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Published on: October 27, 2009
A platelet activation-specific monoclonal antibody that recognizes a receptor-induced binding site on canine
M K Boudreaux1, V S Panangala, C Bourne
1Department of Pathobiology, College of Veterinary Medicine, Auburn University, AL.
Insights
A new antibody, Canine Activated Platelet 1 (CAP1), specifically binds to activated canine platelets. This antibody targets a receptor-induced binding site on canine fibrinogen, offering insights into platelet activation mechanisms.
Area of Science:
- Immunology
- Hematology
- Veterinary Medicine
Background:
- Platelet activation is crucial for hemostasis and thrombosis.
- Specific markers for activated canine platelets are needed for research and diagnostics.
Purpose of the Study:
- To develop and characterize a novel monoclonal antibody (MoAb) that recognizes activated canine platelets.
- To investigate the binding characteristics and target antigen of the MoAb CAP1.
Main Methods:
- Flow cytometry using adenosine diphosphate (ADP) and platelet activating factor (PAF) agonists.
- Ethylenediamine-tetraacetic acid (EDTA) and calcium chloride (CaCl2) inhibition assays.
- Immunoblotting and binding assays with isolated canine fibrinogen.
Main Results:
- MoAb CAP1 binding to canine platelets is activation-dependent and proportional to agonist strength.
- Calcium ions appear to play a role in antigen stabilization or binding.
- MoAb CAP1 binds to canine fibrinogen, suggesting it recognizes a receptor-induced binding site on fibrinogen.
Conclusions:
- MoAb CAP1 is a valuable tool for studying canine platelet activation.
- The findings suggest that MoAb CAP1 targets a conformational epitope on fibrinogen exposed during platelet activation.
Abstract:
An activation-specific monoclonal antibody (MoAb) termed "Canine Activated Platelet 1" (CAP1) has been developed and partially characterized. Flow cytometric studies of isolated canine platelets, using adenosine diphosphate (ADP) and platelet activating factor (PAF) as agonists, demonstrated that CAPI binding site number was proportional to agonist strength and agonist concentration. MoAb CAP1 binding was diminished by ethylenediamine-tetraacetic acid, suggesting that the antigen was either stabilized by calcium or antigen binding to the platelet surface was mediated by calcium. ADP-activated gel-filtered platelets also demonstrated reduced binding of MoAB CAP1 even in the presence of 1 mM CaCl2. Binding of MoAb CAP1 could be partially restored by activating gel-filtered platelets with PAF, suggesting that the antigen was either present within platelet granule membranes or was exposed after binding of released proteins(s) with a platelet receptor. A monoclonal antibody to human platelet glycoprotein IIIa (GPIIIa), which cross-reacts with canine platelet GPIIIa regardless of platelet activation status, did not inhibit binding of MoAb CAP1. MoAb CAP1 bound to isolated canine fibrinogen captured on polystyrene microtiter plates in the absence of platelet proteins. Immunoblots indicated that MoAb CAP1 recognizes nonreduced fibrinogen as well as a plasmin digest of isolated canine fibrinogen. Results of the present studies suggest that MoAb CAP1 recognizes a receptor-induced binding site on canine fibrinogen.
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