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Background: Cancer-related venous thromboembolism (VTE) heralds a poor prognosis, especially in pancreatic adenocarcinoma (PAC). Tissue factor (TF) is implicated as one of the main culprits in PAC-associated VTE and disease progression., Methods: In a prospective cohort study of 79 PAC patients, we measured plasma CA19-9 and microparticle-associated TF activity (MP-TF activity). In addition, we enumerated TF(+)MPs and MUC1(+)MPs in plasma (n=55), and studied the expression of TF, MUC1, CD31 and CD68 in tumour tissue (n=44)., Results: Plasma MP-TF activity was markedly elevated in PAC patients with VTE compared with those without (median: 1925 vs 113 fM Xa min(-1); P<0.001) and correlated with the extent of thromboembolic events, metastatic disease and short survival. Similar results were found for CA19-9. Patients with massively progressing thrombosis and cerebral embolisms despite anticoagulant therapy (n=3) had the highest MP-TF activities (12 118-40 188 fM Xa min(-1)) and CA19-9 (40 730-197 000 kU l(-1)). All tumours expressed MUC1 and TF. MP-TF activity did not correlate with intensity of TF expression in adenocarcinoma cells, but corresponded with numbers of TF(+) macrophages in the surrounding stroma., 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 improvement of survival.

Activated protein C (APC) resistance, often associated with the factor V (FV) Leiden mutation, is the most common risk factor for venous thrombosis. We observed increased APC resistance in carriers of fibrinogen γ gene (FGG) haplotype 2, which is associated with reduced levels of the alternatively spliced fibrinogen γ' chain. This finding prompted us to study the effects of fibrinogen and its γ' chain on APC resistance. Fibrinogen, and particularly the γA/γ' isoform, improved the response of plasma to added APC in the thrombin generation-based assay. Similarly, a synthetic peptide mimicking the C-terminus of the fibrinogen γ' chain, which binds thrombin and inhibits its activities, greatly increased the APC sensitivity of normal and FV Leiden plasma, likely due to its ability to inhibit thrombin-mediated activation of FV and FVIII. Although the fibrinogen γ' peptide also inhibited protein C activation by the thrombin/thrombomodulin complex, it still increased the sensitivity of plasma to endogenously formed APC when thrombin generation was measured in the presence of soluble thrombomodulin. We conclude that fibrinogen, and particularly fibrinogen γ', increases plasma APC sensitivity. The fibrinogen γ' peptide might form the basis for pharmacologic interventions to counteract APC resistance., (© 2014 by The American Society of Hematology.)

Introduction: Inhibition of tissue factor, the primary initiator of coagulation in sepsis, attenuates morbidity in primates infused with Escherichia coli. In a human endotoxemia model, microparticles expressing procoagulant TF (MP-TF) are released in blood concurrently with markers of inflammation and coagulation. We investigated whether the release of MP-TF into blood is accompanied by procoagulant and inflammatory changes in patients with E. coli urinary tract infection., Materials and Methods: In a multicenter cohort study, we determined clinical disease severity using APACHE II scores and measured plasma MP-TF activity, TAT, sE-selectin, sVCAM-1, procalcitonin and monocyte count in blood of 215 patients with community-acquired febrile E. coli urinary tract infections., Results: Plasma MP-TF activity on admission corresponded with clinical disease severity (APACHE II score; P=0.006) and correlated significantly but weakly with plasma markers of disease severity (sE-selectin, sVCAM-1, procalcitonin). Additionally, median plasma MP-TF activity was higher in patients than in healthy controls (197 vs. 79 fM Xa/min; P<0.0001), and highest in bacteremic patients (325 fM Xa/min). MP-TF activity showed a weak inverse correlation with monocyte count (rs -0.22; P=0.016) and a weak correlation with TAT (rs 0.23, P=0.017). After 3 days of antibiotic treatment, upon resolution of the infection, plasma MP-TF activity and TAT concentrations declined., Conclusions: Microparticle-associated procoagulant tissue factor activity is related to disease severity and bacteremia in febrile E. coli UTI patients and may contribute to the prothrombotic state in gram-negative sepsis., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Maggots of the blowfly Lucilia sericata are used for the treatment of chronic wounds. As haemostatic processes play an important role in wound healing, this study focused on the effects of maggot secretions on coagulation and fibrinolysis. The results showed that maggot secretions enhance plasminogen activator-induced formation of plasmin and fibrinolysis in a dose- and time-dependent manner. By contrast, coagulation was not affected by secretions. Biochemical studies indicated that a novel serine protease within secretions, designated Sericase, cleaved plasminogen to several fragments. Recombinant Sericase degraded plasminogen leading amongst others to the formation of the mini-plasminogen like fragment Val454-plasminogen. In addition, the presence of a non-proteolytic cofactor in secretions was discovered, which plays a role in the enhancement of plasminogen activator-induced fibrinolysis by Sericase. We conclude from our in vitro studies that the novel serine protease Sericase, with the aid of a non-proteolytic cofactor, enhances plasminogen activator-induced fibrinolysis.

