Mika Skeppholm

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.

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.

On the monitoring of dabigatran treatment in “real life” patients with atrial fibrillation

INTRODUCTION The oral direct thrombin inhibitor dabigatran is increasingly used to prevent thromboembolic stroke in patients with atrial fibrillation (AF). Routine laboratory monitoring is currently not recommended, but measurements of dabigatran and/or its effect are desirable in certain situations. We studied dabigatran exposure and compared different tests for monitoring of dabigatran in a real-life cohort of AF patients. MATERIAL AND METHODS Ninety AF patients (68 ± 9 years, 67% men, mean CHADS2 score 1.5) were treated with dabigatran 150 (n=73) or 110 mg BID (n=17). Trough plasma concentrations of total and free dabigatran by liquid chromatography-tandem mass-spectrometry (LC-MS/MS) were compared to indirect measurements by Hemoclot thrombin inhibitors (HTI) and Ecarin clotting assay (ECA), as well as PT-INR and aPTT. RESULTS Total plasma dabigatran varied 20-fold (12-237 ng/mL with 150 mg BID) and correlated well with free dabigatran (r(2)=0.93). There were strong correlations between LC-MS/MS and HTI or ECA (p<0.001) but these assays were less accurate with dabigatran below 50 ng/mL. The aPTT assay was not dependable and PT-INR not useful at all. There were weak correlations between creatinine clearance (Cockcroft-Gault) and LC-MS/MS, HTI and ECA (p<0.001 for all). A high body weight with normal kidney function was associated with low dabigatran levels. CONCLUSIONS HTI and ECA reflect the intensity of dabigatran anticoagulation, but LC-MS/MS is required to quantify low levels or infer absence of dabigatran. Most real life patients with a normal creatinine clearance had low dabigatran levels suggesting a low risk of bleeding but possibly limited protection against stroke.

Can both EDTA and citrate plasma samples be used in measurements of fibrinogen and C-reactive protein concentrations?

Abstract Background: Fibrinogen and C-reactive protein (CRP) concentrations are predictors of outcome in the atherosclerotic patient. It is important in risk stratification that these quantities are measured reproducibly in routine and research. Method: In the present study, we compare measurements of fibrinogen and high-sensitivity CRP in EDTA and citrate plasma samples (n=150) using nephelometric immunoassays. Fibrinogen was also measured in citrate plasma using a clotting method. Results: In approximately one-third of the samples, the fibrinogen concentration measured by immunoassay was higher in citrate plasma than in EDTA plasma, in spite of the dilution by citrate. The immunoassay results of fibrinogen concentration measurements in EDTA and citrate plasma differed significantly and also differed from those of functionally measured fibrinogen concentrations. A difference was found between the concentration of CRP in EDTA plasma and citrated plasma which also did not correspond to the dilution. Conclusions: Reproducibility of results is essential in risk stratification by fibrinogen or high-sensitivity CRP concentrations and small differences close to the decision limits may have a decisive impact. Immunological measurements are liable to confounding effects that may be difficult to foresee, qualitatively and quantitatively. Great care should be observed when measuring the concentration of calcium containing analytes in anticoagulated samples. Fibrinogen concentrations should preferably be measured functionally in citrate plasma. Clin Chem Lab Med 2008;46:1175–9.

ADAMTS13 and von Willebrand factor concentrations in patients with diabetes mellitus

The von Willebrand factor (VWF) is elevated in patients with diabetes mellitus and degraded by a metalloprotease, ADAMTS13. We hypothesized that this elevation is due to a decreased function of ADAMTS13. Thus, we investigated ADAMTS13 in patients with diabetes mellitus without and with peripheral artery occlusive disease (PAOD). When treating the latter group with dalteparin, VWF is reported to increase significantly, and we therefore measured ADAMTS13 also in these patients. VWF antigen and ADAMTS13 antigen and activity concentrations were measured in patients with diabetes mellitus but without PAOD (diabetes mellitus; n = 23) and with diabetes mellitus and PAOD (diabetes mellitus + PAOD; n = 65) before and after treatment with dalteparin or placebo. In the diabetes mellitus group, concentration of VWF antigen was significantly higher, whereas that of ADAMTS13 activity was significantly lower than in the healthy controls. In the diabetes mellitus along with PAOD group, VWF antigen was significantly higher, but ADAMTS13 antigen or activity did not differ significantly from those of healthy controls. The ADAMTS13 activity/antigen ratio was lower than in controls only in the diabetes mellitus patient group. VWF antigen increased significantly during dalteraprin treatment, whereas ADAMTS13 activity and antigen remained unchanged. Only patients with diabetes mellitus had significantly lower concentrations of ADAMTS13 activity in plasma than controls, although the diabetes mellitus along with PAOD had a more pronounced VWF antigen elevation than diabetes mellitus patients, illustrating a possible link between ADAMTS13 and microangiopathy in diabetes mellitus patients. The increase in VWF antigen concentration during treatment with dalteparin does not seem to be due to changes in ADAMTS13.

