Jürg H. Beer

Association of two silent polymorphisms of platelet glycoprotein la/lla receptor with risk of myocardial infarction: a case-control study

BACKGROUND The platelet membrane glycoprotein Ia/IIa plays a major part in platelet function as a primary receptor for collagen. A previous report showed a variation of glycoprotein Ia/IIa receptor density and function associated with two silent and linked polymorphisms (807C/T and 873G/A) within the glycoprotein Ia gene. Because platelet thrombus formation is implicated in the pathogenesis of acute myocardial infarction, we investigated these polymorphisms among patients who had had a myocardial infarction. METHODS We did a 2/1 case-control study including 177 patients (median age 57.0 [range 32-72] years) and 89 controls with same age and sex. Distributions of the 807C/T and 873G/A polymorphisms were investigated by genotyping DNA by PCR, single-strand conformation polymorphism analysis, and sequencing. FINDINGS The prevalence of the homozygous 807T/873A genotype was 2.9 times higher among patients with myocardial infarction than among controls (16.4% vs 5.6%, p=0.022). There was an association between patients homozygous for the 807T/873A allele and myocardial infarction (odds ratio 3.3 [95% CI 1.2-8.8]), which was strongest in a subgroup of smokers. The homozygous 807T/873A genotype remained an independent risk factor for myocardial infarction (p=0.005) when age, sex, smoking, hypertension, diabetes, body-mass index, LDL-cholesterol and HDL-cholesterol, and fibrinogen were adjusted for by logistic regression. INTERPRETATION The 807T/873A homozygosity of the platelet glycoprotein Ia/IIa gene polymorphism, associated with differences in surface collagen receptor density and activity, appears to be an independent risk factor for acute myocardial infarction. Our findings need to be confirmed in a larger, prospective study that includes patients from different populations and cardiovascular risk groups.

Prevalence of drug-drug interactions at hospital entry and during hospital stay of patients in internal medicine

OBJECTIVE The aim of this study was to assess potential drug-drug interactions (pDDIs) at hospital admission, during hospitalization and at discharge and to evaluate the number of pDDIs created during hospitalization. METHODS The medication of 851 patients was screened for pDDIs (major and moderate severity) using the screening program Pharmavista. The frequency of pDDIs per patient, per number of drugs and drug pairs was estimated. RESULTS During hospitalization, the frequency of major and moderate pDDIs per patient was 1.11, which was higher compared to hospital admission (0.59) or to hospital discharge (0.60). The frequency of major and moderate pDDIs per drug prescribed (13.7% vs. 9.1%) or per drug pairs analyzed (4.5% vs. 2.3%) was higher at hospital admission compared to hospital discharge. 47% of all major and moderate pDDIs at discharge were due to a medication change during hospitalization. CONCLUSIONS Although the number of major and moderate pDDIs per patient did not increase from hospital admission to discharge, it is important to realize that 47% of all major and moderate DDIs at hospital discharge were created during hospitalization. Prescribing drugs with a low risk for pDDIs as well as careful monitoring for adverse drug reactions are important measures to minimize harm associated with DDIs.

Dietary α-Linolenic Acid Inhibits Arterial Thrombus Formation, Tissue Factor Expression, and Platelet Activation

Objective—Plant-derived &agr;-linolenic acid (ALA) may constitute an attractive cardioprotective alternative to fish-derived n-3 fatty acids. However, the effect of dietary ALA on arterial thrombus formation remains unknown. Methods and Results—Male C57Bl/6 mice were fed a high-ALA or low-ALA diet for 2 weeks. Arterial thrombus formation was delayed in mice fed a high-ALA diet compared with those on a low-ALA diet (n=7; P<0.005). Dietary ALA impaired platelet aggregation to collagen and thrombin (n=5; P<0.005) and decreased p38 mitogen-activated protein kinase activation in platelets. Dietary ALA impaired arterial tissue factor (TF) expression, TF activity, and nuclear factor-&kgr;B activity (n=7; P<0.05); plasma clotting times and plasma thrombin generation did not differ (n=5; P=not significant). In cultured human vascular smooth muscle and endothelial cells, ALA inhibited TF expression and activity (n=4; P<0.01). Inhibition of TF expression occurred at the transcriptional level via the mitogen-activated protein kinase p38 in smooth muscle cells and p38, extracellular signal-regulated kinases 1 and 2, and c-Jun N-terminal kinases 1 and 2 in endothelial cells. Conclusion—ALA impairs arterial thrombus formation, TF expression, and platelet activation and thereby represents an attractive nutritional intervention with direct dual antithrombotic effects.

