Conclusions: Circulating TF(+)MPs and mucins may concertedly aggravate coagulopathy in PAC. Understanding of underlying mechanisms may result in new treatment strategies for VTE prevention and... Show moreConclusions: Circulating TF(+)MPs and mucins may concertedly aggravate coagulopathy in PAC. Understanding of underlying mechanisms may result in new treatment strategies for VTE prevention and improvement of survival. Show less
Woei-A-Jin, F.J.S.H.; Starre, W.E. van der; Tesselaar, M.E.T.; Rodriguez, P.G.; Nieuwkoop, C. van; Bertina, R.M.; ... ; Osanto, S. 2014
Multiple myeloma (MM) is associated with an increased risk of venous thromboembolic (VTE) complications. Aim of this study was to measure microparticle-associated tissue factor (MP-TF) activity in... Show moreMultiple myeloma (MM) is associated with an increased risk of venous thromboembolic (VTE) complications. Aim of this study was to measure microparticle-associated tissue factor (MP-TF) activity in patients with newly diagnosed MM before and after chemotherapy and to investigate whether MP-TF activity is associated with VTE. MP-TF activity was assessed in 122 newly diagnosed MM patients who were eligible for combination chemotherapy. MP-TF activity levels (17.6 fM Xa/min [8.6-33.2] (median [IQR]) were higher in untreated MM patients compared to normal healthy volunteers (4.1 fM Xa/min [2.3-6.6], p <0.001). MP-TF activity prior to the start of treatment was not different between patients who developed a VTE during follow-up (n=15) and those who did not (n=107). In 75 patients in whom plasma was obtained before and after chemotherapy, MP-TF activity decreased significantly (from 17.4 [10.2-32.8] to 12.0 [7.0-18.5] fM Xa/min, P=0.006). MP-TF activity remained, however, elevated in patients who developed VTE (15.1 [10.3-25.2]), in contrast to patients not developing VTE (11.4 [7.0-25.2], P<0.001). In conclusion, MP-TF activity is increased in patients with MM. Whether MP-TF activity has a pathogenetic role in VTE in MM patients remains to be established in future studies. Show less
Results of plasma microparticles (MPs) measurements reported in the literature vary widely. This is clearly not only related to the lack of well-standardised MP assays, but also to variations in... Show moreResults of plasma microparticles (MPs) measurements reported in the literature vary widely. This is clearly not only related to the lack of well-standardised MP assays, but also to variations in pre-analytical conditions. In this review we will discuss the pre-analytical variables related to plasma and MP preparation which may affect MP analysis. Additionally we will address several analytical issues in commonly used MP assays and briefly discuss some novel approaches for the detection and characterisation of MPs. Ideally MP measurements should be performed in plasma, freshly prepared directly after blood withdrawal. As platelet contamination seems to be one of the major pre-analytical problems in processing plasma for MP measurement, the use of platelet-free plasma may be preferred. When frozen-thawed plasma is used, especially PMP and annexinV-positive MP counts should be interpreted with caution. When flow cytometry is chosen as a method for quantification of MPs, some analytical conditions should be standardised, e.g. settings of the flow cytometer, quality of the antibodies, and use of counting beads. Fluorescence-nanoparticle tracking analysis and atomic force microscopy can accurately count nanosized MPs, but unfortunately the operational procedures of both methods are still time consuming and they give no information on the functional properties of MPs. The MP-TF activity assay provides information on MPs carrying active TF, regardless of their parental origin. Ultimately, standardisation of pre-analytical procedures and the introduction of reliable and rapid methods for the measurement of MPs are urgently needed to facilitate their use as biomarker in the pathophysiology of diseases. Show less
Introduction: Microparticles (MPs) carrying active tissue factor (TF) have been detected in the plasma of cancer patients in particular in those presenting with acute deep vein thrombosis (DVT) or... Show moreIntroduction: Microparticles (MPs) carrying active tissue factor (TF) have been detected in the plasma of cancer patients in particular in those presenting with acute deep vein thrombosis (DVT) or pulmonary embolism (PE). Experimental studies in mice have revealed that circulating MPs carrying TF contribute to thrombus formation. Aim: To study whether unselected patients with an acute confirmed PE have elevated TF activity in the MP fraction (MP-TF activity). Materials and Methods: Plasma MP-TF activity was measured in 159 non-selected patients with clinically suspected PE and in 48 healthy controls as previously described. Blood was collected at time of inclusion. The diagnosis of acute PE was confirmed in 54 patients and excluded in 105 patients. Results: Median MP-TF activity in 159 patients with clinically suspected PE was 72 fM Xa/min [range 32-6657] fM Xa/min and higher than in healthy controls (66 [range 28-183] fM Xa/min; P<0.05). There was no significant difference (P=0.169) in MP-TF activity between patients with confirmed PE (median 84.5 fM Xa/min [range 36-2149]) and patients without PE (72 fM Xa/min [range 32-6657]) fM Xa/min). In the 159 patients with clinically suspected PE we observed in an exploratory analysis higher MP-TF activity levels in patients with active cancer (median 137 fM Xa/min [range 36-6657]) and cardiovascular disease (median 131.5 fM Xa/min [range 45-2149]) than in patients without these disorders (P=0.0004 and P=0.014, respectively). Conclusion: In patients presenting with clinically suspected PE plasma MP-TF activity was not associated with confirmed PE. (C) 2010 Elsevier B.V. All rights reserved. Show less