Whole blood coagulation assays ROTEM and T-TAS to monitor dabigatran treatment

BACKGROUND A rapid and reliable assessment of the dabigatran effect is desirable in dabigatran treated patients with uncontrolled bleeding or before acute surgery. OBJECTIVE To evaluate how the viscoelastic point-of-care test Rotational thromboelastometry (ROTEM) and Total Thrombus-formation system (T-TAS), which studies thrombus formation under flowing conditions, correlate with dabigatran concentrations in patients with atrial fibrillation (AF). METHOD ROTEM using the reagents In-tem, Ex-tem, Fib-tem or low tissue factor concentration (TF), and T-TAS with the AR-chip (shear rate 600s-1, representing flow in large arteries) were investigated in whole blood samples. Plasma concentrations were determined by mass spectrometry (LC-MS/MS) at trough and post-dose in 30 patients on dabigatran 150mg BID. RESULTS Median plasma dabigatran concentrations at trough were 86ng/mL (29-150) and post-dose (2.8h after ingestion) 175ng/mL (67-490). The ROTEM clotting time (CT) correlated strongly with dabigatran concentrations when activated with the reagents Ex-tem (r=0.92, p<0.01) and Fib-tem (r=0.93, p<0.01), while with In-tem and low TF the correlation was weaker (r=0.72 and r=0.36, p<0.01). There were significant but weaker correlations also between dabigatran concentrations and T-TAS variables (r-values 0.39-0.41, p<0.01), aPTT (r=0.70, p<0.01) and PT-INR (r=0.43, p<0.01) respectively. CONCLUSIONS ROTEM Ex-tem and Fib-tem CT shows a strong correlation with dabigatran concentrations in real-life AF-patients, and results are obtained within minutes. This could make ROTEM useful in acute situations. T-TAS detect differences in hemostasis caused by dabigatran, but the relationships to plasma concentrations of dabigatran are weaker than for ROTEM CT with the settings used in this study.

Inflammation and thrombin generation cause increased thrombin activatable fibrinolysis inhibitor levels in experimental human endotoxemia.

The thrombin activatable fibrinolysis inhibitor (TAFI) is a 58-kDa carboxypeptidase that is synthesized in the liver and circulates in the plasma as a zymogen, pro-TAFI. The principal mediator of TAFI activation is the thrombin–thrombomodulin complex. The active form of TAFI attenuates fibrinolysis by removing C-terminal lysine and arginine residues from partially degraded fibrin. This inhibits plasminogen binding and subsequently plasmin formation in the fibrin-rich clot [1,2].

Effects of direct oral anticoagulants on lupus anticoagulant assays in a real-life setting

