Rolf P. Kreutz

Impact of Proton Pump Inhibitors on the Effectiveness of Clopidogrel After Coronary Stent Placement: The Clopidogrel Medco Outcomes Study

Study Objective. To investigate the potential impact of proton pump inhibitors (PPIs) on the effectiveness of clopidogrel in preventing recurrent ischemic events after percutaneous coronary intervention (PCI) with stent placement.

Hypercoagulability, platelet function, inflammation and coronary artery disease acuity: results of the Thrombotic RIsk Progression (TRIP) study.

The objective of the study was to determine the relation of platelet reactivity, hypercoagulability and inflammation in various stages of coronary artery disease acuity (CAD). Thrombin-induced platelet-fibrin clot strength (MA), time to initial platelet-fibrin clot formation (R), C-reactive protein (CRP), prothrombotic factors, activated GPIIb/IIIa receptor expression and other biomarkers were studied in patients with asymptomatic stable CAD (AS), in patients undergoing PCI for stable (SA) and unstable angina (UA). MA and R were measured by thrombelastography, GPIIb/IIIa expression by flow cytometry and all other markers by fluorokine multianalyte profiling assays. An overall increase in all measurements from a clinically stable to an unstable disease state was observed. There was a distinct stepwise increment in MA [AS vs. SA (p = 0.02), SA vs. UA (p = 0.02) and AS vs. UA (p < 0.001)]. MA exhibited the strongest correlation with other prothrombotic markers (p ≤ 0.02), with CRP (p < 0.001) at all levels of CAD acuity. A distinct pathophysiological state of heightened platelet function, hypercoagulability and inflammation marks the presence of unstable cardiovascular disease requiring intervention. Further studies are required to investigate the primary mechanisms linking the above processes associated with a prothrombotic state resulting in clinical destabilization of the disease.

Platelet function measured by VerifyNow™ identifies generalized high platelet reactivity in aspirin treated patients

Selected aspirin treated patients may exhibit high platelet reactivity to agonists other than arachidonic acid. This study aimed to determine whether the VerifyNow™ identifies generalized high platelet reactivity supported by correlations with other established methods that stimulate platelets with various agonists. Stable outpatients with coronary artery disease (n = 110) were treated with aspirin in a two 3 × 3 Latin square design (81, 162 and 325 mg/day for 4 weeks each). VerifyNow™ (arachidonic acid (AA) cartridge); light transmittance aggregometry; thrombelastography; PFA-100®; flow cytometry; PlateletWorks®; and urinary 11- dehydro thromboxane levels were measured. Multianalyte profiling measured fibrinogen and von Willebrand factor (vWF). Patients with ≥550 ARU by VerifyNow™ had increased 5 mM AA-, 5 µM ADP-, and 2 µg/mL collagen-induced platelet aggregation compared to patients with <550 ARU (p ≤ 0.001). The highest ADP- and collagen-induced aggregation was present in the upper quartile by VerifyNow™ (p < 0.05). Fibrinogen or vWF levels did not differ between VerifyNow™ quartiles. Patients with high platelet reactivity to arachidonic acid identified by VerifyNow™ also exhibit increased reactivity to other important platelet agonists that is independent of fibrinogen and vWF levels. These data suggest that VerifyNow™ may identify a generalized high platelet reactivity phenotype.

The link between heightened thrombogenicity and inflammation: Pre-procedure characterization of the patient at high risk for recurrent events after stenting

Heightened thrombogenicity and biomarker evidence of inflammation have been independently associated with ischemic risk in patients with coronary artery disease. However, a study examining their relation has not been reported. We analysed the relation between measurements of thrombogenicity and biomarkers in patients undergoing stenting and followed for 24 months recurrent ischemic events. In 84 consecutive patients undergoing stenting, pre-procedure thrombogenicity was measured by thrombelastography (TEG) and conventional aggregometry whereas biomarkers were measured by fluorokine multi-analyte profiling. Patients were stratified into quartiles based on platelet-fibrin clot strength (MA) by TEG and correlated with ischemic event occurrence. Patients in the highest MA quartile (high MA) had greater ADP-induced platelet aggregation (57.5 ± 15.0% vs. 47.9 ± 17.6%, p = 0.05), C-reactive protein (25.0 ± 5.6 vs. 4.2 ± 1.0 μg/mL, p = 0.006) and interleukin-8 (23.8 ± 2.8 vs. 14.1 ± 1.6 pg/mL, p < 0.001) than patients within the lowest MA quartile (low MA). Epidermal growth factor (7.7 ± 2.2 vs. 1.2 ± 0.3 pg/mL, p = 0.006) and vascular endothelial growth factor (296 ± 35 vs. 190 ± 10 pg/mL, p = 0.05) were also higher. Patients with high-MA had an ischemic event more often than patients with low-MA (48% vs. 13%, p = 0.02). Our study suggests that a link is present between inflammation and heightened thrombogenicity measured pre-procedurally in the patient at high risk for recurrent ischemic events after stenting. Larger studies are required to solidify these observations and their clinical relevance.

