calcimycin and Blood-Platelet-Disorders

Topics: Antigens, CD; Blood Platelet Disorders; Calcimycin; Calcium; Cell Differentiation; Dendritic Cells; Humans; Ionophores; Phenotype; Platelet Aggregation

We report three cases of platelet dysfunction characterized by defective Ca2+ ionophore-induced platelet aggregation without impaired production of thromboxane A2 (TXA2). The patients had mild to moderate bleeding tendencies, and their platelet aggregation and secretion induced by ADP, collagen, arachidonic acid, stable TXA2 (STA2) and Ca2+ ionophore A23187 was defective or much reduced. However, ristocetin- or thrombin-induced platelet aggregation was normal. The analysis of second messenger formation showed that inositol 1,4,5-triphosphate formation or Ca2+ mobilization induced by thrombin, STA2 or A23187 was normal. Furthermore, the phosphorylation of 47 kDa protein (pleckstrin) and 20 kDa protein (myosin light chain, MLC) in response to those agonists was normal. These findings suggest that the defective site in the patients' platelets lies in the process distal to or independent of protein kinase C activation, Ca2+ mobilization and MLC phosphorylation.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Adult; Arachidonic Acid; Blood Platelet Disorders; Blood Proteins; Calcimycin; Calcium; Collagen; Female; Humans; Ionophores; Male; Middle Aged; Myosin Light Chains; Phosphoproteins; Phosphorylation; Platelet Aggregation; Platelet Membrane Glycoproteins; Ristocetin; Thrombin; Thromboxane A2

Studies were performed on a patient with a longstanding bleeding disorder whose major defects were impaired platelet prothrombinase activity in the absence of added factor Va, and a platelet factor V value that was either decreased or at the lower limit of normal when assayed on multiple occasions. In contrast, plasma factor V values were consistently normal. Unlike Scott Syndrome, in which platelet prothrombinase activity is decreased in both the presence and absence of added factor V, her platelets appeared to utilize added factor Va normally in supporting the generation of prothrombinase activity. These findings suggest an intrinsic defect in platelet factor V as the basis of her platelet prothrombinase defect. This defect appears to be different than that described in the Quebec platelet disorder (factor V Quebec). Immunoblot analyses of washed platelet lysates demonstrated a pattern of variably sized factor V molecules that was entirely similar to that observed in normal platelets, and both the heavy and light chains of her factor V after thrombin cleavage were of the same size as that observed in normal platelets. In addition, her platelet multimerin was normal and immunoblot analysis excluded the type of generalized granular protein defect and pathological proteolysis that has been suggested to explain the factor V defect in the Quebec platelet disorder. The findings in this patient thus suggest a new type of platelet factor V defect as the basis for the impaired capacity of her activated platelets to support prothrombinase generation. The findings further support an important role for platelet factor V in hemostasis.

Topics: Blood Coagulation Factors; Blood Platelet Disorders; Calcimycin; Dose-Response Relationship, Drug; Factor Va; Female; Hemorrhage; Humans; Kinetics; Thrombin; Thromboplastin

Topics: Adenosine Diphosphate; Blood Platelet Disorders; Calcimycin; Humans; Ionophores; Membrane Proteins; Platelet Aggregation; Receptors, Purinergic P2; Receptors, Purinergic P2Y12

We had postulated that in a patient with defective calcium ionophore (A23187)-induced platelet aggregation, whose platelets showed normal intracellular Ca2+ mobilization in either the presence or absence of extracellular Ca2+ in response to A23187. A defect was present in an intracellular calcium-dependent process. We have now investigated whether the agonist-induced protein-tyrosine phosphorylation (PTP) was altered. Protein-tyrosine phosphorylation (PTP)-induced by A23187 in the patient's platelets was greatly diminished but that induced by thrombin was almost normal. These results suggest that an intracellular calcium-dependent process plays a fundamental role in A23187-induced PTP, whereas it does not in thrombin-induced PTP.

