Localization of thrombospondin in clots formed in situ.
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.
D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D001777:Blood Coagulation; D001792:Blood Platelets / Q000032:analysis; D005353:Fibronectins / Q000276:immunology; D006023:Glycoproteins / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D007124:Immunoenzyme Techniques; D009504:Neutrophils / Q000276:immunology; D019699:Thrombospondins
D000911:Antibodies, Monoclonal; D005353:Fibronectins; D006023:Glycoproteins; D019699:Thrombospondins