Generation Of Platelet-derived Microparticles Research Articles

CARD12: Shear Stress Renders Redistribution of P2Y12 and PAR-1 Receptors from Platelets to Microparticles: A Cue to Limited Effectiveness of Antiplatelet Therapy in MCS

Background: Platelet exposure to hypershear stress generated by mechanical circulatory support (MCS) results in platelet dysfunction, thrombosis, and bleeding coagulopathy. Antiplatelet agents, targeting ADP-evoked P2Y12 and thrombin-evoked PAR1 platelet receptors, have limited effectiveness reducing MCS thrombosis, rather favoring bleeding. Shear-mediated platelet dysfunction is associated with the downregulation of platelet adhesion receptors, decreased aggregation response, and generation of platelet-derived microparticles (PDMPs). We tested the hypothesis that MCS shear stress alters the surface expression of agonist receptors P2Y12 and PAR1 on platelets and PDMPs, thus affecting platelet aggregation response induced by ADP, collagen, and TRAP-6. Methods: Human platelets were exposed to shear stress (30-70 dynes/cm2, 10’) or sonication; PDMPs were isolated via differential centrifugation. P2Y12, PAR1, & αIIbβ3 expression on platelets and PDMPs was visualized by immunostaining and multi-colored flow cytometry. Platelets were distinguished from PDMPs by forward/side scatter vs. standard fluorescent nanobeads SPHEROTM. Platelet aggregation in plasma induced by ADP, collagen, and TRAP-6 was assessed by optical aggregometry. Results: Platelet exposure to increasing shear stress and sonication resulted in a decrease in P2Y12+ and PAR1+ platelet populations (Fig. 1A&B). The baseline surface density of P2Y12 on platelets was very low and slightly increased after 70 dyne/cm2 shear (Fig. 1C). While also low, PAR1 density on platelets tended to decrease with increasing shear stress (Fig. 1D). Shear stress induced generation of P2Y12+ and PAR1+ PDMPs, accounting for 3.5±0.6% and 4.3±0.5% after 70 dyne/cm2 shear, respectively. Remarkably, P2Y12 and PAR1 surface density on PDMPs was between 7 and 10-fold higher than on platelets (Fig. 1C&D). When added to intact platelets, PDMPs significantly decreased platelet aggregation in plasma induced by ADP, collagen, and TRAP-6. Conclusions: Elevated shear stress promotes generation of PDMPs carrying increased amounts of P2Y12 and PAR1 on their surface; the surface density of these receptors on sheared platelets was also affected. Sheared PDMPs reduced platelet aggregation likely acting as competitive inhibitors imposing steric difficulties for platelets to aggregate and thus favoring MCS bleeding phenotype and limiting the effectiveness of antiplatelet agents.

Shear-Mediated Platelet Microparticles Demonstrate Phenotypic Heterogeneity as to Morphology, Receptor Distribution, and Hemostatic Function.

Implantable Cardiovascular Therapeutic Devices (CTD), while lifesaving, impart supraphysiologic shear stress to platelets, resulting in thrombotic and bleeding coagulopathy. We previously demonstrated that shear-mediated platelet dysfunction is associated with downregulation of platelet GPIb-IX-V and αIIbβ3 receptors via generation of Platelet-Derived MicroParticles (PDMPs). Here, we test the hypothesis that sheared PDMPs manifest phenotypical heterogeneity of morphology and receptor surface expression and modulate platelet hemostatic function. Human gel-filtered platelets were exposed to continuous shear stress. Alterations of platelet morphology were visualized using transmission electron microscopy. Surface expression of platelet receptors and PDMP generation were quantified by flow cytometry. Thrombin generation was quantified spectrophotometrically, and platelet aggregation was measured by optical aggregometry. Shear stress promotes notable alterations in platelet morphology and ejection of distinctive types of PDMPs. Shear-mediated microvesiculation is associated with the remodeling of platelet receptors, with PDMPs expressing significantly higher levels of adhesion receptors (αIIbβ3, GPIX, PECAM-1, P-selectin, and PSGL-1) and agonist receptors (P2Y12 and PAR1). Sheared PDMPs promote thrombin generation and inhibit platelet aggregation induced by collagen and ADP. Sheared PDMPs demonstrate phenotypic heterogeneity as to morphology and defined patterns of surface receptors and impose a bidirectional effect on platelet hemostatic function. PDMP heterogeneity suggests that a range of mechanisms are operative in the microvesiculation process, contributing to CTD coagulopathy and posing opportunities for therapeutic manipulation.

