No abstract

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) .

Physiological angiogenesis results from the balance of angiogenesis activators and inhibitors.

No abstract

Thrombospondin is a principal glycoprotein secreted by thrombin- stimulated platelets and has known affinities for fibrinogen and fibrin. We studied the distribution of thrombospondin in clots formed in situ on Formvar-coated coverslips at 37 degrees C for intervals up to 17 hours. The distributions of three other major platelet granular proteins--fibrinogen, fibronectin, and von Willebrand factor (vWF)-- were also determined. The portions of the clots adhering to the coverslips after stripping, washing, and fixation with formaldehyde were stained for the four proteins by the peroxidase-antiperoxidase technique. Monoclonal antibodies were used to localize thrombospondin, fibronectin, and vWF; affinity-purified polyclonal antibodies were used to localize fibrinogen. Platelets stained positively for all four proteins. Thrombospondin was maximally present in the fibrin meshwork from 1 1/2 to 2 hours, after which the intensity of staining decreased until only trace amounts of thrombospondin were detectable between four and 17 hours. Antifibrinogen and, to a lesser extent, antifibronectin stained the fibrin meshwork at all time points. The vWF was not detectable in the fibrin meshwork at any time point. Staining of polymorphonuclear leukocytes (PMNLs) in a fine granular pattern was found with antithrombospondin. The fraction of PMNLs staining positively was 6% to 14% at 1/2 to 4 hours and increased at eight hours to 27%. At 17 hours, 52% of the PMNLs stained for thrombospondin. More than 48% of the PMNLs stained with antifibrinogen at all time points. PMNLs did not stain for either fibronectin or vWF. These studies indicate that thrombospondin is a transient component of the temporary fibrin meshwork and has a unique spatial and temporal distribution in the hemostatic plug.