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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.
Insights
Vasoactive intestinal peptide receptor I (VIPRI) is expressed in megakaryocytes. This suggests VIP may directly influence megakaryocytopoiesis and platelet aggregation.
Area of Science:
- Hematology
- Molecular Biology
- Cell Biology
Background:
- Vasoactive intestinal peptide (VIP) is a peptide hormone with diverse physiological roles.
- The presence and function of VIP receptors in megakaryocytes, the precursors of platelets, are not well understood.
Purpose of the Study:
- To investigate the expression and function of VIP receptors in human megakaryocytes.
- To determine if VIP directly impacts megakaryocytopoiesis and platelet function.
Main Methods:
- Human megakaryocytes were isolated from cord blood and bone marrow.
- Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect VIPRI gene expression.
- Covalent crosslinking techniques identified VIPRI protein on platelet membranes.
Main Results:
- VIPRI, along with VIP, c-mpl, and PF-4, was coexpressed in megakaryocyte mRNA.
- RT-PCR and Southern blot confirmed VIPRI expression.
- Specific binding of VIP to a 50,000 Mr protein on platelet membranes was demonstrated.
Conclusions:
- VIPRI is expressed in human megakaryocytes.
- VIP may exert direct effects on megakaryocytopoiesis.
- These findings support a role for VIP in modulating platelet aggregation.