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To investigate changes in von Willebrand factor (VWF) concentration, function, and multimers during pediatric extracorporeal membrane oxygenation (ECMO) and determine whether routine monitoring of VWF during ECMO would be useful in predicting bleeding.Prospective observational study of pediatric ECMO patients from April 2017 to May 2019.The PICU in a large, tertiary referral pediatric ECMO center.Twenty-five neonates and children (18 yr) supported by venoarterial ECMO.None.Arterial blood samples were collected within 24 hours pre-ECMO, daily for the first 5 days of ECMO, every second day until decannulation, and 24 hours post-ECMO. The STA R Max analyzer was used to measure VWF antigen (VWF:Ag) and ristocetin cofactor (VWF:RCo) activity. VWF collagen binding (VWF:CB) was measured using an enzyme-linked immunosorbent assay. VWF multimers were measured using the semi-automated Hydragel 11 VWF Multimer assay. Corresponding clinical data for each patient was also recorded. A total of 25 venoarterial ECMO patients were recruited (median age, 73 d; interquartile range [IQR], 3 d to 1 yr). The median ECMO duration was 4 days (IQR, 3-8 d) and 15 patients had at least one major bleed during ECMO. The percentage of high molecular weight multimers (HMWM) decreased and intermediate molecular weight multimers increased while patients were on ECMO, irrespective of a bleeding status. VWF:Ag increased and the VWF:RCo/VWF:Ag and VWF:CB/VWF:Ag ratios decreased while patients were on ECMO compared with the baseline pre-ECMO samples and healthy children.Neonates and children on ECMO exhibited a loss of HMWM and lower VWF:CB/VWF:Ag and VWF:RCo/VWF:Ag ratios compared with healthy children, irrespective of major bleeding occurring. Therefore, monitoring VWF during ECMO would not be useful in predicting bleeding in these patients and changes to other hemostatic factors should be investigated to further understand bleeding during ECMO.

We hypothesize that thrombosis with thrombocytopenia syndrome recently described after administration of adenovirus‐vectored vaccines for severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) occurs as a result of the unique properties of the adenovirus vectors, which can have widespread biodistribution throughout the body. The antigen is delivered to megakaryocyte cells, which act as part of the primary immune system and distribute the antigen within progeny platelets, also a key component of the immune system. The interaction of the antigen induces preformed antiplatelet factor 4 (PF4) antibodies to bind to PF4–heparan sulfate complexes in the absence of exogenous heparin, at sites where the heparan sulfate concentration in the vascular glycocalyx is optimal for complex formation, causing thrombosis and thrombocytopenia as observed clinically. This hypothesis is testable in cell culture and animal models, and potentially in vivo, and if proven correct has significant implications for vaccine development and our understanding of the links between the coagulation and immune systems., We hypothesize that thrombosis with thrombocytopenia syndrome recently described after administration of adenovirus‐vectored vaccines for severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) occurs as a result of the unique properties of the adenovirus vectors, which can have widespread biodistribution throughout the body. The antigen is delivered to megakaryocyte cells, which act as part of the primary immune system and distribute the antigen within progeny platelets, also a key component of the immune system. The interaction of the antigen induces preformed antiplatelet factor 4 (PF4) antibodies to bind to PF4–heparan sulfate complexes in the absence of exogenous heparin, at sites where the heparan sulfate concentration in the vascular glycocalyx is optimal for complex formation, causing thrombosis and thrombocytopenia as observed clinically.

Platelets are a key component of the hemostatic system and their roles in inflammation via interactions with leukocytes have also gained attention in recent years. Changes in platelet phenotype and function can cause bleeding and/or thrombosis and, as such, monitoring platelet-specific changes is crucial to assessing hemostasis in the clinical setting. Currently, available platelet function tests such as platelet aggregometry and thromboelastography require a large volume of blood, which is a major limitation for the pediatric population. Whole blood flow cytometric analysis of platelets is increasingly utilized in recent years, primarily due to the sensitivity of this method, but also because it only requires a small amount of blood with minimal sample manipulation. We have developed a whole blood flow cytometry methodological approach that enables the assessment of platelet phenotype, function, and their interactions with monocytes and neutrophils.

Flow cytometry is a valuable tool in determining the phenotype and function of platelets accurately. The emergence of platelet flow cytometry in recent years provides an attractive alternative to other platelet analytical techniques, with advantages such as requiring small volumes and being highly sensitive to minimal changes in receptor function and expression. Here we present a methodical approach encompassing the stages in the development and optimization of platelet flow cytometry panels based on our extensive experience in this area.

To investigate platelet pathophysiology associated with pediatric extracorporeal membrane oxygenation (ECMO).Prospective observational study of neonatal and pediatric ECMO patients from September 1, 2016, to December 31, 2019.The PICU in a large tertiary referral pediatric ECMO center.Eighty-seven neonates and children (18 yr) supported by ECMO.None.Arterial blood samples were collected on days 1, 2, and 5 of ECMO and were analyzed by whole blood flow cytometry. Corresponding clinical data for each patient was also recorded. A total of 87 patients were recruited (median age, 65 d; interquartile range [IQR], 7 d to 4 yr). The median duration of ECMO was 5 days (IQR, 3-8 d) with a median length of stay in PICU and hospital of 18 days (IQR, 10-29 d) and 35 days (IQR, 19-75 d), respectively. Forty-two patients (48%) had at least one major bleed according to a priori determined definitions, and 12 patients (14%) had at least one thrombotic event during ECMO. Platelet fibrinogen receptor expression decreased (median fluorescence intensity [MFI], 29,256 vs 26,544; p = 0.0005), while von Willebrand Factor expression increased (MFI: 7,620 vs 8,829; p = 0.0459) from day 2 to day 5 of ECMO. Platelet response to agonist, Thrombin Receptor Activator Peptide 6, also decreased from day 2 to day 5 of ECMO, as measured by binding with anti-P-selectin, PAC-1 (binds activated GPIIb/IIIa), and anti-CD63 monoclonal antibodies (P-selectin area under the curve [AUC]: 63.46 vs 42.82, respectively, p = 0.0022; PAC-1 AUC: 93.75 vs 74.46, p = 0.0191; CD63 AUC: 55.69 vs 41.76, p = 0.0020).The loss of platelet response over time may contribute to bleeding during ECMO. These novel insights may be useful in understanding mechanisms of bleeding in pediatric ECMO and monitoring platelet markers clinically could allow for prediction or early detection of bleeding and thrombosis.

