Search

Background The role of platelets in the pathogenesis of venous thromboembolism (VTE) is receiving increasing attention; however, limited information is available on platelet function in the acute phase of the disease. Objective To characterize platelet function according to VTE phenotypes. Patients/Methods In total, 154 subjects (isolated pulmonary embolism [iPE], n = 28; isolated deep vein thrombosis [iDVT], n = 35; DVT+PE, n = 91) were included. In this study platelet function analyzer (PFA)-200, light transmission aggregometry (LTA), thrombin generation (TG) in presence (PRP) and absence (PFP) of platelets and platelet flow cytometry were investigated. LASSO regression was used to select clinical and platelet biomarkers that distinguish between VTE phenotypes. Results PFA-200 results did not differ between VTE phenotypes. LTA from DVT+PE subjects showed lowest maximum aggregation after epinephrine and adenosine diphosphate compared to iPE and iDVT. Lower % of PAC-1-positive platelets after in-vitro trigger were present in DVT+PE and iPE compared to iDVT. TG in PRP had lower peak height and velocity in DVT+PE and iPE against iDVT. The results of LASSO regression for the distinction between DVT+PE vs iDVT identified 18 variables (AUC =0.93) of which 72% were platelet biomarkers. For distinction between iPE and iDVT, 10 variables were selected (AUC = 0.96) of which 50% were platelet-related. Obesity was the only variable weakly discriminating between DVT+PE vs iPE (AUC = 0.66). Conclusion This explorative study suggests an important distinction between PE-related phenotypes and iDVT when considering clinical and platelet function data. Lower platelet-dependent TG along with reduced platelet reactivity suggest higher platelet degranulation in PE-dependent phenotypes compared to iDVT.

INTRODUCTION: Vitamin K antagonists (VKA) and non-vitamin K oral antagonist anticoagulants (NOAC) are used in the clinic to reduce risk of thrombosis. However, they also exhibit vascular off-target effects. The aim of this study is to compare VKA and NOAC on atherosclerosis progression and calcification in an experimental setup.MATERIAL AND METHODS: Female Apoe-/- mice (age 12 weeks) were fed Western-type diet as control or supplemented with dabigatran etexilate or warfarin for 6 or 18 weeks. Vascular calcification was measured in whole aortic arches using µCT and [18 F]-NaF. Atherosclerotic burden was assessed by (immuno)histochemistry. Additionally, in vitro effects of warfarin, thrombin, and dabigatran on primary vascular smooth muscle cells (VSMC) were assessed.RESULTS: Short-term treatment with warfarin promoted formation of atherosclerotic lesions with a pro-inflammatory phenotype, and more rapid plaque progression compared with control and dabigatran. In contrast, dabigatran significantly reduced plaque progression compared with control. Long-term warfarin treatment significantly increased both presence and activity of plaque calcification compared with control and dabigatran. Calcification induced by warfarin treatment was accompanied by increased presence of uncarboxylated matrix Gla protein. In vitro, both warfarin and thrombin significantly increased VSMC oxidative stress and extracellular vesicle release, which was prevented by dabigatran.CONCLUSION: Warfarin aggravates atherosclerotic disease activity, increasing plaque inflammation, active calcification, and plaque progression. Dabigatran lacks undesired vascular side effects and reveals beneficial effects on atherosclerosis progression and calcification. The choice of anticoagulation impacts atherosclerotic disease by differential off target effect. Future clinical studies should test whether this beneficial effect also applies to patients.

Atherothrombosis is characterized by the inflammatory process of atherosclerosis combined with a hypercoagulable state leading to superimposed thrombus formation. In atherosclerotic plaques, cell signaling can occur via protease-activated receptors (PARs), four of which have been identified so far (PAR1-PAR4). Proteases that are able to activate PARs can be produced systemically, but also at the sites of lesions, and they include thrombin and activated factor X. After PAR activation, downstream signaling can lead to both proinflammatory effects and a hypercoagulable state. Which specific effect occurs depends on the type of protease and activated PAR, and the site of activation. Hypercoagulable effects are mainly exerted through PAR1 and PAR4, whereas proinflammatory responses are mostly seen after PAR1 and PAR2 activation. PAR signaling pathways contribute to atherothrombosis, suggesting that inhibition of these pathways possibly prevents cardiovascular events based on this pathophysiological mechanism. In this review, we highlight the pathways by which PAR activation leads to proinflammatory responses and a hypercoagulable state. Furthermore, we give an overview of potential pharmacological treatment targets that promote vascular protection.

Summary. Background: Thrombin generation assays are sensitive methods for assessment of the overall clotting potential of plasma, but, despite their common use in thrombosis research, standardization of preanalytic conditions is lacking. In order to set up a standardized protocol, we analyzed different preanalytic variables and validated the calibrated automated thrombogram method. Methods and Results: Thrombin generation was assessed with 0, 1 and 5 pm tissue factor (TF). Variations in thrombin generation were mostly attributable to the type of collection tube, mainly because of variations in contact activation. The collection tube also determined the influence of other preanalytic variables on thrombin generation, e.g. the need for a discard tube, the storage of whole blood, and the centrifugation method. Regarding the collection system, blood drawn through intravenous catheters or butterfly needles showed significantly more hemolysis than blood obtained with venipuncture using conventional needles. The results showed that a discard tube is still needed for thrombin generation measurements. After blood collection, whole blood is best centrifuged immediately, to prevent activation or degradation of coagulation proteins, and a second centrifugation step at 10 000 × g is recommended. After thawing, plasma is best analyzed immediately, as storage resulted in thrombin generation results outside the 10% range of the reference sample. On the basis of these results, we set up an in-house standardized protocol, which was used for validation, resulting in coefficients of variations of

Although the link between blood coagulation and atherogenesis has been long postulated, only recently, and through the extensive work on transgenic mice, crossbred on an atherogenic background, has the direction of this interaction become visible. In general, hypercoagulability in mice tends to increase atherosclerosis, whereas hypocoagulability reduces the atherosclerotic burden, depending on the mouse model used. The information on a direct relationship between coagulation and atherosclerosis in humans, however, is not that clear. Almost all coagulation proteins, including tissue factor, are found in atherosclerotic lesions in humans. In addition to producing local fibrin, a matrix for cell growth, serine proteases such as thrombin may be very important in cell signaling processes, acting through the activation of protease-activated receptors (PARs). Activation of PARs on vascular cells drives many complex processes involved in the development and progression of atherosclerosis, including inflammation, angiogenesis, and cell proliferation. Although current imaging techniques do not allow for a detailed analysis of atherosclerotic lesion phenotype, hypercoagulability, defined either by gene defects of coagulation proteins or elevated levels of circulating markers of activated coagulation, has been linked to atherosclerosis-related ischemic arterial disease. New, high-resolution imaging techniques and sensitive markers of activated coagulation are needed in order to study a causal contribution of hypercoagulability to the pathophysiology of atherosclerosis. Novel selective inhibitors of coagulation enzymes potentially have vascular effects, including inhibition of atherogenesis through attenuation of inflammatory pathways. Therefore, we propose that studying the long-term vascular side effects of this novel class of oral anticoagulants should become a clinical research priority.

See also Shapiro S, Laffan M. Making contact with microparticles. This issue, pp 1352–4. Summary. Background: The procoagulant properties of microparticles (MPs) are due to the of the presence of phosphatidylserine (PS) and tissue factor (TF) on their surface. The latter has been demonstrated especially on MPs derived from monocytes. Objectives: To investigate the relative contribution of TF and factor (F)XII in initiating coagulation on MPs derived from monocytes, platelets and erythrocytes. Methods: Microparticles were isolated from calcium ionophore-stimulated platelets, erythrocytes and monocytic THP-1 cells. MPs were quantified, characterized for cell-specific antigens and analyzed for TF, PS exposure and their thrombin-generating potential. Results: The MP number was not proportional to PS exposure and the majority of the MPs exposed PS. TF activity was undetectable on platelet- and erythrocyte-derived MPs (

Summary. Background: Overt proteinuria is a strong risk factor for thromboembolism, owing to changes in the levels of various coagulation proteins and urinary antithrombin loss. The described coagulation disturbances in these patients are based on outdated studies conducted primarily in the 1970s and 1980s. Whether these coagulation disturbances resolve with antiproteinuric therapy has yet to be studied. Methods: At otal of 32 patients with overt proteinuria (median, 3.7 g day )1 ; interquartile range, 1.5–5.6) were enrolled in this intervention crossover trial designed to assess optimal antiproteinuric therapy with sodium restriction, losartan, and diuretics. Levels of various procoagulant and anticoagulant proteins, and parameters of two thrombin generation assays (calibrated automated thrombogram [CAT] and prothrombin fragment 1 + 2) were compared between the placebo period and the maximum antiproteinuric treatment period. As a secondary analysis, coagulation measurements of the placebo period in these patients were compared with those of 32 age-matched and sex-matched healthy controls. Results: Median proteinuria was significantly lower during the maximum treatment period (median, 0.9 g day )1 ; interquartile range, 0.6–1.4; P < 0.001) than during the placebo period. Similarly, levels of various liver-synthesized procoagulant and anticoagulant proteins, activated protein C resistance and prothrombin fragment 1 + 2 levels were significantly lower during the maximum treatment period than during the placebo period. However, von Willebrand factor and factor VIII levels were similar. On the basis of the higher levels of procoagulant proteins (fibrinogen, FV, FVIII, and von Willebrand factor) and both thrombin generation assays, patients were substantially more prothrombotic than healthy controls (P < 0.004). Conclusions: Antiproteinuric therapy ameliorates the prothrombotic state. Proteinuric patients are in a more prothrombotic state than healthy controls.

Summary. Background: Despite improved treatment options, myocardial infarction is still an important cause of morbidity and mortality. One of the contributing mechanisms in the acute myocardial infarction (AMI) is plasma hypercoagulability. Methods: We investigated hypercoagulability in 135 (first) patients with AMI using thrombin generation (TG) testing. TG testing was performed in plasmas, drawn upon admission and before medication administration, and subsequently after 4 days, 3 and 6 months. Further, we evaluated determinants of thrombin generation using multiple regression analysis of major coagulation proteins and inhibitors. Admission TG results were also related to 1-year outcome: cardiovascular death, recurrent myocardial infarction, a second coronary intervention [percutaneous coronary intervention (PCI) or coronary artery bypass grafting (CABG)] and ischemic stroke. Results: At day 0, the TG parameters peak height, endogenous thrombin potential (ETP) and lag time were increased compared with a reference population. Peak height and lag time stayed persistently increased in patients. The lowest half of the ETP values was statistically not significantly associated with an occurrence of endpoints. The lowest half of the ETP values combined with the upper half of the D-dimer values were associated with endpoints; odds ratio 5.8 (1.1–30.7). Tissue factor pathway inhibitor (TFPI) seems to be an important determinant of TG in AMI and healthy persons. Conclusions: TG reflects acute hypercoagulability during AMI and partly also in the 6-month period after the acute event. TG shows a trend of an inverse association with risk of recurrent ischemic cardiovascular complications. Unraveling mechanisms in TG might improve our understanding of the pathophysiology of AMI and direct future improvements in medical care.

Summary. Background: Concentrations of inflammatory and hemostatic variables are influenced by biological variation, which is the natural within-subject variation over time. Objectives: The aim of this study was to determine fibrinogen, C-reactive protein (CRP), platelet aggregation, thrombin generation and prothrombin time (PT): (i) the number of repeated measurements needed to obtain the true habitual concentration of an individual; (ii) the recommended analytical imprecision for diagnosis and monitoring; (iii) the recommended analytical bias; (iv) the contribution of analytical imprecision to test result variability; (v) the index of individuality; (vi) the reference change value; and (vii) the seasonal variation. Subjects and methods: We collected 520 blood samples over a 1-year period from 40 healthy individuals, and determined the between-subject, within-subject and seasonal variation in fibrinogen, CRP, platelet aggregation, thrombin generation and PT. Results: One or two repeated measurements were sufficient to establish the true habitual concentration, except for platelet aggregation and peak thrombin generation, where at least four and nine repeated measurements were needed, respectively. For diagnosis, the maximal recommended coefficient of analytical variation (CV) was 4%–27%, except for CRP (77.7%). For monitoring, these CVs were on average 3% lower. Recommended analytical bias varied between 1.7% and 33.2%. Finally, seasonal variation was observed in concentrations of fibrinogen and thrombin generation, which could explain approximately 11% of their total variation. Conclusion: This study provides insights into the biological variability of selected inflammatory and hemostatic markers, which can be used for sample size calculations and to determine the analytical quality specifications for their respective assays.

Summary. Background: In hypertensive patients, the activated renin–angiotensin system induces a prothrombotic state resulting from imbalance between coagulation and fibrinolysis. Although blood pressure cannot be regulated in therapy-resistant hypertensive patients, they may still be responsive to medication that attenuates the renin–angiotensin system. Objective: Our objective was to study possible attenuating properties of angiotensin II type 1 receptor blockers (AT1RBs) on the prothrombotic state in therapy-resistant hypertensive patients, focusing on parameters of fibrinolysis and coagulation. Methods: Fourteen therapy-resistant hypertensive patients received AT1RB eprosartan infusion (45 and 150 μg kg–1) (study group), and 33 therapy-resistant hypertensive patients received saline (0.9%) infusion (control group) prior to renal angiography. Baseline values of parameters of coagulation and fibrinolysis were set at 1.00, and relative changes were calculated. Results: Plasminogen activator inhibitor type 1 (PAI-1) antigen showed non-significant decreases in both the study group (arterial 1.00–0.45, venous 1.00–0.42) and control group (arterial 1.00–0.84, venous 1.00–0.88). PAI-1 activity significantly decreased in the study group (arterial 1.00–0.72, venous 1.00–0.71) and control group (arterial 1.00–0.83, venous 1.00–0.94). In the study group, tissue-type plasminogen activator (t-PA) antigen decreased significantly (arterial 1.00–0.62, venous 1.00–0.67), whereas t-PA activity significantly increased (arterial 1.00–6.15, venous 1.00–2.66). In the control group, t-PA antigen remained unchanged. No changes were observed in blood pressure during and after infusion of eprosartan. Conclusion: Therapy-resistant hypertensive patients show beneficial changes in fibrinolytic activity after infusion of a non-pressor dose of AT1RB.