Clopidogrel increases expression of chemokines in peripheral blood mononuclear cells in patients with coronary artery disease: results of a double-blind placebo-controlled study.

BACKGROUND: Chemokines and platelet activation are both important in atherogenesis. Platelet inhibitors are widely used in coronary artery disease (CAD), and we hypothesized that the platelet inhibitor clopidogrel could modify chemokines in CAD patients. OBJECTIVES: We sought to investigate the effect of clopidogrel on the expression of chemokines and chemokine receptors in peripheral blood mononuclear cells (PBMC) in CAD patients. PATIENTS/METHODS: Thirty-seven patients with stable angina were randomized to clopidogrel (n = 18) or placebo (n = 19). PBMC, blood platelets and plasma were collected at baseline and after 7-10 days in the patients, and in 10 healthy controls. mRNA levels of chemokines and chemokine receptors in PBMC were analyzed by ribonuclease protection assays and real-time reverse transcriptase polymerase chain reaction. Platelet activation was studied by flow cytometry. RESULTS: (i) At baseline, the gene expression of the regulated on activation normally T-cell expressed and secreted (RANTES) chemokines and macrophage inflammatory peptide (MIP)-1beta in PBMC, the expression of CD62P and CD63 on platelets and the levels of platelet-derived microparticles (PMP) were elevated in angina patients comparing healthy controls; (ii) markers of platelet activation were either reduced (CD63) or unchanged (CD62P, PMP, beta-thromboglobulin) during clopidogrel therapy; (iii) in contrast, clopidogrel significantly up-regulated the gene expression of RANTES and MIP-1beta in PBMC, while no changes were found in the placebo group; (iv) a stable adenosine 5'-diphosphate metabolite attenuated the release of MIP-1beta, but not of RANTES, from activated PBMC in vitro. CONCLUSIONS: Even if we do not argue against a beneficial role for clopidogrel in CAD, our findings may suggest potential inflammatory effects of clopidogrel in CAD.

Effects of platelets and platelet-derived material on the activated partial thromboplastin time (Cephotest) coagulation test.

OBJECTIVE: The Cephotest is an activated partial thromboplastin time (APTT) test used to measure the activity of the intrinsic pathway of coagulation. To perform this test, blood is usually centrifuged to obtain plasma that is almost without erythrocytes and leucocytes and with only a minimal amount of platelets. MATERIAL AND METHODS: In the present experiments blood was centrifuged at different speeds to produce either platelet-poor plasma (PPP) or platelet-rich plasma (PRP). PPP and PRP obtained from the same whole blood samples from each of several persons were tested in pairs using the standard Cephotest reagent to observe the consequences of Cephotest being performed on plasma containing platelets. The same procedure was used with a Cephotest reagent with a reduced concentration of phosphatidylserine. In succeeding experiments the PRP was preincubated with SFLLRN or Ca-ionophore to activate the platelets, a procedure also known to produce platelet-derived microparticles. PPP and PRP were compared by thrombin time and reptilase time tests to find out at which stage platelets might influence the Cephotest. RESULTS: The results showed that the platelets did influence Cephotest when using both the regular reagent and the phospholipid-reduced agent. When using the regular reagent, PRP showed a tendency towards a longer APTT than PPP. It is suggested that this was caused by platelets consuming some of the first traces of thrombin generated. CONCLUSIONS: When using the phospholipid-reduced reagent, PRP showed a shorter APTT than PPP, probably because the platelets contributed phosphatidylserine to the system. When the platelets were activated before testing, their effects on the tests were increased. Microparticles that formed during platelet activation may have contributed to these effects.

CXC-chemokines in coronary artery disease: possible pathogenic role of interactions between oxidized low-density lipoprotein, platelets and peripheral blood mononuclear cells.

