Reduced platelet response to aspirin in patients with coronary artery disease and type 2 diabetes mellitus

Introduction

Diabetes mellitus is complicated by accelerated atherosclerosis, resulting in an increased risk of coronary artery disease (CAD) and thrombosis. Despite the proven benefits of aspirin, previous studies indicate a reduced cardiovascular protection from aspirin in diabetic patients. We aimed to investigate whether diabetes mellitus influenced the platelet response to aspirin in patients with CAD.

Materials and Methods

Platelet aggregation and activation were evaluated during aspirin treatment in 85 diabetic and 92 non-diabetic patients with CAD. Adherence to aspirin was carefully controlled. All patients had CAD verified by coronary angiography and were taking 75 mg non-enteric coated aspirin daily.

Results

Diabetic patients showed significantly higher levels of platelet aggregation compared to non-diabetic patients evaluated by VerifyNow^® Aspirin (p = 0.03) and Multiplate^® aggregometry using arachidonic acid (AA) 0.5 mM (p = 0.005) and 1.0 mM (p = 0.009). In addition, platelet activation determined by soluble P-selectin was significantly higher in diabetics compared to non-diabetics (p = 0.005). The higher AA-induced aggregation was associated with higher levels of HbA_1c. Compliance was confirmed by low levels of serum thromboxane B₂ (below 7.2 ng/mL). Diabetics had significantly higher levels of serum thromboxane B₂ (p < 0.0001).

Conclusions

Diabetic patients with CAD had significantly higher levels of both platelet aggregation and activation compared to non-diabetic patients with CAD despite treatment with the same dosage of aspirin. These findings may partly explain the reduced cardiovascular protection from aspirin in diabetic patients.     

Genetic determinants of response to aspirin: appraisal of 4 candidate genes

Introduction

Intersubject variability in platelet response to aspirin could be related to genetic factors that regulate platelet enzymes or receptors. This study evaluates the impact of the selected polymorphisms in the COX-1 gene, the CYP5A1 gene, the P2RY1 receptor gene, and the GPIIbIIIa receptor gene on platelet response to aspirin and risk of suffering from major adverse cardiovascular and cerebrovascular events (MACCE).

Materials and methods

192 Caucasian patients with stable coronary artery disease treated with daily aspirin were recruited and followed for 3 years. Platelet aggregation was measured by light transmission aggregometry with arachidonic acid (1.6 mM) and adenosine diphosphate (5, 10 or 20 μM) used as agonists. Genotyping was performed by standard PCR methods.

Results

Arachidonic acid-induced platelet aggregation was unaffected by the COX-1 22C/T and by the Pl^A1/A2 polymorphisms. However, carriers of the 1622 G/G genotype of the P2RY1 gene had significantly higher levels of arachidonic acid-induced platelet aggregation compared with non-carriers (AA 2.0%, AG 2.0% vs. GG 9.0%, p = 0.047). Carrying the 1622 G/G genotype increased the risk of inadequate platelet response to aspirin, defined as arachidonic acid-induced aggregation ≥ 20%, by a factor of 8.5 (1.4 - 53.3, p = 0.022) and the risk of 3-year MACCE by a factor of 7 (1.4 - 34.7, p = 0.017).

Conclusion

The 1622A/G mutation of the P2RY1 gene could contribute to inadequate platelet response to aspirin and is associated with an increased risk of suffering from MACCE.     

Pioglitazone inhibits platelet function and potentiates the effects of aspirin: a prospective observation study

Background

Thiazolidinediones (TZDs) are agonists of PPARγ and exert beneficial metabolic effects in patients with diabetes. They may also affect platelet function.

Objectives

To characterize potential platelet inhibitory effect of pioglitazone alone and in the presence of aspirin.

Methods

20 normal and 20 diabetic subjects were enrolled in a prospective study. On day 1, a blood sample was obtained at baseline and a second one after ingestion of 30 mg of pioglitazone. PRP was prepared and platelet aggregation and release were evaluated using ADP, collagen and arachidonic acid as agonists. Subjects returned at 6-9 days later after ingesting a single 81 mg dose of aspirin and a third blood sample was obtained. The subjects then again ingested 30 mg of pioglitazone and a fourth and final blood sample was obtained. Platelet aggregation and release were measured. PRP was incubated with thrombin to activate platelets, and the serum was separated and assayed for thromboxane B2, TGFβ and CD40L

Results

Pioglitazone alone did not affect aggregation with arachidonic acid. However, following ingestion of both aspirin and pioglitazone aggregation was significantly decreased compared to aspirin alone (P < 0.0001). Pioglitazone also potentiated aspirin-induced inhibition of ATP release using either arachidonic acid or collagen. Following pioglitazone alone, TXB₂ release was 32,719 ± 3,585 pg/ml which was significantly reduced compared to baseline (42,075 ± 4,479, P = 0.0004). Pioglitazone also potentiated the inhibition of TXB₂ release by aspirin.

