Aggregometry In Platelet-rich Plasma Research Articles

Brain-derived gangliosides prime human platelet aggregation and induce platelet-leukocyte aggregate formation

BackgroundPlatelet activation and interaction with leukocytes are crucial in inflammation. Gangliosides, sialic acid–containing glycosphingolipids, have been linked to different inflammatory conditions related to cardio- and neurodegenerative disorders. The role of gangliosides in platelet and leukocyte function, although reported, still needs further investigation. ObjectivesWe aimed to study the role of gangliosides in platelet activation and platelet-leukocyte interaction in vitro. MethodsPlatelet activation was studied through aggregometry in platelet-rich plasma from apparently healthy human volunteers. Signaling protein phosphorylation was analyzed by immunoblotting. Platelet P-selectin expression and platelet-leukocyte aggregate formation were measured by flow cytometry. ResultsThe gangliosides monosialoganglioside GM1, disialoganglioside GD1a, and trisialoganglioside GT1b did not induce by themselves any platelet aggregation. Conversely, when preincubated with platelets, they potentiate platelet aggregation induced by submaximal adenosine diphosphate and collagen concentrations and increased P-selectin expression. Incubation of platelets with free sialic acid and the soluble part of monosialoganglioside GM1 induced a similar potentiating effect on platelet aggregation but not on platelet P-selectin expression. Consistently, analyzing the signaling protein phosphorylation, only the entire gangliosides activated extracellular stimuli–responsive kinase 1/2 suggesting that a complete ganglioside is crucial for its action on platelets. Both the priming effect on platelet aggregation and ERK1/2 activation were prevented by aspirin. Moreover, incubation of citrated whole blood with gangliosides induced platelet-leukocyte aggregate formation accompanied by increased expression of granulocyte and monocyte CD11b compared with untreated blood, suggesting a primary leukocyte activation. ConclusionGangliosides may act in vitro both on platelet and leukocyte activation and on their interaction. The observed effects might contribute to inflammatory processes in clinical conditions.

P1656: EVALUATION OF PLATELET FUNCTION IN RARE OR UNDIAGNOSED BLEEDING DISORDERS

Background: Qualitative and/or quantitative platelet defects promote bleeding and its identification requires a careful clinical evaluation and a rational use of diagnostic laboratory assays. Aims: To study platelet function by different methods in patients with hemorrhagic symptoms who are suspected of thrombopathy to compare results obtained with each system and validate usefulness of methods employed. Methods: We included 72 patients with bleeding suspected of thrombopathy, which in some cases was confirmed by genetic study (Table 1). Patients with coagulation disorders were excluded. We used the following methods to test platelet function: Light aggregometry in platelet-rich plasma (PRP) using ADP, collagen, epinephrine and arachidonic acid as stimulating agonists. Flow cytometry in PRP using TRAP and ADP to induce platelet activation, and PAC1 monoclonal antibody (mAb) to recognize activated fibrinogen receptor, and anti P- selectin and anti-CD63 mAbs to test, respectively, release from alpha and dense granules. PFA-100 ® in whole citrated blood with collagen +ADP and collagen+Epinephrine cartridges. Total Thrombus-formation Analysis System (T-TAS ® 01, Zarcos, Japan) with T-TAS ® PL-chip (capillaries covered with collagen / under flow) in blood samples drawn in BAPA, a potent synthetic anticoagulant which inhibits Factor Xa and thrombin. Results: Some bleeding due to platelet dysfunction was diagnosed by any of the methods used, but others can only be detected with some of the techniques tested (Table 2). This difference in the sensitivity of the methods may be due to the different experimental conditions used in each of them: type of sample used, platelets’ activators, and flow vs static conditions. Forty % of undiagnosed bleedings did not seem to be related to platelet dysfunction. Image:Summary/Conclusion: All methods are complementary and their sensitivity depends on the pathway evaluated by the technique used and the mechanism involved in platelet dysfunction.

