Formation Of Platelet-derived Microparticles Research Articles

Anti-Platelet Activity of Sea Buckthorn Seeds and Its Relationship with Thermal Processing.

Sea buckthorn (Hippophae rhamnoides L.) is a tree or shrub with small, orange berries. Sea buckthorn seeds have shown many properties beneficial to human health, including antioxidant, anti-hypertensive, anti-hyperlipidemic, and retinoprotective activities. Seeds, as a component of food, are often exposed to high temperatures, which can increase or decrease their biological activity. In our previous study, we showed that both raw and roasted sea buckthorn seeds had significant antioxidant activity, which was measured in human plasma in vitro. In this paper, we evaluated the effect of extracts from raw and roasted sea buckthorn seeds on several parameters of hemostasis in vitro, including thrombus formation in full blood (measured by the Total Thrombus formation Analysis System-T-TAS), blood platelet activation (based on the exposition of P-selectin, the active form of GPIIb/IIIa on their surface and platelet-derived microparticles formation), aggregation (measured with impedance aggregometry), adhesion to fibrinogen and collagen, arachidonic acid metabolism in washed platelets stimulated by thrombin, and COX-1 activity. We also measured the levels of free 8-isoprostane in plasma and the total non-enzymatic antioxidant status of plasma. The extract from roasted seeds (50 µg/mL) significantly prolonged the time of occlusion measured by T-TAS-the AUC10 (area under the curve) value was decreased by approximately 18%. Both extracts decreased the exposition of the active form of GPIIb/IIIa on the surface of platelets activated with 10 μM ADP (by 38.4-62.2%) and 20 μM ADP (by 39.7-51.3%). Moreover, the extract from raw seeds decreased the exposition of P-selectin on the surface of platelets stimulated with 20 μM ADP (by 31.2-34.9%). The adhesion of thrombin-stimulated platelets to fibrinogen and collagen was inhibited only by the extract from roasted sea buckthorn seeds (by 20-30%). Moreover, the extract from raw seeds inhibited the level of TBARS (thiobarbituric acid-reactive substances, an indicator of enzymatic peroxidation of arachidonic acid) in washed platelets stimulated with thrombin; the activity of COX-1 was inhibited by both extracts, although the effect of the extract from raw seeds was stronger. These results indicate that sea buckthorn seeds have anti-platelet activity that is not decreased by thermal processing, but more research is needed to determine which exact chemical compounds and mechanisms are responsible for this phenomenon.

MicroRNA-363-3p/sphingosine-1-phosphate receptor 1 axis inhibits sepsis-induced acute lung injury via the inactivation of nuclear factor kappa-B ligand signaling.

Infection-associated inflammation and coagulation are critical pathologies in sepsis-induced acute lung injury (ALI). This study aimed to investigate the effects of microRNA-363-3p (miR-363-3p) on sepsis-induced ALI and explore the underlying mechanisms. A cecal ligation and puncture-induced septic mouse model was established. The results of this study suggested that miR-363-3p was highly expressed in lung tissues of septic mice. Knockdown of miR-363-3p attenuated sepsis-induced histopathological damage, the inflammation response and oxidative stress in lung tissues. Furthermore, knockdown of miR-363-3p reduced the formation of platelet-derived microparticles and thrombin generation in blood samples of septic mice. Downregulation of miR-363-3p suppressed sphingosine-1-phosphate receptor 1 (S1PR1) expression in lung tissues and subsequently inactivated the nuclear factor kappa-B ligand (NF-κB) signaling. A luciferase reporter assay confirmed that miR-363-3p directly targeted the 3’-untranslated region of the mouse S1pr1 mRNA. Collectively, our study suggests that inactivation of NF-κB signaling is involved in the miR-363-3p/S1PR1 axis-mediated protective effect on septic ALI.

The Effects of the Combined Argatroban/Nitric Oxide-Releasing Polymer on Platelet Microparticle-Induced Thrombogenicity in Coated Extracorporeal Circuits.