Topics: Blood Platelet Disorders; Blood Platelets; Calcimycin; Calcium; Extracellular Space; Humans; Phosphorylation; Platelet Aggregation; Protein-Tyrosine Kinases; Thrombin

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.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Diphosphate; Blood Platelet Disorders; Blood Platelets; Calcimycin; Child; Female; GTP Phosphohydrolases; GTP-Binding Proteins; Humans; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Kinetics; Platelet Aggregation; Prostaglandin Endoperoxides, Synthetic; Receptors, Thromboxane; Signal Transduction; Thrombin; Thromboxane A2; Type C Phospholipases

Calcium changes in normal and thrombasthenic platelets were recorded using the PICA-apparatus. Aequorin was loaded in the presence of DMSO, EGTA and PGE1. Platelets of three patients with type I thrombasthenia stimulated with A-23, 187, thrombin, PMA in the presence 1 mM Ca++ and 1 mM Mg++ were able to normally raise their calcium concentrations. The maximal values could be found below the normal range with collagen, ADP and PAF-acether. Calcium mobilization from internal stores in response to thrombin was normal. There were two calcium peaks in normal platelets stimulated with ADP. The second one was suppressed by omitting fibrinogen, stirring, or by adding aspirin, and was absent in thrombasthenic platelets. Thus the GP IIb-IIIa complex is not a prerequisite for calcium fluxes but is involved, when weak agonists such ADP are used, through an aggregation-dependent reinforcement of platelet activation.

Topics: Adenosine Diphosphate; Aequorin; Blood Platelet Disorders; Blood Platelets; Calcimycin; Calcium; Collagen; Humans; In Vitro Techniques; Luminescent Proteins; Platelet Activating Factor; Platelet Aggregation; Tetradecanoylphorbol Acetate; Thrombasthenia; Thrombin

Platelets from dogs with Basset Hound Hereditary Thrombopathy (BHT) initially displayed a thrombasthenia-like aggregation defect but have been shown to have normal amounts of platelet membrane glycoproteins IIb and IIIa (GPIIb-IIIa), and therefore are more accurately described as thrombopathic. The presence of normal quantities of GPIIb-IIIa, however, did not rule out the possibility of a functionally abnormal glycoprotein complex which would be unable to bind radio-labeled fibrinogen. Therefore, fibrinogen binding in BHT platelets was evaluated. Platelets from BHT and normal dogs were activated with 1 x 10(-5) M ADP in the presence of 125I-fibrinogen and the surface-bound radioactivity was quantitated. The amount of fibrinogen bound by BHT dog platelets was not significantly different than that bound by normal dog platelets. Platelets from dogs with BHT bound 30,282 +/- 3,133 and normal dog platelets bound 31,664 +/- 2,772 molecules of fibrinogen per platelet. The quantitatively normal GPIIb-IIIa complex binds fibrinogen in normal amounts and does not seem to represent the abnormality responsible for the aggregation defect in BHT platelets. Therefore, other factors central to normal platelet function and related to platelet aggregation must be considered.

Topics: Animals; Blood Platelet Disorders; Calcimycin; Dog Diseases; Dogs; Fibrinogen; Platelet Aggregation; Platelet Membrane Glycoproteins

A four-year-old Standardbred gelding presented with a 3.5 year history of intermittent epistaxis and spontaneous submucosal petechiae and ecchymoses in the nares and the mouth. Routine haematological and biochemical examinations were unremarkable. A thrombocytopathy was suspected when activated partial thromboplastin time, one stage prothrombin time, plasma fibrinogen and the platelet count were all normal. The patient's platelets failed to aggregate with serotonin, adenosine diphosphate, collagen (at 20 micrograms/ml) or the endoperoxide analogue U46619. Very high levels of collagen (100 micrograms/ml) did cause aggregation. The response to the calcium ionophore A23187 was reduced and although complete degranulation occurred the resulting aggregates were unstable. Thromboxane generation in response to collagen and ADP was inferred from the concentration of its stable metabolite thromboxane B2 and was reduced. A diagnosis of a thrombasthenia-like syndrome possibly equivalent to Type II Glanzmann's thrombasthenia in people was made.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Diphosphate; Animals; Blood Platelet Disorders; Blood Platelets; Calcimycin; Collagen; Epistaxis; Female; Horse Diseases; Horses; Male; Microscopy, Electron; Platelet Aggregation; Prostaglandin Endoperoxides, Synthetic; Serotonin; Thrombasthenia

