Pernille Just Vinholt

The antiplatelet effect of clopidogrel is not attenuated by statin treatment in stable patients with ischemic heart disease

Recent studies suggest that cytochrome P450 (CYP) 3A4 metabolized statins attenuate the antiaggregatory effect of clopidogrel. We evaluated how CYP3A4 metabolized statins and non-CYP3A4 metabolized statins influence platelet aggregation when given concomitantly with clopidogrel. Sixty-six stable patients with ischemic heart disease were included in this parallel group study. All patients were on clopidogrel and aspirin. Thirty-three patients received a CYP3A4 metabolized statin (simvastatin or atorvastatin), and 33 were treated with a non-CYP3A4 metabolized statin (pravastatin). The antiplatelet effect of clopidogrel was assessed at inclusion and 21 days after statin discontinuation. Platelet function was evaluated by two methods 1) optical platelet aggregometry after stimulation with 20 and 30 microM ADP, and 2 and 4 mg/l collagen, respectively, 2) a Platelet FunctionAnalyzer-100. The primary effect measure was final platelet aggregation after stimulation with 20 microM ADP. No difference was observed between patients treated with a CYP3A4 metabolized statin and patients receiving a non-CYP3A4 metabolized statin (30% point (7-42) versus 20% point (9-32), p = 0.83). Platelet aggregation was not improved by discontinuation of statins for 21 days. Indeed, we found that statin treatment given concomitantly with clopidogrel resulted in an improved platelet inhibition when compared to clopidogrel given alone. The antiplatelet effect of clopidogrel is not attenuated by concomitant treatment with a CYP3A4 metabolized statin in patients with clinical stable ischemic heart disease.

An overview of platelet indices and methods for evaluating platelet function in thrombocytopenic patients

Thrombocytopenia is associated with bleeding risk. However, in thrombocytopenic patients, platelet count does not correlate with bleeding risk and other factors are thus likely to contribute to this risk. This review presents currently available platelet‐related markers available on automated haematology analysers and commonly used methods for testing platelet function. The test principles, advantages and disadvantages of each test are described. We also evaluate the current literature regarding the clinical utility of the test for prediction of bleeding in thrombocytopenia in haematological and oncological diseases. We find that several platelet‐related markers are available, but information about the clinical utility in thrombocytopenia is limited. Studies support that mean platelet volume (MPV) can aid diagnosing the cause of thrombocytopenia and low MPV may be associated with bleeding in thrombocytopenia. Flow cytometry, platelet aggregometry and platelet secretion tests are used to diagnose specific platelet function defects. The flow cytometric activation marker P‐selectin and surface coverage by the Cone‐and‐Plate[let] analyser predict bleeding in selected thrombocytopenic populations. To fully uncover the clinical utility of platelet‐related tests, information about the prevalence of platelet function defects in thrombocytopenic conditions is required. Finally, knowledge of the performance in thrombocytopenic samples from patients is essential.

Platelet count is associated with cardiovascular disease, cancer and mortality: A population-based cohort study.

INTRODUCTION Platelet count is used to determine bleeding risk and monitoring thrombopoiesis. While abnormal platelet counts are associated with mortality and morbidity, it is unclear whether it also apply to platelet counts within reference range. We investigated the relationship between platelet count (100-450×109/L) and mortality, development of future cardiovascular disease (myocardial infarction, ischaemic stroke, or peripheral vascular disease), venous thromboembolism, bleeding or cancer in the general population. MATERIAL AND METHODS We conducted a register-based cohort study of 21,252 adults (≥20years) from the Danish General Suburban Population Study (GESUS). Laboratory results from GESUS were linked to information from national registers regarding morbidity and death. Cox proportional hazard regression was conducted with adjustment for age, sex, smoking status, haemoglobin, leukocyte count, C-reactive protein and Charlson comorbidity index. RESULTS We found a U-shaped relationship between mortality and platelet count. Mortality was significantly increased for platelet count <175×109/L or >300×109/L. When categorizing platelet count using the interval 201-250×109/L as reference group, platelet count 301-450×109/L was associated with mortality, adjusted hazard ratio (HR)=1.42(95% CI 1.06-1.90) and cardiovascular disease, adjusted HR=1.32 (95% CI 1.03-1.69). Platelet count 100-200×109/L was associated with future cancer, adjusted HR=1.28(95% CI 1.05-1.57), but not with future bleeding or venous thromboembolism. CONCLUSIONS Platelet count is associated with mortality, future cardiovascular disease, and future cancer.