Conclusion

Pioglitazone inhibits platelet function and potentiates the inhibitory effects of aspirin.     

Plasma and Whole Blood Clot Strength Measured by Thrombelastography in Patients Treated with Clopidogrel during Acute Coronary Syndromes

Introduction

Treatment with clopidogrel, a selective platelet P₂Y₁₂ receptor antagonist, reduces risk of recurrent ischemic events in patients with acute coronary syndrome (ACS), by limiting platelet aggregation and activation. Stable whole blood clot formation requires activation of platelets, generation of fibrin and final fibrin crosslinks. In this study we intended to compare plasma and whole blood thrombelastography (TEG) measurements in patients during ACS.

Materials and Methods

Whole blood and plasma samples from 32 patients with non-ST segment elevation myocardial infarction (NSTEMI) were collected after administration of clopidogrel. Whole blood and plasma fibrin clot strength (MA) were determined by TEG. Platelet aggregation was determined by light transmittance aggregometry (LTA) using adenosine 5'-diphosphate (ADP), thrombin receptor activation peptide (TRAP), or collagen as agonists. Fibrinogen and C-reactive protein (CRP) concentrations were measured by ELISA.

Results

Heightened plasma fibrin clot strength was associated with increased platelet reactivity stimulated by ADP (ρ = 0.536; p = 0.002), TRAP (ρ = 0.481; p = 0.007), and collagen (ρ = 0.538; p = 0.01). In contrast to plasma fibrin MA, whole blood MA did not correlate with platelet aggregation. Platelet count was the primary contributor to the difference in thrombin induced whole blood MA and plasma fibrin MA. Increasing levels of CRP were associated with increased plasma fibrin clot strength and platelet reactivity.

Conclusions

Our data suggest that inflammation is associated with increased plasma fibrin clot strength and lower platelet inhibition by clopidogrel during ACS. Platelet count is a main contributor to additional contractile force of whole blood TEG as compared to plasma TEG during treatment with clopidogrel.     

Aspirin treatment influences platelet-related inflammatory biomarkers in healthy individuals but not in acute stroke patients

Objectives

Platelet-leukocyte aggregation is believed to contribute to acute thrombotic events. While the effect of aspirin on platelet-to-platelet aggregation is well established, the impact of the drug on pro-inflammatory platelet function remains equivocal. Thus we investigated the effect of aspirin on selected platelet-related inflammatory biomarkers in both acute ischaemic stroke patients and healthy volunteers.

Methods

Using five-colour flow cytometry the platelet surface expression of CD62P and CD40L and subpopulations of leukocyte-platelet aggregates were assessed in 63 acute stroke patients and 40 healthy volunteers at baseline and after a 10-day period of aspirin intake at a daily dose of 150 mg. Simultaneously the plasma levels of soluble CD62P and CD40L, serum level of TxB₂, and whole blood impedance platelet aggregation under arachidonic acid (AA) stimulation were investigated.

Results

No differences in values of studied platelet-related inflammatory biomarkers in both resting platelets and those activated with TRAP after 10-day treatment with aspirin were confirmed in stroke subjects. In healthy individuals the resting platelet expression of CD62P, plasma level of soluble CD62P and percentage of circulating monocyte-platelet aggregates were lower after the aspirin intake period (P = 0.009; P = 0.04; P = 0.004, respectively). In both studied groups serum level of TxB₂ and platelet aggregation under AA stimulation were lower than before treatment (P < 0.001).

Conclusion

Despite effective inhibition of COX-1-dependent platelet aggregation, aspirin does not influence the platelet α-granule-derived inflammatory mediators and monocyte-platelet aggregation in acute stroke subjects, although it does in healthy individuals.     

Optimizing thrombelastography (TEG) assay conditions to monitor rFVIIa (NovoSeven) therapy in haemophilia a patients

Introduction

There is no established laboratory method that can predict the most optimal dose of bypassing agents for treatment of haemophilia A. The objectives of the study was to develop an assay that can a) differentiate between the haemostatic capacity in blood from healthy individuals and severe and moderate haemophilia patients; b) show a dose-response correlation to rFVIIa addition; and c) show dose response differences of rFVIIa addition to plasma samples from non-inhibitor patients of different severity.

