Inhibition of protease-activated receptor 4 impairs platelet procoagulant activity during thrombus formation in human blood.

Abstract

UNLABELLED:

Essentials The platelet thrombin receptor, PAR4, is an emerging anti-thrombotic drug target. We examined the anti-platelet & anti-thrombotic effects of PAR4 inhibition in human blood. PAR4 inhibition impaired platelet procoagulant activity in isolated cells and during thrombosis. Our study shows PAR4 is required for platelet procoagulant function & thrombosis in human blood.

SUMMARY:

Background Thrombin-induced platelet activation is important for arterial thrombosis. Thrombin activates human platelets predominantly via protease-activated receptor (PAR)1 and PAR4. PAR1 has higher affinity for thrombin, and the first PAR1 antagonist, vorapaxar, was recently approved for use as an antiplatelet agent. However, vorapaxar is contraindicated in a significant number of patients, owing to adverse bleeding events. Consequently, there is renewed interest in the role of platelet PAR4 in the setting of thrombus formation. Objectives To determine the specific antiplatelet effects of inhibiting PAR4 function during thrombus formation in human whole blood. Methods and Results We developed a rabbit polyclonal antibody against the thrombin cleavage site of PAR4, and showed it to be a highly specific inhibitor of PAR4-mediated platelet function. This function-blocking anti-PAR4 antibody was used to probe for PAR4-dependent platelet functions in human isolated platelets in the absence and presence of concomitant PAR1 inhibition. The anti-PAR4 antibody alone was sufficient to abolish the sustained elevation of cytosolic calcium level and consequent phosphatidylserine exposure induced by thrombin, but did not significantly inhibit integrin αII b β3 activation, α-granule secretion, or aggregation. In accord with these in vitro experiments on isolated platelets, selective inhibition of PAR4, but not of PAR1, impaired thrombin activity (fluorescence resonance energy transfer-based thrombin sensor) and fibrin formation (anti-fibrin antibody) in an ex vivo whole blood flow thrombosis assay. Conclusions These findings demonstrate that PAR4 is required for platelet procoagulant function during thrombus formation in human blood, and suggest PAR4 inhibition as a potential target for the prevention of arterial thrombosis.

• Authors: S L French, J F Arthur, H Lee, W S Nesbitt, R K Andrews, E E Gardiner, J R Hamilton
• Journal: Journal of thrombosis and haemostasis : JTH (J Thromb Haemost) Vol. 14 Issue 8 Pg. 1642-54 (08 2016) ISSN: 1538-7836 [Electronic] England
• PMID: 26878340 (Publication Type: Journal Article)
• Copyright: © 2016 International Society on Thrombosis and Haemostasis.

Chemical References

• Antibodies
• Lactones
• P-Selectin
• Phosphatidylserines
• Platelet Aggregation Inhibitors
• Pyridines
• Receptor, PAR-1
• Receptors, Thrombin
• Fibrin
• Thrombin
• protease-activated receptor 4
• Calcium
• vorapaxar

Topics

• Adult
• Animals
• Antibodies (chemistry)
• Blood Platelets (cytology)
• Calcium (metabolism)
• Cytosol (metabolism)
• Female
• Fibrin (chemistry)
• Fluorescence Resonance Energy Transfer
• Healthy Volunteers
• Humans
• Lactones (therapeutic use)
• Male
• Mice
• Mice, Transgenic
• Middle Aged
• P-Selectin (metabolism)
• Phosphatidylserines (chemistry)
• Platelet Activation (drug effects)
• Platelet Aggregation
• Platelet Aggregation Inhibitors (chemistry, therapeutic use)
• Pyridines (therapeutic use)
• Receptor, PAR-1 (metabolism)
• Receptors, Thrombin (antagonists & inhibitors)
• Signal Transduction
• Thrombin (chemistry)
• Thrombosis (metabolism)
• Young Adult