CXC-chemokines may be involved in atherogenesis. Herein we examined the possible role of CXC-chemokines in the inflammatory interactions between oxidized (ox-) low-density lipoprotein (LDL), platelets and peripheral blood mononuclear cells (PBMC) in 15 patients with coronary artery disease (CAD) without 'traditional' risk factors and 15 carefully matched controls. Our main findings were: (a) ox-LDL stimulated the release of the CXC-chemokines interleukin (IL)-8, ENA-78 and GRO-alpha from PBMC, particularly in CAD. (b) In platelets, ox-LDL induced release of ENA-78 and, when combined with SFLLRN, also of GRO-alpha, with significantly higher response in CAD. (c) Platelet-rich plasma, especially when costimulated with ox-LDL, enhanced the release of IL-8 from PBMC, particularly in CAD patients. (d) Freshly isolated PBMC showed markedly increased IL-8 mRNA expression in CAD patients. Our findings suggest enhanced inflammatory interactions between ox-LDL, platelets and PBMC in CAD patients involving CXC-chemokine related mechanisms, possible contributing to atherogenesis in these and other CAD patients.

Platelet shape change induced by the peptide YFLLRNP.

Platelet shape change represents an early response to activating agents. Whereas the PAR-1-activating peptide SFLLRN induces a total platelet activation, YFLLRNP brings the process only to the shape change step in a process independent of the cytosolic Ca2+ concentration. In this paper, the YFLLRNP-induced shape change has been observed in human citrated platelet-rich plasma (PRP). Scanning electron microscopy of platelets activated by 300 microM YFLLRNP showed platelets that had changed shape and extended long pseudopods. The protein content of the material sedimenting at 13,000 x g from 1% Triton X-100 extracts increased during the shape change, indicating a reorganization of the cytoskeleton. This was supported by electrophoresis. The GP IIb-IIIa complex was not activated, however, and the platelets that had undergone shape change did not support clot retraction. As no increase in binding of FITC-labeled annexin V (FITC-annexin V) was observed, the extensive shape change was not associated with a disturbance of the membrane phospholipid asymmetry. Platelet aggregation was never observed with 300 microM YFLLRNP, but could be seen at much higher concentrations, neither was secretion from dense granules observed at 300 microM as no extracellular ATP could be observed. These studies confirm and extend the concept of the Ca2+-independent shape change as a distinct and sharply delineated process that, in itself, may be of little pathophysiological importance if such "partly activated" platelets occur in the circulation.

Increased concentrations of soluble CD40 ligand, RANTES and GRO-alpha in preeclampsia--possible role of platelet activation.

Activated platelets may release inflammatory mediators that activate leukocytes and trigger inflammatory reactions in endothelial cells. We examined the concentrations of soluble CD40 ligand (sCD40L) and the chemokines RANTES and GRO-alpha in platelet-free plasma (PFP), and unstimulated and SFLLRN-stimulated platelet-rich plasma (PRP), as well as in platelet pellets before stimulation using enzyme immunoassays. Nineteen women with normal and twenty-one with preeclamptic pregnancies were studied, and several differences between these two groups of pregnancies were revealed (1). Women with preeclampsia had significantly increased concentrations of sCD40L and GRO-alpha in PFP (2). Platelets from these patients spontaneously released larger quantities of CD40L and RANTES ex vivo (3). When further activated ex vivo by SFLLRN, platelets from preeclamptic women released lower amounts per platelet of CD40L, RANTES and GRO-alpha (4). The platelet pellets in preeclamptic women contained decreased amounts of CD40L, RANTES and GRO-alpha per platelet. Our findings suggest enhanced platelet activation in vivo during preeclampsia resulting in increased release of inflammatory mediators, possibly contributing to inflammation, leukocyte activation and endothelial dysfunction in this disorder.

Persistently elevated levels of von Willebrand factor antigen in HIV infection. Downregulation during highly active antiretroviral therapy.