Platelets are small but very abundant blood cells that play a key role in hemostasis, contributing to thrombus formation at sites of injury. The ability of platelets to perform this function, as well as functions in immunity and inflammation, is dependent on the presence of cell surface glycoproteins and changes in their quantity and conformation after platelet stimulation. In this article, we describe the characterization of platelet surface markers and platelet function using platelet-specific fluorescent probes and flow cytometry. Unlike traditional platelet tests, immunophenotypic analysis of platelets by flow cytometry allows the analysis of platelet function in samples with very low platelet counts as often encountered in clinical situations. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Immunophenotyping of platelet surface receptors Alternate Protocol: Fix-first method for immunophenotyping of platelet surface receptors Basic Protocol 2: Determination of platelet activation using P-selectin expression and/or PAC1 binding Basic Protocol 3: Determination of procoagulant platelets using annexin V binding or antibodies specific for coagulation factor V/Va or X/Xa Support Protocol: Preparation of isolated platelets.

Evidence-based guidelines recommend exercise therapy for patients with chronic heart failure (CHF). Such patients have increased atherothrombotic risk. Exercise can transiently increase platelet activation and reactivity and decrease vascular function in healthy participants, although data in CHF are scant. Eccentric (ECC) cycling is a novel exercise modality that may be particularly suited to patients with CHF, but the acute impacts of ECC cycling on platelet and vascular function are currently unknown. Our null hypothesis was that ECC and concentric (CON) cycling, performed at matched external workloads, would not induce changes in platelet or vascular function in patients with CHF. Eleven patients with heart failure with reduced ejection fraction (HFrEF) took part in discrete bouts of ECC and CON cycling. Before and immediately after exercise, vascular function was assessed by measuring diameter and flow-mediated dilation (FMD) of the brachial artery. Platelet function was measured by the flow cytometric determination of glycoprotein IIb/IIIa activation and granule exocytosis in the presence and absence of platelet agonists. ECC cycling increased baseline artery diameter (pre: 4.0 ± 0.8 mm vs. post: 4.2 ± 0.7 mm; P = 0.04) and decreased FMD%. When changes in baseline artery diameter were accounted for, the decrease in FMD post-ECC cycling was no longer significant. No changes were apparent after CON. Neither ECC nor CON cycling resulted in changes to any platelet-function measures (all P > 0.05). These results suggest that both ECC and CON cycling, at a moderate intensity and short duration, can be performed by patients with HFrEF without detrimental impacts on vascular or platelet function. NEW & NOTEWORTHY This is the first evidence to indicate that eccentric (ECC) cycling can be performed relatively safely by patients with chronic heart failure (CHF), as it did not result in impaired vascular or platelet function compared with conventional cycling. This is important, as acute exercise can transiently increase atherothrombotic risk, and ECC cycling is a novel exercise modality that may be particularly suited to patients with CHF.

Polycyclic aromatic hydrocarbons are very important components of oil pollution. These pollutants tend to sorb to cell surfaces, exerting toxic effects on organisms. Our study developed a flow cytometric method for the detection of PAHs sorbed to phytoplankton by exploiting their spectral characteristics. We discriminated between cells with PAHs from cells free of PAHs. Clear discrimination was observed with flow cytometer provided with 375 or 405nm lasers in addition to the standard 488nm laser necessary to identify phytoplankton. Using this method, we measured the relationship between the percentages of phytoplankton organisms with PAHs, with the decrease in the growth rate. Moreover, the development of this method could be extended to facilitate the study of PAHs impact on cell cultures from a large variety of organisms.

Platelets are subcellular blood elements with a well-established role in haemostasis. Upon activation platelets undergo granule exocytosis, resulting in α-granule P-Selectin being expressed on the cell membrane. This allows binding of activated platelets to P-Selectin glycoprotein ligand 1 (PSGL-1) expressing leukocytes, forming leukocyte-platelet aggregates (LPAs). Whole blood flow cytometry (FCM) has demonstrated that elevated circulating LPAs (especially monocyte LPAs) are linked to atherothrombosis in high risk patients, and that activated platelet binding influences monocytes towards a pro-adhesive and pro-atherogenic phenotype. However, a limitation of conventional FCM is the potential for coincident events to resemble LPAs despite no tethering. Imaging cytometry can be used to characterize LPA formation and distinguish circulating MPAs from coincidental events. Platelets and leukocyte subsets are identified by expression of surface markers (e.g. the lipopolysachharide receptor CD14 on monocytes, glycoprotein Ib CD42b on platelets). In conventional FCM, all events with both leukocyte and platelet characteristics are designated as LPAs. However, by using an 'internal' mask based on the brightfield image and the fluorescent platelet identifier, imaging flow cytometry is able to distinguish leukocytes with tethered platelets (genuine LPAs) from leukocyte with coincidental, untethered platelets nearby. Mechanisms (e.g. adhesion molecules) or consequences (e.g. signal transduction) can then be separately analysed in platelet tethered and untethered leukocytes. Imaging flow cytometry therefore provides a more accurate approach for both enumeration and analysis of LPAs than conventional FCM.

Advances in science education research have the potential to improve the way students learn to perform scientific interpretations and understand science concepts. We developed active, collaborative activities to teach skills in manipulating flow cytometry data using FlowJo software. Undergraduate students were given compensated clinical flow cytometry listmode output (FCS) files and asked to design a gating strategy to diagnose patients with different hematological malignancies on the basis of their immunophenotype. A separate cohort of research trainees was given uncompensated data files on which they performed their own compensation, calculated the antibody staining index, designed a sequential gating strategy, and quantified rare immune cell subsets. Student engagement, confidence, and perceptions of flow cytometry were assessed using a survey. Competency against the learning outcomes was assessed by asking students to undertake tasks that required understanding of flow cytometry dot plot data and gating sequences. The active, collaborative approach allowed students to achieve learning outcomes not previously possible with traditional teaching formats, for example, having students design their own gating strategy, without forgoing essential outcomes such as the interpretation of dot plots. In undergraduate students, favorable perceptions of flow cytometry as a field and as a potential career choice were correlated with student confidence but not the ability to perform flow cytometry data analysis. We demonstrate that this new pedagogical approach to teaching flow cytometry is beneficial for student understanding and interpretation of complex concepts. It should be considered as a useful new method for incorporating complex data analysis tasks such as flow cytometry into curricula.