Objective: To confirm the effect of the endothelial protein receptor gene (PROCR) haplotypes H1 and H3 on venous thromboembolism (VTE), to study their effect on endothelial protein C receptor (EPCR) expression in human umbilical vein endothelial cells, and to investigate the functionality of H1 tagging single-nucleotide polymorphisms in an in vitro model., Approach and Results: Protein C (PC), activated PC, and soluble EPCR (sEPCR) levels were measured in 702 patients with VTE and 518 healthy individuals. All subjects were genotyped for PROCR H1 and H3. Human umbilical vein endothelial cells isolated from 111 umbilical cords were used to study the relation between PROCR haplotypes, PROCR mRNA, cellular distribution of EPCR, and rate of PC activation. Finally, the functionality of the intragenic PROCR H1 single-nucleotide polymorphisms was analyzed using a luciferase-based method. We confirmed that individuals carrying H1 have reduced VTE risk, increased plasma activated PC levels, and reduced plasma sEPCR levels and that individuals with the H3H3 genotype have an increased VTE risk and increased plasma sEPCR levels. In cultured human umbilical vein endothelial cells, H1 is associated with increased membrane-bound EPCR, increased rate of PC activation, and reduced sEPCR in conditioned medium, but does not significantly influence PROCR mRNA levels. In contrast, H3 is associated with reduced membrane-bound EPCR and increased sEPCR in human umbilical vein endothelial cell-conditioned medium, higher levels of a truncated mRNA isoform, and a lower rate of PC activation. Finally, we identified the g.2132T>C single-nucleotide polymorphism in intron 1 as an intragenic H1-specific functional single-nucleotide polymorphism., Conclusions: These results support a protective role of PROCR H1 against VTE and an increased risk of VTE associated with the H3 haplotype.

Introduction: Extracellular vesicles (EV) are phospholipid bilayer-enclosed vesicles recognized as new mediators in intercellular communication and potential biomarkers of disease. They are found in many body fluids and mainly studied in fractions isolated from blood plasma in view of their potential in medicine. Due to the limitations of available analytical methods, morphological information on EV in fresh plasma is still rather limited., Objectives: To image EV and determine the morphology, structure and size distribution in fresh plasma by cryo-electron microscopy (cryo-EM)., Methods: Fresh citrate- and ethylenediaminetetraacetic acid (EDTA)-anticoagulated plasma or EV isolated from these plasmas were rapidly cryo-immobilized by vitrification and visualized by cryo-EM., Results: EV isolated from fresh plasma were highly heterogeneous in morphology and size and mostly contain a discernible lipid bilayer (lipid vesicles). In fresh plasma there were 2 types of particles with a median diameter of 30 nm (25-260 nm). The majority of these particles are electron dense particles which most likely represent lipoproteins. The minority are lipid vesicles, either electron dense or electron lucent, which most likely represent EV. Lipid vesicles were occasionally observed in close proximity of platelets in citrate and EDTA-anticoagulated platelet-rich plasma. Cryo-electron tomography (cryo-ET) was employed to determine the 3D structure of platelet secretory granules., Conclusions: Cryo-EM is a powerful technique that enables the characterization of EV in fresh plasma revealing structural details and considerable morphological heterogeneity. Only a small proportion of the submicron structures in fresh plasma are lipid vesicles representing EV.

Background: Besides its role in blood clotting, fibrinogen exerts a poorly understood anticoagulant function by binding thrombin and modulating its activity. In particular, the γA/γ' fibrinogen isoform binds with high affinity to thrombin exosite II through the anionic carboxyl-terminal end of the γ' chain. This interaction down-regulates thrombin-mediated factor VIII (FVIII) activation, but its effect on FV activation is unknown., Objectives: To investigate the overall anticoagulant activity of fibrinogen and particularly of fibrinogen γ' in plasma, and to verify whether the fibrinogen γ' carboxyl-terminal peptide affects thrombin-mediated FV activation., Methods: Thrombin generation was measured by calibrated automated thrombography in whole and defibrinated plasma and in plasma supplemented with the (sulfated) fibrinogen γ' carboxyl-terminal peptide (0-500 μmol L(-1) ). The effect of the peptide on thrombin-mediated FV activation was studied in model systems and in plasma., Results: Total fibrinogen prolonged the lag time of thrombin generation at low tissue factor (TF) concentrations. The fibrinogen γ' peptide dose-dependently prolonged the lag time and decreased the peak height of thrombin generation at low TF, whereas a scrambled control peptide was ineffective. These effects persisted in the presence of an anti-FVIII antibody, suggesting that the peptide may also inhibit thrombin-mediated activation of FV. This was confirmed in model systems and in plasma., Conclusions: Total fibrinogen and the fibrinogen γ' peptide have an overall anticoagulant effect on thrombin generation determined at low TF. Inhibition of thrombin-mediated FV activation by the fibrinogen γ' peptide is a novel mechanism of the anticoagulant activity of fibrinogen γ'., (© 2013 International Society on Thrombosis and Haemostasis.)