Levels of circulating von Willebrand factor (vWf) antigen are thought to reflect endothelial involvement in various disorders. In the present study we found markedly elevated plasma levels of vWf in HIV-infected patients demonstrated on both cross-sectional and longitudinal testing. Notably, we found that a persistent rise in vWf antigen was associated with progression of HIV-related disease. This elevation of vWf antigen represented functionally normal vWf as evaluated by plasma FVIII, ristocetin cofactor assay and vWf multimer analyses. While HIV-infected patients showed enhanced platelet activation, platelets did not contribute substantially to the increased vWf levels. The high vWf levels were significantly correlated with high viral load, and during HAART, the pronounced decline in HIV RNA levels was accompanied by a corresponding decrease in vWf. The persistent elevation of functionally normal vWf during HIV infection, most probably reflecting a persistent endothelial cell activation, may have an important role in the pathogenesis of HIV infection.

CXC-chemokines, a new group of cytokines in congestive heart failure--possible role of platelets and monocytes.

OBJECTIVES: The purpose of the present study was to examine the circulating levels of CXC-chemokines in patients with various degree of congestive heart failure (CHF). BACKGROUND: CXC-chemokines may be important mediators in the persistent immune activation observed in CHF patients by activation of circulating neutrophils, T-cells and monocytes and possibly by the recruitment of these cells into the failing myocardium. METHODS: Levels of interleukin (IL)-8, growth-regulated oncogene (GRO) alpha and epithelial neutrophil activating peptide (ENA)-78 were measured both in serum and in platelet-free plasma by enzyme immunoassay in 47 patients with CHF and in 20 healthy controls. RESULTS: (i) CHF patients had significantly elevated levels of all the three CXC-chemokines with IL-8 and GRO alpha showing a gradual increase along with increasing NYHA class. (ii) There was an inverse correlation between IL-8 and left ventricular ejection fraction (EF) and cardiac index (CI). (iii) Both unstimulated and lipopolysaccharide (LPS)-stimulated monocytes from CHF patients released markedly elevated amounts of all three CXC-chemokines. (iv) Platelets from patients with severe CHF were characterised by decreased content of GRO alpha and ENA-78 as well as decreased release of these chemokines upon thrombin receptor stimulation. (v) Activated platelets stimulated peripheral blood mononuclear cells in vitro to enhanced release of IL-8, and neutralising antibodies against ENA-78 inhibited this interaction. CONCLUSIONS: This study demonstrates for the first time elevated levels of CXC-chemokines in CHF, which may be of importance for progression of heart failure. Our findings further suggest that activated monocytes and platelets may contribute to enhanced CXC-chemokine levels in CHF.

The charge-heterogeneity of human fibrinogen as investigated by 2D electrophoresis.

The charge-heterogeneity of human plasma fibrinogen subunit chains was characterized by two-dimensional electrophoresis (2DE). Western blotting with antibodies specific for the gamma-chain demonstrated that the gamma-chains focus at varying isoelectric points (pI). This microheterogeneity was also observed in fibrinogen secreted from hepatocytic cells and in recombinant fibrinogen expressed in Chinese hamster ovary (CHO) cells. Further, covalent gammagamma-dimerization by FXIIIa was not influenced by the charge-heterogeneity, and removal of the carbohydrate did not reduce the number of gamma-chain pI variants. These observations suggest that the microheterogeneity of the gamma-chain is a multifactorial phenomenon that is not due to physiologic modification of the glycoprotein in circulation.

Procoagulant expression in platelets and defects leading to clinical disorders.

