Objective-Coated-platelets are a subset of cells observed during costimulation of platelets with collagen and thrombin.Important characteristics of coated-platelets include retention of multiple ␣-granule proteins and expression of phosphatidylserine on the cell surface. The mitochondrial permeability transition pore (MPTP) is a key step in apoptosis and is suggested to be involved in some forms of platelet activation. The objective of this study was to examine the role of MPTP in the synthesis of coated-platelets. Methods and Results-Flow cytometric analysis of coated-platelet production was used to examine the impact of pharmacological effectors of MPTP formation. Cyclosporin A, coenzyme Q, and bongkrekic acid all inhibit MPTP formation as well as production of coated-platelets. Phenylarsine oxide and diamide, both potentiators of MPTP formation, stimulate coated-platelet synthesis. Atractyloside, another inducer of MPTP formation, does not affect the percentage of coated-platelets synthesized; however, it does increase the level of phosphatidylserine exposed on the surface of coated-platelets. Conclusions-These findings indicate that MPTP formation is an integral event in the synthesis of coated-platelets.Although the exact function of the MPTP remains to be determined, these data support a growing body of evidence that apoptosis-associated events are vital components of the platelet activation process. (Arterioscler Thromb Vasc Biol. 2005;25:467-471.)Key Words: coated-platelet Ⅲ mitochondrial permeability transition pore Ⅲ coenzyme Q Ⅲ phosphatidylserine Ⅲ cyclosporin A Ⅲ phenylarsine oxide Ⅲ diamide C oated-platelets are a subset of platelets resulting from dual agonist activation with collagen and thrombin. 1-4 These unusual cells, formerly known as COAT platelets (collagen and thrombin-activated platelets, see below), are characterized by high-affinity retention on the platelet surface of several ␣-granule proteins, 2 expression of surface phosphatidylserine (PS), 1 and high prothrombinase activity. 1 ␣-Granule proteins bound to coated-platelets are derivatized with serotonin, 2 and binding sites on the cell surface for serotonin-derivatized proteins are provided by fibrinogen and thrombospondin. 3 The putative structure of the coated-platelet surface includes an intertwined network of ␣-granule proteins, each retained on the cell surface through multivalent interactions with membrane receptors and neighboring proteins. 4 Although considerable progress has been made in molecular characterization of coated-platelets, their physiological significance remains largely speculative.Agonist(s) other than collagen plus thrombin can also produce a subpopulation of platelets with many, if not all, the characteristics of coated-platelets; included among these agonists are thrombin plus Fc receptor engagement, 5 high-dose thrombin, 6 and immobilized collagen. 7,8 As mentioned, coated-platelets were referred to previously as COAT platelets, an acronym for the collagen and thrombin agonists used in their formation. 1 H...
Summary. Dual agonist stimulation of platelets with thrombin and convulxin results in generation of coated-platelets, a sub-population of cells known formerly as COAT-platelets (collagen and thrombin). Coated-platelets retain several procoagulant proteins on their surface and express phosphatidylserine (PS). In this report, we utilize a new methodology to demonstrate that coated-platelets also release microparticles. Platelets were prelabeled with 2.5 lM Bodipy-maleimide and then stimulated with convulxin plus thrombin. Microparticles, 0.3-0.5 lm in diameter, were observed by fluorescence confocal microscopy. Confocal microscopy was also used to demonstrate that microparticles were positive for glycoprotein IIb/IIIa, glycoprotein Ib, CD9, and PS, but negative for fibrinogen and thrombospondin. Furthermore, microparticles released from Bodipy-labeled platelets were observed by flow cytometry, and activation with convulxin plus thrombin produced 15 ± 5 microparticles per coated-platelet. In contrast, platelets stimulated with thrombin or convulxin alone produced few microparticles. Phenylarsine oxide and diamide, both of which potentiate the mitochondrial permeability transition pore and coated-platelet production, significantly increased the number of microparticles released per coated-platelet.
