Platelet-leukocyte Conjugates Research Articles

Flow cytometric evaluation of platelet-leukocyte conjugate stability over time: methodological implications of sample handling and processing.

Platelet activation and subsequent aggregation is a vital component of atherothrombosis resulting in acute myocardial infarction. Therefore, quantifying platelet aggregation is a valuable measure for elucidating the pathogenesis of acute coronary syndromes (ACS). Circulating platelet-monocyte conjugates (PMC) as determined by flow cytometry (FCM) are an important measure of in vivo platelet aggregation. However, the influence of sample handling on FCM measurement of PMC is not well-studied. The changes in FCM measurement of PMC with variation in sample handling techniques were evaluated. The stability of PMC concentrations over time with changes in fixation and immunolabeling intervals was assessed. The effect of Time-to-Fix and Time-to-Stain on FCM PMC measurements was investigated in five healthy volunteers. Time-to-Fix (i.e., interval between phlebotomy and sample fixation) was performed at 3, 30, and 60min. Time-to-Stain (i.e., time of fixed sample storage to staining) was performed at 1, 24, and 48h. Increasing Time-to-Stain from 1 to 24 or 48h resulted in lower PMC measures (p < 0.0001). A statistically significant difference in PMC measurement with increasing Time-to-Fix was not observed (p < 0.41). Postponement of sample staining has deleterious effects on the measurement of PMC via FCM. Delays in immunolabeling of fixed samples compromised measurement of PMC by 30% over the first 24h. Staining/FCM should be completed within an hour of collection.

Platelets in Advanced Chronic Kidney Disease: Two Sides of the Coin.

Rates of thrombosis and bleeding episodes are both increased in patients with advanced chronic kidney disease (CKD). The pathogenic mechanisms of thrombosis in these patients include platelet activation, increased formation of platelet-leukocyte conjugates, and platelet-derived microparticles, as well as effects of uremic toxins on platelets. On the other side of the coin, platelet hyporeactivity mediated by uremic toxins and anemia contributes to the increased bleeding risk in advanced CKD. Platelets also contribute to the inflammatory environment, thus increasing the risk of cardiovascular diseases in these patients. This review provides insights into the altered platelet function in advanced stages of CKD and their relationship with risks of thrombosis and bleeding. Particularly, the effect of dialysis on platelets will be discussed. Furthermore, therapeutic options with respect to thrombotic disorders as well as bleeding in patients with CKD are reviewed.

The Involvement of Toll-like Receptor-2 in Arterial Thrombus Formation.

There is emerging evidence for the participation of toll-like receptor-2 (TLR2) expressed on platelets and endothelial cells in the setting of arterial thrombosis. In isolated human platelets, TLR2/1 activation was demonstrated to induce platelet activation, secretion, aggregation, adhesion to collagen coatings and the formation of platelet-leukocyte conjugates, whereas murine platelets were less sensitive to TLR2/1 stimulation. Also, endothelial cells can be activated by stimulation with TLR2 agonists, resulting in increased expression of adhesion molecules, synthesis of inflammatory mediators and Weibel-Palade body exocytosis. Endothelial TLR2 signalling promotes atherosclerotic lesion development in mouse atherosclerosis models. Experiments with germ-free mouse models demonstrated that the presence of commensal microbiota increased endothelial von Willebrand factor synthesis in the liver through TLR2. In the carotid artery ligation model, the elevated von Willebrand factor plasma levels enhanced platelet deposition to the injury site. Furthermore, in the hyperlipidemic ApoE-deficient mouse model, TLR2 deficiency was shown to protect from ferric chloride-induced carotid artery thrombosis. This review article provides an overview on TLR2 signalling in platelets and the vascular endothelium and summarizes how TLR2 signalling contributes to arterial thrombosis.

