calcimycin and Thrombocytopenia

An HIV antibody (Ab) against platelet integrin GPIIIa49-66 induces complement-independent platelet particle formation by the elaboration of reactive oxygen species (ROS) downstream of the activation of the platelet NADPH oxidase by the 12-lipoxygenase (12-LO) product 12(S)-HETE. To determine whether other inducers of platelet particle formation also function via the induction of ROS, we examined the effects of the Ca(2+) ionophore A23187 and phorbol myristate acetate (PMA). Both agents induced oxidative platelet particle formation in an identical fashion as Ab, requiring Ca(2+) flux and 12(S)-HETE production as well as intact NADPH oxidase and 12-LO pathways. Since HIV-ITP patients with this Ab correct their platelet counts with dexamethasone (Dex), we examined the role of this steroid in this unique autoimmune disorder. Dex at therapeutic concentrations inhibited Ab-, A23187-, or PMA-induced platelet particle formation by inhibiting platelet PLA(2), 12-LO, and NADPH oxidase. The operational requirement of translocation of PLA(2), 12-LO, and NADPH oxidase components (p67 phox) from cytosol to membrane for induction of ROS was both inhibited and partially reversed by Dex in platelets. We conclude that (1) platelet particle formation can be induced by the generation of ROS; and (2) platelet PLA(2), 12-LO, NADPH oxidase, and cytosol membrane translocation, requirements for ROS production, are inhibited by Dex.

Topics: Animals; Anti-Inflammatory Agents; Antibodies, Monoclonal; Arachidonate 12-Lipoxygenase; Blood Platelets; Calcimycin; Carcinogens; Cell Membrane; Cytosol; Dexamethasone; Group IV Phospholipases A2; Humans; Integrin beta3; Ionophores; Lipoxygenase Inhibitors; Mice; Mice, Inbred C57BL; Mice, Knockout; NADPH Oxidases; Oxidation-Reduction; Phospholipases A; Phospholipases A2; Protein Transport; Reactive Oxygen Species; Tetradecanoylphorbol Acetate; Thrombocytopenia

Heparin-induced thrombocytopenia (HIT) with thrombosis is a serious complication of heparin use. HIT sera can generate platelet-derived microparticles, which are produced in a heparin-dependent manner and are hypothesized to be important initial pathological participants because they promote vascular occlusion. To date, microparticles have been studied using flow cytometric techniques. However, it is uncertain whether the small-sized material seen in flow cytometric studies represents true platelet microparticles shed from activated platelets or whether they are platelets that have contracted after releasing their internal components. This report describes a morphological investigation of platelet-derived microparticles in HIT using, among other techniques, confocal, scanning electron, and transmission electron microscopy. Following incubation with HIT sera, the existence of small membrane-bound vesicles in the milieu of activated platelets was demonstrated. A population of microparticles, expressing platelet-specific glycoproteins, was separated from platelets by centrifugation over a sucrose layer. These microparticles had identical flow cytometric profiles, size heterogeneity, and GPIb(alpha) and GPIIb/IIIa staining intensity as the microparticle population in unfractionated samples. When microparticles were generated in situ and fixed onto grids, they were demonstrated to be distinct membrane-bound vesicles that originated near the platelet body and terminal ends of pseudopods on activated platelets. These microparticles appeared to be generated by localized swelling, budding, and release. Collectively, these morphological studies document the existence of true microparticles in platelets activated by HIT sera. The microparticles may play an important role in the pathogenesis of HIT.

Topics: Anticoagulants; Blood Platelets; Calcimycin; Flow Cytometry; Heparin; Humans; In Vitro Techniques; Microscopy, Electron; Platelet Activation; Reference Values; Serotonin; Thrombin; Thrombocytopenia

