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Background Acute coronary syndrome is associated with platelet hyperactivity, which in its persistent form, promotes recurrent thrombotic events. Complex cardiac rehabilitation after acute coronary syndrome improves clinical outcome; however, its effect on platelet hyperactivity is unknown. Design and methods We enrolled 84 acute coronary syndrome patients on dual antiplatelet therapy, who underwent a new complex cardiac rehabilitation programme (NovaCord physiotherapy, lifestyle counselling, strict diet, stress management and regular coaching) and 51 control acute coronary syndrome patients with traditional cardiac rehabilitation. Platelet functionality was determined at enrolment and at three months follow-up by aggregometry, serum platelet-derived growth factor levels, total- and platelet-derived microvesicle counts (PMV; CD41a+/CD61+, CD62P+). Results Platelet aggregation parameters and platelet-derived growth factor levels were significantly decreased in the complex cardiac rehabilitation group at three months (1 µg/ml collagen, median (interquartile range): 22 (10-45) vs 14 (7.5-25.5)%, p = 0.0015; 2 µg/ml collagen: 36 (22-60) vs 26.5 (16-37)%, p = 0.0019; 1.25 µM adenosine-diphosphate: 4.5 (1-10) vs 1 (0-3)%, p = 0.0006; 5 µM adenosine-diphosphate: 27 (16-38) vs 22 (12-31)%, p = 0.0078; epinephrine: 33 (15-57) vs 27 (12-43)%, p = 0.01; platelet-derived growth factor: 434.6 (256.0-622.7) vs 224.8 (148.5-374.1) pg/ml, p = 0.0001). In contrast, these changes were absent or did not reach statistical significance in the traditional cardiac rehabilitation group. Platelet-derived microvesicle counts were significantly decreased in both groups, while total microvesicle count was significantly reduced only in the complex cardiac rehabilitation group (median (interquartile range): 3945.5 (2138-5661) vs 1739 (780-2303) count/µl; p = 0.0001). Conclusions Platelet hyperactivity three months after acute coronary syndrome significantly decreased in patients undergoing complex cardiac rehabilitation. Besides dual antiplatelet therapy, effective management and comprehensive control of cardiovascular risk factors might represent a new, non-pharmacological approach to influence platelet functionality.

High on-clopidogrel platelet reactivity (HPR) is a predictor of ischemic events after percutaneous coronary intervention. We conducted a prospective cohort study to identify variables related to HPR in acute coronary syndrome patients who are at high thrombotic risk. We enrolled 463 patients undergoing urgent coronary angiography. Platelet reactivity was measured 12-36 hours after 600 mg clopidogrel loading with multiple electrode aggregometry (Multiplate® analyzer, Roche, Basel, Switzerland, 6.4 µM ADP). HPR was defined by the consensus cut-off area under the curve >46 U. The rate of HPR was 16.0%. We analyzed simple clinical and laboratory parameters with backward multivariate logistic regression and identified the following predictors of HPR: platelet count (per G/L, OR: 1.0073, 95% CI: 1.0035-1.0112, p = 0.0002), CRP level (per mg/L, OR: 1.0077, 95% CI: 1.0016-1.01372, p = 0.01), and active smoking (OR: 0.51, 95% CI: 0.29-0.89, p = 0.02). We developed and internally validated a risk prediction model demonstrating moderate discriminative capacity (area-under-the-receiver operating characteristic curve = 0.67). In conclusion, we found a relatively low rate of high on-clopidogrel platelet reactivity (16.0%) even in an acute patient cohort. HPR measured by Multiplate was associated with high platelet count and CRP level on admission and was inversely related to active smoking. The model with rapidly available simple parameters might help to identify individuals at risk for HPR in the acute setting.