Clotting, anticoagulation, platelet consumption, and poor platelet function are major factors in clinical extracorporeal circulation (ECC). We have shown that nitric oxide-releasing (NOReL) coatings prevent thrombosis in a rabbit model of ECC without systemic anticoagulation. Nitric oxide-releasing prevents platelet adhesion and activation, resulting in preserved platelet count and function. Previous work has shown that activated platelets form platelet-derived microparticles (PMPs). These experiments were designed to determine if PMPs can identify platelet function during ECC. The objective of this study is to investigate the effects of NOReL on platelet activation and PMP formation during ECC. Uncoated ECCs, including with and without systemic heparin, and NOReL-coated ECCs, including DBHD/N2O2 and argatroban (AG)/DBHD/N2O2-coated ECCs without systemic heparin, were tested in a 4-hour rabbit thrombogenicity model. Before and after ECC exposure, platelets were stimulated with collagen, and PMPs were measured using flow cytometry. The uncoated ECCs clotted within the first hour, while the NOReL-coated ECCs circulated for 4 hours. During pre-ECC blood exposure, platelets stimulated with collagen produced PMPs. With post-ECC exposure, platelets from uncoated circuits generated less PMPs than baseline (mean ± SDs: 23246 ± 3611 baseline vs. 1300 ± 523 uncoated post circuit, p = 0.018) when stimulated with collagen. However, platelets from the AG/DBHD/N2O2-coated ECCs generated a greater number of PMPs as baseline values (23246 ± 3611 baseline vs. 37040 ± 3263 AG/DBHD/N2O2 post 4 hours circuit, p = 0.023). Blood exposure during ECC results in platelet activation and clotting in uncoated ECCs. The remaining circulating platelets have lost function, as demonstrated by the low PMP formation in response to collagen. AG/DBHD/N2O2-coated ECCs prevented significant platelet activation and clotting, while DBHD/N2O2 trended towards prevention of platelet activation. In addition, function of the circulating platelets was preserved, as demonstrated by PMP formation in response to collagen. These results indicate that PMPs may be an important measure of platelet activation during ECC. Platelet-derived microparticles may provide a simplified way to measure platelet function during clinical ECC.

P6006Pathogenesis of symptomatic crises in patients with coronary artery spasm: evidence for acute damage to vascular glycocalyx and to circulating platelets

Abstract Background Coronary artery spasm (CAS) is known to occur commonly even in a “pure culture”, but little is known regarding the precipitation of the symptomatic crises which characterize this disorder. There have been recent reports of the occurrence of plaque erosion and intracoronary thrombosis during CAS crises. We have recently shown that (1) the anti-aggregatory effects of nitric oxide (NO) are impaired in CAS patient and (2) release into plasma of the endothelial glycocalyx component syndecan-1 (SD-1) occurs during acute episodes of CAS, suggesting glycocalyx erosion by inflammatory enzyme release. Objective In the current study, we sought to determine whether this phenomenon is accompanied by damage to circulating platelets and whether activation of mast cells may represent a possible precipitant. Methods In patients with acute episodes and chronic phases of CAS (n=10), as well as normal subjects (n=12), plasma concentrations of SD-1, of platelet-derived microparticles (PMPs), of the mast cell enzyme tryptase and of malondialdehyde (MDA), a measure of oxidative stress, were evaluated. Results The results are summarized in the table. Symptomatic crises were associated with substantial elevation of SD-1 concentrations and also those of PMPs and of tryptase, relative to chronic status. However, MDA concentrations did not vary significantly during acute episodes. Impact of acute exacerbation of CAS on markers of glycocalyx shedding (SD-1), platelet activation (PMP counts), mast cell activation (tryptase) and oxidative stress (MDA) Parameter Acute CAS Chronic CAS p* Normal subjects** SD-1 (μg/L) 50.3±3.9 14.3±4.7 <0.0001 12.7±2.4 PMPs (counts) 24200±8100 4800±1010 0.02 10400±2900 Tryptase (μg/L) 4.4±0.4 4.1±0.4 0.03 <12.0 MDA (μM) 3.2±0.2 3.1±0.1 NS 3.09±0.15 *Acute vs. chronic; **provided for reference only. Conclusion (1) The combination of SD-1 release and formation of PMPs suggests that CAS crises reflect both glycocalyx “shedding” and platelet activation/apoptosis, a combination which would facilitate coronary thrombosis. (2) CAS crises are associated with mast cell activation, which may contribute to the above vascular/platelet damage.