Objectives: Stimulation of arginine vasopressin 2 receptor (V2R) with arginine vasopressin (AVP) results in a rise in von Willebrand factor (VWF) and factor VIII plasma levels. We hypothesized that... Show moreObjectives: Stimulation of arginine vasopressin 2 receptor (V2R) with arginine vasopressin (AVP) results in a rise in von Willebrand factor (VWF) and factor VIII plasma levels. We hypothesized that gain-of-function variations in the V2R gene (AVPR2) would lead to higher plasma levels of VWF and FVIII. Methods and Results: We genotyped the control populations of two population-based studies for four AVPR2 variations: a-245c, G12E, L309L, and S331S. Rare alleles of a-245c, G12E, and S331S, which were in linkage disequilibrium, were associated with higher VWF propeptide, VWF and FVIII levels. The functionality of the G12E variant was studied in stably transfected MDCKII cells, expressing constructs of either 12G-V2R or 12E-V2R. Both V2R variants were fully glycosylated and expressed on the basolateral membrane. The binding affinity of V2R for AVP was increased three-fold in 12E-V2R-green fluorescent protein (GFP) cells, which is in accordance with increased levels of VWF propeptide associated with the 12E variant. The dissociation constant (K-D) was 4.5 nm [95% confidence interval (CI) 3.6-5.4] for 12E-V2R-GFP and 16.5 nm (95% CI 10.1-22.9) for 12G-V2R-GFP. AVP-induced cAMP generation was enhanced in 12E-V2R-GFP cells. Conclusions: The 12E-V2R variant has increased binding affinity for AVP, resulting in increased signal transduction, and is associated with increased levels of VWF propeptide, VWF, and FVIII. Show less
The procoagulant function of activated factorV (FVa) is inhibited by activated Protein C (APC) through proteolytic cleavages at R306, R506 and R679. Recombinant FVa mutated at all three APC... Show moreThe procoagulant function of activated factorV (FVa) is inhibited by activated Protein C (APC) through proteolytic cleavages at R306, R506 and R679. Recombinant FVa mutated at all three APC-cleavage sites, FVa-GQA, was still inactivated by APC through at least two cleavages in the heavy chain of FVa; relatively rapid cleavage at R-x1 close to residue 506 and slower cleavage at R-x2 nearby residue 306. We investigated the exact location of these two cleavages, by substitution of arginines by glutamine within the R-x1-region (R501, R505 or R510) and the R-x2-region (R313, R316, R317 or R321). Immunoblot and kinetic analyses of the inactivation of activated R-x1-mutants by APC revealed that using mutant FVa-GQA-505Q no R-x2-R-x1 fragment was formed and that the inactivation reaction was first order with a rate constant of 1.0x10(4) M-1 s(-1), similar to the rate constant of R-x2 cleavage (k(2) = 1.3 x 10(4) M-1 s(-1)). No single arginine could be pinpointed identified as R-x2. Individual replacement of arginine by glutamine at positions 313, 316, 317 or 321 in FV-GQA-505Q did not result in the disappearance of R-x2 as judged from kinetic and immunoblot analyses. However, replacement of all four arginines by glutamine completely prevented formation of the R-x2-R-709 fragment. We conclude that substitution of arginine 506 by glutamine as in FV-Leiden, leads to the detection of a novel cleavage site at arginine 505 (R-x1). Substitution of arginine 306 by glycine, like in FV-Cambridge, reveals several alternative cleavage sites near arginine 306, which together constitute a secondary cleavage site. (C) 2009 Elsevier Ltd. All rights reserved. Show less
Background: Microparticles (MPs) are small vesicles released from cells of different origin, bearing surface antigens from parental cells. Elevated numbers of blood MPs have been reported in ... Show moreBackground: Microparticles (MPs) are small vesicles released from cells of different origin, bearing surface antigens from parental cells. Elevated numbers of blood MPs have been reported in (cardio) vascular disorders and cancer. Most of these MPs are derived from platelets. Objectives: To investigate whether atomic force microscopy (AFM) can be used to detect platelet-derived MPs and to define their size distribution. Methods: Blood MPs isolated from seven blood donors and three cancer patients were immobilized on a modified mica surface coated with an antibody against CD41 prior to AFM imaging. AFM was performed in liquid-tapping mode to detect CD41-positive MPs. In parallel, numbers of CD41-positive MPs were measured using flow cytometry. Mouse IgG(1) isotype control was used as a negative control. Results: AFM topography measurements of the number of CD41-positive MPs were reproducible (coefficient of variation = 16%). Assuming a spherical shape of unbound MPs, the calculated diameter of CD41-positive MPs (d(sph)) ranged from 10 to 475 nm (mean: 67.5 +/- 26.5 nm) and from 5 to 204 nm (mean: 51.4 +/- 14.9 nm) in blood donors and cancer patients, respectively. Numbers of CD41-positive MPs were 1000-fold higher than those measured by flow cytometry (3-702 x 10(9) L-1 plasma vs. 11-626 x 10(6) L-1 plasma). After filtration of isolated MPs through a 0.22-mu m filter, CD41-positive MPs were still detectable in the filtrate by AFM (mean d(sph): 37.2 +/- 11.6 nm), but not by flow cytometry. Conclusions: AFM provides a novel method for the sensitive detection of defined subsets of MPs in the nanosize range, far below the lower limit of what can be measured by conventional flow cytometry. Show less
