Summary.Epidemiological studies have shown that women who use third-generation oral contraceptives (OC) containing desogestrel, gestodene or norgestimate have a higher risk of venous thrombosis than women who use second-generation OC containing levonorgestrel. It is also known that a mutation in factor V (factor V Leiden ), which results in resistance to activated protein C (APC) and which is the most common cause of hereditary thrombophilia, potentiates the prothrombotic effect of OC.Effects of APC on thrombin generation in the plasma of women using OC were compared to the response to APC in non-OC users and in individuals that were heterozygous or homozygous for factor V Leiden . The response towards APC was evaluated on basis of the ratio (APC-sr) of the time integrals of thrombin formation determined in the presence and absence of APC.Compared with women not using OC, women who used OC exhibited a significantly decreased sensitivity to APC (P < 0 . 001), independent of the kind of OC used. Women who used third-generation monophasic OC were significantly less sensitive to APC than women using second-generation OC (P < 0 . 001) and had APC-sr that did not significantly differ from heterozygous female carriers of factor V Leiden who did not use OC. Women who were heterozygous for factor V Leiden and used OC had APC-sr in the range of homozygous carriers of factor V Leiden . Two women who started OC therapy had significantly elevated APC-sr within 3 d.Acquired APC resistance may explain the epidemiological observation of increased risk for venous thrombosis in OC users, especially in women using third-generation OC.

The lag time, ETP and peak height of thrombin generation depend on the levels of multiple coagulation factors and inhibitors. The specific assay determinants vary with the experimental conditions.

SwedenTo cite this article: van der Meijden PEJ, van Schilfgaarde M, van Oerle R, Renné T, ten Cate H, Spronk HMH. Platelet-and erythrocyte-derived microparticles trigger thrombin generation via factor XIIa. J Thromb Haemost 2012; 10: 1355-62.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 cellspecific 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 (< 1 fM nM )1 PS), whereas monocyte-derived MPs exposed TF (32 fM nM )1 PS). Platelet-, erythrocyte-and monocyte-derived MPs, but not purified phospholipids, initiated thrombin generation in normal plasma in the absence of an external trigger (lag time < 11 min). Deficiency or inhibition of FVII had no effect on thrombin generation induced by platelet-and erythrocyte-derived MPs, but interfered with monocyte MP-triggered coagulation. Platelet-and erythrocyte-derived MPs completely failed to induce thrombin generation in FXII-deficient plasma. In contrast, monocyte-derived MPs induced similar thrombin generation in normal vs. FXII-deficient plasma. Conclusion: MPs from platelets and erythrocytes not only propagate coagulation by exposing PS but also initiate thrombin generation independently of TF in a FXII-dependent manner. In contrast, monocyte-derived MPs trigger coagulation predominantly via TF.

Thrombin generation was greatly influenced by preanalytic conditions, demonstrating the need for an international standardized protocol.

Background-Thrombin generation in vivo may be important in regulating atherosclerotic progression. In the present study, we examined for the first time the activity and presence of relevant coagulation proteins in relation to the progression of atherosclerosis. Methods and Results-Both early and stable advanced atherosclerotic lesions were collected pairwise from each individual (nϭ27) during autopsy. Tissue homogenates were prepared from both total plaques and isolated plaque layers, in which the activity of factors (F) II, X, and XII and tissue factor was determined. Microarray analysis was implemented to elucidate local messenger RNA synthesis of coagulation proteins. Part of each specimen was paraffin embedded, and histological sections were immunohistochemically stained for multiple coagulation markers with the use of commercial antibodies. Data are expressed as median (interquartile range [IQR] Key Words: atherosclerosis Ⅲ hypercoagulability Ⅲ immunohistochemistry Ⅲ plaque Ⅲ thrombosis A therosclerosis is widely recognized as a chronic inflammatory disease. 1 Rupture of an atherosclerotic plaque is considered the predominant underlying cause of acute atherothrombotic events such as myocardial infarction, ischemic stroke, and vascular death. A close relation between blood coagulation and atherosclerosis 2,3 is supported by studies revealing the presence of specific coagulation proteins within an atherosclerotic lesion. Tissue factor (TF) and factor (F) VII, of which the complex is the principal initiator of coagulation in vivo, are expressed on macrophages and vascular smooth muscle cells (SMC) within the arterial vessel wall and atherosclerotic lesion. 4,5 Both proteins potentially participate in multiple proatherogenic processes such as migration and proliferation of SMC, 6 inflammation, and angiogenesis. 7 In addition to the single effects of each protein, the local interaction between macrophage/SMC-derived TF and FVII may provide a catalytic complex for subsequent generation of thrombin and fibrin, of which the latter is also detectable in atherosclerotic lesions. 8,9 The procoagulant condition of the atherosclerotic lesion may be further enhanced by the presence of various proinflammatory cytokines (eg, tumor necrosis factor-␣, interleukin-1 10 ), which may downregulate local expression of anticoagulant proteins such as thrombomodulin and the endothelial protein C receptor on endothelial cells. 11 Received September 4, 2009; accepted June 28, 2010 Clinical Perspective on p 830Thrombin, a key enzyme in blood coagulation, may also play a critical role in many processes related to the development, progression, and atherothrombotic potential of atherosclerotic plaques. 12 Direct evidence for the role of thrombin in the atherogenic process comes from experiments showing reduced progression of atherosclerosis in apolipoprotein E Ϫ/Ϫ mice on pharmacological inhibition of thrombin. 13 Moreover, decreased expression of TF pathway inhibitor (TFPI) on an apolipoprotein E Ϫ/Ϫ background increased the atheroscle...