Objective: We examined the vascular system, from the microvasculature to the aorta, in diabetes mellitus, using non-invasive methods. Methods: We enrolled patients with type 1 diabetes: 17 patients without complications (DMW) and 19 patients with clinically manifest complications (DMC). Control group was represented by 34 healthy volunteers (C). We examined microvascular function with laser-Doppler flowmetry, using post-occlusive reactive hyperemia test and local heating. Arterial stiffness was studied by arteriograph, determining augmentation index and pulse wave velocity. We measured serum levels of sE-selectin and sICAM-1, markers of endothelial dysfunction. Results: Microvascular reactivity was significantly reduced in DMC-, and tendentiously in DMW groups. sE-selectin level was significantly higher in DMC group than in controls. Arterial stiffness was the highest in the DMC group and the lowest in the DMW group. Heart rate was significantly higher in both diabetic groups compared to controls. Time to maximum flow during PORH test tended to be the shortest in DMW group. Conclusions: Our results confirm impairment of the microvascular system in diabetic patients, even in early, uncomplicated stage of the disease, and might demonstrate diffuse hyperkinesis in the vascular system, resulting from the insulin effect or refering to the “vasodilation phase” of diabetes mellitus.

The benefit of adjusted antiplatelet therapy in patients with myocardial infarction after primary percutaneous coronary intervention is not well elucidated. We aimed to identify patients with high on treatment platelet reactivity and to gradually adjust antiplatelet therapy. Materials and Methods We enrolled 133 acute myocardial infarction and 67 stable angina patients undergoing intracoronary stenting into our study. Maximal aggregation was determined with light transmission aggregometry. Aggregation > 50% induced by 5 μM ADP was indexed with high on-clopidogrel treatment platelet reactivity. In these cases 75 mg clopidogrel was doubled and control test was performed. Patients effectively inhibited with 150 mg clopidogrel were defined as clopidogrel pseudo non-responders. Patients with high platelet reactivity even on 150 mg clopidogrel were considered as clopidogrel real non-responders and were switched to ticlopidine. Results Aggregations (5ADP; p = 0.046) and the ratio of real non-responders (p = 0.013) were significantly higher in the myocardial infarction group. Most real non-responders were effectively treated with switch of therapy. The ratio of pseudo non-responders also tended to be higher in myocardial infarction. Platelet reactivity remained constant during follow-up; however, a new appearance of high platelet reactivity was observed at 6 and at 12 months. Conclusions Patients with acute myocardial infarction undergoing percutaneous coronary intervention may benefit from prospective platelet function testing, because of higher platelet reactivity and much higher ratio of clopidogrel real non-response. Switch of therapy may effectively overcome clopidogrel non-response. A new appearance of high platelet reactivity with unknown clinical significance is observed in both groups among the patients on clopidogrel.

Background Drug-eluting coronary stent implantation emerged as a safe and effective therapeutic approach by preventing coronary restenosis and reducing the need for further revascularization. However, in contrast to bare metal stents, recent data suggest a unique underlying pathology, namely late coronary stent thrombosis and delayed endothelial healing. Objective To report a case of very late coronary stent thrombosis (834 days after implantation) requiring repeat urgent target-vessel revascularization. Importantly, six days before the acute coronary event, combined nonsteroidal anti-inflammatory drug therapy was initiated. Results Although a dual antiplatelet regimen was continuously maintained, aggregation measurements indicated only partial antiplatelet effect, which returned to the expected range when nonsteroidal antiinflammatory drugs were omitted. Conclusions The observation indicates that, even 834 days after drug-eluting stent implantation, effective combined antiplatelet therapy might be crucial in certain individuals and the possible impact of drug interactions should not be underestimated. Further efforts should focus on the challenging task of identifying patients or medical situations with prolonged, increased risk of stent thrombosis.