Platelet activation, including the formation of monocyte platelet aggregates (MPAs), contributes to atherosclerosis, thrombus formation, and acute coronary syndromes. Regular participation in exercise can lower cardiovascular risk, but little is known regarding the impact of exercise training on platelet function. We investigated the effect of 6 mo of walking exercise on platelet function in sedentary older adults without significant cardiovascular disease. Twenty-seven participants were randomly allocated to 6 mo of either: no-exercise ( n = 13) or 3 × 50 min/wk of supervised center-based walking ( n = 14). Circulating and agonist-induced MPAs were assessed using flow cytometry before [ month 0 (0M)] and after [ month 6 (6M)] the intervention. Circulating MPAs increased from 0M (3.7 ± 1.0%) to 6M (4.7 ± 1.6%) in the no-exercise group ( P = 0.009), whereas a nonsignificant decrease was observed in the walking group (0M 4.3 ± 1.7 vs. 6M 3.7 ± 1.2 %, P = 0.052). The change in MPAs between groups was significant ( P = 0.001). There were no differences between groups in platelet responses to agonists across the interventions (all P > 0.05). Collectively, these data suggest that the absence of regular exercise may increase MPAs, which are cellular mediators involved in atherosclerosis, while regular walking inhibits such increases. The thrombotic function of platelets appears to be relatively unaltered by exercise training. This study provides novel data related to the cardioprotective effects associated with participation in exercise.NEW & NOTEWORTHY Monocyte-platelet aggregates contribute to atherosclerosis and exercise can lower cardiovascular risk. This is the first study to discover that a lack of regular physical activity is associated with increased monocyte-platelet aggregates over a 6-mo intervention period. In contrast, walking exercise inhibits increased monocyte-platelet aggregates in the circulation. This study highlights a novel pathway by which regular participation in exercise exerts its cardioprotective effects.

Polycystic ovarian syndrome (PCOS) affects 12 to 19% of women and has reproductive and metabolic features (endothelial dysfunction, increased diabetes, and cardiovascular risk factors). It also appears to have altered coagulation and fibrinolysis with a prothrombotic state with epidemiological evidence of increased venous thromboembolism. We aimed to comprehensively assess hemostasis in women with PCOS versus control women. In an established case-control cohort of lean, overweight, and obese women with (n = 107) and without PCOS (n = 67), with existing measures of plasminogen activator inhibitor 1 (PAI-1), asymmetric dimethylarginine (ADMA), hormonal, and metabolic markers, we also assessed prothrombin fragments 1 and 2 (PF1 & 2), plasminogen, tissue plasminogen activator (tPA), and thrombin generation (TG). Higher levels of ADMA (0.70 vs. 0.39 µmol/L, p

Platelets are subcellular blood elements with a well-established role in haemostasis. Upon activation platelets express P-Selectin (CD62P) on the cell membrane and bind to P-Selectin glycoprotein ligand 1 expressing monocytes, influencing them toward a pro-adhesive and inflammatory phenotype. It is well established that elevated circulating monocyte-platelet aggregates (MPAs) are linked to atherothrombosis in high risk patients. However, whole blood flow cytometry (FCM) has recently shown that circulating MPAs may also occur in the absence of platelet activation, particularly in healthy children. A potential limitation of conventional FCM is the potential for coincident events to resemble monocyte platelet aggregates. Here we report a novel imaging cytometry approach to further characterize monocyte-platelet aggregate formation by P-Selectin dependent and P-Selectin independent mechanisms and distinguish circulating MPAs from coincidental events. Monocytes were identified by expression of the lipopolysachharide receptor (CD14 BV421), while platelets were identified by expression of the glycoprotein Ib (CD42b APC). Differentiation of P-Selectin dependent and P-Selectin independent binding was achieved with AF488 labeled CD62P. Overall analysis of circulating and in vitro generated MPAs by conventional and imaging cytometry methods showed very strong correlation (r2 = >0.99, P

We tested whether the quality of spermatozoa from mature male sheep would be affected during nutrition-induced changes in testicular mass. Merino rams were fed for 65 days with diets that increased, maintained or decreased body and testis mass (n=8 per group). In semen collected on Days 56 and 63, underfed rams had less sperms per ejaculate than well-fed rams (P

An important measure of male quality is sperm viability; i.e., the percentage of live sperm within an ejaculate, as this provides an accurate measure of the number of sperm potentially available for egg fertilization. Sperm viability is often determined by fluorescence microscopy using dyes that differentially stain viable and nonviable sperm, but the technique has a number of limitations. Here, a flow cytometry (FCM) method was developed, which allows the rapid determination of honeybee sperm viability, facilitating high throughput analyses. Using samples with known sperm viabilities, it was found that data obtained from FCM were more accurate and less variable compared with data obtained for the same samples using fluorescence microscopy. It was also found that a previously reported additional population of honeybee sperm found in datasets using FCM is caused by freeze–thawing samples. In conclusion, the method described here allows to quantify sperm viability of honeybees quickly and with high accuracy. This will be of great value for future scientific research and could also be of value to guide future bee breeding programs, given the agricultural importance of honeybees as pollinators. © 2014 International Society for Advancement of Cytometry

Diabetes is associated with increased cardiovascular risk. We have recently shown that the naturally occurring flavonol quercetin (Que) or the synthetic flavonol 3′,4′-dihydroxyflavonol (DiOHF) inhibits platelet function and delays thrombus formation in healthy mice. Therefore, the aim of this study was to investigate the effect of Que or DiOHF treatment on platelet function and ferric chloride–induced carotid artery thrombosis in a mouse model of type 1 diabetes. Diabetic mice treated with Que or DiOHF maintained blood flow at a significantly higher level than untreated diabetic mice at the end of the recording period. In addition, treatment with Que or DiOHF significantly reduced diabetes-induced platelet hyper-aggregability in response to platelet agonist stimulation. Furthermore, treatment with Que or DiOHF significantly inhibited dense, but not alpha, granule exocytosis in diabetic and control mice. Our demonstration that flavonols delay thrombus formation in diabetes suggests a potential clinical role for these compounds in anti-platelet therapy.

Background: Whilst resistance training has been proven to convey considerable benefits to olderpeople; immediately post-exercise there may be elevated transient risks for cardiac events and falls. Objectivesand Measurements:We assessed the acute effects of eccentrically-biased (EB) and conventional (CONV)resistance exercise on: platelet number, activation and granule exocytsosis; and mean velocity of centre ofpressure displacement (Vm). Design, Setting, Participants and Intervention:Ten older adults (7 males, 3females; 69 ± 4 years) participated in this randomised controlled cross-over study in which they performed EBand CONV training sessions that were matched for total work and a control condition. Results:Immediately post-exercise there was a statistically significant difference in platelet count between the control condition, in which ithad declined (pre 224 ± 35 109/L; post 211 ± 30 109/L: P < 0.05) and CONV in which it had increased (pre 236 ±55 109/L; post 242 ± 51 109/L: P > 0.05). There was no change in platelet activation and granule exocytsosis orVm following EB and CONV. Conclusions:Overall, while minor differences between regimens were observed,no major adverse effect on parameters of platelet function or centre of pressure displacement were observedacutely following either regimen. Eccentrically-biased and conventional resistance exercise training regimens donot appear to present an elevated acute risk in the context of changes to platelet function contributing to a cardiacevent or postural stability increasing falls risk for apparently healthy older adults.

