Alexander O. Spiel

Effects of pantoprazole and esomeprazole on platelet inhibition by clopidogrel

BACKGROUND Clopidogrel is activated by CYP2C19, which also metabolizes proton pump inhibitors (PPI). As proton pump inhibitors are metabolized to varying degrees by CYP2C19, we hypothesized that the reported negative omeprazole-clopidogrel drug interaction may not be a class effect. METHODS Responsiveness to clopidogrel was assessed by the vasodilator-stimulated phosphoprotein phosphorylation (VASP) assay and aggregometry (Multiplate Analyzer) in 300 patients with coronary artery disease (CAD) undergoing percutaneous coronary intervention (PCI). RESULTS The mean platelet reactivity index (PRI, assessed by the VASP assay) was nearly the same in patients with (n = 226; PRI = 51%) or without PPI treatment (n = 74; PRI = 49%; P = .724). Likewise, the adenosine diphosphate-induced platelet aggregation did not differ significantly between patients with or without PPI treatment (45 vs. 41 U; P = .619). Similarly, there was no difference in the PRI or the adenosine diphosphate-induced platelet aggregation between patients with pantoprazole (n = 152; PRI = 50%; aggregation = 47 U), esomeprazole (n = 74; PRI = 54%; aggregation = 42 U), or without PPI (n = 74; PRI = 49%; aggregation = 41 U; P = .382). CONCLUSION In contrast to the reported negative omeprazole-clopidogrel drug interaction, the intake of pantoprazole or esomeprazole is not associated with impaired response to clopidogrel.

Von Willebrand Factor in Cardiovascular Disease Focus on Acute Coronary Syndromes

von Willebrand factor (VWF) plays a pivotal role in platelet adhesion and aggregation at sites of high shear rates (eg, in coronary arteries that have stenotic or ruptured atherosclerotic plaque lesions). Numerous studies have investigated the relationship between VWF plasma levels and thromboembolic cardiovascular events. In contrast to the rather weak association in the general population, in patients with preexisting vascular disease, VWF is significantly predictive for adverse cardiac events, including death. Likewise, VWF typically rises during the course of acute coronary syndrome, and the extent of this VWF release is an independent predictor of adverse clinical outcome in these patients. Various lines of evidence indicate that VWF is not only a marker but also actually an important effector in the pathogenesis of myocardial infarction. This central role of VWF in thrombogenesis has made it a promising target for research into new antiplatelet therapies that specifically inhibit VWF. This review focuses on the role of VWF in acute coronary syndrome and further outlines the relevance of therapeutic interventions targeting VWF for acute coronary syndrome patients.

Validation of rotation thrombelastography in a model of systemic activation of fibrinolysis and coagulation in humans

Summary.  Background: Thrombelastography (TEG) is a whole blood assay to evaluate the viscoelastic properties during blood clot formation and clot lysis. Rotation thrombelastography (e.g. ROTEM®) has overcome some of the limitations of classical TEG and is used as a point‐of‐care device in several clinical settings of coagulation disorders. Endotoxemia leads to systemic activation of the coagulation system and fibrinolysis in humans. Objectives: We validated whether ROTEM® is sensitive to endotoxin induced, tissue factor‐triggered coagulation and fibrinolysis and if its measures correlate with biohumoral markers of coagulation and fibrinolysis. Patients and methods: Twenty healthy male volunteers participated in this randomized placebo‐controlled trial. Volunteers received either 2 ng kg−1 National Reference Endotoxin or saline. Results: Endotoxemia significantly shortened ROTEM® clotting time (CT) by 36% (CI 0.26–0.46; P < 0.05) with a strong inverse correlation with the peak plasma levels of prothrombin fragments (F1 + 2) (r = −0.83, P < 0.05). Additionally, endotoxin infusion enhanced maximal lysis (ML) 3.9‐fold (CI: 2.5–5.2) compared with placebo or baseline after 2 h (P < 0.05). Peak ML and peak tissue plasminogen activator (t‐PA) values correlated excellently (r = 0.82, P < 0.05). ROTEM® parameters clot formation time and maximal clot firmness were not affected by LPS infusion, whereas platelet function analyzer (PFA‐100) closure times decreased. Conclusions: Rotation thrombelastography (ROTEM®) detects systemic changes of in vivo coagulation activation, and importantly it is a point of care device, which is sensitive to changes in fibrinolysis in humans. The ex vivo measures CT and ML correlate very well with established in vivo markers of coagulation activation (F1 + 2) and fibrinolysis (t‐PA), respectively.