The typical data presentation by flow cytometry of platelet suspensions stimulated with calcium ionophore A-23187, thrombin, C5b-9, or other agonists shows a unimodal decrease (or 'left-shift') in forward angle light scatter. Many reports in the literature interpret these findings as indicative of the appearance of small membranous microparticles generated from the platelets as part of the activation response. Investigators may attempt to quantify the microparticles by calculation of the percentage of counts falling below a gate set around the forward angle light scatter distribution of fresh, non-activated platelets. We believe that this approach can lead to erroneous results unless the total particle count in the sample is also determined. The true change in total particle count in a platelet sample is easily estimated on the flow cytometer by adding a known amount of fluorescent beads to the platelet suspension and noting a change in the ratio of bead versus non-bead event counts as a result of the stimulus added. Using this technique under settings considered routine for platelet analysis on a FACScan flow cytometer (Becton-Dickinson), we have found that particle counts increased very little (less than a doubling) in platelet suspensions stimulated with 1-10 microM A-23187 or 0.01-0.5 U/ml thrombin or with sera from patients with diagnosed heparin-induced thrombocytopenia (HIT). Only by employing high sensitivity settings for signal thresholding on orthogonal light scatter, combined with fluorescence gating on high prevalence surface antigens, were we able to detect significant increases (5- or 6-fold) in total particle count in the same experiments. The new events we observed were separated by a decade or more (log scale) from intact platelets on the light scatter plots and fluorescence histograms in a bimodal distribution. We postulate that the unimodal-shifted population of events seen under routine settings after stimulation with ionophore is really degranulated platelets, and only the much smaller new modal subpopulation represents microparticles released as new entities into the platelet suspension. We conclude that without high sensitivity settings for data acquisition, it is most likely incorrect to claim that the left-shifted events on flow cytometry light scatter plots appearing contiguous with the distribution of activated platelets are released microparticles.

Topics: Blood Platelets; Calcimycin; Cell Membrane; Flow Cytometry; Heparin; Humans; Ionophores; Platelet Activation; Sensitivity and Specificity; Thrombin; Thrombocytopenia

The Wiskott-Aldrich syndrome (WAS) is an inherited platelet/T-lymphocyte disease characterized by small platelets, thrombocytopenia and immunodeficiency. Because degradative events have a significant role, we directly examined calpain (Ca(2+)-dependent neutral protease), a prominent protease in the affected cells, by functional and antigenic quantitation. Calpain activity in platelets of seven WAS patients was decreased to 59 +/- 3.7% (P < 0.01) relative to platelets of 11 normals. Platelets of two patients with immune thrombocytopenia had normal calpain activity. By immunoblotting, mu-procalpain, the mu-calpain species in resting (unstimulated) blood cells, was decreased in platelets of nine WAS patients to 58 +/- 14.6% (P < 0.01) relative to paired normals. In contrast, mu-procalpain levels in lymphocytes of seven WAS patients did not differ from normal lymphocytes. Normal platelets and lymphocytes have different mechanisms for Ca(2+)-dependent mu-procalpain activation. On addition of ionophore and Ca2+ to stirred platelets, 80kD mu-procalpain was rapidly (0.5 min) and quantitatively converted to 76 kD active mu-calpain; this process was the same in WAS platelets. In lymphocytes, mu-procalpain activation was slow, only partially complete (40 min), and the active species was 78 kD. The marked depletion of calpain in WAS platelets demonstrated in this study may result from inappropriate stimulation of platelets and be related to the severe thrombocytopenia that characterizes this disease.

Topics: Autoimmune Diseases; Blood Platelets; Calcimycin; Calcium; Calpain; Enzyme Precursors; Humans; Immunoblotting; Lymphocytes; Male; Thrombocytopenia; Wiskott-Aldrich Syndrome

To determine whether platelet-activating factor (PAF)-induced release of cyclooxygenase products might be dependent on G proteins in vivo, we administered pertussis toxin (PTX) (9.7-10.0 micrograms/kg iv) to conscious pigs approximately 48 h before bolus infusions of PAF (10 ng/kg). Autoradiography of ADP-ribosylated lung cell membrane proteins from PTX-treated pigs demonstrated marked reduction in the amount of radiolabel ([32P]NAD) incorporated, indicating that PTX induced ADP-ribosylation of G proteins in vivo. PAF, infused at hourly intervals from 0-4 h, caused increases in plasma concentrations of thromboxane B2 (TxB2) concomitant with pulmonary hypertension and vasoconstriction in anesthetized pigs. These physiological changes were blocked or markedly attenuated by indomethacin, indicating they were dependent on cyclooxygenase products. In PTX-treated pigs, the PAF-induced pulmonary hypertension and vasoconstriction were modestly attenuated, whereas the increases in plasma TxB2 were markedly attenuated. PTX prevented PAF-induced aggregation of platelets in vivo as evidenced by blockade of thrombocytopenia. However, in vitro, PAF-induced aggregation of platelets was independent of PTX. Moreover, incubation of platelet-rich plasma with 50 microM PAF failed to increase TxB2 levels. These findings suggested that a PTX-sensitive cell other than the platelet was responsible for triggering release of TxA2 and thrombocytopenia in vivo. We conclude that PAF-induced release of TxA2, pulmonary vasoconstriction, and thrombocytopenia in anesthetized pigs are dependent on a PTX-sensitive G protein; however, the residual hemodynamic effects indicate involvement of a PTX-insensitive G protein, or alternatively, G protein independent pathways.

