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Identification of thrombospondin as a high molecular mass protein released from activated equine platelets


, : Identification of thrombospondin as a high molecular mass protein released from activated equine platelets. American Journal of Veterinary Research 58(9): 954-960

Objective-To establish the existence of platelet-derived proteins in equine plasma, with the future goal of developing an assay for the detection of in vivo platelet activation. Animals-5 mature healthy horses. Procedure-Platelet-rich plasma and platelet-poor plasma were prepared from anticoagulated blood. Platelets were separated from plasma proteins by gel filtration, then activated with 0.5 mu-M platelet-activating factor. Protease inhibitors were added, and the released platelet proteins were harvested. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed on the released platelet proteins and platelet-poor plasma, and the resultant silver-stained bands were compared. Immunoblot analysis was performed on released platelet proteins, using an antibody to human thrombospondin; human platelet-derived proteins served as the positive control for the antibody. Results-Released platelet proteins in the presence of beta-mercaptoethanol (reduced samples) contained several proteins that were not observed in plasma including (mean +- SEM) 194 +- 2, 159 +- 2, 151 +- 2, 104 +- 2, and 95 +- 1 kd. Immunoblots of released platelet proteins had a prominent 180 +- 2-kd protein in reduced samples that was recognized by an antibody to human thrombospondin, and with prolonged color development, 2 additional less prominent proteins (166 +- 1 and 155 +- 1 kd) were observed. Conclusions-Several proteins are released from activated equine platelets that are not detectable in normal equine plasma. Thrombospondin is one of the high molecular mass proteins released by activated equine platelets. Clinical Relevance-An assay can be developed for detection of thrombospondin in equine plasma and may be useful for detection of in vivo platelet activation in horses.

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Accession: 002863933

PMID: 9284998

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Related references

Aiken M.L., 1991: An amplified signal of limited platelet activation detection of thrombospondin tsp released from activated cells on resting platelets. FASEB Journal 5(4): A516

Clezardin, P.; McGregor, J.L.; Manach, M.; Robert, F.; Dechavanne, M.; Clemetson, K.J., 1984: Isolation of thrombospondin released from thrombin-stimulated human platelets by fast protein liquid chromatography on an anion-exchange Mono-Q column. Thrombospondin, a glycoprotein found in human platelet alpha granules, is thought to play a major role in platelet haemostatic functions. A rapid method to isolate thrombospondin for functional and structural studies was developed. Freshly prepare...

Legrand C.; Thibert V.; Dubernard V.; Begault B.; Lawler J., 1992: Molecular requirements for the interaction on thrombospondin with thrombin activated human platelets modulation of platelet aggregation. We have investigated the molecular requirements for thrombospondin (TSP) to bind to the platelet surface and to support the subsequent secretion-dependent platelet aggregation. For this, we used two distinct murine monoclonal antibodies (MoAbs), d...

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Nieuwenhuis, H.K.; van Oosterhout, J.J.; Rozemuller, E.; van Iwaarden, F.; Sixma, J.J., 1987: Studies with a monoclonal antibody against activated platelets: evidence that a secreted 53,000-molecular weight lysosome-like granule protein is exposed on the surface of activated platelets in the circulation. To define the role of activated platelets we have attempted to prepare monoclonal antibodies specific for activated platelets. The IgG2b antibody of one of the clones, designated 2.28, was studied in more detail. Native platelets from normal indiv...

Nieuwenhuis H.K.; Van Oosterhout J.J.G.; Rozemuller E.; Van Iwaarden F.; Sixma J.J., 1987: Studies with a monoclonal antibody against activated platelets evidence that a secreted 53000 molecular weight lysosome like granule protein is exposed on the surface of activated platelets in the circulation. To define the role of activated platelets we have attempted to prepare monoclonal antibodies specific for activated platelets. The IgG2b antibody of one of the clones, designated 2.28, was studied in more detail. Native platelets from normal indiv...

Ando, Y., 1985: Substances released by platelets. 2. Thrombospondin. Rinsho Byori. Japanese Journal of Clinical Pathology Spec No 62: 60-68

Hayes, H.; Commissaire, J.; Bergere, J.L., 1984: A high-molecular-mass cell wall protein released from Clostridium tyrobutyricum by heat treatment. Analysis of the cell wall of 4 strains of Clostridium tyrobutyricum reveals an unusually high protein content (35-40% dry weight). Brief heat treatment of whole cells of these stains causes release of two proteins, flagellin and a cell wall compon...

Hayes, H.; Commissaire, J.; Bergere, J.L., 1984: A high-molecular-mass cell wall protein released from Clostridium tyrobutyricum by heat treatment. Analysis of the cell wall of 4 strains of Clostridium tyrobutyricum reveals an unusually high protein content (35-40% dry weight). Brief heat treatment of whole cells of these stains causes release of two proteins, flagellin and a cell wall compon...

D'Angelo, A.; Lockhart, M.S.; D'Angelo, S.V.; Taylor, F.B., 1987: Protein S is a cofactor for activated protein C neutralization of an inhibitor of plasminogen activation released from platelets. Platelets stimulated with thrombin release an inhibitor of plasminogen activator (PAI), which has been shown previously to be neutralized by activated protein C (APC). The requirements for optimal neutralization of PAI activity were investigated....