SummaryCombined antiplatelet therapy reduces recurrent atherothrombotic events in stable coronary disease patients; however, high residual platelet reactivity measured ex vivo still raises concerns as a condition related to treatment failure. Alpha-2 adrenoceptor enhances platelet reactivity and might contribute to this phenomenon. For the present study, 121 stable angina patients on standard dual antiplatelet therapy (75 mg clopidogrel and 100 mg acetylsalicylic acid) were recruited. Born aggregometry was performed with adenosine diphosphate (ADP),collagen and epnephrine. To verify platelet adrenergic activity, potentiation by low-dose epinephrine and inhibition by selective alpha-2 receptor blocker atipamezole were determined. To assess the P2Y12-specific residual activity, cangrelor was used. Plasma norepinephrine, soluble CD40-ligand, high-sensitivity-C-reactive protein (hsCRP) - and in 24 subjects platelet P-selectin positivity were measured. Epinephrine - at very low concentration (10-9g/ml) - significantly potentiates (1.25 µM ADP: 26.5% vs. 43%; 5 µM ADP: 53% vs. 64.5%; collagen: 17% vs 42%, p

Systemic factors and blood flow velocity related to atherosclerosis have been examined mainly separately or by in vitro studies. The aim of our study was to investigate the association between local coronary blood flow (corrected TIMI frame count, CTFC) and systemic atherosclerosis-related inflammatory parameters such as soluble intercellular adhesion molecule-1 (sICAM-1), interleukin-6 (Il-6), high sensitivity C-reactive protein (hsCRP) and von Willebrand factor (vWF) in humans. We enrolled the following groups of ischemic heart disease (IHD) patients: patients with coronary stenosis and stable (CAD, n = 96) or unstable angina (ACS, n = 27), patients with documented myocardial ischemia and normal coronary angiogram (NEG, n = 68). Patient groups showed only marginal differences in CTFC or sICAM-1 levels. In contrast, when IHD patients were studied individually, general positive correlation was found between CTFC and sICAM-1 level (r = 0.33; in NEG r = 0.25; in CAD r = 0.37; in ACS r = 0.61), being the strongest in ACS. The relation was independent from age, gender, BMI, smoking, hypertension, diabetes, previous myocardial infarction, family history of IHD, medication, hsCRP, IL-6 and vWF levels. (odds ratio, OR = 6.4; CI 95%: 2.43-16.84; p < 0.05). Nevertheless, correlation between CTFC and IL-6, hsCRP, vWF levels was not found. These results indicate inverse correlation between coronary blood flow and adhesion molecule production independently from conventional cardiovascular risk factors and inflammatory markers.

Reactive free radical and oxidant production leads to DNA damage during myocardial ischemia/reperfusion. Consequent overactivation of poly(ADP-ribose) polymerase (PARP) promotes cellular energy deficit and necrosis. We hypothesized that PARP is activated in circulating leukocytes in patients with myocardial infarction and reperfusion during primary percutaneous coronary intervention (PCI). In 15 patients with ST segment elevation acute myocardial infarction, before and after primary PCI and 24 and 96 h later, we determined serum hydrogen peroxide concentrations, plasma levels of the oxidative DNA adduct 8-hydroxy-2'-deoxyguanosine (8OHdG), tyrosine nitration, PARP activation, and translocation of apoptosis-inducing factor (AIF) in circulating leukocytes. Plasma 8OHdG levels and leukocyte tyrosine nitration were rapidly increased by PCI. Similarly, poly(ADP-ribose) content of the leukocytes increased in cells isolated just after PCI, indicating immediate PARP activation triggered by reperfusion of the myocardium. In contrast, serum hydrogen peroxide concentrations and the translocation of AIF gradually increased over time and were most pronounced at 96 h. Reperfusion-related oxidative/nitrosative stress triggers DNA damage, which leads to PARP activation in circulating leukocytes. Translocation of AIF and lipid peroxidation occurs at a later stage. These results represent the first direct demonstration of PARP activation in human myocardial infarction. Future work is required to test whether pharmacological inhibition of PARP may offer myocardial protection during primary PCI.