Materials and Methods

Citrated whole blood from 25 haemophilia A patients was used in four thrombelastography (TEG) assays initiated with: 1) kaolin, 2) Tissue Factor (TF, Innovin 1:42,500), 3) TF 1:42,500 + 1.2nM tPA (tissue plasminogen activator) or 4) TF 1:200,000. rFVIIa was added to give a final concentration in the range of 0.02-4.8 µg/ml.

Results

The TEG assays showed large differences in clot formation demonstrated by prolonged clotting time (R-time), decreased maximum thrombus generation (MTG) between severe and moderate haemophilia A patients and between haemophilia patients and healthy males. The maximal amplitudes (MA) of the clot and resistance against fibrinolysis were only compromised when TF with tPA was added.

Conclusion

In vitro addition of rFVIIa improved all TEG profiles significantly in a dose-dependent manner; but only the TEG assay containing kaolin could differentiate between the rFVIIa doses, showing that blood from severe patients need higher doses of rFVIIa to normalize the clot formation profile compared to blood from moderate patients. Kaolin seems to be the most useful TEG assay for monitoring rFVIIa treatment.     

Synergistic effect of a factor Xa inhibitor, TAK-442, and antiplatelet agents on whole blood coagulation and arterial thrombosis in rats

Introduction

Activated platelets facilitate blood coagulation by providing factor V and a procoagulant surface for prothrombinase. Here, we investigated the potential synergy of a potent factor Xa/prothrombinase inhibitor, TAK-442, plus aspirin or clopidogrel in preventing arterial thrombosis and whole blood coagulation.

Methods

Thrombus formation was initiated by FeCl₃-induced rat carotid injury. Bleeding time was evaluated with the rat tail transection model. Whole blood coagulation was assessed by thromboelastographic examination (TEG) for which blood obtained from control, aspirin-, or clopidogrel-treated rats was transferred to a TEG analyzer containing, collagen or adenosine diphosphate (ADP), and TAK-442 or vehicle.

Results

TAK-442 (3 mg/kg, po), aspirin (100 mg/kg, po) or clopidogrel (3 mg/kg, po) alone had no significant effect on thrombus formation, whereas the combination of TAK-442 with aspirin and clopidogrel remarkably prolonged the time to thrombus formation without additional significant prolongation of bleeding time. TEG demonstrated that the onset of collagen-induced blood coagulation were slightly longer in aspirin-treated rats than control; however, when the blood from aspirin-treated rats was subsequently treated in vitro with 100 nM TAK-442, the onset of clotting was significantly prolonged. In contrast, only marginal prolongation was observed with TAK-442 treatment of blood from control animals. The onset time of ADP-induced blood coagulation was slightly longer in clopidogrel-treated rats compared with control, and it was further extended by TAK-442 treatment.

Conclusion

These results demonstrate that blood coagulation can be markedly delayed by the addition of TAK-442 to antiplatelets treatment which could contribute to synergistic antithrombotic efficacy in these settings.     

The risk of false results in the assessment of platelet function in the absence of antiplatelet medication: Comparision of the PFA-100, multiplate electrical impedance aggregometry and verify now assays

Objectives

Evaluation of aspirin (ASA) responsiveness with platelet function tests varies by the choice of blood mixture and functional test and cut off values for defining the the treatment used. Addition to that we also aimed to determine aggregement between three tests and to research whether there is any necessity to measure baseline platelet activity.

Methods

The study group comprised of 52 patients with multiple risk factors receiving primary prophylaxis of ASA (100 mg/day). For each patient inhibition of platelet aggregation with aspirin was determined using three different whole blood tests: Multiplate electrical impedance aggregometry , Verify Now Aspirin, and collagen-epinephrine closure time PFA-100. Platelet aggregation was assessed with multiplate electrical impedance aggregometry,and was defined as the area under curve (AUC,AUxmin).Maximal 6,4 microM collagen-induced AUC were used to quantify platelet aggregation due to ASA.The ASA response was defined as > 30 % reduction in basal platelet aggregation with multiplate electrical impedance aggregometry.Collagen induced platelet aggregation at the Verify Now Aspirin assay quantitated the ASA-induced platelet inhibition as aspirin reaction units (ARU).According to manifacturer insert ARU > 550 indicates aspirin resistance. ASA platelet function studies were assessed twice at baseline (pre-aspirin), and after 7 day(post-aspirin) were performed.

