Håkan Wallén

Improved screening for silent atrial fibrillation after ischaemic stroke

OBJECTIVES To what extent silent paroxysmal atrial fibrillation (AF) is present in ischaemic stroke patients has not been established. We hypothesized that brief intermittent long-term electrocardiogram (ECG) recordings at regular time intervals are more effective than short-term continuous ECG monitoring in detecting silent AF episodes. METHODS AND RESULTS Consecutive patients who had suffered an ischaemic stroke/transient ischaemic attack (TIA) and were without known AF underwent a 24 h continuous ECG recording and performed 10 s rhythm registrations using a hand-held ECG recorder twice daily for 30 days and when arrhythmia symptoms occured. Two hundred and forty-nine stroke patients were included. Mean National Institute of Health Stroke Scale (NIHSS) score was 0.9 (0-10). In total, 17 patients were diagnosed with AF. One hundred and eight AF episodes were diagnosed in 15 patients using intermittent recording, out of which 22% where unscheduled symptom triggered episodes. In three patients AF was diagnosed with both methods and in two patients AF was detected exclusively with 24 h Holter monitoring. A significant difference in favour of the hand-held ECG was shown between the two methods (P = 0.013). The total prevalence of AF was 6.8% and increased to 11.8% in patients ≥75 years. No AF was found in patients <65 years. CONCLUSIONS Prolonged brief intermittent arrhythmia screening substantially improves the detection of silent paroxysmal AF in patients with a recent ischaemic stroke/TIA, and thus facilitates the detection of patients who should receive oral anticoagulant treatment.

Incidence of pulmonary and venous thromboembolism in pregnancies after in vitro fertilisation: cross sectional study

Objective To estimate the risk of pulmonary embolism and venous thromboembolism in pregnant women after in vitro fertilisation. Design Cross sectional study. Setting Sweden. Participants 23 498 women who had given birth after in vitro fertilisation between 1990 and 2008 and 116 960 individually matched women with natural pregnancies. Main outcome measures Risk of pulmonary embolism and venous thromboembolism (identified by linkage to the Swedish national patient register) during the whole pregnancy and by trimester. Results Venous thromboembolism occurred in 4.2/1000 women (n=99) after in vitro fertilisation compared with 2.5/1000 (n=291) in women with natural pregnancies (hazard ratio 1.77, 95% confidence interval 1.41 to 2.23). The risk of venous thromboembolism was increased during the whole pregnancy (P<0.001) and differed between the trimesters (P=0.002). The risk was particularly increased during the first trimester, at 1.5/1000 after in vitro fertilisation versus 0.3/1000 (hazard ratio 4.22, 2.46 to 7.26). The proportion of women experiencing pulmonary embolism during the first trimester was 3.0/10 000 after in vitro fertilisation versus 0.4/10 000 (hazard ratio 6.97, 2.21 to 21.96). Conclusions In vitro fertilisation is associated with an increased risk of pulmonary embolism and venous thromboembolism during the first trimester. The risk of pulmonary embolism is low in absolute terms but because the condition is a leading cause of maternal mortality and clinical suspicion is critical for diagnosis, an awareness of this risk is important. Trial registration ClinicalTrials.gov NCT01524393.

Atorvastatin reduces thrombin generation and expression of tissue factor, P-selectin and GPIIIa on platelet-derived microparticles in patients with peripheral arterial occlusive disease

