Platelet-monocyte Interactions Research Articles

Interferon-α increases monocyte migration via platelet–monocyte interaction in murine intestinal microvessels

The aim of this study was to investigate the effect of interferon (IFN)-α on recruitment of platelets and monocytes within the murine small intestinal venular endothelium. Monocytes were isolated from bone marrow of C57B6 mice. Platelets were collected from murine blood. Rolling and adhesion to submucosal microvessels in the small intestine were examined under an intravital fluorescence microscope after injection of fluorescein-labelled monocytes or platelets. In some mice, IFN-α (5×10(5) U/kg) was administered intraperitoneally. After treatment with an antibody against P-selectin, changes in monocyte and platelet migration were also investigated. Changes in monocyte migration under the condition of thrombocytopenia were also investigated. Platelets and monocytes interacted with murine intestinal microvessels, although only few platelets and monocytes showed migration behaviour. Intraperitoneal injection of IFN-α enhanced the migration of both platelets and monocytes in the intestinal microvessels. Pretreatment with anti-P-selectin attenuated the increase in migration of platelets and monocytes induced by administration of IFN-α. Thrombocytopenia decreased the rolling ratio of monocytes, suggesting that the effect of IFN-α on migration was P-selectin-dependent, derived from both the endothelium of microvessels and platelets. The results of this study suggest that IFN-α acts as a potent proinflammatory agent via its stimulatory effect on the endothelium-platelet-monocyte interaction in intestinal microvessels by a P-selectin-dependent mechanism.

023 Platelet activation induces phenotypic and functional changes in circulating monocytes as a consequence of monocyte–platelet aggregate formation

<h3>Background and Hypothesis</h3> Circulating levels of monocyte-platelet aggregates (MPAs) increase in response to platelet activation, and have previously been shown to be elevated in patients presenting with acute cardiovascular (CV) events and in healthy subjects who have underlying CV risk factors. MPAs are also believed to be crucial mediators involved in the pathophysiology of atherosclerosis. Since circulating monocytes comprise different sub-populations which can infiltrate and migrate into tissues to differing degrees, we hypothesised that activated platelets may enhance their differentiation towards a pro-atherogenic phenotype. <h3>Methods</h3> MPAs and monocytes in peripheral blood obtained from healthy volunteers were measured and characterised by FACS analysis. Additionally, monocytes were isolated through immunomagnetic negative selection for CD14+/CD16− cells and co-incubated with autologous platelets. Flow cytometry and adhesion assays on pre-activated endothelial cells (treated with TNF-α 10 ng/ml for 3 h) were performed at different time points. <h3>Results</h3> Three monocytic subsets were identified in blood: classical CD14+/CD16−, CD14high/CD16+ and CD14low/CD16+ cells. They displayed different expression patterns of the adhesion molecules CD11b and CD11c, although no difference was found in their ability to form MPAs in vivo. Platelet co-incubation enhanced monocyte phenotypic change toward CD14+/CD16+ double positivity in a time-dependent manner (33.33±7.79% vs 16.76±6.34% at 48h in the presence or absence of platelets respectively; p=0.0001), with CD16 expression being directly correlated to MPA levels (r²=0.746; p=0.005). This phenotypic change was associated with enhanced monocyte adhesion to activated endothelium (11.5±1.7 vs 5.7±1.5 cells per field, when co-incubated or not with platelets respectively; p=0.005). Adhesion assay demonstrated that only cells positive for both CD14 and CD16 could bind to endothelium, while classical CD14+/CD16− monocytes could not (Abstract 023 Figure 1a). In keeping with this, the sub-population of CD14+/CD16+ monocytes exhibited higher levels of both CD11b and CD11c on the cell surface compared to the monocyte sub-population only positive for CD14. PSGL-1 blocking antibody, which abrogates monocyte-platelet interaction, abolished all these effects (Abstract 023 Figure 2). Moreover, induction of cyclooxygenase 2 (COX-2) was observed in monocytes at 18 h of co-incubation with platelets (Abstract 023 Figure 1b), and specific blockade of COX-2 with NS-398 (10 μM) reduced platelet-dependent CD16 upregulation in CD14+ monocytes, without affecting MPA formation in vitro (Abstract 023 Figure 2). <h3>Conclusion</h3> Platelet interaction upregulates CD16 and expression of adhesion molecules on monocytes, in a COX-2-dependent manner, resulting in greater adhesion to the endothelium.