Results

After ASA intake none of the patients was found aspirin resistant with PFA-100. (CEPI-CT (129 ± 36 vs 289 ± 18 ). None of the patients was found aspirin resistant with PFA-100. As > 30 % reduction in bazal platelet aggregation with multiplate electrical impedance aggregometry is selected all of the patients have been stratified as responders.(COL TEST 688±230 vs 169 ± 131 AU) None of the patients with Verify Now Aspirin found resistance to ASA(594 ± 62 vs 446 ± 43).Prior to ASA intake 15 of all patients with VN(501 ± 16) and 2 of all patients with multiplate electrical impedance aggregometry (223 ± 40 AUC )aggregation levels below the cut off label before ingestion of ASA.None of the patients was above the cut off label with PFA -100 (129 ± 36).

Conclusions

Verify Now ASA assay, multiplate electrical impedance aggregometry and PFA-100 seem to be reliable tests in reflecting ASA effect on platelets. Cut off labels for the defining the responsiveness given by man?facturer may show significant interindividual variabiliy with Verify Now ASA assay and multiplate electrical impedance aggregometry, and these test may show platelet inhibition despite the absence of ASA intake. Consideration of the pretreatment values may eliminate the risk of overestimation in the assessment of platelet inhibtion by ASA.     

Baseline platelet size is increased in patients with acute coronary syndromes developing early stent thrombosis and predicts future residual platelet reactivity. A case-control study

Introduction

Pre-procedural predictors of early stent thrombosis (ST) and future response to platelet inhibitors are in demand. We sought to evaluate the impact of baseline platelet indices on the occurrence of early ST and future residual platelet reactivity.

Materials and methods

Hundred and eight patients with acute coronary syndromes (ACS) in whom stents were implanted were included: 36 consecutive ST cases and 72 matched controls. Platelet indices assessed with flow cytometry before stent implantation were retrieved from the department's data base. Residual platelet reactivity specific to aspirin (aspirin reaction units-ARU) and clopidogrel (P2Y12 reaction units-PRU) was assessed prospectively with VerifyNow^® under dual antiplatelet treatment.

Results

Platelet size reported as mean platelet volume (MPV) or proportion of large platelets (LPLT) was significantly higher in ST cases compared with controls (10.4, 95% confidence intervals [CI], 10.1-10.8 vs. 9.7, CI, 9.5-9.9, P = 0.0004 and 35.8, CI, 34.2-37.3 vs. 33.3, CI, 32.2-34.3, P = 0.007, respectively). Dual aspirin and clopidogrel poor-responsiveness was diagnosed significantly more often in ST cases than in controls (19.6% vs. 1.4%, P = 0.004), whereas no difference was observed for single aspirin or clopidogrel poor-responsiveness. A strong correlation was found between MPV and both, ARU (r = 0.66, P < 0.0001) and PRU (r = 0.55, P < 0.0001). Similarly, higher LPLT was associated with higher ARU (r = 0.47, P < 0.0001) and PRU (r = 0.38, P = 0.0001).

Conclusions

Baseline platelet size is increased in patients with ACS developing early ST and correlates with future residual platelet reactivity under aspirin and clopidogrel therapy. Dual but not isolated aspirin or clopidogrel poor-responsiveness appears to be associated with early ST.     

Platelet aggregation is dependent on platelet count in patients with coronary artery disease

Introduction

Platelet function testing in whole blood is widely used to evaluate the effect of antiplatelet agents, but it is not known whether results are affected by whole blood parameters. This study investigated the importance of platelet count, haematocrit, red blood cells (RBC), and white blood cells in whole blood platelet aggregometry.

Materials and methods

We included 417 patients with coronary artery disease on aspirin mono-therapy and 21 aspirin-naïve healthy individuals. Blood sampling was performed one hour after aspirin ingestion. The antiplatelet effect of aspirin was evaluated using the VerifyNow® Aspirin assay and multiple electrode aggregometry (MEA, Multiplate®) induced by collagen (1.0 μg/mL) and arachidonic acid (1.0 or 0.75 mmol/L). Measurements of whole blood parameters were performed to evaluate the three major cell lines in circulating blood.

