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INTRODUCTION: Dabigatran (Dabi) is not routinely monitored. However, in emergency cases quantitative assessment is required and laboratories must provide suitable tests at all hours. Little is known about Dabi effects on thrombin generation. MATERIALS AND METHODS: Patient samples (n=241) were analyzed for functional Dabi concentrations (Dabi-TT) using a combination of the Hemoclot Thrombin Inhibitors assay (HTI®) and, for samples with low levels, undiluted thrombin time (TT). Results were compared to prothrombin time (PT) and activated partial thromboplastin time (APTT). In 49 samples Dabi effects were further investigated with Calibrated Automated Thrombogram (CAT®) for thrombin generation and with Russell's viper venom time (RVVT), prothrombinase-induced clotting time (PiCT®), chromogenic Anti-IIa® and ecarin clotting assay (ECA®). Fibrinogen and D dimer were assessed to reflect the coagulation status of the patient. A subset of these samples (n=21) were also analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: Dabi-TT correlated with RVVT (R(2)=0.49), PiCT® (R(2)=0.73), ECA® (R(2)=0.89), Anti-IIa® (R(2)=0.90) and LC-MS/MS (R(2)=0.81). APTT correlated curvi-linearly with Dabi-TT (R(2)=0.71), but was normal in many cases (18/70) despite Dabi-TT>40ng/mL. There was no association between Dabi-TT and fibrinogen or D dimer levels. Increasing Dabi concentrations prolonged lag time (R(2)=0.54) and, surprisingly, elevated the ETP and Peak of CAT® (p<0.001). CONCLUSIONS: Thrombin-specific tests measure Dabi accurately, whereas coagulation time based assays depend more on other factors. The enhanced thrombin generation in Dabi-treated patients may predict clinically relevant hypercoagulability and warrants further investigation. [ABSTRACT FROM AUTHOR]

Introduction: Current guidelines recommend acetylsalicylic acid (ASA) treatment after coronary artery bypass grafting (CABG) to reduce thrombotic vein graft occlusion. The optimal dosage of ASA is not known., Materials and Methods: Forty-two patients undergoing elective CABG were randomized to receive either ASA 75mg or 160mg once daily (OD) or 75mg twice daily (BID) after the operation. Platelet function testing was performed before, and one and three months after the operation., Results: White blood cell counts increased during the initial postoperative days whereas platelet counts were initially slightly reduced after the operation but increased after one month without any major changes of mean platelet volumes. Serum thromboxane B 2 was more effectively suppressed at one and three months after the operation with ASA 75mg BID or 160mg OD than with 75mg OD (p<0.001). ASA 75mg BID and 160mg OD were equally effective. Adenosine diphosphate stimulated platelet aggregation in whole blood (Multiplate®) was increased one and three months after the operation, and this was counteracted by ASA 75mg BID but not by 75 or 160mg OD. Arachidonic acid-induced aggregation was more effectively inhibited by 75mg BID or 160mg OD compared to 75mg OD at three months., Conclusions: Less effective inhibition of platelet activation was obtained with ASA 75mg OD than with ASA 160mg OD or 75mg BID up to three months after CABG. Especially the latter dose is of interest for further studies of efficacy and clinical outcomes after CABG., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Aim: Postprandial platelet activation was related to postprandial insulin rather than glucose levels in a previous meal insulin study in type 2 diabetes mellitus (T2DM). We therefore compared postprandial platelet activation in type 1 (T1DM) patients without insulin secretion and T2DM patients with high postprandial insulin levels., Material and Methods: Patients with T1DM (n=11) and T2DM (n=12) were studied before and 90min after a standardized meal without premeal insulin. Five T1DM patients volunteered for a restudy with their regular premeal insulin. Platelet activation was assessed by flow cytometry, with and without the thromboxane analogue U46619 or ADP, and by whole blood aggregometry (Multiplate®). Effects of insulin (100μU/mL) in vitro were also studied., Results: Before the meal, glucose, insulin and platelet activation markers other than platelet-leukocyte aggregates (PLAs) were similar in T1DM and T2DM; PLAs were higher in T1DM. Postprandial glucose levels increased more markedly in T1DM (to 22.1±1.4 vs. 11.2±0.6mmol/L) while