Rac1 regulates platelet microparticles formation and rheumatoid arthritis deterioration

Platelets play important roles in blood clotting, hemostasis and wound repair, while more and more research show that platelets also have significant contributions in the process of inflammation. Rheumatoid arthritis is a chronic systemic inflammatory autoimmune disease. Platelet microparticles, which are membrane vesicles shed by activated platelets, are reported to amplify inflammation in Rheumatoid arthritis. Here we show that either platelet-specific deletion of Rac1 (Rac1−/-) or Rac1-specific inhibitor NSC23766 dramatically inhibit platelet-derived microparticles formation. As we all know, collagen-induced arthritis (CIA) mouse model is the most common autoimmune model of rheumatoid arthritis. Interestingly, NSC23766 alleviated the process of collagen-induced arthritis of DBA mice in vivo, including the reduced hind paw thickness and ankle stiffness, the reduction of arthritic scores and incidence of arthritis. Our work also found that NSC23766-treated CIA mouse spleen is less swollen and contains less enlarged white pulp than PBS control. The histological analysis shows that NSC23766-treated but not solvent control improve the cartilage erosion symptom in the joint of CIA mouse. Interestingly, platelet microparticles in the peripheral blood of NSC23766-treated CIA mice were decreased significantly compared with PBS-treated CIA mice. In conclusion, our work demonstrated that Rac1 inhibition alleviates collagen-induced arthritis through the decrease of platelet microparticles’ release. In short, Rac1 aggravate the rheumatoid arthritis deterioration through the regulation of platelet microparticles formation.

Rac1 regulates sepsis-induced formation of platelet-derived microparticles and thrombin generation

Dysfunctional coagulation aggravates clinical outcome in patients with sepsis. The aim of this study was to define the role of Rac-1 in the formation of platelet-derived microparticles (PMPs) and thrombin generation (TG) in abdominal sepsis. Male C57BL/6 mice underwent cecal ligation and puncture (CLP). Scanning electron microscopy and flow cytometry were used to quantify PMPs. TG was determined by use of a fluorimetric assay. It was found that CLP increased Rac1 activity in platelets, which was abolished by administration of the Rac1 inhibitor NSC23766. Sepsis-induced TG in vivo was reflected by reduced capacity of plasma from septic animals to generate thrombin ex vivo. Administration of NSC23766 increased peak and total TG in plasma from CLP mice indicating that Rac-1 regulates sepsis-induced formation of thrombin. The number of circulating PMPs was markedly elevated in animals with abdominal sepsis. Treatment with NSC23766 significantly decreased formation of PMPs in septic mice. Platelet activation in vitro caused release of numerous MPs. Notably, NSC23766 abolished PMP formation in activated platelets in vitro. These findings suggest that Rac-1 regulates PMP formation and TG in sepsis and that inhibition of Rac1 activity could be a useful target to inhibit dysfunctional coagulation in abdominal sepsis.

Time-dependent inhibitory effects of cGMP-analogues on thrombin-induced platelet-derived microparticles formation, platelet aggregation, and P-selectin expression

