UBC Theses and Dissertations
Platelet membrane surface heterogeneity detected by partition in two-polymer phase systems Webber, Timothy James
Platelets are heterogeneous with respect to a variety of functional and physical properties, as a result of different phenomena. Megakaryocytes confer upon their progeny different platelet properties which then change during circulation. Since platelet survival time analyses of platelet production and platelet ageing have provided diagnostic information, efforts have been expended to obtain comparable information regarding circulating platelet heterogeneity through the use of methods which reflect platelet properties, e.g., function, size, density, and membrane surface characteristics, which are believed to change during the survival of platelets within the circulation. Platelet membrane properties were studied here because cell surface charge is thought to be altered during circulation, and because countercurrent distribution (CCD) in two-polymer aqueous phase systems has been shown to be capable of detecting such membrane changes during erythrocyte maturation and ageing. This technique, CCD in phase systems sensitive to membrane charge, was therefore assessed for application to the problem of determining circulating platelet age distribution information in an investigation of platelet heterogeneity. Aqueous two-polymer phase systems were shown to be charged, demonstrating, between the upper and lower phases, a potential difference which decreased with decreasing phosphate and increasing chloride content. Platelet surface property distributions were heterogeneous based on the shape and spread of the CCD curves for these blood elements in comparison to expected theoretical distributions for uniform particles. Platelets from rabbit, monkey, and human demonstrated species specific differences in membrane surface properties as reflected by the single step partition measurement. The species specific differences found by single step parititon were also confirmed by CCD. Platelets, from rabbits, monkeys, and humans, separated according to density which has been shown to be age related, were found to exhibit differences in size, and membrane surface properties by CCD. The partition distributions of individual density separated platelet subpopulations, low density, intermediate density, and high density platelets, were each heterogeneous and all overlapped (partitions coincided), indicating that within each subpopulation there were platelets of different membrane surface properties, and that each subpopulation was in part similar to the others. However, the relative partitions of these density separated platelets were also different based on statistical comparisons of the CCD parameters. CCD and density analyses of in vitro ⁵¹Cr-labeled rabbit platelets revealed a platelet subpopulation, found to be enriched in high density platelets, which exhibited enhanced uptake for ⁵¹Cr, but not for ¹¹¹In, ¹⁴C- or ³H-serotonin, nor for ³H-adenine. This platelet subpopulation was of low partition relative to the platelet population, and was no longer apparent after in vivo injection and circulation for 24 to 48 hours. Combinations of ³⁵S-sulphate cohort labeling in vivo, with ³H-serotonin labeling in vitro, density gradient centrifugation, and CCD, indicated that platelets which were newly released into the circulation were of relatively high density and low partition. Although this subpopulation was heterogeneous, its average density and surface properties changed with platelet maturation and age in the circulation.
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