The pituitary adenylate cyclase–activating polypeptide is a physiological inhibitor of platelet activation
Kathleen Freson, Hitoshi Hashimoto, Chantal Thys, Christine Wittevrongel, Sophie Danloy, Yoshiko Morita, Norihito Shintani, Yoshiaki Tomiyama, Jos Vermylen, Marc F. Hoylaerts, Akemichi Baba, Chris Van Geet
Kathleen Freson, Hitoshi Hashimoto, Chantal Thys, Christine Wittevrongel, Sophie Danloy, Yoshiko Morita, Norihito Shintani, Yoshiaki Tomiyama, Jos Vermylen, Marc F. Hoylaerts, Akemichi Baba, Chris Van Geet
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Article Hematology

The pituitary adenylate cyclase–activating polypeptide is a physiological inhibitor of platelet activation

Abstract

The pituitary adenylate cyclase–activating polypeptide (PACAP) is a neuropeptide of the vasoactive intestinal peptide/secretin/glucagon superfamily. Studies in two related patients with a partial trisomy 18p revealed three copies of the PACAP gene and elevated PACAP concentrations in plasma. The patients suffer from severe mental retardation and have a bleeding tendency with mild thrombocytopenia, and their fibroblasts show increased PACAP mRNA levels. The PACAP receptor (vasoactive intestinal peptide/pituitary adenylate cyclase–activating peptide receptor 1 [VPAC1]) in platelets and fibroblasts is coupled to adenylyl cyclase activation. Accordingly, we found increased basal cAMP levels in patients’ platelets and fibroblasts, providing a basis for the reduced platelet aggregation in these patients. Megakaryocyte-specific transgenic overexpression of PACAP in mice correspondingly increased PACAP release from platelets, reduced platelet activation, and prolonged the tail bleeding time. In contrast, the PACAP antagonist PACAP(6-38) or a monoclonal PACAP antibody enhanced the collagen-induced aggregation of normal human platelets, and in PACAP knockout mice, an increased platelet sensitivity toward collagen was found. Thus, we found that PACAP modulates platelet function and demonstrated what we believe to be the first hemostatic defect associated with PACAP overexpression; our study suggests the therapeutic potential to manage arterial thrombosis or bleeding by administration of PACAP mimetics or inhibitors, respectively.

Authors

Kathleen Freson, Hitoshi Hashimoto, Chantal Thys, Christine Wittevrongel, Sophie Danloy, Yoshiko Morita, Norihito Shintani, Yoshiaki Tomiyama, Jos Vermylen, Marc F. Hoylaerts, Akemichi Baba, Chris Van Geet

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PACAP detection in fibroblasts and plasma. (A) Semiquantitative RT-PCR u...
PACAP detection in fibroblasts and plasma. (A) Semiquantitative RT-PCR using 20 and 17 cycles showed PACAP(1–38) overexpression in fibroblasts from patient VI:1 compared with the control. RT-PCR was performed on two separate fibroblast samples, and β-actin is the internal control. (B) PACAP mRNA detection in fibroblasts, megakaryocytic cell lines DAMI, MEG-01, and K562, and platelets was performed by RT-PCR. (C) Immunoblot analysis of the VPAC1 receptor (58 kDa) in platelets from two unrelated controls and patient VI:1. (D) PACAP detections by ELISA in plasma from citrate (left panel) or ACD (right panel) blood from VI:1 (squares) and V:4 (triangles) or IV:5 (circles) and V:3 (filled diamonds) versus a citrated plasma pool (asterisks) or IV:6 (open diamonds). (E) The left panel shows the dose-dependent stimulation by PACAP(6–38) of the collagen-induced (0.2 μg/ml) aggregation of normal human platelets; representative tracings of five separate experiments are shown. The right panel illustrates the effect of PACAP(6–38) on collagen-induced (2 μg/ml) platelet aggregation in patient VI:1. Coll, collagen.