In platelets, nitric oxide (NO) activates cGMP/PKG signalling, whereas prostaglandins and adenosine signal through cAMP/PKA. Cyclic nucleotide signalling has been considered to play an inhibitory role in platelets. However, an early stimulatory effect of NO and cGMP-PKG signalling in low dose agonist-induced platelet activation have recently been suggested. Here, we investigated whether different experimental conditions could explain some of the discrepancy reported for platelet cGMP-PKG-signalling. We treated gel-filtered human platelets with cGMP and cAMP analogues, and used flow cytometric assays to detect low dose thrombin-induced formation of small platelet aggregates, single platelet disappearance (SPD), platelet-derived microparticles (PMP) and thrombin receptor agonist peptide (TRAP)-induced P-selectin expression. All four agonist-induced platelet activation phases were blocked when platelets were costimulated with the PKG activators 8-Br-PET-cGMP or 8-pCPT-cGMP and low-doses of thrombin or TRAP. However, extended incubation with 8-Br-PET-cGMP decreased its inhibition of TRAP-induced P-selectin expression in a time-dependent manner. This effect did not involve desensitisation of PKG or PKA activity, measured as site-specific VASP phosphorylation. Moreover, PKG activators in combination with the PKA activator Sp-5,6-DCL-cBIMPS revealed additive inhibitory effect on TRAP-induced P-selectin expression. Taken together, we found no evidence for a stimulatory role of cGMP/PKG in platelets activation and conclude rather that cGMP/PKG signalling has an important inhibitory function in human platelet activation.

TMEM16F Plays a Vital Role In Platelet Microparticle Generation and Thrombosis

Transmembrane (TMEM)16f is member of the TMEM16 family of ion channels, recently shown to be essential for optimal Ca2+-dependent phospholipid scrambling in platelets, platelet-dependent pro-coagulant activity and thrombosis. However, relatively little is known about the effect of TMEM16f on platelet signaling, functional activity, and microparticle formation. TMEM16f-/- mice were obtained from Andrea Vortkamp and the platelets of these mice were isolated, stimulated with either dual agonist (thrombin + convulxin) or calcium ionophore (A23187), and platelet surface exposure of phosphatidylserine (PS) and microparticle generation were assessed by both flow cytometry and high resolution immunofluorescent confocal microscopy. To measure PS exposure, annexin V binding to platelet surfaces was quantified: In response to thrombin and convulxin, only 10.65% ± 1.35% of TMEM16f-/- platelets exposed PS after dual agonist exposure, compared to 16.1% ± 2.3%PS+ WT platelets when analyzed by high resolution microscopy. When analyzed by flow cytometry, dual agonist exposure of WT platelets yielded 52.4% ± 7.0% of maximal annexin V binding achieved by 10μM A23187; in contrast, there was no significant increase in annexin V binding detected in TMEM16f-/- platelets treated with dual agonist. Responses to calcium ionophore were also reduced in TMEM16f-/- platelets relative to WT (17.3% ± 8.3% PS+ TMEM16f-/- platelets in response to 1μM A23187 compared to 40% ± 1.4% PS+ WT platelets, by high resolution microscopy). Microparticle generation from TMEM16f-/- platelets compared to WT platelets was also evaluated by high resolution immunofluorescent microscopy and flow cytometry. On average, approximately 5.7 ± 0.33 microparticles were generated by untreated WT platelets however thrombin and convulxin treated platelets yielded 10.66 ± 0.92 microparticles per platelet whereas no significant increase in microparticle generation was observed in TMEM16f-/- platelets after dual agonist treatment. These data suggest that TMEM16f is required not only for PS exposure on platelet membranes, but also for the shedding of PS+ microparticles. TMEM16f-/- mice also have a significant defect in stable occlusive thrombus formation following 10% ferric chloride injury of the carotid artery (0/6 TMEM16f-/- mice form stable thrombi compared to 5/6 WT mice forming stable thrombi under the same conditions). TMEM16f-/- platelets had a slight (15%) reduction in total fibrinogen binding stimulated by PAR4 peptide agonist compared to WT, but showed no significant differences in aggregation to PAR4 agonist peptide or ADP compared to WT control platelets, suggesting that the defect in thrombus formation is likely due to PS- or microparticle-dependent procoagulant activity. This hypothesis is supported by preliminary results demonstrating that when microparticles isolated from ionophore-stimulated WT platelets were injected into TMEM16f knockout mice, 3/3 TMEM16f-/- mice formed stable thrombi, compared to 5/6 WT mice injected with vehicle control. In conclusion, TMEM16f-/- mice are deficient in platelet PS exposure, platelet-derived microparticle formation, and injury-induced thrombus formation, and platelet-derived microparticles appear to contribute to the defect in thrombus formation. Disclosures:No relevant conflicts of interest to declare.