Mean platelet volume (MPV) is associated with various diseases. Several authors reported anticoagulant and time dependency. Therefore, standardized laboratory methods are essential. The aim of this study was to standardize the MPV measurement. Blood was collected in potassium-ethylenediaminetetra-acid (EDTA) and sodium-citrate tubes. First, MPV and platelet count were determined every half hour for 4 hours in 20 healthy volunteers. The same parameters were acquired from a second group of 100 healthy donors. We measured at the point of highest stability determined in the first step and aimed to determine a reference range. Citrate samples revealed significantly smaller MPV (7.0 fL ± 0.69 standard deviation [SD]) than EDTA (8.0 fL ± 0.8 SD). Platelets swell until 120 minutes in EDTA and until 60 minutes in citrate. Mean platelet count changed significantly in citrate. In the second group, no inverse correlation between MPV and platelet count was seen. A reference range was calculated (EDTA, 7.2-10.8 fL; citrate, 6.1-9.5 fL). Platelets stored in citrate are significantly smaller compared to those stored in EDTA. Timing is important when measuring platelet volume. Optimal measuring time should be 120 minutes after venipuncture. For this we depicted a reference range. Platelet count is most stable in EDTA. There was no inverse relation between MPV and platelet count.

BackgroundVariations in the blood coagulation activity, determined genetically or by medication, may alter atherosclerotic plaque progression, by influencing pleiotropic effects of coagulation proteases. Published experimental studies have yielded contradictory findings on the role of hypercoagulability in atherogenesis. We therefore sought to address this matter by extensively investigating the in vivo significance of genetic alterations and pharmacologic inhibition of thrombin formation for the onset and progression of atherosclerosis, and plaque phenotype determination.Methodology/Principal FindingsWe generated transgenic atherosclerosis-prone mice with diminished coagulant or hypercoagulable phenotype and employed two distinct models of atherosclerosis. Gene-targeted 50% reduction in prothrombin (FII−/WT:ApoE−/−) was remarkably effective in limiting disease compared to control ApoE−/− mice, associated with significant qualitative benefits, including diminished leukocyte infiltration, altered collagen and vascular smooth muscle cell content. Genetically-imposed hypercoagulability in TMPro/Pro:ApoE−/− mice resulted in severe atherosclerosis, plaque vulnerability and spontaneous atherothrombosis. Hypercoagulability was associated with a pronounced neutrophilia, neutrophil hyper-reactivity, markedly increased oxidative stress, neutrophil intraplaque infiltration and apoptosis. Administration of either the synthetic specific thrombin inhibitor Dabigatran etexilate, or recombinant activated protein C (APC), counteracted the pro-inflammatory and pro-atherogenic phenotype of pro-thrombotic TMPro/Pro:ApoE−/− mice.Conclusions/SignificanceWe provide new evidence highlighting the importance of neutrophils in the coagulation-inflammation interplay during atherogenesis. Our findings reveal that thrombin-mediated proteolysis is an unexpectedly powerful determinant of atherosclerosis in multiple distinct settings. These studies suggest that selective anticoagulants employed to prevent thrombotic events may also be remarkably effective in clinically impeding the onset and progression of cardiovascular disease.

We used the thrombin generation assay to evaluate the hypercoagulable state according to JAK2 V617F mutational status in essential thrombocythemia (ET) and polycythemia vera (PV) patients. Thrombin generation was determined in the presence and absence of activated protein C (APC), and APC resistance was expressed as normalized APC sensitivity ratio (nAPCsr). Tissue factor pathway inhibitor (TFPI), total and free protein S (PS), prothrombin (