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.

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.

Thesis (MSc(Genetics))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: The hypertensive conditions of pregnancy (including pre-eclampsia (PE)) is the leading cause of primary obstetric death in South Africa and affects at least five percent of pregnancies in the Western Cape province. Reduced levels of placental protein 13 (PP13) early in pregnancy are associated with a higher incidence of PE in later gestation. PP13 and annexin II have been co-localised to the brush border membrane of syncytiotrophoblasts, and form a complex that is transported to the maternal circulation. It is speculated that genetic variation in the gene encoding annexin II (ANXA2) could underlie the reduced PP13 levels. The aim of this study was to screen the ANXA2 gene, including the proximal promoter region, in two South African population groups, (Mixed Ancestry and Black) from the Western Cape, to identify whether variants in the ANXA2 gene confer susceptibility to PE. The study cohort comprised of 120 pre-eclamptic maternal, 94 pre-eclamptic fetal and 54 healthy control individuals. Genomic DNA of patient and control individuals was extracted for PCR amplification of ANXA2 and Multiphor SSCP/HD analysis was performed for mutation detection. The conformational variants identified were subjected to automated DNA sequencing and subsequently to RFLP analysis, to confirm the genotypes in the remainder of the cohort. Nine previously identified variants (c.-31 T>C, c.292 G>T; p.Val98Leu, c.975 C>T;p.Gly325Gly, c.-12+75 C>A, c.-11-43 G>A, c.-11-13 A>T, c.48+67 C>T, c.449-17 G>A, c.683-56 G>A) and 16 novel variants (c.-442 C>G, c.-191 G>C, c.-189_-188insGCCGG, c.-135 C>G, c.-92 A>T, c.222 C>T; p.Ala74Ala, c.600 C>T; p.Asp110Asp, c.934 G>A; p.Gly312Ser, c.244-42 G>C, c.244-76 C>G, c.528+38 C>T, c.589-5 C>T, c.682+49 C>T, c.961-30 A>G, c.961-24 C>G, c.*1057 A>G) were identified upon screening the ANXA2 gene. Statistical analysis identified significant association at five loci: SNP c.-92 A>T located within the ANXA2 5‟UTR, exonic SNP c.222 C>T; p.Ala74Ala and three intronic SNPs c.244-76 C>G, c.449-17 G>A and c.589-5 C>T. Three of the five variants (c.-92 A>T, c.244-76 C>G, c.589-5 C>T) were significantly associated with PE (P<0.05) and could contribute to PE susceptibility in these two SA iv populations, whereas the other two variants (c.222 C>T; p.Ala74Ala, c.449-17 G>A) revealed a possible protective effect, suggesting a reduced risk of developing PE. In silico analysis predicted the disruption and creation of several putative transcription factor binding sites by three SNPs in the ANXA2 gene, which could subsequently affect ANXA2 functioning. This study provides evidence for genetic variation in the ANXA2 gene, which warrants functional experimental validation in an attempt to investigate the function of these SNPs in molecular, cellular and physiological processes underlying PE. Identifying an association between variants in the ANXA2 gene and PE could contribute to the development of an additional early biomarker. The early identification of PE would promote the South African health system by providing the appropriate health care support and monitoring of high risk pregnancies, which could ultimately result in improved pregnancy outcome. / AFRIKAANSE OPSOMMING: Die hipertensiewe siektes van swangerskap (insluitende pre-eklampsie (PE)) is die belangrikste direkte oorsaak van moedersterftes in Suid-Afrika en dit kom voor by ongeveer 5% van swangerskape in die Wes-Kaap provinsie. Verlaagde plasentale proteïen 13 (PP13) vlakke tydens vroeë swangerskap word verbind met „n hoër voorkoms van PE in latere swangerskap. PP13 en anneksin II kom albei op die borselgrens membraan van synsitiotrofoblaste voor waar hulle „n kompleks vorm wat na die moederlike sirkulasie vervoer word. Daar word gespekuleer dat die onderliggende oorsaak vir laer PP13 vlakke as gevolg van genetiese variasie in die geen wat anneksin II kodeer (ANXA2) kan wees. Die doel van hierdie studie was om die ANXA2 geen, insluitende die proksimale promoter area, in twee Suid-Afrikaanse populasie groepe, (Kleurling en Swart) van die Wes-Kaap, te skandeer met die doel om variante in die ANXA2 geen te identifiseer en ‟n moontlike assosiasie met die vatbaarheid vir PE te bepaal. Hierdie studie populasie het bestaan uit 120 pre-eklamptiese vroue, 94 neonate van pre-eklamptiese ma‟s en 54 gesonde kontrole individue. Genomiese DNS van die pasiënte en kontrole individue is geëkstraeer vir polimerase kettingreaksie amplifikasie van die ANXA2 geen, waarna Multiphor enkelstring konformasie polimorfisme heterodupleks analise uitgevoer is met die doel om DNS variante te identifiseer. Die verskillende konformasies waargeneem is onderwerp aan semi-geoutomatiseerde DNS volgorde bepalingsanalise en gevolglik restriksie fragment lengte polimorfisme analise om genotipes in die res van die studiegroep te bevestig. Vyf-en-twintig variante is geïdentifiseer met die skandering van die ANXA2 geen, waarvan nege voorheen geïdentifiseer is (c.-31 T>C, c.292 G>T; p.Val98Leu, c.975 C>T;p.Gly325Gly, c.-12+75 C>A, c.-11-43 G>A, c.-11-13 A>T, c.48+67 C>T, c.449-17 G>A, c.683-56 G>A) en 16 nuwe variante is (c.-442 C>G, c.-191 G>C, c.-189_-188insGCCGG, c.-135 C>G, c.-92 A>T, c.222 C>T; p.Ala74Ala, c.600 C>T; p.Asp110Asp, c.934 G>A; p.Gly312Ser, c.244-42 G>C, c.244-76 C>G, c.528+38 C>T, c.589-5 C>T, c.682+49 C>T, c.961-30 A>G, c.961-24 C>G, c.*1057 A>G). Statistiese analise het „n statisties beduidende assosiasie met vyf SNPs geïdentifiseer: SNP c.-92 A>T geleë in die ANXA2 5‟UTR, die koderende SNP c.222 C>T; p.Ala74Ala en drie SNPs c.244-76 C>G, c.449-17 G>A and c.589-5 C>T geleë in die nie-koderende areas. Drie van hierdie vyf SNPs (c.-92 A>T, c.244-76 C>G, c.589-5 C>T) het statisties beduidende assosiasie met PE (P<0.05) getoon en kan bedra tot die vatbaarheid vir PE in hierdie twee Suid-Afrikaanse populasies, terwyl die ander twee SNPs (c.222 C>T; p.Ala74Ala, c.449-17 G>A) „n moontlike beskermende effek gedui het, wat „n verlaagde risiko vir die ontwikkeling van PE voorstel. In silico analise het voorspel dat verskeie voorgestelde transkripsiefaktor bindingsetels onderbreek of geskep sal word in die teenwoordigheid van drie SNPs in die ANXA2 geen, wat gevolglik die funksionering van ANXA2 kan affekteer. Hierdie studie verskaf bewyse vir genetiese variasie in die ANXA2 geen, wat verdere funksionele eksperimentele ondersoeke vereis om die funksie van hierdie SNPs in molekulêre, sellulêre en fisiologiese prosesse onderliggend aan PE te bepaal. Die identifisering van ‟n assosiasie tussen variante in die ANXA2 geen en PE kan bydra tot die ontwikkeling van ‟n addisionele vroeë genetiese merker. Die vroeë identifisering van PE kan die Suid-Afrikaanse gesondheidsisteem geweldig baat deurdat die geskikte gesondheidsorg en ondersteuning asook deurgaanse monitering van hoë risiko swangerskappe verskaf sal kan word. Dit kan uiteindelik lei tot ‟n verbeterde uitkoms vir swangerskappe in Suid-Afrika.

