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

Clézardin, Philippe; McGregor, John L.; Lyon, M; Clemetson, Kenneth J.; Huppert, J.

doi:10.1111/j.1432-1033.1986.tb09363.x

Cited by 27 publications

(36 citation statements)

References 26 publications

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“…The remaining steps were carried out as described in Materials and Methods 34.4% and 6% respectively. Autoradiograms of radiolabeled thrombospondin and osteonectin showed no evidence of irreversible aggregation or denaturation [24] (and Fig. 1, lane E).…”

Section: Determination O J the Thrombospondin And Osteonectin Standarmentioning

confidence: 99%

“…bovine serum albumin as previously indicated. After removing the washing buffer, an anti-thrombospondin mouse monoclonal antibody (PIO) [24], diluted at l/SOOOOO in Tris/Tween buffer, and an anti-osteonectin polyclonal antibody [22], diluted at l/lOOOO, were added to thrombospondincoated and osteonectin-coated wells, respectively (100 pl/ well), and incubated for 1 h at 37°C. The remaining steps were carried out as described above.…”

Section: Determination O J the Thrombospondin And Osteonectin Standarmentioning

confidence: 99%

“…After washing with a Tris/Tween buffer (15 mM Tris, 2 mM CaCl,, 0.05% Twen 20, pH 7.4) containing 0.5% bovine serum albumin, the nitrocellulose sheet was incubated with purified thrombospondin (50 pg/ml in Tns/ Tween buffer) overnight at room temperature. After incubation, the nitrocellulose sheet was washed twice in Tris/ Tween buffer (10 min/wash) and was then immersed under gentle agitation for 2 h at room temperature in Tris/Tween buffer containing an anti-thrombospondin monoclonal antibody (PIO) [24], diluted at I/lOOO. The washing procedure was repeated then a goat anti-(mouse IgG) coupled to horseradish peroxidase was added for a further 1 h incubation at room temperature.…”

Section: Dot Blottingmentioning

confidence: 99%

“…An anti-thrombospondin monoclonal antibody (PIO) [24], diluted in bicarbonate coating buffer to a concentration of 10 pg/ml, was added to microtitre plates (100 pl/well) and incubated for 3 h at 37°C. Addition of albumin instead of monoclonal antibody P10 was used as the first negative control.…”

Section: Fused-rocket Ii~2munoelectrophoresismentioning

confidence: 99%

“…After incubation, the contents of the wells were aspirated and rinsed three times with Tris/Tween buffer. A purified anti-thrombospondin mouse monoclonal antibody (P10) [24], diluted to I/SOOOOO in Tris/Tween buffer, was reacted for 3 h at 37 "C (100 pl/well). Alternatively monospecific rabbit anti-fibrinogen (1/70000) or anti-fibronectin (l/lOOOOO), antibodies were used.…”

Section: Fused-rocket Ii~2munoelectrophoresismentioning

confidence: 99%

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Complex formation of human thrombospondin with osteonectin

Clézardin

Malaval

Ehrensperger

et al. 1988

European Journal of Biochemistry

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Human thrombospondin, a 450-kDa glycoprotein isolated from platelets and endothelial cells, specifically interacts with osteonectin, a protein of 30 kDa isolated from bovine bones and human platelets. Using ELISA, purified osteonectin binds to solid-phase-adsorbed thrombospondin with a dissociation constant (&) of 0.7 nM. Binding of thrombospondin to solid-phase-adsorbed osteonectin was also observed (& = 0.86 nM). The interaction of thrombospondin with solid-phase-adsorbed osteonectin was significantly decreased (81 % inhibition) when using an excess of fluid-phase osteonectin. Thrombospondin-osteonectin complex formation was calciumdependent as shown by a 50 -80% inhibition in the presence of EDTA. None of the proteins known to interact with thrombospondin (fibrinogen, fibronectin, collagen, plasminogen) had a significant inhibitory effect on thrombospondin-osteonectin complex formation. This selective interaction was confirmed by affinity chromatography. Iodinated osteonectin, previously incubated with purified thrombospondin, specifically bound to an anti-thrombospondin monoclonal antibody (P10) linked to protein-A -Sepharose 4B. Elution of the antithrombospondin antibody from protein A allowed the recovery of the thrombospondin-osteonectin complex in the eluate, as judged by SDS/polyacrylamide gel electrophoresis and autoradiography. Blotting of purified thrombospondin to osteonectin adsorbed onto nitrocellulose further confirmed complex formation. In addition, when released from thrombin-stimulated platelets, thrombospondin and osteonectin bound to anti-thrombospondin IgG-coated plates indicating that osteonectin was complexed to thrombospondin once the platelet-release reaction has occurred.Thrombospondin is a platelet a-granule glycoprotein which is secreted in response to thrombin [l]. This glycoprotein has a molecular mass of 450 kDa and is composed of three equivalent disulphide-linked chains of 150 -160 kDa [2]. Each thrombospondin chain is made up of several protease-resistant domains, which bind specifically with heparin, fibrinogen, fibronectin, collagen, calcium, histidine-rich glycoprotein and plasminogen (for review see [3]).Thrombospondin is synthesized by a wide range of cultured cells [3 -71 including fibroblasts, monocytes, macrophages, endothelial and smooth muscle cells [3] and is incorporated into their extracellular matrices [8,9]. Source-specific differences in fragmentation patterns between thrombospondin molecules were reported following exposure to proteolytic enzymes [lo -121, suggesting thrombospondin polymorphism [ll].The exact physiological function(s) of thrombospondin is unknown; however, there is growing evidence that it takes part in the platelet aggregation process [13, 141 and promotes cell adhesion [6, 71. Recently human platelets have been shown to contain and secrete a 30-kDa phosphoprotein [15], namely osteonectin, which is a major protein of mineralized bone [16, 171. Platelet osteonectin is identical to bovine bone osteonectin in terms of molecular mass and immuno...

