---
title: "Endothelial dysfunction and platelet activation in systemic sclerosis–related pulmonary hypertensi"
id: "plos-one-22-endothelial-and-platelet-dysfunction-in-systemic-sclerosis-with-pulmonary"
canonical_url: "https://medichelpline.com/clinical-feed/plos-one-22-endothelial-and-platelet-dysfunction-in-systemic-sclerosis-with-pulmonary"
content_type: "clinical_feed_article"
specialty: "Rheumatology"
source_name: "PLOS ONE (Medicine)"
source_url: "https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0357987"
published_at: "2026-09-16T14:00:00.000Z"
evidence_level: "Journal Feed"
license: "CC-BY-NC-4.0 / Informational Use"
---
# Endothelial dysfunction and platelet activation in systemic sclerosis–related pulmonary hypertensi
## Provenance & Clinical Metadata
- **Canonical URL:** https://medichelpline.com/clinical-feed/plos-one-22-endothelial-and-platelet-dysfunction-in-systemic-sclerosis-with-pulmonary
- **Specialty:** [Rheumatology](https://medichelpline.com/clinical-feed/rheumatology.md)
- **Primary Source:** PLOS ONE (Medicine)
- **Source URL:** [Original Journal Publication](https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0357987)
- **Published At:** 2026-09-16T14:00:00.000Z
- **Evidence Rating:** Journal Feed
## Executive GIST (TL;DR)
- This PLOS One study evaluated endothelial function and platelet activity in clinically stable systemic sclerosis (**SSc**) patients stratified by echocardiographic pulmonary hypertension (**PH**) probability and compared them with age-matched healthy controls. - Endothelial function was assessed by brachial artery **flow-mediated dilation (FMD)** and blood nitrite measured by chemiluminescence; platelet activity was measured by light transmission aggregometry in response to ADP and **TRAP-6**. - PH probability was determined by echocardiography using TRVmax and additional echocardiographic signs per 2022 ESC/ERS guidelines; PH was confirmed by right heart catheterization (mPAP > 20 mmHg). - FMD was reduced in SSc patients compared with controls and decreased progressively across groups: controls 13.3 ± 3.9%, low PH probability 5.6 ± 1.7%, intermediate PH probability 4.6 ± 0.4%, and confirmed PH 3.8 ± 0.4%. - Blood nitrite levels were higher in SSc patients with confirmed PH than in controls or patients with low PH probability. - TRVmax correlated inversely with FMD and positively with platelet aggregation; in multivariable regression, **TRAP-6–induced platelet aggregation** and FMD were independently associated with TRVmax. - The authors reported two cases in which nebulized sodium nitrite produced a transient reduction in TRVmax. - Exclusion criteria included major comorbidities and use of NO donors or platelet-affecting drugs; NSAIDs and other platelet agents were stopped two weeks before testing. - The study ran from February 16, 2023, to January 31, 2026; protocol registered in the Thai Clinical Trials Registry (TCTR20150518002). Data are not publicly available due to privacy but may be requested from the ethics committee.
## Clinical Analysis & Structured Key Points
Endothelial and platelet dysfunction in systemic sclerosis with pulmonary hypertension | PLOS One Browse Subject Areas ? Click through the PLOS taxonomy to find articles in your field. For more information about PLOS Subject Areas, click here . Article Authors Metrics Comments Media Coverage Reader Comments Figures Figures Abstract Systemic sclerosis (SSc) is an autoimmune connective tissue disease characterized by vasculopathy, inflammation, and fibrosis, in which pulmonary hypertension (PH) is a major cause of mortality. In SSc, the relationship between endothelial and platelet functions with echocardiographic findings have not been evaluated. Here, we evaluated the endothelial and platelet functions in SSc patients with different PH probability and tested their associations with echocardiographic parameters, including the maximal tricuspid regurgitant velocity (TRV max ). Endothelial function was assessed by flow-mediated dilation (FMD) of brachial artery and blood nitrite levels, measured by chemiluminescence method. Platelet activity was measured by light transmission aggregometry in response to stimulation by adenosine diphosphate (ADP) and thrombin receptor–activating peptide-6 (TRAP-6). PH probability was assessed by echocardiography, and PH diagnosis was confirmed by right heart catheterization. SSc patients with PH probability had lower FMD than age-matched controls: FMD decreased from 13.3 ± 3.9% in controls to 5.6 ± 1.7%, 4.6 ± 0.4%, and 3.8 ± 0.4% in SSc patients with low PH probability, intermediate PH probability, and confirmed PH, respectively. SSc patients with PH had higher blood nitrite levels than controls or the patients with low PH probability. TRV max correlated inversely with FMD and positively with platelet aggregation. In multivariable regression analysis, TRAP-6-induced platelet aggregation and FMD were independently associated with TRV max . Furthermore, we demonstrated in two patients that nebulized sodium nitrite transiently reduced TRV max . In conclusion, SSc patients have endothelial dysfunction and increased platelet activity, which exhibits correlation with TRV max . Citation: Imerbtham T, Sriwantana T, Ngamjanyaporn P, Sotananusak T, Ngammisri P, Trerayapiwat K, et al. (2026) Endothelial and platelet dysfunction in systemic sclerosis with pulmonary hypertension. PLoS One 21(9): e0357987. https://doi.org/10.1371/journal.pone.0357987 Editor: Barbora Piknova, National Institutes of Health, National institute of Diabetes and Digestive and Kidney Diseases, UNITED STATES OF AMERICA Received: May 29, 2026; Accepted: August 24, 2026; Published: September 16, 2026 Copyright: © 2026 Imerbtham et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The datasets generated and analyzed during the current study cannot be publicly shared due to patient privacy and regulations. Anonymized data may be available upon reasonable request to researchers who meet the criteria for access to confidential data. Data requests should be directed to the