Platelet-derived microparticles, released as a result of platelet activation, promote coagulation through the surface exposure of phosphatidylserine, acting as the catalytic site for the conversion of prothrombin to thrombin by the activated coagulation factors X and V. Although elevated numbers of circulating platelet-derived microparticles can be detected in a number of clinical disorders, the methods for the detection of these microparticles are far from standardised. In addition, recent reports have also speculated that not all microparticles may expose phosphatidylserine, demonstrating that the binding of Annexin V, a phosphatidylserine-specific binding protein, is not detectable on a population of microparticles. The initial stage of this thesis was to establish a flow cytometric method for the detection and enumeration of microparticles based on their capacity to bind Annexin V and to utilise this assay to investigate a number of the issues that have limited assay standardisation. The assay could be performed on either stimulated or unstimulated plasma or whole blood samples. Interestingly, plasma microparticle counts were significantly higher than whole blood microparticle counts. The effects of centrifugation alone could not be attributed as the sole source of this discrepancy. The antigenic characteristics of platelet-derived microparticles were also investigated, with platelet-derived microparticles demonstrated to express the platelet glycoproteins CD31, CD41a, CD42a and CD61. Platelet-derived microparticles also expressed CD42b, and this expression was significantly decreased when compared to their progenitor platelets. The expression of the platelet activation markers CD62p, CD63, CD40L and PAC-1 was dependent upon the sample milieu, suggesting that the centrifugation conditions required to generate platelet-poor plasma may lead to artefactual increases in the expression of platelet activation markers. An investigation of the role of the GpIIb/IIIa complex on the formation of platelet-derived microparticles was also performed. A monoclonal antibody to the GpIIb/IIIa complex (Abciximab) significantly inhibited in vitro collagen-stimulated platelet-derived microparticle formation. Interestingly, platelets obtained from two subjects with impaired GpIIb/IIIa activation, demonstrated normal microparticle formation following collagen stimulation, suggesting that the presence of GpIIb/IIIa complex, but not its activation, is required for collagen-induced microparticle formation. A novel mechanism for microparticle formation was also investigated, with platelet-derived microparticles demonstrated to form in response to the sclerosing agents sodium-tetradecyl sulphate and polidocanol. Interestingly, the removal of plasma proteins by the washing of platelets left platelets more susceptible to sclerosant-induced microparticle formation, suggesting that plasma proteins may protect platelets from microparticle formation. The procoagulant activity of platelet-derived microparticles was also investigated using a novel coagulation assay (XACT) specific for the procoagulant phospholipid. An evaluation of this assay demonstrated a significant correlation between Annexin V binding microparticle counts and procoagulant activity in both whole blood and plasma samples. There was more procoagulant activity in whole blood samples than in plasma samples, suggesting that the procoagulant phospholipid activity was also associated with erythrocytes or leukocytes. To further investigate this phenomenon, a whole blood flow cytometric assay was developed to assess Annexin V binding to erythrocytes, leukocytes, platelets and microparticles. This assay demonstrated that a large proportion of Annexin V binding (51.0%) was associated with erythrocytes. Interestingly, a proportion of the Annexin V binding erythrocytes (24.5%) and leukocytes (78.8%) were also associated with platelet CD61 antigen, suggesting that they also bound a platelet or platelet-derived microparticle. The effect of sample anticoagulant on microparticle procoagulant activity was investigated. Microparticle counts were most stable in EDTA anticoagulated samples, but were stable in sodium citrate for up to 15 minutes following sample collection. The procoagulant activity of microparticles was significantly inhibited by EDTA in collagen-stimulated platelet-rich plasma samples, when compared to sodium citrate anticoagulated samples. Although the initial method used to investigate microparticles was based upon their ability to bind Annexin V, it was consistently observed that a large proportion of events in the size region of a microparticle were Annexin V negative. An investigation was therefore commenced into the procoagulant activity of microparticles based on their capacity to bind Annexin V. The presence of Annexin V negative microparticles was confirmed by flow cytometry and the proportion of microparticles that bound Annexin V was dependent upon type of agonist used to stimulate microparticle formation. Varying the assay constituents (calcium concentration / Annexin V concentration / buffer type) did not alter the proportion of Annexin V binding microparticles. When compared to Annexin V positive microparticles, Annexin V negative microparticles expressed significantly higher levels of CD42b on their surface, but possessed significantly decreased expressions of CD62p, and CD63. A significant correlation between the percentage of Annexin V binding and XACT procoagulant activity was found (p=0.03). Furthermore, Annexin V binding inhibited greater than 98% of procoagulant phospholipid activity, suggesting that Annexin V binding was a true reflection of procoagulant activity. Microparticles could be sorted using either a flow cytometric or magnetic sorting strategy. By electron microscopy, Annexin V negative events isolated following magnetic sorting were vesicular structures and not small platelets or the remnants of activated platelets. In summary, this thesis has demonstrated the ability of the flow cytometer and XACT assays to detect microparticles and their procoagulant activity. It has also shown that the use of Annexin V to detect microparticles may warrant further investigation.
| Identifer | oai:union.ndltd.org:ADTP/243017 |
| Date | January 2007 |
| Creators | Connor, David Ewan, Clinical School - St Vincent's Hospital, Faculty of Medicine, UNSW |
| Publisher | Awarded by:University of New South Wales. |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | Copyright David Ewa Connor, http://unsworks.unsw.edu.au/copyright |
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