Reversible platelet aggregation in the presence of calcium ions: mechanisms and potential value

Disorders in the functions of platelets – blood cells responsible for the blood clots formation and prevention – are observed as independent diseases, as a complication of cancer and hematological diseases or as a result of a therapy. Nowadays, a test of platelet aggregation by aggregometry is the only diagnostic method for assessing the platelets functions. There are several varieties of aggregometry, which differ both in the method of recording the formation of platelet aggregates and in the method of preparing platelets for the experiment. In most laboratories, it is customary to conduct aggregometry in platelet-rich plasma in the presence of citrate ions. In this case, the concentration of calcium ions in plasma decreases, it prevents the thrombin formation and the plasma coagulation. On the other hand, it has long been known that platelet aggregation in response to ADP in the presence of calcium ions (in blood plasma collected in heparin or hirudin tubes, also blocking plasma clotting) is reversible: after 1-5 minutes after the addition of the activator, the disaggregation begins until the light transmission of the solution (platelet concentration) returns to its original level. This phenomenon is called "reversible” platelet aggregation. Reversible aggregation (“disaggregation”) is sometimes observed in aggregometry of citrate plasma, especially in pediatric patients. However, it is usually not considered normal and is considered a sign of platelet dysfunction. This review considers the known mechanisms of disaggregation in the presence or absence of calcium ions in the medium. The role of secondary activation of platelets as a potential cause of irreversible aggregation is discussed, as well as possible versions for explaining the results of aggregometry, when reversible platelet aggregation is observed.

Evaluation of platelet function in essential thrombocythemia under different analytical conditions

Background. Studies of platelet aggregation (PA) in essential thrombocythemia (ET) reported contrasting results, likely due to differences in analytical conditions.Objective. We investigated platelet aggregation using different techniques and analytical conditions.Patients and Methods. PA was studied by light-transmission aggregometry (LTA) in platelet-rich plasma (PRP) and impedance aggregometry in PRP and whole blood (WB). ADP, collagen, thrombin receptor activating peptide (TRAP-14) and adrenaline were used as agonists. Since ET patients (n = 41) were on treatment with aspirin (100 mg/d), healthy controls (n = 29) were given aspirin (100 mg/d) for 5 days before testing: therefore, thromboxane A2-independent PA was tested in all subjects. Blood samples were collected in citrate (C) [low Ca2+] or lepirudin (L) [physiological Ca2+]; platelet count was adjusted to 250 x 109/L in a set of C-PRP (adjusted C-PRP) and left unmodified in the other samples.Results. Results of PA in 17 ET patients who were poor responders to aspirin (high serum thromboxane B2 levels) were not included in the analysis. With LTA, PA in ET was lower than in controls in adjusted C-PRP and normal in native C-PRP and L-PRP. With impedance aggregometry, PA in L-PRP and L-WB tended to be higher in ET than in controls. Platelet serotonin and ADP contents were reduced in ET. The percentages of circulating platelets expressing P-selectin and platelet-leukocyte hetero-aggregates were higher in ET.Conclusions. Analytical conditions dramatically affect in vitro PA of ET patients, which appears defective under the least physiological conditions and normal/supranormal under conditions that are closer to the physiological.

Exome-chip meta-analysis identifies association between variation in ANKRD26 and platelet aggregation

ABSTRACTPrevious genome-wide association studies (GWAS) have identified several variants associated with platelet function phenotypes; however, the proportion of variance explained by the identified variants is mostly small. Rare coding variants, particularly those with high potential for impact on protein structure/function, may have substantial impact on phenotype but are difficult to detect by GWAS. The main purpose of this study was to identify low frequency or rare variants associated with platelet function using genotype data from the Illumina HumanExome Bead Chip. Three family-based cohorts of European ancestry, including ~4,000 total subjects, comprised the discovery cohort and two independent cohorts, one of European and one of African American ancestry, were used for replication. Optical aggregometry in platelet-rich plasma was performed in all the discovery cohorts in response to adenosine diphosphate (ADP), epinephrine, and collagen. Meta-analyses were performed using both gene-based and single nucleotide variant association methods. The gene-based meta-analysis identified a significant association (P = 7.13 × 10–7) between rare genetic variants in ANKRD26 and ADP-induced platelet aggregation. One of the ANKRD26 SNVs - rs191015656, encoding a threonine to isoleucine substitution predicted to alter protein structure/function, was replicated in Europeans. Aggregation increases of ~20–50% were observed in heterozygotes in all cohorts. Novel genetic signals in ABCG1 and HCP5 were also associated with platelet aggregation to ADP in meta-analyses, although only results for HCP5 could be replicated. The SNV in HCP5 intersects epigenetic signatures in CD41+ megakaryocytes suggesting a new functional role in platelet biology for HCP5. This is the first study to use gene-based association methods from SNV array genotypes to identify rare variants related to platelet function. The molecular mechanisms and pathophysiological relevance for the identified genetic associations requires further study.