Dietary α-linolenic acid diminishes experimental atherogenesis and restricts T cell-driven inflammation

Aims Epidemiological studies report an inverse association between plant-derived dietary α-linolenic acid (ALA) and cardiovascular events. However, little is known about the mechanism of this protection. We assessed the cellular and molecular mechanisms of dietary ALA (flaxseed) on atherosclerosis in a mouse model. Methods and results Eight-week-old male apolipoprotein E knockout (ApoE−/−) mice were fed a 0.21 % (w/w) cholesterol diet for 16 weeks containing either a high ALA [7.3 % (w/w); n = 10] or low ALA content [0.03 % (w/w); n = 10]. Bioavailability, chain elongation, and fatty acid metabolism were measured by gas chromatography of tissue lysates and urine. Plaques were assessed using immunohistochemistry. T cell proliferation was investigated in primary murine CD3-positive lymphocytes. T cell differentiation and activation was assessed by expression analyses of interferon-γ, interleukin-4, and tumour necrosis factor α (TNFα) using quantitative PCR and ELISA. Dietary ALA increased aortic tissue levels of ALA as well as of the n−3 long chain fatty acids (LC n−3 FA) eicosapentaenoic acid, docosapentaenoic acid, and docosahexaenoic acid. The high ALA diet reduced plaque area by 50% and decreased plaque T cell content as well as expression of vascular cell adhesion molecule-1 and TNFα. Both dietary ALA and direct ALA exposure restricted T cell proliferation, differentiation, and inflammatory activity. Dietary ALA shifted prostaglandin and isoprostane formation towards 3-series compounds, potentially contributing to the atheroprotective effects of ALA. Conclusion Dietary ALA diminishes experimental atherogenesis and restricts T cell-driven inflammation, thus providing the proof-of-principle that plant-derived ALA may provide a valuable alternative to marine LC n−3 FA.

Multicentre validation of the Geneva Risk Score for hospitalised medical patients at risk of venous thromboembolism. Explicit ASsessment of Thromboembolic RIsk and Prophylaxis for Medical PATients in SwitzErland (ESTIMATE).

There is a need to validate risk assessment tools for hospitalised medical patients at risk of venous thromboembolism (VTE). We investigated whether a predefined cut-off of the Geneva Risk Score, as compared to the Padua Prediction Score, accurately distinguishes low-risk from high-risk patients regardless of the use of thromboprophylaxis. In the multicentre, prospective Explicit ASsessment of Thromboembolic RIsk and Prophylaxis for Medical PATients in SwitzErland (ESTIMATE) cohort study, 1,478 hospitalised medical patients were enrolled of whom 637 (43%) did not receive thromboprophylaxis. The primary endpoint was symptomatic VTE or VTE-related death at 90 days. The study is registered at ClinicalTrials.gov, number NCT01277536. According to the Geneva Risk Score, the cumulative rate of the primary endpoint was 3.2% (95% confidence interval [CI] 2.2-4.6%) in 962 high-risk vs 0.6% (95% CI 0.2-1.9%) in 516 low-risk patients (p=0.002); among patients without prophylaxis, this rate was 3.5% vs 0.8% (p=0.029), respectively. In comparison, the Padua Prediction Score yielded a cumulative rate of the primary endpoint of 3.5% (95% CI 2.3-5.3%) in 714 high-risk vs 1.1% (95% CI 0.6-2.3%) in 764 low-risk patients (p=0.002); among patients without prophylaxis, this rate was 3.2% vs 1.5% (p=0.130), respectively. Negative likelihood ratio was 0.28 (95% CI 0.10-0.83) for the Geneva Risk Score and 0.51 (95% CI 0.28-0.93) for the Padua Prediction Score. In conclusion, among hospitalised medical patients, the Geneva Risk Score predicted VTE and VTE-related mortality and compared favourably with the Padua Prediction Score, particularly for its accuracy to identify low-risk patients who do not require thromboprophylaxis.

