ABSTRACT
We previously showed that the MRP4 (ABCC4) transporter is expressed in human platelet delta-granules and may be involved in ADP transport. We now demonstrate by immunoblotting and immunofluorescence microscopy that platelet MRP4 is absent in two patients with a platelet delta-storage pool deficiency (delta-SPD)-like phenotype with reduced platelet adenine nucleotide (AN) but normal serotonin levels, whereas their other membrane marker proteins of platelet granules were normally expressed and localized. In these patients, MRP4 was present in lymphocytes, and the coding region of their MRP4/ABCC4 gene did not show any mutation that explained the lack of expression. In platelets with "classic" delta-SPD (low AN and serotonin levels), MRP4 was quantitatively (immunoblot) normal, but, like other delta-granules membrane marker proteins (eg, LAMP2), was mostly displaced from delta-granules to patches at the plasma membrane, suggesting that platelets with classic delta-SPD have an abnormality that impairs the assembly of normal delta-granules. Thus, defective expression of platelet MRP4 is associated with selective defect in AN storage. The genetic basis of the new delta-SPD phenotype remains to be elucidated.
Subject(s)
Adenine Nucleotides/metabolism , Blood Platelets/metabolism , Multidrug Resistance-Associated Proteins/metabolism , Platelet Storage Pool Deficiency/metabolism , Adolescent , Adult , Female , Gene Expression Regulation , Humans , Leukocytes/metabolism , Male , Multidrug Resistance-Associated Proteins/genetics , Phenotype , Platelet Function Tests , Platelet Storage Pool Deficiency/genetics , Platelet Storage Pool Deficiency/pathology , Platelet Storage Pool Deficiency/physiopathology , Protein Transport , RNA, Messenger/genetics , RNA, Messenger/metabolism , Young AdultABSTRACT
OBJECTIVE: Hereditary thrombocytopenias characterized by mutations in the gene for non-muscle myosin heavy chain IIA (NMMHC-IIA) are known as MYH9-related hereditary macrothrombocytopenia, and include the May-Hegglin anomaly, Sebastian platelet syndrome, Fechtner syndrome, and Epstein syndrome. Despite the presence of thrombocytopenia, these patients often have only mild or non-bleeding phenotypes. A major risk for these patients can be inappropriate treatment with long-term corticosteroids or splenectomy for misdiagnosed chronic autoimmune thrombocytopenia, as well as inadequate peri- and postoperative management. METHODS: Using the case of a 44-yr-old male with Fechtner syndrome (macrothrombocytopenia, leukocyte inclusions, sensorineural deafness, glomerulonephritis) who underwent neurosurgery for an intracerebral arteriovenous malformation, we describe current methods to diagnose hereditary MYH9-related macrothombocytopenia by analysis of the blood smear, immunofluorescence staining of the NMMHC-IIA in leucocytes, and by MYH9-gene sequencing. RESULTS: Clusters of NMMHC-IIA in granulocytes and a R1165C mutation in the MYH9-gene in two macrothrombocytopenic family members confirmed the diagnosis of a MYH9-related disease. The patient had no bleeding diathesis by history or physical examination. Thus no perioperative prohemostatic pharmacologic therapies or transfusions were given, with only minimal bleeding observed. Postoperative antithrombotic thromboprophylaxis was not given because of anticipated enhanced risk for bleeding. However, the patient developed symptomatic pulmonary embolism on postoperative day 6, which was successfully managed with 8 months of anticoagulation. CONCLUSION: MYH9-related hereditary macrothrombocytopenia does not necessarily protect against postoperative venous thromboembolism, and affected patients who do not evince bleeding diathesis should be considered for routine postoperative pharmacologic thromboprophylaxis.
Subject(s)
Molecular Motor Proteins/genetics , Myosin Heavy Chains/genetics , Perioperative Care , Thrombocytopenia/etiology , Adult , Humans , Male , Mutation , Premedication , Pulmonary Embolism/drug therapy , Pulmonary Embolism/etiology , Syndrome , Thrombocytopenia/drug therapyABSTRACT
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. Furthermore, an altered distribution of MRP4 was observed in platelets from a patient with Hermansky-Pudlak syndrome with defective delta-granules. Adenosine triphosphate (ATP)-dependent cyclic guanosine monophosphate (cGMP) transport codistributed with MRP4 detection in subcellular fractions, with highest activities in the dense granule and plasma membrane fractions. This transport was inhibited by dipyramidole, indomethacin, and MK571 with median inhibitory concentration (IC(50)) values of 12, 22, and 43 microM, and by ibuprofen. Transport studies with [(3)H]ADP indicated the presence of an orthovanadate-sensitive ADP transporting system, inhibited by dipyramidole, MK571, and cyclic nucleotides. The results indicate a function of MRP4 in platelet mediator storage and inhibition of MRP4 may represent a novel mechanism for inhibition of platelet function by some anti-inflammatory drugs.
