calcimycin and Hemorrhagic-Disorders

Topics: Biological Transport; Blood Platelets; Blotting, Western; Calcimycin; Calcium Signaling; Fluorescent Dyes; Gene Expression Regulation; Hemorrhagic Disorders; Humans; Ion Transport; Ionophores; Large-Conductance Calcium-Activated Potassium Channels; Membrane Lipids; Phosphatidylserines; Potassium; Small-Conductance Calcium-Activated Potassium Channels; Thapsigargin

In contrast to other agents able to induce apoptosis of cultured cells, Ca2+ ionophore A23187 was shown to elicit direct activation of intracellular signal(s). The phenotype of the cells derived from patients having the hemorrhagic disease Scott syndrome, is associated with an abnormally high proportion of apoptotic cells, both in basal culture medium and upon addition of low ionophore concentrations in long-term cultures. These features are presumably related to the mutation also responsible for the defective procoagulant plasma membrane remodeling. We analyzed the specific transcriptional re-programming induced by A23187 to get insights into the effect of this agent on gene expression and a defective gene regulation in Scott cells.. The changes in gene expression upon 48 hours treatment with 200 nM A23187 were measured in Scott B lymphoblasts compared to B lymphoblasts derived from the patient's daughter or unrelated individuals using Affymetrix microarrays. In a similar manner in all of the B cell lines, results showed up-regulation of 55 genes, out of 12,000 represented sequences, involved in various pathways of the cell metabolism. In contrast, a group of 54 down-regulated genes, coding for histones and proteins involved in the cell cycle progression, was more significantly repressed in Scott B lymphoblasts than in the other cell lines. These data correlated with the alterations of the cell cycle phases in treated cells and suggested that the potent effect of A23187 in Scott B lymphoblasts may be the consequence of the underlying molecular defect.. The data illustrate that the ionophore A23187 exerts its pro-apoptotic effect by promoting a complex pattern of genetic changes. These results also suggest that a subset of genes participating in various steps of the cell cycle progress can be transcriptionally regulated in a coordinated fashion. Furthermore, this research brings a new insight into the defect in cultured Scott B lymphoblasts, leading to hypothesize that a mutated gene plays a role not only in membrane remodeling but also in signal transduction pathway(s) leading to altered transcriptional regulation of cell cycle genes.

Topics: Apoptosis; B-Lymphocytes; Calcimycin; Calcium; Cell Cycle; Cell Line; Cell Membrane; Cluster Analysis; Coagulants; Down-Regulation; Gene Expression Regulation; Hemorrhagic Disorders; Histones; Humans; Ionophores; Microarray Analysis; Mutation; Oligonucleotide Array Sequence Analysis; Phenotype; RNA, Complementary; Signal Transduction; Syndrome; Transcription, Genetic; Up-Regulation

A platelet disorder characterized by the absence of thromboxane A2 (TXA2)-induced platelet aggregation is a new clinical entity of platelet dysfunction. The platelets of three patients had the ability to bind exogenous TXA2, but synthetic TXA2 mimetic-induced postreceptor biochemical events, such as IP3 formation, Ca2+ mobilization, phosphatidic acid formation, and GTPase activities, were selectively defective, suggesting impaired coupling between the TXA2 receptor and phospholipase C activation. Gene analysis of the TXA2 receptor showed a substitution of Leu for Arg60 in the first cytoplasmic loop in all patients, and this mutation seemed to be responsible for this platelet disorder.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Amino Acid Substitution; Animals; Blood Platelets; Calcimycin; Calcium Signaling; CHO Cells; Codon; Cricetinae; Cricetulus; DNA, Complementary; Enzyme Activation; GTP Phosphohydrolases; Hemorrhagic Disorders; Humans; Phosphatidic Acids; Phosphatidylinositol Diacylglycerol-Lyase; Platelet Aggregation; Point Mutation; Receptors, Thromboxane; Recombinant Proteins; Sodium Fluoride; Thromboxane A2; Transfection; Type C Phospholipases

Signal transduction on platelet activation involves phosphoinositide-specific phospholipase C (PLC)-mediated hydrolysis of phosphatidylinositides and formation of inositol-1,4,5-triphosphate [I(1,4,5)P3], which mediates Ca2+ mobilization, and diacylglycerol (DG), which activates protein kinase C (PKC) to phosphorylate a 47-kD protein (Pleckstrin). We studied these events in two related patients previously reported (Blood 74:664, 1989) to have abnormal aggregation and 14C-serotonin secretion, and impaired intracellular Ca2+ mobilization in response to several agonists. Thrombin-induced I(1,4,5)P3 and phosphatidic acid formation were diminished. Pleckstrin phosphorylation was impaired on activation with thrombin, platelet-activating factor, and ionophore A23187, but was normal with PKC activator 1,2-dioctonyl-sn-glycerol (DiC8). Ca2+ mobilization induced by guanosine triphosphate (GTP) analog guanosine 5'-0-(3 thiotriphosphate) (GTP gamma S) was diminished. Pretreatment with either A23187 or DiC8 did not correct the impaired adenine diphosphate-induced secretion; however, upon stimulation with A23187 plus DiC8, pleckstrin phosphorylation and secretion were normal, indicating that both PKC activation and Ca2+ mobilization are essential for normal secretion. We conclude that these patients have a unique inherited platelet defect in formation of two key intracellular mediators [I(1,4,5)P3 and DG] and in the responses mediated by them due to a defect in postreceptor mechanisms of PLC activation.