DNA Origami-Platelet Adducts: Nanoconstruct Binding without Platelet Activation.

Objective. Platelets are small, mechanosensitive blood cells responsible for maintaining vascular integrity and activatable on demand to limit bleeding and facilitate thrombosis. While circulating in the blood, platelets are exposed to a range of mechanical and chemical stimuli, with the platelet membrane being the primary interface and transducer of outside-in signaling. Sensing and modulating these interface signals would be useful to study mechanochemical interactions; yet, to date, no methods have been defined to attach adducts for sensor fabrication to platelets without triggering platelet activation. We hypothesized that DNA origami, and methods for its attachment, could be optimized to enable nonactivating instrumentation of the platelet membrane. Approach and Results. We designed and fabricated multivalent DNA origami nanotile constructs to investigate nanotile hybridization to membrane-embedded single-stranded DNA-tetraethylene glycol cholesteryl linkers. Two hybridization protocols were developed and validated (Methods I and II) for rendering high-density binding of DNA origami nanotiles to human platelets. Using quantitative flow cytometry, we showed that DNA origami binding efficacy was significantly improved when the number of binding overhangs was increased from two to six. However, no additional binding benefit was observed when increasing the number of nanotile overhangs further to 12. Using flow cytometry and transmission electron microscopy, we verified that hybridization with DNA origami constructs did not cause alterations in the platelet morphology, activation, aggregation, or generation of platelet-derived microparticles. Conclusions. Herein, we demonstrate that platelets can be successfully instrumented with DNA origami constructs with no or minimal effect on the platelet morphology and function. Our protocol allows for efficient high-density binding of DNA origami to platelets using low quantities of the DNA material to label a large number of platelets in a timely manner. Nonactivating platelet-nanotile adducts afford a path for advancing the development of DNA origami nanoconstructs for cell-adherent mechanosensing and therapeutic agent delivery.

Avatrombopag, a Novel Thrombopoietin Receptor Agonist, Increases Platelet Counts without Increasing Platelet Activation in Patients with Thrombocytopenia Due to Chronic Liver Disease

