211/223. Characterization of a novel bleeding disorder with isolated prolonged bleeding time and deficiency of platelet microvesicle generation. Platelet prothrombinase activity and microvesicle (MV) generation were measured in four patients from three unrelated families with a life-long bleeding disorder associated with slightly prolonged bleeding time and isolated defective serum prothrombin consumption, without platelet function abnormality or von Willebrand factor defect. MV generation was reduced in all the patients either after thrombin plus collagen or A23187 calcium ionophore stimulation, whereas, at variance with Scott syndrome, prothrombinase activity was normal. This abnormality constitutes a new bleeding disorder, which provides new insights into the possible role of platelet microvesicles in health and disease. Furthermore, the results of this study suggest that MV generation should be investigated in patients with a bleeding history and apparently isolated prolonged bleeding time when prothrombin consumption in serum is defective and all other investigations are normal. ( info) |
212/223. Molecular characterization of a dominantly inherited bleeding disorder with impaired platelet responses to thromboxane a2. thromboxane a2 (TXA2) is a major arachidonic acid metabolite of platelets and induces platelet functions by binding to specific receptors on the membrane. We have found patients with hemostatic defects due to impaired platelet responses to TXA2, and molecular characterization of the patients has been carried out. Platelets from these two unrelated patients showed impaired aggregation responses to TXA2 and its analogues despite the normal response to thrombin. Although the patients' platelets exhibited normal binding activities to TXA2 analogues, they showed decreased GTPase activity and second messenger formation when stimulated by STA2, a stable TXA2 agonist. To understand the molecular basis of this abnormality, we determined the cDNA sequence of the TXA2 receptor by reverse transcription-polymerase chain reaction (RT-PCR) from the patient's platelet RNA, and identified a single amino acid substitution (Arg60 for Leu) in the first cytoplasmic loop of the receptor. This mutation was found in both isoforms of the platelet TXA2 receptor which we have recently found: TXR alpha with the same structure as the placental TXA2 receptor and TXR beta with the same structure as the endothelial TXA2 receptor, and was detected exclusively in affected members of two unrelated families with the disorder. The mutant TXR alpha and TXR beta expressed in COS-m6 cells showed decreased agonist-induced phospholipase C activation despite their normal ligand binding affinities. These results suggest that the Arg60 for Leu mutation is responsible for the disorder and imply a critical role for the first cytoplasmic loop in the interaction of the TXA2 receptor with the G protein. ( info) |
213/223. Missense mutations of the glycoprotein (GP) Ib beta gene impairing the GPIb alpha/beta disulfide linkage in a family with giant platelet disorder. We describe here the molecular basis of an isolated hereditary giant platelet disorder (GPD) which is not accompanied with thrombocytopenia or leukocyte inclusion. platelet aggregation with ristocetin and botrocetin was almost normal in this patient. Flow cytometric analysis showed that the glycoprotein (GP) Ib/IX complex was expressed on the platelet membranes at decreased levels. The amount of platelet GPIb alpha and the plasma glycocalicin concentration, the water-soluble extracellular portion of GPIb alpha, were also decreased. The anti-GPIb alpha antibody coprecipitated GPIb beta and GPIX, although the ratios of these polypeptides to GPIb alpha was greatly decreased compared with the ratio in normal platelets. Immunoblot analysis under nonreduced conditions showed that most of the GPIb alpha in the patient's platelets was not disulfide linked with GPIb beta. dna sequencing analysis showed compound heterozygosity for two independent single nucleotide substitutions: from Tyr (TAC) to Cys (TGC) at residue 88, and from Ala (GCC) to Pro (CCC) at residue 108 in her GPIb beta gene. These substitutions were not found in genomic dna samples from 108 normal individuals. These mutations might result in decreased expression of the GPIb/IX complex and may influence the association of the complex with the membrane skeleton, consequently impairing normal platelet morphology. Furthermore, the phenotype caused by mutations in the subunits of the GPIb/IX complex could span the spectrum from a normal phenotype, to isolated GPD, to a full-blown bleeding disorder, such as bernard-soulier syndrome. ( info) |