Platelet function in patients with acute coronary syndrome (ACS) predicts recurrent ACS

Summary.  Background: Platelet hyperfunction contributes to acute coronary syndromes (ACS). Thus, we hypothesized that platelet function under high shear stress predicts recurrent ACS during long‐term follow‐up of ACS patients. Patients and methods: Consecutive ACS patients (n = 208) were prospectively followed‐up for an average of 28 months. Platelet function was measured with the platelet function analyzer (PFA‐100®; Dade Behring, Marburg, Germany) at baseline for collagen/adenosine diphosphate closure times (CADP‐CT) and for collagen/epinephrine closure times (CEPI‐CT) after infusion of a uniform dose of 250 mg aspirin. Results: Of the conventional risk factors, only the prevalence of diabetes was higher in ACS patients with re‐events. However, use of clopidogrel and use of beta blockers were also slightly lower in patients with re‐events (P < 0.05). The unadjusted risk hazard ratio (HR) for re‐events was 3.3 [95% confidence interval (95% CI): 1.4–7.4; P = 0.005] in those patients with the shortest CADP‐CT values (lowest quartile). Similarly, the risk was 2.0‐fold higher (95% CI: 1.1–3.6; P = 0.02) in ACS patients with CEPI‐CT < 300 s as compared with CEPI‐CT ≥ 300 s. Inclusion of diabetes, clopidogrel and beta blockers in a multivariate Cox regression model enhanced the predictive value of CEPI‐CT (HR: 2.7). Inclusion of von Willebrand factor levels did not alter the HR for recurrent ACS (HR: 2.1; 95% CI: 1.1–5.2; P = 0.03) for CEPI‐CT < 300 s, but reduced the HR for CADP‐CT (HR: 2.8, 95% CI: 0.8–9.8; P = 0.11). Conclusion: Shortened CT values reflect biologically relevant platelet hyperfunction in patients with ACS because they predict recurrent ACS.

Interspecies differences in coagulation profile

Many animals are used in research on blood coagulation and fibrinolysis, but the relevance of animal models to human health is often questioned because of differences between species. The objective was to find an appropriate animal species, which mimics the coagulation profile in humans most adequately. Species differences in the coagulation profile with and without thrombin stimulation in vitro were assessed in whole blood by Rotation Thromboelastometry (ROTEM). Endogenous thrombin generation was measured in platelet-poor plasma. Measurements were performed in blood from five different species: humans, rats, pigs, sheep and rabbits. In humans and sheep, the clotting time (ROTEM) was in the same range with or without thrombin stimulation and a 100-fold lower dose of thrombin (0.002 IU) was required to cause a shortening in the clotting time as compared to rats, pigs and rabbits (0.2 IU) (p<0.05). Similarly, the endogenous thrombin potential (ETP) was in the same range in humans and sheep. The maximum clot firmness with or without thrombin stimulation was similar in rabbits and humans. The maximum lysis with or without thrombin stimulation was similar in humans and pigs. Significant species differences exist in the coagulation profile with or without thrombin stimulation. Most importantly, sheep had a clotting time most similar to humans and could thus be a suitable species for translational coagulation studies. Moreover, our findings confirm the potential usefulness of pigs as an experimental species to study fibrinolytic pathway and support the usefulness of rabbits as a species for examining platelets.

In vivo profile of the human leukocyte microRNA response to endotoxemia.