Background: The thrombopoietin (TPO) receptor c-mpl is expressed on the surface of megakaryocytes and platelets. In addition to its effect on increasing megakaryocyte growth and maturation, TPO also lowers the threshold for platelet activation in vivo and in vitro . TPO receptor agonists (TPO-RA) may, perhaps to differing extents, also lead to platelet activation. Indeed, in a recent study of patients with chronic liver disease (CLD), the TPO-RA eltrombopag reduced the need for platelet transfusions, but was associated with an increased incidence of thrombosis. Therefore, the goal of this study was to evaluate the effect of avatrombopag, a novel TPO-RA, on in vivo platelet activation and in vitro platelet reactivity in response to agonists in patients with CLD.Methods: CLD patients participating in the Phase 3 E5501-G000-310 and -311 studies (Randomized, Global, Double-blind, Placebo-controlled, Parallel-group Studies to Evaluate the Efficacy and Safety of Once-daily Oral Avatrombopag for the Treatment of Adults with Thrombocytopenia Associated with Liver Disease Prior to an Elective Procedure) could participate in a substudy to evaluate the effects of avatrombopag on platelet function. IRB-approved informed consent was obtained prior to sample collection. Patients (n = 30) were randomized to treatment on Days 1 - 5 with placebo or avatrombopag: 60 mg daily in patients with platelet counts <40 x 109/L, and 40 mg daily in patients with platelet counts 40 to ≤50 x 109/L. Samples were collected at Baseline (Day 1, pre-drug) and after treatment (Day 4 and Day 10). No patient received a platelet transfusion within the 10 days prior to these blood draws.Platelet activation was evaluated by whole blood flow cytometry (which, unlike other methods, is accurate in samples with low platelet counts) with and without in vitro stimulation with adenosine diphosphate (ADP) and thrombin receptor activating peptide (TRAP). Endpoints included platelet surface activated glycoprotein (GP) IIb-IIIa (as reported by the activation-dependent monoclonal antibody PAC1), platelet surface P-selectin, platelet surface GPIb, platelet phosphatidylserine expression and generation of platelet-derived microparticles. Citrate-anticoagulated whole blood was incubated (15 min, room temperature) with an antibody cocktail containing fluorescein isothiocyanate (FITC)-conjugated PAC1, phycoerythrin (PE)-conjugated anti-P-selectin, and PE-Cy5-conjugated anti-CD42b (GPIb) with or without ADP 0.5 µM or 20 µM or TRAP 1.5 µM or 20 µM. Samples were fixed with 1% formaldehyde, then shipped cold together with the remaining blood sample (for analysis of phosphatidylserine expression and microparticles) to the Center for Platelet Research Studies for analysis. Phosphatidylserine expression on platelets was determined by staining with FITC-conjugated annexin V. Platelet-derived microparticles were identified by light scattering properties and positive staining for CD41 and CD42b. Changes from Baseline for drug-treated (60 mg and 40 mg avatrombopag cohorts combined, n=20) vs. placebo-treated patients (n=10) were compared using a mixed effects model with p<0.05 being considered significant.Results: Avatrombopag, 60 mg and 40 mg treatment, but not placebo treatment, resulted in significant increases in platelet counts at Day 10 (Table). Circulating activated platelets were not increased in avatrombopag-treated patients compared with placebo-treated patients (Table, “no agonist” data), as measured by platelet surface activated GPIIb-IIIa and platelet surface P-selectin. Platelet reactivity, as measured by platelet surface activated GPIIb-IIIa and platelet surface P-selectin after low and high concentrations of ADP and TRAP, was also not increased in avatrombopag-treated patients compared with placebo-treated patients (Table). Similarly, there was no evidence of increased platelet activation in avatrombopag-treated patients compared with placebo-treated patients as assessed by platelet surface GPIb, platelet phosphatidylserine expression and generation of platelet-derived microparticles.Conclusions: In this randomized, double-blind, placebo-controlled, parallel-group study of patients with thrombocytopenia due to CLD, treatment with avatrombopag resulted in increased platelet counts, but did not increase either platelet activation in vivo or platelet reactivity in vitro . [Display omitted] DisclosuresFrelinger:Ionis: Research Funding; Sysmex: Research Funding; Pfizer: Research Funding; Ironwood: Research Funding; GLSynthesis: Research Funding; Baxalta: Research Funding. Michelson:Elsevier: Patents & Royalties; Eisai: Research Funding; GLSynthesis: Research Funding; Instrumentation Laboratory: Consultancy; Baxalta: Research Funding; Ionis: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Sysmex: Research Funding; Pfizer: Research Funding; Ironwood: Research Funding.

Effect of DDAVP on Platelet Activation and Platelet-Derived Microparticle Generation.

The way by which 1-deamino-8-D-arginine vasopressin (DDAVP) acts on platelets remains unclear. Data from the literature tend to show that there is no definite effect on platelet activation, but recent work has suggested that a subtype of platelets, activated by the combined action of collagen and thrombin, was triggered by DDAVP. Moreover, platelet microparticles (PMPs), which have been shown to be procoagulant, have rarely been studied in this context. The goal of this study was to analyze the effects of DDAVP on PMPs' release through platelet activation. Fifteen out of 18 consecutive patients undergoing a therapeutic test with DDAVP were included. They were suffering from factor VIII deficiency or from von Willebrand disease. The expression of P-selectin and PAC-1 binding on platelets and the numbers of circulating PMPs were evaluated ex vivo before and after DDAVP infusion. Peripheral blood was collected on CTAD to limit artifactual platelet activation. DDAVP induced a significant decrease of platelet counts and volume. Only small changes of P-selectin expression and PAC-1 binding were observed. Considering PMPs, two populations of patients could be defined, respectively, with (120%, n = 6) or without (21%, n = 7) an increase of PMPs after DDAVP. The decrease in platelet counts and volume remained significant in the group of responders. This study shows that DDAVP induces the generation/release of PMPs in some patients with factor VIII deficiency and von Willebrand disease 1 hour after DDAVP infusion.

