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Platelets store self. agonists such as ADP and serotonin in dense core granules. Although exocytosis of these granules is crucial for hemostasis and thrombosis, the underlying mechanism is not fully understood. Here, we show that incubation of permeabilized platelets with unprenylated active mutant Rab27A-Q78L, wild type Rab27A, and Rab27B inhibited the secretion, whereas inactive mutant Rab27A-T23N and other GTPases had no effects. Furthermore, we affinity-purified a GTPRab27A-binding protein in platelets and identified it as Munc13-4, a homologue of Munc13-1 known as a priming factor for neurotransmitter release. Recombinant Munc13-4 directly bound to GTP-Rab27A and -Rab27B in vitro, but not other GTPases, and enhanced secretion in an in vitro assay. The inhibition of secretion by unprenylated Rab27A was rescued by the addition of Munc13-4, suggesting that Munc13-4 mediates the function of GTP-Rab27. Thus, Rab27 regulates the dense core granule secretion in platelets by employing its binding protein, Munc13-4. [ABSTRACT FROM AUTHOR]

Platelets play critical roles in hemostasis and thrombosis through their aggregation following activation of integrin α[sub IIb]β[sub 3]. However, the molecular mechanism of the integrin activation inside platelets remains largely unknown. Pharmacological experiments have demonstrated that protein kinase C (PKC) plays an important role in platelet aggregation. Because PKC inhibitors can have multiple substrates and given that non-PKC-phorbol ester-binding signaling molecules have been demonstrated to play important roles, the precise involvement of PKC in cellular functions requires re-evaluation. Here, we have established an assay for analyzing the Ca[sup 2+]-induced aggregation of permeabilized platelets. The aggregation of platelets was inhibited by the addition of the arginine-glycine-aspartate-serine peptide, an integrin-binding peptide inhibitor of α[sub IIb]β[sub 3], suggesting that the aggregation was mediated by the integrin. The aggregation was also dependent on exogenous ATP and platelet cytosol, indicating the existence of essential cytosolic factors required for the aggregation. To examine the role of PKC in the aggregation assay, we immunodepleted PKCα and β from the cytosol. The PKC-depleted cytosol lost the aggregation-supporting activity, which was recovered by the addition of purified PKCα. Furthermore, the addition of purified PKCα in the absence of cytosol did not support the aggregation, whereas the cytosol containing less PKC supported it efficiently, suggesting that additional factors besides PKC would also be required. Thus, we directly demonstrated that PKCα is involved in the regulation of Ca[sup 2+]-induced platelet aggregation. [ABSTRACT FROM AUTHOR]