Results

In patients, platelet count correlated significantly with platelet aggregation (MEA_collagen, p < 0.0001; MEA_{arachidonic acid}, p < 0.0001; VerifyNow®, p = 0.03). Haematocrit and RBC correlated inversely with MEA induced by collagen (p_haematocrit < 0.001; p_RBC = 0.07) and with VerifyNow® (p_haematocrit < 0.0001; p_RBC < 0.0001), but not with MEA induced by arachidonic acid (p_haematocrit = 1; p_RBC = 0.87). White blood cells correlated significantly with platelet aggregation (MEA_collagen, p < 0.001; MEA_{arachidonic acid}, p < 0.0001; VerifyNow®, p = 0.05). Similar associations were observed in aspirin-naïve healthy individuals.

Conclusions

Whole blood aggregometry is dependent on all major cell lines in whole blood. Importantly, platelet aggregation is significantly associated with platelet count even within the normal range.     

Acute cigarette smoke exposure reduces clot lysis -- association between altered fibrin architecture and the response to t-PA

Background

Enhanced thrombolysis is a proposed mechanism for reduced mortality in cigarette smokers with STEMI (“smoker's paradox”). The mechanisms remain unclear but studies suggest fibrin architecture (FA) may affect thrombolysis. Our group has previously shown that acute cigarette smoke exposure (CSE) alters FA. This study was done to evaluate the association between FA, thrombolysis and CSE.

Methods and Results

Otherwise healthy smokers (n = 22) were studied before and after smoking two cigarettes. Non-smokers (n = 22) served as controls. Two ex-vivo models were used to evaluate clot lysis of venous blood and these data were compared to FA as determined by SEM. In the first model, clot lysis in a glass tube at 60 minutes after addition of t-PA was measured. The second model quantified lysis utilizing thromboelastography. With the latter, after a clot reached maximum strength, t-PA was added and clot lysis at 60 min was noted. SEM studies were performed on platelet poor plasma mixed with thrombin and FA was examined at 20 K.Clot lysis was similar in both groups except that post-smoking, TEG showed a significantly lower lysis compared to pre- and non-smoking clots. SEM analysis showed significantly thinner fibers and denser clots post-smoking.

Conclusions

Venous clots from smokers failed to show an enhanced lysis when exposed to t-PA. In fact, acute CSE was associated with changes in FA and increased resistance to thrombolysis. These findings in part may explain enhanced thrombogenicity but suggest that mechanisms other than enhanced fibrinolysis are likely to be responsible for “smoker's paradox.”     

Hemostatically distinct FFPs equally improve abnormal TEG variables in an in vitro dilutional coagulopathy model

Introduction

To improve fresh frozen plasma (FFP) availability, thawed plasma is stored at 4 °C for up to 5 days and considered equivalent to freshly thawed FFP. However, we have shown that hemostatic potential of thawed plasma is highly variable between donors and significantly diminished during storage. We hypothesized that smaller volumes of plasma with higher hemostatic potential (FFP-H) would be needed to restore normal thrombelastogram (TEG) values compared to plasma with lower hemostatic potential (FFP-L).

Materials and Methods

A dilutional coagulopathy model was established from whole blood by diluting plasma with saline to 23%, while cellular components were kept unchanged. Saline was gradually replaced with equal volumes of FFPs with distinctive hemostatic potentials, which was evaluated by the calibrated automated thrombogram. Clot formation in the presence of tissue factor was evaluated by TEG at baseline and after addition of increasing concentrations of FFP-H and FFP-L.

Results

Blood dilution with saline in the presence of tissue factor resulted in abnormal TEGs that resemble a pattern observed in severely bleeding trauma patients. All FFPs produced similar improvements in TEG variables despite different hemostatic potentials. TEG changes were solely dependent on FFP volume and reached the normal reference range when plasma concentration increased to 40%.

Conclusion

Plasma dilution and tissue factor in whole blood results in an abnormal TEG with a hyperfibrinolytic pattern. A plasma concentration of at least 40% was necessary for TEG normalization after dilution with saline. An effect of FFPs’ hemostatic potential on clot formation could not be detected by TEG in this in vitro model.     

P2Y12 platelet reactivity after thrombolytic therapy for ST-segment elevation myocardial infarction

Introduction

Thrombolysis, as reperfusion therapy for ST segment elevation myocardial infarction (STEMI), induces a pro-thrombotic status with enhanced platelet activity; this study aims to evaluate P2Y12 platelet reactivity and response to clopidogrel in the post-thrombolysis scenario.

