Lysosomes, dense granules, and granules share a cohort of SNAREs and associated proteins that facilitate activation-induced fusion with the plasma membrane.11-13,32,49 Although HPS model platelets had normal levels of these proteins and we were unable to detect consistent alterations in SNAP-23 phosphorylation,83 it is likely that 1 of these components is mistargeted or fails to become activated in HPS megakaryocytes or platelets. that granule secretion elicited by low agonist doses is impaired in all 3 HPS models. High agonist doses or supplemental adenosine 5-diphosphate (ADP) restored normal granule secretion, suggesting that the impairment is secondary to absent DES dense granule content release. Colchicine Intravital microscopy following laser-induced vascular injury showed that defective hemostatic thrombus formation in HPS mice largely reflected reduced total platelet accumulation and affirmed a reduced area of granule secretion. Agonist-induced lysosome secretion ex vivo was also impaired in all 3 HPS models but was incompletely rescued by high agonist doses or excess ADP. Our results imply that (1) AP-3, BLOC-1, and BLOC-3 facilitate protein sorting to lysosomes to support ultimate secretion; (2) impaired secretion of granules in HPS, and to some degree of lysosomes, is secondary to impaired dense granule secretion; and (3) diminished granule and lysosome secretion might contribute to pathology in HPS. Introduction Effective thrombus formation by platelets at sites of blood vessel injury requires the stimulus-dependent release of effectors from membrane-enclosed dense granules, granules, and lysosomes.1-3 Dense granules harbor small molecules that upon release amplify platelet activation and adhesion, blood vessel constriction, and wound repair.4-6 granules store protein factors that facilitate platelet adhesion, clot stabilization, fibrinolysis, angiogenesis, wound repair, and inflammation.7-9 Lysosomes store proteolytic enzymes that likely contribute to thrombus remodeling.3 Granule contents are normally released upon platelet stimulation. 10 Disorders of granule secretion11-13 or granule formation1,14 result in excessive bleeding. Hermansky-Pudlak syndrome (HPS) is a group of autosomal recessive disorders characterized by prolonged bleeding, oculocutaneous albinism, and other symptoms.15,16 Clinically significant bleeding diathesis in HPS has been ascribed to platelet dense granule malformation.5,16 Platelets in HPS patients and mouse models17 lack detectable dense granules by electron microscopy18 and do not effectively store serotonin and adenine nucleotides or release them upon stimulation.19-21 Consequently, platelet aggregation in vitro is impaired.22 These defects likely reflect impaired delivery of membrane contents to nascent dense granules within megakaryocytes or proplatelets. The genes that are mutated in the 9 known HPS variants and in 12 of 15 mouse HPS models encode subunits of distinct protein complexes (adaptor protein-3 [AP-3] and biogenesis of lysosome-related organelles complex [BLOC]-1, -2, and -3) that function in transmembrane cargo delivery to lysosome-related organelles (LROs) in other cell types.15,23-25 AP-3 sorts cargoes from endosomes into transport carriers toward lysosomes or LROs.26 BLOC-3 is a guanine nucleotide exchange factor for the tissue-restricted Rab GTPases RAB32 and RAB38,27 which function with BLOC-1 and BLOC-2 in as yet unclear ways to deliver cargoes from endosomes to LROs in melanocytes.25 RAB32 and RAB38 regulate cargo localization to dense granuleClike compartments in a megakaryocytoid cell line,28 but how AP-3 and BLOCs function in megakaryocytes and platelets is not known. Dense granules and granules are both LROs29 and, like lysosomes, are proposed to derive from similar multivesicular precursors30,31 and to employ similar fusion machinery for secretion.11-13,32 Nevertheless, the formation of and dense granules is differentially controlled. For example, NBEAL2, VPS16B, and VPS33B regulate the biogenesis of granules but not dense granules.33-37 In platelets of HPS patients, the number, morphology, and content levels of granules and lysosomes are normal.17,38,39,40 Thrombin-induced secretion of granule and lysosome contents was impaired in platelets from 1 uncharacterized HPS patient,41 but has not been systematically analyzed in different HPS subtypes. Thrombin-induced lysosome secretion from platelets in mouse HPS models was reported to vary from modestly impaired to hyperactive.17,39 To assess platelet granule and lysosome secretion in HPS variants, we exploited 3 congenic mouse HPS models with defects in different protein complexes: pearl (or BLOC-1?/?), a model for HPS type 9 that lacks BLOC-144,45; and light ear (or BLOC-3?/?), a model for HPS type 4 that lacks BLOC-3.46,47 Platelets from each model were analyzed for agonist-dependent secretion from granules and lysosomes using ex vivo assays and intravital Colchicine imaging. We provide evidence that AP-3, BLOC-1, and BLOC-3 impact the release of multiple platelet granule types. Materials and methods Mouse strains Experiments used 12- to 13-week-old male C57BL/6J (wild-type [WT]) and congenic B6-Cg-Web site. For intravital imaging, antibodies or F(ab)2 fragments were labeled using Alexa Fluor monoclonal antibody labeling kits according to the manufacturers instructions (Invitrogen, Carlsbad, CA). Chemicals were from Sigma-Aldrich (St Louis, MO) and reagents were from Invitrogen unless otherwise specified. The DuoSet ELISA Development kit was from R&D Systems (Minneapolis, MN). Platelet preparation and flow cytometry analysis Platelets were isolated from blood by differential centrifugation as described by Pang et al48 (supplemental Methods). Platelets resuspended in Tyrodes solution buffered with values were determined by the Bonferroni multiple comparison test. Results Activation-induced cell surface expression of CD62p in response to low-dose agonist is Colchicine impaired in HPS model platelets To assess granule secretion, we first probed for cell surface exposure of CD62p (P-selectin) after fusion.