insulin levels increased only in T2DM (from 24.4±4.4 to 68.8±12.3μU/mL). Platelet P-selectin expression, fibrinogen binding and PLA formation stimulated by U46619 were markedly enhanced (approximately doubled) and whole blood aggregation stimulated by U46619 was increased (p<0.05 for all) after the meal in T2DM patients but not in T1DM patients. The pilot study with premeal insulin in T1DM patients showed postprandial platelet activation when postprandial insulin levels increased. In vitro insulin mildly activated platelets in both groups., Conclusion: Postprandial platelet activation via the thromboxane pathway is related to postprandial hyperinsulinemia and not to postprandial hyperglycaemia in patients with diabetes., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Dilute Russell Viper Venom Time (DRVV-T) might be useful in urgent settings for screening patients on Non-VKA Oral Anticoagulants (NOACs)., Aim: To compare the accuracy of DRVV-T with gold standard assays for the assessment of pharmacodynamics of dabigatran, rivaroxaban and vitamin K antagonist (VKA) in plasma samples from patients., Methods: Sixty rivaroxaban, 48 dabigatran and 50 VKA samples from patients were included. DRVV-T was performed in all groups using STA®-Staclot®DRVV-Screen and -Confirm. For NOACs, PT and aPTT were performed using different reagents while plasma drug concentrations were measured by liquid mass-spectrometry (LC-MS/MS). For VKA, INR was performed using RecombiPlasTin 2G®., Results: For NOACs, correlations between calibrated STA®-Staclot®DRVV-Confirm and LC-MS/MS (rs=0.88 and 0.97 for rivaroxaban and dabigatran, respectively) were higher than the ones obtained with STA®-Staclot®DRVV-Screen (rs=0.87 and 0.91), PT (rs=0.83 to 0.86) or aPTT (rs=0.84 to 0.89). Bland Altman analyses showed that calibrated DRVV-T methods tend to overestimate plasma concentrations of NOACs. ROC curves revealed that cut-off to exclude supra-therapeutic levels at Ctrough (i.e. 200ng/mL) are different for dabigatran and rivaroxaban. Neither STA®-Staclot®DRVV-Screen nor -Confirm correlated sufficiently with the intensity of VKA therapy (rs=0.35 and 0.52)., Conclusions: STA®-Staclot®DRVV-Confirm provides a rapid estimation of the intensity of anticoagulation with rivaroxaban or dabigatran without specific calibrators. At Ctrough, thresholds for rivaroxaban and dabigatran can be used to identify supra-therapeutic plasma level. However, this test cannot differentiate the nature of the NOACs. The development of a point-of-care device optimising this method would be of particular interest in emergency situations., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Introduction: Previous data from our group show that acetylsalicylic acid (ASA), especially at low dose, alters the network structure of fibrin, rendering it more porous. The present study was performed to extend the dose-response curve for effects of ASA on fibrinogen clotting properties and to examine the variability of these effects during a 24-h dose interval., Material and Methods: Fifteen healthy volunteers received ASA 37.5 mg daily (low dose) for 10 days and, after an interval of 2 weeks, 320 mg daily (medium dose) for 7 days, followed by a single bolus dose of 640 mg (high dose). The plasma fibrinogen concentrations were determined and the permeability of fibrin gels (Ks) was assayed with a recently modified flow measurement technique. Three-dimensional (3D) structure of the fibrin network was studied by confocal microscopy., Results: ASA therapy did not influence fibrinogen concentrations. Compared to baseline, Ks levels were increased by 21% and 31% in samples during medium and high dose ASA treatment (p<0.01) and, even more markedly, by 44% (p<0.0001) with very low dose ASA treatment (p<0.01, compared to the higher doses). The effects of ASA on fibrin gel permeability were stable over a 24-h dose interval. During ASA treatment, thicker fibrin fibers and larger network pores with irregular structure were observed by confocal microscopy., Conclusions: Acetylation of lysine residues in the fibrinogen molecule may explain the alterations in its clotting property, resulting in altered fibrin gel permeability. The mechanism(s) behind the greater increase in fibrin gel permeability and alterations in 3D structure of the fibrin network observed, and why this phenomenon is more pronounced at low compared to intermediate or high ASA doses, deserve further investigations.

Despite undoubtful clinical evidence of anti-platelet effects of aspirin, previous studies demonstrate little inhibition by aspirin treatment of single platelet activation as measured by flow cytometry. This discrepancy was further evaluated using flow cytometric measurements of circulating platelet-leukocyte aggregates (PLAs), which may be a more sensitive marker of platelet activation in vivo than measurements of activation markers on single platelets. Blood samples were obtained from 15 healthy subjects before and after aspirin treatment (75 and 500 mg daily for 1 week). Platelet (P-selectin expression) and leukocyte (CD11b expression) activation and platelet-leukocyte aggregation were monitored by whole blood flow cytometry. Approximately 1% platelets in unstimulated samples were P-selectin-positive, i.e., circulating activated platelets, and about 3% of leukocytes were circulating as PLAs; neither of these parameters were reduced by aspirin treatment. Circulating platelet micro-aggregates were not influenced by aspirin either. In vitro stimulation with ADP, thrombin, or PAF increased platelet P-selectin expression and thus PLA formation, but these responses were not affected by aspirin. Leukocyte CD11b expression, a marker of leukocyte secretion, was not significantly influenced by aspirin either in unstimulated samples or upon in vitro stimulation. Thus, the present data support the concept that thromboxane generation is of little importance for the activation of single platelets; the platelet inhibiting effect of aspirin is seen mainly in the presence of close cell-cell contact, which enhances the importance of thromboxane. Multiple mechanisms may contribute to the remarkable clinical anti-thrombotic effect of aspirin.

Diabetes mellitus (DM) is associated with platelet and leukocyte dysfunction. Previous observations with regard to insulin effects on platelet and leukocyte function are less than consistent. We thus investigated the effects of insulin on platelets and leukocytes, as well as on platelet-leukocyte interactions in whole blood. Hirudinized whole blood from 20 healthy subjects was preincubated at 37 degrees C in the absence or presence of insulin (30 and 300 microU/ml), and further incubated without or with adenosine diphosphate (ADP) (10(-5) M) or N-formyl-methionyl-leucyl-phenylalanine (fMLP) (10(-7) M), respectively. Platelet P-selectin expression, platelet fibrinogen binding, single platelet and platelet-platelet aggregate (PPA) counts, and leukocyte CD11b expression and superoxide anion production were monitored by flow cytometry. Insulin decreased single platelet counts (P<0.05) and increased PPAs (P<0.01) at 300 microU/ml in unstimulated samples, but did not significantly influence the P-selectin expression or fibrinogen binding of single platelets. Insulin also enhanced ADP-induced platelet aggregation, seen as an augmented decrease of single platelet counts. Insulin (30 microU/ml) increased leukocyte CD11b mean fluorescence intensity (MFI) in unstimulated, as well as fMLP- and ADP-stimulated samples (P<0.05 for all). fMLP-induced superoxide anion (O(2)(-)) production was, however, attenuated by insulin. Furthermore, fMLP-activation of leukocytes was associated with enhanced platelet fibrinogen binding and P-selectin expression. In conclusion, clinically relevant concentrations of insulin enhance platelet aggregability and leukocyte CD11b expression, but attenuate leukocyte respiratory burst activity. Our results suggest that insulin may modulate thrombotic and inflammatory processes in vivo in a complex manner.