Materials and Methods

Observational, prospective study, including consecutive patients with elective angiography after thrombolytic therapy for STEMI. Every patient received antiplatelet therapy with loading doses of 250 mg aspirin and 300 mg clopidogrel on admission followed by 100 mg aspirin and 75 mg clopidogrel daily. P2Y12-dependent platelet reactivity (expressed in P2Y12-Reaction Units, PRU) was assessed with VerifyNow® device on admission, daily after thrombolysis and pre-angiography.

Results

41 patients fulfilled the inclusion criteria. Median time between thrombolysis and angiography was 2,5 days (IQR 1,8-4,1). Post-treatment platelet reactivity (PPR) showed poor correlation with time on clopidogrel treatment (r2 = 0.04) and reached a maximum value of 274 ± 84 PRU during the first 24 h after thrombolysis (Day + 1 determination). After this, values showed a progressive reduction until the point of angiography (249 ± 82 PRU), without significant differences between consecutive time-points (p = 0,549).Inhibition of platelet aggregation (IPA) assessed as a percentage of P2Y12 receptor blockage was poor, increasing gradually from 0 ± 4% on admission to 11 ± 6% the day of the angiography (p = 0,001). 71,4% of patients showed PPR ≥ 208 PRU during angiography.

Conclusions

Platelet reactivity, as assessed by post-treatment P2Y12 mediated reactivity, is heightened after thrombolytic therapy during STEMI management. In this scenario, standard doses of clopidogrel did not achieve significant inhibition of ADP-mediated platelet reactivity.     

Influence of renal function and platelet turnover on the antiplatelet effect of aspirin

Introduction

Kidney disease predisposes to cardiovascular events. This study investigated the influence of renal function and platelet turnover on the antiplatelet effect of aspirin in patients with coronary artery disease.

Materials and Methods

We included 124 aspirin-treated patients with coronary artery disease and normal to moderately reduced renal function. All tests were performed one hour after aspirin ingestion. Renal function was assessed using creatinine, estimated glomerular filtration rate (eGFR), and cystatin C. The antiplatelet effect of aspirin was evaluated using the VerifyNow® Aspirin assay and multiple electrode aggregometry (MEA, Multiplate®) induced by collagen (1.0 μg/mL) and arachidonic acid (1.0 mmol/L). Von Willebrand factor was measured as a marker of endothelial dysfunction. Platelet turnover was evaluated by measurements of immature, reticulated platelets.

Results

Renal function did not influence the antiplatelet effect of aspirin evaluated by MEA (r = − 0.2-0.09, p = 0.03-0.77) or the VerifyNow® (r = − 0.12-0.11, all p-values > 0.1). In contrast, renal function correlated inversely with von Willebrand factor levels (r_creatinine = 0.48, p < 0.0001; r_eGFR = − 0.46, p < 0.001; r_{cystatin C} = 0.54, p < 0.0001). The number of immature platelets correlated with platelet aggregation according to MEA (r = 0.20-0.39, all p-values < 0.03), but not according to VerifyNow® (r = − 0.07, p = 0.50).

Conclusions

A reduced antiplatelet effect of aspirin may be explained by an increased number of immature platelets. Moderately impaired renal function was associated with high levels of von Willebrand factor, but not with a reduced antiplatelet effect of aspirin.     

Markers of endothelial and platelet activation are associated with high on-aspirin platelet reactivity in patients with stable coronary artery disease

Introduction

Aspirin inhibits the cyclooxygenase-1 (COX-1) mediated thromboxane A2 synthesis. Despite COX-1 inhibition, in patients with coronary artery disease (CAD), platelets can be activated through other mechanisms, like activation by thrombin.

Materials and Methods

At baseline in this cross-sectional substudy of the ASCET trial, 1001 stable CAD patients, all on single aspirin treatment, were classified by the PFA100® method, as having high on-aspirin residual platelet reactivity (RPR) or not. Markers of hypercoagulability, endothelial and platelet activation as related to RPR, were evaluated to explore the potential mechanisms behind high on-aspirin RPR.

Results

Altogether, 25.9% (n = 259) of the patients were found to have high on-aspirin RPR. S-thromboxane B₂ levels were very low and did not differ between patients having high on-aspirin RPR or not. Patients with high on-aspirin RPR had significantly higher levels of von Willebrand Factor (vWF) (124 vs 100%, p < 0.001, platelet count (236 vs 224 × 10⁹/l, p = 0.008), total TFPI (68.4 vs 65.5 ng/ml, p = 0.005) and ß-thromboglobulin (ß-TG) (33.3 vs 31.3 IU/ml, p = 0.041) compared to patients with low on-aspirin RPR. No significant differences between the groups were observed in levels of endogenous thrombin generation (ETP), pro-thrombin fragment 1+2 (F1+2), D-dimer, soluble TF (sTF) or P-selectin (all p > 0.05).