Topics: Animals; Blood Platelets; Calcimycin; GTP-Binding Proteins; Hemodynamics; Indomethacin; Injections, Intravenous; Lung; Pertussis Toxin; Phosphorylation; Platelet Activating Factor; Prostaglandin-Endoperoxide Synthases; Sodium Chloride; Swine; Thrombocytopenia; Thromboxane B2; Time Factors; Vasoconstriction; Virulence Factors, Bordetella

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

When ethanol is administered to rats chronically by inhalation for 5-7 days to produce a final ethanol concentration in plasma of about 50 mM platelet aggregation in vitro to collagen is markedly inhibited. Platelet aggregation to ADP is relatively unaffected and there is no evidence of thrombocytopenia or morphological change in platelets on scanning and transmission electron microscopy. The changed sensitivity to collagen is not simply due to the presence of ethanol in plasma during the in vitro tests because in vitro addition of a similar concentration of ethanol produces a much smaller inhibition of platelet aggregation to collagen in platelet-rich plasma from control animals. The acute or subacute administration of ethanol either by injection (3 g/kg-1 intraperitoneally) or by inhalation (4-6 h) produced very variable results, platelets from some animals being inhibited with respect to collagen as much as those from animals chronically exposed to ethanol, whereas other showed no inhibition other than that attributable to the presence of ethanol in plasma. When ethanol was administered chronically by inhalation for 30 days, thrombocytopenia was apparent and platelets from these animals were unresponsive to all aggregating agents tested. It is concluded that even relatively short periods of ethanol intake may lead to significant inhibition of platelet function, specifically aggregation to collagen. This may be of relevance to the suggested protective effect of ethanol intake in cardiovascular disease.

Topics: Adenosine Diphosphate; Animals; Blood Platelets; Calcimycin; Collagen; Epinephrine; Ethanol; In Vitro Techniques; Platelet Aggregation; Rats; Rats, Inbred Strains; Thrombocytopenia

Thrombin-induced platelet aggregation has been generally believed to be irreversible. However, thrombin-induced aggregation of washed platelets is reversible if fibrin formation is prevented or the fibrin which binds the platelets together is removed from the platelet aggregates. After treatment with high concentrations of thrombin (0.5 units/ml) single platelets can be recovered that have lost practically all of their releasable serotonin and adenine nucleotides. These platelets are able to aggregate upon addition of low concentrations of ADP in the presence of fibrinogen. They aggregate in response to the ionophore A23, 187 in the absence of added fibrinogen, whereas sodium arachidonate-induced aggregation requires added fibrinogen. Thrombin-treated platelets change their shape in response to collagen in the absence of fibrinogen, and will aggregate upon the addition of collagen providing fibrinogen is present. This response to collagen can be blocked with aspirin but not with a mixture of creatine phosphate/creatine phosphokinase. Upon a second exposure to thrombin, thrombin-pretreated platelets do not change their shape and do not undergo aggregation. Thrombin-pretreated platelets will not retract a thrombin-induced fibrin clot unless ADP, sodium arachidonate, the ionophore A23, 187 or collagen are added together with thrombin. The ability of thrombin-treated platelets to adhere to the exposed subendothelial surface of the rabbit aorta is reduced, compared with untreated control platelets. The thrombin-treated platelets shorten the bleeding time of thrombocytopenic rabbits. However, the are not as effective in shortening the bleeding time as normal control platelets. When injected into rabbits with a normal platelet count, the thrombin-treated platelets that circulate after infusion survive for the same length of time as untreated control platelets. These findings indicate that thrombin-induced platelet aggregation with extensive release of granule constituents is not irreversible and that thrombin treatment does not cause irreversible damage of all platelets that would lead to their immediate elimination from the circulation. Furthermore, these platelets can still be haemostatically effective. It is conceivable that platelets that have lost their amine storage granule contents during a release reaction in vivo, such as may occur in certain cases of intravascular coagulation and repeated episodes of thrombosis, may be found in the circulation of man.

Topics: Adenosine Diphosphate; Animals; Arachidonic Acids; Blood Platelets; Calcimycin; Cell Survival; Clot Retraction; Collagen; Fibrinogen; Platelet Adhesiveness; Platelet Aggregation; Rabbits; Serotonin; Thrombin; Thrombocytopenia