Antibodies against a 23Kd heparin binding fragment of thrombospondin inhibit platelet aggregation

Gartner, T. Kent; Walz, Daniel A.; Aiken, Martha L.; Starr-Spires, Linda; Ogilvie, M L

doi:10.1016/0006-291x(84)90950-1

Cited by 59 publications

(38 citation statements)

References 20 publications

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“…Similar results were obtained by Gartner et al using a monospecific antithrombospondin antibody [24]. Since the thrombospondin domain implicated in haemagglutination could play a role in platelet aggregation [26], these two well characterized monoclonal antibodies (P10 and P12) could be useful tools to investigate the role of thrombospondin in platelet aggregation.…”

Section: Discussionsupporting

confidence: 82%

Characterization of two murine monoclonal antibodies (P10, P12) directed against different determinants on human blood platelet thrombospondin

et al. 1986

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Thrombospondin, a 450-kDa glycoprotein composed of three disulphide linked chains, is located in human blood platelet alpha-granules and is released from platelets upon stimulation. This glycoprotein is thought to play a major role in platelet aggregation. The aim of this study was to characterize two monoclonal antibodies (P10 and P12) directed against human blood platelet thrombospondin. When the released material obtained after stimulation of platelets with thrombin in the presence of 2 mM calcium was immediately treated with EDTA, labelled with ' ' ' I and incubated with monoclonal antibodies PI0 and P12, both immunoprecipitated a major labelled protein band with a molecular mass of 160 kDa and a weaker band at 146 kDa, as analysed on reduced dodecyl sulphate/polyacrylamide gels. The major band corresponds in molecular mass to the thrombospondin subunits. If, however, the released material was left in the presence of Ca2+ for 48 h, then the main band was at 130 kDa and in addition one minor protein band (75 kDa) was immunoprecipitated by P10 whereas P12 recognized two minor protein bands (75 and 60 kDa). When P10 and P12 were incubated with '251-labelled platelet releasates treated for 48 h at 4°C with 10 mM EDTA, three major protein bands (160, 146 and 130 kDa) were immunoprecipitated in addition to the minor bands mentioned above. These results indicate that thrombospondin is probably degraded by the endogenous platelet calcium-dependent protease. Investigation of tryptic peptide fragments of thrombospondin isolated by fast protein liquid chromatography showed that 251-labelled antibody P10 bound to 400-kDa and 120-kDa fragments whereas '2SI-labelled PI2 only recognized a 400-kDa fragment. Competition studies involving solid-phase antibody binding and double antibody sandwich assays showed that P10 and P12 were directed against different determinants of thrombospondin. Purified thrombospondin, isolated in the presence of calcium, either directly or after treatment with EDTA, haemagglutinated trypsinized, formaldehyde-fmed sheep erythrocytes identically. The haemagglutination activity of EDTA-treated thrombospondin was inhibited by P10 and enhanced by P12. On the other hand, P10 and P12, despite their binding to calciumtreated thrombospondin, had no effect on its haemagglutination activity. Monoclonal antibodies PI0 and P12 could be useful tools to investigate the role of thrombospondin in platelet aggregation.Thrombospondin, a calcium-sensitive glycoprotein located in human blood platelet alpha-granules, is released from platelets upon stimulation [l]. Human platelet thrombospondin is composed of three equivalent disulphide-linked chains of 150 -160 kDa [2]. Isolated human platelet thrombospondin has been shown to interact with several proteins [2 -81. These thrombospondin-protein interactions suggest that thrombospondin may have several structural domains with independent functions. However, the functional domains of thrombospondin interacting with most of these proteins have still to be elucidated. The h...

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Section: Discussionsupporting

confidence: 82%

Characterization of two murine monoclonal antibodies (P10, P12) directed against different determinants on human blood platelet thrombospondin

et al. 1986

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“…Obviously, other mechanisms are involved in platelet aggregation induced by high concentrations of agonists, as Gray platelets, which lack a-granule constituents, aggregate nearly normally in response to ^0.15 U/mL thrombin. 44 Interestingly in our study, the inhibitory effect of recombinant proteins on platelet aggregation induced by a low concentration of ADP or thrombin was accompanied by an inhibition of platelet secretion, an effect that we 11 and others 9 have also observed with antibodies directed against the heparin-binding domain of TSP. These results strongly suggest that the binding of secreted TSP to the platelet surface during the activation process in some way contributes to amplification of the release reaction.…”

Section: Discussionsupporting

confidence: 60%

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

Legrand

Morandi

Mendelovitz

et al. 1994

Arterioscler Thromb

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Thrombospondin (TSP) is a platelet a-granule adhesive protein that plays a critical role in the stabilization of thrombus by promoting the formation of platelet macroaggregates. We have recently shown that a monoclonal antibody (mAb) to the NH 2 -terminal heparin-binding domain of TSP, MAE, inhibits platelet aggregation induced by thrombin in a dose-dependent manner. In this study, we have expressed in Escherichia coli two recombinant proteins comprising residues 1 to 174 (TSP18) and 1 to 242 (TSP28) of TSP. After purification, both proteins reacted equally well with mAb MAII, whereas the reactivity of TSP18 for heparin was lower than that of TSP28 or native TSP. At micromolar concentrations, TSP18 and TSP28 inhibited the second wave of platelet aggregation and the concomitant release of [

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“…The ability of anti-TSP antibodies to inhibit platelet aggregation has been confirmed by several investigators using both monoclonal 89 and polyclonal 49 ' n -71 antibodies. It appears that a carboxy-terminal 18kD TSP fragment may be involved in mediating the effect of TSP on platelet aggregation.…”

Section: Antibodies To Thrombospondin Inhibit Platelet Aggregationmentioning

confidence: 88%

“…The best characterized domain is the 23-35 kD N-terminal heparin-binding fragment 10 that has been isolated by Dixit et al 45 and Raugi et al 46 Fibrinogen binding 47 and hemaglu-tinating domains 48 have also been partially characterized, as has a fragment important for platelet aggregation. 49 '…”

mentioning

confidence: 99%

Thrombospondin: a versatile multifunctional glycoprotein.

Silverstein¹,

Leung²,

Nachman³

1986

Arteriosclerosis

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Antibodies against a 23Kd heparin binding fragment of thrombospondin inhibit platelet aggregation

Cited by 59 publications

References 20 publications

Characterization of two murine monoclonal antibodies (P10, P12) directed against different determinants on human blood platelet thrombospondin

Characterization of two murine monoclonal antibodies (P10, P12) directed against different determinants on human blood platelet thrombospondin

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

Thrombospondin: a versatile multifunctional glycoprotein.

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