Since the introduction of tyrosine kinase inhibitors, the overall survival of patients with chronic myeloid leukemia has markedly improved. However long term use of these drugs results in various adverse events. Treatment with second generation dasatinib is often complicated by hemorrhagic events. Previous lumi-aggregometry studies have shown impaired platelet function in patients on dasatinib therapy. Dual agonist activated platelets (coated-platelets) are also sensitive indicators of platelet function. We hypothesized that dual activation with convulxin and thrombin of platelets in a flow cytometric assay could be a more sensitive method for detecting platelet dysfunction as compared to single agonist studies used in lumi-aggregometer. Platelets of healthy volunteers incubated with dasatinib as well as platelets from patients on dasatinib therapy were investigated. Low therapeutic plasma level dasatinib concentrations at which a considerable reduction in coated-platelet generation was observed in vitro, did not cause detectable change in platelet aggregation response. Coated-platelet assay and lumi-aggregometry were also investigated at 0, 1 and 4 hours after drug administration in dasatinib treated CML patients. Significant decrease was observed at 1 hour in maximal aggregation by collagen. Although the aggregation curves became normalized by 4 hours, coated-platelet generation was still inhibited in dasatinib treated patients. Nilotinib, another second generation tyrosine kinase inhibitor, had no effect on aggregation and on coated-platelet formation neither in vitro nor in ex vivo samples. At therapeutic plasma levels coated-platelet assay is more sensitive than lumi-aggregometry studies for the demonstration of the inhibitory effect of dasatinib on platelet function.
Acute promyelocytic leukemia (APL) is generally characterized by t(15;17)(q24;q21). In some cases, the classic translocation cannot be identified by conventional methods, since the PML-RARA fusion protein results from complex, variant, or cryptic translocation. The diagnostic algorithm of APL starts with screening methods, such as flow cytometry (FC), followed by fluorescence in situ hybridization or polymerase chain reaction to confirm the diagnosis. Our aim was to develop a novel protocol for analyzing APL samples based on multidimensional dot-plots that can provide comprehensive information about several markers at the same time. The protocol included four optimized multidimensional dot-plots, which were tested by retrospective reanalysis of FC results in APL ( n = 8) and non-APL ( n = 12) acute myeloid leukemia (AML) cases. After predicting the potential position of hypergranular- and microgranular-type aberrant promyelocytes, the percentages of blast populations were examined within the gates in all AML cases. The percentage of blasts in each predefined gate was well above the cut-off value (95%) in APL cases in all tubes. In non-APL AML cases, the percentage of blasts in the same gates never reached the cut-off value in all investigated tubes, and even when it did in a single tube, the pattern was markedly different from that observed in APL cases. In conclusion, multidimensional dot-plots can be used for screening APL even in cryptic APL cases, although reproducibility across several laboratories would require standardization of antibodies and fluorochromes. This easy-to-use and quick method can support the diagnosis of APL and the prompt initiation of the appropriate treatment. Electronic supplementary material The online version of this article (10.1007/s00277-019-03642-w) contains supplementary material, which is available to authorized users.
CD9, a member of the tetraspanin superfamily, is the third most abundant protein on the platelet surface, but its function remains unknown. In this report, we demonstrate that CD9 is required for the release of microparticles from coated-platelets. Coated-platelets are formed as a result of dual agonist activation with collagen and thrombin, and each coated-platelet releases 15-25 microparticles averaging 0.4 microm in diameter. We report here that four separate monoclonal antibodies against CD9 inhibited microparticle release from coated-platelets by 72-102% with an IC(50) of approximately 500 ng/mL for ALB6 and SN4. In addition, the anti-alpha(IIb)beta(3) monoclonal antibody AP2 also inhibited microparticle release although additional anti-alpha(IIb)beta(3) monoclonals did not. These data support participation of the tetraspanin CD9, together with the integrin alpha(IIb)beta(3), in the membrane vesiculation process associated with platelet microparticle release.
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