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Human platelet osteonectin: release, surface expression, and partial
characterization
RJ Kelm and KG Mann
University of Vermont, College of Medicine, Department of Biochemistry,
Burlington 05405.
Our laboratory has previously shown that osteonectin, an abundant
noncollagenous bone protein, is contained in and secreted from human
platelets. In this study, the distribution of osteonectin both in the
supernatant and on the platelet surface after activation was measured by
fluid-phase and solid-phase radioimmunoassay, respectively. Total cellular
osteonectin was determined by RIA of guanidinium chloride extracted
platelets and ranged from 0.65 to 2.2 micrograms/10(8) platelets or 135,000
to 457,000 molecules/platelet. Platelets treated with varying
concentrations of collagen and thrombin released osteonectin in a
dose-dependent fashion. Approximately 61% of the total platelet osteonectin
was secreted at saturating concentrations of collagen and thrombin. A small
fraction of platelet osteonectin is expressed on the surface of platelets
in an activation-specific manner as evidenced by the specific and saturable
binding of [125I]-anti- osteonectin monoclonal antibody, IIIA3A8, to
thrombin-activated platelets. Based on a non-linear least squares
regression analysis of the antibody binding, 2,200 IIIA3A8 molecules, or
0.8% of the total platelet osteonectin, is expressed on the platelet
surface on activation. Platelet osteonectin was purified from the
supernatant of thrombin-activated platelets by immunoaffinity
chromatography. Western blotting of proteins secreted by washed,
thrombin-stimulated platelets with IIIA3A8 indicated that the osteonectin
molecule released from the platelet is a single chain polypeptide.
Comparison of immunopurified platelet osteonectin with isolated bovine bone
osteonectin and isolated human bone osteonectin by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis indicated that platelet
osteonectin has a greater apparent molecular weight than bone osteonectin.
The NH2-terminal sequence of immunopurified platelet osteonectin was
obtained by automated Edman degradation and is identical to the sequence of
human bone osteonectin derived from the cDNA of SaOS-2 cells. Collectively,
these data suggest that platelet osteonectin is structurally distinct from
bone osteonectin in a region of the molecule at a distance from the
NH2-terminus.
Volume 75,
Issue 5,
pp. 1105-1113,
03/01/1990
Copyright © 1990 by The American Society of Hematology
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