Abstract 1738: Platelet activation and heterotypic platelet leukocyte conjugate formation in the blood of glioblastoma patients

Abstract Objective Glioblastoma patients (GBM) suffer from an increased incidence of cardiovascular events. Platelets are well known as main player of the primary hemostasis, but have a broad range of additional functions. The formation of heterotypic conjugates between platelets and leukocytes (PLC) represents a pro-inflammatory surrogate marker and is usually increased after platelet activation. The aim of the present study was to evaluate the platelet activation status and the rate of circulating PLC in GBM. Methods Blood samples were drawn of consecutive patients before surgery for a suspected glioblastoma. The formation of PLC and several parameters of platelet activation were determined by flow cytometry before and after stimulation with either ADP or the thrombin receptor-activating peptide (TRAP) in vitro: expression of P-Selectin, CD63, CD40L and fibrinogen-binding to the activated GPIIb/ IIIa. Blood samples from age and gender matched healthy volunteers were used as controls. Statistical analysis was done with the Mann-Whitney-Test. Results Final analysis included 22 patients with histopathological proven virgin glioblastoma (9f, 13m, mean age 67.5 years, range from 55 to 86 years) and their respective controls. Basal platelet activation and in vitro platelet reactivity was increased in GBM. The difference got significant in the basal expression of CD63 (2.8% versus 1.9%, p=0.008), the Fibrinogen-binding after ADP-stimulation (110.3 MFI versus 63.1 MFI, p=0.04) and the CD63 expression after TRAP-stimulation (38.4% versus 33.3%, p=0.04). Furthermore, a reduced number of circulating PLC and in vitro PLC formation was seen in GBM without getting statistically significant. Conclusions In this preliminary report, we show for the first time an increased level of platelet activation and agonist-induced platelet reactivity in GBM. Both could be a reflection of the pro-thrombotic status in these patients. Interestingly, the formation of PLC was not increased, but in tendency decreased. Whether this observation potentially mirrors the intratumoral, anti-inflammatory microenviroment in GBM remains unclear. Citation Format: Sascha Marx, Maximilian Splittstöhser, Heiko Paland, Carolin Seifert, Madlen Juettner, Andreas Boehm, Christoph A. Ritter, Sandra Bien-Moeller, Henry W. Schroeder, Bernhard Rauch. Platelet activation and heterotypic platelet leukocyte conjugate formation in the blood of glioblastoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1738. doi:10.1158/1538-7445.AM2017-1738

Effect of intravenous lidocaine on the transcerebral inflammatory response during cardiac surgery: a randomized-controlled trial.

Postoperative cognitive dysfunction (POCD) occurs frequently after cardiac surgery. The pathophysiology of POCD remains elusive, but previous work showed that intravenous lidocaine may be protective against POCD, possibly by modulating cerebral inflammation. We hypothesized that intravenous lidocaine would attenuate the cerebral inflammatory response to cardiopulmonary bypass (CPB) by reducing the transcerebral activation gradients of platelets, leukocytes, and/or platelet-leukocyte conjugates. We studied 202 patients undergoing cardiac surgery with CPB in this prospective randomized double-blinded placebo-controlled trial. Subjects were randomized to receive either intravenous lidocaine (bolus + 48-hr infusion) or placebo (identical infusion volume and duration). Paired jugular venous and radial arterial blood samples were drawn at several time points and analyzed by fluorescence-activated cell sorting to identify activated platelets and platelet-leukocyte conjugates. Transcerebral activation gradients were calculated by subtracting arterial values from venous values and were compared between groups using repeated measures regression models with covariate adjustment for age, sex, surgery type, and CPB duration. Beginning after aortic cross-clamp release and peaking ten minutes after the termination of CPB, the mean (SD) transcerebral activation gradient of platelet-monocyte conjugates decreased in lidocaine-treated vs placebo-treated patients [-1.84 (11.47) mean linear fluorescence intensity (MLFI) vs 1.46 (13.88) MLFI, respectively; mean difference, -4.08 MLFI; 95% confidence interval, -7.86 to -0.29; P = 0.03). No difference was seen at any time point for activated platelets or for platelet-neutrophil conjugates. While lidocaine did not affect the systemic or transcerebral activation of platelets or leukocytes, we did observe a reduction in the transcerebral activation of platelet-monocyte conjugates after aortic cross-clamp release. This may be a manifestation of reduced cerebral inflammation during cardiopulmonary bypass in response to treatment with lidocaine. This trial was registered at ClinicalTrials.gov (NCT00938964).

