Use of a monoclonal antibody to measure the surface expression of thrombospondin following platelet activation

Abstract

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 α‐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.

Increased platelet activation and abnormal membrane glycoprotein content and redistribution in myeloproliferative disorders

“…CLB-gran12 (anti-CD63, Immunotech), 18´2 mg/ml, is directed against the lysosomal membrane protein gp53 exposed on the external platelet membrane upon platelet stimulation (Nieuwenhuis et al, 1987). P10 (anti-TSP, Immunotech), 4´55 mg/ml, is directed against TSP, an alpha-granule glycoprotein, that binds to GPIV after degranulation (Legrand et al, 1988). PAC-1 (Becton Dickinson), 4´55 mg/ml, is specific for the activated fibrinogen receptor on GPIIb/IIIa (Shattil et al, 1985).…”

Selective inhibition of platelet macroaggregate formation by a recombinant heparin-binding domain of human thrombospondin.

“…21 We anticipated that TSP18 and TSP28 would inhibit expression of the endogenous protein by blocking its interaction with membrane receptors. On the contrary, we found either no decrease or only a slight increase in the binding of mAb 5G11 when we activated the platelets in the presence of TSP18 or TSP28 at concentrations that inhibited platelet aggregation (Fig 4).…”

Induction of thrombospondin-1 by all-trans retinoic acid modulates growth and differentiation of HL-60 myeloid leukemia cells

“…The purified protein gave a single band (M r 180 000) on electrophoresis in the presence of SDS and ␤-mercaptoethanol. Anti-TSP antibodies, used in this study as purified IgGs, were the rat monoclonal antibody 5G11 prepared in our laboratory 33 and the polyclonal antibodies R1 and R5, a kind gift of Pr J Lawler (Harvard Medical School, Boston, MA, USA). The specificity of these antibodies has been previously demonstrated by immunoblotting using platelet lysates.…”

“…The specificity of these antibodies has been previously demonstrated by immunoblotting using platelet lysates. 33,34 The R5 antibody, which has been prepared against a fusion protein encompassing residues 784-932 of human TSP-1, inhibits platelet aggregation. 34 The R1 antibody has been used to immunolocalize TSP in MCF-7 cells and HUVEC.…”