Background: Venous thromboembolism (VTE) is a multicausal disorder involving environmental and genetic risk factors. In many thrombophilic families the clustering of thrombotic events cannot be explained by known genetic risk factors, indicating that some remain to be discovered., Objectives: We aimed to identify novel thrombosis susceptibility alleles in a large panel of small thrombophilic families: the Genetics In Familial Thrombosis (GIFT) study., Patients/methods: In the GIFT study, 201 families were recruited consisting of 438 siblings with an objectively confirmed VTE at a young age. Multipoint linkage analysis (402 SSR markers) and fine mapping were performed, followed by genotyping of tagging SNPs in positional candidate genes., Results: Established genetic risk factors such as factor V Leiden, ABO blood group non-O, prothrombin 20210A, fibrinogen gamma 10034T and deficiencies of antithrombin, protein C and protein S were more frequent in GIFT patients than in unselected VTE patients. Linkage supported the presence of novel thrombosis susceptibility loci on 7p21.3-22.2 (LOD score = 3.23) and Xq24-27.3 (LOD score = 1.95). Simulation analysis showed that the chr7 signal was genome-wide statistically significant (P = 0.022). Tagging SNPs (n = 157) in eight positional candidate genes (LOD drop 1.5 regions) were genotyped in GIFT patients and 332 healthy controls. Five chr7 SNPs associated with VTE. SNP THSD7A rs2074597 was responsible for part of the chr7 signal., Conclusions: The GIFT panel is rich in established genetic risk factors for VTE, but genetic factors remain unidentified in many families. Genome-wide linkage failed to identify the previously established genetic risk factors for VTE, but identified a novel VTE susceptibility locus on chr7., (© 2013 International Society on Thrombosis and Haemostasis.)

Background: Tissue factor (TF) expression by tumors contributes to tumor growth. Release of TF-positive microparticles (MPs) may contribute to venous thromboembolism (VTE)., Objectives: To conduct a prospective cohort study to determine whether elevated MP-associated TF (MP-TF) activity is predictive of VTE and mortality in four cancer types., Patients/methods: We determined MP-TF activity in pancreatic, gastric, colorectal and brain cancer patients. We used a chromogenic endpoint assay for all patients and also a chromogenic kinetic assay for patients with pancreatic and brain cancer., Results: During follow-up, 12/60 (20%) pancreatic, 6/43 (14%) gastric, 12/126 (10%) colorectal and 19/119 (16%) brain cancer patients developed VTE; 46/60 (77%), 30/43 (70%), 47/126 (37%) and 67/119 (56%), respectively, died. MP-TF activity levels were highest in pancreatic cancer. We did not find a statistically significant association of MP-TF activity with the risk of VTE in any of the four cancer types by using two statistical methods. An association of MP-TF activity with the risk of mortality was detected in pancreatic cancer with the endpoint assay (hazard ratio [HR] 1.8 and 95% confidence interval [CI] 1.4-2.3 per doubling of activity, P < 0.001) and the kinetic assay (HR 1.2, 95% CI 1.1-1.4, P < 0.001); adjustment for type of treatment was not performed. In pancreatic cancer, MP-TF activity correlated with D-dimer level (endpoint assay, r = 0.51; chromogenic assay, r = 0.48), and a correlation between assays (r = 0.61) was found., Conclusion: MP-TF activity was not associated with future VTE in pancreatic, gastric, colorectal and brain cancer. However, we found a strong association of MP-TF activity with mortality in pancreatic cancer. MP-TF activity might be reflective of an aggressive pancreatic cancer phenotype., (© 2012 International Society on Thrombosis and Haemostasis.)

Increased microparticle tissue factor (TF) activity is not only found in cancer patients, but also in patients with cardiovascular and inflammatory diseases. Methods such as flow cytometry and impedance-based flow cytometry allow the analysis of microparticle subsets but provide no insight on which microparticles carry active TF. Conversely, the microparticle-TF activity itself does not reveal the cellular origin of the microparticles carrying the active TF.For this reason, we developed an immuno-magnetic bead method to capture subsets of microparticles directly from plasma. The method was optimized for capture of platelet-derived microparticles (PMPs) from plasma. Only 100 μl platelet-poor plasma (PPP) was needed in combination with 135 μl (27 μg) of biotinylated antihuman CD41 monoclonal antibody (MoAb) and 200 μl of streptavidin beads to achieve complete separation of PMPs from plasma. As a control, biotinylated mouse IgG1 isotype control MoAb was used instead of the anti-CD41 MoAb. Using biotinylated anti-CD14 MoAb, CD14-positive microparticles were captured from normal plasma spiked with microparticles isolated from the supernatant of lipopolysaccharide-stimulated monocytes (MoMPs). TF activity was found both in the positive (selected) and negative (depleted) fractions indicating that both CD14-positive and negative MoMPs carry active TF. We propose that this method can be used in the future to investigate the source of microparticles carrying active TF in plasma of patients with cancer and other diseases.

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 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.