Thesis (M. Med. Sc.)--University of Hong Kong, 2005.

Thesis (Ph. D.)--Drexel University, 2006. / Includes abstract and vita. Includes bibliographical references (leaves 146-156).

Thesis (MTech (Biomedical Technology))--Cape Peninsula University of Technology, 2016. / Type 2 diabetes mellitus (T2DM) is a metabolic disorder which is characterised by insulin resistance, defective insulin secretion or both. The chronic state of hyperglycemia in diabetes is associated with microvascular complications and macrovascular complications which account for an estimated 80% of deaths as a result of cardiovascular complications. Type 2 diabetes mellitus is an inflammatory disease and pro- inflammatory stimuli in the form of activated endothelial cells, render the vascular endothelial surface attractive for both platelets and leukocytes. Activated platelets bind to the exposed extracellular matrix (ECM) and also to each other in both physiological haemostasis and pathological thrombosis. They can also adhere to leukocytes. Platelet leukocyte interactions are divided into 3 stages; these include the initiation of the interaction, stabilization of the aggregates and amplification of leukocyte activation. This study attempts to contribute to the current knowledge of T2DM by investigating the percentage of monocytes and neutrophils forming aggregates with platelets in pre-diabetes and diabetes and comparing this to non- diabetes individuals as well as the up regulation of pro-thrombotic and activation antigens on the surface of monocytes and neutrophils (Tissue Factor and CD69). Levels of platelet activation and function will be determined by both plate monocyte aggregates (PMA) and platelet neutrophil aggregates (PNA). A total of 124 individuals were recruited from Bellville South, Cape Town, South Africa. This comprised of diabetes (DM) (n=15), pre-diabetes (pre-T2DM) (n=25) and controls (n=84). All individuals were screened for diabetes using the oral glucose tolerance test (OGTT). Platelet leukocyte measurements were performed using the Navios 8-colour flow cytometer. The median percentage of circulating platelets bound to monocytes (%PMAs) was significantly increased in the T2DM 49.04[36.78-62] and the Pre-T2DM 48.96[36.72-61.2],groups, compared to the control group 7.2[5.4-9], p<0.0001. The median %PNAs, which show interactions between neutrophils and platelets, were significantly increased in the T2DM group 13.56[10.17-16.95] compared to the control group 6.01[4.51-7.51] p<0.0001. Platelet monocyte aggregates (PMAs) were higher in both the pre-T2DM and T2DM groups when compared to the control group indicating increased interactions between platelets and monocytes. In addition to forming aggregates with leukocytes, the platelets were able to initiate activation and phenotypic change to the leukocytes by increasing the expression of CD69 and TF (CD142). This finding provides further evidence that there is a link between the inflammatory process and the prothrombotic activity evident in diabetes and pre-diabetes individuals. Furthermore, we describe elevated levels of circulating activated neutrophils which directly correlate with increased PNA formation in both the pre-T2DM and T2DM group. / South African Medical Research Council

Previous work on the binding of resting platelets to the basement membrane glycoprotein, laminin, has identified the Ic/IIa integrin c01aplex (CD49f/CD29), also known as VLA-6, as the receptor. There exists however, another protein with a molecular weight of 67kDa, that mediates this function on other cells. It is abundantly expressed on the membranes of breast cancer cells, where it plays a key role in both the localisation at, and penetration of vascular beds, by metastases. The objectives of this study were: * The development of a micro-titre assay similar to those used in previous studies, standardised and calibrated to characterize the adhesion of unstimulated normal human platelets to laminin-coated surfaces. * To determine the effect on adhesion of platelet activation, enzymatic surface-glycoprotein removal, antibodies to specific receptors and interaction with other adhesive proteins known to bind to platelet membranes. * To establish the in vivo relevance of the experimental findings, by the assay of adhesion of glycoprotein IIb/IIIa-deficient platelets of two patients with Glanzmann's Thrombasthenia. These studies serve d to distinguish specific binding sites for laminin from the known surface receptors of platelets. The methodology used to isolate laminin receptors from the membranes of breast carcinoma cells was then applied to platelet concentrates. Membranes were obtained by centrifuging the ultrasonic lysate of a unit of platelets. These were solubilized and passed over a laminin-Sepharose column. The bound components were eluted and identified by means of SDS-gel electrophoresis, after which a concentrate was tested for laminin binding by means of dot-blot methodology. The principle contribution of this work is the finding of a 67kDa receptor for laminin on the surface membranes of platelets. The combination of the various approaches applied to characterise the adhesion of platelets to laminin, show that this is a specific, Mg²⁺-dependent process, inhibited by Ca²⁺ and not enhanced by platelet activation. Adhesion was decreased by proteolysis with trypsin and chymotrypsin, showing that the adhesion is mediated by a surface glycoprotein. Proteolysis with the Serratia marcescens metalloprotease, which cleaves off glycoprotein lb, did not affect adhesion, proving that this well-known receptor for platelet adhesion is not involved in the adhesion. The receptors GPIV and glycocalicin were also excluded, as the presence of antibodies to these receptors had no effect. Prior incubation with fibrinogen or von Willebrand factor, which binds to specific receptors on the platelet membrane, inhibited adhesion, most likely due to spatial interference with the receptor site for laminin. The presence of the tetrapeptide recognised by the membrane receptors for many adhesive proteins, RGDS, at concentrations of up to 1mM, had no effect. The platelets of the two subjects with Glanzmann's Thrombasthenia adhered normally, definitively ruling out the involvement of GPIIb/IIIa, which is absent from these platelets. The isolation process recovered a membrane component from the laminin-Sepharose column with an elution pattern identical to that for the well characterised 67kDa receptor for laminin on the surface of breast carcinoma cells. They have the same molecular weights in both the reduced (67kDa) and non-reduced (53kDa) states. Blot identification demonstrated laminin binding by the eluate. In the last part of the work, collaborative studies using more sophisticated methodology have confirmed that platelet receptors for laminin play a role in their adhesion to living tissue. Anti-laminin Fab antibodies significantly decreased the adhesion when whole blood was perfused over isolated rabbit aortic segments. That these receptors are identical to the 67kDa receptor of breast carcinoma cells was shown by the specific, high affinity binding of antibodies directed at the carcinoma receptors to the surface of platelets when examined by flow cytometry. In addition, they inhibit platelet adhesion by 50-60% in the micro-titre assay. It is proposed that both the VLA-6 and the 67kDa receptors are required for platelet adhesion to laminin, possibly as a two-stage process, similar to the systems for adhesion to von Willebrand factor, where binding is initially to GPIb, followed by binding to GPIIb/IIIa. The possible relevance of this receptor in the pathophysiology of the metastatic process is discussed.

