Protease-activated receptor 4 activity promotes platelet granule release and platelet-leukocyte interactions

Abstract

Human platelets express two protease-activated receptors (PARs), PAR1 (F2R) and PAR4 (F2RL3), which are activated by a number of serine proteases that are generated during pathological events and cause platelet activation. Recent interest has focused on PAR4 as a therapeutic target, given PAR4 seems to promote experimental thrombosis and procoagulant microparticle formation, without a broadly apparent role in hemostasis. However, it is not yet known whether PAR4 activity plays a role in platelet-leukocyte interactions, which are thought to contribute to both thrombosis and acute or chronic thrombo-inflammatory processes. We sought to determine whether PAR4 activity contributes to granule secretion from activated platelets and platelet-leukocyte interactions. We performed in vitro and ex vivo studies of platelet granule release and platelet-leukocyte interactions in the presence of PAR4 agonists including PAR4 activating peptide, thrombin, cathepsin G, and plasmin in combination with small-molecule PAR4 antagonists. Activation of human platelets with thrombin, cathepsin G, or plasmin potentiated platelet dense granule secretion that was specifically impaired by PAR4 inhibitors. Platelet-leukocyte interactions and platelet P-selectin exposure the following stimulation with PAR4 agonists were also impaired by activated PAR4 inhibition in either a purified system or in whole blood. These results indicate PAR4-specific promotion of platelet granule release and platelet-leukocyte aggregate formation and suggest that pharmacological control of PAR4 activity could potentially attenuate platelet granule release or platelet-leukocyte interaction-mediated pathological processes.

Keywords: PAR4; granulocytes; platelet activation; platelet dense granule release; platelet-leukocyte interactions; protease-activated receptor 4.

Figure 1.

PAR4 inhibitors impair platelet dense granule release. Washed human platelets (2 × 108/ml) were pretreated with the PAR1 inhibitor SCH 79797 (3 μM), PAR4 inhibitors VU0652925 (10 μM) and VU0661245 (10 μM), Src kinase inhibitor PP2 (10 μM, only shown in CRP condition), or vehicle (0.2% DMSO), then stimulated with the agonists (A) PAR4 AP (activating peptide, 200 μM), (B) TRAP-6 (20 μM), (C) human α-thrombin (5 nM = 0.7 U/ml), (D) human cathepsin G (340 nM = 0.1 U/ml), (E) human plasmin (260 nM = 0.3 U/ml), or (F) CRP (10 μg/ml) and assessed for dense granule release by luminescent ATP assay. Basal = no agonist; n ≥ 3–6 independent experiments; * designates p < 0.05 versus vehicle.

Figure 2.

PAR4 inhibitors diminish platelet-granulocyte interactions and platelet alpha granule release. Washed human platelets (2 × 108/ml) and purified granulocytes (2 × 106/ml) from the same donor were combined and pretreated with the PAR1 inhibitor SCH 79797 (3 μM), PAR4 inhibitors VU0652925 (10 μM) and VU0661245 (10 μM), or vehicle (0.2% DMSO), then stimulated with the agonists PAR4 AP (activating peptide, 200 μM), TRAP-6 (20 μM), human α-thrombin (5 nM = 0.7 U/ml), human cathepsin G (340 nM = 0.1 U/ml), human plasmin (260 nM = 0.3 U/ml), or CRP (10 μg/ml) and stained with markers for granulocytes (CD66b), platelets (CD41), and activated platelets (CD62P). (A) All samples were gated for platelets by CD41+ and granulocytes by CD66b+. (B) Granulocytes (CD66b+) were plotted against CD41, and a line was drawn to designate the CD41+ region. Platelets (CD41+) are shown for comparison. Bar graphs designate (C) percent of CD41+ granulocytes and (D) percent of activated (CD62P+) platelets for each treatment. Basal = no agonist; n ≥ 3 independent experiments; * designates p < 0.05 versus vehicle.

Figure 3.

PAR4 inhibitors diminish platelet-leukocyte interactions and platelet alpha granule release in whole human blood. Whole human blood was pretreated with the PAR1 inhibitor SCH 79797 (3 μM), PAR4 inhibitors VU0652925 (10 μM) and VU0661245 (10 μM), or vehicle (0.2% DMSO), then stimulated with the agonists PAR4 AP (activating peptide, 200 μM), TRAP-6 (20 μM), or CRP (10 μg/ml) and stained with markers for granulocytes (CD66b), platelets (CD41), activated platelets (CD62P), and monocytes (CD14). (A) All samples were gated for platelets by CD41+, granulocytes by CD66b+, and monocytes by CD14+. (B) Granulocytes (CD66b+) or monocytes (CD14+, not shown) were plotted against CD41, and a line was drawn to designate the CD41+ region. Platelets (CD41+) are shown for comparison. Bar graphs designate (C) percent of CD41+ granulocytes, (D) percent of CD41+ monocytes, and (E) percent of activated (CD62P+) platelets for each treatment. Basal = no agonist; n ≥ 3 independent experiments; * designates p < 0.05 versus vehicle.

Figure 4.

PAR4 inhibitors diminish platelet-leukocyte interactions and platelet alpha granule release in whole baboon (Papio anubis) blood. Whole baboon blood was pretreated with the PAR1 inhibitor SCH 79797 (3 μM), PAR4 inhibitors VU0652925 (10 μM) and VU0661245 (10 μM), or vehicle (0.2% DMSO), then stimulated with the agonists PAR4 AP (activating peptide, 200 μM), TRAP-6 (1 mM), or CRP (10 μg/ml) and stained with markers for leukocytes (CD45), platelets (CD41), and activated platelets (CD62P). (A) All samples were gated for platelets by CD41+ and granulocytes by CD45+ and high side scatter (SSC, granularity). (B) Granulocytes (CD45+, high SSC) were plotted against CD41, and a line was drawn to designate the CD41+ region. Platelets (CD41+) are shown for comparison. Bar graphs designate (C) percent of CD41+ granulocytes and (D) percent of activated (CD62P+) platelets for each treatment. Basal = no agonist; n ≥ 3–4 independent experiments; * designates p < 0.05 versus vehicle.