Does C-reactive protein independently predict mortality in adult community-acquired bacteremia patients with known sepsis severity?

We evaluated whether sepsis severity and C‐reactive protein (CRP) level on admission prognostically corroborated or annulled each other in adult patients with incident community‐acquired bacteremia (Funen, Denmark, 2000–2008). We used logistic regression and area under the receiver operating characteristic curve (AUC) to evaluate 30‐day mortality in four models: (i) age, gender, comorbidity, bacteria, and ward. (ii) Model 1 and sepsis severity. (iii) Model 1 and CRP. (iv) Model 1, sepsis severity, and CRP. Altogether, 416 of 1999 patients died within 30 days. CRP independently predicted 30‐day mortality [Model 4, odds ratio (95% CIs) for 100 mg/L: 1.16 (1.06–1.27)], but it did not contribute to the AUC (Model 2 vs Model 4: p = 0.31). In the 963 non‐severe sepsis patients, CRP independently predicted 30‐day mortality [Model 4: 1.42 (1.20–1.69)] and it increased the AUC (Model 2 vs Model 4: p = 0.06), thus CRP contributed as much as sepsis severity to prognosis.

The effect of centrifugation speed and time on pre-analytical platelet activation.

Abstract Background: The results of laboratory analyses are affected by pre-analytical variables, and in particular can platelets be activated by shear handling stress and secrete granular substances. We therefore evaluated the effect of centrifugation speed and time on pre-analytical platelet activation. Methods: Citrate- and EDTA-anticoagulated blood from healthy volunteers were centrifuged at 80–10,000 g for 5–15 min to prepare plasma and platelet-rich plasma. Pre-analytical platelet activation was assessed by flow cytometric measurement of platelet P-selectin (CD62p) expression. Blood cell counts, mean platelet volume (MPV), immature platelet fraction (IPF), and platelet distribution width (PDW) were measured. Platelet aggregation in platelet-rich plasma induced by arachidonic acid (AA), ADP or thrombin receptor activator peptide-6 (TRAP) was tested by 96-well aggregometry. Results: The median percentage of platelets expressing P-selectin in citrate- and EDTA-plasma centrifuged at 2000 g for 10 min were 43% [interquartile range (IQR), 38%–53%] and 56% (IQR, 31%–78%), respectively (p=0.82). Platelet-rich plasma prepared at 100–250 g for 10 min had significantly lower platelet P-selectin expression (11%–15%), p<0.001. Platelet count in plasma samples decreased with increasing speed but platelets were only completely removed if plasma was re-centrifuged. In platelet-rich plasma, increasing centrifugation speed significantly increased platelet yield but decreased contamination from other blood cells, platelet composition was altered as platelet parameters (MPV, IPF, and PDW) was lowered. Platelet aggregation was not affected by the centrifugation speed platelet-rich plasma was prepared. Conclusions: Proportional to centrifugation speed, platelets in plasma and platelet-rich plasma were activated with centrifugation speed, cell content and composition changed while platelet aggregation was unaltered.

Measurement of platelet aggregation, independently of patient platelet count: A flow-cytometric approach

Essentials Platelet function may influence bleeding risk in thrombocytopenia, but useful tests are needed. A flow cytometric platelet aggregation test independent of the patient platelet count was made. Platelet aggregation was reduced in thrombocytopenic patients with hematological cancer. High platelet aggregation ruled out bleeding tendency in thrombocytopenic patients.