Cilostazol, a specific PDE-3 inhibitor, ameliorates chronic ileitis via suppression of interaction of platelets with monocytes

Excessive migration of monocytes to a site of intestinal inflammation contributes to tissue damage in Crohn's disease. It is known that cilostazol, a specific phosphodiesterase-3 (PDE-3) inhibitor of platelets, decreases monocyte recruitment to intestinal mucosa through suppression of platelet-monocyte interactions. The objective of this study was to clarify whether cilostazol ameliorates murine ileitis by suppression of monocyte migration. Significant inflammation was induced in the ileum of SAMP1/Yit mice at 23 wk of age after piroxicam treatment for 3 wk. Weight of the terminal ileum of mice was significantly greater with inflammatory cell infiltration in SAMP1/Yit mice than in control mice (AKR-J). Treatment of SAMP1/Yit mice with cilostazol-containing food (200 ppm) for 3 wk significantly attenuated the increase in intestinal weight and the histological changes, including invasion of F4/80-positive macrophages. A significant increase in migration of monocytes and platelets to microvessels of the ileal mucosa was observed in SAMP/Yit mice in vivo by using an intravital fluorescence microscope. Pretreatment with cilostazol significantly attenuated the increased migration of monocytes, possibly through suppression of platelet-monocyte interactions. In conclusion, a PDE-3 inhibitor ameliorates murine ileitis through attenuating migration of monocytes to the intestinal mucosa, suggesting a potential usefulness of antiplatelet drugs for treatment of Crohn's disease.

S1647 IFN-α Increases Rolling of Monocytes via Platelet-Monocyte Interaction in Murine Intestinal Microvessels

Background: IFN-α has both inhibiting and promoting effects on immune system, based on regulation and downmodulation of Th2 cytokines. In some reports, IFN-α was used to treat UC with therapeutic effectiveness. However, induction and exacerbation of UC or CD by IFN-α during the therapy of chronic hepatitis C was also reported. So the immunological influence of IFN-α for IBD is still a controversial issue. In addition, IFN-α has an effect on microcirculation with thrombocytopenia as an adverse effect. Recently we demonstrated platelets contribute to the inflammatory condition in which monocytes are involved via platelet-monocyte interaction in LPS-induced acute ileitis (Microcirculation 2008; 15: 441). However, there is no report whether IFN-α could modulate the monocyte migration in the intestinal mucosa. The objective of this study was to assess the influence of IFN-α on platelet and monocyte interactions with venular endothelium and to investigate the mechanism by which these interactions are induced in intestinal microvessels. Methods: Monocytes were isolated from bone marrow of C57B6 by magnetic cell sorting with beads-conjugated anti CD 11b antibody. Platelets were collected from murine blood by using acid citrate dextrose buffer. Rolling and adherence to submucosal microvessels in the small intestine was examined under an intravital fluorescence microscope after injection of fluorescein-labelled monocytes or platelets to donor mice. In some mice, IFN-α (5×105U/Kg) was intraperitoneally administered. In addition, after treatment with antibody against P-selectin or GPIbα (CD42b), changes of monocytes and platelet migration were also investigated. Results: Intraperitoneal injection of IFN-α enhanced the migration of both platelets and monocytes in the intestinal microvessels. Pretreatment with anti-P-selectin attenuated the increase of migration in platelet and monocyte induced by administration of IFN-α. Thrombocytopenia induced by GPIbα antibody decreased the rolling ratio of monocyte, suggesting that the effect of IFN-α on migration was P-selectin dependent, derived from both endothelium of microvessels and platelets. Conclusions: Interaction of platelets and monocytes was observed in intestinal microvessels. Enhanced interaction of platelets and monocytes to these microvessels was demonstrated after intraperitoneal injection of IFN-α by P-selectin dependent mechanism, derived from endothelium of microvessels and activated plateles. This study implicates IFN-α as a potent proinflammatory agent via its stimulatory effect on endothelial-plateletmonocytes interaction in intestinal microvessels.