Increased Platelet-leukocyte Aggregates Are Associated With Myocardial No-reflow in Patients With ST Elevation Myocardial Infarction

ObjectiveWe aimed to investigate the association between platelet-leukocyte aggregates (PLA) levels on admission and the risk of myocardial no-reflow in patients with ST elevation myocardial infarction (STEMI) who underwent primary percutaneous coronary intervention (PCI). MethodsA total of 83 patients with STEMI undergoing primary PCI were included in the current study. Platelet-leukocyte conjugates (PLA), including platelet-monocyte aggregates (PMA), platelet-neutrophil aggregates (PNA) and platelet-lymphocyte aggregates were studied by flow cytometry in peripheral venous blood. No-reflow was defined as coronary blood flow grade thrombolysis in myocardial infarction ≤2 or thrombolysis in myocardial infarction 3 and myocardial blush grade ≤2. ResultsNo-reflow was observed in 19 patients (22.9%). Compared with the reflow group, the level of PNA (76.5 ± 13.3) and PMA (90.3 ± 5.2) before PCI no-reflow group was significantly higher than that in normal reflow (P < 0.001). Using multiple logistic regression analysis, PNA (odds ratio [OR] = 1.179; 95% CI: 1.035-1.342; P = 0.013) and PMA (OR = 1.248; 95% CI: 1.040-1.498; P = 0.017) were found to be a significant predictor of no-reflow together with pain to balloon time (OR = 1.022; 95% CI: 1.002-1.041; P = 0.028), estimated glomerular filtration rate (OR = 1.311; 95% CI: 1.009-1.856; P = 0.047) and higher thrombus burden (OR = 0.061; 95% CI: 0.006-0.658; P = 0.021). Receiver operating characteristic curve analysis revealed that PNA (area under the curve = 0.881; 95% CI: 0.809-0.952; P < 0.001), PMA (area under the curve = 0.794; 95% CI: 0.699-0.889; P < 0.001) have important predictive value for the myocardial no-reflow. ConclusionsOur study indicated that preprocedural increased PLA levels display a significantly independent association with no-reflow phenomenon after PCI. Increased PLA levels may predict the development of no-reflow phenomenon in patients with STEMI who underwent PCI.

Abstract 535: Trem Like Transcript-1 (TLT-1) Regulates Both Leukocytes and Endothelial Cells to Mediate Immunohemostasis

TLT-1 is a 35kd receptor stored in the α-granules of platelets and released upon platelet activation like p-selectin. A soluble form is also released. A case has been made that the protein or proteins that regulate inflammatory bleeding is stored in the platelet α-granules. We hypothesized that TLT-1 is one of these proteins and we investigated TLT-1’s role in inflammatory bleeding. Using two different models of inflammation and three different models of transmigration we show that TLT-1 mediates neutrophil transmigration across the vessel wall. In the absence of TLT-1 transmigration leads to bleeding that can be partially rescued by soluble TLT-1 (sTLT-1: see poster J. Morales). Based on these results we hypothesized that TLT-1 enhances the progression of atherosclerosis and cancer. To test this hypothesis we crossed the treml1 -/- mice with apoe -/- mice creating the DKO mouse and evaluated lesion progression over 20 weeks. Our results demonstrate that the DKO has significantly less lesions in the aortic sinus than their littermate controls (n=8/group; p &lt;0.05). The smaller lesions are associated with less platelet activation and fewer platelet-leukocyte conjugates. Parallel studies investigating TLT-1 in the α-granules revealed a significant co-localization with pro-angiogenic VEGF and higher sTLT-1 release with PAR1 vs PAR4 platelet activation; suggestive of a role for TLT-1 in angiogenesis. We subsequently asked if, “sTLT-1 has an effect on endothelial cells?” We show angiogenic effects of sTLT-1 on HUVECs that is blocked by the addition of anti-TLT-1, demonstrating it’s a sTLT-1 mediated process. Further studies using the Lewis lung carcinoma model demonstrate that the tumors in the null mouse have significantly fewer vessels than wildtype tumors and lower infiltration of macrophages (n=15). We subsequently show that sTLT-1 partially rescues the phenotype by increasing macrophage infiltration and increasing vessel growth (n=13) and that antibodies to TLT-1 recapitulate the treml1 -/- phenotype (n=10) suggesting anti-TLT-1 may be used as an intervention. We conclude that TLT-1 regulates both leukocytes and endothelial cells to mediate vascular integrity and is a plausible target for therapeutic interventions in cancer and atherosclerosis.

