Ryutaro Shirakawa

Small GTPase Rab4 Regulates Ca2+-induced α-Granule Secretion in Platelets

Upon activation, platelets release many active substances stored in α- and dense-core granules. However, the molecular mechanisms governing regulated exocytosis are not yet fully understood. Here, we have established an assay system using permeabilized platelets to analyze the Ca2+-induced exocytosis of both types of granules, focusing on RabGTPases. Incubation with Rab GDP dissociation inhibitor, an inhibitory regulator of RabGTPases, reduced membrane-bound RabGTPases extensively, and caused strong inhibition of the Ca2+-induced secretion of von Willebrand factor (vWF) stored in α-granules, but not that of [3H]5-hydroxytryptamine (5-HT) in dense-core granules. Specifically, Rab4 co-fractionated with vWF and P-selectin (an α-granule marker) upon separation of platelet organelles by density gradient centrifugation. Incubation of the permeabilized platelets with cell extracts expressing the dominant negative mutant of His-tagged Rab4S22N, but not with those of similar mutant His-Rab3BT36N, inhibited the vWF secretion, whereas neither of the cell extracts affected the [3H]5-HT secretion. Importantly, the inhibition of vWF secretion was rescued by depleting the cell extracts of the His-Rab4S22N with nickel beads. Thus, in platelets, the regulatory mechanisms governing α- and dense-core granule secretions are distinct, and Rab4 is an essential regulator of the Ca2+-induced exocytosis of α-granules.

Constitutive GDP/GTP Exchange and Secretion-dependent GTP Hydrolysis Activity for Rab27 in Platelets

We have previously demonstrated that Rab27 regulates dense granule secretion in platelets. Here, we analyzed the activation status of Rab27 using the thin layer chromatography method analyzing nucleotides bound to immunoprecipitated Rab27 and the pull-down method quantifying Rab27 bound to the GTP-Rab27-binding domain (synaptotagmin-like protein (Slp)-homology domain) of its specific effector, Slac2-b. We found that Rab27 was predominantly present in the GTP-bound form in unstimulated platelets due to constitutive GDP/GTP exchange activity. The GTP-bound Rab27 level drastically decreased due to enhanced GTP hydrolysis activity upon granule secretion. In permeabilized platelets, increase of Ca2+ concentration induced dense granule secretion with concomitant decrease of GTP-Rab27, whereas in non-hydrolyzable GTP analogue GppNHp (β-γ-imidoguanosine 5′-triphosphate)-loaded permeabilized platelets, the GTP (GppNHp)-Rab27 level did not decrease upon the Ca2+-induced secretion. These data suggested that GTP hydrolysis of Rab27 was not necessary for inducing the secretion. Taken together, Rab27 is maintained in the active status in unstimulated platelets, which could function to keep dense granules in a preparative status for secretion.

Crystal structure of human DAAM1 formin homology 2 domain

Reorganization of the actin filament is an essential process for cell motility, cell–cell attachment and intracellular transport. Formin proteins promote nucleation and elongation of the actin filament, and thus are key regulators for this process. The formin homology 2 (FH2) domain forms a head‐to‐tail ring‐shaped dimer, and processively moves towards the barbed end. Dishevelled‐associated activator of morphogenesis (DAAM) is a Rho‐regulated formin implicated in neuronal development. Here, we present the crystal structure of human DAAM1 FH2 dimer at 2.8 Å resolution. This is the first dimeric structure of the mammalian formin. The core structure of human DAAM1 is similar to those of mouse mDia1 and yeast Bni1p, whereas the orientations of the FH2 dimeric rings are different between human DAAM1 and yeast Bni1p, despite their similar dimer interactions. This difference supports the previous prediction that the dimer architecture of the formin is highly flexible in the actin‐free state. The results of the actin assembly assays using the DAAM1 mutants demonstrated that the length of the linker connecting the N‐terminal domain and the core region is crucial for the activity.

Molecular Dissection of α- and Dense-Core Granule Secretion of Platelets

Abstract: Upon activation, platelets release many active substances stored in α‐ and dense‐core granules. However, the molecular mechanisms governing the regulated exocytosis are not yet fully understood. We have established an assay system using streptolysin‐O‐permeabilized platelets to analyze the Ca2+‐induced secretions of von Willebrand factor stored in α‐granules and [3H]5‐hydroxytryptamine (5‐HT) in dense‐core granules. Using the assay, we found that small GTPase Rab4 regulates α‐, but not dense‐core, granule secretion in platelets. Furthermore, we purified a cytosolic essential protein and currently are analyzing its function.

Familial hemophagocytic lymphohistiocytosis with the MUNC13-4 mutation: a case report

A 44-day-old male infant with familial hemophagocytic lymphohistiocytosis (FHL) associated with the MUNC13-4 mutation is reported. He presented with fever and poor feeding, lymphocytosis with thrombocytopenia and CSF pleocytosis without virological explanation. On the basis of progressive hyperferritinemia (1323xa0ng/ml), anemia (hemoglobin: 5.2xa0g/dl), hypertriglyceridemia (547xa0mg/dl) and increased LDH (1063xa0IU/l) with hemophagocytosis in the bone marrow, hemophagocytic lymphohistiocytosis was diagnosed. He showed a good response to corticosteroid therapy and the disease was stable for more than 5 months. Thereafter, he suffered from central nervous system complications, and successfully underwent unrelated cord blood stem cell transplantation. A remission was observed for more than 2 years, with mild mental retardation. Genetic analysis revealed that he had a compound heterozygous mutation of MUNC13-4; namely a novel 2163G>A mutation resulting in W721X, and 754-1G>C resulting in a premature stop codon in this gene. Western blot analysis showed the complete loss of the MUNC13-4 protein, whereas other molecules associated with the SNARE systems were detected at normal levels. Conclusion. FHL may have a broad clinical spectrum, and further analysis on its phenotype-genotype association is required to establish an appropriate treatment strategy, including immunochemotherapy and stem cell transplantation in the future.

Elucidation of the molecular mechanism of platelet activation: Dense granule secretion is regulated by small guanosine triphosphate‐binding protein Rab27 and its effector Munc13‐4

Cardiovascular diseases such as myocardial and cerebral infarction are common critical diseases occurring more frequently in the elderly. The trigger of the diseases is platelet activation following plaque rupture or erosion. Investigation of the molecular mechanism in platelet activation has been exclusively performed pharmacologically. We have succeeded in establishing the granule secretion and aggregation assays using permeabilized platelets. These systems enabled us to examine the molecular mechanism in platelet activation with molecular biological and biochemical methods. Using these assay systems, we have been investigating the molecular mechanism of platelet activation. With a support grant from the Novartis Foundation for Gerontological Research, we found several molecules involved in the regulation. In this report, I present the progress in the research of the granule secretion mechanism in activated platelets, which was reported in the Japanese Geriatric Society Meeting in 2005.