Monocyte Subtypes Predict Clinical Course and Prognosis in Human Stroke

The number of circulating monocytes increases after stroke. In this study, we assessed the time course and phenotype of monocyte subsets and their relationship with the clinical course and outcome in 46 consecutive stroke patients and 13 age-matched controls. The proportion of the most abundant 'classical' CD14(high)CD16- monocytes did not change after stroke, whereas that of CD14(high)CD16+ monocytes increased and CD14(dim)CD16+ monocytes decreased. CD14(high)CD16+ monocytes had the highest expression of TLR2, HLA-DR and the angiogenic marker, Tie-2; CD14(dim)CD16+ monocytes had the highest expression of costimulatory CD86 and adhesion molecule CD49d. Platelet-monocyte interactions were highest in CD14(high)CD16- monocytes and lowest in CD14(dim)CD16+ monocytes. In adjusted models, 1/CD14(high)CD16- monocytes were associated with poor outcome (OR: 1.38), higher mortality (OR: 1.40) and early clinical worsening (OR: 1.29); 2/CD14(high)CD16+ monocytes were inversely related to mortality (OR: 0.32); and 3/CD14(dim)CD16+ monocytes were inversely related to poor outcome (OR: 0.74) and infarction size (r=-0.45; P=0.02). These results illustrate that the predominant monocyte subtype conveys harmful effects after stroke, which include stronger interaction with platelets. Alternatively, rarer subpopulations of monocytes are beneficial with a phenotype that could promote tissue repair and angiogenesis. Therefore, monitoring of monocyte subtypes may emerge as a useful tool at the bedside for stroke patients.

Levels and value of platelet activation markers in different subtypes of acute non-cardio-embolic ischemic stroke

Introduction Platelet activation and its interaction with leukocytes are important in the pathophysiology of ischemic stroke. This study aimed to evaluate the value of platelet activation and platelet-leukocyte interaction in different subtypes of acute, non-cardio-embolic ischemic stroke. Methods Fifty-four patients with acute, non-cardio-embolic ischemic stroke, including 32 small-vessel and 22 large-vessel diseases, were evaluated. Platelet activation markers (CD62P, CD63, and CD40L) and platelet-leukocyte interaction were measured by flow cytometry at different time points (< 48 hours and Days 7, 30, and 90 post-ischemic stroke). Markers were also evaluated in 28 other stroke patients in the convalescent stage (3 to 9 months after acute stroke) and in 28 control subjects. Results Patients with ischemic stroke had significantly increased circulating CD62P, CD63, platelet-monocyte interaction, and platelet-lymphocyte interaction in the acute stage compared with the convalescent stage and control groups. Levels of CD62P and CD63 were significantly higher in the large-vessel disease group than in the small-vessel disease group, and differences in CD62P were significant even at one month. The CD40L level in the poor outcome group was significantly higher than that in the good outcome group. Stroke patients with diabetes mellitus and large-vessel disease were associated with poor outcome. Conclusions Patients with large-vessel cerebral infarction elicit higher platelet activation and platelet-leukocyte interaction compared to small-vessel infarction. Further large scale trials are warranted to evaluate the relationship between platelet activation markers and outcome in stroke patients under different anti-platelet therapies, and to clarify optimal treatment.

Platelet-Monocyte Interactions - A Dangerous Liaison Linking Thrombosis, Inflammation and Atherosclerosis

Atherosclerosis is an inflammatory disease. Monocyte-platelet interactions may play a key role in this process by various pathways. They "inflame" each other as well as adjacent cell types at the vascular wall by direct physical interactions, autocrine and paracrine activation processes. These processes promote monocyte recruitment (in)to the vascular wall--a key mechanism in atherogenesis. This article highlights the molecular basis and the inflammatory pathways initiated by platelet-monocyte interactions.

Tirofiban preserves platelet loss during continuous renal replacement therapy in a randomised prospective open-blinded pilot study

IntroductionApproximately one third of all patients with cardiogenic shock suffer from acute kidney injury. Percutaneous coronary intervention, intra-aortic balloon pump, and continuous renal replacement therapy (CRRT) require effective antiplatelet therapy and anticoagulation, resulting in a high risk for platelet loss and bleeding events. The reversible platelet glycoprotein IIb/IIIa receptor inhibitor tirofiban was investigated to preserve platelet number and activation in a prospective open-blinded endpoint evaluation study.MethodsForty patients with cardiogenic shock and acute kidney injury requiring CRRT were randomly assigned to two groups receiving unfractioned heparin (UFH) (n = 20) or a combined anticoagulation with UFH and tirofiban (n = 20). The primary endpoint was platelet loss during CRRT. Secondary endpoints were urea reduction, haemofilter life span, bleeding events, and necessity for platelet transfusions.ResultsIn UFH-treated patients, the percentage of platelet-monocyte aggregates significantly increased (P < 0.001) and consecutively platelet cell count significantly decreased (P < 0.001). In contrast, combined treatment with UFH and tirofiban significantly decreased platelet-monocyte aggregates and platelet numbers (P < 0.001).ConclusionsThis pilot study provides evidence that the use of tirofiban in addition to UFH prevents platelet loss and preserves platelet function in patients with cardiogenic shock and acute kidney injury requiring CRRT. The pathophysiological inhibition of platelet aggregation and platelet-monocyte interaction appears to be causally involved.

