title: Use of arabinogalactan to obtain washed murine platelets free of contaminating plasma proteins and appropriate for studies of function, morphology, and thrombopoiesis.
abstract: A simplified procedure for preparing washed murine platelets, free of contaminating plasma proteins, has been developed. Platelet-rich plasma (PRP) was prepared from diluted whole blood from C57BL/6N mice by two centrifugations at 100 x g for 12 minutes. Platelets were concentrated and then washed by centrifugation through isosmolar 10% arabinogalactan (Stractan). Platelet recovery was 85%  +/- 6% (1 SD) (n = 10) from whole blood to PRP and 86% +/- 4% (1 SD) (n = 6) from PRP to Stractan-washed platelets. Overall recovery of platelets with this technique was 73% +/- 10% (1 SD). Contamination of platelets with plasma proteins could not be detected with use of unlabeled platelets that had been incubated with radiolabeled plasma proteins followed by washing with Stractan. The Stractan-washed platelets were assessed for function by using aggregometry. The response of Stractan-washed platelets to collagen and thrombin was identical to that of unwashed platelets. Stractan-washed platelets did not respond to 20 mumol/L adenosine diphosphate unless supplemented with 12% platelet-free plasma. The morphology of the Stractan-washed platelets indicated that degranulation had not occurred. With use of antibodies directed against the alpha granule membrane protein GMP-140 or fibrinogen, no evidence of secretion or plasma protein contamination was observed. The use of this method resulted in an improved assay for the rate of thrombopoiesis, based on detection of radioactive proteins in newly synthesized platelets, by eliminating contamination by radioactive plasma proteins. Our results indicate that this procedure is a convenient method for the separation of platelets from platelet-rich plasma, free of plasma proteins, which are suitable for bioassays, functional studies, and morphologic investigations.
authors.affiliation: Department of Laboratory Medicine, University of California School of Medicine, San Francisco.
authors.forename: R J
authors.initials: RJ
authors.lastname: Hill
authors.forename: P E
authors.initials: PE
authors.lastname: Stenberg
authors.forename: P M
authors.initials: PM
authors.lastname: Sullam
authors.forename: J
authors.initials: J
authors.lastname: Levin
mesh_terms: D000818:Animals; D001781:Blood Component Removal / Q000379:methods*; D001792:Blood Platelets / Q000032:analysis / Q000502:physiology* / Q000648:ultrastructure; D001798:Blood Proteins / Q000032:analysis*; D002469:Cell Separation / Q000379:methods*; D005685:Galactans; D006410:Hematopoiesis; D007150:Immunohistochemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D010974:Platelet Aggregation; D010976:Platelet Count; D010979:Platelet Function Tests
chemical_list: D001798:Blood Proteins; D005685:Galactans; C005653:arabinogalactan