CYP2C19*17 increases clopidogrel-mediated platelet inhibition but does not alter the pharmacokinetics of the active metabolite of clopidogrel†

The aim of the present study was to determine the impact of CYP2C19*17 on the pharmacokinetics and pharmacodynamics of the active metabolite of clopidogrel and the pharmacokinetics of proguanil. Thus, we conducted an open‐label two‐phase cross‐over study in 31 healthy male volunteers (11 CYP2C19*1/*1, 11 CYP2C19*1/*17 and nine CYP2C19*17/*17). In Phase A, the pharmacokinetics of the derivatized active metabolite of clopidogrel (CAMD) and platelet function were determined after administration of a single oral dose of 600 mg clopidogrel (Plavix; Sanofi‐Avensis, Horsholm, Denmark). In Phase B, the pharmacokinetics of proguanil and its metabolites cycloguanil and 4‐chlorphenylbiguanide (4‐CPB) were determined in 29 of 31 subjects after a single oral dose of 200 mg proguanil given as the combination drug Malarone (GlaxoSmithKline Pharma, Brondby, Denmark). Significant correlations were found between the area under the time–concentration curve (AUC0–∞) of CAMD and both the absolute ADP‐induced P2Y12 receptor‐activated platelet aggregation (r = −0.60, P = 0.0007) and the percentage inhibition of aggregation (r = 0.59, P = 0.0009). In addition, the CYP2C19*17/*17 and CYP2C19*1/*17 genotype groups had significantly higher percentage inhibition of platelet aggregation compared with the CYP2C19*1/*1 subjects (geometric mean percentage inhibition of 84%, 73% and 63%, respectively; P = 0.014). Neither the absolute ADP‐induced P2Y12 receptor‐activated platelet aggregation, exposure to CAMD nor the pharmacokinetic parameters of proguanil, cycloguanil and 4‐CPB exhibited any significant differences among the genotype groups. In conclusion, carriers of CYP2C19*17 exhibit higher percentage inhibition of platelet aggregation, but do not have significantly lower absolute P2Y12 receptor‐activated platelet aggregation or higher exposure to the active metabolite after a single oral administration of 600 mg clopidogrel.

An ELISA for the quantitation of von Willebrand Factor: Osteoprotegerin complexes in plasma

BACKGROUND Von Willebrand factor (VWF) is pivotal in arterial thrombosis, and osteoprotegerin (OPG) is besides being a bone protein also related to cardiovascular diseases. OPG can bind VWF, but the significance of this interaction is not known. OBJECTIVES The aim was to develop an assay for measurement of von Willebrand factor-osteoprotegerin complex (VWF:OPG) in human plasma. Furthermore, the significance of VWF:OPG complex as a marker of cardiovascular disease (CVD) was evaluated. PATIENTS/METHODS A sandwich ELISA for quantification of VWF:OPG was developed using a polyclonal rabbit anti-human VWF capturing antibody and a monoclonal anti-human OPG detecting antibody. Samples were quantified relative to a standard curve obtained from dilutions of a plasma pool from healthy individuals. The assay was evaluated in two groups of patients with CVD and two groups of asymptomatic individuals with and without documented coronary calcification (total n=118). RESULTS AND CONCLUSIONS The assay detected VWF:OPG complexes in human plasma, while no significant signal was observed when testing solutions containing VWF or recombinant OPG alone. Importantly, the ELISA assay was able to detect in vitro formed complexes between human VWF and recombinant OPG in a dose-dependent manner. There was a large inter-individual variation in plasma VWF:OPG levels, but we found no significant differences in the level of VWF:OPG complexes between the four groups. Thus, we conclude that increasing OPG plasma levels in atherosclerotic CVD are not derived from increased levels of complexed form of VWF and OPG, but are more likely due to increased amounts of OPG secreted into the circulation.