To gain insight into microRNAs (miRNAs) involved in the regulation of the human innate immune response, we screened for differentially expressed miRNAs in circulating leukocytes in an in vivo model of acute inflammation triggered by Escherichia coli lipopolysaccharide (LPS) infusion. Leukocyte RNA was isolated from venous blood samples obtained from healthy male volunteers before and 4h after LPS-infusion. After fluorescence labeling, RNA samples were hybridized to microarrays containing capture probes for measuring the abundance of more than 600 human miRNAs. Target genes were predicted for differentially expressed miRNAs and then compared to changes in genome-wide expression levels, which had been established in a previous study. Data analysis revealed that five miRNAs consistently responded to LPS-infusion, four of which were down-regulated (miR-146b, miR-150, miR-342, and let-7g) and one was up-regulated (miR-143). The miR-150 and mir-342 response was confirmed by real-time PCR. By correlating to measured LPS-induced changes of the leukocyte transcriptome, we next searched for predicted target genes, whose stability might be under (co-) control by these miRNAs. We found that the rapid transcriptional activation during acute inflammation of select genes, such as the gene encoding interleukin-1 receptor-associated kinase 2 (IRAK2) might be facilitated by decreased levels of LPS-responsive miRNAs. The increased level of miR-143 might be associated with the pronounced down-regulation of the B-cell CLL/lymphoma 2 (BCL2) gene expression during LPS endotoxemia, and could further be involved in the translational silencing of several other predicted inflammation-related target genes. This is the first in vivo study to demonstrate relative abundance of miRNA levels in peripheral blood leukocytes during acute LPS-induced inflammation. The miRNAs and their potential target genes identified herein contribute to the understanding of the complex transcriptional program of innate immunity initiated by pathogens.

Duffy antigen modifies the chemokine response in human endotoxemia

Objective:The Duffy receptor is a promiscuous receptor for chemokines and selectively binds CXC and CC chemokines with high affinity. Preclinical data show that presence of the Duffy receptor on red blood cells may influence plasma levels of proinflammatory cytokines and chemokines and be protective during inflammation. This trial was designed to investigate the influence of the Duffy antigen on human inflammation in vivo. Design:Prospective, analyst-blinded clinical trial. Patients:A total of 32 healthy male volunteers: 16 Duffy-positive white subjects and 16 Duffy-negative subjects of African descent. Measurements and Main Results:All subjects received an intravenous bolus of 2 ng/kg endotoxin (lipopolysaccharide). Cytokines, chemokines, and their receptors were quantified by enzyme immunoassay, reverse transcriptase–polymerase chain reaction, and flow cytometry. Results:Plasma levels of tumor necrosis factor, interleukin-6, interleukin-10, and whole blood growth-related oncogen-α, monocyte chemoattractant protein-1, and interleukin-8 messenger RNA increased similarly in both groups after lipopolysaccharide infusion. Monocyte chemoattractant protein-1 peak plasma levels were roughly two-fold higher in Duffy-positive subjects compared with Duffy-negative subjects (16 ng/mL vs. 7 ng/mL, p < .0001). Similarly, growth-related oncogen-α levels were 2.5-fold higher in Duffy-positive subjects 2 hrs after lipopolysaccharide infusion (210 pg/mL vs. 85 pg/mL; p < .001). Erythrocyte-bound monocyte chemoattractant protein-1, growth-related oncogen-α, and interleukin-8 increased 20- to 50-fold in Duffy-positive subjects (p < .00001 vs. baseline). Conclusion:The Duffy antigen substantially alters chemokine concentrations in blood, but it does not have a protective effect during human endotoxemia.

Hemostasis in cardiac arrest patients treated with mild hypothermia initiated by cold fluids

AIM OF THE STUDY Application of mild hypothermia (32-33 degrees C) has been shown to improve neurological outcome in patients with cardiac arrest. However, hypothermia affects hemostasis, and even mild hypothermia is associated with bleeding and increased transfusion requirements in surgery patients. On the other hand, crystalloid hemodilution has been shown to induce a hypercoagulable state. The study aim was to elucidate in which way the induction of mild therapeutic hypothermia by a bolus infusion of cold crystalloids affects the coagulation system of patients with cardiac arrest. METHODS This was a prospective pilot study in 18 patients with cardiac arrest and return of spontaneous circulation (ROSC). Mild hypothermia was initiated by a bolus infusion of cold 0.9% saline fluid (4 degrees C; 30ml/kg/30min) and maintained for 24h. At 0h (before hypothermia), 1, 6 and 24h we assessed coagulation parameters (PT, APPT), platelet count and performed thrombelastography (ROTEM) after in vitro addition of heparinase. RESULTS A total amount of 2528 (+/-528)ml of 0.9% saline fluid was given. Hematocrit (p<0.01) and platelet count (-27%; p<0.05) declined, whereas APTT increased (2.7-fold; p<0.01) during the observation period. All ROTEM parameters besides clotting time (CT) after 1h (-20%; p<0.05) did not significantly change. CONCLUSION Mild hypothermia only slightly prolonged clotting time as measured by rotation thrombelastography. Therefore, therapeutic hypothermia initiated by cold crystalloid fluids has only minor overall effects on coagulation in patients with cardiac arrest.

