CHARACTERIZATION OF VASOACTIVE-INTESTINAL-PEPTIDE RECEPTORS ON HUMAN MEGAKARYOCYTES AND PLATELETS

Vasoactive intestinal peptide receptor I (VIPRI) expression was examin ed in megakaryocytes using reverse transcriptase-polymerase chain reac tion (RT-PCR). VIPRI protein was characterized in platelet membranes u sing covalent crosslinking techniques. Human megakaryocytes were isola ted from suspension cultures of cord blood and adult bone marrow monon uclear cells using a murine monoclonal antibody to human platelet glyc oprotein IIB/IIIA (CD41) and immunomagnetic beads. RT-PCR primers were constructed for the VIP, VIPRI, and VIPRII genes as well as for megak aryocyte specific genes, c-mpl and platelet factor 4 (PF-4). VIP, VIPR I, c-mpl, and PF-4 were coexpressed in megakaryocyte mRNA. Southern bl ot analysis confirmed the expression of VIPRII. I-125-VIP was covalent ly cross-linked to human platelet membranes using the homobifunctional reagent disuccinimidyl suberate, followed by polyacrylamide gel elect rophoresis and autoradiography. A I-125-VIP-protein complex of M(r) = 50,000 was identified. Labeling of the M(r) = 50.000 component was com pletely abolished by unlabeled VIP, but not by peptide histidine methi onine or growth hormone releasing factor, indicating specific binding of VIP to the platelet membranes. Taken together, these results sugges t that VIP may have direct effects on megakaryocytopoiesis and support our earlier observations of VIP modulation of platelet aggregation. ( C) 1996 by The American Society of Hematology.