Clopidogrel reduces the inflammatory response of lung in a rat model of decompression sickness

Inflammation and platelet activation are critical phenomena in the setting of decompression sickness. Clopidogrel (Clo) inhibits platelet activation and may also reduce inflammation. The goal of this study was to investigate if Clo had a protective role in decompression sickness (DCS) through anti-inflammation way. Male Sprague–Dawley rats (n=111) were assigned to three groups: control+vehicle group, DCS+vehicle, DCS+Clo group. The experimental group received 50mg/kg of Clo or vehicle for 3 days, then compressed to 1,600kPa (150msw) in 28s, maintained at 150msw for 242s and decompressed to surface at 3m/s. In a control experiment, rats were also treated with vehicle for 3 days and maintained at atmospheric pressure for an equivalent period of time. Clinical assessment took place over a period of 30min after surfacing. At the end, blood samples were collected for blood cells counts and cytokine detection. The pathology and the wet/dry ratio of lung tissues, immunohistochemical detection of lung tissue CD41 expression, the numbers of P-selectin positive platelets and platelet–leukocyte conjugates in blood were tested. We found that Clo significantly reduced the DCS mortality risk (mortality rate: 11/45 with Clo vs. 28/46 in the untreated group, P<0.01). Clo reduced the lung injury, the wet/dry ratio of lung, the accumulation of platelet and leukocyte in lung, the fall in platelet count, the WBC count, the numbers of activated platelets and platelet–leukocyte complexes in peripheral blood. It was concluded that Clo can play a protective role in decompression sickness through reducing post-decompression platelet activation and inflammatory process.

215 Ticagrelor Inhibits Release of Pro-inflammatory Cytokines TNF and IL-6 during Human Endotoxaemia

IntroductionIn the PLATelet inhibition and patient Outcomes (PLATO) study, ticagrelor was associated with fewer pulmonary infection events and subsequent deaths but slightly higher inflammatory markers compared to clopidogrel. Ticagrelor and...

Platelet-Leukocyte Conjugation Mediated By GPIbα-αXβ2 (CD11c/CD18) Interaction

BackgroundThe formation of platelet-leukocyte conjugates has been implicated in a variety of pathologies, including thrombosis, atherosclerosis, glomerulonephritis, multiple sclerosis and other inflammatory diseases. In prior studies (Simon et al., J. Exp Med, 2000, 193-204), platelet GPIbα and leukocyte αMβ2 (CD11b/CD18) had been implicated as receptor:counter-receptor pairing in mediating formation of platelet:leukocyte complexes. A specific segment (αM201-217) in the I domain of αM (CD11b) subunit of αMβ2 engaged GPIbα, and a peptide (M2) corresponding to this segment blocked neutrophil recognition of platelets and anti-M2 blocked conjugate formation (Ehlers et al., J. Exp. Med, 2003, 1077-88). The I-domains of αM and αX are highly conserved (74% homology), but αX has different amino acids at two positions shown to be critical for recognition of GPIbα by the αM I-domain. This study considers the role of a particular receptor:counter-receptor pairing, GPIb on platelets and αXβ2 (CD11c/CD18) on leukocytes, in mediating this heterotypic cell interaction. Methods and ResultsTo directly address whether αXβ2 recognizes GPIbα, αXβ2 was expressed in HEK cells and its corresponding I-domain was expressed and purified. αXβ2 cells adhered specifically to GPIbα, and purified αX I-domain bound to platelets by flow cytometry and to GPIbα by surface plasmon resonance. However, this interaction was readily distinguished from that of αM I-domain binding to GPIbα. αX binding to GPIbα was not inhibited by: 1) M2 or anti-M2; 2) a peptide from a region of GPIbα implicated in vWF interaction and that blocked adhesion of αMβ2 cells to GPIbα; 3) small molecules that blocked αM I-domain binding; and 4) mutations in GPIbα that blocked αM I-domain binding. Furthermore, by mutational analyses, a region of αX I domain was implicated binding GPIbα that is structurally distant and unrelated to the αM I domain binding site.Next, we examined the role of αXβ2 in vivo in a cytokine- and neutrophil-dependent inflammatory response. The local Shwartzman-like reaction (LSR), induced by successive LPS and cytokine injections into the skin, produces a thrombo-hemorrhagic vasculitis, which depends on leukocytes and their interaction with activated platelets and endothelial cells. In prior work, we have shown that the LSR is dependent upon leukocyte αM and platelet GPIba. Leukocyte-derived microparticles promote the accumulation of fibrin, generation of occlusive thrombi, and eventual hemorrhage from inflamed blood vessels. The LSR produces hemorrhage in the intact skin 24 hours after TNF-a injection. CD11c-/- mice subjected to LSR exhibited a significant reduction in the development of hemorrhagic lesions. In analysis blinded to genotype, CD11c-/- mice had a 76% reduction in lesion severity (lesion hemorrhage score: WT, 3.4 ± 1.0, n=12 vs. CD11c-/-, 0.8 ± 1.0, n=11; P<0.001). Histopathology of vasculitic lesions demonstrated substantial reductions in erythrocyte extravasation, edema, leukocyte accumulation, and occlusive thrombi in CD11c-/- mice. ConclusionsLeukocyte αXβ2 (CD11c/CD18) recognizes GPIbα on platelets in a way distinct from αMβ2 (CD11bCD18). This interaction appears to be functionally significant in vivo and could be targeted for suppressing adverse inflammatory responses. Disclosures:Simon:Cordis/J&J: Consultancy; Janssen/J&J: Consultancy; Medtronic Vascular: Consultancy; Merck: Consultancy; Medtronic Foundation: Research Funding.