Enhanced platelet-derived microparticle formation is associated with carotid atherosclerosis in convalescent stroke patients

Platelets participate in the development and progression of atherosclerosis. During this process they interact with endothelial cells and leukocytes. Therefore, we investigated the associations between carotid atherosclerosis and platelet reactivity markers. The platelet surface expression of P-selectin (CD62P) and the activated GPIIb/IIIa receptor (corresponding to increased binding of PAC-1), as well as the fraction of platelet-derived microparticles (PMPs) prior to and after platelet stimulation with TRAP or ADP, were determined using flow cytometry in 94 subjects in the convalescent phase of ischaemic stroke and in 76 disease controls. The mean common carotid intima-media thickness (CCAmean IMT), maximal common carotid IMT (CCAmax IMT) and maximal bifurcation IMT (BIFmax IMT) were measured bilaterally using B mode, colour Doppler ultrasonography. In stroke subjects IMT within CCA and BIF were greater than in disease controls and the percentage of PMPs prior to and after ex vivo stimulation with agonists was significantly higher than in controls. Multiple regression analysis revealed that PMPs were positively and independently correlated with both CCAmean IMT ( β = 0.23; p < 0.01) and stroke ( β = 0.21; p<0.01), while PAC-1 binding to platelets activated with ADP was negatively and independently associated with CCAmean IMT ( β = −0.29; p<0.001) and atherosclerotic carotid plaque presence ( β = −0.28, p = 0.003). We found a positive association between enhanced PMP formation and atherosclerotic thickening of carotid intima-media or carotid plaque in patients after ischaemic stroke. We demonstrated that diminished expression of active GPIIb/IIIa in the ADP-activated platelets is associated with increased carotid IMT, independently of stroke.

Reactive leptin resistance and the profile of platelet activation in acute ischaemic stroke patients

Leptin is an adipokine that in vitro enhances agonist-induced platelet aggregation and adipokine expression. Hyperleptinaemia represents a risk factor for cardiovascular disease. We conducted a prospective evaluation of the potential link between blood platelet activation and plasma leptin levels in post-stroke patients. Using five-colour flow cytometry, the platelet surface expression of CD40L, CD62P, the subpopulations of monocyte-platelet aggregates and platelet-derived microparticles (PMPs) as well as the plasma leptin, soluble leptin receptor (sOb-R), leptin/sOb-R ratio, the plasma adiponectin, and leptin/adiponectin ratio were assessed in 98 stroke patients on the first (V₀), 10th (V₁ ) and 90th (V₂) day after stroke and once in 78 age-, gender- and vascular risk factor-matched disease controls. We demonstrated that at V0 leptin resistance, defined as leptin/sOb-R ratio, was higher than in the controls [1.1 (0.5-1.8 vs. 0.5 (0.2-1.1); p=0.02]. After adjustment according to the factors which influence platelet activation, we confirmed the relationship between percentage of circulating PMPs and plasma leptin level (B=0.18; p=0.02) or the leptin/sOb-R ratio (B=0.23; p=0.02) in normal-weight subjects in the acute phase of stroke. No correlation could be demonstrated between the adipokine parameters and the percentage of monocyte-platelet aggregates or expression of platelet pro-inflammatory glycoproteins. In conclusion, formation of PMPs on the first day following an ischaemic stroke shows a positive correlation with leptin levels and with resistance to leptin. Leptin level does not seem to affect the expression of platelet surface proinflammatory glycoproteins.

Platelet apoptosis in paediatric immune thrombocytopenia is ameliorated by intravenous immunoglobulin