Morbid obesity and metabolic syndrome in Ossabaw miniature swine are associated with increased platelet reactivity.

Background: Metabolic syndrome (MetS) and type 2 diabetes mellitus in humans are associated with increased platelet activation and hyperreactivity of platelets to various agonists. Ossabaw swine develop all the hallmarks of MetS including obesity, insulin resistance, hypertension, dyslipidemia, endothelial dysfunction, and coronary artery disease when being fed excess calorie atherogenic diet. We hypothesized that Ossabaw swine with MetS would exhibit increased platelet reactivity compared with lean pigs without MetS. Materials and methods: Ossabaw swine were fed high caloric, atherogenic diet for 44 weeks to induce MetS (n = 10) and were compared with lean controls without MetS that had been fed normal calorie standard diet (n = 10). Light transmittance aggregometry was performed using adenosine diphosphate (ADP), collagen, thrombin, and arachidonic acid (AA) at different concentrations. Dose response curves and EC50 were calculated. Glucose tolerance testing and intravascular ultrasound study of coronary arteries were performed. Results: MetS pigs compared with lean controls were morbidly obese, showed evidence of arterial hypertension, elevated cholesterol, low-density lipoprotein/high-density lipoprotein, and triglycerides, and insulin resistance. Platelets from MetS pigs were more sensitive to ADP-induced platelet aggregation than leans (EC50: 1.83 ± 1.3 μM vs 3.64 ± 2.2 μM; P = 0.02). MetS pigs demonstrated higher platelet aggregation in response to collagen than lean pigs (area under the curve: 286 ± 74 vs 198 ± 123; P = 0.037) and a trend for heightened response to AA (AUC: 260 ± 151 vs 178 ± 145; P = 0.13). No significant difference was found for platelet aggregation in response to thrombin. Conclusions: MetS in Ossabaw swine is associated with increased reactivity of platelets to ADP and collagen. The Ossabaw swine may be a practical, large animal model for the study of certain aspects of platelet pathophysiology and examine vascular devices in a metabolic environment comparable to humans with MetS.

Inflammatory changes during the 'common cold' are associated with platelet activation and increased reactivity of platelets to agonists.

Epidemiologic studies have shown increased rates of myocardial infarction after upper respiratory tract infections. We hypothesized that changes in platelet activation and reactivity and inflammation occur during the ‘common cold’. Previously healthy individuals with viral upper respiratory tract infections were studied (n = 18). Venous blood samples were obtained during the time of infection and again after 6 weeks. Platelet reactivity was higher during the ‘common cold’ as measured by low-dose ADP-induced aggregation (46 ± 28 versus 27 ± 21% 6 weeks after presentation, P = 0.003) and was higher than control individuals (22 ± 8%, P = 0.003). Platelet P-selectin expression increased during illness (2.3 ± 0.2% CD62-positive platelets versus 1.8 ± 0.1% at 6 weeks after presentation, P = 0.017; and 1.7 ± 0.2% in the control group, P = 0.03). C-reactive protein (3.7 ± 1.3 versus 2.2 ± 1.5 mg/l, P = 0.004) and tumor necrosis factor-α (27.6 ± 28 versus 12.7 ± 9 pg/ml, P = 0.03) were increased during the ‘common cold’. There were no significant differences in levels of soluble P-selectin (P = 0.18), soluble vascular adhesion molecule-1 (P = 0.59) and soluble intercellular adhesion molecule-1 (P = 0.23). Increased platelet reactivity and activation during the ‘common cold’ are associated with inflammation as measured by increased levels of C-reactive protein and tumor necrosis factor-α, but not increased levels of endothelial markers. These findings support a pro-aggregatory state that, in part, explains the thrombotic events shown by epidemiological studies.

