Use of a monoclonal antibody to measure the surface expression of thrombospondin following platelet activation.
The radiolabelled monoclonal antibody, 5G11, directed against native thrombospondin, has been used to assess the surface expression of secreted thrombospondin on human blood platelets. Emphasis has been placed on studying the role of fibrinogen in this process. Unstimulated platelets bound low amounts of 5G11 (about 2000 molecules/platelet). Binding increased 2-fold and 5-7-fold after stimulation of platelets with ADP or thrombin (or ionophore A23187) respectively. Unstimulated platelets from patients deficient in alpha-granule proteins (gray platelet syndrome) bound baseline levels of 5G11. However, binding was not increased after activation. Thrombospondin expression on thrombin-stimulated normal platelets was for a large part divalent-cation-dependent and was not affected by AP-2, a monoclonal antibody to GPIIb-IIIa complexes. However, binding of 5G11 was some 50% lower when platelets were stimulated in the presence of Fab fragments of a polyclonal rabbit antibody to fibrinogen. This suggested either a direct binding of thrombospondin to surface-bound fibrinogen or a steric inhibition due to a close proximity of the two proteins. The fact that binding of 5G11 was at the lower limit of the normal range to the stimulated platelets of an afibrinogenaemic patient specifically lacking detectable fibrinogen favoured the latter explanation. Thus, a major fibrinogen-independent pathway for thrombospondin expression must exist.
D000347:Afibrinogenemia / Q000503:physiopathology; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001778:Blood Coagulation Disorders / Q000503:physiopathology; D001792:Blood Platelets / Q000502:physiology*; D004492:Edetic Acid / Q000494:pharmacology; D005340:Fibrinogen / Q000502:physiology*; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D019699:Thrombospondins
D000911:Antibodies, Monoclonal; D006023:Glycoproteins; D019699:Thrombospondins; D005340:Fibrinogen; D004492:Edetic Acid