Measurement of the activation of equine platelets by use of fluorescent-labeled annexin V, anti-human fibrinogen antibody, and anti-human thrombospondin antibody.
OBJECTIVE
To investigate the potential use of fluorescent-labeled annexin V, anti-human fibrinogen antibody, and anti-human thrombospondin antibody for detection of the activation of equine platelets by use of flow cytometry.


METHODS
Platelets obtained from 6 Thoroughbreds.


METHODS
Flow cytometry was used to assess platelet activation as indicated by detection of binding of fluorescent-labeled annexin V, anti-human fibrinogen antibody, and anti-thrombospondin antibody to unactivated and ADP-, collagen-, platelet activating factor (PAF)-, and A23187-activated equine platelets. Human platelets were used as control samples. Determination of 14C-serotonin uptake and release was used to assess the extent of platelet secretion.


RESULTS
Anti-human thrombospondin antibody failed to bind to equine platelets. Annexin V bound to platelets activated with PAF or A23187 when platelets had undergone secretion. Anti-human fibrinogen antibody bound to ADP-, PAF-, and A23817-activated platelets, but binding was not dependent on platelet secretion. The extent of binding of anti-fibrinogen antibody was less in equine platelets, compared with that for human platelets, despite maximal stimulation.


CONCLUSIONS
Activation of equine platelets can be detected by use of fluorescent-labeled annexin V and anti-human fibrinogen antibody but not by use of anti-human thrombospondin antibody. These flow cytometric techniques have the potential for detection of in vivo platelet activation in horses at risk of developing thrombotic disorders.
Kingston
Janene K
JK
Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman 99164-6610, USA.
Bayly
Warwick M
WM
Sellon
Debra C
DC
Meyers
Kenneth M
KM
Wardrop
K Jane
KJ
D000818:Animals; D017304:Annexin A5 / Q000097:blood* / Q000276:immunology; D000906:Antibodies / Q000097:blood*; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology*; D000001:Calcimycin / Q000494:pharmacology; D005340:Fibrinogen / Q000276:immunology / Q000378:metabolism*; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D006736:Horses / Q000097:blood*; D007476:Ionophores / Q000494:pharmacology; D010972:Platelet Activating Factor / Q000276:immunology; D015539:Platelet Activation / Q000502:physiology*; D019699:Thrombospondins / Q000276:immunology / Q000378:metabolism*
D017304:Annexin A5; D000906:Antibodies; D005456:Fluorescent Dyes; D007476:Ionophores; D010972:Platelet Activating Factor; D019699:Thrombospondins; D000001:Calcimycin; D005340:Fibrinogen