Laboratory diagnosis of lupus anticoagulant (LA) is based on prolongation in at least one coagulation assay (diluted Russells viper venom time - dRVVT or activated partial thromboplastin time - aPTT), which normalises after addition of phospholipids. Both assays may be influenced by anticoagulants and therefore LA should not be tested during warfarin or heparin treatment. It has been shown (primarily in vitro) that direct oral anticoagulants (DOACs - dabigatran [DAB], rivaroxaban [RIV] and apixaban [API]) may also influence LA testing. We tested the effects of DOACs on assays routinely used for the diagnosis of LA in patients treated with these drugs in a real-life setting. Plasma from patients with atrial fibrillation treated with DAB (n=30), RIV (n=20) and API (n=17) and not known to have LA were tested using dRVVT (LA-screen and LA-confirm, Life Diagnostics) and aPTT (PTT-LA, Diagnostica Stago and aPTT Actin FS, Siemens Healthcare Diagnostics) assays. According to the diagnostics algorithm, dRVVT and aPTT ratios of <1.2 were considered negative, ratios of >1.4 positive, while if the ratios were 1.2-1.4 LA could not be ruled out. Plasma concentrations varied between 8-172 µg/l for DAB, 8-437 µg/l for RIV and 36-178 µg/l for API. LA diagnosis was negative in only eight (27 %) plasma samples from patients treated with DAB, and in five (25 %) and four samples (24 %) from patients treated with RIV and API, respectively. LA Positivity (dRVVT and aPTT ratios >1.4) was found in 5 cases (17 %) among patients treated with DAB, in 10 cases (50 %) treated with RIV and in 7 cases (41 %) treated with API. A concentration-dependent effect of DOACs on dRVVT-based parameters was observed, particularly as regards DAB. At lower concentrations, RIV and API had only minor effects on the confirmatory tests (below 100 µg/l and 70 µg/l, respectively). Our results suggest that a risk of overestimation of LA detection is present in samples from patients treated with DOACs. Therefore, LA testing should not be performed during treatment with DOACs. Prolongation in confirmatory assays may be helpful for the recognition of false positivity, especially as regards DAB.

FRI0593 Effects of New (Direct) Oral Anticoagulants on Lupus Anticoagulant Assays

Background Laboratory diagnosis of lupus anticoagulant (LA) as a part of the diagnosis of antiphospholipid syndrome (APS) is based on prolongation of at least one coagulation assay (diluted Russels viper venom (dRVVT) or activated partial thromboplastin time (APTT)) which normalizes after addition of phospholipids. Both assays may be influenced by anticoagulants and therefore LA should not be tested during warfarin or heparin treatment. New (direct) oral anticoagulants (N(D)OAC based on direct inhibition of thrombin (dabigatran (DAB)) or factor Xa (rivaroxaban (RIV) and apixaban (API)) are approved for the treatment of venous thromboembolism (VTE). Therefore N(D)OAC may be used in the treatment of patients with APS where LA diagnosis is critical for the decision about the VTE treatment. It has been shown (primarily in-vitro) that N(D)OAC may influence LA testing. Objectives We have tested the effects of N(D)OAC on the assays routinely used for the diagnosis of LA in patients treated with these drugs in real life settings. Methods Plasmas from patients with atrial fibrillation treated with DAB (n=30), RIV (n=20) and API (n=17) and known not to have LA were tested using dRVVT (LA screen and LA confirm Life Diagnostics) and APTT (PTT LA Diagnostica Stago and Actin FS Siemens Healthcare Diagnostics) assays. According to the diagnostics algorithm, the dRVVT and APTT ratio <1.2 was considered negative, >1.4 positive, while if the ratio was 1.2-1.4 LA could not be ruled out. Plasma concentrations of drugs varied between 8-172 μg/L for DAB, 8-437 μg/L for RIV and 36-178 μg/L for API. Results Only 8 (27%) out of 30 plasmas from patients treated with DAB were negative, while 4 (13%) plasmas were positive for LA In all but 2 samples confirmation tests were also prolonged. DAB concentration correlated with both dRVVT (r=0.75 p<0.0001) and both APTT (r=0.55, p<0.005) assays, while ratio >1.2 was observed even with the lowest concentration. Negative LA was observed only in one sample from the patients treated with RIV (drug concentration 8 μg/L), while half of the samples diagnosed positive for LA. Confirmation tests were prolonged with RIV concentration above 100 μg/l. RIV concentration correlated with both dRVVT assays (r=0.86 p<0.0001) and both dRVVT and APTT ratio (r=0.56, p=0.01). LA diagnosed positive in 7 (41%) samples from patients treated with API. Eight samples (47%) were diagnosed negative for the presence of LA. Correlation between API concentration and dRVVT confirm test was observed (r=0.83, p<0.001) and it seems that a concentration >100 μg/L was associated with the prolongation of this confirmatory assay. Conclusions Our results suggest that the risk of overestimation of LA detection is present in samples from patients treated with N(D)OAC, particularly RIV. Therefore LA testing should not be performed in patients during the treatment with N(D)OAC. Prolongation of confirmation assays may be helpful for the recognition of false positivity, especially in the case of DAB. Disclosure of Interest None declared