Effects on Anti-Human Platelet Antigen 1 (HPA-1a) Antibodies on Neonatal Megakaryocytopoiesis

Neonatal alloimmune thrombocytopenia (NAIT) is the most common cause of severe thrombocytopenia and of intracranial hemorrhage (ICH) in term newborn infants. NAIT is caused by fetomaternal incompatibility in one of the platelet-specific surface antigens, and is mediated by maternally produced antibodies against the fetal antigen. The platelet antigen most commonly involved is human platelet antigen-1 (HPA-1, also known as PLA-1), which is responsible for approximately 75% of cases. While the mechanisms underlying the thrombocytopenia in fetuses and neonates have not been thoroughly studied, it is widely accepted that it is predominantly due to antibody-mediated platelet destruction. However, in adults with immune thrombocytopenic purpura (ITP), studies have shown that the anti-platelet antibodies also act by suppressing megakaryocyte (MK) proliferation and maturation, which contributes to the thrombocytopenia.

This study was designed to test the hypothesis that anti-HPA-1 antibodies would affect MK proliferation and maturation, in a manner similar to anti-platelet antibodies in ITP.

To test this hypothesis, we obtained serum samples from pregnant women known to have anti-HPA-1a antibodies and a history of previously affected pregnancies (NAIT sera; n=5), and from healthy pregnant women without anti-HPA-1 antibodies (control sera; n=2). We then obtained cord blood samples from healthy full-term infants delivered by elective C-section, and isolated CD34+ cells from HPA-1a/1a (PLA-1 positive) samples. These CD34+ cells (>90% purity) were then cultured in a liquid culture system, in the presence of thrombopoietin and 10% NAIT or control serum. To avoid any potential confounding effects from anti-A or anti-B antibodies on megakaryocytopoiesis, only blood type O cord blood samples were used for this study. The number of cells in each culture was quantified on days 7, 11, and 14. At the end of the culture period (day 14), the percentage of MKs (CD41+ cells) and their maturational status were evaluated by flow cytometry, using CD42b (GPIb alpha) expression as a marker of mature MKs.

These studies revealed a significant reduction in the number of cells generated in the presence of NAIT compared to control sera on days 7, 11, and 14 of culture (approximately 40% of control counts at all time points). This finding was associated with an increased percentage of dead cells (by trypan blue exclusion assay) in NAIT vs. control cultures, particularly on day 7 (19.4±3.1% vs. 12.2±5.3% dead cells; p=0.03) and day 11 of culture (12.8±1.3 vs. 6.5±2.0% dead cells; p=0.002). At the end of the culture period, there were no significant differences in the percentages of MKs between NAIT and control cultures (94.3±0.7% vs. 95.2±0.7% CD41+ cells, respectively). In regard to the maturational level, only one of the five NAIT sera induced a reduction in the percentage of CD42b+ MKs (to 83%), which was also associated with the strongest reduction in cell number. All other NAIT serum samples did not affect MK maturation, as evidenced by a percentage of CD42b+ MKs similar to that in control cultures (93.7±0.6% vs. 94.2±0.25% in NAIT vs. control cultures, respectively). Given the combination of reduced cell number and increased cell death in the NAIT cultures, we then evaluated the rate of apoptosis (by TUNEL assay) and proliferation (using Ki67 as a marker of proliferation) in two additional NAIT and two control cultures. In these experiments, the percentage of cells undergoing apoptosis was approximately 2-fold higher in NAIT compared to control cultures, on both day 7 and day 11 of culture, while there were no significant differences in Ki67 expression.

In conclusion, our studies indicate that human serum containing anti-HPA-1 antibodies reduces the number of MKs generated in culture, without affecting MK differentiation. Preliminary studies suggest that the decreased number of MKs is associated with increased apoptosis. Additional studies are in progress to establish the specific mechanisms underlying these observations.

Bussel:Portola: Consultancy; Eisai: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; GlaxoSmithKline: Consultancy, Equity Ownership, Research Funding, Speakers Bureau; Amgen Inc.: Equity Ownership, Research Funding, Speakers Bureau; Cangene: Research Funding; Genzyme: Research Funding; Immunomedics: Research Funding; Ligand: Research Funding, Speakers Bureau; Shionogi: Research Funding, Speakers Bureau; Sysmex: Research Funding.