Comparing light transmission aggregometry and PFA100 for monitoring antiplatelet therapy in patients with coronary heart disease

Abstract Objectives Many patients undergoing antiplatelet therapy continue to experience thrombotic events and failure of therapy leading to so-called ‘resistance’ to antiplatelet therapy. Recently, there has been an increasing focus on in vitro laboratory monitoring of platelet functions, which has the promise of identifying these patients. This study aimed to document the prevalence of laboratory evidence of resistance to aspirin and clopidogrel therapy in Saudi patients with stable coronary heart disease (CHD). Methods Light transmission aggregometry in platelet-rich plasma was performed in response to adenosine diphosphate (ADP), arachidonic acid (AA), collagen and adrenaline. In addition, a platelet function analyser (PFA100) was employed using both collagen/ADP and collagen/epinephrine cartridges. Results Light transmission aggregometry (LTA) identified the resistance to aspirin and clopidogrel therapy, according to the persistence of the aggregation response to AA aggregation, to be 13% and 26%, respectively. By PFA100 testing, closure times within the limits of laboratory reference values indicated residual platelet reactivity, and the prevalence of resistance to anti-platelet therapy was reported to be 33% for collagen/ADP cartridges and 30.7% for collagen/epinephrine. A concordance between LTA and PFA100 CT was noted in only 22.6% of patients. Conclusion This study showed a wide prevalence of on-treatment platelet reactivity in patients with CHD on dual platelet therapy ( aspirin and clopidogrel ). In addition, the global whole blood test of platelet function by PFA100 estimated a much higher prevalence of resistance than LTA when compared to the gold standard and more specific LTA analysis.

C3435T polymorphism of the ABCB1 gene is associated with poor clopidogrel responsiveness in a Mexican population undergoing percutaneous coronary intervention

BackgroundClopidogrel is a pro-drug and its intestinal absorption is limited by the P-glycoprotein encoded by the ABCB1 gene. It is metabolized hepatically by cytochrome P450 enzymes encoded by CYP genes to produce an active metabolite that antagonizes the P2Y12 platelet receptor. Some patients exhibit poor clopidogrel responsiveness due to polymorphisms, resulting in thrombotic events. The aim of this study was to determine the relationship between poor clopidogrel responsiveness and the ABCB1, CYP and P2RY12 gene polymorphisms among patients undergoing percutaneous coronary intervention (PCI). Methods and resultsTwo hundred seventy-six patients who underwent PCI were included in this study. Clopidogrel responsiveness was determined via optical aggregometry in platelet-rich plasma using 10μM ADP. Patients exhibiting a platelet aggregation response higher than 70% were classified as poor responders. The genetic polymorphisms were analyzed via real-time PCR.Poor responsiveness to clopidogrel was noted in 22.1% of the patients. The TT genotype of the C3435T polymorphism of the ABCB1 gene and omeprazole usage were each associated with poor clopidogrel responsiveness (Exp (β) 2.73, p=0.009 and Exp (β) 3.86, p=0.04, respectively). ConclusionPoor clopidogrel responsiveness is associated with the TT genotype of the C3435T polymorphism of the ABCB1 gene.

Platelet factor XIIIa release during platelet aggregation and plasma clot strength measured by thrombelastography in patients with coronary artery disease treated with clopidogrel

It has been estimated that up to half of circulating factor XIIIa (FXIIIa) is stored in platelets. The release of FXIIIa from platelets upon stimulation with adenosine diphosphate (ADP) in patients with coronary artery disease treated with dual antiplatelet therapy has not been previously examined. Samples from 96 patients with established coronary artery disease treated with aspirin and clopidogrel were examined. Platelet aggregation was performed by light transmittance aggregometry in platelet-rich plasma (PRP), with platelet-poor plasma (PPP) as reference, and ADP 5 µM as agonist. Kaolin-activated thrombelastography (TEG) was performed in citrate PPP. PRP after aggregation was centrifuged and plasma supernatant (PSN) collected. FXIIIa was measured in PPP and PSN. Platelet aggregation after stimulation with ADP 5 µM resulted in 24% additional FXIIIa release in PSN as compared to PPP (99.3 ± 27 vs. 80.3 ± 24%, p < 0.0001). FXIIIa concentration in PSN correlated with maximal plasma clot strength (TEG-G) (r = 0.48, p < 0.0001), but not in PPP (r = 0.15, p = 0.14). Increasing quartiles of platelet-derived FXIIIa were associated with incrementally higher TEG-G (p = 0.012). FXIIIa release was similar between clopidogrel responders and non-responders (p = 0.18). In summary, platelets treated with aspirin and clopidogrel release a significant amount of FXIIIa upon aggregation by ADP. Platelet-derived FXIIIa may contribute to differences in plasma TEG-G, and thus, in part, provide a mechanistic explanation for high clot strength observed as a consequence of platelet activation. Variability in clopidogrel response does not significantly influence FXIIIa release from platelets.