Agonist-induced platelet cytoplasmic Ca2+ concentrations ([Ca2+]i) in patients with congenital cyclo-oxygenase deficiency (A) and with impaired aggregation to A23187 (B) were measured with aequorin in the presence or absence of extracellular Ca2+. The influence of TMB-8 or ONO3708 on agonist-induced [Ca2+]i in those platelets was also investigated. In Patient 1, there was a single aequorin luminescence peak in response to arachidonate, which was a thromboxane A2(TXA2) independent Ca2+ influx. The luminescence peak due to the formation of TXA2 was not detectable. The A23187-induced [Ca2+] i was decreased in the presence of extracellular Ca2+, but was within normal limits in the absence of extracellular Ca2+. A thrombin or STA2-induced elevation of [Ca2+] i was always within normal limits under any conditions. These results suggest that cyclo-oxygenase activity (CO activity) contributes to the A23187-induced Ca2+ influx, but does not contribute to the Ca2+ release from intracellular stores, and that the thrombin or STA2-induced Ca2+ influx and release do not depend on the CO activity. In Patient 2, the time lag from the addition of A23187 to the aequorin luminescence peak was found both in the presence and absence of extracellular Ca2+, which was more obvious in the latter. This A23187-induced elevation of [Ca2+] i disappeared after treatment of the platelets with TMB-8 in the absence of extracellular Ca2+, which is rarely seen in normal platelets. The most striking finding was that the thrombin-induced rise in [Ca2+] i in the absence of extracellular Ca2+ was not detectable. These findings might be closely related to abnormal platelet function in this patient.

Topics: Blood Platelet Disorders; Blood Platelets; Calcimycin; Calcium; Cytoplasm; Humans; Platelet Aggregation; Prostaglandin-Endoperoxide Synthases

Phosphoinositide/polyphosphoinositide (PI/PPI) metabolism, measured by the increase of 3H-phosphatidic acid (PA) and the decrease of 3H-phosphatidylinositol (PI) in 3H-arachidonate-labeled platelet suspensions, was assessed in five patients whose platelet functional defects included impaired initial rates of ADP, epinephrine and U44069 aggregation in platelet-rich plasma (PRP). In one patient, 3H-PA formation induced by collagen and thrombin was reduced or absent on two of three occasions, and the decrease in 3H-PI was reduced on one of these two occasions in response to collagen and A23187, and on all 3 occasions in response to thrombin. The variations in the formation of 3H-PA in this patient on different occasions broadly paralleled the variations in the initial rates of ADP and U44069 aggregation and in epinephrine aggregation seen in PRP. No such abnormalities of PI metabolism were found in four other patients with similar, but not identical, functional defects. These results suggest an impairment affecting metabolism of PI/PPI via the PI/PPI cycle in this patient's platelets. The association of abnormalities of PI metabolism with defects of initial platelet responses provides further support for a physiological role of phosphoinositide metabolism in the early activation mechanism of platelets.

Topics: Arachidonic Acid; Arachidonic Acids; Blood Platelet Disorders; Blood Platelets; Calcimycin; Collagen; Humans; In Vitro Techniques; Phosphatidic Acids; Phosphatidylinositol Phosphates; Phosphatidylinositols; Serotonin; Thrombin

Storage pool-deficient (SPD) platelets, which have decreased amounts of dense-granule and/or alpha-granule constituents, contain normal amounts of lysosomal acid hydrolases, but in some cases exhibit impaired secretion of these enzymes. We examined this impaired secretion response in SPD patients with varying extents of granule deficiencies, and determined the effects of added dense-granule constituents. Acid hydrolase secretion was impaired in patients with severe dense-granule deficiencies, but not in patients with lesser dense-granule deficiencies, including those with alpha-granule deficiencies as well. When dense-granule constituents (ADP, ATP, serotonin, Ca+2, pyrophosphate) were added to gel-filtered platelets, ADP, but none of the other constituents, completely corrected the impairment of thrombin and A23187-induced secretion in SPD platelets. The concentration of ADP required to normalize thrombin-induced secretion varied markedly, from 0.01 to 10 microM, among the individual patients. Fixation of platelets with formaldehyde before centrifugation did not prevent the enhancement of secretion by ADP. Excess ATP, which acts as a specific antagonist of ADP-mediated responses, completely blocked this enhancement of secretion in SPD platelets by ADP, and partially inhibited acid hydrolase secretion induced by low, but not high, concentrations of thrombin in normal platelets as well. Treatment of normal platelets with acetylsalicylic acid in vivo, but not in vitro, produced an impairment of acid hydrolase secretion similar in extent to that in SPD platelets, but which could not be completely corrected by added ADP. One possible explanation of these results is that the impairment of acid hydrolase secretion may be secondary to the dense-granule deficiency in SPD platelets, and that secreted ADP may potentiate the lysosomal secretion response in normal platelets as well.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Aspirin; beta-N-Acetylhexosaminidases; Blood Platelet Disorders; Blood Platelets; Calcimycin; Calcium; Dose-Response Relationship, Drug; Epinephrine; Formaldehyde; Humans; Hydrolases; Platelet Storage Pool Deficiency; Thrombin

