Working Group on Vascular Biology

Saturday, 6 August 2005
11:00 to 14:30
Ballroom 1
Sydney Convention and Exhibition Centre

 Chairman : Peter J. Newman, ( USA )
SSC Organizing Committee: Michael C. Berndt ( Australia ), John Griffin ( USA ), Irène Juhan-Vague (France), Klaus T. Preissner ( Germany )

The program was divided into two parts: (I) Detection and characterization of circulating endothelial cells and their progenitors, and (II) Determination and characterization of (circulating) microparticles.

Approximately 160 people attended the session.

Session I : Detection and characterization of circulating endothelial cells and their progenitors (Chairs: K.T. Preissner & P.J. Newman)

Andrew Blann ( UK): “Circulating endothelial cells (CECs) and endothelial progenitor cells (EPCs): Two sides of the same coin or two different coins”? That endothelial cells detectable in blood are not a homogeneous population, but rather represent more than one species of endothelial cells was discussed. CECs are thought to arise from the vessel wall, whereas EPCs are mobilized from the bone marrow. Thus, although originally defined according to different criteria, there are also some common characteristics. The lack of consensus regarding definition and methodologies remains an important area of future work for this Working Group. The use of CD146-coated magnetic beads to identify and purify CECs was discussed, as well as the extent to which the presence of CECs reflects endothelial cell damage. The origin of CD34+ EPCs remains controversial as they are identifiable in peripheral blood and capable of forming in vitro colonies. Improving the array of surface markers allowing to discriminate CECs from EPCs remains an important problem for further study.

Alexander Woywodt ( Germany ): “Detection of circulating endothelial cells by immuno-magnetic separation (IMS) assays.” That circulating endothelial cells are a novel marker of microvascular damage was reinforced. CD146 driven-immunomagnetic isolation appears to be the technique of choice to isolate and enumerate these cells. However, several variables influence isolation of CECs by IMS (both at the pre-analytical and analytical levels). In this respect, a standardized methodology represents an important step towards consensus regarding CECs. Moreover, given the variable phenotype of these rare cells in peripheral blood, this technique still has several pitfalls, and precautions taken to avoid them were discussed. The second part of the talk focused on the clinical utility of detecting circulating endothelial cells as a marker of ANCA-associated small-vessel vasculitis.

Françoise Dignat-George ( France ): “Detection of circulating endothelial cells in the vascular compartment”. A historical perspective of the development of CD146 mAbs as selective markers for detecting CEC was provided, followed by a description of clinical disorders that have been associated with increased circulating CECs, including infection, malignancies, transplantation, and immune disorders like TTP. A working definition of CECs was proposed, and the clinical utility of CECs as biomarkers of endothelial damage was discussed. It was concluded that a consensual definition of the most appropriate technique is a key issue to be addressed in order to validate CECs in large cohorts of patients. The future potential for proteomic analysis to provide selective markers allowing to discriminate CECs (from damaged vessels) versus EPCs (bone marrow) was also discussed.

Session II : Determination and characterization of (circulating) microparticles (Chairs: J Griffin & J-M Freyssinet)

Françoise Dignat-George and Jean-Marie Freyssinet (France): “Questionnaire on microparticle detection and characterization : a retrospective analysis ”. Since MP are increasingly being viewed as markers for various pathophysiological processes, and may in addition have therapeutic applications, Drs. Dignat-George and Freyssinet have undertaken the large, important, and challenging task of surveying how investigators in this growing field prepare and characterize MP. The results of their ISTH-sponsored questionnaire were presented, and the results divided into pre-analytical and analytical procedures. Relative consensus was reached that blood should be anticoagulated with citrate, and that minimal manipulation should be involved to avoid cellular activation and inadvertent production of MP. Outstanding issues remaining on the pre-analytical side include whether whole blood or plasma is used to prepare MP, and if/how MP should be stored prior to analysis. On the analytical side, standardization of methods of quantitation, surface markers, and functional properties of MP remains an important goal. Current technologies used in this regard include flow cytometry, ELISA, determination of pro-coagulant and anti-coagulant activities, and the beginning of adoption of proteomic technologies.

Rienk Nieuwland ( Amsterdam , The Netherlands): “Detection and characterization of microparticles by flow cytometry”. Circulating cell-derived microparticles most often have procoagulant properties due to exposure of negatively charged phospholipids. Most studies on microparticles, however, have been performed on microparticles after in vitro manipulations, e.g. pelleting/resuspending or freezing/thawing, thereby possibly influencing microparticle structure. This group investigated whether non-manipulated microparticles expose phosphatidylserine (PS), whether this exposure is affected by in vitro manipulations, and if so, whether this changes their procoagulant properties. Surprisingly, in their hands only very few non-manipulated microparticles from venous blood of healthy individuals, or from pericardial blood of patients undergoing cardiac surgery exposed PS, as evaluated by annexin V binding. Upon pelleting/resuspending, freezing/thawing, or both, however, the fraction of PS-exposing microparticles increased, which was accompanied by fragmentation, change in the size of MPs, and and/or loss of particular microparticle populations. Interestingly, the extent of PS exposure did not affect the procoagulant activity or the mechanism of coagulation activation, allowing them to conclude that even low exposure of PS is sufficient to support coagulation. They cautioned that microparticles in fresh samples should not be quantified based on PS exposure. In the discussion, however, it was emphasized that the conditions for using annexin V have to be better defined with respect to the biochemistry of this PS probe.

Johan W. M. Heemskerk ( Maastricht , The Netherlands): “Phosphatidylserine-dependent procoagulant potential of microparticles”. Tissue factor-induced thrombin generation with PRP and with platelet-derived MP similarly relies on phosphatidylserine exposure. Thrombin generation in PRP is enhanced by integrin αIIbβ3-mediated shedding of MP. Platelets shed phosphatidylserine-exposing MP in the absence of activation (coagulation). This shedding is thought to be (1) secondary to F actin degradation, (2) mediated by integrins, and (3) negatively regulated by PKA (cAMP). It was concluded that integrin αIIbβ3 signaling accomplishes destabilization of the membrane cytoskeleton, negatively controlled by PKA, and resulting in MP shedding from the plasma membrane.

Yasushi Ozeki ( Japan ): Described a new ELISA method for detecting platelet-derived microparticles that utilized an anti-GPIX mAb for capture, and an anti-GPIb mAb to detect. This commercially-available assay might be detecting very small MP that flow cytometric analysis misses.

Nigel S. Key ( Minneapolis , USA ): “Tissue factor-dependent procoagulant potential of microparticles”. Evidence for TF-dependent procoagulant activity on MP derived from platelets and monocytes was presented, and the concept of encrypted versus de-encrypted TF exposure and its associated pro-coagulant activity was discussed, as were assay variables of TF procoagulant assays and the need for standardization and normalization.

Thomas Exner ( Sydney , Australia ): Presented the so-called XACT assay aimed at detecting procoagulant phospholipids in plasma, based on factor Xa-activated clotting time. One advantage of this assay is that it can be performed with whole plasma samples and is insensitive to the presence of most lupus anticoagulants. Hence, it can be anticipated that procoagulant MP are also detected.

Cheng Hock Toh ( Liverpool , UK ): “Anticoagulant potential of microparticles”. Dr Toh highlighted the fact that microparticles, depending on their cellular source, are able to display not only procoagulant properties, but also/instead express EPCR-bound activated protein C, which often functions to initiate anticoagulant pathways. The quantitation and functional analysis of these particular microparticles were described.

Bruce Furie ( Harvard , USA ): “ Impedance-based flow cytometry for measuring microparticles: New instrument, new answers”. TF-bearing, PSGL-1-bearing MP, likely derived from monocytes, bind to laser-damaged vessels in a P-selectin-dependent manner, and deliver TF in such a way as to promote fibrin deposition and thrombus growth. This model is currently thought to reflect inflammatory injury. Quantitation and detection of at least a sub-population of very small MP is unfortunately complicated by the fact that the particle size is on the same order of magnitude as the wavelength of light (488 nm) used for their detection. To overcome this limitation, an impedance-based instrument, which measures electronic volume and has a 10X signal:noise ratio has been developed, and found to be able to detect up to 1.6 x 10 6/ml TF-bearing MP in the blood of individuals with certain forms of cancer, including pancreatic, colon, breast, and ovarian. It is postulated that delivering TF in this way might contribute to the incidence of thromboembolism prevalent in the later stages of many cancers.

Eric F. Grabowski ( Boston , USA ): Microparticles in flowing blood”. The hemolytic uremic syndrome (HUS) results from Shiga-toxin-producing strains of E. coli, and causes acute renal failure in children. Though Shiga-toxin is able to increase TF activity 2-3 fold on the surface of activated endothelium, it does not appear to be able to similarly activate TF on EC-derived MP, perhaps due to downregulation of MP TF activity by TFPI.

Pudur Jagadeeswaran ( San Antonio , USA ): “Zebrafish microparticles from thrombocytes and their role in hemostasis”. Thrombin and collagen were found to induce the formation of annexin V-positive MP from zebrafish thrombocytes, and these MP were capable of accumulating at sites of laser-induced arterial injury.

Jean-Marie Freyssinet (Paris & Strasbourg, France) - “Round table discussion of an action plan for the standardization of the determination of microparticles”. There was broad agreement that microparticles can generally be defined as 0.1-1 m M cell-derived vesicular structures that lack a nucleus or synthetic capability. They can, and often do, however, contain a membrane skeleton. Microparticles have their origins in a variety of blood and vascular cell types, and mAb and proteomic analysis is likely to shed important clues as to their varied origins – much work remains to be done in this regard. MP contain varying amounts of surface-exposed PS, depending on their origins and mode of preparation/shedding. Both the phenotype and function of MP vary according to cellular origins and inducers of vesiculation. Hence, MP can be pro-coagulant or anti-coagulant, but when the balance is disrupted in favor of the former population, this reflects an increased thrombotic risk. Preferential approaches to their preparation and analysis was felt to be premature, but remains a worthy goal of future VB Workshop activities.