We investigated the effects of statin treatment on platelet-derived microparticles (PMPs) and thrombin generation in atherothrombotic disease. Nineteen patients with peripheral arterial occlusive disease were randomised to eight weeks of treatment with atorvastatin or placebo in a cross-over fashion. Expression of GPIIIa (CD61), P-selectin (CD62P), tissue factor (TF, CD142) and phosphatidylserine (PS; annexin-V or lactadherin binding) was assessed on PMPs. Thrombin generation in vivo was assessed by measurement of prothrombin fragment 1+2 in plasma (F1+2) and ex vivo by using the calibrated automated thrombogram (CAT). During atorvastatin treatment, expression of TF, P-selectin and GPIIIa was significantly reduced vs. placebo (p<0.001 for all). No effect on annexin-V or lactadherin binding was seen. Thrombin generation was significantly reduced during atorvastatin as assessed by both the CAT assay (p<0.001) and by measurements of F1+2 (p<0.01). Subsequent in vitro experiments showed that when TF on microparticles (MPs) was blocked by antibodies, the initiation of thrombin generation was slightly but significantly delayed. Blocking PS on MPs using annexin-V or lactadherin resulted in almost complete inhibition of thrombin generation. In conclusion, atorvastatin reduces thrombin generation and expression of TF, GPIIIa and P-selectin on PMPs in patients with peripheral vascular disease. Microparticle-bound TF slightly enhances initiation of thrombin generation whereas negatively charged surfaces provided by MPs or lipoproteins could reinforce thrombin generation. Statins may inhibit initiation of thrombin generation partly through a microparticle dependent mechanism but the main effect is probably through reduction of lipoprotein levels.

Effects of ex vivo platelet supplementation on platelet aggregability in blood samples from patients treated with acetylsalicylic acid, clopidogrel, or ticagrelor

BACKGROUND Transfusion of platelet concentrate is often used to treat bleeding in patients on platelet inhibitors, but little is known about its efficacy between different inhibitors. We assessed the effect of ex vivo platelet supplementation on platelet aggregability in blood samples from patients treated with acetylsalicylic acid (ASA), clopidogrel, or ticagrelor. METHODS Platelet aggregability was investigated with multiple electrode aggregometry with adenosine diphosphate (ADP), arachidonic acid (to assess ASA-dependent aggregability), and thrombin receptor activating peptide-6 (TRAP) as activators in whole-blood samples from patients treated with ASA (n=10), ASA+clopidogrel (n=15), or ASA+ticagrelor (n=15), and from healthy controls (n=10). Aggregability was measured before and after supplementation of AB0-compatible fresh apheresis platelets (+46, +92, and +138×10(9) litre(-1)). RESULTS Both ASA-dependent and ADP-dependent aggregability improved in a dose-dependent fashion after platelet supplementation. ASA-dependent aggregability was completely restored in all patient groups, but there was only a small improvement in ADP-dependent aggregability in patients on dual antiplatelet therapy. There was less effect of platelet supplementation on ADP- and ASA-dependent aggregability in ticagrelor-treated patients than in clopidogrel-treated patients [3.9 (95% confidence interval 1.6-6.3) vs 9.0 (5.2-12.8) AU×min (P=0.021) and 48 (36-59) vs 69 (60-78) AU×min (P=0.004), respectively, at the highest platelet dose]. CONCLUSIONS Platelet supplementation improved platelet aggregability independently of antiplatelet therapy. The effect on ADP-dependent platelet inhibition was limited however. Reduced effect of platelet transfusion is more likely within 2 h of drug intake in patients treated with ASA+ticagrelor compared with ASA+clopidogrel.

Effects on fibrin network porosity of anticoagulants with different modes of action and reversal by activated coagulation factor concentrate

Orally available direct thrombin inhibitors (DTI) and direct activated factor X inhibitors (DFXaI) may replace vitamin K antagonists in patients needing long‐term anticoagulant treatment. We investigated the influence on the fibrin network of anticoagulants with different modes of action: AR‐H067637 (DTI), the active metabolite of AZD0837, apixaban (DFXaI), fondaparinux (indirect FXaI) and warfarin. Counteraction of the anticoagulant effect by FEIBA® (Factor Eight Inhibitor Bypass Activity) was also investigated. Tissue factor, phospholipids and calcium were used to initiate coagulation in human platelet poor plasma. The permeability constant (Ks), reflecting the amount of buffer passing through the coagulum, was calculated and the fibrin network was visualized by 3D confocal microscopy. Warfarin (International Normalized Ratio 2‐3) increased Ks in plasma by 28–50% compared with control. ‘Therapeutic’ plasma concentrations of AR‐H067637 (0·3–0·6 μmol/l), apixaban (0·2–0·4 μmol/l) and fondaparinux (0·1–0·3 μmol/l) increased Ks by 72–91%, 58–76% and 36–53% respectively. Addition of FEIBA® totally reversed the warfarin effect but only partially reversed effects of the other anticoagulants at concentrations that increased Ks by 50% or more. Fibrin network observed with 3D confocal microscopy agreed well with the permeability results. In conclusion, all examined anticoagulants rendered the fibrin network more porous. FEIBA® reversed the increased permeability in warfarin plasma but had only partial effects on the other anticoagulants.

