VPAC1 Receptor-Mediated Regulation of Megakaryopoiesis.

Identification of the regulatory pathways that direct megakaryopoiesis and platelet production is essential for the development of novel strategies to treat life threatening bleeding complications in bone marrow suppressed patients. We demonstrated that megakaryocytes and platelets express the Gs-coupled VPAC1 receptor, for which both PACAP and VIP are specific agonists. We have further identified a bleeding tendency and found three copies of the PACAP gene in two related patients with severe mental retardation, responsible for elevated PACAP plasma levels and associated increased platelet cAMP concentrations, resulting in strongly reduced platelet aggregation (JCI, 2004, 113, 905). In this study, we have further demonstrated a fundamental role for the VPAC1 signalling pathway during megakaryocyte maturation and platelet formation. Patients with PACAP overexpression have mild thrombocytopenia, a normal platelet survival and relatively small platelets with an MPV of 8.2 fL (normal MPV 9-13 fL). FACS analysis of the patients’ platelets showed reduced expression of GPIX and GPIIIa. Electron microscopy of bone marrow of patients and of mice, specifically overexpressing PACAP in megakaryocytes revealed the presence of early megakaryocyte progenitors but almost not of mature megakaryocytes. Immature megakaryoblasts seemed to have reduced levels of rough endoplasmic reticulum cisternae and free ribosomes. To further study the modulating role of VPAC1 in thrombopoiesis, control mice were therefore subcutaneously injected with neutralizing polyclonal or monoclonal anti-PACAP, anti-VIP or anti-VPAC1 antibodies. Injection of these antibodies in all cases led to increased platelet counts, compared to control antibodies (monoclonal anti-PACAP antibody: 1194 ± 237 x 10⁹ plt/L; control antibody: 722 ± 178 x 10⁹ plt/L; p=0.01, unpaired t-test at day 7 after injection). This strategy was also capable of reducing the drop in platelet count in busulfan treated mice (polyclonal anti-PACAP antibody: 561 ± 121 x 10⁹ plt/L; control antibody: 349 ± 65 x 10⁹ plt/L; p=0.033, unpaired t-test at day 29 or day 18 after respectively antibody and busulfan injection). In addition, bone marrow examination of mice injected with monoclonal anti-PACAP or anti-VPAC1 antibodies revealed an increase in megakaryocyte numbers and showed a marked expansion and mobilization of megakaryocyte progenitor cells. Mice injected with a monoclonal anti-VPAC1 antibody showed an increase of about 50% in bone marrow CFU-MK. In conclusion, we provide evidence that the VPAC1 pathway modulates normal megakaryopoiesis. Further studies are needed to evaluate whether this pathway can be safely manipulated in man in the treatment of thrombocytopenia.