Post-ischaemic silencing of p66Shc reduces ischaemia/reperfusion brain injury and its expression correlates to clinical outcome in stroke

AIM Constitutive genetic deletion of the adaptor protein p66(Shc) was shown to protect from ischaemia/reperfusion injury. Here, we aimed at understanding the molecular mechanisms underlying this effect in stroke and studied p66(Shc) gene regulation in human ischaemic stroke. METHODS AND RESULTS Ischaemia/reperfusion brain injury was induced by performing a transient middle cerebral artery occlusion surgery on wild-type mice. After the ischaemic episode and upon reperfusion, small interfering RNA targeting p66(Shc) was injected intravenously. We observed that post-ischaemic p66(Shc) knockdown preserved blood-brain barrier integrity that resulted in improved stroke outcome, as identified by smaller lesion volumes, decreased neurological deficits, and increased survival. Experiments on primary human brain microvascular endothelial cells demonstrated that silencing of the adaptor protein p66(Shc) preserves claudin-5 protein levels during hypoxia/reoxygenation by reducing nicotinamide adenine dinucleotide phosphate oxidase activity and reactive oxygen species production. Further, we found that in peripheral blood monocytes of acute ischaemic stroke patients p66(Shc) gene expression is transiently increased and that this increase correlates with short-term neurological outcome. CONCLUSION Post-ischaemic silencing of p66(Shc) upon reperfusion improves stroke outcome in mice while the expression of p66(Shc) gene correlates with short-term outcome in patients with ischaemic stroke.

Platelet microparticle formation and thrombin generation under high shear are effectively suppressed by a monoclonal antibody against GPIbα

We studied the inhibition of platelet microparticle (MP) formation and thrombin generation under high shear forces. We hypothesized that an inhibitor of the GPIb a -von Willebrand factor (vWF) interaction would be more effective in suppressing MP formation and thrombin generation than GPIIb/IIIa inhibitors. Platelet-rich plasma (PRP) anticoagulated with PPACK (D-Phe-Pro-Arg chloromethyl ketone) was exposed in a cone-and-plate viscometer (shear: 5,000 s(-1) for 5 min) in the presence of antagonists to GPIb a (the monoclonal antibody [Mab] Ib-23) or to GPIIb/IIIa (abciximab, tirofiban, eptifibatide) at their IC90 determined in platelet aggregometry with ristocetin or ADP, respectively. We used double labeling (CD41-PE and annexin-V-FITC) for flow cytometric detection of MP and their aminophospholipid exposure. Thrombin generation was measured using PRP prepared from ACD anticoagulated blood. About 40% of the thrombin generation was found to be mediated by the MP fraction of the PRP. Blockade of GPIb a with Mab Ib-23 reduced MP formation and thrombin generation by 50%, and was more effective than any GPIIb/IIIa antagonist. The combination of Mab Ib-23 with one of the GPIIb/IIIa inhibitors further reduced the MP formation to ~ 30%. The antibody also partially inhibited thrombin induced platelet aggregation. Epitope mapping suggested that Mab Ib-23 binds between the amino acids 201 and 268 of GPIb a , explaining the interference with vWF and thrombin interaction. In contrast to the commonly used GPIIb/IIIa antagonists, the blockade of GPIb a with Mab Ib-23 effectively reduces the prothrombotic MP generation and thrombin formation at shear rates typically found in arterial stenoses.

PI3K/p110α inhibition selectively interferes with arterial thrombosis and neointima formation, but not re-endothelialization: potential implications for drug-eluting stent design

BACKGROUND Impaired re-endothelialization and stent thrombosis are a safety concern associated with drug-eluting stents (DES). PI3K/p110α controls cellular wound healing pathways, thereby representing an emerging drug target to modulate vascular homoeostasis after injury. METHODS AND RESULTS PI3K/p110α was inhibited by treatment with the small molecule inhibitor PIK75 or a specific siRNA. Arterial thrombosis, neointima formation, and re-endothelialization were studied in a murine carotid artery injury model. Proliferation and migration of human vascular smooth muscle cell (VSMC) and endothelial cell (EC) were assessed by cell number and Boyden chamber, respectively. Endothelial senescence was evaluated by the β-galactosidase assay, endothelial dysfunction by organ chambers for isometric tension. Arterial thrombus formation was delayed in mice treated with PIK75 when compared with controls. PIK75 impaired arterial expression and activity of tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1); in contrast, plasma clotting and platelet aggregation did not differ. In VSMC and EC, PIK75 inhibited expression and activity of TF and PAI-1. These effects occurred at the transcriptional level via the RhoA signalling cascade and the transcription factor NFkB. Furthermore, inhibition of PI3K/p110α with PIK75 or a specific siRNA selectively impaired proliferation and migration of VSMC while sparing EC completely. Treatment with PIK75 did not induce endothelial senescence nor inhibit endothelium-dependent relaxations. In line with this observation, treatment with PIK75 selectively inhibited neointima formation without affecting re-endothelialization following vascular injury. CONCLUSION Following vascular injury, PI3K/p110α inhibition selectively interferes with arterial thrombosis and neointima formation, but not re-endothelialization. Hence, PI3K/p110α represents an attractive new target in DES design.