Clinical Development Phases I-III Regulatory, Quality, Manufacturing Platelets are subcellular fragments in blood whose activation is central to atherothrombosis and cardiovascular events. Platelet activation is complex, with multiple pathways of initiation and amplification involved. Antiplatelet drugs may target any of these pathways to diminish the propensity of platelets to become activated. The most well-established antiplatelet drugs, aspirin and thienopyridines, target the thromboxane and adenosine diphosphate auto-amplification pathways of platelet activation, respectively. These are very effective medicines, but response varies and residual on-treatment platelet function is associated with poorer clinical outcomes. Alternative antiplatelet therapies are available and more are in development. Therefore, tailoring antiplatelet therapy to on-treatment platelet activation is an appealing strategy and the subject of investigation. There are many different approaches to measuring platelet activation and response to stimulation with antiplatelet therapy, but there is very little agreement between them regardless of the cutoffs used. Therefore, selection of the appropriate platelet function test is important in assessing on-treatment platelet activation for guided therapy. Recent research has shown that tailoring antiplatelet therapy can improve these laboratory measures of platelet activation, but clinical studies that assess cardiovascular benefit against potential for increased bleeding are required.

AB Purpose: Hyperactive platelets contribute to the thrombotic response in humans, and exercise transiently increases platelet function. Caffeine is routinely used by athletes as an ergogenic aid, but the combined effect of exercise and caffeine on platelet function has not been investigated. Methods: Twelve healthy males were randomly assigned to one of four groups and undertook four experimental trials of a high-intensity aerobic interval training (AIT) bout or rest with ingestion of caffeine (3 mg[middle dot]kg-1) or placebo. AIT was 8 x 5 min at approximately 75% peak power output (approximately 80% V[spacing dot above]O2peak) and 1-min recovery (approximately 40% peak power output, approximately 50% V[spacing dot above]O2peak) intervals. Blood/urine was collected before, 60, and 90 min after capsule ingestion and analyzed for platelet aggregation/activation. Results: AIT increased platelet reactivity to adenosine diphosphate (placebo 30.3%, caffeine 13.4%, P < 0.05) and collagen (placebo 10.8%, caffeine 5.1%, P < 0.05) compared with rest. Exercise placebo increased adenosine diphosphate-induced aggregation 90 min postingestion compared with baseline (40.5%, P < 0.05), but the increase when exercise was combined with caffeine was small (6.6%). During the resting caffeine protocol, collagen-induced aggregation was reduced (-4.3%, P < 0.05). AIT increased expression of platelet activation marker PAC-1 with exercise placebo (P < 0.05) but not when combined with caffeine. Conclusion: A single bout of AIT increases platelet function, but caffeine ingestion (3 mg[middle dot]kg-1) does not exacerbate platelet function at rest or in response to AIT. Our results provide new information showing caffeine at a dose that can elicit ergogenic effects on performance has no detrimental effect on platelet function and may have the potential to attenuate increases in platelet activation and aggregation when undertaking strenuous exercise.

Flavonols are polyphenolic compounds with broad-spectrum kinase inhibitory, as well as potent anti-oxidant and anti-inflammatory properties. Anti-platelet potential of quercetin (Que) and several related flavonoids have been reported; however, few studies have assessed the ability of flavonols to inhibit exocytosis of different platelet granules or to inhibit thrombus formation in vivo. 3',4'-Dihydroxyflavonol (DiOHF) is a flavonol which is structurally related to Que and has been shown to have greater anti-oxidant capacity and to improve the endothelial function in the context of diabetes and ischaemia/reperfusion injury. While the structural similarity to Que suggests DiOHF may have a potential to inhibit platelet function, no studies have assessed the anti-platelet potential of DiOHF. We therefore investigated platelet granule inhibition and potential to delay arterial thrombosis by Que and DiOHF. Both Que and DiOHF showed inhibition of collagen, adenosine diphosphate and arachidonic acid stimulated platelet aggregation, agonist-induced GPIIb/IIIa activation as demonstrated by PAC-1 and fibrinogen binding. While both flavonols inhibited agonist-induced granule exocytosis, greater inhibition of dense granule exocytosis occurred with DiOHF as measured by both ATP release and flow cytometry. In contrast, while Que inhibited agonist-induced P-selectin expression, as measured by both platelet surface P-selectin expression and upregulation of surface GPIIIa expression, inhibition by DiOHF was not significant for either parameter. C57BL/6 mice treated with 6 mg kg(-1) IV Que or DiOHF maintained greater blood flow following FeCl3-induced carotid artery injury when compared to the vehicle control. We provide evidence that Que and DiOHF improve blood flow following arterial injury in part by attenuating platelet granule exocytosis.

People with diabetes have an increased risk of life-threatening cardiovascular disease compared to the general population. Furthermore, people with diabetes are at greatly increased risk of not responding to standard anti-platelet therapy, such as aspirin, for the prevention of atherothrombotic events. This phenomenon is often referred to as treatment failure. Those who are at increased risk of such events despite aspirin therapy can be prospectively identified by a variety of laboratory measures of residual on-treatment platelet function, known as aspirin resistance. However, there is little agreement among laboratories on the approaches to these measurements, and insufficient data to guide the clinical management of people with diabetes-associated aspirin resistance if it is prospectively identified. This review provides a critical appraisal of the different approaches to the detection and evidence of mechanisms which contribute to this phenomenon, as well evidence for the potential effectiveness of different clinical approaches to overcoming aspirin treatment failure in diabetes. Potential mechanisms of aspirin resistance in diabetes include elevated platelet turnover that results in an immature platelet fraction able to synthesise the uninhibited therapeutic target of aspirin, cyclooxygenase-1 (COX-1); residual thromboxane production by both COX-1-dependent and COX-1-independent pathways; up-regulation of aspirin-insensitive pathways of platelet function, such as adenosine diphosphate signalling; and increased underlying atherosclerotic disease burden that results in elevated underlying platelet hyper-reactivity. High on-aspirin platelet reactivity in diabetes may be related to glycemic control. Potential approaches to treatment include controlling modifiable risk factors to achieve effective glycemic control, guided increases in aspirin dose or frequency of administration, or the use of additional antiplatelet therapies. While evidence suggests that altering antiplatelet therapy, particularly by increasing frequency of aspirin administration, can overcome incomplete inhibition of thromboxane synthesis, no clinical studies to date have assessed the effectiveness of these in preventing breakthrough atherothrombosis. While some clinicians currently alter therapy on the basis of theoretical potential benefit of these strategies following identification of aspirin resistance in the laboratory, this is not yet supported by clinical evidence of a benefit, and clear clinical guidelines for the management of aspirin resistance are lacking.

Taurine, 2-aminoethanesulphonic acid is a common ingredient of energy drinks which is very popular with young adults. Taurine in energy drinks is known to enhance muscular performance in athletes. However, caffeine in high concentrations as found in most energy drinks have also been implicated to play an adverse role leading to cardiovascular diseases (CVD). Hyper activation of platelets is one of the major risk factors of CVD. The aim of the study was to evaluate effect of taurine alone on platelet aggregation and activation of platelet surface antigens by flowcytometry. It is hypothe- sized that taurine's antioxidant property would inhibit platelet activity. Twelve healthy, male and female, volunteers aged 20-60 years were recruited for this study. A statistically significant inhibition of platelet aggregation was observed upon stimulation with agonists adenosine diphosphate (ADP), collagen and arachidonic acid (AA) (p

The term resistance has been applied to interindividual variability in platelet reactivity during antiplatelet therapy or to thrombosis despite appropriate therapy. In particular "aspirin resistance" and "clopidogrel resistance" have been the subject of intense investigation for their association with poor cardiovascular outcomes. Several mechanisms have been investigated including resistance arising from poor bioavailability, especially in clopidogrel therapy as resulting from a loss of function variant in hepatic metabolism required for prodrug activation. A limitation of studies linking on-treatment reactivity and clinical outcome is that they have been performed in high-risk patients with recent atherothrombotic disease. On-treatment platelet reactivity correlates with acuity of recent atherothrombosis, and variability in pretreatment platelet function predicts on-treatment platelet function for both aspirin and clopidogrel. It is therefore likely that high on-treatment platelet function at the time of testing may often result from underlying platelet hyperreactivity related to acute atherothrombosis, rather than true pharmacological resistance. The association of high on-treatment platelet reactivity with poor clinical outcomes may therefore be attributed to variability in underlying burden of disease instead of, or in addition to, pharmacological resistance to antiplatelet therapy.

Polycystic ovarian syndrome (PCOS), diagnosed based on hyperandrogenism, ovulatory dysfunction, and polycystic ovaries, is one of the most common disorders of reproductive-aged females. Etiology includes both genetic and environmental/lifestyle factors contributing to both insulin resistance and hyperandrogenism. Clinically, PCOS has reproductive, psychological, and metabolic features, the latter predisposing to cardiovascular disease (CVD). Hemostatic abnormalities have an association with and a demonstrated pathophysiological role in CVD in non-PCOS populations but have not been adequately explored in PCOS. This review focuses on the hemostatic system in PCOS, exploring also relationships to the metabolic and hormonal abnormalities of the syndrome, and aims to identify whether hemostatic abnormalities are present as potential contributors to increased cardiovascular risk. Ultimately, this area may reveal preventative and therapeutic opportunities, which could improve the cardiovascular health of women with PCOS.

Platelets are small, anucleate blood elements of critical importance in cardiovascular disease. The ability of platelets to activate and aggregate to form blood clots in response to endothelial injury, such as plaque rupture, is well established. These cells are therefore important contributors to ischaemia in atherothrombosis, and antiplatelet therapy is effective for this reason. However, growing evidence suggests that platelets are also important mediators of inflammation and play a central role in atherogenesis itself. Interactions between activated platelets, leukocytes and endothelial cells trigger autocrine and paracrine activation signals, resulting in leukocyte recruitment at and into the vascular wall. Direct physical interaction may contribute also, through platelet adhesion molecules assisting localization of monocytes to the site of atherogenesis and platelet granule release contributing to the chronic inflammatory milieu which leads to foam cell development and accelerated atherogenesis. Recent studies have shown that antiplatelet therapy in animal models of accelerated atherogenesis can lead to decreased plaque size and improve plaque stability. This review examines the complexity of platelet function and the nature of interactions between activated platelets, leukocytes and endothelial cells. We focus on the growing body of evidence that platelets play a critical role in atherogenesis and contribute to progression of atherosclerosis.

Release of serotonin and activation of serotonin 5HT2A receptors on platelet surfaces is a potent augmentative stimulus for platelet aggregation. However, earlier-generation serotonin receptor antagonists were not successfully exploited as antiplatelet agents, possibly owing to their lack of specificity for the 5HT2A receptor subtype.To assess whether targeted inhibition of the serotonin 5HT2A receptor attenuates recurrent thrombosis and improves coronary patency in an in vivo canine model mimicking unstable angina.In protocol 1, anesthetized dogs were pretreated with a novel, selective inverse agonist of the 5HT2A receptor (APD791) or saline. Recurrent coronary thrombosis was then initiated by coronary artery injury+stenosis, and coronary patency was monitored for 3 h. Protocol 2 was similar, except that: (i) treatment with APD791 or saline was begun 1 h after the onset of recurrent thrombosis; (ii) template bleeding time was measured; and (iii) blood samples were obtained for in vitro flow cytometric assessment of platelet responsiveness to serotonin.APD791 attenuated recurrent thrombosis, irrespective of the time of treatment: in both protocols, flow-time area (index of coronary patency; normalized to baseline coronary flow) averaged 58-59% (P0.01) following administration of APD791 vs. 21-28% in saline controls. Moreover, the in vivo antithrombotic effect of APD791 was not accompanied by increased bleeding, but was associated with significant and selective inhibition of serotonin-mediated platelet activation.5HT2A receptor inhibition with APD791, even when initiated after the onset of recurrent thrombosis, improves coronary patency in the in vivo canine model.

Background— Poor clinical outcome in aspirin-treated patients has been termed aspirin resistance and may result from inadequate inhibition of platelet cyclooxygenase-1 (COX-1) by aspirin. The objectives of this study were to determine prospectively whether COX-1-dependent and other platelet function assays correlate with clinical outcomes in aspirin-treated patients. Methods and Results— Blood was collected before percutaneous coronary intervention from 700 consecutive aspirin-treated (81 or 325 mg for ≥3 days) patients. Platelet function was tested by (1) serum thromboxane B 2 ; (2) arachidonic acid-stimulated platelet surface P-selectin and activated glycoprotein IIb/IIIa and leukocyte-platelet aggregates; and (3) platelet function analyzer (PFA)-100 collagen-epinephrine and collagen-ADP closure time (CT). Adverse clinical outcomes of all-cause death, cardiovascular death, and major adverse cardiovascular events (cardiovascular death, myocardial infarction, hospitalization for revascularization, or acute coronary syndrome) were assessed by telephone interview and/or medical record review. Clinical outcomes information was obtained at 24.8±0.3 months after platelet function testing. By univariate analysis, COX-1-dependent assays, including serum thromboxane B 2 level, were not associated with adverse clinical outcomes, whereas the COX-1-independent assay, PFA-100 collagen-ADP CT P =0.0149). After adjustment for covariables (including sex, aspirin dose, Thrombolysis in Myocardial Infarction risk score, clopidogrel use), both serum thromboxane B 2 >3.1 ng/mL and PFA-100 collagen-ADP CT Conclusions— In this prospective study of 700 aspirin-treated patients presenting for angiographic evaluation of coronary artery disease, residual platelet COX-1 function measured by serum thromboxane B 2 and COX-1-independent platelet function measured by PFA-100 collagen-ADP CT, but not indirect COX-1-dependent assays (arachidonic acid-stimulated platelet markers, shortened PFA-100 collagen-epinephrine CT), correlate with subsequent major adverse cardiovascular events. This study suggests that multiple mechanisms, including but not confined to inadequate inhibition of COX-1, are responsible for poor clinical outcomes in aspirin-treated patients, and therefore the term aspirin resistance is inappropriate.