Evaluation of resistance to anti-platelet therapy in patients with coronary heart disease in a developing country.

Resistance to anti-platelet therapy whether defined by the laboratory detection of platelet function or the recurrence of cardiovascular events, has received extensive coverage in the literature coming predominantly from developed countries and very scanty information comes from developing countries. We aim to document the prevalence of aspirin and clopidogrel resistance in Saudi patients with coronary heart disease (CHD) and to probe the possible responsible mechanism[s]. 238 patients with CHD were enrolled from the outpatient clinic and wards of King Khalid University Hospital, Riyadh. Platelet function testing was undertaken using both optical aggregometry in platelet rich plasma as well as the Platelet Function Analyzer [PFA100] which uses whole blood. Agonist-induced platelet aggregation in response to arachidonic acid-induction: The prevalence of residual activity was detected in 12.6% patients. Resistance to clopidogrel as reflected by the residual aggregation responses to ADP was detected in 25.7%. PFA100 closure time: Closure times for collagen and epinephrine cartridges that are shorter than the maximum for local reference of 84 - 198 seconds were detected in around 30% of patients on therapy. There was no significant relationship between diabetes mellitus, smoking, dyslipidemia, or consumption of non-steroidal anti-inflammatory drugs and the resistance to anti-platelet therapy. Resistance to anti-platelet therapy is quite prevalent among patients with CHD in Saudi Arabia and compares favorably with reports in the literature. However, the lack of a significant relationship between resistance to antiplatelet therapy and the above-mentioned conditions could be due to small numbers.

Simple and accessible screening method for congenital thrombopathies using an impedance haematology counter – reply: The differences between impedance aggregometry in whole blood versus aggregometry in PRP. Is there a need for caution?

Abstract Brahimi et al. in this journal formed a hypothesis that “the platelet count is underestimated, by an automated cell counter, each time platelet aggregates are present in the sample tube.” The addition of a platelet agonist to a stimulated sample tube will lead to the formation of platelet aggregates and hence to a drop in the platelet count. In the case of a hereditary platelet dysfunction, platelet aggregates cannot be formed upon addition of a platelet agonist and the platelet count will remain unchanged. The authors propose a hypothesis to develop “a more accessible screening technique for these hereditary platelet dysfunctions.” In our reply, we critically evaluate this screening method and focus on the importance of the differences between impedance aggregometry in whole blood versus aggregometry in platelet-rich plasma.

EV-077 in vitro inhibits platelet aggregation in type-2 diabetics on aspirin

IntroductionThis study aimed to characterize the in vitro effect of EV-077, a compound that antagonises the binding of prostanoids and isoprostanes to the thromboxane receptor (TP) and inhibits the thromboxane synthase (TS), on platelet aggregation of patients with type-2 diabetes and coronary artery disease (CAD) on chronic aspirin treatment. The effect of EV-077 on 8-iso-PGE2-mediated TP receptor contraction of human arteries was also investigated. Materials and MethodsFifty-two type-2 diabetics with CAD on chronic aspirin (100mg) treatment were studied. Arachidonic acid-induced platelet aggregation was measured by impedance aggregometry in platelet-rich plasma (PRP) and whole blood anticoagulated with hirudin, and by light transmission aggregometry in citrate-anticoagulated PRP following 10-min in vitro exposure to EV-077 (100nmol/l) or control. The effect of EV-077 was measured on isometric contraction of 24 human umbilical arteries induced by isoprostane 8-iso-PGE2. ResultsArachidonic acid (1mmol/l) induced substantial aggregation in hirudin-anticoagulated whole blood (63±4AU), which was significantly reduced by in vitro exposure to EV-077 (38±3AU, P<0.001). Virtually no arachidonic acid-induced aggregation in citrate-anticoagulated or hirudin-anticoagulated PRP was observed. EV-077 potently, competitively and reversibly inhibited TP mediated contraction of umbilical arteries by 8-iso-PGE2 (P<0.01). ConclusionsAspirin did not completely inhibit arachidonic acid-induced platelet aggregation in whole blood from type-2 diabetics with CAD. This aggregation is likely induced by prostanoids and/or isoprostanes produced by leukocytes, because it was significantly reduced by EV-077. The TP receptor-mediated contraction of human arteries induced by isoprostane 8-iso-PGE2 was effectively inhibited by EV-077.