To Bleed or Not to Bleed: MCS-Related Shear Stress Promotes Redistribution of GPIb and αIIbβ3 from Platelets to Microparticles Thus Impairing Platelet Hemostatic Function

Purpose Bleeding is a leading cause of readmission and 1-year mortality for mechanical circulatory support (MCS) patients. Mechanical damage of platelets and coagulation proteins induced by MCS-generated high shear stress is the main driver of bleeding. We previously demonstrated that hypershear promotes platelet pro-apoptotic behavior resulting in membrane reorganization and generation of platelet-derived microparticles (PDMP). Here, we test the hypothesis that hypershear alters surface expression and shedding of adhesion receptors GPIb and αIIbβ3 from platelets and PDMPs thus affecting platelet aggregability - all potential bleeding contributors. Methods Human platelets were exposed to shear stress in a hemodynamic shearing device (30-70 dyn/cm2, 10 min). Integrin GPIb and αIIbβ3 surface expression and PDMP generation were quantified by flow cytometry. Single platelets and PDMPs were distinguished by FS/SS characteristics. Shedding of soluble GPIb and αIIbβ3 fragments was quantified by ELISA. Platelet aggregation was measured by optical aggregometry. Results Platelet exposure to hypershear led to a reduction of platelet GPIb surface expression, with a significant drop of GPIb+ platelet number and fluorescence (73.5 ± 2.9% and 953.1 ± 101.0 AU vs. 94.3 ± 0.7% and 1620 ± 72.4 AU for intact platelets; M ± SEM, ANOVA, p ≤ 0.05). Yet, no shear-mediated shedding of soluble GPIb fragment or β3 subunit was found. Downregulation of platelet GPIb expression coincided with a progressive increase of GPIb- & αIIbβ3-positive PDMPs after shear (up to 3.3 ± 0.6% & 12.5 ± 2.8%, correspondingly). Surface density of GPIb and αIIbβ3 on PDMPs was 2-4x higher than on platelets, post-shear. Platelet aggregation to increasing concentrations of ADP and TRAP-6 was significantly impaired by 70 dyn/cm2 shear stress. Conclusion MCS-mediated hypershear and platelet stress accumulation 1) induces downregulation of platelet adhesion receptors via ejection of receptor-enriched PDMPs; 2) does not induce shedding of soluble fragments of GPIb and β3, and; 3) mechanistically limits platelet aggregatory and hemostatic responses. Shear-mediated redistribution of primary adhesion receptors from platelets to PDMPs coupled with impaired aggregability may mechanistically contribute to MCS-related bleeding.

Platelet Dysfunction During Mechanical Circulatory Support: Elevated Shear Stress Promotes Downregulation of αIIbβ3 and GPIb via Microparticle Shedding Decreasing Platelet Aggregability.

[Figure: see text].

Impact of high mechanical shear stress and oxygenator membrane surface on blood damage relevant to thrombosis and bleeding in a pediatric ECMO circuit.

The roles of the large membrane surface of the oxygenator and the high mechanical shear stress (HMSS) of the pump in the extracorporeal membrane oxygenation (ECMO) circuit were examined under a pediatric support setting. A clinical centrifugal pump and a pediatric oxygenator were used to construct the ECMO circuit. An identical circuit without the oxygenator was constructed for comparison. Fresh human blood was circulated in the two circuits for 4 hours under the identical pump speed and flow. Blood samples were collected hourly for blood damage assessment, including platelet activation, generation of platelet-derived microparticles (PDMP), losses of key platelet hemostasis receptors (glycoprotein (GP) Ibα (GPIbα) and GPVI), and high molecular weight multimers (HMWM) of von Willebrand factor (VWF) and plasma free hemoglobin (PFH). Platelet adhesion on fibrinogen, VWF, and collagen was further examined. The levels of platelet activation and generation of PDMP and PFH exhibited an increasing trend with circulation time while the expression levels of GPIbα and GPVI receptors on the platelet surface decreased. Correspondingly, the platelets in the blood samples exhibited increased adhesion capacity to fibrinogen and decreased adhesion capacities on VWF and collagen with circulation time. Loss of HMWM of VWF occurred in both circuits. No statistically significant differences were found in all the measured parameters for blood damage and platelet adhesion function between the two circuits. The results indicate that HMSS from the pump played a dominant role in blood damage associated with ECMO and the impact of the large surface of the oxygenator on blood damage was insignificant.