By specifically analysing for the various active principles of plasma, platelets, tissues and their fractions, much new information has been obtained concerning the role of lipoids and platelets in blood coagulation and in the hemostatic mechanisms in health and disease. Analysed components are studied in artificial clotting systems, especially a two-stage thrombin- forming system. Some 86 cases of bleeding disorders, 32 new born normal infants and their mothers, and many normal adult bloods have been analysed with respect to components of the clotting and hemostatic functions. The detailed considerations embodied in the thesis are encompassed under the following heads: 1) the importance of certain lipoids, especially cephalin 2) the normal need, in plasma clotting, for platelets, 3) the particular significance of a platelet component, which has many analogies to cephalin, in the thromboplastic system, 4) potentiation of the thromboplastic actions of cephalin, of platelets, and of tissue thromboplastin (to some extent) by a variety of experimental additives. Part of this may be explained as a 'thromboplastin generation' through co-participation of certain plasmatic components (antihemophilic globulin, PTC" etc. ) . Part, however, may be the result of certain proteolytic enzymes, particularly trypsin, 'disaggregating' lipoproteins and thus rendering their phospholipid (and sometimes calcium) available for participation in the clotting reactions, 5) possible Ca-containing and lipid-containing 'intermediates’ in the thrombin-forming reactions, 6) myelin figure formation as an explanation of ‘alterations' of platelets and certain other formed elements such as thrombocytes, megakaryocytes, and stromatolytic erythrocytes 7) the multiplicity of factors which platelets may contribute to the blood clotting and hemostatic mechanisms 8) the occurrence of many clinical disorders due to deficiency of platelet functions. Thrombocytopenias denote deficient numbers ('counts' and total bulk in body). Thrombocytopathies are deficiencies of specific platelet components, e . g. thromboplastic factor, accelerator, vasoconstrictor (5-hydroxy tryptamine), or retractor factor. Such deficiencies can be clinically significant even when the platelet count is normal. Bleeding in leukaemias, uremias, etc. may often be accounted for in these terms, 9) the nature and modes of action of heparin and other 'antithromboplastic’ inhibitors, and of some antiproteases, in relation to the mechanisms discussed 10) the ‘cephalin availability theory' of the author, as a useful working hypothesis to explain the importance of the natural thromboplastic phospholipid. Lipid release from platelet, tissue, or possibly plasma sources may very well be the long-obscure 'trigger mechanism' which initiates blood coagulation.

A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, in partial fulfilment of the requirements for the degree of Master of Medicine in the branch of Anaesthesiology Johannesburg, 2019 / Background Anaesthesia frequently involves administration of refrigerated intravenous drugs to patients. Often overlooked, maintenance of the cold chain forms a key component of pharmacovigilance for anaesthetists. However the South African national Department of Health guidelines on: “Cold Chain and Immunisation Operations Manual”, does not detail specific requirements for medically validated cold boxes. Consequently the risk of iatrogenic harm to patients from heat-sensitive pharmaceuticals in inappropriately temperature regulated cold boxes exists. Methods The research design was that of a descriptive, prospective and contextual study. Part I study population comprised the ambient air temperatures of the refrigerator and cold boxes used for storage of heat-sensitive pharmaceuticals in theatre at CHBAH taken at one minute intervals over eight hours. Part II study population was newly purchased cold boxes and cold packs for the purpose of assessing individual cold box thermal performance over eight hours. Results In Part I, only a single cold box (polystyrene box number 19) was able to maintain the recommended temperature range of 2 − 8°C for the eight hour period (4.35%). The refrigerator temperature time plot showed a significant deviation of temperature at approximately 30 minutes. In Part II, only fabric and polystyrene cold boxes with three cold packs in situ were able to maintain the recommended temperature of 2 − 8°C. Conclusion This study highlighted the failure of non-medically validated cold boxes to reliably maintain the temperature of heat-sensitive pharmaceuticals. / E.K. 2019

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