Adenine nucleotides, ADP and ATP, are coreleased from dense granules during platelet activation, as well as from endothelial cells and damaged red blood cells following vascular injury. Through autocrine and paracrine mechanisms, these extracellular signaling molecules interact with the platelet P2 receptors to amplify ongoing platelet activation. Two receptors for ADP, the G(q)-protein-coupled P2Y1 and G(i)-protein-coupled P2Y12 and one receptor for ATP, the P2X1 ion channel, have been identified on platelets. Due to distinct pharmacological properties and differential regulation, the P2Y and P2X receptors essentially operate on different scales of time and distance and trigger selective intracellular signaling cascades. Recent advances in the understanding of the P2Y receptor physiology have reinforced the concept of these receptors as useful targets for antithrombotic therapy. The function of P2X1 in platelet activation only recently started to be unraveled. This review focuses on recent findings on the physiology of these platelet ADP and ATP receptors, their distinct downstream intracellular signaling pathways as well as on the available agonists, antagonists and inhibitors that allow their pharmacological discrimination.

The functional significance of extracellular signal-regulated kinase 2 (ERK2) activation was investigated during shear induced human platelet aggregation (SIPA) in vitro and during shear controlled thrombosis in vivo in intestinal arterioles and venules of wild type (WT) and transgenic (TG) mice with platelet-specific overexpression of human P2X(1) (TG). In SIPA, ERK2 was rapidly phosphorylated during GPIb stimulation, its activation contributing to SIPA for 50%, independently of P2X(1) regulation. Thrombotic occlusion of injured arterioles occurred considerably faster in TG (4.3 +/- 2.3 min) than in WT (38 +/- 8 min) arterioles, but occlusion times in TG (19 +/- 12) and WT (48 +/- 4.5 min) venules differed less. Both the alphabeta-meATP triggered desensitization of platelet P2X(1), as well as P2X(1) antagonism by NF279 or NF449 prolonged mean occlusion to about 75 min in WT and 65 min in TG arterioles, but venular occlusion times were less affected. Preventing ERK2 activation by U0126 prolonged occlusion times in TG (41 +/- 10 min) and WT (51 +/- 17) arterioles more than in TG (46 +/- 5 min) and WT (56 +/- 6 min) venules, uncovering a role for ERK2 in shear controlled thrombosis. Antagonism of GPIb by a recombinant murine von Willebrand factor (VWF)-A1 fragment prolonged occlusion times to comparable values, ranging from 55 to 58 min, both in TG and WT arterioles and venules. Further inhibition strategies, combining VWF-A1, U0126 and NF449 in WT and TG mice and resulting in occlusion in various time windows, identified that inhibition by VWF-A1 largely abrogated the ERK2 contribution to thrombosis. In conclusion, P2X(1) and ERK2 both participate in shear stress controlled thrombosis, but ERK2 activation is initiated predominantly via GPIb-VWF interactions.

The ATP-gated P2X1 ion channel is the only P2X subtype expressed in human platelets. Via transmission electron microscopy, we found that P2X1 mediates fast, reversible platelet shape change, secretory granule centralization, and pseudopodia formation. In washed human platelets, the stable P2X1 agonist alpha,beta-methylene ATP (alpha,beta-meATP) causes rapid, transient (2-5 s), and dose-dependent myosin light chain (MLC) phosphorylation, requiring extracellular Ca2+. Phosphorylation was inhibited by the calmodulin (CaM) inhibitor W-7, but not by the Rho kinase inhibitor HA-1077, i.e. it is exclusively regulated by Ca2+/CaM-dependent MLC kinase. Correspondingly, the P2X1-induced platelet shape change was inhibited by W-7 and by the MLC kinase inhibitor ML-7 but not by HA-1077. W-7, ML-7, the protein kinase C inhibitor GF109203-X, and the Src family kinase inhibitor PP1 inhibited the collagen and convulxin-induced early platelet degranulation, shape change, and subsequent aggregation, indicating a role for Ca2+/CaM and MLC kinase in these glycoprotein VI-related platelet responses. The secreted ATP-mediated P2X1-dependent ERK2 activation induced by low collagen concentrations contributes to MLC kinase activation since P2X1 desensitization or blockade of ERK2 phosphorylation by U0126 strongly attenuated MLC phosphorylation, degranulation, and aggregation. We therefore conclude that at low doses of collagen, glycoprotein VI activation leads to early protein kinase C- and MLC kinase-dependent degranulation. Rapidly released ATP triggers P2X1 -mediated Ca2+ influx, activating ERK2, in turn amplifying platelet secretion by reinforcing the early MLC kinase phosphorylation. Hence, the P2X1-ERK2-MLC axis contributes to collagen-induced platelet activation by enhancing platelet degranulation.