Hemostasis is a result of interactions between fibrillar structures in the damaged vessel wall, soluble components in plasma, and cellular elements in blood represented mainly by platelets and platelet-derived material. During formation of a platelet plug at the damaged vessel wall, factors IXa and VIIIa form the "tenase" complex, leading to activation of factor X on the surface of activated platelets. Subsequently, factors Xa and Va form the "prothrombinase" complex, which catalyzes the formation of thrombin from prothrombin, leading to fibrin formation. An enhanced expression of negatively charged phosphatidylserine in the outer membrane leaflet resulting from a breakdown of the phospholipid asymmetry is essential for the formation of the procoagulant surface. An ATP-driven and inward-acting aminophospholipid "translocase" and a "floppase" counterbalancing this have been postulated to maintain the dynamic state of phospholipid asymmetry. A phospholipid-nonspecific "scramblase," believed to be responsible for the fast breakdown of the asymmetry during cell activation, has recently been isolated from erythrocytes, cloned, and characterized. An intracellular calcium-binding segment and one or more thioesterified fatty acids are probably of importance for calcium-induced activation of this transporter protein. Cytosolic calcium ions also activate the calcium-dependent protease calpain associated with shedding of microvesicles from the transformed platelet membrane. These are shed with a procoagulant surface and with surface-exposed P-selectin from the alpha-granules. Theoretically, therefore, microvesicles can be involved in both coagulation and inflammation. Scott syndrome is probably caused by a defect in the activation of an otherwise normal scramblase, resulting in a relatively severe bleeding tendency. In Stormorken syndrome, the patients demonstrate a spontaneous surface expression of aminophospholipids. Activated platelets and the presence of procoagulant microvesicles have been demonstrated in several clinical conditions, such as thrombotic and idiopathic thrombocytopenia, disseminated intravascular coagulation, and HIV-1 infection, and have been found to be associated with fibrin in thrombosis. Procoagulant microvesicles may also be formed from other cells as a result of apoptosis.

Studies on the dual effects on platelets of a monoclonal antibody to CD9, and on the properties of platelet CD9.

The article describes effects on human platelets of a murine monoclonal antibody of the IgG2a subtype (clone FN99) directed against the membrane glycoprotein CD9. This antibody exerts a dual action on human platelets in plasma depending on whether the complement system can be activated or not, resulting either in membrane permeabilization or a true platelet aggregation. Secretion from the alpha-granules during permeabilisation was not observed in the sense that the granule-located protein thrombospondin was retained in the platelets, as opposed to what was seen with platelets that had undergone an antibody-induced aggregation. Only a small fraction of P-selectin was found on the surface of the permeabilised platelets. The cytoskeletal protein actin-binding protein (filamin) was profoundly degraded during membrane permeabilisation, however, and scanning electron microscopy showed platelets that were swollen with only a few pseudopodia. Preincubation of platelets with three different antibodies to CD9 showed strong inhibition of a subsequent binding of FITC-labelled Fab fragment of FN99 indicating that antibodies tend to bind in the same area of the CD9 molecule. No association of CD9 to the platelet actin-based cytoskeleton was observed. CD9 was present on the surface of microvesicles derived from calcium ionophore-treated platelets.

Enhanced levels of soluble and membrane-bound CD40 ligand in patients with unstable angina. Possible reflection of T lymphocyte and platelet involvement in the pathogenesis of acute coronary syndromes.

BACKGROUND: The CD40 ligand (CD40L) on activated T cells and platelets may be activating matrix metalloproteinases, inducing procoagulant activity, and be involved in the pathogenesis of acute coronary syndromes by promoting plaque rupture in atheroma. METHODS AND RESULTS: To study the role of CD40L-CD40 interaction in coronary disease, we analyzed levels of soluble (s) and membrane-bound CD40L in the peripheral blood from 29 patients with stable angina, 26 with unstable angina, and 19 controls. Our main findings follow. (1) Patients with unstable angina had significantly raised serum levels of sCD40L when compared with patients with stable angina and controls. (2) Platelets could release large amounts of sCD40L when stimulated ex vivo with the thrombin receptor-agonist peptide SFLLRN in both patients and controls. (3) Platelets in patients with unstable angina were characterized ex vivo by decreased intracellular levels and decreased SFLLRN-stimulated release of sCD40L, which may possibly represent a higher percentage of degranulated platelets in these patients. (4) T cells in patients with unstable angina had enhanced surface expression of CD40L and increased release of sCD40L on anti-CD3/anti-CD28 stimulation in vitro when compared with patients with stable angina and controls. (5) Recombinant CD40L and serum from patients with unstable angina who had high sCD40L levels induced enhanced release of monocyte chemoattractant peptide-1 from mononuclear cells, a CC-chemokine involved in the pathogenesis of atherosclerosis. CONCLUSIONS: This first demonstration of enhanced levels of soluble and membrane-bound forms of CD40L in angina patients, with particularly high levels in patients with unstable angina, suggests that CD40L-CD40 interaction may play a pathogenic role in both the long-term atherosclerotic process and in the triggering and propagation of acute coronary syndromes.