Inhibition of Platelet Activation by Clopidogrel Prevents Hypertension-Induced Cardiac Inflammation and Fibrosis

PurposePlatelets are essential for primary hemostasis; however, platelet activation also plays an important proinflammatory role. Inflammation promotes the development of cardiac fibrosis and heart failure induced by hypertension. In this study, we aimed to determine whether inhibiting platelet activation using clopidogrel could inhibit hypertension-induced cardiac inflammation and fibrosis.MethodsUsing a mouse model of angiotensin II (Ang II) infusion (1,500 ng/[kg·min] for 7 days), we determined the role of platelet activation in Ang II infusion-induced cardiac inflammation and fibrosis using a P2Y12 receptor inhibitor, clopidogrel (50 mg/[kg·day]).ResultsCD41 staining showed that platelets accumulated in Ang II-infused hearts. Clopidogrel treatment inhibited Ang II infusion-induced accumulation of α-SMA+ myofibroblasts and cardiac fibrosis (4.17 ± 1.26 vs. 1.46 ± 0.81, p < 0.05). Infiltration of inflammatory cells, including Mac-2+ macrophages and CD45+Ly6G+ neutrophils (30.38 ± 4.12 vs. 18.7 ± 2.38, p < 0.05), into Ang II-infused hearts was also suppressed by platelet inhibition. Real-time PCR and immunohistochemical staining showed that platelet inhibition significantly decreased the expression of interleukin-1β and transforming growth factor-β. Acute injection of Ang II or PE stimulated platelet activation and platelet-leukocyte conjugation, which were abolished by clopidogrel treatment.ConclusionThus, inhibition of platelet activation by clopidogrel prevents cardiac inflammation and fibrosis in response to Ang II. Taken together, our results indicate Ang II infusion-induced hypertension stimulated platelet activation and platelet-leukocyte conjugation, which initiated inflammatory responses that contributed to cardiac fibrosis.

Effects of outdoor air pollutants on platelet activation in people with type 2 diabetes

Exposure to air pollution is associated with increased morbidity and mortality from cardiovascular disease. We hypothesized that increases in exposure to ambient air pollution are associated with platelet activation and formation of circulating tissue factor-expressing microparticles. We studied 19 subjects with type 2 diabetes, without clinical evidence of cardiovascular disease, who had previously participated in a human clinical study of exposure to ultrafine particles (UFP). Blood was obtained for measurements of platelet activation following an overnight stay in the Clinical Research Center, prior to each of their two pre-exposure visits. Air pollution and meteorological data, including UFP counts, were analyzed for the 5 days prior to the subjects’ arrival at the Clinical Research Center. Contrary to expectations, increases in UFP were associated with decreases in surface expression of platelet activation markers. The number of platelet-leukocyte conjugates decreased by −80 (95% confidence interval (CI) −123 to −37, p = 0.001) on the first lag day (20–44 h prior to the blood draw) and by −85 (CI −139 to −31, p = 0.005) on combined lag days 1 to 5, per interquartile range (IQR) increase in UFP particle number (2482). However, levels of soluble CD40L increased 104 (CI 3 to 205, p = 0.04) pg/ml per IQR increase in UFP on lag day 1, a finding consistent with prior platelet activation. We speculate that, in people with diabetes, exposure to UFP activates circulating platelets within hours of exposure, followed by an increase in soluble CD40L and a rebound reduction in circulating platelet surface markers.