Following exposure to calcium ionophore A23187, washed peripheral blood mononuclear cells (MNC) aggregated and formed thromboxane, like platelets. However, while aspirin strongly inhibited platelet aggregation and thromboxane formation, it had a little effect on the aggregation of MNC. In about 50% of the samples studied, aggregation of MNC was associated with the secretion of ATP. However studies in which exogenous ATP or ADP were used, suggested that the aggregation of MNC is independent of the secretion of nucleotides. MNC from 2 thrombasthenic patients failed to aggregate and bound 9-10 fold less radiolabelled fibrinogen than those from normals when challenged with A23187. However, fibrinogen, which plays a major role in the aggregation of platelets, did not appear to be involved in the aggregation of MNC. A differing behavior of these two types of cells was also found when the effect of plasma was studied on the aggregation response to A23187. Indeed, citrated plasma greatly enhanced the aggregation of platelets while it suppressed the response to MNC. This inhibitory effect of plasma was not detected when heparinized plasma was substituted for citrated plasma. We conclude that the aggregation of MNC in response to A23187 does not involve basic events known to play a major role in the aggregation of platelets. The response to A23187 may be an important probe for understanding basic mechanisms and pathophysiological significance of the aggregation of MNC.

Topics: Adult; Blood Physiological Phenomena; Blood Platelet Disorders; Calcimycin; Cell Aggregation; Fibrinogen; Humans; Leukocytes, Mononuclear; Platelet Aggregation; Thrombasthenia

Platelets from a patient with the Hermansky-Pudlak syndrome were studied. These platelets had decreased amounts of serotonin and adenine nucleotides, and a decreased number of mepacrine-labeled dense bodies. beta-Thromboglobulin and acid hydrolases contained in alpha-granules and lysosomes respectively were present in normal amount. Platelets in platelet-rich plasma did not respond to collagen, but arachidonic acid and ionophore A 23187 induced normal aggregation and normal thromboxane (TX) synthesis. Alpha-granule release was found impaired and remained subnormal even with high doses of inducers. In response to thrombin aggregation, release and TX synthesis of isolated metrizamide gradient platelets were found at lower than normal levels. Phosphorylation of P20 and P43 proteins was normal. Only a combination of ADP plus thrombin could restore a normal aggregation, with normal alpha-granule and lysosome release and normal TX synthesis. These results indicated that in the absence of dense bodies: the release of other granules is impaired; the TX synthesis is delayed except when induced by arachidonic acid and A 23187 ionophore; the absence of dense bodies could be compensated for by the addition of ADP which restores the impaired release reaction and TX formation; and P20 and P43 polypeptides were phosphorylated as rapidly as those in normal platelets.

Topics: Adenine Nucleotides; Adenosine Diphosphate; Adolescent; Albinism; Blood Platelet Disorders; Blood Platelets; Calcimycin; Drug Synergism; Humans; Male; Phosphorylation; Platelet Aggregation; Platelet Storage Pool Deficiency; Serotonin; Syndrome; Thrombin; Thromboxanes

A patient is described who presented a thrombocytopenia with thrombocytopathy followed by the development of a leukaemia. The disorder was characterized by decreased aggregation in the presence of ADP, and a lack of aggregation in the presence of arachidonic acid, natural endoperoxide or collagen. In parallel, 14C-serotonin release was severely decreased or nil in response to these inducers. Thrombin induced a slightly decreased aggregation and a normal 14C-serotonin release. Thromboxane B2 (T X B2) synthesis was normal after stimulation by arachidonic acid, natural endoperoxide or thrombin showing a normal arachidonate metabolism. In addition, the mepacrine test showed no significant decrease of the number of dense bodies with an average of 4.6 per platelet (versus 5.4 +/- 0.8 sd in controls). Stimulation by ionophore A 23187 failed to induce aggregation, 14C-serotonin release, or T X B2 synthesis. Furthermore, in the presence of EDTA, A 23187 did not provoke activation as reflected by 14C-serotonin release or T X B2 synthesis. Thus, in this case of thrombocytopathy, the hypothesis of abnormal intracellular Ca++ fluxes responsible for the defective platelet release phenomenon, was suggested.