The aptamer ARC1779 is a potent and specific inhibitor of von willebrand factor mediated ex vivo platelet function in acute myocardial infarction

ARC1779 is an aptamer, which blocks binding of the von Willebrand Factor (VWF) A1 domain to platelet GPIb receptors. VWF is increased in the elderly an in the setting of acute myocardial infarction (AMI), as reflected by increased shear-dependent platelet function. We hypothesized that ARC1779 concentration-dependently inhibits ex vivo platelet function, and that this concentration effect relationship may be shifted in patients with AMI. We studied ex vivo dose response curves for ARC1779 on VWF activity, shear-dependent platelet function, and agonist-induced platelet aggregation. We included patients with AMI on standard treatment (n = 40), young (n = 20) and elderly controls (n = 20) in this ex vivo dosing study. AMI patients displayed ∼2-fold increased plasma levels of VWF activity as compared to controls. ARC1779 inhibited VWF activity (IC90: ∼3–4 µg/mL) and shear dependent platelet function (Platelet Function Analyzer (PFA-100), IC50: ∼0.5–0.9 µg/mL and Cone and Plate Analyzer (CPA), IC50: ∼0.1–0.4 µg/mL in citrated blood) at comparable concentrations in all groups. In contrast to GPIIb/IIIa antagonists, ARC1779 did not inhibit platelet aggregation induced by ADP, collagen or arachidonic acid at concentrations (10 µg/mL) that fully inhibited VWF dependent platelet function. ARC1779 potently and specifically inhibits VWF activity and VWF dependent platelet function, even in the setting of AMI where VWF activity is increased.

Increased platelet aggregation and in vivo platelet activation after granulocyte colony-stimulating factor administration. A randomised controlled trial.

Granulocyte colony-stimulating factor (G-CSF) stimulates the bone marrow to produce granulocytes and stem cells and is widely used to accelerate neutrophil recovery after chemotherapy. Interestingly, specific G-CSF receptors have been demonstrated not only on myeloid cells, but also on platelets. Data on the effects of G-CSF on platelet function are limited and partly conflicting. The objective of this study was to determine the effect of G-CSF on platelet aggregation and in vivo platelet activation. Seventy-eight, healthy volunteers were enrolled into this randomised, placebo-controlled trial. Subjects received 5 μg/kg methionyl human granulocyte colony-stimulating factor (r-metHuG-CSF, filgrastim) or placebo subcutaneously for four days. We determined platelet aggregation with a whole blood impedance aggregometer with various, clinically relevant platelet agonists (adenosine diphosphate [ADP], collagen, arachidonic acid [AA], ristocetin and thrombin receptor activating peptide 6 [TRAP]). Filgrastim injection significantly enhanced ADP (+40%), collagen (+60%) and AA (+75%)-induced platelet aggregation (all p<0.01 as compared to placebo and p<0.001 as compared to baseline). In addition, G-CSF enhanced ristocetin-induced platelet aggregation (+18%) whereas TRAP-induced platelet aggregation decreased slightly (-14%) in response to filgrastim. While baseline aggregation with all agonists was only slightly but insignificantly higher in women than in men, this sex difference was enhanced by G-CSF treatment, and became most pronounced for ADP after five days (p<0.001). Enhanced platelet aggregation translated into a 75% increase in platelet activation as measured by circulating soluble P-selectin. G-CSF enhances platelet aggregation and activation in humans. This may put patients suffering from cardiovascular disease and cancer at risk for thrombotic events.