Heritability, genetic correlation and linkage to the 9p21.3 region of mixed platelet–leukocyte conjugates in families with and without early myocardial infarction

Background and aimsVariations in mixed platelet–leukocyte conjugate formation in human whole blood could be genetically determined. We quantified platelet and leukocyte activation and interaction in families with or without early myocardial infarction and evaluated their heritability, genetic correlation and linkage to the 9p21.3 region. Methods and resultsThe study population included 739 subjects (≥15 years old) from 54 large pedigrees, 23 with and 31 without familial myocardial infarction. Mixed platelet–leukocyte conjugates and markers of platelet or leukocyte activation (P-selectin, CD11b and L-selectin surface expression) were measured both before and after in vitro blood stimulation with collagen-ADP.All traits had significant genetic components (17.5–65.3% of the phenotypic variability), while shared household effects (0–39.6%) and environmental covariates (0–10.2%) tended to be smaller. Stimulated platelet-polymorphonuclear leukocyte (PMN) and platelet–monocyte conjugates showed the highest linkage to the 9p21.3 region (LOD = 0.94 and 1.33, respectively; empirical p value = 0.017 and 0.009). PMN markers resulted strongly genetically correlated between them in bivariate analysis among pairs of quantitative traits. ConclusionThis study supports a genetic regulation of human mixed platelet–leukocyte conjugates.

Postprandial cell inflammatory response to a standardised fatty meal in subjects at different degree of cardiovascular risk

A fatty meal may represent a challenge of in vivo acute inflammatory reaction. We evaluated the acute effects of a standardised fatty meal administration on leukocytes and platelets and on their interactions on 61 subjects at different degree of cardiovascular risk, without any clinical event. Before and 2 hours after a fatty meal, blood cells were counted and markers of leukocyte (intracellular myeloperoxidase [MPO] and Mac-1) and platelet (P-selectin and microparticles) activation and mixed platelet-leukocyte conjugates measured by flow-cytometry. After the fatty meal, both white blood cell and platelet count significantly increased, more markedly in subjects with lower cardiovascular risk score. Mac-1 expression too increased (from 32.2 ± 27.2% to 45.6 ± 29.0%, p=0.0016), while MPO decreased (from 83.1 ± 16.3% to 64.5 ± 23.1%, p<0.0001). A trend for increased platelet activation and interaction with leukocytes was also observed. Women were more markedly susceptible to fatty meal challenge, as compared to men, while age did not seem to affect any cell response to fatty meal. Waist-to-hip ratio and body mass index influenced polymorphonuclear cells (PMN) degranulation and platelet count increase, respectively. Cellular responses to the fatty meal, in particular PMN degranulation, were attenuated in subjects at higher degree of cardiovascular risk, who showed a basal mild inflammatory activation status. In conclusion, a fatty meal consumption may represent a model of acute inflammatory response and appears to be modulated by different demographic and cardiovascular risk degree. This model could be applied to study the effect of food-derived antioxidants or nutritional supplements, but its relevance remains to be demonstrated.