Reduction of blood coagulation and monocyte-platelet interaction following the use of a minimal extracorporeal circulation system (Synergy®) in coronary artery bypass grafting (CABG)

Cardiovascular surgery with cardiopulmonary bypass (CPB) induces activation of blood coagulation and systemic inflammation involved in post-operative complications. Our study evaluated the impact of the minimal extracorporeal circulation (mini-CPB) system (Synergy, Sorin Group) on these functional aspects. Twenty patients were randomly assigned to standard CPB (n = 10) or to Synergy (n = 10). Platelet expression of PAC-1, and monocyte/granulocyte-platelet conjugates were evaluated by flow cytometry. A leukocyte-platelet adhesion index was calculated after cell number normalization. ELISAs were performed to measure IL-6 and TNF-alpha, thrombin-antithrombin III complexes (TAT), prothrombin fragments (F1+2), beta-thromboglobulin (beta-TG) and sP-selectin (sCD62P). Blood samples were drawn at the time of anesthesia (T1), at the end of CPB (T2), and at 4 (T3) and 24 hours (T4) after weaning from CPB. All patients were similar for clinical characteristics. When compared to standard CPB, the Synergy showed lower levels of the monocyte-platelet adhesion index at T2 (0.023 +/- 0.005 vs 0.063 +/- 0.013, P = 0.0092) and T4 (0.031 +/- 0.003 vs 0.055 +/- 0.005, P = 0.0017), TAT complexes at T2 (27.175 +/- 5.967 vs 86.592 +/- 5.415, P = 0.0005) and T3 (26.977 +/- 2.468 vs 45.146 +/- 4.365, P = 0.0041), F1+2 fragments at T2 (2.222 +/- 0.226 vs 4.249 +/- 0.292, P = 0.0009), and sP-selectin at T3 (115.17 +/- 19.623 vs 169.554 +/- 19.709, P = 0.0703) and T4 (108.542 +/- 6.429 vs 140.799 +/- 14.771, P = 0.0833). In summary, the Synergy exhibited a lower post-operative activation of blood coagulation, together with a reduced interaction between circulating monocytes and platelets.

Monocyte–platelet complexes on CD14/CD16 monocyte subsets: relationship with ApoA-I levels. A preliminary study

The adhesion of the monocytes to the endothelium and their extravasation into the intima are key steps in atherogenesis. Studies showed the essential role of L-selectin (CD62-L), expressed by the monocytes, and the platelets by forming complexes with monocytes. The delipided apolipoprotein (Apo) A or high-density lipoprotein (HDL) has antiinflammatory effects on monocytes and can bind platelets (monocyte-platelet complexes [MPCs]). The aim of this study was to identify a possible relationship between the MPCs, the monocyte subset, and ApoA-I/HDL serum levels in vivo. Platelet-monocyte complexes were estimated by flow cytometry in 16 volunteers. Monocyte-platelet interaction was characterized by the percentage of monocytes coexpressing the constitutive platelet marker, glycocalicin gpIb-alpha (CD42b; CD42b+monocytes in %, MPC%). Monocytes were divided into four subsets based on lipopolysaccharide receptor (CD14) and FcgammaIII receptor (CD16) expression (CD14++/CD16-, G1; CD14++/CD16+, G2; CD14+/CD16-, G3; and CD14+/CD16+, G4). HDL and ApoA-I levels were measured by routine laboratory techniques. MPC% in the different subsets were G1=8.1+/-3.4%, G2=21.2+/-14%, G3=18+/-12.6%, and G4=22.3+/-14.3% (analysis of variance: P<.001). MPC% in the entire monocyte population was negatively correlated to ApoA-I (R=-0.71, P=.001). The relationship between ApoA-I and MPC% was found mainly in the subsets G1 (R=-0.67, P=.001) and G2 (R=-0.61, P=.01). MPC% was not correlated with any other lipids or lipoprotein or high-sensitivity C-reactive protein. When whole blood was incubated with HDL/ApoA-I, no modification of platelet CD42b fluorescence was observed, indicating that there is no direct interaction between the HDL/ApoA-I and the CD42b fluorescence. Among the monocytes, the G2 subset appeared to have the highest extravasation potential. Indeed, we previously showed that those cells overexpressed CD62-L, and we observed in this work that they were coated with platelets more than the G1 cells. The G2 subset could be more directly involved in the development of atherosclerotic lesions.