214/223. Deficiency of (33P)2MeS-ADP binding sites on platelets with secretion defect, normal granule stores and normal thromboxane a2 production. Evidence that ADP potentiates platelet secretion independently of the formation of large platelet aggregates and thromboxane a2 production. By the term "Primary Secretion Defect" (PSD), we mean a common heterogeneous group of congenital defects of platelet secretion, characterized by a normal primary wave of platelet aggregation induced by ADP and other agonists, a normal concentration of platelet granule contents, and normal production of thromboxane A2. The biochemical abnormalities responsible for PSD are not well known. Since a secretion defect similar to PSD is found in platelets that are severely deficient of binding sites for the ADP analogue 2MeS-ADP and do not aggregate in response to ADP, we tested the hypothesis that PSD platelets have moderately decreased 2MeS-ADP binding sites, which may be sufficient for normal ADP-induced aggregation but not for potentiating platelet secretion. The specific binding of [33P]2MeS-ADP to platelets from 3 PSD patients (347, 443 and 490 sites/platelet; KD 2.8-3.9 nM) was lower than to platelets from 24 normal subjects (647 [530-1102]; KD = 3.8 [2.3-7.3]) (median [range]). Normal values were found in a fourth PSD patient (710; KD 3.7). The degree of inhibition of PGE1-induced cAMP increase by 0.1 microM ADP was lower in patients than in controls. The secretion induced by the endoperoxide analogue U46619 from normal, acetylsalicylic acid-treated platelets under conditions that prevented the formation of large aggregates was potentiated by 1 mumol/l ADP and inhibited by apyrase. These findings indicate that a partial deficiency of the platelet ADP receptor(s) might be responsible for the defect of platelet secretion in some PSD patients and that ADP potentiates platelet secretion independently of the formation of large aggregates and thromboxane a2 production. ( info) |
215/223. Defective signal transduction through the thromboxane a2 receptor in a patient with a mild bleeding disorder: deficiency of the inositol 1,4,5-triphosphate formation despite normal G-protein activation. We describe an 11-year-old girl with a mild bleeding disorder since early childhood. The disorder was characterized by a prolonged bleeding time, and the patient's platelets showed defective aggregation responses to thromboxane a2 (TXA2) mimetic U46619 and arachidonic acid. In contrast, the platelets showed normal responses to thrombin and Ca ionophore A23187. When the platelet TXA2 receptor was examined with the [3H]-labeled TXA2 agonist U46619, the equilibrium dissociation rate constants (kd) and the maximal concentration of binding sites (Bmax) of the patient's platelets were within normal ranges. Normal GTPase activity was also induced in the patient's platelets by stimulation with U46619, however, inositol 1,4,5-triphosphate (IP3) formation was not induced by U46619. These results suggests that the patient's platelets had a defect in phospholipase C activation beyond TXA2 receptors. ( info) |
Two patients receiving preoperative carbenicillin manifested platelet dysfunction and severe wound hemorrhage. Platelet transfusions appeared beneficial. Investigations indicate that hemostatic abnormalities, especially platelet aggregation defects, may be produced by carbenicillin. Extreme caution and awareness of possible hemorrhage are advised when carbenicillin is used preoperatively. ( info) |
217/223. Familial pulmonary fibrosis associated with oculocutaneous albinism and platelet function defect. A new syndrome. A family was studied in which four siblings had oculocutaneous albinism. In three of these a platelet function defect characterized by poor response to collagen was found. The fourth had previously died from cryptogenic fibrosing alveolitis. Two of the survivors had cryptogenic fibrosing alveolitis and the third had physiological lung function disturbance. bone marrow examination of one showed pigment laden macrophages (Hermansky-Pudlak Syndrome). Three other normally pigmented family members were found to have normal platelets and no evidence of cryptogenic fibrosing alveolitis, although one had pulmonary disease attributable to occupational dust exposure. To elucidate the aetiological factors involved, three unrelated normally pigmented patients with known platelet function defect were studied. One proved to have cryptogenic fibrosing alveolitis. Four unrelated albinos were also studied and had normal lungs and platelets. It is suggested that, in addition to the known association between platelet function defect and albinism, there is an association between a platelet function defect and cryptogenic fibrosing alveolitis. ( info) |