Rivaroxaban for the treatment of venous thromboembolism: The SWIss Venous ThromboEmbolism Registry (SWIVTER)

We investigated three-month clinical outcomes in patients with venous thromboembolism (VTE) treated with rivaroxaban or conventional anticoagulation in routine clinical practice. Between November 2012 and February 2015, 2,062 consecutive patients with VTE from 11 acute care hospitals in Switzerland were enrolled in the SWIss Venous ThromboEmbolism Registry (SWIVTER). Overall, 417 (20 %) patients were treated with rivaroxaban. In comparison to 1,645 patients on conventional anticoagulation, patients on rivaroxaban were younger (56 ± 18 vs. 65 ± 17 years; p<0.001), less often had pulmonary embolism (38 % vs 66 %; p<0.001), hypertension (26 % vs 41 %; p<0.001), cancer (10 % vs 28 %; p<0.001), congestive heart failure (10 % vs 17 %; p=0.001), diabetes (8 % vs 15 %; p<0.001), chronic lung disease (7 % vs 13 %; p=0.001), renal insufficiency (7 % vs 13 %; p=0.001), recent surgery (7 % vs 14 %; p<0.001), and acute coronary syndrome (1 % vs 4 %; p=0.009). VTE reperfusion therapy was more frequently used (28 % vs 9 %; p<0.001) and indefinite-duration anticoagulation treatment less often planned (26 % vs 39 %; p<0.001), respectively. In the propensity score-adjusted population, the risk of recurrent VTE was similar in patients on rivaroxaban vs conventional anticoagulation (1.2 % vs 2.1 %, hazard ratio [HR] 0.55, 95 % confidence interval [CI] 0.18-1.65; p=0.29); the risk of major bleeding was also similar, respectively (0.5 % vs 0.5 %, HR 1.00, 95 %CI 0.14-7.07; p=1.00). Conventional anticoagulation is still frequently used for the treatment of VTE, particularly in the elderly and those with comorbidities. Early clinical outcomes were comparable between propensity score-adjusted patient populations on rivaroxaban and conventional anticoagulation.

High-density lipoprotein from patients with coronary heart disease loses anti-thrombotic effects on endothelial cells: impact on arterial thrombus formation.

Thrombus formation is determined by the balance between pro- thrombotic mediators and anti-thrombotic factors.High-density lipoprotein (HDL) from healthy subjects exerts anti-thrombotic properties. Whether this is also the case for HDL from patients with stable coronary heart disease (CHD) or acute coronary syndrome (ACS) is unknown.In human aortic endothelial cells in culture,HDL (50 µg/ml) from healthy subjects (HS) inhibited thrombin-induced tissue factor (TF) expression and activity, while HDL (50 µg/ml) from CHD and ACS patients did not. Similarly, only healthy HDL increased endothelial tissue factor pathway inhibitor (TFPI) expression and tissue plasminogen activator (tPA) release, while HDL from CHD and ACS patients had no effect. Healthy HDL inhibited thrombin-induced plasminogen activator inhibitor type 1 (PAI-1) expression, while HDL from ACS patients enhanced endothelial PAI-1 expression. Inhibition of nitric oxide (NO) formation with L-NAME (100 µmol/l) abolished the anti-thrombotic effects of healthy HDL on TF, TFPI, and tPA expression. The exogenous nitric oxide donor, DETANO, mimicked the effects of healthy HDL and counterbalanced the loss of anti-thrombotic effects of HDL from CHD and ACS patients in endothelial cells. In line with this observation, healthy HDL, in contrast to HDL from CHD and ACS patients, increased endothelial NO production. In the laser-injured carotid artery of the mouse, thrombus formation was delayed in animals treated with healthy HDL compared with mice treated with vehicle or HDL from patients with CHD or ACS. In conclusion, HDL from CHD and ACS patients loses the ability of healthy HDL to suppress TF and to increase TFPI and t-PA and instead enhances PAI-1 and arterial thrombus formation.