Extracellular Matrix Metalloproteinase Inducer (CD147) Is a Novel Receptor on Platelets, Activates Platelets, and Augments Nuclear Factor κB–Dependent Inflammation in Monocytes

In atherosclerosis, circulating platelets interact with endothelial cells and monocytes, leading to cell activation and enhanced recruitment of leukocytes into the vascular wall. The invasion of monocytes is accompanied by overexpression of matrix metalloproteinases (MMPs), which are thought to promote atherosclerosis and trigger plaque rupture. Following interaction with itself, the extracellular matrix metalloproteinase inducer (EMMPRIN) induces MMP synthesis via a little-known intracellular pathway. Recently, we showed upregulation of EMMPRIN on monocytes during acute myocardial infarction. EMMPRIN also stimulates secretion of MMP-9 by monocytes and of MMP-2 by smooth muscle cells, indicating that it may be an important regulator of MMP activity. Expression of EMMPRIN on platelets has not been described until now. Here, we demonstrate that resting platelets show low surface expression of EMMPRIN, which is upregulated by various platelet stimulators (flow cytometry). EMMPRIN is located in the open canalicular system and in alpha granules of platelets (according to electron microscopy and sucrose gradient ultracentrifugation). Platelet stimulation with recombinant EMMPRIN-Fc induced surface expression of CD40L and P-selectin (according to flow cytometry), suggesting that EMMPRIN-EMMPRIN interaction activates platelets. Coincubation of platelets with monocytes induced EMMPRIN-mediated nuclear factor kappaB activation (according to Western blot) in monocytes with increased MMP-9 (zymography), interleukin-6, and tumor necrosis factor-alpha secretion (according to ELISA) by monocytes. In conclusion, EMMPRIN displays a new platelet receptor that is upregulated on activated platelets. Binding of EMMPRIN to platelets fosters platelet degranulation. Platelet-monocyte interactions via EMMPRIN stimulate nuclear factor kappaB-driven inflammatory pathways in monocytes, such as MMP and cytokine induction. Thus, EMMPRIN may represent a novel target to diminish the burden of protease activity and inflammation in atherosclerosis.

Expression of COX-2 in platelet-monocyte interactions occurs via combinatorial regulation involving adhesion and cytokine signaling

Tight regulation of COX-2 expression is a key feature controlling eicosanoid production in atherosclerosis and other inflammatory syndromes. Adhesive interactions between platelets and monocytes occur in these conditions and deliver specific signals that trigger inflammatory gene expression. Using a cellular model of monocyte signaling induced by activated human platelets, we identified the central posttranscriptional mechanisms that regulate timing and magnitude of COX-2 expression. Tethering of monocytes to platelets and to purified P-selectin, a key adhesion molecule displayed by activated platelets, induces NF-kappaB activation and COX-2 promoter activity. Nevertheless, COX-2 mRNA is rapidly degraded, leading to aborted protein synthesis. Time-dependent signaling of monocytes induces a second phase of transcript accumulation accompanied by COX-2 enzyme synthesis and eicosanoid production. Here, generation of IL-1beta, a proinflammatory cytokine, promoted stabilization of COX-2 mRNA by silencing of the AU-rich mRNA decay element (ARE) in the 3'-untranslated region (3'UTR) of the mRNA. Consistent with observed mRNA stabilization, activated platelets or IL-1beta treatment induced cytoplasmic accumulation and enhanced ARE binding of the mRNA stability factor HuR in monocytes. These findings demonstrate that activated platelets induce COX-2 synthesis in monocytes by combinatorial signaling to transcriptional and posttranscriptional checkpoints. These checkpoints may be altered in disease and therefore useful as targets for antiinflammatory intervention.

Expression of COX-2 in platelet-monocyte interactions occurs via combinatorial regulation involving adhesion and cytokine signaling.