Les annexines sont définies comme étant des protéines qui se lient de manière calcium-dépendante aux phospholipides membranaires chargés négativement. Elles ont été associées à différents processus biologiques tels les réponses des plantes aux stress biotiques et abiotiques. Nous avons identifié une annexine végétale, appelée Ntann12, dont l’expression est induite après infection des plantes par la bactérie Rhodococcus fascians. <p> Ntann12 possède les domaines caractéristiques des annexines et se lie aux phospholipides chargés négativement, de manière calcium-dépendante. L’expression de Ntann12 est très abondante dans les cellules différentiées des racines, où la protéine a été détectée par immunolocalisation dans le cytosol et dans le noyau. Des analyses par western blot ont montré que l’accroissement relatif de la quantité de protéines liées aux membranes est positivement corrélé à l’augmentation de la concentration en Ca2+. <p> Au niveau physiologique, l'expression de Ntann12 est induite par l’apport exogène d’auxine. Elle est contrôlée dans les racines par un signal induit par la lumière, et provenant des parties aériennes. Le transport polaire de l'auxine a été identifié comme étant le processus cellulaires nécessaires à l'expression de Ntann12 dans les racines. En outre, cette expression est réprimée par les stress salin, osmotique et hydrique. Ces résultats suggèrent que l’annexine Ntann12 est impliquée dans le métabolisme de l’auxine.<p><p>/<p><p>Annexins are defined as calcium-binding proteins, and they have been associated in plants with different biological processes such as responses to biotic and abiotic stress. Ntann12 expression is induced upon infection of tobacco plant by R. fascians. <p> Ntann12 possesses the conserved annexin repeat with the sequence for type II Ca2+-binding site and recombinant as well as native Ntann12 binds to negatively charged phospholipids in a Ca2+-dependent manner. It is mainly expressed in root differentiated cells where the protein was immunolocalized in the cytosol and in the nucleus. Ntann12 was examined by western blot in both microsomal and cytosolic fractions from tobacco roots cells, and was detected in both the cytosol and microsome. The relative increase of Ntann12 proteins associated with the microsome is coupled with an increase in Ca2+ concentration.<p> At the physiological level, Ntann12 expression is induced by exogenous application of auxin, and was found to be regulated in the root system by a light-induced signal coming from plant aerial part and polar auxin transport was identified to be the cellular process required for Ntann12 expression in root cells. Furthermore, Ntann12 expression is down-regulated by salt, osmotic and water stress. These results collectively suggest that the annexin Ntann12 is implicated in auxin metabolism. <p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished