Osteonectin is an alpha-granule component involved with thrombospondin in platelet aggregation.
We previously showed that thrombospondin, a major alpha-granule glycoprotein of human platelets, forms a specific complex with osteonectin, a phosphoglycoprotein originally described in bone that is also present in human platelets. The storage organelles and the function of osteonectin in platelets are still unknown. In this study, using electron microscopy in combination with immunogold staining, the major storage organelle for platelet-secreted proteins, the alpha-granules. Furthermore, osteonectin was qualitatively and quantitatively assessed by studying normal platelets and the platelets from a patient with gray platelet syndrome. Gray platelet syndrome is a rare congenital bleeding disorder characterized by a selective deficiency in morphologically recognizable platelet alpha-granules and in the alpha-granule secretory proteins. Binding of an iodinated antiosteonectin monoclonal antibody to gray platelet proteins transferred to nitrocellulose from SDS-polyacrylamide gels showed no band corresponding to osteonectin compared to control platelets. Using a polyclonal antiosteonectin antibody-based radioimmunoassay, gray platelets contained 0.2 +/- 0.03 ng osteonectin per 10(6) platelets, which is only 20% of the normal platelet content of osteonectin (0.93 +/- 0.16 ng per 10(6) platelets). Study of the localization of osteonectin to the surface of human platelets demonstrated that a radioiodinated antiosteonectin polyclonal antibody bound specifically to thrombin-stimulated platelets but not to resting platelets. Binding was concentration-dependent, saturable (1710 +/- 453 binding sites per platelet, Kd = 1 microM), and inhibited by an excess of cold antiosteonectin polyclonal antibody. No binding was observed on the surface of thrombin-stimulated gray platelets. To gain further insights into the role of osteonectin released from activated platelets, the effect of an antiosteonectin polyclonal antibody was tested on the aggregation of washed platelets. F(ab')2 fragments from the antiosteonectin polyclonal antibody inhibited in a dose-dependent manner the aggregation of collagen-stimulated, washed human platelets without affecting collagen-induced platelet serotonin release. To characterize the mechanism through which antiosteonectin F(ab')2 fragments inhibit platelet aggregation, the expression of endogenous thrombospondin (TSP) on the surface of thrombin-activated platelets was studied using 125I-labeled anti-TSP monoclonal antibody P10. The endogenous surface expression of TSP to thrombin-stimulated platelets was significantly inhibited in the presence of antiosteonectin F(ab')2 fragments (6286 +/- 2065 molecules of P10 per platelet) compared to 11,230 +/- 766 molecules of P10 per platelet in the presence of nonimmune F(ab')2 fragments.(ABSTRACT TRUNCATED AT 400 WORDS)
Clezardin
P
P
INSERM U 234, Laboratoire de Biochimie des Protéines Osseuses, Hôpital Edouard Herriot, Lyon, France.
Malaval
L
L
Morel
M C
MC
Guichard
J
J
Lecompte
T
T
Trzeciak
M C
MC
Dechavanne
M
M
Breton-Gorius
J
J
Delmas
P D
PD
Kaplan
C
C
D000328:Adult; D000911:Antibodies, Monoclonal; D001791:Blood Platelet Disorders / Q000097:blood*; D015153:Blotting, Western; D003094:Collagen / Q000494:pharmacology; D003594:Cytoplasmic Granules / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology; D008854:Microscopy, Electron; D015676:Osteonectin / Q000097:blood* / Q000276:immunology; D010974:Platelet Aggregation; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D013577:Syndrome; D013917:Thrombin / Q000494:pharmacology; D019699:Thrombospondins
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D015676:Osteonectin; D010980:Platelet Membrane Glycoproteins; D019699:Thrombospondins; D003094:Collagen; D013917:Thrombin