Tight regulation of COX-2 expression is a key feature controlling eicosanoid production in atherosclerosis and other inflammatory syndromes. Adhesive interactions between platelets and monocytes occur in these conditions and deliver specific signals that trigger inflammatory gene expression. Using a cellular model of monocyte signaling induced by activated human platelets, we identified the central posttranscriptional mechanisms that regulate timing and magnitude of COX-2 expression. Tethering of monocytes to platelets and to purified P-selectin, a key adhesion molecule displayed by activated platelets, induces NF-kappaB activation and COX-2 promoter activity. Nevertheless, COX-2 mRNA is rapidly degraded, leading to aborted protein synthesis. Time-dependent signaling of monocytes induces a second phase of transcript accumulation accompanied by COX-2 enzyme synthesis and eicosanoid production. Here, generation of IL-1beta, a proinflammatory cytokine, promoted stabilization of COX-2 mRNA by silencing of the AU-rich mRNA decay element (ARE) in the 3'-untranslated region (3'UTR) of the mRNA. Consistent with observed mRNA stabilization, activated platelets or IL-1beta treatment induced cytoplasmic accumulation and enhanced ARE binding of the mRNA stability factor HuR in monocytes. These findings demonstrate that activated platelets induce COX-2 synthesis in monocytes by combinatorial signaling to transcriptional and posttranscriptional checkpoints. These checkpoints may be altered in disease and therefore useful as targets for antiinflammatory intervention.

Platelet–monocyte pro-coagulant interactions in on-pump coronary surgery

Platelets and monocytes possess haemostatic properties, but the clinical effect of platelet-monocyte interactions on haemostasis following coronary surgery is not known. The study characterises the platelet and monocyte responses in cardiac surgery and its impact on haemostasis. In 1342 patients, changes in white blood cell counts (WBC), monocyte counts and platelet counts were measured. PMC formation was analysed by flow-cytometry using monoclonal antibodies against pan-leucocyte marker CD45, monocyte marker CD14 and platelet marker CD42. TF expression was determined using monoclonal antibodies against, CD45, CD14 and human-TF. Continuous variables were expressed as mean+/-SD. Changes in monocyte and platelet counts over time were considered as repeated measures data, and analysed using Generalised Estimating Equations (GEE). Multivariate regression analysis was used to evaluate the effect of several factors on blood loss. A monocytosis occurs with on-pump coronary surgery, but is less pronounced than with off-pump surgery. No difference was seen in patients having redo-surgery or more complex cardiac surgery. Factors associated with monocytosis on multivariate analysis were higher body mass index (p=0.02), diabetes (p=0.035) and smoking (p=0.01). Older patients manifested a lower response (p<0.001). Cross-clamp fibrillation was associated with a lower (p=0.048) monocytic response than was cardioplegia. PMC formation dropped following administration of heparin, peaked at 5 min of CPB, and declined by 2h of CPB (p=0.04). A return towards preoperative levels was found during postoperative days 1-5. No significant change in monocyte TF expression occurred. The mean postoperative blood loss was 581.2+/-292.8 ml, and inversely related to increasing preoperative platelet counts (p<0.001), and to higher monocyte % counts (p=0.012). Patients, who were female (p<0.001), had higher body mass indices (p<0.001), and higher core body temperatures during surgery (p=0.013), as well as patients having perioperative aprotinin (p<0.001) related to less blood loss. A higher postoperative platelet count as well as monocyte% significantly and independently decreases postoperative blood loss following cardiac surgery.

Treadmill exercise in claudicants on aspirin results in improved antioxidant status but only minimal platelet activation

The consequence of exercise on platelets remains controversial and adverse effects may result from repeated ischaemia reperfusion injury. We investigated platelet activation (platelet P-selectin (PS), and activated glycoprotein (Gp) IIb/IIIa), platelet–monocyte aggregates (PMA) and total plasma antioxidant status (TPAS) in claudicants after exercise. Twenty claudicants, taking 75 mg of aspirin daily, were subjected to repeated treadmill testing (3 km/h, 10% inclination). Blood was sampled before and after exercise. Activated GpIIb/IIIa, PS and PMA were quantified with flow cytometry. TPAS was quantified using a decolourisation assay. Percent positive cells for PS (pre-exercise 3.76% vs. 40 min post-exercise 4.10%; P < 0.05) and platelet–monocyte aggregates (pre-exercise: 25.31% vs. 40 min post-exercise 26.99%; P < 0.05) were significantly higher after exercise. Relative median fluorescence (RMF) for activated GpIIb/IIIa was significantly higher 40 min after exercise (pre-exercise: 3.04 vs. 40 min post-exercise: 4.01; P < 0.05). TPAS was significantly higher post-exercise (pre-exercise: 1.31 mmol/l vs. 1 min post-exercise: 1.40 mmol/l and 40 min post-exercise: 1.38 mmol/l; P < 0.01). Following moderate exercise, ‘aspirin treated claudicants’ show marginal platelet activation, PMA formation and a favourable improvement in antioxidant status. Further studies are required to assess the effect of additional antiplatelet agents and the significance of platelet–monocyte interactions. The possibility that aspirin contributes to the TPAS changes following exercise needs to be investigated.