Summary. Background: Prasugrel is a novel antiplatelet prodrug of the same thienopyridine class as clopidogrel and ticlopidine. Metabolism of prasugrel generates the active metabolite R-138727, an antagonist of the platelet P2Y12 adenosine diphosphate (ADP) receptor, leading to inhibition of ADP-mediated platelet activation and aggregation. ADP also enhances the platelet response to collagen, and these two agonists contribute to the generation of platelet procoagulant activity. We therefore examined whether R-138727 inhibits ADP- and collagen-triggered platelet procoagulant activities.Methods and results: As shown by whole blood flow cytometry, R-138727 inhibited surface phosphatidylserine expression on ADP plus collagen-stimulated platelets and tissue factor (TF) expression on ADP-, collagen-, and ADP plus collagen-stimulated monocyte–platelet aggregates. R-138727 reduced monocyte–platelet aggregate formation, thereby further inhibiting TF expression. ADP, collagen, and ADP plus collagen accelerated the kinetics of thrombin generation in recalcified whole blood and R-138727 significantly inhibited this acceleration. Clot strength in a modified thromboelastograph system was also inhibited by R-138727 (IC50 0.7 ± 0.1 μm). Conclusions: In addition to its previously known inhibitory effects on platelet activation and aggregation, the active metabolite of prasugrel, R-138727, inhibits platelet procoagulant activity in whole blood (as determined by phosphatidylserine expression on platelets and TF expression on monocyte–platelet aggregates), resulting in the functional consequences of delayed thrombin generation and impaired clot development.

Introduction Adenosine A2 agonists improve arterial patency in experimental models of recurrent thrombosis, an effect purportedly triggered by stimulation of platelet A2 receptors and subsequent down-regulation of platelet function. However: (i) there is no direct evidence to substantiate this premise; and (ii) given the recognized differences among species in platelet signaling, it is possible that the mechanisms of A2 receptor stimulation may be model-dependent. Accordingly, we applied an integrated in vivo and in vitro approach, using both canine and human models, to test the hypothesis that the anti-thrombotic effects of A2 agonist treatment are due in part to inhibition of platelet activation. Methods In Protocol 1, recurrent coronary thrombosis was triggered in anesthetized dogs by application of a stenosis at a site of arterial injury. Coronary patency and flow cytometric indices of platelet activation (P-selectin expression; formation of heterotypic aggregates) were compared in dogs pre-treated with the A2 agonist CGS 21680 versus controls. In Protocols 2 and 3, blood samples were obtained from dogs and human volunteers. In vitro aggregation and platelet activation (assessed by impedance aggregometry and flow cytometry, respectively) were quantified in paired aliquots pre-incubated with CGS versus vehicle. Results In the canine models, CGS improved in vivo coronary patency and attenuated in vitro aggregation but, contrary to our hypothesis, did not evoke a down-regulation in platelet activation. In contrast, in human blood samples, CGS attenuated both in vitro aggregation and flow cytometric markers of platelet activation–aggregation. Conclusion The mechanisms contributing to the anti-thrombotic effect of A2 agonist treatment are species-dependent: adenosine A2 receptor stimulation inhibits platelet activation in human, but not canine, models.

Introduction: We have previously demonstrated platelet hyperreactivity in cystic fibrosis (CF) patients. Carriers of one CF mutation (heterozygotes) have been shown to have abnormalities related to the presence of only one-half the normal amount of CF transmembrane conductance regulator protein. Platelet hyperreactivity in CF heterozygotes would be an important cardiovascular risk factor, since approximately 1 in 25 Caucasians is a CF carrier. Materials and methods: We used highly sensitive assays of platelet activation to assess the difference between 16 CF heterozygotes and 16 age- and sex-matched healthy controls without CF mutations. Results: We found no difference in platelet activation between CF heterozygotes and controls. Conclusions: The 50% reduction in the CF transmembrane conductance regulator protein in heterozygotes is insufficient to cause platelet activation.

Summary. Background: Clopidogrel is a widely used antithrombotic agent that inhibits the platelet P2Y12 adenosine diphosphate (ADP) receptor. There is increasing interest in ‘clopidogrel resistance’. Objectives: To determine whether ‘clopidogrel resistance’ is accounted for by a pre-existent variability in platelet response to ADP. Methods: Platelet response to 20 μm ADP was analyzed by four independent whole blood flow cytometric assays: platelet surface activated GPIIb-IIIa, platelet surface P-selectin, monocyte-platelet aggregates and neutrophil-platelet aggregates. In 25 consecutive, non-aspirin-treated healthy subjects, we studied platelet response before and after clopidogrel administration. In addition, we studied the platelet response in 613 consecutive aspirinated patients with or without coronary artery disease (CAD, as determined by angiography) who had or had not been treated with clopidogrel. In these patients, we tested for homogeneity of variance across all durations of clopidogrel exposure and severity of CAD by estimating the ‘goodness of fit’ of two independent models. Results: In the healthy subjects, pre-clopidogrel response to ADP predicted post-clopidogrel response to ADP. In the patients, clopidogrel, as expected, inhibited the platelet response to ADP. However, irrespective of the duration of clopidogrel administration, the severity of CAD, and the dose of aspirin, clopidogrel did not increase the variance in the platelet response to ADP in any of the four assays of platelet response. Conclusions: These studies provide evidence that ‘clopidogrel resistance’ is accounted for by a pre-existent variability in platelet response to ADP and this variability is not increased by clopidogrel administration.

