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2016 Annual Minutes
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7/12/2016 at 7:15:23 PM GMT
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2016 Annual Minutes

SSC MINUTES VASCULAR BIOLOGY 2016 MONTPELLIER

 

Extracellular vesicles. Chairs: Chris Gardiner (UK) and Pia Siljander (Finland)

 

Role of EV-bound polyphosphate in prostate cancer driven thrombosis (Katrin Nickel; Hamburg, Germany).

Katrin Nickel described a novel mechanism for cancer-related thrombosis. Prostate cancer cells release prostasomes, heterogeneous extracellular vesicles (30-400 nm) which expose not only tissue factor but also long chain polyphosphates (polyP). PolyP co-localises with factor (F)XIIa and initiates FXII-dependent coagulation in vitro. Mice injected with prostasomes develop lethal pulmonary embolism (PE) within 30 minutes. In contrast, mice deficient in FXI, FXII, or high-molecular-weight kininogen, are protected from lethal prostasome-induced PE. Pre-treatment of wild type mice with the anti-FXIIa 3F7 prior to injection of prostasomes prevents PE but does not increase the bleeding time. Pre-treatment of prostasomes with polyP inhibitors also prevents PE. The plasma from patients with prostate cancer shows increased thrombin generation, which is abolished by the removal of prostasomes. The isolated prostasomes trigger thrombin generation in normal plasma. Katrin concluded that targeting factor XIIa and/or polyphosphate may provide a novel opportunity for safe anticoagulation therapy in patients with cancer.

 

TF-EV in clinical trials and the need for TF-EV standardisation (Johannes Thaler; Vienna, Austria)

Johannes Thaler stated the need for clarification regarding the performance of tissue factor (TF)-extracellular vesicles (EV) assays. He referred to the work of Butenas et al., who showed that in plasma of normal healthy individuals the concentration of functional TF does not exceed 20fM. In clinical trials investigating the role of TF-EV in predicting thrombotic risk in cancer patients, two methods are commonly used: flow cytometry (FCM) and activity assays. The specificity of antibodies used to detect TF by FCM may be assessed by testing unstimulated and lipopolysaccharide-stimulated monocytes. Anti-TF antibodies that bind to unstimulated monocytes or antibodies failing to detect a substantial increase in TF-EV following lipopolysaccharide stimulation are not suitable. Further standardisation of FCM measurement of EVs are necessary, and this work is now being performed the Vascular Biology SSC in collaboration with ISEV, ISAC and METVES (www.evflowcytometry.org). There is better agreement between functional TF-EV assays, but most methods cannot reliably detect TF levels of < 0.5 pg/mL. Consequently, TF-EV is only detectable in specific patient groups e.g. disseminated intravascular coagulation and pancreatic cancer. More studies will be essential to investigate the performance of the various TF-EV assays.

 

Desirable properties of a TF-EV reference preparation (Chris Gardiner; London, UK)

There are many different methods for measuring tissue factor (TF) antigen and activity by extracellular vesicles (EV), and it is clear that these methods are not all measuring the same thing. Chris Gardiner presented data from the laboratory of Nigel Mackman, who demonstrated that detection of TF antigen by either flow cytometry or ELISA do not correlate with TF-coagulant activity. It was concluded from these results that antigenic assays lack sensitivity and specificity. The importance of determining the specificity of assays by using reliable positive controls, e.g. lipopolysaccharide-stimulated monocytes, and negative controls, e.g. an inhibitory antibody against TF or FVIIai were discussed. There are several methodological approaches for measuring TF-EV activity and each  approach has its own advantages and limitations. At present, the only available material to standardize these assays is commercial thromboplastin. Unfortunately, the amount of TF varies between batches, and, as the TF is in soluble form, it cannot be used in most capture assays and cannot be pelleted by centrifugation. Consequently, thromboplastin can only be used to calibrate/standardise the final measurement. Chris proposed to use TF-EV from the human primary pancreatic adenocarcinoma cell line BxPC-3, which constitutively releases EV exposing coagulant TF. Application of reconstituted EV from BxPC-3 in citrate-anticoagulated plasma would allow a “like with like” approach to facilitate standardization of TF-EV activity assays.

 

Consideration and challenges in generating reference materials –tissue factor extracellular vesicles (Elaine Gray; London, UK)

Elaine Gray asked “Why do we need reference materials?” She explained the role of bioassays and the concept of biological standardisation. The use of a common reference standard improves agreement between laboratories. A previous working party on tissue factor (TF) standardization in cancer was initiated 2005 under the Haemostasis and Malignancy Subcommittee of the ISTH. Two TF preparations and several cell lysates were compared and although all showed linearity, assay parallelism was not observed between either TF preparation and the cell lysates. However, the cell lysates showed linearity and parallelism. Ultimately, the SSC did not proceed with the TF standard. The challenges of manufacturing reference standards were described Elaine outlined the questions that the subcommittee would need to address. Pilot studies will be required to determine the purpose of the reference standard and whether the standard will support a single method or multiple methods. The SSC must decide on the type of reference preparation (i.e. SSC or WHO), the methods for which it will be used and who will coordinate large collaborative studies to qualify the suitability. Funding will be required to produce and run a collaborative study and ultimately the production of the reference standard.

 

Endothelial progenitor cells. Chairs: Anna Randi (UK), Florence Sabatier (France)

 

The BOEC as a model for organ-specific endothelial assessment – apples versus oranges (M Toshner)

 

Standardization of measurements of extracellular vesicles. Chair: Rienk Nieuwland (Netherlands)

Standardization of microparticle enumeration across different flow cytometry platforms: results of a multicentre collaborative workshop and future directions (Romaric Lacroix, Marseille, France)

Romaric presented the final results of a collaborative workshop on platelet microparticle (PMP) standardization by flow cytometry. This workshop evaluated a forward or scatter bead-based strategy adapted to different flow cytometry platform. The study included 44 laboratories and 52 cytometers of 14 different types. Using resolution capability and background noise level as criteria to qualify the instruments, the standardization strategy proved to be compatible with 85% (44/52) instruments. The inter-laboratory variability of PMP counts was 37% and 28% for each sample. No difference was found between instruments using forward or side scattered light as the relative sizing parameter. Romaric concluded that despite remaining limitations, this study demonstrates a potential of bead-based strategies to standardize of MP enumeration. However, additional standardization efforts such as standardization of fluorescence signals, development of sub-micron reference materials and MP-dedicated reagents, are still mandatory to evaluate the clinical relevance of (P)MP at a multicentre level.

 

Size and refractive index determination by flow cytometry discriminate extracellular vesicles from lipoprotein particles (Leonie de Rond; Amsterdam, Netherlands)

Flow cytometry is the most widely used method to study single extracellular vesicles (EV). However, flow cytometry data have arbitrary units, which hampers data interpretation and comparison. Here we present Flow-SR, a label-free method to obtain the diameter (d) and refractive index (RI) from the light scattering signals of a flow cytometer.

Mie theory and reference beads were used to obtain the relation between the measured forward and side scattering of a flow cytometer (A50-Micro; Apogee, UK) and d and RI of a particle. We validated this approach by determining d and RI of a mixture with 5 populations of silica and polystyrene beads <500 nm. For each population in the beads mixture, d and RI were determined with measurement errors on the mean <8% and <2%, respectively. We, subsequently, applied  Flow-SR on platelet-depleted supernatant of outdated platelet concentrates containing EV and lipoproteins. Two populations with different RI were clearly discernable. Using fluorescent labeling, we confirmed that the population with RI ≤1.42 contains EV and the population with RI>1.42 contains lipoproteins.

Flow-SR is the first method that converts the scattering signals of a commercial flow cytometer to the comparable measurement units of dimension and RI. We have demonstrated that this method is accurate and that it allows label-free discrimination between EV and lipoprotein particles in human plasma.

Quantitative light scattering to standardize flow cytometry measurements of extracellular vesicles (Edwin van der Pol; Amsterdam Netherlands)