Platelet-induced expression of tissue factor procoagulant activity in freshly isolated human mononuclear cells: implications for experimental use.

Monocytes express tissue factor (TF) as a result of cytokine stimulation or endothelial adherence. We evaluated monocyte-platelet interaction in vitro as another trigger for monocyte TF enhancement in human mononuclear cells isolated by density gradient centrifugation from peripheral blood. Cell TF procoagulant activity (TF-PCA) was quantitated by a one-stage recalcification clotting time assay. Platelets were counted and identified by whole blood flow cytometry as CD61 positive particles, activated platelets were characterized by P-Selectin (CD62) expression, and monocytes by surface CD14 expression. A significant correlation between normalized TF-PCA of isolated mononuclear cells and platelet count was shown (r = 0.43, P < 0.001). Percentage of activated platelets in baseline samples was 4.2 +/- 3.5 while adenosine diphosphate (ADP) increased platelet positivity to 34 +/- 17% (P < 0.001). After isolation, 52 +/- 12% of platelets within suspensions were activated (P < 0.0001). Percentage of CD62-positive monocytes (CD14+ particles) increased from baseline 5% to 13 +/- 6% in ADP-stimulated samples to 53 +/- 17% after isolation (P < 0.001). These findings suggest that density gradient centrifugation activates platelets and that an adhesive interaction between monocytes and platelets may promote TF-PCA expression in isolated mononuclear suspensions. Enhanced monocyte TF expression as a result of an activated platelet-monocyte interaction seems to be an important laboratory effect requiring consideration when utilizing this technique in an experimental setup.

Increased Platelet Binding to Circulating Monocytes in Acute Coronary Syndromes

Present therapies for acute coronary syndromes aim toward limiting platelet-platelet adhesion and aggregation processes. However, platelet-leukocyte interactions may contribute importantly to disease progression in the arterial wall. Recent studies suggest that prevention of platelet-leukocyte binding via P-selectin glycoprotein ligand-1 (PSGL-1) may be beneficial in animal models of vascular injury. P-selectin-PSGL-1 interactions were found to account for most platelet-monocyte binding observed in peripheral blood samples from healthy donors. However, a significant component of observed adhesion was calcium independent, involving neither PSGL-1 nor P-selectin. Platelet-monocyte interactions were examined in 52 patients admitted within 14 hours of symptom onset, with acute coronary syndromes defined as unstable angina (n=12) and acute myocardial infarction (n=13) or noncardiac chest pain (n=27). When compared with patients with noncardiac chest pain, significantly elevated levels of platelet-monocyte binding were found in patients with acute myocardial infarction (70.1+/-15.4% versus 45.4+/-23.3%; P<0.01) and unstable angina (67.4+/-12.9% versus 45.4+/-23.3%; P>0.01). Calcium-independent platelet-monocyte binding was significantly elevated in myocardial infarction patients alone (14.7+/-7.7% versus 6.1+/-5.96%; P<0.001). There is evidence for a significant P-selectin-independent molecular component to the platelet-monocyte conjugation observed in peripheral blood. Patients with myocardial infarction and unstable angina demonstrate increased total binding of platelets to monocytes. Additionally, calcium-independent adhesion was significantly elevated in patients with evidence of myocardial infarction. These findings demonstrate that novel cation-independent adhesion mechanisms may mediate platelet-monocyte binding, representing a new therapeutic target after vascular injury associated with myocardial infarction.

Postprandial lipemia is associated with platelet and monocyte activation and increased monocyte cytokine expression in normolipemic men.

The activation of platelets and monocytes has been implicated in the development of cardiovascular diseases. We asked the question if postprandial lipemia following a fat- containing meal is associated with platelet and monocyte activation and increased platelet-monocyte interaction. Thirteen healthy, normal weight, normolipemic males, 20 to 49 years, consumed a 40% fat meal of whole foods. Blood samples were obtained at fasting and 3 1/2 and 6 hours after ingestion. Triglyceride levels increased to 48% over baseline at 3 1/2 hours postconsumption and returned to fasting levels by 6 hours. Multiparameter flow cytometry using monoclonal antibodies showed that the percentage of platelets expressing surface P-selectin and the activated conformation the GPIIb-IIa receptor was significantly higher at 3 1/2 hours compared to fasting. The percentage of platelet-monocyte aggregates increased by 36% at 3 1/2 hours and 43% at 6 hours postconsumption. The percentage of monocytes expressing intracellular tumor necrosis factor-alpha (TNF-alpha) increased seven and eightfold at 3 1/2 and 6 hours, respectively. The expression of interleukin-1beta (IL-1beta increased in a similar manner. These data suggest activation of platelets and monocytes after a moderate fat meal. Repetitive activation of platelets and monocytes could be an early event in the initiation and development of atherosclerosis.

Antiplatelet effects of abciximab, tirofiban and eptifibatide in patients undergoing coronary stenting

OBJECTIVESWe sought to investigate whether abciximab, tirofiban and eptifibatide achieve comparable antiplatelet effects with coronary stenting.BACKGROUNDThe glycoprotein (GP) IIb/IIIa antagonists abciximab, tirofiban and eptifibatide differ in chemical structure, binding site and pharmacokinetics.METHODSSixty patients undergoing coronary stenting were randomly assigned to abciximab (bolus 0.25 mg/kg body weight, infusion 10 μg per min for 12 h), tirofiban (bolus 10 μg/kg, infusion 0.15 μg/kg per min for 72 h) or eptifibatide (bolus 180 μg/kg, infusion 2 μg/kg per min for 72 h). We took serial blood samples to analyze platelet function by using flow cytometry, turbidimetric aggregometry and the rapid platelet-function assay (RPFA).RESULTSAs assessed by RPFA, platelet aggregation after 2 h of infusion was reduced to 5.9 ± 7.8% (mean ± SD) of baseline by abciximab, to 5.0 ± 5.4% by tirofiban and to 7.8 ± 7.1% by eptifibatide (p = 0.42). Turbidimetric aggregometry with adenosine diphosphate stimulation yielded similar results, whereas percent inhibition of platelet aggregation after thrombin receptor stimulation was 45.8 ± 16.8% with abciximab, 51.3 ± 17.6% with tirofiban and 52.9 ± 14.8% with eptifibatide (p = 0.37). Tirofiban and eptifibatide maintained their level of platelet inhibition during infusion. Flow cytometry revealed that the reduction in the monocyte-platelet interaction by abciximab, tirofiban and eptifibatide was not significantly different (20.0 ± 21.9%, 23.8 ± 18.2% and 21.0 ± 19.8%, respectively; p = 0.87).CONCLUSIONSAbciximab, tirofiban and eptifibatide, at currently recommended doses, achieved similar levels of inhibition of platelet aggregation and a similar reduction in the platelet–monocyte interaction.

Effect of glycoprotein IIb/IIIa receptor blockade on platelet-leukocyte interaction and surface expression of the leukocyte integrin Mac-1 in acute myocardial infarction

OBJECTIVESThis prospective randomized study investigated platelet-induced upregulation of Mac-1 on monocytes and its inhibition by glycoprotein (GP) IIb/IIIa blockage in patients with acute myocardial infarction (AMI).BACKGROUNDIn experimental AMI, Mac-1 on leukocytes is the pivotal adhesion molecule for detrimental inflammatory responses. In vitro, platelet adhesion to monocytes upregulates Mac-1.METHODSPatients undergoing stenting in AMI within 48 h after onset of symptoms were randomly assigned to receive either standard-dose heparin (n = 50) or abciximab plus low-dose heparin (n = 50). In serial blood samples, we assessed platelet-monocyte interaction and Mac-1 surface expression by triple color immunofluorescence flow cytometry.RESULTSCompared with platelet-negative monocytes, Mac-1 surface expression on monocytes with attached platelets was upregulated (median fluorescence intensity [interquartile range]: 259 [179 to 367] vs. 135 [78 to 195] arbitrary units, p < 0.001). As an indicator of platelet-monocyte interaction, mean fluorescence of the platelet marker GP Ibα in the monocytes population decreased after abciximab, although it remained unaffected by heparin alone. Abciximab achieved this effect by a reduction in platelet mass attached to monocytes (GP Ibα fluorescence intensity of heterotypic aggregates at 24 h [arbitrary units]: 187 [143 to 236] after abciximab vs. 228 [156 to 332] after heparin, p = 0.02), whereas it did not affect the percentage of monocytes with adherent platelets. Reduction of platelet-monocyte interaction resulted in decreased Mac-1 surface expression (fluorescence intensity at 24 h [arbitrary units]: 116 [68 to 153] after abciximab vs. 162 [117 to 239] after heparin, p = 0.001).CONCLUSIONSIn patients with AMI, platelet-leukocyte interactions modulate Mac-1 expression on monocytes. Glycoprotein IIb/IIIa blockade is a therapeutic option to interfere with this mechanism.