218/223. Defect of platelet function associated with chronic hypoglycaemia. Two patients are described with chronic hypoglycaemia; the first having glucose-6-phosphatase deficiency (type I glycogen storage disease), and the second fructose 1:6-diphosphatase deficiency. Both cases were associated with a bleeding diathesis, a defect of platelet aggregation, and a deficiency of platelet adenine nucleotides. The effect on the platelet abnormalities of a period of normoglycaemia was studied in both patients. Correction of the platelet abnormalities occurred rapidly after stabilization of the blood glucose within the normal range. Normal function persisted for the duration of the normoglycaemia, facilitating diagnostic liver biopsy and surgical procedures. A biochemical explanation for the nucleotide deficiency is suggested. ( info) |
219/223. Aminophospholipid exposure, microvesiculation and abnormal protein tyrosine phosphorylation in the platelets of a patient with Scott syndrome: a study using physiologic agonists and local anaesthetics. The Scott syndrome is a rare inherited haemorrhagic disorder characterized by the inability of blood cells to expose aminophospholipids and to shed microparticles. We have had the opportunity to study a recently reported French patient with this syndrome and have confirmed by means of a fluorescence assay for transbilayer lipid movement a reduced aminophospholipid exposure when platelets were stimulated with the calcium-ionophore ionomycin, in spite of a normal elevation of intracellular Ca2 . Secretion and calpain activation were also shown to be normal. Significantly, the level of phosphotyrosine-labelled proteins in platelets treated with thrombin or a thrombin collagen mixture and in particular the phosphorylation of a 40 kD band were severely reduced. Furthermore, inhibition of thiol-containing enzymes. including tyrosine-phosphatases, by N-ethyl maleimide did not lead to aminophospholipid exposure in the patient's platelets, in spite of increased tyrosine protein phosphorylation. In contrast, amphiphilic membrane drugs such as tetracaine and propranolol induced both surface aminophospholipid exposure in Scott platelets and the shedding of microparticles, thereby showing that membrane perturbation can lead to loss of phospholipid asymmetry in this syndrome. Our results provide the first insight that the lack of expression of procoagulant phospholipids and microparticle formation in Scott syndrome platelets is associated with a defect of intracellular signalling. ( info) |
220/223. necrosis, haemorrhage and complement depletion following bites by the spitting cobra (Naja nigricollis). The Spitting Cobra, Naja nigricollis, is widely and densely distributed in africa. Fourteen patients with proven N. nigricollis bites, who were seen in the savanna region of nigeria, did not exhibit the neurological signs, such as cranial nerve lesions and respiratory paralysis, expected following Elapid poisoning. All had local swelling, in eight cases involving the entire limb, and ten developed local tissue necrosis. Spontaneous haemorrhage was detected in three cases and was the probable cause of death in one of them; the other death in this series was unexplained. Haematological abnormalities included prolonged clot lysis anf failure of clot retraction due to a platelet defect. There was no specific deficit in clotting factors and a delayed rise in fibrin degradation products was attributed to extensive tissue damage at the site of the bit. Most patients showed depletion of complement component C3 and glycine-rich beta-glycoprotein (GBG), suggesting activation of the alternative pathway of complement fixation. There was evidence of hepatocellular damage in two out of six patients investigated. There was no evidence that specific polyvalent antivenoms, used in doses of up to 80 ml, prevented any of the effects of N. nigricollis venom. Clinical laboratory diagnosis is discussed. In the past many bites were wrongly classified as viper bites on the basis of clinical findings. Immunodiagnosis is a promising method for assessing the true importance of N. nigricollis bite in West africa. ( info) |