To evaluate the role of intravenous immunoglobulin (IVIg) in platelet apoptosis in paediatric immune thrombocytopenia, we investigated the platelets of 20 paediatric patients with acute immune thrombocytopenia (ITP), before and after IVIg treatment. Healthy children with platelet counts in the normal range and children with thrombocytopenia due to chemotherapy were enrolled as controls. All ITP patients presented with platelet counts <20 × 10(9) /l and bleeding symptoms. Markers of apoptosis, including activated caspase-3, -8 and -9, phosphatidylserine (PS) exposure, mitochondrial inner membrane potential (ΔΨm), as well as platelet-derived microparticle formation, were analysed by flow cytometry. After IVIg treatment, platelet counts increased to >20 × 10(9) /l in all patients. ITP patients had significantly increased proportions of platelets with activated caspase-3, -8 and -9, with PS exposure, and with decreased ΔΨm, and demonstrated increased microparticle formation. Except for ΔΨm, these markers for apoptosis were reduced by IVIg treatment. Platelets of children with thrombocytopenia after chemotherapy also demonstrated increased microparticle formation and decreased ΔΨm, but no activation of caspases 3, 8 and 9 or PS exposure. In conclusion, in acute paediatric ITP, enhanced platelet apoptosis is seen at diagnosis that normalizes after IVIg treatment.

Protein phosphatase inhibitor calyculin – A modulates activation markers in TRAP-stimulated human platelets

Platelet activation is accompanied with the phosphorylation of a number of proteins on serine (Ser) and threonine (Thr) residues. The phosphorylation level of these proteins is dependent upon the protein kinase/phosphatase activity ratio. The aim of this study was to investigate the consequences of inhibiting protein phosphatase 1 (PP1) and 2A (PP2A) on platelet functions. Protein phosphatases were inhibited by preincubation of platelet rich plasma (PRP) samples with calyculin-A (CLA). Subsequently, platelets were activated by thrombin-receptor activating peptide (TRAP) and platelet aggregation, platelet-derived microparticle (PMP) formation, surface expressions of P-selectin (CD62), lysosome-associated membrane protein (CD63), glycoprotein Ib and IIb were examined. Phosphatase activity was determined by using phosphorylated 20 kDa myosin light chain (P-MLC20) as substrate. In CLA-treated platelets substantial decrease of P-MLC20 phosphatase activity was observed. CLA significantly suppressed TRAP-induced surface expression of P-selectin and CD63 in a concentration-dependent manner as compared to non-treated samples and moderately decreased platelet aggregation. In TRAP-activated samples, 50 nM of CLA pretreatment completely abolished the level of PMPs and the prevention of GPIb downregulation was also observed; however, no difference was found in GPIIb expression. In conclusion, PP1 and PP2A-catalyzed dephosphorylation processes have crucial roles in PMP formation and in the regulation of alpha-granule and lysosome secretion in human platelets.

Platelet-Derived Microparticles and Platelet Count in Preterm Newborns

Objective: Does formation of platelet-derived microparticles correspond to platelet activation? Methods: The study was performed in 51 preterm newborns, 25 girls and 26 boys. The control group consisted of 55 term newborns, 25 girls and 30 boys. Blood samples were collected from the umbilical artery. The percentage of platelet-derived microparticles and platelet count were determined using flow cytometric analysis based on the CD61-positive antigen. Results: The percentage of platelet-derived microparticles was higher in preterm newborns (5.46) in comparison to term newborns (4.22, p < 0.01). We found 4.61% of platelet-derived microparticles in preterm female newborns and 6.28% in preterm boys (p < 0.0070). The platelet count was 256 × 10³ µl in girls and 238 × 10³ µl in boys. Female healthy term newborns presented higher values of platelet-derived microparticles (4.4%) than male newborns (4.07%, p = 0.4725, table 1). The platelet count in girls was found to be 308 × 10³ µl and in boys 270 × 10³ µl. Conclusions: Higher percentage of platelet-derived microparticles in preterm newborns may provide a compensatory mechanism for the hemostatic system.

