Thombospondin, the major glycoprotein released from a-granules of thrombinstimulated platelets, is a disulfide-bonded trimer of 160 kilodalton subunits and apparently functions as a platelet lectin . Because cultured human umbilical vein endothelial cells synthesize and secrete a glycoprotein (GP-160) which is a disulfide-bonded multimer of 160 Walton subunits, the possibility that GP-160 is thrombospondin was investigated . Tritiated GP-160 could be immunoisolated from [3H]leucine-labeled endothelial cell postculture medium using a rabbit antiserum to human platelet thrombospondin . Thrombospondin and GP-160 comigrated in two different two-dimensional electrophoretic systems . Both proteins are disulfidebonded trimers of acidic 160-kdalton subunits . A competitive radioimmunoassay for binding of ' 25 1-thrombospondin to the rabbit antibodies indicated that 49 l-rg of thrombospondin antigen per 106 confluent endothelial cells accumulated in postculture medium over 24 h . Thus, endothelial cells secrete large amounts of a glycoprotein that is identical or very similar to platelet thrombospondin .Thrombospondin, also known as thrombin-sensitive protein (2, 3) or glycoprotein G (4), is a major platelet a-granule glycoprotein that is secreted and then partially bound to platelet membranes when human platelets aggregate in response to thrombin (5-9). Studies by Lawler and co-workers indicate that thrombospondin is a 450-kdalton filamentous protein of dimensions 7 X 65 run, has a pl of 4 .7, and is composed of three large disulfide-linked subunits (10, 11) . During the process of aggregation, thrombin-stimulated platelets develop a membrane-bound lectin activity (12-14) that originates from a-granules and appears to play an important role in mediating platelet aggregation by binding to a specific receptor on other platelets (15, 16) . We recently found that purified human platelet thrombospondin has lectin activity (i .e., agglutinates fixed trypsinized sheep erythrocytes) and blocks the agglutination of thrombin-treated platelets . Therefore, we suggested that thrombospondin is the endogenous lectin of human platelets (17) . This may explain why platelets from patients with the gray platelet syndrome, which lack a-granules and a-granule constituents, including thrombospondin, aggregate poorly in response to thrombin (18) .

Thrombospondin, a major glycoprotein released from a granules of thrombin-stimulated platelets, is a disulfidebonded trimer of 160-kilodalton subunits. Cultured human foreskin and fetal lung fibroblasts secreted thrombospondin (determined by enzyme-linked immunosorbent assay) into the culture medium in a time-dependent manner (15.7 and 5.8 Thrombospondin is a major platelet a granule glycoprotein that is secreted and then partially bound to platelet membranes when human platelets aggregate in response to thrombin (1)(2)(3)(4)(5)(6)(7)(8). Thrombospondin is a 450-kilodalton (kDal) protein and is composed of three large disulfide-linked subunits (9, 10). During platelet aggregation, thrombin-stimulated platelets develop a membrane-bound lectin-like activity (11-13), which originates from a granules and appears to play a role in mediating platelet aggregation by binding to a specific receptor on other platelets (14,15 3.7 X 10'°Bq). The radioactive postculture medium was removed, centrifuged at 8,000 x g for 2 min to remove cells and debris, and frozen at -35°C. The radioactive cell layers were washed twice, removed with a rubber policeman, pelleted by centrifugation, dissolved by boiling for 5 min in 2% NaDodSO4 containing protease inhibitors as described (20), and frozen until analyzed.In experiments in which the accumulation of thrombospondin antigen was measured by enzyme-linked immunosorbent assay (ELISA), fibroblasts were cultured in minimal essential medium containing 20% rabbit serum in 2-cm2 wells of multiwell plates. When the cells were confluent, the cells were washed, and the medium was replaced with 1 ml of fresh minimal essential medium containing 20% rabbit serum. At various times after the medium change, the postculture medium was removed, centrifuged at 8,000 X g for 2 min, and frozen until assayed. 998The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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