The comparison of flow cytometry results between laboratories remains a challenge. At present, most laboratories select extracellular vesicles (EV) by setting an inclusion gate based on the scatter signal of two polystyrene bead sizes. However, due to the refractive index (RI) difference between EV (RI≈1.4) and polystyrene (RI≈1.6) and the variety of optical configurations among flow cytometers, the current standard leads to poor reproducibility. A well-defined polystyrene beads mixture (www.metves.eu) and an EV reference sample (CD235a-FITC labeled erythrocyte EV) were measured on 46 instruments in 33 laboratories worldwide. Stand-alone software (www.exometry.com) provided the relation between particle diameter, RI, and scatter for the specific optical configuration of each instrument. This relation was used to set EV size gates of 300-600 nm, 600-1200 nm, and 1200-3000 nm. Each site applied the size gates to their own analyses software and independently reported the EV concentration within each size gate. The polystyrene beads mixture contains a population of 400 nm FITC-labeled polystyrene beads, which could not be detected by 34% of the instruments. For comparison, a 400 nm polystyrene bead scatters light more efficiently than a 1 µm EV. Thus, roughly one third of the flow cytometers used by laboratories with expertise in EV detection detect EV or particles >1 µm. Among the instruments capable of detecting 1200-3000 nm EV, the established standard resulted in a CV of 74%, whereas our approach resulted in a CV of 59%. Among the other size gates, no reproducibility was obtained. Final data and results will be submitted to JTH in 2016. Taken together, well-defined beads and Mie theory were used to standardize the size range detected by flow cytometry. Roughly one third of the instruments do not detect EV. Within the 1200-3000 nm size range, an EV size gate by Mie theory (CV=59%) leads to a better reproducibility than a gate on beads (CV=74%). We expect that knowledge on the RI of EV may further improve our approach. Hitherto, standardization of EV <1200 nm is ineffective and requires more flow cytometers with nanoparticle detection sensitivity.

 

Receptor shedding from platelets in pathological environments. Chair: Elizabeth Gardiner (Australia)


Soluble GPVI as a potential biomarker of platelet activation in patients with thermal injury and sepsis (Paul Harrison; Birmingham, UK)

Paul measured soluble glycoprotein VI (sGPVI), a platelet activation biomarker, in plasma samples which were collected as part of the Scientific Investigation of the Biological Pathways Following Thermal Injury Study (SIFTI) study. Whilst platelets are traditionally recognized for their central role in haemostasis, numerous studies now highlight how platelets can be potent protagonists in an inflammatory response. Platelets directly recognize, sequester and kill pathogens to activate and recruit leukocytes to sites of infection and inflammation, and to modulate leukocyte behaviour, enhancing their ability to phagocytose and kill pathogens. Work from Paul’s laboratory showed that whilst sGPVI is not increased in the first few days post thermal injury, there are significantly higher sGPVI levels at later time points in patients that developed sepsis compared to non-septic patients. Systemic platelet activation during septic episodes may contribute to the pathogenesis of multi-organ failure. Future work will assess the advantage of stratifying patient groups based on plasma sGPVI levels in samples collected within 4 days post thermal injury to help predict the onset of sepsis and multi-organ failure and aid clinical decisions on possible early therapeutic intervention.

 

Platelet receptors and coagulopathy (Robert Andrews; Melbourne, Australia)

GPVI, which binds collagen, and GPIbα (the ligand-binding subunit of GPIb-IX-V) that binds von Willebrand factor(VWF), form a unique adhesion-signaling complex on the platelet surface that initiates platelet adhesion, spreading, activation and secretion, and integrin αIIbβ3-mediated platelet aggregation (thrombus formation) at high shear rates in flowing blood. Shedding of GPVI generates a ~55-kDa soluble GPVI ectodomain fragment (sGPVI) and a ~10-kDa platelet-associated remnant fragment, which provide a sensitive read-out of receptor engagement, since all GPVI is intact on the surface of resting platelets, unless shedding is induced. Depletion or blockade of platelet GPVI in experimental models results in profoundly decreased arterial thrombosis or stroke, impaired procoagulant activity and thrombin generation ex vivo or in vivo, and protection from tissue factor-induced thromboembolism. Robert showed that FXa induces sGPVI release since shedding could occur in recalcified whole blood or anticoagulated blood treated with Russell Viper Venom (a strong specific activator of FX). Shedding was strongly blocked by the FXa-inhibitors rivaroxaban and enoxaparin, but unaffected by the thrombin inhibitors dabigatran, lepirudin and hirudin. Whether FXa or FXa inhibitors affect ADAM10-mediated processing of other substrates on human platelets and other vascular cells is unknown. Defining the mechanism of FXa-mediated activation of ADAM10/sheddase activity and GPVI shedding may identify new therapeutic targets regulating thrombus growth and stability, determine the relationship between coagulation, platelet activation and ADAM10-mediated substrate processing, and also enable potential clinical consequences of anticoagulation to be recognized.

 



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