Generation of platelet-derived microparticles through the activation of the toll-like receptor 4

IntroductionInfection from different bacterial may increase the risk of thrombosis and atherosclerosis risk by production and secretion of many proinflammatory factors. Human platelets have toll-like receptor 4 (TLR4), the principal receptor for lipopolysaccharide (LPS). The activation of platelet produces Platelet-derived Microparticles (PDMPs) measuring less than 1.0 micron (that are very abundant in circulation >90%), which are associated with the development of Cardiovascular Diseases (CVDs), the leading cause of death in the world. ObjectivesExperiments were designed to evaluate the generation of pro-thrombogenic microparticles in vitro on platelets via TLR4 activation. MethodsPlatelet-rich plasma and washed platelets from healthy volunteers were incubated for the generation of PDMPs. The best source for the generation of microparticles was washed platelets. Then the washed platelets were incubated for 15 minutes with ultrapure Escherichia coli LPS (0–9 μg/mL) followed by activation with ADP (1 μM, subaggregant concentration), centrifuged for 60 minutes and analyzed by flow cytometry. ResultsIncubating platelets with LPS (9 μg/mL) and ADP (1 μM) produced a 34-fold increase in PDMPs generation. Finally, we evaluated this protocol to detect the inhibition of PDMPs generation, washed platelets were incubated with acetylsalicylic acid (10 μM) and an inhibition of 7.7-fold in PDMPs generation for activation of TLR4 was found. ConclusionA new and easy protocol for PDMPs generation and analysis by Flow Cytometry is established. In the future it could be used to determine the association of PDMPs with different pathologies.

Abstract 149: Phosphatidylserine Promotes Thrombosis in Pediatric CPB Model

Over 18,000 children per year receive cardiopulmonary bypass (CPB) surgery. Unfortunately, common CPB-related thrombotic complications continue to result in significant mortality and morbidity. Previous ex-vivo CPB studies using animal blood document an increase in platelet-derived microparticles (PMPs), which are small (0.1-1 micron) membrane vesicles that may be 50 to 100-fold more pro-coagulant than activated platelets. Our hypothesis is that increased duration and magnitude of shear stress in an ex-vivo pediatric CPB circuit increases the generation of PMP expressing pro-thrombotic phosphatidylserine. We constructed an ex-vivo CPB circuit that circulates heparinized human blood from healthy adult volunteers for six hours at pediatric flow rates ( e.g., 0.3, 0.5, and 0.7 L/min). Our protocol normalizes each run through the circuit to a normal hematocrit, pH, ionized calcium, and an activated clotting time of 180 to 220 sec. An aliquot of static blood controls is maintained in a similar test environment without CPB circuitry. PMP-PS (CD41a+/phosphatidylserine[PS]) concentration and pro-coagulant function are measured in platelet-depleted plasma using high-resolution flow cytometry (BS FACS Canto II with PMT) and STA®-Procoag-PPL. Thrombin generation ( e.g. , calibrated automated thrombogram) and clot formation ( e.g. , thromboelastography) further define the coagulation function of pump-produced PMP-PS. At 0.5 L/min the circuit generates an exponential increase in PMP-PS and decreasing PPL clot time compared to static blood control ( p &lt;0.001, Figure 1, n=4). Platelet count, prothrombin time, and activated partial thromboplastin time do not change over time. Results also document an increase in peak thrombin potential and clot formation that correlate strongly with the increase in PMP-PS. PMP-PS may be a clinically relevant biomarker and therapeutic target to decrease life-threatening CPB surgery coagulation complications.

