Abstract
Platelets were isolated from human blood by Percoll density gradient centrifugation in a low Ca2+/high Mg2+ buffer. The buffer reversibly inactivates the cells during separation. The purity of the isolated cells (> 99%) was determined by flow cytometry, their viability was confirmed by fluorescein diacetate hydrolysis, and their morphology was studied with TEM. Plasma proteins were adsorbed onto hydrophobic glass surfaces, and pure platelets were added and incubated for up to 30 min at 37°C. Platelet activation was determined by cell spreading, formation of microparticles and surface exposure of CD62P indicating the release of α-granules. Surface-immobilized IgG was shown to cause the release of microparticles and cell lysis, in accordance with data published by others. Surface-immobilized vWF was shown to induce CD62P exposure on the platelet cell surface. The specificity of this response was demonstrated by adsorbing plasma proteins from normal and factor VIII-deficient plasma.
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