Platelet-derived microparticles during and after acute coronary syndrome.

As microparticles are shedded upon platelet activation, and may be used to assess platelet function, we measured plasma concentrations of platelet-derived microparticles (PMPs) during and after an acute coronary syndrome (ACS). Fifty-one patients with ACS were investigated at admission, within 24 hours (before coronary angiography), and six months later. Sixty-one sex- and age-matched healthy controls were investigated once. PMPs were defined as particles <1.0 μm in size, negative to phalloidin (labels cell-fragments), and positive to CD61. Exposure of phosphatidylserine (PS+), CD62P and CD142 were also measured. Plasma concentrations of PS+PMPs exposing CD61, CD62P and CD142 were elevated 2.5, 6.0-, and 5.0-fold at admission (p<0.001 for all, compared to controls; aspirin only), decreased significantly 24 hours later following initiation of treatment with clopidogrel and subcutaneous anticoagulation (p<0.001 for all), and decreased even further six months later (p<0.01 for all). However, PS+PMPs exposing CD62P or CD142 were still between 1.2-and 2.3-fold higher than in controls (p<0.001 for both). The pattern for PS-PMPs during and after the ACS was very similar to that for PS+PMPs although the numbers were approximately 1/3 lower. In conclusion, PMP concentrations follow the pattern of platelet activation during and after an ACS. Decreased concentrations are observed after initiation of antithrombotic treatment, but PMP exposing CD62P or CD142 are still elevated after six months. Flow cytometric measurements of PMP in frozen-thawed samples enable studies of platelet function in larger clinical trials.

Long-term effects of an expanded cardiac rehabilitation programme after myocardial infarction or coronary artery bypass surgery: a five-year follow-up of a randomized controlled study

Objective: To investigate the long-term effect of expanded cardiac rehabilitation on a composite end-point, consisting of cardiovascular death, myocardial infarction or readmission for cardiovascular disease, in patients with coronary artery disease. Design: Single-centre prospective randomized controlled trial. Setting: University hospital. Subjects: Two hundred and twenty-four patients with acute myocardial infarction or undergoing coronary artery by-pass grafting. Intervention: Patients were randomized to expanded cardiac rehabilitation (a one-year stress management programme, increased physical training, staying at a ‘patient hotel’ for five days after the event, and cooking sessions), or to standard cardiac rehabilitation. Main measures: Data on cardiovascular death, myocardial infarction, readmission for cardiovascular disease and days at hospital for cardiovascular reasons were obtained from national registries of the Swedish National Board of Health and Welfare. Results: The primary end-point occurred in 121 patients altogether (54%). The number of cardiovascular events were reduced in the expanded rehabilitation group compared with the standard cardiac rehabilitation (53 patients (47.7%) versus 68 patients (60.2%); hazard ratio 0.69; P = 0.049). This was mainly because of a reduction of myocardial infarctions in the expanded rehabilitation group. During the five years 12 patients (10.8%) versus 23 patients (20.3%); hazard ratio 0.47; P = 0.047 had a myocardial infarction. Days at hospital for cardiovascular reasons were significantly reduced in patients who received expanded cardiac rehabilitation (median 6 days) compared with standard cardiac rehabilitation (median 10 days; P = 0.02). Conclusion: Expanded cardiac rehabilitation after acute myocardial infarction or coronary artery bypass grafting reduces cardiovascular morbidity and days at hospital for cardiovascular reasons.