Generation of Platelet Microparticles after Cryopreservation of Apheresis Platelet Concentrates Contributes to Hemostatic Activity.

Objective:In the last decade, substantial evidence has accumulated about the use of cryopreserved platelet concentrates, especially in trauma. However, little reference has been made in these studies to the morphological and functional changes of platelets. Recently platelets have been shown to be activated by cryopreservation processes and to undergo procoagulant membrane changes resulting in the generation of platelet-derived microparticles (PMPs), platelet degranulation, and release of platelet-derived growth factors (PDGFs). We assessed the viabilities and the PMP and PDGF levels of cryopreserved platelets, and their relation with thrombin generation.Materials and Methods:Apheresis platelet concentrates (APCs) from 20 donors were stored for 1 day and cryopreserved with 6% dimethyl sulfoxide. Cryopreserved APCs were kept at -80 °C for 1 day. Thawed APCs (100 mL) were diluted with 20 mL of autologous plasma and specimens were analyzed for viabilities and PMPs by flow cytometry, for thrombin generation by calibrated automated thrombogram, and for PDGFs by enzyme-linked immunosorbent assay testing.Results:The mean PMP and PDGF levels in freeze-thawed APCs were significantly higher (2763±399.4/µL vs. 319.9±80.5/µL, p<0.001 and 550.9±73.6 pg/mL vs. 96.5±49 pg/mL, p<0.001, respectively), but the viability rates were significantly lower (68.2±13.7% vs. 94±7.5%, p<0.001) than those of fresh APCs. The mean endogenous thrombin potential (ETP) of freeze-thawed APCs was significantly higher than that of the fresh APCs (3406.1±430.4 nM.min vs. 2757.6±485.7 nM.min, p<0.001). Moreover, there was a significant positive poor correlation between ETP levels and PMP levels (r=0.192, p=0.014).Conclusion:Our results showed that, after cryopreservation, while levels of PMPs were increasing, significantly higher and earlier thrombin formation was occurring in the samples analyzed despite the significant decrease in viability. Considering the damage caused by the freezing process and the scarcity of evidence for their in vivo superiority, frozen platelets should be considered for use in austere environments, reserving fresh platelets for prophylactic use in blood banks.

Involvement of Platelet-Derived Microparticles in Tumor Progression and Thrombosis

Platelet-derived microparticles (PMPs) represent the most abundant microparticle (MP) subtype. Their presence reflects platelet activity, physiopathology, and the thrombotic state of cancer patients. The quantity and composition of PMPs strictly depends on the way MPs were generated. Because platelets play a key role in cancer progression, as well as formation of metastasis, PMPs also may be important in the proliferation of cancer cells, cancer cell interactions, metastatic progression, angiogenesis, and inflammation. Alternatively, the concentration of circulating PMPs may differ according to the stage of a cancer and thus potentially could be used as a biomarker. Here we review the mechanisms underlying the generation and composition of PMPs and the clinical and experimental studies describing the involvement of PMPs in cancer and the Trousseau syndrome. Lastly, we focus on their clinical relevance, as well as their potential application as biomarkers in cancer.

Platelet hyperreactivity explains the bleeding abnormality and macrothrombocytopenia in a murine model of sitosterolemia

AbstractSitosterolemia is a rare, autosomal recessive disease caused by mutations in the adenosine triphosphate-binding cassette transporter genes ABCG5 or ABCG8 that result in accumulation of xenosterols in the body. Clinical manifestations include tendon xanthomas, premature coronary artery disease, hemolytic anemia, macrothrombocytopenia, and bleeding. Although the effect of sterol accumulation on the predisposition for atherosclerosis is evident, how xenosterol accumulation leads to defects in platelet physiology is unknown. Sitosterolemia induced in Abcg5- and Abcg8-deficient mice fed a high plant sterol diet resulted in accumulation of free sterols in platelet plasma membranes, leading to hyperactivatable platelets characterized by constitutive binding of fibrinogen to its αIIbβ3 integrin receptor, internalization of the αIIbβ3 complex, generation of platelet-derived microparticles, and changes in the quantity and subcellular localization of filamin. The latter was associated with macrothrombocytopenia, shedding of GPIbα, impaired platelet adhesion to von Willebrand factor, and inability to form stable thrombi. Plasma levels of soluble GPIbα were strongly correlated with plasma sitosterol levels in samples from human sitosterolemic patients, implicating a similar mechanism of sterol-induced platelet passivation in the human disease. Intercalation of plant sterols into the plasma membrane therefore results in dysregulation of multiple platelet activation pathways, leading to macrothrombocytopenia and bleeding.