Serotonin antagonism improves platelet inhibition in clopidogrel low-responders after coronary stent placement: an in vitro pilot study.

Increased residual platelet reactivity remains a burden for coronary artery disease (CAD) patients who received a coronary stent and do not respond sufficiently to treatment with acetylsalicylic acid and clopidogrel. We hypothesized that serotonin antagonism reduces high on-treatment platelet reactivity. Whole blood impedance aggregometry was performed with arachidonic acid (AA, 0.5 mM) and adenosine diphosphate (ADP, 6.5 µM) in addition to different concentrations of serotonin (1–100 µM) in whole blood from 42 CAD patients after coronary stent placement and 10 healthy subjects. Serotonin increased aggregation dose-dependently in CAD patients who responded to clopidogrel treatment: After activation with ADP, aggregation increased from 33.7±1.3% to 40.9±2.0% in the presence of 50 µM serotonin (p<0.05) and to 48.2±2.0% with 100 µM serotonin (p<0.001). The platelet serotonin receptor antagonist ketanserin decreased ADP-induced aggregation significantly in clopidogrel low-responders (from 59.9±3.1% to 37.4±3.5, p<0.01), but not in clopidogrel responders. These results were confirmed with light transmission aggregometry in platelet-rich plasma in a subset of patients. Serotonin hence increased residual platelet reactivity in patients who respond to clopidogrel after coronary stent placement. In clopidogrel low-responders, serotonin receptor antagonism improved platelet inhibition, almost reaching responder levels. This may justify further investigation of triple antiplatelet therapy with anti-serotonergic agents.

Twice daily dosing of aspirin improves platelet inhibition in whole blood in patients with type 2 diabetes mellitus and micro- or macrovascular complications

The efficacy of low-dose aspirin in type 2 diabetes mellitus (T2DM) has been questioned. We tested if twice daily dosing of aspirin would be more effective in T2DM, possibly due to increased platelet turnover. A randomised cross-over study compared 75 mg aspirin OD, 75 mg BID and 320 mg OD (≥ 2 week treatment periods) in 25 patients with T2DM and micro- or macrovascular complications. Platelet responses were examined by impedance aggregometry (WBA) and the IMPACT-R aspirin test in whole blood, light transmittance aggregometry in platelet-rich plasma (LTA), and urinary 11-dehydro-thromboxane B2 (TxM). Aspirin 75 mg BID decreased arachidonic acid (AA)-induced WBA compared to 75 mg OD (9.7 ± 4.5 vs. 12.6 ± 3.5 ohm; p = 0.003) or to 320 mg OD (11.5 ± 4.2 Ohms; p = 0.049). WBA responses to collagen were similarly attenuated by BID or high dosing (by 12-14%; p = 0.02 for both). The IMPACT-R showed a better response to 75 mg BID compared to 75 mg OD (p = 0.049), but not to 320 mg OD. AA-induced aggregation by LTA was <6.5% on all occasions, with no differences between aspirin dosages. TxM was reduced after 320 mg OD (p = 0.002), but not 75 mg BID (p = 0.07). Reticulated platelets were highly correlated with mean platelet volume (MPV; r2 = 0.74, p<0.0001). Both markers for platelet turnover were correlated with AA-induced WBA, but neither identified patients who benefited from BID dosing dependably. In conclusion, twice daily dosing improved laboratory responses to aspirin in high risk T2DM patients. Studies of whether BID dosing of aspirin can improve clinical outcomes in such patients are of interest.

CAMP/CREB-mediated Transcriptional Regulation of Ectonucleoside Triphosphate Diphosphohydrolase 1 (CD39) Expression