We have generated transgenic mice overexpressing the human P2X1 ion channel in the megakaryocytic cell lineage. Platelets from transgenic mice exhibited a gain of P2X1ionotropic activity as determined by more prominent P2X1-mediated Ca2+ influx and platelet shape change. P2X1 overexpression enhanced platelet secretion and aggregation evoked by low doses of collagen, convulxin, or the thromboxane A2 mimetic U46619. In contrast, transgenic platelet responses to adenosine diphosphate (ADP) or thrombin were normal. Perfusing whole blood from transgenic mice over collagen fibers at a shear rate of 1000 seconds−1 resulted in increased P2X1-dependent aggregate formation and phosphatidylserine exposure. Platelet hyperreactivity to collagen was correlated with up-regulated extracellular signal-regulated kinase 2 (ERK2) phosphorylation. Accordingly, the MEK1/2 inhibitor U0126 potently inhibited the collagen-induced aggregation of transgenic platelets when stirred or when perfused over a collagen surface. In a viscometer, shear stress caused potent aggregation of transgenic platelets under conditions in which wild-type platelets did not aggregate. In an in vivo model of thromboembolism consisting of intravenous injection of a low dose of collagen plus epinephrine, transgenic mice died more readily than wild-type mice. Preinjection of U0126 not only fully protected transgenic mice against thrombosis, it also enhanced the survival of wild-type mice injected with a higher collagen dose. Hence, the platelet P2X1 ion channel plays a role in hemostasis and thrombosis through its participation in collagen-, thromboxane A2-, and shear stress–triggered platelet responses. Activation of the ERK2 pathway is instrumental in these processes.

SummaryATP is a potent agonist of the P2X1 ion channel, mediating a rapid, quickly desensitized influx of Ca2+. In hirudinized PRP, containing apyrase, the two stable selective P2X1 agonists, α,β-methylene ATP, and L-β, γ-methylene ATP induced extracellular Ca2+-dependent fast and reversible platelet shape change, leading to desensitization of the P2X1 ion channel. Preincubation with HPLC-purified ADP potently antagonized the subsequent α, β-methylene ATP- and L-β, γ-methylene ATP-evoked platelet shape change. Accordingly, upon heterologous expression of P2X1 in Xenopus oocytes, HPLC-purified ADP acted as an antagonist of the ATP-induced current, but was inactive itself. Since ATP and ADP are co-released from dense granules during platelet activation, we investigated whether the P2X1 ion channel is involved in the response of platelets to collagen. We found that platelet shape change and aggregation induced by low concentrations of collagen were strongly inhibited after selective desensitization of P2X1 with its agonists or by pretreating the platelets with a low concentration of ADP (0.5 μM), that antagonizes the P2X1 channel without desensitizing the P2Y1 receptor. Our data suggest that, during collagen-initiated platelet activation, the early secretion of ATP results in the activation of the P2X1 ion channel, which plays a role as a positive regulator of further platelet responses.

In a recent issue of Blood, Greco et al[1][1]reported on the expression of a novel structurally altered P2X1 receptor in platelets and in megakaryocytic cell lines. This P2X1 variant lacks 17 amino acids in its extracellular domain due to a deletion within exon 6 of the P2X1 gene (GenBank accession

ADP plays a central role in platelet aggregation. Activation of platelets via ADP proceeds via three membrane receptor proteins, two of which are coupled to a G protein and the third one constituting an ion channel mediating rapid Ca2+-influx. Via Ca2+-mobilization, the Gq-coupled P2Y1 receptor acts in concert with the Gi-coupled P2TAC receptor, which functions by lowering intracellular cAMP levels. The importance of Ca2+-influx via the P2X1 ion channel remains to be elucidated.