A global assay of haemostasis which uses recombinant tissue factor and tissue-type plasminogen activator to measure the rate of fibrin formation and fibrin degradation in plasma

The global assay of Overall Haemostasis Potential we previously described has been refined. The coagulation cascade in platelet-poor plasma is triggered by adding a minimal dose of recombinant tissue factor together with purified phospholipids and calcium; fibrinolysis is initiated by adding recombinant tissue type-plasminogen activator in a concentration similar to what can be obtained during thrombolysis. Numerical differentials of optical densities reflecting rates of fibrin formation and degradation are calculated by a new software, and the Coagulation Profile (Cp) and the Fibrinolysis Profile (Fp) are determined. The combined effect of these counteractive systems is expressed as a ratio of Cp to Fp, called the Overall Haemostasis Index. Commercially available coagulant-deficient patient plasma samples and plasma with various amounts of added PAI-1 are examined; changes of fibrin turbidity demonstrate that this assay can determine Cp and Fp in a physiologically relevant way. Increased Cp and decreased Fp in prothrombotic patients, as well as expected effects of heparin or a thrombin inhibitor on Cp and Fp, suggest that our method can detect hypercoagulability and assist in monitoring antithrombotic treatment. Ongoing studies will show whether this simple assay can be of value in clinical routine.

Thrombin activatable fibrinolysis inhibitor and its relationship to fibrinolysis and inflammation during the acute and convalescent phase of ischemic stroke.

To investigate thrombin activatable fibrinolysis inhibitor (TAFI) in ischemic stroke and its relationship to fibrinolysis and inflammation, we investigated 32 patients with ischemic stroke during the acute phase and after 60 days. TAFI antigen levels, global markers of hemostasis (coagulation and fibrinolysis) and inflammatory markers were measured in plasma. TAFI antigen levels were significantly elevated at admission (128%; 109–151%) and at day 1 (129%; 109–152%) compared with day 60 (108%; 91–127%; both P < 0.01) and with healthy control individuals (99%; 76–122%; P < 0.05). In parallel, fibrinolysis assessed as the overall fibrinolysis potential (OFP), part of the overall hemostatic potential assay (OHP), was decreased at all time points compared with control individuals (P < 0.01 for all) and was found to be inversely related to TAFI (r = −0.40; P = 0.0008; n = 20). The OFP and the overall coagulation potential (another part of the OHP assay), and to a lesser degree TAFI, showed significant relationships to C-reactive protein and fibrinogen. In conclusion, elevated TAFI antigen levels may be a consequence of an acute phase reaction, and together with a depressed OFP suggest impaired fibrinolysis in patients with acute ischemic stroke. The OHP method may be useful as a complement to standard hemostatic variables in evaluating hemostasis in stroke patients.

Detection of elevated INR by thromboelastometry and thromboelastography in warfarin treated patients and healthy controls

INTRODUCTION The diagnostic potential of whole blood viscoelastic tests thromboelastography (TEG®) and thromboelastometry (ROTEM®) to detect warfarin-induced INR elevation remains elusive. METHODS Viscoelastic tests were performed in 107 patients on warfarin and 89 healthy controls. Tests were activated by kaolin for TEG, and ellagic acid (INTEM) or tissue factor (EXTEM) for ROTEM. RESULTS Viscoelastic tests revealed significant differences in clotting profiles between controls and warfarin-treated patients. Compared with healthy controls, patients treated with warfarin had prolonged EXTEM clotting and TEG reaction time (p<0.001), both of which were also increased beyond the reference range. Increased INR values correlated with EXTEM CT (Spearman rho=0.87) and TEG R-time (rho=0.73). EXTEM CT had a sensitivity and specificity of 0.89 and 1.00, respectively, to detect elevated INR above 1.2 units, with a positive and negative predictive values (PPV and NPV) of 1.00 and 0.88, respectively. Similarly, TEG R-time had a sensitivity and specificity of 0.86 and 0.87, respectively, with a PPV of 0.89 and a NPV of 0.83. The corresponding receiver operator characteristic area under the curve was 0.99 (95% confidence interval [CI], 0.99 - 1.00) for EXTEM CT and 0.94 (95% CI, 0.91 - 0.97) for TEG R-time. CONCLUSIONS Tissue factor-activated viscoelastic testing (EXTEM) revealed individuals with warfarin-induced INR elevation accurately, while TEG - activated through the intrinsic pathway - still was of acceptable diagnostic value. Further studies are required to evaluate the diagnostic potential of viscoelastic tests in relation to standard laboratory tests in other mixed patient populations, where the PPV and NPV may be inferior.