Conclusions

The high on-aspirin RPR as defined by PFA100® seems not to be due to increased thrombin activity as evaluated with ETP, sTF, F1+2 or D-dimer. The elevated levels of platelet count, ß-TG, TFPI and especially vWF might be explained by increased endothelial and platelet activation in these patients.     

Platelet inhibition assessed with VerifyNow, flow cytometry and PlateletMapping in patients undergoing heart surgery

Introduction

A substantial number of patients with coronary artery disease undergo cardiac surgery within five days of discontinuing anti-platelet treatment with aspirin and clopidogrel. The aims of this study were to describe the degree of platelet inhibition in patients with dual anti-platelet treatment scheduled for coronary artery bypass graft (CABG) surgery and to investigate whether the measured platelet inhibition correlated to intra- and postoperative risk for bleeding and transfusion requirements.

Material and Methods

Sixty patients were included. Platelet inhibition was analysed with flow cytometry including phosphorylation status of the vasodilator-stimulated phosphoprotein (VASP-assay) and two bed-side analyzers, VerifyNow-System and PlateletMapping, a modified thrombelastograph. All 60 patients were analysed with VerifyNow and PlateletMapping, and 48 were analysed with flow cytometry and VASP-assay.

Results

There was a correlation between the ADP-receptor inhibition as measured by VASP-assay and VerifyNowP2Y₁₂ (r = - 0.29, p < 0.05), and between VASP-assay and the expression of P-selectin (r = 0.29, p < 0.05) as measured by flow cytometry when platelets were stimulated with 5 µM ADP. VerifyNowP2Y₁₂ was the only measurement of platelet inhibition correlated to total blood loss (Spearman r = 0.29, p = 0.03) and red blood cell transfusion (Spearman r = 0.43, p < 0.01) requirements, although this might be confounded by aprotinin treatment.

Conclusion

We found a modest agreement between the methods for preoperative platelet inhibition, though not for PlateletMapping-MA_ADP. There was a correlation between preoperative platelet inhibition measured by VerifyNowP2Y₁₂ and surgical blood loss or transfusion requirements. However, for the individual patient, preoperative use of VerifyNowP2Y₁₂ as an instrument to decide bleeding and transfusion risk does not seem helpful.     

The effect of clopidogrel besylate and clopidogrel hydrogensulfate on platelet aggregation in patients with coronary artery disease: a retrospective study

Background

Recently several alternative forms of the original clopidogrel hydrogensulfate (CHS) were spread worldwide. A large amount of such drugs turned out to be clopidogrel besylate (CB). Only three studies, involving healthy volunteers, investigated the antiplatelet effect of CB, whereas its attribute remained unexplored in the case of patients with cardiovascular diseases. This retrospective study aimed to evaluate the difference between the antiplatelet effects of two clopidogrel formulas, CHS and CB, on patients with coronary artery diseases.

Methods

Data of 150 patients with previous CHS treatment were investigated. According to the documentations, the CHS therapy was shifted to CB. 94 patients of the selected population received dual antiplatelet therapy, clopidogrel and aspirin. The antiplatelet effects of CHS and CB were compared by ADP induced platelet aggregation measurements using light transmission aggregometry.

Results

Irrespective of the therapeutic combinations the performed statistical investigations failed to show significant difference (p = 0.30) between the effect of CB (AGGmax_CB: 27.6 ± 13.7%) or CHS (AGGmax_CHS: 29.0 ± 15.3%) on the ADP induced platelet aggregation. Insignificant deviations were found in both forms of clopidogrel salts, either in the lack (AGGmax_CB : 32.5 ± 14,2%; AGGmax_CHS: 34,0 ± 16,1%; p = 0,29) or in the presence of aspirin (AGGmax_CB: 24.7 ± 12,5%; AGGmax_CHS: 26,0 ± 14,1%; p = 0,31).

Conclusion

Our results indicated that both CB and CHS had an identical inhibitory effect on ADP induced platelet aggregation in patients with cardiovascular diseases. Moreover their efficiency showed no overall significant difference in the case of dual antiplatelet therapy with aspirin as well. However there might be an inter- and intraindividual variability between the two clopidogrel formulas.     