Background Percutaneous coronary intervention (PCI) is an important therapeutic strategy in patients with ischaemic heart disease. Our aim was to clarify the extent of endothelial injury induced by PCI in stable angina (SA) or in acute ST-elevation myocardial infarction (STEMI). Methods Circulating endothelial cell (CEC) count, von Willebrand factor (vWF) and soluble intercellular adhesion molecule-1 (sICAM-1) levels were determined pre-, post-, 24 and 96h after PCI in patients with SA ( n =23) and with STEMI ( n =28). To provide control data regarding the effect of angiography itself stable angina patients with coronarography only ( n =23) were enrolled. Results PCI and coronarography in stable angina patients caused measurable, but only non-significant elevation of CEC count and plasma vWF ( p =NS). In STEMI, significantly higher baseline CEC count ( p =0.019) and vWF plasma levels ( p =0.046) were found compared to SA with PCI/or coronarography. After PCI, explicit increase in CEC count was observed (significant peak at 24h) ( p =0.036). Positive correlation was found between baseline CKMB and CEC count at 24h ( r =0.51, p Conclusion Both coronary angiography and elective PCI cause only mild endothelial injury. However, in patients with STEMI, not only the procedure itself but myocardial ischemia and the ongoing atherothrombotic process might be responsible for the prolonged and more pronounced endothelial damage.

Adenosine triphosphate (ATP) and its stable analog, alpha,beta-methylene ATP, activate the platelet P2X(1) ion channel, causing a rapid Ca(++) influx. Here, we show that, in washed apyrase-treated platelets, alpha,beta-methylene ATP elicits reversible extracellular signal-regulated kinase 2 (ERK2) phosphorylation through a Ca(++)- and protein kinase C-dependent pathway. In contrast, high-performance liquid chromatography-purified adenosine diphosphate (ADP) did not trigger ERK2 phosphorylation. alpha,beta-Methylene ATP also activated the ERK2 pathway in P2X(1)-transfected HEK293 cells but not in cells expressing mutated P2X(1)delL nonfunctional channels. Because ATP released from the dense granules during platelet activation contributes to platelet aggregation elicited by low doses of collagen, and because collagen causes ERK2 phosphorylation, we have investigated the role of P2X(1)-mediated ERK2 activation in these platelet responses. We found that the antagonism of P2X(1) with ADP or desensitization of this ion channel with alpha,beta-methylene ATP both resulted in impaired ERK2 phosphorylation, ATP secretion, and platelet aggregation induced by low concentrations of collagen (< or = 1 microg/mL) without affecting the minor early dense granule release. Selective MEK1/2 inhibition by U-0126 and Ca(++) chelation with EGTA (ethyleneglycoltetraacetic acid) behaved similarly, whereas the PKC inhibitor GF109203-X totally prevented collagen-induced secretion and ERK2 activation. In contrast, when elicited by high collagen concentrations (2 microg/mL), platelet aggregation and secretion no longer depended on P2X(1) or ERK2 activation, as shown by the lack of their inhibition by alpha,beta-methylene ATP or U-0126. We thus conclude that mild platelet stimulation with collagen rapidly releases ATP, which activates the P2X(1)-PKC-ERK2 pathway. This process enhances further degranulation of the collagen-primed granules allowing platelet aggregation to be completed.

The P2Y1 Receptor Antagonist Adenosine-2’,5’-Diphosphate Non-selectively Antagonizes the Platelet P2X1 Ion Channel

The four highly conserved intracellular tyrosine residues of the P2X(1) ion channel were mutated into phenylalanine. Simultaneous electrophysiological and calcium measurements in transfected human embryonic kidney (HEK 293) cells indicated that Y362F and Y370F mutants were non-functional, despite their proper plasma membrane expression. The Y16F and Y363F mutants retained 2.2% and 26% of the wild-type P2X(1) activity, respectively. However, no tyrosine phosphorylation was detected on Western blots of P2X(1) immunoprecipitates derived either from HEK 293 cell lysates or from human platelets, expressing P2X(1) endogenously. Thus, Y16, Y362, Y363 and Y370 are required for the appropriate three-dimensional structure and function of the intracellular P2X(1) domains.