J Vet Sci.  2019 Jan;20(1):10-15. 10.4142/jvs.2019.20.1.10.

Characterization of the distinct mechanism of agonist-induced canine platelet activation

• Chaudhary
• Kim S

• KMID: 2434772
• DOI: http://doi.org/10.4142/jvs.2019.20.1.10

Abstract

Platelet activation has a major role in hemostasis and thrombosis. Various agonists including adenosine diphosphate (ADP) and thrombin interact with G protein-coupled receptors (GPCRs) which transduce signals through various G proteins. Recent studies have elucidated the role of GPCRs and their corresponding G proteins in the regulation of events involved in platelet activation. However, agonist-induced platelet activation in companion animals has not been elucidated. This study was designed to characterize the platelet response to various agonists in dog platelets. We found that 2-methylthio-ADP-induced dog platelet aggregation was blocked in the presence of either P2Y₁ receptor antagonist MRS2179 or P2Y₁₂ receptor antagonist AR-C69931MX, suggesting that co-activation of both the P2Y₁ and P2Y₁₂ receptors is required for ADP-induced platelet aggregation. Thrombin-induced dog platelet aggregation was inhibited in the presence of either AR-C69931MX or the PKC inhibitor GF109203X, suggesting that thrombin requires secreted ADP to induce platelet aggregation in dog platelets. In addition, thrombin-mediated Akt phosphorylation was inhibited in the presence of GF109203X or AR-C69931MX, indicating that thrombin causes Gi stimulation through the P2Y₁₂ receptor by secreted ADP in dog platelets. Unlike human and murine platelets, protease-activated receptor 4 (PAR4)-activating peptide AYPGKF failed to cause dog platelet aggregation. Moreover, PAR1-activating peptide SFLLRN or co-stimulation of SFLLRN and AYPGKF failed to induce dog platelet aggregation. We conclude that ADP induces platelet aggregation through the P2Y₁ and P2Y₁₂ receptors in dogs. Unlike human and murine platelets, selective activation of the PAR4 receptor may be insufficient to cause platelet aggregation in dog platelets.

Keyword

Platelets; Dogs; Thrombin; Protease-activated receptors; Adenosine diphosphate

MeSH Terms

Adenosine Diphosphate
Animals
Blood Platelets*
Dogs
GTP-Binding Proteins
Hemostasis
Humans
Pets
Phosphorylation
Platelet Activation*
Platelet Aggregation
Receptors, Proteinase-Activated
Thrombin
Thrombosis
Adenosine Diphosphate
GTP-Binding Proteins
Receptors, Proteinase-Activated
Thrombin

Figure

• 

  Fig. 1 2-Methylthio-adenosine diphosphate (2-MeSADP)-induced platelet aggregation in canine platelets. (A) Washed platelets from dog were stimulated with different concentrations of 2-MeSADP for 3.5 min under stirring conditions. (B) 2-MeSADP-induced platelet aggregation was measured in washed canine platelets in the presence of 100 nM AR-C69931MX or 100 µM MRS2179. Platelet aggregation was measured by using a Lumi-aggregometer (Chrono-Log). The arrow indicates when agonist is added. Tracings are representative of three independent experiments.

• 

  Fig. 2 The effect of secreted adenosine diphosphate on thrombin-induced platelet aggregation in canine platelets. Thrombin-induced platelet aggregation was measured in canine platelets in the presence of 10 µM GF109203X or 100 nM AR-C69931MX. The arrow indicates when agonist is added. Tracings are representative of three independent experiments.

• 

  Fig. 3 Role of secreted adenosine diphosphate and Gi-coupled P2Y12 receptor signaling in Akt phosphorylation in response to thrombin in canine platelets. Canine platelets preincubated with 100 nM AR-C69931MX or 10 µM GF109203X were stimulated at 37℃ for 3 min with thrombin (1 unit/mL). Samples were separated by SDS-PAGE, transferred onto polyvinylidene difluoride membranes, and phosphorylation of Akt was measured by western blotting using an anti-phospho-Akt (Ser473) antibody. β-Actin was used as a lane-loading control. Results are representative of three independent experiments.

• 

  Fig. 4 AYPGKF- and SFLLRN-induced platelet aggregation in canine platelets. Washed platelets from dog were stimulated with different concentrations of the protease-activated receptor 4 (PAR4)-activating peptide AYPGKF (A), the PAR1-activating peptide SFLLRN (B), or combination of AYPGKF and SFLLRN (C) for 3.5 min under stirring conditions. The arrow indicates when agonist is added. Data are representative of three independent experiments.

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