Mastoparan promotes exocytosis and increases intracellular cyclic AMP in human platelets. Evidence for the existence of a Ge-like mechanism of secretion.
Recent studies have shown that mastoparan, an amphiphilic peptide derived from wasp venom, accelerates guanine nucleotide exchange and GTPase activity of purified GTP-binding proteins. In the present study we have examined the functional consequences of exposure of intact human platelets to mastoparan. Mastoparan promoted rapid (less than or equal to 1 min) dose-dependent increases in 5-hydroxy[14C]tryptamine and beta-thromboglobulin release from dense-granule and alpha-granule populations respectively. The exocytotic response did not result from a lytic effect of mastoparan and occurred in the complete absence of platelet shape change and aggregation. Liberation of [3H]arachidonate and increases in cytosolic [Ca2+] (detected with fura 2) were not observed in platelets stimulated with mastoparan. Similarly, in platelets preloaded with [3H]inositol during reversible electroporation, mastoparan did not cause the accumulation of [3H]inositol phosphates. Mastoparan-induced secretion was unaffected by preincubation with either the protein kinase C inhibitor staurosporine (10 nM-10 microM) or prostacyclin (PGI2; 100 ng/ml) and was not accompanied by phosphorylation of the 45 kDa protein kinase C substrate or the 20 kDa protein normally associated with platelet activation. The G-protein inhibitor guanosine 5'-[beta-thio]diphosphate (GDP[S]; 1 mM) attenuated the secretion induced by mastoparan in both intact and saponin-permeabilized platelets. Encapsulation of GDP[S] during reversible permeabilization inhibited mastoparan-induced secretion, providing evidence for an intracellular action of GDP[S]. In all these studies thrombin (0.05-0.2 unit/ml) elicited characteristic responses, and thrombin-induced secretion was inhibited by staurosporine, PGI2 and GDP[S]. Mastoparan also increased intra-platelet cyclic AMP in a dose-dependent manner. Mastoparan and PGI2 increased 32P incorporation into a protein of approx. 24 kDa, whereas phosphorylation of a 50 kDa substrate was only seen in PGI2-stimulated platelets. These results indicate that mastoparan promotes secretion by a mechanism which does not involve stimulation of phospholipase C and suggest that the secretory event may result either from a direct fusogenic action of mastoparan and/or from stimulation of the putative exocytosis-linked G-protein, Ge.
Wheeler-Jones
C P
CP
Platelet Section, Thrombosis Research Institute, London, U.K.
Saermark
T
T
Kakkar
V V
VV
Authi
K S
KS
D000470:Alkaloids / Q000494:pharmacology; D000595:Amino Acid Sequence; D001095:Arachidonic Acids / Q000378:metabolism; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism*; D003594:Cytoplasmic Granules / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D011464:Epoprostenol / Q000494:pharmacology; D005089:Exocytosis / Q000187:drug effects*; D019204:GTP-Binding Proteins / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D036341:Intercellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D010455:Peptides; D010716:Phosphatidylinositols / Q000378:metabolism; D015539:Platelet Activation; D012503:Saponins / Q000494:pharmacology; D019311:Staurosporine; D013873:Thionucleotides / Q000494:pharmacology; D013917:Thrombin / Q000378:metabolism; D014862:Wasp Venoms / Q000737:chemistry / Q000494:pharmacology*
D000470:Alkaloids; D001095:Arachidonic Acids; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D010716:Phosphatidylinositols; D012503:Saponins; D013873:Thionucleotides; D014862:Wasp Venoms; D006153:Guanosine Diphosphate; C023427:guanosine 5'-O-(2-thiodiphosphate); C037500:mastoparan; D011464:Epoprostenol; D000242:Cyclic AMP; D013917:Thrombin; D019204:GTP-Binding Proteins; D019311:Staurosporine; D002118:Calcium