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

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 ß3 integrin, respectively. By contrast, type III anti-HPA-1a antibodies reacting with αvß3 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.

Generation of induced pluripotent stem cell line derived from FNAIT patient with CD36 deficiency mutations.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) caused by anti-CD36 isoantibodies is a common disease and the frequency of type I CD36 deficiency is relatively high in eastern Asian populations.Currently, patient-specific induced pluripotent stem cells (hiPSC) are believed to be useful tools for studying anti-CD36 mediated FNAIT and finding new therapeutic approaches to the disease.We generated an iPSC line from peripheral blood mononuclear cells of a patient carrying a 329-330delAC of the CD36 gene.The iPSC expressed pluripotency markers, gave rise to derivatives of three germ layers during spontaneous differentiation, had a normal karyotype, and retained the patient-specific mutation.

Antiendothelial αvß3 Antibodies Are a Major Cause of Intracranial Bleeding in Fetal/Neonatal Alloimmune Thrombocytopenia.

OBJECTIVE: Fetal/neonatal alloimmune thrombocytopenia is a severe bleeding disorder, which can result in intracranial hemorrhage (ICH), leading to death or neurological sequelae. In whites, maternal anti-human platelet antigen-1a (HPA-1a) antibodies are responsible for the majority of cases. No predictive factors for ICH are available to guide prophylactic treatment during pregnancy. In this study, we investigated antibodies from mothers with ICH-positive fetal/neonatal alloimmune thrombocytopenia and with ICH-negative fetal/neonatal alloimmune thrombocytopenia to identify serological and functional differences between the groups. APPROACH AND RESULTS: In an antigen capture assay, we observed a stronger binding of +ICH antibodies to endothelial cell (EC)-derived αvß3. By absorption experiments, we subsequently identified anti-HPA-1a antibodies of anti-αvß3 specificity in the +ICH but not in the -ICH cohort. Only the anti-αvß3 subtype, but not the anti-ß3 subtype, induced EC apoptosis of HPA-1a-positive ECs by caspase-3/7 activation, and mediated by reactive oxygen species. In addition, only the anti-αvß3 subtype, but not the anti-ß3 subtype, interfered with EC adhesion to vitronectin and with EC tube formation. CONCLUSIONS: We conclude that the composition of the anti-HPA-1a antibody subtype(s) of the mother may determine whether ICH occurs. Analysis of anti-HPA-1a antibodies of the anti-αvß3 subtype in maternal serum has potential in the diagnostic prediction of ICH development and may allow for modification of prophylactic treatment in fetal/neonatal alloimmune thrombocytopenia.