Single test isolated lupus anticoagulant positivity is associated with increased plasma levels of inflammatory markers and dyslipidemia

Objective To investigate whether a single positive test for lupus anticoagulant (LA) is associated with levels of inflammatory markers and traditional cardiovascular risk factors, independent of autoimmune disease, thrombophilia and occurrence of other antiphospholipid antibodies. Methods In a retrospective observational study we included persons referred for thrombophilia testing during 2011−2014. Persons with autoimmune disease, thrombophilia or presence of specific anti-phospholipid antibodies were excluded. Multivariate logistic regression analyses adjusted for age and sex was performed and odds ratios (ORs) with 95% confidence intervals (95% CI) calculated. Results Of 381 individuals tested, 271 fulfilled the criteria, of whom 22 (8%) were LA positive and 249 (92%) LA negative. LA positivity was associated with higher body mass index (BMI) (OR 1.12, 95% CI: 1.03–1.23, p = 0.01); C-reactive protein (OR 1.08 95% CI:1.04–1.11, p < 0.001); fibrinogen (OR 1.51 95% CI: 1.27–1.78, p < 0.001); coagulation factor VIII (FVIII) (OR 1.73 95% CI: 1.01–2.96, p = 0.046), low high density lipoprotein (HDL) (OR 0.03 95% CI: 0.00–0.19, p < 0.001) and high triglyceride (OR 1.81 95% CI: 1.12–2.92, p = 0.02) compared with LA negative individuals. Conclusion This study shows that single test isolated LA positivity is associated with increased levels of inflammatory markers, low HDL cholesterol, elevated triglyceride and high BMI.

Prediction of bleeding and prophylactic platelet transfusions in cancer patients with thrombocytopenia

Abstract Studies on markers for bleeding risk among thrombocytopenic cancer patients are lacking. This prospective observational cohort study investigated whether platelet parameters and a standardised bleeding questionnaire predicted bleeding or prophylactic platelet transfusions in patients with cancer and thrombocytopenia. Admitted adult patients with cancer and platelet count <80 × 109/L were enrolled, but excluded if they experienced surgery or trauma within 7 days or platelet transfusion within 14 days. Patients were interviewed, blood samples collected and, subsequently, spontaneous bleeding and prophylactic platelet transfusion within 30 days were registered. Of 197 patients enrolled, 56 (28%) experienced bleeding. In multivariate analyses, predictors of bleeding were infection (adjusted odds ratio (OR) = 2.65 and 95% confidence interval (95% CI) 1.04–6.74); treatment with platelet inhibitors, heparin or warfarin OR = 2.34, 95% CI 1.23–4.48; urea nitrogen OR = 1.15, 95% CI 1.07–1.25; creatinine OR = 1.01, 95% CI 1.01–1.01; and haemoglobin OR = 0.62, 95% CI 0.41–0.93. Specific information regarding previous gastrointestinal bleeding OR = 3.33, 95% CI 1.19–9.34 and haematuria OR = 3.00, 95% CI 1.20–7.52 predicted bleeding whereas the standardised bleeding questionnaire did not. Prophylactic platelet transfusions were administered to 97 patients. Predictors of prophylactic platelet transfusions were: platelet count OR = 0.96, 95% CI 0.94–0.97; fibrinogen OR = 0.88, 95% CI 0.83–0.95; mean platelet volume OR = 0.69, 95% CI 0.49–0.97; platelet aggregometry with OR = 2.48, 95% CI 1.09–5.64 for collagen-induced platelet aggregation within the lowest quartile; and albumin OR = 1.07, 95% CI 1.01–1.15. In conclusion, except for immature platelet fraction (IPF), platelet parameters predicted prophylactic platelet transfusion but not bleeding. Bleeding risk factors were previous haematuria or gastrointestinal bleeding, infection, antiplatelet or anticoagulant treatment, high urea nitrogen, low haemoglobin or high creatinine.