Characterization of vasoactive intestinal peptide receptors on human megakaryocytes and platelets.

ABSTRACT

Vasoactive intestinal peptide receptor I (VIPRI) expression was examined in megakaryocytes using reverse transcriptase-polymerase chain reaction (RT-PCR). VIPRI protein was characterized in platelet membranes using covalent crosslinking techniques. Human megakaryocytes were isolated from suspension cultures of cord blood and adult bone marrow mononuclear cells using a murine monoclonal antibody to human platelet glycoprotein IIB/IIIA (CD41) and immunomagnetic beads. RT-PCR primers were constructed for the VIP, VIPRI, and VIPRII genes as well as for megakaryocyte specific genes, c-mpl and platelet factor 4 (PF-4). VIP, VIPRI, c-mpl, and PF-4 were coexpressed in megakaryocyte mRNA. Southern blot analysis confirmed the expression of VIPRI. 125I-VIP was covalently cross-linked to human platelet membranes using the homobifunctional reagent disuccinimidyl suberate, followed by polyacrylamide gel electrophoresis and autoradiography. A 125I-VIP-protein complex of Mr = 50,000 was identified. Labeling of the Mr = 50,000 component was completely abolished by unlabeled VIP, but not by peptide histidine methionine or growth hormone releasing factor, indicating specific binding of VIP to the platelet membranes. Taken together, these results suggest that VIP may have direct effects on megakaryocytopoiesis and support our earlier observations of VIP modulation of platelet aggregation.