Aspirin response evaluated by the VerifyNow Aspirin System and light transmission aggregometry

Introduction

Patients with inadequate platelet inhibition by aspirin, referred to as aspirin resistance, might have an increased risk of suffering cardiovascular events. Therefore, identification of these patients by measuring platelet function is of great interest. Our objectives were to evaluate performance parameters of VerifyNow™ and to determine the agreement between VerifyNow™ and light transmission aggregometry (LTA) ad modum Born.

Materials and Methods

We included 21 healthy volunteers and 40 patients with stable coronary artery disease. Duplicate measurements of platelet aggregation were performed using VerifyNow™ and LTA (arachidonic acid 1.0 mM) in healthy volunteers before aspirin and in all participants on four consecutive days during treatment with non-enteric-coated aspirin 75 mg daily. VerifyNow™ test results were expressed in Aspirin Reaction Units (ARU) and LTA test results in percent of maximal aggregation. The cut-off for determination of aspirin resistance was ≥ 550 ARU and ≥ 20%, respectively.

Results

All participants were compliant, confirmed by complete suppression of serum-thromboxane B₂. VerifyNow™ was highly repeatable with a coefficient of variance of 0.5% at baseline and 3.0% during aspirin treatment. No individuals were identified as aspirin resistant with VerifyNow™, whereas seven (12%) individuals were identified with LTA. ROC analysis using LTA as the gold standard showed poor sensitivity and good specificity with a cut-off at 550 ARU.

Conclusion

VerifyNow™ was highly repeatable, but further studies are needed to investigate the relevance of the cut-off level at 550 ARU for detecting aspirin resistance.     

Aspirin response variability after major orthopedic surgery

Introduction

Variability in platelet response to aspirin has been reported in patients undergoing cardiac surgery but has rarely been described in other operative settings and its mechanism remains uncertain. We performed a prospective cohort study to investigate the variability in platelet response to aspirin and to explore its mechanism in patients undergoing major orthopedic surgery.

Materials and Methods

Twelve aspirin-treated patients undergoing elective hip or knee replacement were recruited. Once-daily aspirin was continued throughout the perioperative period. We measured platelet function using light transmission aggregation (LTA) in response to arachidonic acid (PL_AA) and serum thromboxane B₂ (TXB₂) at baseline (before surgery) as well as on days 1, 2, 3, 4, 5, 6, and 8 after surgery. We defined aspirin low response as a PL_AA > 20%.

Results

Six patients exhibited aspirin low response, which typically started on post-operative days 3 or 4; the remaining 6 patients had normal response to aspirin. Compared to aspirin responders, patients with aspirin low response showed significantly higher serum TXB₂ levels, a more pronounced early decrease in platelet count, and a significantly more rapid recovery of the platelet count after surgery.

Conclusion

Aspirin response variability occurred in patients after major orthopedic surgery, with one-half of the patients in our study exhibiting post-operative aspirin low response. Increased platelet turnover might be a contributor to aspirin response variability after orthopedic surgery.     

Evaluation of platelet function and pharmacological platelet inhibition in patients with myeloproliferative disorders using multiple electrode aggregometry

Background

The aim of this study was to describe platelet aggregation characteristics by multiple electrode aggregometry (MEA) and to evaluate MEA for its potential to detect platelet dysfunction and response to anti-aggregatory drugs in patients with myeloproliferative disorders (MPD).

Methods

We compared the platelet response to arachidonic acid (ASPI test), adenosine diphosphate (ADP test) and thrombin receptor activating peptide (TRAP test) in hirudin-anticoagulated blood of 55 patients with polycythaemia vera and essential thrombocythaemia and 75 controls.

Results

Comparing MPD patients and controls no statistically significant difference indicative of platelet dysfunction was found in MPD patients. Analysis of covariance revealed platelet- and leukocyte count as a significant influencing factor on MEA function. Furthermore we could demonstrate that ASA and clopidogrel treatment results in a statistically significant lower ASPI (Controls: p < 0.0001, MPD: p < 0.0001) and ADPtest value (MPD: p = 0.00125) compared to untreated patients thereby validating the method for monitoring of anti-aggregatory therapy.

Conclusion

In this study MEA was confirmed as a valid method for monitoring of ASA and clopidogrel treatment in patients with MPD and normal control subjects. The platelet and leukocyte count were identified as major influencing factors on MEA aggregation tests both in MPD patients and controls. No functional platelet abnormalities were detected in MPD patients.