal antibodies, a reliable laboratory parameter as a prognostic tool for FNAIT severity is still lacking. In this study, we developed whole blood platelet phagocytosis assay (WHOPPA), a flow cytometry-based phagocytosis assay that uses a pH-sensitive fluorescent dye (pHrodo-SE) to analyze anti-HPA-1a-dependent platelet phagocytosis in whole blood. WHOPPA revealed a high phagocytosis rate for the anti-HPA-1a opsonized platelets by monocytes but not by neutrophils. Analysis of different monocyte populations showed that all monocyte subsets, including classical (CD14++CD16−), intermediate (CD14++CD16+), and nonclassical (CD14+CD16++) monocytes, were able to engulf opsonized platelets. A unique monocyte subset, termed shifted monocytes (CD14+CD16−), showed the highest phagocytosis rate and was detected after platelet engulfment. FcγR inhibition tests revealed that except for FcγRIIa, FcγRI and FcγRIII on monocytes were responsible for the phagocytosis of anti-HPA-1a opsonized platelets. Analysis of anti-HPA-1a antibodies from FNAIT cases (n = 7) showed the phagocytosis of HPA-1aa but not of HPA-1bb platelets by monocytes. The phagocytosis rate was highly correlated with bound antibodies measured by flow cytometry (p < 0001; r = 0.9214) and MAIPA assay (p < 0.001; r = 0.7692). The phagocytosis rates were equal for type I and II anti-HPA-1a antibodies recognizing the plexin–semaphoring–integrin (PSI) domain and PSI/epidermal growth factor 1 domain of β3 integrin, respectively. By contrast, type III anti-HPA-1a antibodies reacting with αvβ3 integrin did not induce platelet phagocytosis. Furthermore, effector-silenced mAbs against HPA-1a inhibited the phagocytosis of anti-HPA-1a opsonized platelets. In conclusion, WHOPPA is a reliable in vitro platelet phagocytosis assay that mimics the phagocytosis of anti-HPA-1a opsonized platelets in whole blood. This assay allows to prove platelet phagocytosis ex vivo and evaluate the inhibitory capacity of different inhibitors as therapeutically strategies for the prevention of fetal thrombocytopenia in FNAIT in the future.

胎儿和新生儿同种免疫性血小板减少症（Fetal and neonatal alloimmune thrombocytopenia, FNAIT）是一种严重的出血性疾病，其主要病因是母体抗HPA-1a抗体与胎儿血小板发生反应，该反应可引发Fc依赖的血小板吞噬作用。目前虽已开发出多种用于检测母体抗体的血清学方法，但仍缺乏可作为FNAIT严重程度预后工具的可靠实验室指标。本研究开发了全血血小板吞噬试验（WHOPPA）——一种基于流式细胞术的吞噬检测方法，该方法使用pH敏感性荧光染料（pHrodo-SE）来分析全血中抗HPA-1a依赖的血小板吞噬过程。WHOPPA结果显示，单核细胞可对经抗HPA-1a调理的血小板产生较高吞噬率，而中性粒细胞则无此能力。对不同单核细胞群的分析表明，所有单核细胞亚群——包括经典型（CD14++CD16−）、中间型（CD14++CD16+）及非经典型（CD14+CD16++）单核细胞——均能够吞噬经调理的血小板。一类被称为移位单核细胞（CD14+CD16−）的独特单核细胞亚群展现出最高的吞噬率，且该细胞群可在血小板被吞噬后被检测到。Fcγ受体抑制试验结果显示，除FcγRIIa外，单核细胞表面的FcγRI与FcγRIII参与了抗HPA-1a调理血小板的吞噬过程。对7例FNAIT患者的抗HPA-1a抗体进行分析后发现，单核细胞可吞噬HPA-1aa型血小板，但无法吞噬HPA-1bb型血小板。吞噬率与流式细胞术检测的结合抗体量（p < 0.0001；r = 0.9214）及单克隆抗体特异性捕获血小板抗原试验（MAIPA assay）结果（p < 0.001；r = 0.7692）均呈高度相关。分别识别β3整合素的plexin-semaphorin-integrin（PSI）结构域与PSI/表皮生长因子1结构域的I型和II型抗HPA-1a抗体，其吞噬率无显著差异。与之相反，针对αvβ3整合素的III型抗HPA-1a抗体则无法诱导血小板吞噬。此外，靶向HPA-1a的效应功能沉默单克隆抗体（mAbs）可抑制抗HPA-1a调理血小板的吞噬过程。综上，WHOPPA是一种可靠的体外血小板吞噬试验，可模拟全血中抗HPA-1a调理血小板的吞噬过程。该试验能够在离体条件下验证血小板吞噬作用，并可在未来用于评估各类抑制剂的抑制能力，以作为预防FNAIT相关胎儿血小板减少症的治疗策略。