Topics: Adenosine Diphosphate; Adult; Blood Platelets; Blood Proteins; Calcimycin; Calcium; Diglycerides; Enzyme Activation; Female; Hemorrhagic Disorders; Humans; Inositol 1,4,5-Trisphosphate; Male; Middle Aged; Myosin Light Chains; Phosphatidic Acids; Phosphatidylinositol Diacylglycerol-Lyase; Phosphoproteins; Phosphoric Diester Hydrolases; Phosphorylation; Platelet Activating Factor; Platelet Aggregation; Protein Processing, Post-Translational; Serotonin; Signal Transduction; Thrombin

Platelet glycoprotein VI (GPVI), a 62kD membrane protein, has been identified as one of the platelet receptors for collagen, since GPVI-deficient platelets exhibit abnormal responses to collagen and an abnormal bleeding tendency. We report a female patient with a mild bleeding history whose platelets expressed 10% GPVI of normal platelets. Shape change, aggregation and ATP release of the patient's platelets were completely absent in response to 1-5 micrograms/ml collagen but present normally in response to ADP and Ca2+ ionophore A23187. Adhesion of the patient's platelets to coated collagen was mildly affected (40-60% of normal platelets) in spite of only 10% expression of GPVI. Flow cytometrical studies revealed that the patient's platelets expressed normal amounts of the GPIa/IIa complex. These results suggest that platelet GPVI is less involved in adhesion to collagen than shape change and aggregation induced by collagen.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Antigens, CD; Blood Platelets; Calcimycin; CD36 Antigens; Cell Adhesion; Cells, Cultured; Collagen; Female; Hemorrhagic Disorders; Humans; Immunoblotting; Middle Aged; Platelet Aggregation; Platelet Membrane Glycoproteins; Thrombasthenia

A severe hereditary hemorrhagic diathesis in Simmental cattle has been identified in North America. Platelet numbers and coagulation profiles of affected cattle are normal. We have further characterized the severe dysfunction of platelet aggregation. All agonists tested elicited normal shape change. Aggregations in response to ADP, A23187, and collagen were absent. Aggregations were decreased or required more time for completion in response to PAF and thrombin. No ultrastructural abnormalities were observed in transmission electron micrographs. Dense granule release of ATP in response to PAF was normal. Thrombin-induced aggregation was dependent upon external calcium concentration in normal but not affected animals. Clot retraction in the blood from affected animals was abnormal. The data implicate a defect of Ca++ mobilization or utilization.

Topics: Adenosine Diphosphate; Animals; Blood Platelets; Calcimycin; Calcium; Cattle; Cattle Diseases; Collagen; Hemorrhagic Disorders; Inbreeding; Microscopy, Electron; Platelet Aggregation; Thrombin

Platelet aggregation and secretion have been described to be associated with phosphorylation reactions. Thrombasthenic and EDTA-treated control platelets undergo a normal serotonin release in the absence of aggregation. We now studied the phosphorylation of specific proteins associated with platelet secretion. In the presence of ionophore, significant increases occurred in the phosphorylation of two polypeptides of 43,000 and 20,000 molecular weight ( P43 and P20) in a concentration dependent manner, and this was accompanied by an increase in the 14C-5HT release. The 32P-labelling of P43 and P20 reaches a peak within 1 min of platelet activation and is followed by a rapid dephosphorylation over the next 2-10 min. While the P20 is identified as the myosin light chain, the identity and the function of the P43 remain unknown. Isoelectric focusing separates 4 proteins from P43 during two dimensional electrophoresis, but only one of them is phosphorylated by A 23187. Chlorpromazine (CPZ) and trifluoperazine (TFP) inhibit the P43 and P20 phosphorylation as well as the 14C-5HT release in a dose dependent manner. The inhibitory action of the drugs is more pronounced for P43 than for P20, especially when the reactions are carried out at 20 degrees C instead of at 37 degrees C, while the release reaction is still inhibited under these conditions. These results allow different hypotheses for the relationship of phosphorylation-secretion and indicate the importance of one of these proteins ( P43 ) for the release reaction.

Topics: Blood Platelets; Blood Proteins; Calcimycin; Chlorpromazine; Electrophoresis, Polyacrylamide Gel; Hemorrhagic Disorders; Humans; Molecular Weight; Myosins; Peptides; Phosphorylation; Platelet Aggregation; Serotonin; Trifluoperazine