SummaryThe novel thienopyridine prodrug prasugrel, a platelet P2Y12 ADP receptor antagonist, requires in vivo metabolism for activity. Although pharmacological data have been collected on the effects of prasugrel on platelet aggregation,there are few data on the direct effects of the prasugrel’s active metabolite, R-138727, on other aspects of platelet function. Here we examined the effects of R-138727 on thrombo-inflammatory markers of platelet activation, and the possible modulatory effects of other blood cells, calcium, and aspirin. Blood (PPACK or citrate anticoagulated) from healthy donors pre- and post-aspirin was incubated with R-138727 and the response to ADP assessed in whole blood or platelet-rich plasma (PRP) by aggregometry and flow cytometric analysis of leukocyte-platelet aggregates,platelet surface P-selectin, and GPIIb-IIIa activation. Low-micromolar concentrations of R-138727 resulted in a rapid and consistent in-hibition of these ADP-stimulated thrombo-inflammatory markers.These rapid kinetics required physiological calcium levels, but were largely unaffected by aspirin. Lower IC50 values in whole blood relative to PRP suggested that other blood cells affect ADP-induced platelet activation and hence the net inhibition by R-138727. R-138727 did not inhibit P2Y12-mediated ADP-induced shape change, even at concentrations that completely inhibited platelet aggregation, confirming the specificity of R-138727 for P2Y12. In conclusion, R-138727, the active metabolite of prasugrel, results in rapid, potent, consistent, and selective inhibition of P2Y12-mediated up-regulation of thromboinflammatory markers of platelet activation.This inhibition is enhanced in the presence other blood cells and calcium,but not aspirin.

All cells are covered by glycans, an individually unique layer of oligo- and polysaccharides that are critical moderators of self-recognition and other cellular-level interactions (e.g. fertilization). The functional similarity between these processes suggests that gamete surface glycans may also have an important, but currently overlooked, role in sexual selection. Here we develop a user-friendly methodological approach designed to facilitate future tests of this possibility. Our proposed method is based on flow cytometric quantification of female-induced sperm acrosome reaction and sperm surface glycan modifications in the Mediterranean mussel Mytilus galloprovincialis. In this species, as with many other taxa, eggs release water-soluble factors that attract conspecific sperm (chemoattraction) and promote potentially measurable changes in sperm behavior and physiology. We demonstrate that flow cytometry is able to identify sperm from other seawater particles as well as accurately measure both acrosome reaction and structural modifications in sperm glycans. This methodological approach can increase our understanding of chemically-moderated gamete-level interactions and individual-specific gamete recognition in Mytilus sp. and other taxa with similar, easily identifiable acrosome structure. Our approach is also likely to be applicable to several other species, since carbohydrate-mediated cellular-level interactions between gametes are universal among externally and internally fertilizing species.

Major age-related diseases such as cardiovascular disease and cancer are the primary causes of morbidity and mortality in Australia and worldwide. In our recent study characterising differences in the plasma proteome between healthy children and adults, a large number of proteins differentially expressed with age were found to be of platelet origin. This study aimed to characterise differences in the resting platelet proteome and the platelet releasate of healthy children compared to healthy adults. This study represents the setup of a procedure for the proteomic analysis of platelets from children. Differentially expressed platelet proteins were identified using Two-dimensional Differential In-Gel Electrophoresis and mass spectrometry. Significant differences in the expression of nine proteins (1.1%) in the resting platelet proteome were observed in children compared to adults. Serotransferrin, fibrinogen alpha chain, glyceraldehyde-3 phosphate dehydrogenase, serum albumin, transgelin-2, calponin-2/LIM and SH3 domain protein 1 and human chorionic gonadotropin 2039797 were up - regulated , whereas thrombospondin-1 was down - regulated in children. Eleven proteins (1.5%) were differentially expressed in the platelet releasate of children compared to adults, where transferrin was also upregulated and TSP-1 was down regulated . Identified proteins are involved in processes including tissue and organ development, cell proliferation regulation and angiogenesis. Our results provide novel insights into platelet physiology as well as growth, development and ageing in healthy individuals. Biological significance The incidence of major diseases such as cardiovascular disease (CVD) and cancer increase with increasing age and are the major causes of morbidity and mortality both in Australia and worldwide. As the aged population continues to increase dramatically, so too will the financial strains associated with the long term care of the elderly population. Compared to adults, children have a significantly lower incidence of major diseases such as thromboembolic disease. This suggests that children have a protective mechanism against the development of disease. Therefore, studies focussing on the molecular changes of proteins, the machinery of the cell, between children and adults are the key to determining the underlying mechanisms responsible for the onset of major diseases. A well-defined example of how protein expression can change with age is that of the plasma proteome. Significant differences in the expression of numerous plasma proteins between healthy children and adults have been recently demonstrated. Interestingly, a large number of differentially expressed proteins were found to be of platelet origin. This finding forms the basis for the current study, presenting as strong evidence for the age-specific differences of the platelet proteome.

Summary. Background: Previous studies have shown that ischemic preconditioning (PC) not only limits infarct size, but also improves arterial patency in models of recurrent thrombosis. We hypothesize that this enhanced patency is presumably because of a PC-induced attenuation of platelet-mediated thrombosis. However, there is, at present, no direct evidence that PC acts on the platelets per se and favorably down-regulates platelet reactivity. Objectives: Our goal was to test the concept that PC ischemia attenuates molecular indices of platelet activation-aggregation. Methods: Anesthetized dogs were randomly assigned to receive 10 min of PC ischemia followed by 10 min of reperfusion or a time-matched control period. Spontaneous recurrent coronary thrombosis was then initiated in all dogs by injury + stenosis of the left anterior descending coronary artery. Coronary flow was monitored for 3 h poststenosis, and molecular indices of platelet activation-aggregation were quantified by whole blood flow cytometry. Results: Coronary patency was, as expected, better-maintained following injury + stenosis in the PC group vs. controls (53% ± 5%* vs. 23% ± 5% of baseline flow, respectively; *P

Background— Thrombotic events still occur in aspirin-treated patients with coronary artery disease. Methods and Results— To better understand aspirin “resistance,” serum thromboxane B 2 (TXB 2 ) and flow cytometric measures of arachidonic acid–induced platelet activation (before and after the ex vivo addition of aspirin and indomethacin) were analyzed in 700 consecutive aspirin-treated patients undergoing cardiac catheterization. In 680 of 682 evaluable patients, serum TXB 2 concentrations were reduced compared with nonaspirinated healthy donors. Twelve patients had serum TXB 2 that was lower than nonaspirinated healthy donors but >10 ng/mL. Arachidonic acid stimulated greater platelet activation in patients with high serum TXB 2 (>10 ng/mL) than in patients with low serum TXB 2 . Addition of ex vivo aspirin reduced arachidonic acid–induced platelet activation to similar levels regardless of serum TXB 2 concentrations, which suggests that patients with high residual serum TXB 2 concentrations were either noncompliant or underdosed with aspirin. Among the remaining 98% of patients, ex vivo administration of either aspirin or indomethacin failed to prevent platelet activation across all degrees of arachidonic acid–induced platelet activation and aspirin doses. Although the patients were not randomized with respect to clopidogrel treatment, multivariate analysis showed that arachidonic acid–induced platelet activation was less in patients receiving clopidogrel. Conclusions— There is a residual arachidonic acid–induced platelet activation in aspirin-treated patients that (1) is caused by underdosing and/or noncompliance in only &2% of patients and (2) in the remaining patients, occurs via a cyclooxygenase-1 and cyclooxygenase-2 independent pathway, in direct proportion to the degree of baseline platelet activation, and is mediated in part by adenosine diphosphate–induced platelet activation.