CD39 is a transmembrane enzyme that inhibits platelet reactivity and inflammation by phosphohydrolyzing ATP and ADP to AMP. Cyclic AMP (cAMP), an essential second messenger, is particularly important in regulating genes controlling vascular homeostasis. These experiments test the hypothesis that cAMP might positively regulate the expression of CD39 and thereby modulate important vascular homeostatic properties. Cd39 mRNA was induced by 13.8- fold in RAW cells treated with a membrane-permeant cAMP analogue (8-bromo-cyclic AMP; 8-Br-cAMP), stimulation of adenylate cyclase, or prostanoids known to drive cAMP response. Fluorescence-activated cell sorting, immunofluorescence, and TLC assays demonstrated that both CD39 protein expression and enzymatic activity were increased in cells treated with 8-Br-cAMP but not in cells transfected with short hairpin RNA against CD39. This analogue drove a significant increase in transcriptional activity at the Cd39 promoter although not when the promoter's cAMP-response element sites were mutated. Pretreatment with cAMP-dependent protein kinase (PKA), phosphoinositide 3-kinase (PI3K), or ERK inhibitors nearly obliterated the cAMP-driven increase in Cd39 mRNA, protein expression, and promoter activity. 8-Br-cAMP greatly increased the phosphorylation of CREB1 (Ser(133)) and ATF2 (Thr(71)) in a PKA-, PI3K-, and ERK-dependent fashion. Chromatin immunoprecipitation assays demonstrated that binding of phosphorylated CREB1 and ATF2 to cAMP-response element-like sites was significantly increased with 8-Br-cAMP treatment and that binding was reduced with PKA, PI3K, and ERK inhibition, whereas transfection of Creb1 and Atf2 overexpression constructs enhanced cAMP-driven Cd39 mRNA expression. Transfection of RAW cells with mutated Creb1 (S133A) reduced cAMP-driven Cd39 mRNA expression. Furthermore, the cAMP-mediated induction of Cd39 mRNA, protein, and phosphohydrolytic activity was replicated in primary peritoneal macrophages. These data identify cAMP as a crucial regulator of macrophage CD39 expression and demonstrate that cAMP acts through the PKA/CREB, PKA/PI3K/ATF2, and PKA/ERK/ATF2 pathways to control a key vascular homeostatic mediator.

Detrimental Effects of Energy Drink Consumption on Platelet and Endothelial Function

Background Energy drink consumption has been anecdotally linked with sudden cardiac death and, more recently, myocardial infarction. As myocardial infarction is strongly associated with both platelet and endothelial dysfunction, we tested the hypothesis that energy drink consumption alters platelet and endothelial function. Methods Fifty healthy volunteers (34 male, aged 22 ± 2 years) participated in the study. Platelet aggregation and endothelial function were tested before, and 1 hour after, the consumption of 250 mL (1 can) of a sugar-free energy drink. Platelet function was assessed by adenosine diphosphate-induced (1 μmol/L) optical aggregometry in platelet-rich plasma. Endothelial function was assessed via changes in peripheral arterial tonometry and expressed as the reactive hyperemia index (RHI). Results Compared with baseline values, there was a significant increase in platelet aggregation following energy drink consumption, while no change was observed with control (13.7 ± 3.7% vs 0.3 ± 0.8% aggregation, respectively, P <.01). Similarly, RHI decreased following energy drink consumption (−0.33 ± 0.13 vs 0.07 ± 0.12 RHI [control], P <.05). Mean arterial pressure significantly increased following energy drink consumption, compared with control ( P <.05). Heart rate was unaffected by energy drink consumption. Conclusion Energy drink consumption acutely increases platelet aggregation and decreases endothelial function in healthy young adults.

Fortschritte in der Thrombozytenfunktionsdiagnostik

Both for diagnosis of congenital and acquired platelet dysfunction as well as for therapy monitoring after application of platelet function inhibitors various methods have been established for evaluation of platelet function. In contrast to the gold standard of platelet function testing, the light transmission aggregometry in platelet rich plasma the Point-of-care (POC) analyzers allow fast analysis of platelet function without extensive laboratory work up. The conditions of the pre-analytical phase, however, are still of enormous importance in the prevention of medical errors. There is increasing clinical data in monitoring the effect of platelet aggregation inhibitors, showing that quantitative determination of the platelet function degree correlates with risk of increased bleeding or stent thrombosis. However, it is still unclear, which is the optimal test system, to predict the clinical outcome of these patients.

Whole blood aggregometry for evaluation of the antiplatelet effects of clopidogrel

Introduction The marked interindividual variability in platelet inhibition even after administration of high loading doses of clopidogrel raised the question whether monitoring of antiplatelet effects in patients undergoing percutaneous coronary intervention (PCI) can improve clinical outcome. Established methods for monitoring antiplatelet drug activity such as optical aggregometry and determination of surface protein expression are not suitable for routine bedside testing. Material and methods We therefore compared the applicability of whole blood impedance aggregometry (20 μmol/L ADP) and the whole blood bedside ULTEGRA assay with ADP-cartridges (20 μmol/L) with optical aggregometry in platelet-rich plasma and determination of surface protein expression (P-Selectin and activated GPIIb/IIIa) by flow cytometry. We analyzed samples obtained from 27 patients scheduled for elective PCI who received a loading dose of 600 mg of clopidogrel. Blood samples were withdrawn before clopidogrel, before PCI and 24 h thereafter. Results Platelet aggregation assessed by optical aggregometry (20 μmol/L ADP) declined from 65 ± 9% (baseline) to 42 ± 12% (PCI) and 45 ± 13% (24 h; p < 0.01). Expression of surface proteins displayed a similar time course. Platelet aggregation determined by impedance aggregometry decreased from 4.6 ± 4.0 Ω (baseline) to 0.1 ± 0.3 Ω (PCI) and 0.5 ± 1.1 Ω (24 h) with no detectable residual platelet aggregation during PCI in 88% of patients. The ULTEGRA assay showed only slight changes after administration of clopidogrel. Correlation analysis between the various assays revealed significant correlations only between optical aggregometry and flow cytometry. Conclusions The results indicate that both of the whole blood assays cannot substitute for optical aggregometry or determination of surface proteins in the assessment of clopidogrel-induced platelet inhibition.

The Genotypic Combination of Homozygous Carriers of Both H1 Haplotype and 34C Shows a Significantly Lower Responsiveness to ADP-Induced Platelet Aggregation Than Others in Healthy Volunteers.

BACKGROUND: The adenosine diphosphate (ADP) receptor, P2Y12, plays a central role in platelet activation and its blocker clopidogrel reduces the incidence of cardiovascular events. Recently, the polymorphisms of the P2Y12 gene (H1 and H2 haplotype) was shown to be associated with increased ADP-induced platelet aggregation in healthy volunteers. In addition, the polymorphism (C34T) of the P2Y12 gene was reportedly associated with higher numbers of cerebrovascular events in patients with PAD receiving clopidgrel therapy. However, it is totally unknown if there is any association between polymorphisms combination and platelet reactivity. In this study, we aimed to investigate in detail about the association between ADP-induced platelet aggregation and the polymorphisms of P2Y12 gene, haplotype and C34T, in healthy volunteers.METHODS AND RESULTS: We determined P2Y12 genotype and platelet reactivity in 267 healty volunteers. Genotyping was performed using direct sequencing or a single nucleotide primer extension (SNuPE). In previous study, H1 and H2 haplotype were defined by 4 polymorphisms, C139T, T744C and insertion801A in intron1 and G52T in exon2, and there were in complete linkage disequilibrium. Result of genotyping of haplotype, however, G52T was found not to be in complete linkage disequilibrium. Allele frequencies of H1 and H2 were 0.83 and 0.17, respectively. For C34T, allele frequencies of 34C and 34T were 0.75 and 0.25, respectively. Platelet reactivity was evaluated by optical aggregometry in platelet-rich plasma. Aggregation was induced by 2 or 5 μmol/L ADP to evaluate maximal aggregation (%) and the area under the aggregate curve (AUC) for 6 min. Maximal aggregation induced by 2 μmol/L ADP was similar in homozygous carriers of H1 haplotype and carriers of H2 haplotype (p=0.262). AUC induced by 2 μmol/L ADP was lower in homozygous carriers of H1 haplotype than carriers of H2 haplotype (p=0.074). However, haplotype was not associated with platelet reactivity induced by 5 μmol/L ADP. For C34T, whereas, maximal aggregation and AUC induced by 2 or 5 μmol/L ADP was lower in homozygous carriers of 34C than carriers of 34T (p=0.009 and p=0.006 or p=0.002 and p=0.003, respectively). Furthermore, for the genotypic combination of haplotype and genotype (C34T), maximal aggregation and AUC induced by 2 or 5 μmol/L ADP was significantly lower in homozygous carriers of both H1 haplotype and 34C than others (p=0.003 and p<0.001 or p=0.05 and p=0.007, respectively). When platelet aggregation was induced by 1 mg/mL Collagen, the same trends were observed.CONCLUSIONS: We demonstrated that polymorphisms of the P2Y12 gene are associated with increased ADP-induced platelet aggregation, moreover, that group with genotypic combination of homozygous carriers of both H1 haplotype and 34C shows a significantly lower responsiveness to ADP-induced platelet aggregation than others in healthy volunteers. This suggests the possibility that polymorphisms of the P2Y12 gene are related to the receptor expression of P2Y12.

Recurrent Vascular Access Site Thrombosis in Patients on Hemodialysis - a Problem of Thrombophilia?.

