Platelet dense granule storage pool disease (SPD) is a bleeding disorder characterized by a lack of normal platelet dense granule function, as evidenced by decreased platelet aggregation in response to ADP, epinephrine and collagen. Platelet SPD has been studied most extensively in humans and rodents with Hermansky-Pudlak syndrome (HPS), whose phenotype is a result of defects in granule trafficking, leading to oculocutanous albinism, lysosomal storage diseases, and platelet dysfunction. We have been characterizing the fawn-hooded hypertensive (FHH) rat, which has been previously shown to have a bleeding disorder consistent with a platelet SPD and some of the features of HPS. While the platelets in the FHH rat have normal alpha granules and lysosomes, they lack dense granules as assessed by transmission electron microscopy. Platelet flow cytometric analysis of GPIb and GPIIb indicated that the FHH platelets have normal surface expression of these adhesion proteins. The FHH rat has a mutation in the Rab38 gene at the ATG start site, which is associated with the bleeding disorder. Rab38 is part of a large family of GTPases, which are involved in granule formation and secretion. Western blotting of FHH tissues revealed that there is no expression of Rab38 protein. We have used confocal immunomicroscopy to assess Rab38 in platelet formation and function. In normal rat and human platelets, there was punctate expression of Rab38. There was no Rab38 staining detected in FHH platelets. In human megakaryocytic cell lines, Dami and HEL cells, there was punctate staining of Rab38 that was mainly in the periphery of the cells, with a variable amount of perinuclear staining. There was partial colocalization of Rab38 with serotonin and VWF, and with Lamp-3, a marker of lysosomes. The degree of colocalization varied between cells. There was no clear association of Rab38 with actin and tubulin in megakaryocytes. We also examined a cohort of patients with SPD, but not HPS, for mutations in Rab38. The entire coding region and intron-exon boundaries of the Rab38 gene were sequenced in 18 patient samples collected at Emory University for the CDC Women with Bleeding Disorders and Menorrhagia Study. Ten of the patients had platelet function defects documented by standard platelet aggregation studies, and eight had no identifiable platelet function defect. No mutations in Rab38 were detected. Whereas numerous known polymorphisms were identified and confirmed, there was no association of any of them with platelet function abnormalities. In conclusion, Rab38 is expressed in platelets and megakaryocytes and may interact with other granule proteins during megakaryocyte development. Failure to express Rab38 is associated with platelet dysfunction. Further studies are needed to determine its function in megakaryocytes and platelets, and to determine whether defects in Rab38 are a cause of platelet SPD in humans.

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