Aspirin, the most widely used antiplatelet agent, irreversibly acetylates the enzyme cyclooxygenase 1 (COX-1), thereby inhibiting platelet thromboxane synthesis and subsequent platelet aggregation. Although aspirin has been demonstrated to reduce the odds of serious atherothrombotic events and death in high-risk patients by 25%, subsets of patients fail to respond to therapy and continue to suffer atherothrombotic events. This aspirin treatment failure may be due to sub-optimal bioavailability (e.g. because of non-compliance or under-dosing) or may be a consequence of the as yet poorly understood phenomenon of aspirin resistance. In this review, we summarize the current laboratory methods used to identify aspirin-resistant patients, outline the cellular mechanisms that may contribute to aspirin resistance, and discuss the clinical implications of this important issue.

Summary. Background: Monocytes and neutrophils form heterotypic aggregates with platelets initially via engagement of platelet surface P-selectin with leukocyte surface P-selectin glycoprotein ligand-1 (PSGL-1). The resultant intracellular signaling causes the leukocyte surface expression of tissue factor and activation of leukocyte surface Mac-1 (integrin αMβ2, CD11b/CD18). The activation-dependent conformational change in monocyte surface Mac-1 results in the binding of coagulation factor Xa (FXa) and/or fibrinogen to Mac-1. The aim of this study was to develop whole blood flow cytometry assays of these procoagulant activities and to investigate the effects of platelet binding to monocytes and neutrophils. Methods: Citrate or D-Phe-Pro-Arg-chloromethylketone (PPACK) anticoagulated whole blood was incubated with monoclonal antibodies against CD14 (PECy5), CD42a (PE), FITC-conjugated test antibody and an agonist, and then fixed with FACS lyse. Appropriate isotype negative controls were prepared in parallel. A BD FACSCalibur was used to analyze monocytes and neutrophils, which were identified based on CD14 fluorescence, forward and 90° light scatter. These populations were further gated into CD42a-positive (platelet-bound) and CD42a-negative (platelet-free). Geometric mean fluorescence and per cent positive data were collected for each subpopulation to measure the binding of test antibodies directed at CD42a, tissue factor, coagulation FXa, bound fibrinogen, activated Mac-1, and CD11b. Compensation controls were prepared on six normal donors prior to the study and these settings were used throughout the 10 donor study. Negative controls verified the lack of cross talk, particularly in the quantified FITC and PE parameters. Results: The physiologic agonists collagen and ADP increased monocyte-platelet and neutrophil-platelet aggregates and increased leukocyte surface Mac-1/CD11b and surface-bound tissue factor, FXa and fibrinogen. Whereas the increases in Mac-1/CD11b were mainly independent of leukocyte-platelet binding, the increases in surface-bound tissue factor, FXa and fibrinogen were mainly dependent on leukocyte-platelet binding. Conclusions: (i) We have developed novel whole blood flow cytometry assays to measure bound tissue factor, coagulation FXa, fibrinogen, activated Mac-1 and CD11b on the surface of monocytes and neutrophils, allowing independent analysis of monocytes and neutrophils with and without surface-adherent platelets. (ii) The monocyte and neutrophil surface binding of tissue factor, FXa and fibrinogen is mainly dependent on platelet adherence to monocytes and neutrophils, whereas the monocyte and neutrophil surface expression of CD11b and activated Mac-1 is mainly independent of platelet adherence to monocytes and neutrophils.

Objectives The purpose of this study was to examine the effects of glycoprotein (GP) IIb/IIIa antagonists (abciximab, eptifibatide, and tirofiban) and other inhibitors on translocation of CD40L from intraplatelet stores to the platelet surface and on the release of soluble CD40L (sCD40L) from platelets. Background CD40L is a proinflammatory and prothrombotic ligand in the tumor necrosis factor family. Methods Platelet surface CD40L was measured by flow cytometry, and sCD40L was measured by enzyme-linked immunosorbent assay. Results Translocation of CD40L from intraplatelet stores to the platelet surface was not inhibited by GP IIb/IIIa antagonists. However, release of sCD40L from the surface of activated platelets was inhibited by GP IIb/IIIa antagonists in a dose-dependent manner, in concert with inhibition of PAC1 binding to platelets (a surrogate marker for fibrinogen binding). Release of sCD40L from activated platelets was also markedly reduced in Glanzmann platelets (deficient in GP IIb/IIIa). Ethylenediaminetetraacetic acid was an effective inhibitor of sCD40L release, but only when added before platelet activation. Both cytochalasin D (an inhibitor of actin polymerization) and GM6001 (an inhibitor of matrix metalloproteinases [MMPs]) inhibited the release of sCD40L from platelets when added before, as well as 3 min after, platelet activation. However, neither cytochalasin D nor GM6001 affected translocation of CD40L to the platelet surface. Conclusions The GP IIb/IIIa antagonists inhibit release of sCD40L from activated platelets. Release of sCD40L from platelets is regulated, at least in part, by GP IIb/IIIa, actin polymerization, and an MMP inhibitor-sensitive pathway. In addition to their well-characterized inhibition of platelet aggregation, GP IIb/IIIa antagonists may obviate the proinflammatory and prothrombotic effects of sCD40L.

Cardiac surgery involving cardiopulmonary bypass is a common yet complex procedure that results in considerable disruption of hemostasis during and following surgery. Despite the relatively common and widespread use of this procedure, there remains a significant peri-operative risk of both thrombosis and hemorrhage in some patients. This is known as the hemostatic defect of cardiopulmonary bypass. Strategies including the use of pharmacological agents, hemodilution, autologous blood transfusion, rapid in-theatre monitoring of hemostatic potential with fine-tuning of the degree of heparinization, minimally invasive surgery and the use of biologically coated cardiopulmonary bypass equipment have been employed to ameliorate the effects of cardiopulmonary bypass on hemostasis. However there exists a fine line between preventing hemorrhage and promoting thrombosis. Likewise attempts to prevent thrombosis may result in increased hemorrhage. Research into many strategies for minimizing the hemostatic defect of cardiopulmonary bypass is incomplete, with safety and efficacy the subjects of intensive investigation.