Introduction: Insulin treatment is essential in Type 1 diabetes mellitus (DM). However, previous studies have shown complex effects of insulin on platelet function. Proinsulin C-peptide has shown beneficial effects in Type 1 DM, but it is not known if it can affect platelet activation. We thus investigated how insulin, C-peptide, and their combination influence platelets from DM patients and healthy subjects., Materials and Methods: Hirudinized blood from patients (n = 10) and healthy subjects (n = 10) was preincubated in the absence or presence of insulin (10 and 100 microU/ml), C-peptide (0.3, 1, and 10 nM), or the combination (1 nM C-peptide + 100 microU/ml insulin or 10 nM C-peptide + 100 microU/ml insulin) and further incubated without or with 10(-6) M ADP. Platelet activation was monitored by platelet fibrinogen binding and P-selectin expression using whole blood flow cytometry. Data are presented as binding index (BI), which integrates the percentage of activated cells and their mean fluorescence intensity., Results: Insulin enhanced ADP-induced platelet fibrinogen binding in both Type 1 DM patients and healthy subjects. For example, ADP-stimulated platelet fibrinogen BI increased from 4.25 +/- 0.74 to 8.63 +/- 2.00 with 10 microU/ml insulin (P < .05) in Type 1 DM patients. However, insulin did not increase platelet P-selectin expression. Proinsulin C-peptide did not influence platelet fibrinogen binding or P-selectin expression in either Type 1 DM patients or healthy subjects. The combination of C-peptide and insulin had similar effects as insulin alone., Conclusions: Insulin at physiological concentrations enhances platelet fibrinogen binding in both Type 1 DM patients and healthy subjects, whilst C-peptide does not influence platelet activation., (Copyright 2002 Elsevier Science Ltd.)

The urinary excretion of stable metabolites of thromboxane A2, such as 11-dehydro-thromboxane B2, reflects platelet activity in vivo. Efficient sample purification is required before analysis of thromboxane metabolites, due to the presence of large amounts of interfering material in urine. Analysis by gas chromatography-mass spectrometry after extensive sample work-up procedures provides the most reliable data, but detection by enzyme immunoassay may be reliable if sample cleanup is adequate. We describe an improved immunoassay procedure for 11-dehydro-thromboxane B2, which is based on a simple one-step solid phase extraction, by using Bond-Elut Certify II columns, followed by enzyme immunoassay by using commercially available reagents. 11-Dehydro-thromboxane B2 exists in two forms, with different chemical and immunological characteristics, which are in pH-dependent equilibrium. We kept 11-dehydrothromboxane B2 in its open ring form throughout the assay, by incubating and handling samples at pH 8.6. The extraction step achieved a recovery of 83% (95% confidence interval 74-92%), the sensitivity of the enzyme immunoassay was doubled, and the reproducibility of the assay improved under these conditions. Intra- and interassay coefficients of variation were 3 and 13.8%, respectively. A single 500-mg dose of aspirin reduced the excretion of 11-dehydro-thromboxane B2 by 77+/-14%, suggesting good specificity. Comparison with gas chromatography-mass spectrometry in 28 urine samples showed excellent agreement between the two methods (r2 = 0.94; p<0.0001), and a regression line with a slope close to 1.0. The presently modified enzyme immunoassay for 11-dehydro-thromboxane B2 is suitable for clinical studies evaluating platelet function in vivo and has the advantage of being simpler and less expensive to use than gas chromatography-mass spectrometry.

Blood platelet activation in vivo was evaluated by measuring beta-thromboglobulin in plasma and high molecular weight beta-thromboglobulin in urine in hypertensive smoking and nonsmoking middle-aged men (n=36) and in normotensive age-matched controls (n=40). We found no significant linear relationships between nocturnal or resting urinary high molecular weight beta-thromboglobulin and plasma beta-thromboglobulin in the combined hypertensive and normotensive groups. The excretion of high molecular weight beta-thromboglobulin correlated significantly with diastolic blood pressure when all subjects were pooled. After 60 minutes supine rest, nonsmokers had higher excretion of high molecular weight beta-thromboglobulin than smokers. Plasma beta-thromboglobulin levels tended to be higher in hypertensives. In multivariate analyses, resting high molecular weight beta-thromboglobulin excretion was positively related to diastolic blood pressure and negatively related to smoking, whereas plasma beta-thromboglobulin was positively related to diastolic blood pressure and inversely related to apolipoprotein A1 and B. We conclude that urinary high molecular weight beta-thromboglobulin and plasma beta-thromboglobulin are not closely related, but are complementary analyses, as there are methodological confounders for both variables.