Introduction: Vascular access site thrombosis in patients receiving hemodialysis is a major cause of hospital admission and recurrent surgery. The underlying pathologic cause is often stenosis of the venous vessel due to fibromuscular hyperplasia. But in the case of early failure occasional studies have investigated that hypercoagulability could play an important role in this context.Aim of the study: Is there a higher prevalence of hereditary and acquired thrombophilic risk factors in patients with vascular access thrombosis in comparison to patients without?Patients: In 2002 and 2003 we examined 52 consecutive patients (mean age 66,1 years) receiving hemodialyisis. 27 patients (pts) in group 1 had a history of vascular access site thrombosis and 25 pts in group 2 had not and an open vascular access for longer than at least six months. All pts in group 1 had a history of at least two occlusions of vascular access. 10/27 pts in group 1 with prosthetic grafts had a history of thrombosis of arteriovenous fistula before implantation of PFTE graft.Methods: In every patient hereditary and acquired thrombophilic risk factors were determined including antithrombin (AT), protein C (PC), protein S (PS), factor V-G1691A-mutation (FVM), prothrombin-G20210A-mutation (FIIM), homocysteine, lipoproteine (a) (Lpa), lupus anticoagulant (LA), cardiolipin antibodies IgG and IgM (ACA), fibrinogen and factor VIII. Platelet hyperreactivity was studied by light transmittance aggregometry in platelet rich plasma (Aggregometer PAP 4, moelab inc.). Aggregation was recorded as the maximum percentage change in light transmittance from baseline using platelet poor plasma as a reference. We defined sticky platelets as platelet aggregation > 30% after induction with different concentrations of ADP (10, 1 and 0,5 μmol) in platelet rich plasma.Results: We found in 14/27 pts with vascular access site thrombosis antiphospholipd antibodies (LA and/or ACA) in comparison to only 2/25 in pts without thrombosis. Activated platelets like the sticky platelets syndrome was shown in 11/27 pts in group 1 and 4/25 pts in group 2. In both groups hyperhomocysteinaemia (23/27 pts and 21/25 resp.), factor VIII elevation (21/27 pts and 22/25 resp.), fibrinogen elevation (22/27 pts and 21/25 resp.) and high levels of Lpa (7/27 pts and 6/25 resp.) were quite similar. There were no significant differences in the number of hereditary risk factors like AT, PC, PS, FVM and FIIM in both groups.Conclusions: In patients receiving hemodialysis we found a high prevalence of acquired thrombophlic risk factors like elevation of factor VIII, homocysteine and fibrinogen. There seems to be causal relation between vascular access site thrombosis and espacially antiphospholpid antibodies and activated platelets (sticky platelets syndrome). The evaluation of these thrombophilic risk factors in patients with recurrent vascular access site thrombosis could lead to an improved antithrombotic therapy.

Clopidogrel Resistance and Aspirin Resistance - a Cause of Coronary Artery Stent Thrombosis?.

Background: Occlusions of coronary artery stents are a seldom but often fatal complication after coronary artery intervention. With aggressive platelet inhibition therapy (combination of Aspirin (ASS) and clopidogrel) a reduction of stent thrombosis to less than 2% was achieved. The inhibition of platelet function has great interindividual variability and resistance against ASS and/or clopidogrel were previously described. Therefore we studied the relation between coronary artery stent thrombosis and insufficient inhibition of platelet function in patients after coronary artery intervention.Methods: Between 2000 and 2003 we examined 20 patients (pts) with coronary artery stent thrombosis within 4 weeks after coronary artery intervention. All patients received a clopidogrel loading dose of 300mg followed by 75mg/die and ASS 100mg/die. Platelet inhibition was studied by light transmittance aggregometry in platelet rich plasma (Aggregometer PAP 4, moelab inc.). Aggregation was recorded as the maximum percentage change in light transmittance from baseline using platelet poor plasma as a reference. We defined ASS resistance as platelet aggregation > 30% after induction with 0,5mg/ml arachidonic acid and clopidogrel resistance as platelet aggregation > 30% after induction with 20 μmol ADP. We compared these pts with 20 pts without stent thrombosis within 4 weeks after coronary artery intervention.Results: In the group with coronary artery stent thrombosis we found 13/20 patients with insufficient platelet inhibition. 2/20 pts in this group were resistent against both ASS and clopidogrel. In 2/20 pts we observed ASS resistance and in 9/20 pts clopidgrel resistance. In the group of patients without stent thrombosis we found only 1/20 pt with ASS resistance and 1/20 pt with clopidogrel resistance.Conclusions: Insufficient platelet inhibition could be one cause of early stent thrombosis after coronory artery intervention. Platelet function testing is able to detect insufficient platelet inhibition and early testing could be an instrument to prevent coronary artery stent thrombosis.