Mechanism of platelet adhesion to von Willebrand factor and microparticle formation under high shear stress

We describe here the mechanism of platelet adhesion to immobilized von Willebrand factor (VWF) and subsequent formation of platelet-derived microparticles mediated by glycoprotein Ibalpha (GPIbalpha) under high shear stress. As visualized in whole blood perfused in a flow chamber, platelet attachment to VWF involved one or few membrane areas of 0.05 to 0.1 microm(2) that formed discrete adhesion points (DAPs) capable of resisting force in excess of 160 pN. Under the influence of hydrodynamic drag, membrane tethers developed between the moving platelet body and DAPs firmly adherent to immobilized VWF. Continued stretching eventually caused the separation of many such tethers, leaving on the surface tube-shaped or spherical microparticles with a diameter as low as 50 to 100 nm. Adhesion receptors (GPIbalpha, alphaIIbbeta3) and phosphatidylserine were expressed on the surface of these microparticles, which were procoagulant. Shearing platelet-rich plasma at the rate of 10,000 s(-1) in a cone-and-plate viscosimeter increased microparticle counts up to 55-fold above baseline. Blocking the GPIb-VWF interaction abolished microparticle generation in both experimental conditions. Thus, a biomechanical process mediated by GPIbalpha-VWF bonds in rapidly flowing blood may not only initiate platelet arrest onto reactive vascular surfaces but also generate procoagulant microparticles that further enhance thrombus formation.

Current methods of assessing platelet function: relevance to transfusion medicine

Current methods of assessing platelet function: relevance to transfusion medicine

Cleavage of platelet endothelial cell adhesion molecule‐1 (PECAM‐1) in platelets exposed to high shear stress

Platelet endothelial cell adhesion molecule-1 (PECAM-1, CD31) is a 130 kDa transmembrane glycoprotein that belongs to the immunoglobulin superfamily and is expressed on the surface of endothelial cells, platelets, and other blood cells. Although the importance of this adhesion molecule in various cell-cell interactions is established, its functional role in platelets remains to be elucidated. In this study, we examined whether PECAM-1 underwent changes in platelets exposed to high shear stress. Platelet PECAM-1 was cleaved under high shear stress and was released into the extracellular fluid as a fragment with an approximate molecular weight of 118 kDa. The cleavage was inhibited by an anti-VWF MoAb, but not by recombinant VWF A1 domains. These findings suggest that the GPIb-VWF interaction is involved in PECAM-1 cleavage under high shear stress, and that the cleavage is independent of GPIb clustering by VWF multimers. Furthermore, EGTA or calpeptin inhibited PECAM-1 cleavage. This finding provides evidence for the involvement of calpain in PECAM-1 cleavage. Flow-cytometric analysis revealed that PECAM-1 expression on the platelet surface was decreased under high shear stress. This reduction occurred exclusively in a specific population of platelets, which corresponded to platelet-derived microparticles (PMP). In conclusion, PECAM-1 cleavage under high shear stress is closely related to the activation of calpain and the process of PMP formation mediated by the GPIb-VWF interaction.

Collagen-induced generation of platelet-derived microparticles in whole blood is dependent on ADP released from red blood cells and calcium ions

We have evaluated the effects of different anti-coagulants or agonists on the generation of platelet-derived microparticles (PMPs) using flow cytometry. Twenty μg/ml of collagen induced significantly greater PMP formation in whole blood anti-coagulated with argatroban, a selective thrombin inhibitor, as compared with platelet-rich plasma, or whole blood anti-coagulated with citrate. Thus, whole blood kept at the physiological Ca2+ concentration provides an optimal condition for the formation of PMP. Convulxin, a GPVI-selective agonist, also induced PMP formation at the magnitude which far exceeds those of other agonists, such as thrombin receptor-activating peptide, ADP or epinephrine. These findings suggest that GPVI-mediated platelet activation plays a key role in the formation of PMP in the presence of physiological Ca2+ in whole blood. The addition of red blood cells to PRP potentiated PMP formation induced by collagen. Pretreatment of whole blood with the combination of creatine phosphate and creatine phosphokinase reduced PMP formation induced by collagen. Blockade of ADP receptors, P2Y12 with AR-C69931MX and P2Y1 with A3P5P, respectively, further suppressed collagen-induced PMP formation. We conclude that ADP released from red blood cells enhances PMP formation induced by collagen, and that both P2Y12 and P2Y1 contribute to ADP-potentiation of PMP generation induced by collagen.

