Prostacyclin Analogues Inhibit Platelet Reactivity, Extracellular Vesicle Release and Thrombus Formation in Patients with Pulmonary Arterial Hypertension

Abstract

(1) Background: Prostacyclin analogues (epoprostenol, treprostinil, and iloprost) induce vasodilation in pulmonary arterial hypertension (PAH) but also inhibit platelet function. (2) Objectives: We assessed platelet function in PAH patients treated with prostacyclin analogues and not receiving prostacyclin analogues. (3) Methods: Venous blood was collected from 42 patients treated with prostacyclin analogues (49.5 ± 15.9 years, 81% female) and 38 patients not receiving prostacyclin analogues (55.5 ± 15.6 years, 74% female). Platelet reactivity was analyzed by impedance aggregometry using arachidonic acid (AA; 0.5 mM), adenosine diphosphate (ADP; 6.5 µM), and thrombin receptor-activating peptide (TRAP; 32 µM) as agonists. In a subset of patients, concentrations of extracellular vesicles (EVs) from all platelets (CD61⁺), activated platelets (CD61⁺/CD62P⁺), leukocytes (CD45⁺), and endothelial cells (CD146⁺) were analyzed by flow cytometry. Platelet-rich thrombus formation was measured using a whole blood perfusion system. (4) Results: Compared to controls, PAH patients treated with prostacyclin analogues had lower platelet reactivity in response to AA and ADP (p = 0.01 for both), lower concentrations of platelet and leukocyte EVs (p ≤ 0.04), delayed thrombus formation (p ≤ 0.003), and decreased thrombus size (p = 0.008). Epoprostenol did not affect platelet reactivity but decreased the concentrations of platelet and leukocyte EVs (p ≤ 0.04). Treprostinil decreased platelet reactivity in response to AA and ADP (p ≤ 0.02) but had no effect on the concentrations of EVs. All prostacyclin analogues delayed thrombus formation and decreased thrombus size (p ≤ 0.04). (5) Conclusions: PAH patients treated with prostacyclin analogues had impaired platelet reactivity, EV release, and thrombus formation, compared to patients not receiving prostacyclin analogues.

Keywords: extracellular vesicles; platelet reactivity; prostacyclin analogues; pulmonary arterial hypertension; thrombus formation.

Figure 1

Trial schedule (A) and patients’ flow diagram (B). PAH—pulmonary arterial hypertension, ERA—endothelin receptor antagonists, PGE-5i—phosphodiesterase-5 inhibitors.

Figure 2

(A) Platelet reactivity in blood in response to arachidonic acid (ASPI test), adenosine diphosphate (ADP test), and thrombin receptor-activating peptide-6 (TRAP test). Unstimulated platelets (no agonist) were used as a negative control. Panel A: Patients receiving prostacyclin analogues and not treated with prostacyclin analogues. Values were compared using the Mann–Whitney U test and shown as the median and interquartile range. (B) Comparison between no prostacyclin analogues, epoprostenol, and treprostinil). Values were compared using the Kruskal—Wallis test with Dunn’s correction for multiple comparisons and shown as the median and interquartile range. AUC—area under the curve.

Figure 3

Concentrations of extracellular vesicles (EVs) from platelets (CD61, CD62P), leukocytes (CD45), and endothelial cells (CD146) measured with flow cytometry in platelet-depleted plasma. (A) Patients receiving prostacyclin analogues and not treated with prostacyclin analogues. Values were compared using the Mann–Whitney U test and shown as the median and interquartile range. (B) Comparison between no prostacyclin analogues, epoprostenol, and treprostinil. Values were compared using the Kruskal–Wallis test with Dunn’s correction for multiple comparisons and shown as the median and interquartile range.

Figure 4

Platelet-rich thrombus formation measured in a whole blood perfusion system using the microchip containing capillaries coated with type 1 collagen under arterial shear rate (2000 s⁻¹). (A,B) Patients receiving prostacyclin analogues and not treated with prostacyclin analogues. Values were compared using the Mann–Whitney U test and shown as the median and interquartile range. (C,D) Comparison between no prostacyclin analogues, epoprostenol, and treprostinil. Values were compared using the Kruskal–Wallis test with Dunn’s correction for multiple comparisons and shown as the median and interquartile range. T10—onset of thrombus formation. Occlusion time—complete occlusion of the capillary. Area under curve (AUC)—parameter reflecting platelet-rich thrombus size.

Figure 5

Examples of pictures obtained during thrombus formation in the whole blood perfusion scheme. (A) (i) A control patient who achieved total occlusion of the microchip after 3 min 40 s, (B) (ii) a patient treated with epoprostenol who achieved clotting after 9 min 39 s, and (C) (iii) a patient treated with treprostinil who did not achieve clotting during the measurement time of 12 min. All pictures were made at 3 min. The full movies are attached in the online Supplementary Materials.