Rab27a activity is affected in several mouse models of human disease including Griscelli (ashen mice) and Hermansky-Pudlak (gunmetal mice) syndromes. A loss of function mutation occurs in the Rab27a gene in ashen (ash), whereas in gunmetal (gm) Rab27a dysfunction is secondary to a mutation in the α subunit of Rab geranylgeranyl transferase, an enzyme required for prenylation and activation of Rabs. We show here that Rab27a is normally expressed in cytotoxic T lymphocytes (CTLs), but absent in ashen homozygotes (ash/ash). Cytotoxicity and secretion assays show that ash/ash CTLs are unable to kill target cells or to secrete granzyme A and hexosaminidase. By immunofluorescence and electron microscopy, we show polarization but no membrane docking of ash/ash lytic granules at the immunological synapse. In gunmetal CTLs, we show underprenylation and redistribution of Rab27a to the cytosol, implying reduced activity. Gunmetal CTLs show a reduced ability to kill target cells but retain the ability to secrete hexosaminidase and granzyme A. However, only some of the granules polarize to the immunological synapse, and many remain dispersed around the periphery of the CTLs. These results demonstrate that Rab27a is required in a final secretory step and that other Rab proteins also affected in gunmetal are likely to be involved in polarization of the granules to the immunological synapse.

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Few molecular events important to platelet biogenesis have been identified. Mice homozygous for the spontaneous, recessive mutation gunmetal (gm) have prolonged bleeding, thrombocytopenia, and reduced platelet ␣-and ␦-granule contents. Here we show by positional cloning that gm results from a G3 A substitution mutation in a splice acceptor site within the ␣-subunit of Rab geranylgeranyl transferase (Rabggta), an enzyme that attaches geranylgeranyl groups to Rab proteins. Most Rabggta mRNAs from gm tissues skipped exon 1 and lacked a start codon. Rabggta protein and Rab geranylgeranyl transferase (GGTase) activity were reduced 4-fold in gm platelets. Geranylgeranylation and membrane association of Rab27, a Rab GGTase substrate, were significantly decreased in gm platelets. These findings indicate that geranylgeranylation of Rab GTPases is critical for hemostasis. Rab GGTase inhibition may represent a new treatment for thrombocytosis and clotting disorders. P latelets play a pivotal role in acute myocardial infarction, unstable angina, deep venous thrombosis, and stroke. About 4 million individuals are hospitalized each year in the United States with these disorders. Treatment with agents that prevent platelet activation reduces the risk of myocardial infarction and stroke by about 30% and death by about 15% (1). Novel platelet antagonists are needed, however, because platelet activation is not always inhibited by current drugs.Mice homozygous for gunmetal (gm), a spontaneous, recessive mutation, have prolonged bleeding caused by defects in platelets and megakaryocytes (2, 3). These mice also have macrothrombocytopenia and reduced platelet ␣-and ␦-granule contents (storage pool deficiency, SPD). Megakaryocytes, the progenitors of platelets, are more plentiful in gm mice, but have abnormal intracellular membranes, increased emperipolesis, and decreased platelet synthesis. In addition, gm homozygotes have partial cutaneous albinism (Fig. 1). These features closely resemble the rare human disorders gray platelet syndrome (GPS) and platelet ␣,␦-SPD. Positional cloning of gm was undertaken both to shed light on the mechanism of disease in platelet SPD disorders and to identify a possible target for antiplatelet drug development. Materials and MethodsMice and Genetic Mapping. Mice obtained from The Jackson Laboratory were bred at Roswell Park Cancer Institute. Backcross mice were phenotyped by coat color and genotyped for simple sequence length polymorphisms (SSLPs) and restriction fragment length polymorphism (RFLP) using standard techniques. Linkage relationships were determined by segregation analysis, and best locus order was decided by minimizing crossovers and eliminating double crossovers (4). Inverse Repetitive Element-Direct cDNA Selection. Expressed sequences were isolated from YACs by inverse repetitive elementdirect cDNA selection (6). Briefly, outward-oriented, biotinlabeled, B1 repetitive element-specific primers were used for long-range PCR of YAC clones (5). Denatured PCR products were incubated with rib...

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