Microvesicles bind soluble fibrinogen, adhere to immobilized fibrinogen and coaggregate with platelets.

In the present study we have investigated whether platelet derived microvesicles can bind soluble fibrinogen, bind to immobilized fibrinogen, and coaggregate with platelets. Flow cytometry was used for studies on binding of soluble fibrinogen and coaggregation, whereas ELISA wells were used to study binding of microvesicles to immobilized fibrinogen. Biotinylated microvesicles produced by stimulation with A23187, thrombin or SFLLRN of platelets which had been surface-labelled with biotin, were used both for the coaggregation experiments and for the binding studies with immobilized fibrinogen. Unlabelled microvesicles and biotinylated fibrinogen were employed when studying binding of soluble fibrinogen to the microvesicles. For the flow cytometry, the biotinylated proteins were reacted with avidin or streptavidin which was PE-conjugated, whereas the same substances were conjugated with alkaline phosphatase for the ELISA studies. The microvesicles formed after stimulation of platelets by SFLLRN or A23187 clearly bound the soluble, biotinylated fibrinogen. Moreover, isolated biotinylated microvesicles added to washed platelets prior to activation, were associated to the microaggregates that formed after stimulation. A significant binding of biotinylated microvesicles to immobilized fibrinogen could also be detected. The binding of microvesicles to soluble and immobilized fibrinogen and association to platelets was clearly specific and at least partly dependent on the GPIIb-IIIa complex, as all of these phenomena could be prevented or reduced by addition of the c7E3 Fab which blocks the activated form of this receptor complex. From these in vitro results it is clear that microvesicles can bind to immobilized fibrinogen, bind soluble fibrinogen and are able to coaggregate with platelets. It may be speculated that these results also reflect a haemostatic role of microvesicles in vivo.

Enhanced activation of platelets with abnormal release of RANTES in human immunodeficiency virus type 1 infection.

Besides their role in hemostasis, platelets are involved in inflammatory and immunological processes, and we hypothesize that platelet activation may play an immunopathogenetic role in HIV-1 infection. Blood was drawn from 15 controls and 20 HIV-1-infected patients with normal platelet counts, classified into groups of non-AIDS and AIDS. Platelet activation was detected using flow cytometry with mAbs against the release markers P-selectin and CD63, mAb against GPIb, and the probe annexin V detecting surface exposure of aminophospholipids. The amount of microvesicles was measured using mAb against GPIIIa. Compared to controls, blood samples from HIV-1-infected patients showed significantly enhanced levels of microvesicles and activated platelets as detected by their exposure of P-selectin, CD63, and aminophospholipids, as well as reduction in GPIb expression. Increased expression of P-selectin and amounts of microvesicles were most pronounced in advanced clinical and immunological disease. When studying the effect of HIV-1 protease inhibitor therapy (indinavir) on platelet activation, we found that concomitant with a profound decrease in plasma viral load, there was a near normalization of several of the parameters reflecting enhanced platelet activation. Finally, we demonstrated that platelets may be an important source of the chemokine RANTES in HIV-1-infected patients. Although both unstimulated and SFLLRN-stimulated platelets from asymptomatic patients had enhanced release of RANTES, platelets from AIDS patients were characterized by markedly enhanced spontaneous, but decreased SFLLRN-stimulated release of this chemokine. Taken together, these results, which demonstrate for the first time increased platelet activation in HIV-1-infected patients with normal platelet counts, may represent a previously unrecognized immunopathogenic factor in HIV-1 infection.

Shear-induced platelet activation and platelet microparticle formation in native human blood.