The platelet hyporesponsiveness to clopidogrel in acute coronary syndrome patients treated with 75 mg/day clopidogrel may be overcome within 1 month of treatment

Platelets are involved in thrombus formation and inflammation following vascular injury, while clopidogrel exerts antithrombotic and anti-inflammatory actions. We investigated various platelet-derived prothrombotic and proinflammatory mediators as well as the platelet aggregatory response in patients with acute coronary syndromes (ACS) receiving clopidogrel, as a function of the patient responsiveness to drug treatment. Blood samples were obtained from 40 patients with recent (<24 h) ACS before clopidogrel loading 600 mg (followed by a maintenance dose of 75 mg/day) as well as 5-days and 30-days afterwards. Twelve patients exhibited platelet reactivity index (PRI) values higher than 50% evaluated by the Vasodilator Stimulated Phosphoprotein (VASP) test at 5 days and were characterized as nonresponders. The platelet response to adenosine diphosphate (ADP) and thrombin receptor agonist peptide-14 (TRAP) was studied by flow cytometry and light transmission aggregometry. A maximum reduction of ADP- or TRAP-induced platelet aggregation in 28 clopidogrel responding patients was observed at 5 days postclopidogrel loading, whereas in nonresponders, it was achieved at 30-days along with a significant decrease in the PRI values. Similar results were obtained for the membrane expression of CD40L and the production of platelet-derived microparticles. By contrast, the maximum inhibition of P-selectin expression and platelet–leukocyte conjugate formation was observed at 30-days in both patient groups. A maintenance dose of 75 mg clopidogrel differentially affects the platelet aggregation and platelet-derived prothrombotic and proinflammatory mediators in ACS patients within the first month of the treatment, a phenomenon that is highly influenced by the drug response variability. Since these factors may be involved in the major adverse cardiovascular events in ACS patients, especially in those undergoing percutaneous coronary intervention, the above findings may be clinically important.

Novel Role of Platelets in Mediating Inflammatory Responses and Ventricular Rupture or Remodeling Following Myocardial Infarction

The goal of this study was to investigate the role of platelets in systemic and cardiac inflammatory responses and the development of postinfarct ventricular complications, as well as the efficacy of antiplatelet interventions. Using a mouse myocardial infarction (MI) model, we determined platelet accumulation and severity of inflammation within the infarcted myocardium by immunohistochemistry and biochemical assays, analyzed peripheral blood platelet-leukocyte conjugation using flow cytometry, and tested antiplatelet interventions, including thienopyridines and platelet depletion. Platelets accumulated within the infarcted region early post-MI and colocalized with inflammatory cells. MI evoked early increase in circulating platelet-leukocyte conjugation mediated by P-selectin/P-selectin glycoprotein ligand-1. Antiplatelet interventions inhibited platelet-leukocyte conjugation in peripheral blood, inflammatory infiltration, content of matrix metalloproteinases or plasminogen activation, and expression of inflammatory mediators in the infarcted myocardium (all P<0.05) and lowered rupture incidence (P<0.01). Clopidogrel therapy alleviated the extent of chronic ventricular dilatation by serial echocardiography. Platelets play a pivotal role in promoting systemic and cardiac inflammatory responses post-MI. Platelets accumulate within the infarcted myocardium, contributing to regional inflammation, ventricular remodeling, and rupture. Antiplatelet therapy reduces the severity of inflammation and risk of post-MI complications, demonstrating a previously unrecognized protective action.

Heritability of platelet-leukocyte conjugates and cell activation markers in families with and without juvenile myocardial infarction

Heritability of platelet-leukocyte conjugates and cell activation markers in families with and without juvenile myocardial infarction

Vascular Effects of Ultrafine Particles in Persons with Type 2 Diabetes

BackgroundDiabetes confers an increased risk for cardiovascular effects of airborne particles.ObjectiveWe hypothesized that inhalation of elemental carbon ultrafine particles (UFP) would activate blood platelets and vascular endothelium in people with type 2 diabetes.MethodsIn a randomized, double-blind, crossover trial, 19 subjects with type 2 diabetes inhaled filtered air or 50 μg/m3 elemental carbon UFP (count median diameter, 32 nm) by mouthpiece for 2 hr at rest. We repeatedly measured markers of vascular activation, coagulation, and systemic inflammation before and after exposure.ResultsCompared with air, particle exposure increased platelet expression of CD40 ligand (CD40L) and the number of platelet-leukocyte conjugates 3.5 hr after exposure. Soluble CD40L decreased with UFP exposure. Plasma von Willebrand factor increased immediately after exposure. There were no effects of particles on plasma tissue factor, coagulation factors VII or IX, or D-dimer.ConclusionsInhalation of elemental carbon UFP for 2-hr transiently activated platelets, and possibly the vascular endothelium, in people with type 2 diabetes.