Topics: Adenosine Diphosphate; Blood Platelet Disorders; Blood Platelets; Calcimycin; Calcium; Edetic Acid; Female; Follow-Up Studies; Humans; Leukemia; Middle Aged; Platelet Aggregation; Quinacrine; Serotonin; Thrombocytopenia; Thromboxane B2

The secretion of the dense granule constituents ATP, ADP, calcium, pyrophosphate (PPi), and orthophosphate (Pi), and the release of magnesium induced by thrombin and the divalent cation ionophore A23187 have been quantitated directly in gel-filtered platelets from patients with storage pool deficiency (SPD). Both the contents and the maximal amounts of the dense granule constituents secretable by thrombin were decreased in all the patients studied, while the nonsecretable, retained amounts of these substances were identical in SPD and normal platelets. In response to both thrombin and A23187, the amounts of secretable ATP and ADP were strongly correlated in the platelets of individual patients; in contrast, secretable calcium showed no correlation with the nucleotides, and significant amounts of calcium were secreted in the total absence of nucleotide secretion in the platelets of several patients. The contents of magnesium were normal in all patients, and approximately 12% of platelet magnesium was liberated by thrombin in both SPD and normal platelets. A23187 induced the release of up to 70% of the magnesium content of normal platelets, but released significantly less (46%) magnesium from SPD platelets. Platelet aggregation induced by A23187 in platelet-rich plasma was also markedly decreased in SPD platelets. The correlations among secretable dense granule constituents suggest the presence in SPD platelets of abnormal dense granule structures that sequester calcium and other constituents but little or no adenine nucleotides, and are thus consistent with a hypothesis that impaired nucleotide transport and/or storage may be the primary dense granule defect in this disorder. In addition, these results demonstrate that certain responses to A23187 are impaired in SPD platelets.

Topics: Adenine Nucleotides; Biological Transport; Blood Platelet Disorders; Blood Platelets; Calcimycin; Calcium; Chromatography, Gel; Cytoplasmic Granules; Humans; Magnesium; Thrombin

A patient with a lifelong bleeding disorder is presented with a prolonged bleeding time and abnormal aggregation and secretion responses to arachidonic acid, thromboxane A2, PAF-acether and the divalent calcium ionophore A23187. Platelet alpha and dense granule contents and morphology appear normal. The proposed defect is due to an abnormality of a platelet intracellular calcium dependent process.

Topics: Arachidonic Acids; Bleeding Time; Blood Platelet Disorders; Calcimycin; Calcium; Female; Humans; Middle Aged; Nucleotides; Platelet Activating Factor; Platelet Aggregation; Thromboxane A2

Decreased responsiveness to adrenaline has been observed in five apparently normal unrelated human donors. In four of the donors this trait is inherited. Three of the donors, as well as their affected relatives, also exhibited depressed responsiveness to collagen and vasopressin but normal responsiveness to ADP and thrombin. The other two affected donors exhibit normal responsiveness to most other agonists. Normal responsiveness can be restored in all instances either by incubating the platelet-rich plasma at 20 degrees C or by addition of a low concentration of the divalent cation ionophore, A-23187. All affected platelets which have been examined have ATP and ADP contents, cholesterol to phospholipid ratios, and phospholipid class compositions within the normal range. Both the resting level of cyclic-3'5'-AMP and the ability of adrenaline to prevent elevation of cyclic-3',5'-AMP levels by prostaglandin E1 are normal. Mixing experiments demonstrate the absence of a circulating inhibitor of platelet function and suggest that the defect resides in the platelets. We conclude that the depressed responsiveness of human platelets to adrenaline may result from a defect in Ca2+ mobilization to the cytosol.

Topics: Adenosine Diphosphate; Adult; Blood Platelet Disorders; Blood Platelets; Calcimycin; Collagen; Epinephrine; Female; Humans; Male; Pedigree; Platelet Aggregation; Vasopressins

Topics: Arachidonic Acids; Blood Platelet Disorders; Blood Platelets; Calcimycin; Calcium Metabolism Disorders; Child, Preschool; Female; Humans; Male; Middle Aged; Platelet Aggregation

Topics: Adenosine Diphosphate; Aged; Animals; Aorta; Arachidonic Acids; Blood Platelet Disorders; Blood Platelets; Calcimycin; Humans; Male; Muscle Contraction; Platelet Aggregation; Rabbits; Thromboxane A2

Topics: Anti-Bacterial Agents; Blood Platelet Disorders; Blood Platelets; Calcimycin; Centrifugation, Density Gradient; Humans; L-Lactate Dehydrogenase; Light; Metrizamide; Oxygenases; Platelet Aggregation; Serotonin; Thromboxanes