Shear-induced platelet activation and platelet microparticle formation are triggered in native human blood by high arterial shear or by a sudden increase in shear as introduced by a stenosis with potential consequences for collagen-induced platelet thrombus formation. Blood was drawn from healthy volunteers and directly perfused ex vivo over various well-defined eccentric stenoses. Shear-induced platelet activation was determined by using flow cytometry to assess: 1) GPIIb-IIIa activation by fluorescein isothiocyanate (FITC)-labeled Mab PAC-1; and 2) translocation of membrane aminophospholipids (procoagulant activity) by FITC-labeled Annexin V. Microparticle formation was measured by flow cytometry and FITC-labeled Mab Y2/51 directed against GPIIIa. Significant platelet activation and platelet microparticle formation were elicited when the wall shear rate reached 10,500 sec-1 for a period of 0.075 sec. Prolonged exposure to or a rapid increase in shear further enhanced activation and microparticle formation. Shear-induced platelet activation was associated with significantly increased collagen-induced platelet thrombus formation that was insensitive to aspirin ingestion. Exposure of native blood to very high shear thus activates platelets to express GPIIb-IIIa, renders the platelet membrane procoagulant and stimulates microparticle formation. These responses are associated with enhanced collagen-induced thrombus formation by prostaglandin-independent mechanisms.

Shear-induced platelet activation and platelet microparticle formation at blood flow conditions as in arteries with a severe stenosis.

In the present study, we investigated whether high arterial shear stresses at various exposure times or a sudden increase in shear stress introduced by a stenosis affect platelet activation and platelet microparticle formation in native human blood. We used a parallel-plate perfusion chamber device through which nonanticoagulated human blood was drawn (10 mL/min) by a pump directly from an antecubital vein through the flow channel of a perfusion chamber at wall shear rates of 420, 2600, and 10500 s-1. In another set of experiments, an eccentric stenosis was introduced into the flow channel. Wall shear rates of 2600 or 10500 s-1 at the stenosis apex were maintained at the same flow rate. The wall shear rate upstream and downstream of these stenoses was 420 s-1. A shear rate of 420 s-1 is within the range of those encountered in healthy small coronary arteries, whereas those of 2600 and 10500 s-1 are representative for vessels with various degrees of stenotic lesions. The blood was exposed to these shear rates for periods varying from 0.075 to 3.045 seconds. Platelet activation was assessed as activated glycoprotein (GP) IIb/IIIa by FITC-labeled monoclonal antibody (MAb) PAC-1 and aminophospholipid translocation by FITC-labeled annexin V. Microparticle formation was quantified by FITC-labeled MAb Y2/51 directed against GP IIIa. Significant platelet activation and formation of microparticles were observed at 10500 s-1 only (P < .008). This shear-induced platelet activation and microparticle formation were enhanced by introduction of a thrombus-promoting surface consisting of type III human collagen fibrils. Introduction of the most severe stenosis at 10500 s-1 further increased platelet activation (P < .017). The collagen-induced thrombus formation increased the platelet thrombus volume at 10500 s-1 from 16.5 to 33.8 microns3/microns2 (P < .003) on the stenosis apex when the most severe stenosis was used. A correlation (P < .0001) between platelet thrombus volume and platelet microparticle formation was observed in the presence of the eccentric stenoses. Apparently, high shear stress (315 dynes/cm2 at 10500 s-1), as encountered in severe atherosclerotic arteries, activated platelets and triggered platelet microparticle formation. In contrast, no significant platelet activation or formation of platelet microparticles was observed at physiological shear (420 s-1) or at the shear condition simulating shear in arteries with a less severe stenosis (2600 s-1). The data imply that platelets are activated and form microparticles in native blood at very high shear stresses. These events are potentiated by prolonged exposure to the high shear or by a sudden change of increasing shear due to the stenosis. The latter situation apparently enhances platelet thrombus formation at the stenosis.

Glycoprotein IIb-IIIa on platelet-derived microparticles, and microparticle structures studied by electron microscopy, confocal laser microscopy and crossed radio-immunoelectrophoresis.

