Abnormal platelet aggregation in idiopathic pulmonary arterial hypertension: role of nitric oxide

Abstract

Idiopathic pulmonary arterial hypertension (IPAH) is a rare and progressive disease. Several processes are believed to lead to the fatal progressive pulmonary arterial narrowing seen in IPAH including vasoconstriction, cellular proliferation inflammation, vascular remodeling, abnormalities in the lung matrix, and in situ thrombosis. Nitric oxide (NO) produced by NO synthases (NOS) is a potent vasodilator and plays important roles in many other processes including platelet function. Reduced NO levels in patients with IPAH are known to contribute to the development of pulmonary hypertension and its complications. Platelet defects have been implied in IPAH, but original research supporting this hypothesis has been limited. Normal platelets are known to have NOS activity, but little is known about NOS expression and NO production by platelets in patients with IPAH. Here we characterized the phenotype of the platelets in IPAH and show a defect in their ability to be activated in vitro by thrombin receptor activating protein but not adenosine diphosphate. We also show that endothelial NOS (eNOS) levels in these platelets are reduced and demonstrate that NO is an important regulator of platelet function. Thus reduced levels of eNOS in platelets could impact their ability to regulate their own function appropriately.

Fig. 1.

Platelet counts (platelets × 10⁹/ml) were determined in 154 patients with idiopathic pulmonary arterial hypertension (IPAH) and 105 healthy controls. Gray area shows the normal range of platelets counts in our clinical laboratory.

Fig. 2.

Cyclooxygenase-1 (COX-1) expression (A) is similar in both IPAH (3 representative samples from a total n = 13; lanes 1, 2, and 3) and control (3 representative samples from a total n = 12; lanes 3, 4, and 5) platelets, and COX-2 mRNA expression was not detected in either (B). The housekeeping gene GAPDH was used to normalize (C).

Fig. 3.

Western analysis of endothelial nitric oxide synthase (eNOS) protein from the platelets of the patients with IPAH (3 representative samples from a total n = 9; A, lanes 1, 2, and 3) and control (3 representative samples from a total n = 9; A, lanes 4, 5, and 6). β-actin was used to normalize (B). Quantitative analysis showed the significant difference of eNOS protein between IPAH and control (C). Inducible NOS (iNOS; D) and neuronal NOS (nNOS; E) proteins could not be detected in either IPAH or control platelets protein lysates. eNOS mRNA expression was not detected in platelets in either IPAH or control platelets (F). Three IPAH patients (lanes 1, 2, and 3) and 3 controls (lanes 4, 5, and 6) are shown. The PCR product size of eNOS mRNA is 468 bp, and human umbilical vein endothelial cell (HUVEC) lines were used as positive control (F). The housekeeping gene GAPDH was used to normalize (G).

Fig. 4.

Isolated platelets from the patients with IPAH and control were stained for eNOS protein (red) and platelet marker, CD42b, (green). eNOS and CD42b were detected both in IPAH (A and B, respectively) and control (E and F, respectively). Colocalization of CD42b and eNOS is shown in G for control and in C for IPAH platelets. D and H: negative controls of the same sections stained by the secondary antibody only.

Fig. 5.

Platelets were incubated with various concentrations of N^G-nitro-l- arginine methyl ester (l-NAME; A) or aminoguanadine (AG; B) at the end of a 1 h incubation they were tested for aggregation using 5 μM receptor-activating protein (TRAP) activation as described in the text. Platelet rich plasma was isolated from IPAH patients and from controls. Platelet were then counted and diluted to give the same concentration between patient and control. Dose-response curve using 0–10 mM ADP and 0–30 μM TRAP was carried out. Typical responses to TRAP (C) or ADP (D) are demonstrated on 3 patients and same day controls are shown (n = 3 IPAH; n = 3 controls).

Fig. 6.

Expression levels of CD63 protein in platelet. CD63 expression was measured by Western blot in 3 healthy individuals and 3 IPAH patients (A). Expression levels of PAR1 protein in platelet. PAR1 expression was measured by Western blot in 3 healthy individuals and three IPAH patients (B; representative sample from a total n = 9 IPAH; n = 9 controls).

Fig. 7.

Activation of platelets by thrombin leads to binding and activation of the protease-activated receptor-1 (PAR1) receptor on platelets. This causes platelet degranulation and release of factors such as ADP which potentiates the signal causing a strong aggregation response. Normally NO causes activation of soluble guanylyl cyclase (sGC), which prevents premature activation of platelets. However, in pulmonary hypertension (PH) the level of NO is reduced thus reducing the threshold of platelet activation.