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Section: Determination O J the Thrombospondin And Osteonectin Standarmentioning

confidence: 99%

Section: Determination O J the Thrombospondin And Osteonectin Standarmentioning

confidence: 99%

Section: Dot Blottingmentioning

confidence: 99%

Section: Fused-rocket Ii~2munoelectrophoresismentioning

confidence: 99%

Section: Fused-rocket Ii~2munoelectrophoresismentioning

confidence: 99%

See 3 more Smart Citations

Complex formation of human thrombospondin with osteonectin

Clézardin

Malaval

Ehrensperger

et al. 1988

European Journal of Biochemistry

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Flow‐cytometric detection of surface membrane alterations and concomitant changes in the cytoskeletal actin status of activated platelets

et al. 1990

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Occlusive vascular diseases are promoted by a “prethrombotic state” with increased platelet activity. Polymerization of cytoskeletal proteins and exposure of subcellular structures or rebinding of secreted proteins have been characterized as early reactions after platelet activation preceding adhesion and aggregation. Here, we demonstrate the kinetic increase in specific binding of monoclonal antibodies to thrombospondin (P10) and to platelet membrane activation markers CD63 (GP53, a 53 kD lysosomal protein) and CD62 (GMP140, a 140 kD alpha granule protein) by using a flow‐cytometric bio‐assay and the related change in the actin status by using the DNase‐I inhibition assay after stimulation of normal human platelets with 0.2 U/ml thrombin. F‐actin was raised from 41% to 51% of total platelet actin content 30 s after stimulation and remained thereafter constant (50% at 60 s). Simultaneously, the percentage of P10, CD63, and CD62 positive platelets was elevated from 5.4%, 24.4%, and 9.1% to 67.4%, 80.2%, and 82.3% respectively. The mean number of P10, CD63, and CD62 antibody binding sites increased from 3,300, 1,715, and 2,146 to 6,400, 6,800, and 9,016 per platelet. Conclusively, changes in the organization of the cytoskeletal protein “actin” and exposure of subcellular structures indicating platelet secretion can be regarded as markers of early platelet activation. Thus, the parallel response in both analytical systems provides further support for the diagnostic concept of flow‐cytometric detection of preactivated platelets in the peripheral blood by using fluochrome staining procedures detecting activation dependent structural alterations directly at the cellular level.

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Osteonectin is an α-granule component involved with thrombospondin in platelet aggregation

Clézardin

Malaval

Trzeciak

et al. 1991

Journal of Bone and Mineral Research

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We previously showed that thrombospondin, a major alpha-granule glycoprotein of human platelets, forms a specific complex with osteonectin, a phosphoglycoprotein originally described in bone that is also present in human platelets. The storage organelles and the function of osteonectin in platelets are still unknown. In this study, using electron microscopy in combination with immunogold staining, the major storage organelle for platelet-secreted proteins, the alpha-granules. Furthermore, osteonectin was qualitatively and quantitatively assessed by studying normal platelets and the platelets from a patient with gray platelet syndrome. Gray platelet syndrome is a rare congenital bleeding disorder characterized by a selective deficiency in morphologically recognizable platelet alpha-granules and in the alpha-granule secretory proteins. Binding of an iodinated antiosteonectin monoclonal antibody to gray platelet proteins transferred to nitrocellulose from SDS-polyacrylamide gels showed no band corresponding to osteonectin compared to control platelets. Using a polyclonal antiosteonectin antibody-based radioimmunoassay, gray platelets contained 0.2 +/- 0.03 ng osteonectin per 10(6) platelets, which is only 20% of the normal platelet content of osteonectin (0.93 +/- 0.16 ng per 10(6) platelets). Study of the localization of osteonectin to the surface of human platelets demonstrated that a radioiodinated antiosteonectin polyclonal antibody bound specifically to thrombin-stimulated platelets but not to resting platelets. Binding was concentration-dependent, saturable (1710 +/- 453 binding sites per platelet, Kd = 1 microM), and inhibited by an excess of cold antiosteonectin polyclonal antibody. No binding was observed on the surface of thrombin-stimulated gray platelets. To gain further insights into the role of osteonectin released from activated platelets, the effect of an antiosteonectin polyclonal antibody was tested on the aggregation of washed platelets. F(ab')2 fragments from the antiosteonectin polyclonal antibody inhibited in a dose-dependent manner the aggregation of collagen-stimulated, washed human platelets without affecting collagen-induced platelet serotonin release. To characterize the mechanism through which antiosteonectin F(ab')2 fragments inhibit platelet aggregation, the expression of endogenous thrombospondin (TSP) on the surface of thrombin-activated platelets was studied using 125I-labeled anti-TSP monoclonal antibody P10. The endogenous surface expression of TSP to thrombin-stimulated platelets was significantly inhibited in the presence of antiosteonectin F(ab')2 fragments (6286 +/- 2065 molecules of P10 per platelet) compared to 11,230 +/- 766 molecules of P10 per platelet in the presence of nonimmune F(ab')2 fragments.(ABSTRACT TRUNCATED AT 400 WORDS)

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

Cited by 27 publications

References 26 publications

Complex formation of human thrombospondin with osteonectin

Complex formation of human thrombospondin with osteonectin

Flow‐cytometric detection of surface membrane alterations and concomitant changes in the cytoskeletal actin status of activated platelets

Osteonectin is an α-granule component involved with thrombospondin in platelet aggregation

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