Protein disulfide isomerase-A1 regulates intraplatelet reactive oxygen species-thromboxane A2 -dependent pathway in human platelets

Abstract

Background: Platelet-derived protein disulfide isomerase 1 (PDIA1) regulates thrombus formation, but its role in the regulation of platelet function is not fully understood.

Aims: The aim of this study was to characterize the role of PDIA1 in human platelets.

Methods: Proteomic analysis of PDI isoforms in platelets was performed using liquid chromatography tandem mass spectometry, and the expression of PDIs on platelets in response to collagen, TRAP-14, or ADP was measured with flow cytometry. The effects of bepristat, a selective PDIA1 inhibitor, on platelet aggregation, expression of platelet surface activation markers, thromboxane A₂ (TxA₂ ), and reactive oxygen species (ROS) generation were evaluated by optical aggregometry, flow cytometry, ELISA, and dihydrodichlorofluorescein diacetate-based fluorescent assay, respectively.

Results: PDIA1 was less abundant compared with PDIA3 in resting platelets and platelets stimulated with TRAP-14, collagen, or ADP. Collagen, but not ADP, induced a significant increase in PDIA1 expression. Bepristat potently inhibited the aggregation of washed platelets induced by collagen or convulxin, but only weakly inhibited platelet aggregation induced by TRAP-14 or thrombin, and had the negligible effect on platelet aggregation induced by arachidonic acid. Inhibition of PDIA1 by bepristat resulted in the reduction of TxA₂ and ROS production in collagen- or thrombin-stimulated platelets. Furthermore, bepristat reduced the activation of αIIbβ3 integrin and expression of P-selectin.

Conclusions: PDIA1 acts as an intraplatelet regulator of the ROS-TxA₂ pathway in collagen-GP VI receptor-mediated platelet activation that is a mechanistically distinct pathway from extracellular regulation of αIIbβ3 integrin by PDIA3.

Keywords: PDIs expression; bepristat; flow cytometry; platelet activation; proteomics.

FIGURE 1

Levels of PDI enzymes in platelet lysates. Lysates of resting platelets were analyzed with LC‐MS/MS, and PDI isoforms were semiquantified using emPAI % calculation algorithm. Data represent individual values with medians and interquartile ranges, n = 4–6

FIGURE 2

The expression of PDI enzymes on activated platelets compared with the expressions of platelet surface activation markers. Whole blood samples were stimulated with platelet agonists including (A i, A ii) collagen, (B i, B ii) TRAP‐14, or (C i, C ii) ADP. Expressions of PDIA1, PDIA3, PDIA6, P‐selectin, and the activated αIIbβ3 receptor were measured by flow cytometry. Data show medians and interquartile ranges, n = 10. The highest concentrations of agonists, &&&, ***, ###, $$$, or ^^^ symbols are relevant for PDIA1, PDIA3, PDIA6, P‐selectin, and the activated αIIbβ3 receptor, respectively; p < .001 vs. corresponding resting platelets; one‐way ANOVA with Dunnett's post hoc test

FIGURE 3

Effects of rutin and bepristat on platelet aggregation or agglutination. (A‐E) Washed human platelets (2 × 10⁸/mL) were preincubated with rutin for 2 min and then stimulated with collagen (A), or preincubated with bepristat (Bep) and then stimulated with (B) collagen (col), (C) convulxin (cvx), (D) TRAP‐14, or (E) thrombin (thr) to induce aggregation. (F) Platelets in PRP (2 × 10⁸/mL) were preincubated with bepristat (Bep) for 2 min and then stimulated with ristocetin (rist) or collagen (col) to induce agglutination and aggregation, respectively. Data are medians and interquartile ranges, n = 4–20. # or $ p < .05; ## or ** p < .01; ***, ### or $$$ p < .001 vs. corresponding vehicle control; one‐way ANOVA with Tukey's post hoc test or Kruskal‐Wallis test with Dunn's post‐hoc test

FIGURE 4

The effect of bepristat on expression of platelet surface activation markers. Gel‐filtered platelets at 2 × 10⁸/mL were preincubated with bepristat (Bep) for 10 min, subsequently stimulated with collagen for 5 min, and then expression of activated αIIbβ3 receptor and P‐selectin, as well as the binding of von Willebrand factor were measured. Data are medians and interquartile ranges, n = 6. *p < .05, **p < .01 vs. corresponding vehicle control; one‐way ANOVA with Tukey's post‐hoc test

FIGURE 5

(A) The effect of bepristat on thromboxane A₂ production in platelets compared with reference antiplatelet drugs. Washed platelets at 2 × 10⁸/mL were preincubated with bepristat (Bep), aspirin (ASA), or eptifibatide (Ept) for 2 min and then stimulated for 6 min with collagen (col) or thrombin (thr). TxB₂ (a stable metabolite of TxA₂) was measured in the supernatant with an ELISA kit. Data are individual values with medians and interquartile ranges, n = 4–6. (B) The effect of bepristat on platelet aggregation compared with reference antiplatelet drugs. Washed human platelets (2 × 10⁸/mL) were preincubated with bepristat (Bep), aspirin (ASA), or eptifibatide (Ept) for 2 min and then stimulated with collagen (col) or thrombin (thr) to induce aggregation. Data are individual values with medians and interquartile ranges, n = 5–6. (C) The effect of bepristat on platelet aggregation induced by arachidonic acid compared with collagen. Human platelets in PRP (2 × 10⁸/mL) were preincubated with bepristat (Bep) or aspirin (ASA) for 2 min and then stimulated with arachidonic acid (AA) or collagen (col) to induce aggregation. Data are individual values with medians and interquartile ranges, n = 6–7. *p < .05, **p < .01, ***p < .001 vs. corresponding vehicle control; for A, B, and C Student's t‐test or Mann‐Whitney test

FIGURE 6

(A–B) Effects of bepristat on ROS production in platelets compared with aspirin, apocynin and ML171. Washed human platelets (2 × 10⁸/mL) loaded with H2DCF‐DA were preincubated with (A) bepristat (Bep) or aspirin (ASA), and (B) apocynin (Apo) or ML171 for 5 min and then stimulated with collagen (20 μg/mL) to induce ROS generation. Data are individual values with medians and interquartile ranges, n = 3–4. *p < .05, ***p < .001 vs. corresponding vehicle control; #p < .05 vs. Bep 30; for A, one‐way ANOVA followed by Tukey's post hoc test; for B, Kruskal‐Wallis test with Dunn's post hoc test or one‐way ANOVA followed by Dunnett's post‐hoc test for apocynin and ML171, respectively. (C–D) Effects of apocynin and ML171 on platelet aggregation. Washed human platelets (2 × 10⁸/mL) were preincubated with (C) apocynin (Apo) or (D) ML171 for 2 min and then stimulated with collagen (4 μg/mL) to induce aggregation. Data are medians and interquartile ranges, n = 6–9. ***p < .001 vs. corresponding vehicle control; for C and D, one‐way ANOVA followed by Tukey's post‐hoc test