A Randomized Clinical Trial Investigating the Relationship Between Aprotinin and Hypercoagulabilityin Off-Pump Coronary Surgery

Off-pump coronary artery bypass (OPCAB) surgery is associated with a hypercoagulable state in which the platelet thrombin receptor, protease-activated receptor-1 (PAR-1), helps propagate a thrombin burst within saphenous vein grafts. Aprotinin, used in cardiothoracic surgery mainly for its antifibrinolytic properties, also spares platelet PAR-1 activation due to thrombin. We hypothesized that this PAR-1 antagonistic property provides an antithrombotic benefit during OPCAB surgery. Patients were randomly assigned to receive saline (n = 38) or a modified full-dose regimen of aprotinin (n = 37) IV during OPCAB surgery. Blood sampled perioperatively from the coronary sinus, skin wounds, and systemic circulation was analyzed to test coagulation and platelet function. Major adverse cardiovascular events were monitored by obtaining troponin I at 24 h (myocardial infarction), predischarge computed tomography angiography (vein graft thrombosis), and by clinical examination for stroke. Coronary sinus blood obtained immediately after OPCAB surgery showed significantly less activation in the aprotinin group, as judged by reduced formation of platelet-leukocyte conjugates (P < 0.02) and platelet-derived microparticles (P < 0.05). The aprotinin group showed inhibition of platelet aggregation induced by thrombin (P = 0.007) but not adenosine diphosphate. Thrombin generation, defined by F1.2 levels, was significantly reduced by aprotinin in the coronary sinus but not in skin wound incisions. Major adverse cardiovascular events were significantly reduced in aprotinin-treated patients (5.4% vs 29.7%, P < 0.05). Aprotinin also demonstrated antifibrinolytic properties through diminished red blood cell transfusion (P < 0.04) and reduced blood loss postoperatively (603 +/- 330 vs 810 +/- 415 mL, P < 0.004). This study demonstrates that aprotinin protects patients undergoing OPCAB surgery from a hypercoagulable state by diminishing thrombin-induced platelet activation and thrombin generation within saphenous vein grafts, while maintaining systemic hemostatic and antifibrinolytic benefits. These results support further investigation of aprotinin and other PAR-1 antagonists in OPCAB surgery.

Elevated plasma concentration of NO and cGMP may be responsible for the decreased platelet aggregation and platelet leukocyte conjugation in platelets hypo-responsive to catecholamines

Impaired responsiveness to epinephrine and other catecholamines (CA) were previously reported in platelets of 20 ∼ 30% healthy Japanese and Koreans. In the present study, the possible mechanisms of different responsiveness to CA in platelets of CA hypo-responders (CA-HY) and CA good-responders (CA-GR) were investigated. Increased platelet-leukocyte conjugate (PLC) formations were observed with whole blood of CA-GR than with that of CA-HY in both non-stimulated [mean fluorescence intensity (MFI) values: 1.33 ± 0.26 vs. 1.16 ± 0.19] and ADP (MFI: 5.54 ± 3.46 vs. 2.15 ± 1.13) or TRAP (MFI: 5.11 ± 2.32 vs. 3.38 ± 1.47) activated states. The platelets of CA-GR, when stimulated with ADP (10 µM), released approximately twice the amount of ATP than those of CA-HY (0.88 ± 0.65 and 0.45 ± 0.36 nmole, respectively). Nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) levels were significantly higher in non-stimulated PRP of CA-HY than in that of CA-GR (70.3 ± 24.1 µM and 14.1 ± 4.9 nM vs. 41.1 ± 15.8 µM and 6.7 ± 2.4 nM, respectively). The platelet-monocyte conjugation induced with either ADP or TRAP was significantly reduced in CA-GR with the addition of linsidomine, a NO donor, (MFI: 2.78 ± 0.43 vs. 3.73 ± 0.90, or 4.28 ± 0.95 vs. 5.76 ± 1.33, respectively). Moreover, the degree of platelet aggregation and the ATP secretion induced by epinephrine in CA-GR were significantly retarded with the addition of either linsidomine or 8-Bromo-cGMP (a cGMP analog) with more substantial effects on ATP release than aggregation. The results suggested that elevated NO and/or cGMP plasma levels may be responsible for the lower platelet aggregation and PLC formation observed in CA-HY than that in CA-GR.