An ultrastructural analysis of platelets, erythrocytes, white blood cells, and fibrin network in systemic lupus erythematosus

The study suggests that patients with systemic lupus erythematosus (SLE) present with distinct inflammatory ultrastructural changes such as platelets blebbing, generation of platelet-derived microparticles, spontaneous formation of massive fibrin network and fusion of the erythrocytes membranes. Lupoid platelets actively interact with other inflammatory cells, particularly with white blood cells (WBCs), and the massive fibrin network facilitates such an interaction. It is possible that the concerted actions of platelets, erythrocytes and WBC, caught in the inflammatory fibrin network, predispose to pro-thrombotic states in patients with SLE.

Platelet hyperprocoagulant activity in Type 2 diabetes mellitus: attenuation by glycoprotein IIb/IIIa inhibition.

Platelets are hyperactive in Type 2 diabetes mellitus (T2DM), and antiplatelet treatment with glycoprotein (GP) IIb/IIIa inhibitors provides better thrombotic protection in DM than in non-diabetic subjects. We hypothesized that diabetic platelets are hyperprocoagulant, and that this hyperactivity can be inhibited by GPIIb/IIIa blockade. Patients with T2DM and gender/age/body mass index-matched non-diabetic controls were recruited (n = 12 for both) to study the effect of GPIIb/IIIa blockade on platelet procoagulant activity. Platelet phosphotidylserine (PS), factor (F) Va expression, and platelet-derived microparticle (PDMP) generation were measured by whole blood flow cytometry. Platelet-dependent thrombin generation and plasma clotting time were monitored in recalcified platelet-rich plasma. Compared to controls, basal platelet activation was similar, while thrombin receptor activating peptide stimulated activation was enhanced in patients with T2DM. Diabetic platelets also displayed more profound elevations of platelet PS exposure, FVa binding, and PDMP generation upon stimulation. These alterations resulted in a hyperprocoagulant state, as evidenced by a marked increase in the platelet procoagulant index, enhanced thrombin generation, and a shortened plasma clotting time. GPIIb/IIIa blockade by c7E3 or SR121566 decreased platelet PS exposure and FVa binding, and diminished platelet procoagulant activity in patients with T2DM. Platelets have increased procoagulant activity in patients with T2DM. The hyperprocoagulant activity is counteracted by GPIIb/IIIa blockade.

Role of P2Y receptor subtypes in platelet-derived microparticle generation

Microparticles are shed from the platelet membrane upon platelet activation by strong agonists, and they aid in clot formation. As the P2Y1 and the P2Y12 receptors differentially contribute to different platelet functions, we studied the relative contribution of the P2Y1 and P2Y12 receptors to microparticle formation from platelets. The P2Y12 receptor antagonist AR-C 69931MX, but not the P2Y1 receptor antagonist MRS2179, caused a significant decrease in the number microparticles formed by convulxin and thrombin. In addition, there was no significant decrease in microparticle formation in P2Y1 knockout mouse blood when compared to the wild type mice. These results illustrate that the P2Y12 receptor contributes to microparticle formation from activated platelets by a strong agonist, without any significant involvement of the P2Y1 receptor. We also conclude that there is no correlation in the number of microparticles circulating in vivo between the P2Y1 receptor null mice and the wild type mice under unstimulated conditions. Finally, we conclude that the increased bleeding time in the P2Y1 null mice is due to overall platelet dysfunction and not due to the decrease of circulating microparticles.