Race and sex differences in thrombogenicity : risk of ischemic events following coronary stenting

Race and sex affect thrombogenicity. We have demonstrated that platelet–fibrin clot characteristics can be used to stratify patients for risk of ischemic events following percutaneous coronary intervention. We investigated race and sex differences in thrombogenicty and the relation to ischemic risk in 252 consecutive African–American and Caucasian men and women undergoing elective percutaneous coronary intervention. Platelet–fibrin clot characteristics were measured using the Thrombelastograph Hemostasis System. The incidence of adverse ischemic events was assessed over a 6-month follow-up period. Overall, 40 ischemic events (15.9%) occurred. Adverse events were higher in African–Americans than Caucasians (P = 0.14), and in women than men (P = 0.004). The incidence was highest in African–American women (37.5%) and lowest in African–American men (6.5%). Measured Thrombelastograph parameters were significantly different between ischemic and nonischemic patients (P < 0.05). African–American women in the ischemic group exhibited higher thrombogenicity than the other race and sex groups (P < 0.05). Multivariate logistic regression identified platelet–fibrin mediated clot strength (relative risk 2.52, P = 0.017) and sex (relative risk 2.56, P = 0.009) as significant independent predictors of ischemic events 6 months postpercutaneous coronary intervention. Thrombogenicity is a novel measurable cardiovascular risk factor that varies by race and sex, is highest in African–American women, and independently predicts the frequency of ischemic events following percutaneous coronary intervention. Point-of-service measurements of platelet–fibrin clot characteristics may lead to more intensified antithrombotic therapy and reduced mortality in selected patients.

Implementation of a pharmacogenomics consult service to support the INGENIOUS trial

Hospital systems increasingly utilize pharmacogenomic testing to inform clinical prescribing. Successful implementation efforts have been modeled at many academic centers. In contrast, this report provides insights into the formation of a pharmacogenomics consultation service at a safety‐net hospital, which predominantly serves low‐income, uninsured, and vulnerable populations. The report describes the INdiana GENomics Implementation: an Opportunity for the UnderServed (INGENIOUS) trial and addresses concerns of adjudication, credentialing, and funding.

C-reactive protein and fibrin clot strength measured by thrombelastography after coronary stenting.

Inflammation is implicated in the progression of coronary artery disease and the molecular processes of inflammation and thrombosis are closely intertwined. Elevated levels of C-reactive protein (CRP) have been associated with an elevated risk of adverse ischaemic events after coronary stenting and hypercoagulability. Heightened whole blood clot strength measured by thrombelastography (TEG) has been associated with adverse ischaemic events after stenting. We intended to examine the relationship of CRP to plasma fibrin clot strength in patients after coronary stenting. Plasma fibrin clot strength was measured by TEG in 54 patients 16–24 h after undergoing elective percutaneous coronary intervention (PCI). Coagulation was induced in citrated plasma by addition of kaolin and CaCl2. Plasma levels of CRP and fibrinogen were measured by enzyme-linked immunoassay. Increasing quartiles of CRP were associated with increasing levels of maximal plasma fibrin clot strength measured by TEG (P < 0.001) and increasing BMI (P = 0.04). Patients in the highest quartile of CRP had significantly higher maximal fibrin clot strength (G) than the patients in the lowest quartile (G: 3438 ± 623 vs. 2184 ± 576 dyn/cm2, P < 0.0001). Fibrinogen concentration was not significantly different across quartiles of CRP (P = 0.97). Patients with established coronary artery disease undergoing coronary stenting who have elevated CRP after PCI exhibit heightened maximal plasma fibrin clot strength as compared with those with low CRP. Thrombotic risk associated with elevated CRP may be linked to procoagulant changes and high tensile fibrin clot strength independent of fibrinogen concentration.

Influence of paraoxonase-1 Q192R and cytochrome P450 2C19 polymorphisms on clopidogrel response

Background The metabolic activation of clopidogrel is a two-step process. It has been suggested that paraoxonase-1 (PON1) is a rate-limiting enzyme in the conversion of 2-oxo- clopidogrel to an active thiol metabolite. Conflicting results have been reported in regard to (1) the association of a common polymorphism of PON1 (Q192R) with reduced rates of coronary stent thrombosis in patients taking clopidogrel and (2) its effects on platelet inhibition in patient populations of European descent. Methods Blood samples from 151 subjects of mixed racial background with established coronary artery disease and who received clopidogrel were analyzed. Platelet aggregation was determined with light transmittance aggregometry and VerifyNow® P2Y12 assay. Genotyping for cytochrome P450 2C19 (CYP2C19)*2 and *3 and PON1 (Q192R) polymorphisms was performed. Results Carriers of CYP2C19*2 alleles exhibited lower levels of platelet inhibition and higher on-treatment platelet aggregation than noncarriers. There was no significant difference in platelet aggregation among PON1 Q192R genotypes. Homozygous carriers of the wild-type variant of PON1 (QQ192) had similar on-treatment platelet reactivity to carriers of increased-function variant alleles during maintenance clopidogrel dosing, as well as after administration of a clopidogrel 600 mg loading dose. Conclusion CYP2C19*2 allele is associated with impaired platelet inhibition by clopidogrel and high on-treatment platelet aggregation. PON1 (Q192R) polymorphism does not appear to be a significant determinant of clopidogrel response.