Platelet activation and antiplatelet therapy in patients with ischemic stroke

In patients with ischemic stroke, findings of platelet activation are frequent in platelet function tests. They include increases in plasma levels of beta-thromboglobulin and platelet factor 4, shortening of platelet survival and increasing of platelet lysis, enhancing of shear-induced platelet aggregation (SIPA), and increasing of reticulated platelets as well as platelet fibrinogen binding (PFB) and P-selectin expression (PSE). Aspirin can reduce vascular events including stroke, myocardial infarction and vascular death in high-risk patients with occlusive vascular disease. Aspirin's effect on vascular events is J-shaped, which appeared to be related to the effects on platelet aggregation and release reaction as well as prostaglandin synthesis. Thienopyridines can reduce vascular events even more than aspirin. SIPA was inhibited by thienopyridines but not by aspirin. Combination of aspirin and dipyridamole has an additive effect on preventing subsequent stroke. Aspirin enhanced an inhibitory effect of dipyridamole on SIPA in whole blood. PFB and PSE were not inhibited by aspirin but were partially inhibited by ticlopidine, and markedly inhibited by aspirin plus ticlopidine. Glycoprotein (GP) IIb/IIIa inhibitors can block the final common pathway of platelet aggregation. However, PSE and platelet-derived microparticle formation induced by shear stress were never inhibited at lower concentrations and were rather enhanced at higher concentrations of GP IIb/IIIa inhibitors. These results might be related to the fact that many oral GP IIb/IIIa inhibitors fail to show efficacy in patients with acute coronary syndrome.

P-Selectin expression, platelet aggregates, and platelet-derived microparticle formation are increased in peripheral arterial disease.

Platelet volume has been reported to be increased in vascular disease. Therefore, we studied the relationship of mean platelet volume and platelet count as well as flow cytometrically measured platelet size and platelet function in 50 patients with peripheral arterial disease and 50 healthy volunteers. Platelet activation was measured by P-selectin expression analysis on resting and on stimulated platelets, and the determination of platelet aggregates and platelet-derived microparticles using flow cytometry. P-Selectin expression on platelets was significantly elevated in patients suffering from peripheral arterial disease (all P<0.0001). Platelet aggregates (P<0.0001) and platelet-derived microparticles (P<0.0001) were significantly higher in the patient group compared with controls, whereas mean platelet volume and platelet count showed no significant differences. Platelet count was inversely related to mean platelet volume in patients and controls (r = -0.43, P<0.001). The present study supports the hypothesis of platelet hyperreactivity and circulating activated platelets in peripheral arterial disease. Mean platelet volume, and platelet count cannot be used as predictive markers for platelet activation in peripheral arterial disease patients.

Identification of critical antigen-specific mechanisms in the development of immune thrombocytopenic purpura in mice

AbstractThe pathogenic effects of antiplatelet antibodies were investigated in mice. Monoclonal antibodies (mAbs) of different immunoglobulin G subclass directed against mouse GPIIbIIIa, GPIIIa, GPIbα, GPIb-IX, GPV, and CD31 were generated and characterized biochemically. MAbs against GPIb-IX, GPV, CD31, and linear epitopes on GPIIIa had mild and transient effects on platelet counts and induced no spontaneous bleeding. Anti-GPIbα mAbs induced profound irreversible thrombocytopenia (< 3% of normal) by Fc-independent mechanisms but only had minor effects on hematocrits. In contrast, injection of intact mAbs, but not F(ab)2 fragments, against conformational epitopes on GPIIbIIIa, induced irreversible thrombocytopenia, acute systemic reactions, hypothermia, decreased hematocrits, and a paradoxical loss of surface GPIIbIIIa on platelets in vivo, the latter suggesting the formation of platelet-derived microparticles. Blockage of platelet-activating factor receptors inhibited the acute reactions, but not thrombocytopenia, loss of GPIIbIIIa, and decreases in hematocrits. Repeated injections of low doses of anti-GPIIbIIIa antibodies resulted in profound thrombocytopenia and bleeding, whereas no acute systemic reactions were observed. These data strongly suggest that the identity of the target antigen recognized by antiplatelet antibodies determines the mechanisms of platelet destruction and the severity of bleeding in mice, the latter depending on previously unrecognized anti-GPIIbIIIa-specific inflammatory mechanisms.