Inhibition of VPAC1 Signaling Raises Platelet Numbers after Myelosuppressive Therapy and in GATA1 Congenital Thrombocytopenia.

Thrombocytopenia is a serious complication in patients undergoing myelosuppressive chemotherapy. Therefore, the development of new strategies to reduce thrombocytopenia or accelerate platelet recovery without adverse effect on the bone marrow (BM) is still warranted. Previously we found that the pituitary adenylate cyclase activating peptide (PACAP) is a physiological inhibitor of platelet activation (JCI, 2004). The receptor for PACAP on megakaryocytes (MKs) and platelets is the Gs coupled receptor VPAC1. We have presently investigated whether the neutralization of the regulatory role of VPAC1 can affect thrombopoiesis, independently of thrombopoietin (TPO). We found that inhibition of VPAC1 signaling stimulates in vitro megakaryopoiesis, since more MKs could be generated from Sca1+ cells, isolated from normal mouse BM by incubation with a neutralizing anti-VPAC1 antibody (Ab) (23A11). 23A11 was also capable of enhancing the later stages of MK proliferation of human cord blood derived CD34+ cells. These in vitro findings were validated in two in vivo animal models of myelosuppressive therapy. The subcutaneous injection of a neutralizing Ab directed against PACAP (PP1A4) or 23A11 (1 mg/kg on days 0,3 and 7) boosted platelet recovery in mice after chemotherapy-induced thrombocytopenia (20 mg/kg busulfan intraperitoneally on days 7 and 10) in a TPO-independent manner. Platelet recovery in mice, injected with PP1A4, was also significantly increased compared to non-treated mice exposed to 8 Gy total body irradiation. Plasma TPO concentrations did not differ between the two groups. We next questioned whether VPAC1 inhibition could rescue the GATA1-defective phenotype due to a developmental arrest in the megakaryoblast stage. FACS analysis of BM Sca1+ cells from GATA1 deficient mice cultured during 12 days in MK differentation medium in vitro showed an increased MK DNA ploidy when 23A11 was added to the culture. A similar observation was made with CD34+ cells from BM of a GATA1 deficient patient with severe thrombocytopenia. GATA1 deficient mice are thrombocytopenic, with a platelet count of 50 +/− 15 x 10(9) plt/L. This platelet number increases to 150 +/− 40 x 10(9) plt/L after subcutaneous injection with 23A11 whereas injection with PBS had no effect. Again, TPO plasma levels in these injected GATA1 deficient mice showed no association between anti-PACAP or anti-VPAC1 treatment. Thus, our data indicate that VPAC1 signaling tempers normal megakaryopoiesis and that elimination of this pathway facilitates platelet recovery after myelosuppressive therapy and in GATA1 deficiency.