Platelet aggregation is initiated by the release of mediators as adenosine diphosphate (ADP) stored in platelet granules. Possible candidates for transport proteins mediating accumulation of these mediators in granules include multidrug resistance protein 4 (MRP4, ABCC4), a transport pump for cyclic nucleotides and nucleotide analogs. We investigated the expression of MRP4 in human platelets by immunoblotting, detecting a strong signal at 170 kDa. Immunofluorescence microscopy using 2 MRP4-specific antibodies revealed staining mainly in intracellular structures, which largely colocalized with the accumulation of mepacrine as marker for delta-granules and to a lower extent at the plasma membrane. IntroductionThe critical role played by platelets in hemostasis and thrombosis is related to their function as exocytotic cells that secrete effector molecules at the side of vascular injury. Platelets contain at least 3 types of intracellular granules, in which these mediators are stored and concentrated, known as alpha, dense, and lysosomal granules. 1 While alpha granules contain mainly polypeptides, as fibrinogen, von Willebrand factor, growth factors, and protease inhibitors, dense granules contain small molecules, specifically adenosine diphosphate (ADP), adenosine triphosphate (ATP), serotonin, and calcium. 2 Humans with defective dense granule exocytosis suffer from delta storage pool disease associated with a moderate bleeding tendency. The most severe delta storage pool disease is observed in Hermansky-Pudlak syndrome (HPS), a rare autosomal recessive disorder in which oculocutaneous albinism, bleeding, and lysosomal ceroid storage result from defects of melanosomes, platelet-dense granules, and lysosomes. [3][4][5] Little is known, however, about transport proteins mediating accumulation of the effector molecules in granules or their transport across the plasma membrane. Possible candidates include the multidrug resistance protein 4 (MRP4/ABCC4) and MRP5 (ABCC5). These belong to the C-branch of the human ATP-binding cassette (ABC) transporter superfamily, which consists of 12 members, 9 of which comprise the group of multidrug resistance proteins (MRP1-9; ABCC1-6 and ABCC10-12). 6,7 MRPs are integral membrane glycoproteins that mediate the primary active unidirectional export of organic anions from cells. Conjugates of lipophilic compounds with glutathione, glucuronate, and sulfate are preferred substrates of MRP1-3, [8][9][10] while cyclic purine nucleotides and nucleotide analogs have been identified as substrates for MRP4 and MRP5. 11-15 MRP5 mRNA has been detected in many tissues, 16,17 and the MRP5 protein could be localized in erythrocytes, 12 in smooth muscle cells of the genitourinary tract, 18 and in human heart cardiomyocytes, vascular endothelial, and smooth muscle cells. 19 MRP4 mRNA was detected in prostate, liver, testis, ovary, brain, kidney, and adrenal gland. 16,[20][21][22] Studies in membrane vesicles containing recombinant MRP4 indicate that it represents a transporter with a relatively br...

Hermansky-Pudlak syndrome (HPS) is a rare, autosomal recessive disorder in which oculocutaneous albinism, bleeding, and lysosomal ceroid storage result from defects of multiple cytoplasmic organelles-melanosomes, platelet-dense granules, and lysosomes. As reported elsewhere, we mapped the human HPS gene to chromosome segment 10q23, positionally cloned the gene, and identified three pathologic mutations of the gene, in patients from Puerto Rico, Japan, and Europe. Here, we describe mutation analysis of 44 unrelated Puerto Rican and 24 unrelated non-Puerto Rican HPS patients. A 16-bp frameshift duplication, the result of an apparent founder effect, is nearly ubiquitous among Puerto Rican patients. A frameshift at codon 322 may be the most frequent HPS mutation in Europeans. We also describe six novel HPS mutations: a 5' splice-junction mutation of IVS5, three frameshifts, a nonsense mutation, and a one-codon in-frame deletion. These mutations define an apparent frameshift hot spot at codons 321-322. Overall, however, we detected mutations in the HPS gene in only about half of non-Puerto Rican patients, and we present evidence that suggests locus heterogeneity for HPS.

Hermansky-Pudlak syndrome (HPS) is a disorder of organelle biogenesis in which oculocutaneous albinism, bleeding and pulmonary fibrosis result from defects of melanosomes, platelet dense granules and lysosomes. HPS is common in Puerto Rico, where it is caused by mutations in the genes HPS1 and, less often, HPS3 (ref. 8). In contrast, only half of non-Puerto Rican individuals with HPS have mutations in HPS1 (ref. 9), and very few in HPS3 (ref. 10). In the mouse, more than 15 loci manifest mutant phenotypes similar to human HPS, including pale ear (ep), the mouse homolog of HPS1 (refs 13,14). Mouse ep has a phenotype identical to another mutant, light ear (le), which suggests that the human homolog of le is a possible human HPS locus. We have identified and found mutations of the human le homolog, HPS4, in a number of non-Puerto Rican individuals with HPS, establishing HPS4 as an important HPS locus in humans. In addition to their identical phenotypes, le and ep mutant mice have identical abnormalities of melanosomes, and in transfected melanoma cells the HPS4 and HPS1 proteins partially co-localize in vesicles of the cell body. In addition, the HPS1 protein is absent in tissues of le mutant mice. These results suggest that the HPS4 and HPS1 proteins may function in the same pathway of organelle biogenesis.

Factor X deficiency is a rare haemorrhagic condition, normally inherited as an autosomal recessive trait, in which a variable clinical presentation correlates poorly with laboratory phenotype. The factor X (F10) genes of 14 unrelated individuals with factor X deficiency (12 familial and two sporadic cases) were sequenced yielding a total of 13 novel mutations. Family studies were performed in order to distinguish the contributions of individual mutant F10 alleles to the clinical and laboratory phenotypes. Missense mutations were studied by means of molecular modelling, whereas single basepair substitutions in splice sites and the 5' flanking region were examined by in vitro splicing assay and luciferase reporter gene assay respectively. The deletion allele of a novel hexanucleotide insertion/deletion polymorphism in the F10 gene promoter region was shown by reporter gene assay, to reduce promoter activity by approximately 20%. One family manifesting an autosomal dominant pattern of inheritance possessed three clinically affected members who were heterozygous for a splice-site mutation that was predicted to lead to the production of a truncated protein product. A model which accounts for the dominant negative effect of this lesion is presented. Variation in the antigen level of heterozygous relatives of probands was found to be significantly higher between families than within families, consistent with the view that the nature of the F10 lesion(s) segregating in a given family is a prime determinant of the laboratory phenotype. By contrast, no such relationship could be discerned between laboratory phenotype and polymorphism genotype.

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