Shedding of microparticles from the platelet surface is usually associated with exposure of platelet procoagulant activity. Platelet-derived microparticles have been detected in blood in various disease states. In vitro, platelet stimulation with a number of different agonists results in formation of microparticles. In the present study, microparticles induced by platelet stimulation by calcium ionophore or by membrane incorporation of the terminal complement complex C5b-9 were studied using electron microscopy, confocal laser microscopy, flow cytometry and radio-immunoelectrophoresis. When studied by electron microscopy, microparticle morphology was found to be dependent upon the induction method. Platelet stimulation with the calcium ionophore resulted in smaller, more homogeneous and electron dense microparticles than those induced by insertion of the terminal complement complex. With flow cytometry and confocal laser immunofluorescence microscopy, microparticle GPIIb-IIIa was demonstrated using a FITC-conjugated antibody to GPIIIa. Surface-bound GPIIb-IIIa was demonstrated on the microparticles by immunoelectron microscopy. Crossed immunoelectrophoresis of detergent-solubilized microparticles visualized a very prominent GPIIb-IIIa immunoprecipitate arc, and binding of [(125)1]fibrinogen to microparticle GPIIb-IIIa was demonstrated by radio-immunoelectrophoresis. This suggests that the activated GPIIb-IIIa complex is preserved intact during the shedding of microparticles from the platelet surface.

Stimulated Glanzmann's thrombasthenia platelets produced microvesicles. Microvesiculation correlates better to exposure of procoagulant surface than to activation of GPIIb-IIIa.

The mechanism of formation of platelet-derived microvesicles remains controversial. The aim of the present work was to study the formation of microvesicles in view of a possible involvement of the GPIIb-IIIa complex, and of exposure of negatively charged phospholipids as procoagulant material on the platelet surface. This was studied in blood from three Glanzmann's thrombasthenia patients lacking GPIIb-IIIa and healthy blood donors. MAb FN52 against CD9 which activates the complement system and produces microvesicles due to a membrane permeabilization, ADP (9.37 microM), and the thrombin receptor agonist peptide SFLLRN (100 microM) that activates platelets via G-proteins were used as inducers. In a series of experiments platelets were also preincubated with PGE1 (20 microM). The number of liberated microvesicles, as per cent of the total number of particles (including platelets), was measured using flow cytometry with FITC conjugated antibodies against GPIIIa or GPIb. Activation of GPIIb-IIIa was detected as binding of PAC-1, and exposure of aminophospholipids as binding of annexin V. With normal donors, activation of the complement system induced a reversible PAC-1 binding during shape change. A massive binding of annexin V was seen during shape change as an irreversible process, as well as formation of large numbers of microvesicles (60.6 +/- 2.7%) which continued after reversal of the PAC-1 binding. Preincubation with PGE1 did not prevent binding of annexin V, nor formation of microvesicles (49.5 +/- 2.7%), but abolished shape change and PAC-1 binding after complement activation. Thrombasthenic platelets behaved like normal platelets after activation of complement except for lack of PAC-1 binding (also with regard to the effect of PGE1 and microvesicle formation). Stimulation of normal platelets with 100 microM SFLLRN gave 16.3 +/- 1.2% microvesicles, and strong PAC-1 and annexin V binding. After preincubation with PGE1 neither PAC-1 nor annexin V binding, nor any significant amount of microvesicles could be detected. SFLLRN activation of the thrombasthenic platelets produced a small but significant number of microvesicles (6.4 +/- 0.8%). Incubation of thrombasthenic platelets with SFLLRN after preincubation with PGE1, gave results identical to those of normal platelets. ADP activation of normal platelets gave PAC-1 binding, but no significant annexin V labelling, nor production of microvesicles. Thus, different inducers of the shedding of microvesicles seem to act by different mechanisms. For all inducers there was a strong correlation between the exposure of procoagulant surface and formation of microvesicles, suggesting that the mechanism of microvesicle formation is linked to the exposure of aminophospholipids. The results also show that the GPIIb-IIIa complex is not required for formation of microvesicles after activation of the complement system, but seems to be of importance, but not absolutely required, after stimulation with SFLLRN.