Impact of weight reduction on production of platelet-derived microparticles and fibrinolytic parameters in obesity

Introduction Generation of platelet-derived microparticle (PMP) is implicated in cardiovascular disease (CVD). However, the influence of adiposity and weight reduction on PMP generation remains to be fully elucidated. We compared PMP generation and fibrinolytic parameters between 49 non-diabetic obese (obese group) and 37 age-matched non-obese subjects (control group), and compared the effects of weight reduction on the parameters between a 12-week calorie restricted diet and diet with aerobic exercise in obese subjects. Materials and methods PMP, plasma levels of plasminogen activator inhibitor-1 (PAI-1) activity and tissue-type plasminogen activator (t-PA) antigen were measured before and after intervention. Results Before intervention, PMP, PAI-1 activity and t-PA antigen values were elevated in the obese group compared with the control group. In all 86 subjects of both groups, these three parameters correlated with body mass index, waist circumference and fat tissue mass. There was a positive correlation between plasma levels of fibrinolytic parameters and visceral fat area (VFA). PMP values correlated with subcutaneous fat area (SFA). The intervention significantly reduced PMP, PAI-1 activity and t-PA antigen levels. There was a significant correlation between percentages of changes in PMP values and those in BMI, fat tissue mass and VFA in the obese group. No additional effect of exercise on PMP or fibrinolytic parameters was observed. Conclusions Overproduction of PMP and fibrinolytic abnormalities may be associated with excessive adipose tissue. Weight reduction by either calorie restriction with or without exercise improves fibrinolytic abnormalities and PMP overproduction, probably through reduction of adipose tissue.

Effect of remnant-like particle on shear-induced platelet activation and its inhibition by antiplatelet agents

Remnant-like particles (RLPs) have been reported to promote atherosclerosis and to have effects on platelet function. We studied the effects of RLP on shear-induced platelet activation and their inhibition by antiplatelet agents in vitro. RLP were separated using two monoclonal antibodies, anti apo B-100 and anti apo A-I. These RLP fractions were added to whole blood (WB) or platelet-rich plasma (PRP) in serial dilution of 1, 10 or 100 μg RLP triglyceride (TG) per ml of total sample volume. These samples were incubated, and then stimulated with a high shear stress of 108 dyn/cm 2. Shear-induced platelet aggregation (SIPA) was calculated from the percentage of single platelet loss. P-selectin expression on platelet surface and platelet-derived microparticle (PMP) generation were measured before and after stimulation with shear stress using flow cytometer. SIPA was significantly enhanced by RLP in WB but not in PRP. This enhancing effect was not dose-dependent and was greatest at 10 μg TG/ml. P-selectin expression induced by shear stress was only enhanced by RLP at a concentration of 100 μg TG/ml in both WB and PRP, while generation of PMP induced by shear stress was only enhanced by RLP at a concentration of 100 μg TG/ml in WB. Aspirin inhibited only the enhancement of SIPA by RLPs, while cilostazol inhibited the enhancement of not only SIPA but also p-selectin expression and PMP generation by RLPs.

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.

Cardiology patient page. Platelets and cardiovascular disease.

Platelets are specialized disk-shaped cells in the blood stream that are involved in the formation of blood clots that play an important role in heart attacks, strokes, and peripheral vascular disease. In most people, the more than 200 million platelets in a milliliter of blood act as tiny building blocks to form the basis of a clot to stop bleeding from cuts or injuries. Platelets can detect a disruption in the lining of a blood vessel and react to build a wall to stop bleeding (Figure 1). Figure 1. Platelets form a platelet plug to stop bleeding from an injured blood vessel. In cardiovascular disease, abnormal clotting occurs that can result in heart attacks or stroke. Blood vessels injured by smoking, cholesterol, or high blood pressure develop cholesterol-rich build-ups (plaques) that line the blood vessel; these plaques can rupture and cause the platelets to form a clot. Even though no bleeding is occurring, platelets sense the plaque rupture and are confused, thinking that an injury has taken place that will cause bleeding. Instead of sealing the vessel to prevent bleeding as would occur with a cut, a clot forms in an intact blood vessel, causing a blockage of blood flow (Figure 2). Without …