Ramathibodi Hospital Ethics Committee at Mahidol University (Ethics Committee contact: race.mahidol@gmail.com ). Funding: The author(s) received no specific funding for this work. Competing interests: The authors have declared that no competing interests exist. Introduction SSc is a chronic autoimmune connective tissue disease characterized by immune dysregulation, vasculopathy secondary to endothelial dysfunction, and progressive fibrosis of the skin and visceral organs, including the heart and lungs [ 1 ]. Pulmonary and cardiac complications are the leading causes of morbidity and mortality in SSc. PH is a serious complication that worsens the prognosis of SSc patients. SSc-related PH (SSc-PH) has poorer prognosis and treatment outcome than idiopathic PH [ 2 ]. Understanding the pathogenesis and development of an assessment tool to monitor SSc-PH progression are urgently required to change the course of the disease. Vasculopathy and endothelial dysfunction are key characteristics of SSc and play a vital role in PH development. The endothelium regulates vascular tone, inflammation, thrombosis, and smooth muscle proliferation by synthesizing endogenous vasodilators and platelet inhibitors, including nitric oxide (NO). In SSc, the endothelial dysfunction can lead to abnormal vasoconstriction, inflammation, and vascular remodeling. Even in the absence of PH, the SSc patients had lower FMD than healthy subjects [ 3 , 4 ]. SSc patients have elevated serum or plasma NO, measured as total nitrate and nitrite (NO x ) [ 5 – 7 ]. Yet, the specific levels of nitrite, a bioactive product of NO, or its association with SSc-PH has not been reported. As endothelial dysfunction is a key characteristic contributing to pathogenesis of PH, in this study we investigated the endothelial function in SSc patients, measured as FMD, blood nitrite, and platelet activity, and evaluated their association with echocardiographic findings. Methods Study setting and participants This study obtained the ethical approval from the Ramathibodi Hospital Ethics Committee at Mahidol University (ID 2024/646) and all participants provided written informed consent, in accordance with the principles of the Declaration of Helsinki. The recruitment period was from February 16, 2023, to January 31, 2026. To ensure transparency, the study protocol was recorded in the Thai Clinical Trials Registry (TCTR20150518002). The study involved SSc patients who were clinically stable, along with age-matched healthy controls ( Fig 1 ). According to the 2022 ESC/ERS guidelines [ 8 ], SSc patients were prospectively categorized into three groups according to their PH probability. All SSc patients underwent echocardiographic for screening of PH. By echocardiography, PH was suggestive when TRV max > 3.4 m/s or a TRV max of 2.9 to 3.4 m/s accompanied by at least two additional echocardiographic signs of PH which included right ventricular enlargement (right ventricular/left ventricular basal diameter ratio >1.0), interventricular septal flattening (D-shaped left ventricle), main pulmonary artery diameter >25 mm, right ventricular outflow tract acceleration time 2.2 m/s (when assessable), right atrial area >18 cm², and inferior vena cava diameter >21 mm with reduced inspiratory collapse. These additional parameters were evaluated during standard 2D transthoracic echocardiography. If the patients had the echocardiographic criteria for PH, PH was ultimately confirmed by right heart catheterization (RHC) demonstrating a mean pulmonary arterial pressure (mPAP) over 20 mmHg at rest. Intermediate probability for PH was defined when TRV max of 2.9 to 3.4 m/s without additional echocardiographic signs, or a TRV max ≤ 2.8 m/s accompanied by at least two echocardiographic signs of PH. Low probability for PH was assigned to patient with a TRV max ≤ 2.8 m/s and no additional echocardiographic features suggestive of PH. Download: PNG larger image TIFF original image Fig 1. Study workflow. Clinically stable systemic sclerosis (SSc) patients and healthy controls were enrolled. SSc patients were categorized into low pulmonary hypertension (PH) probability, intermediate PH probability, and confirmed PH. https://doi.org/10.1371/journal.pone.0357987.g001 The exclusion criteria included patients with major comorbidities, including heart failure, ischemic heart disease, chronic obstructive pulmonary disease, chronic kidney disease, active infection, malignancy, and NO donor use. Nonsteroidal anti-inflammatory drugs or agents affecting platelet function were discontinued for at least two weeks before the study. Baseline characteristics and laboratory parameters Demographic and clinical data, including age, sex, body weight, height, body mass index (BMI), disease duration, comorbidities, and current medications, were obtained through structured interviews and review of electronic medical records. The clinical laboratory investigations; for example, complete blood count, creatinine, bilirubin, and lipids were performed at the Pathology Laboratory, Faculty of Medicine Ramathibodi Hospital. All the patients had undergone spirometry and the six-minute walk distance (6MWD) test as the guidelines [ 9 , 10 ]. High-resolution computed tomography (HRCT) of chest was used to assess interstitial lung disease. FMD of the right brachial artery FMD was assessed using a portable ultrasound system (UProbe-C5D, Universal Diagnostic Solutions, Vista, CA, USA) [ 11 ]. Participants fasted overnight (10–12 h) and rested supine for approximately 20 minutes in a quiet, temperature-controlled room before image acquisition. The brachial artery was imaged longitudinally above the antecubital fossa, and baseline brachial artery diameter was recorded continuously for 30 seconds before cuff inflation. A blood pressure cuff was positioned on the distal forearm immediately below the medial epicondyle to minimize direct mechanical effects on the imaged brachial artery. The cuff was inflated to 50 mmHg above systolic blood pressure for 5 minutes and then rapidly deflated to induce reactive hyperemia. Continuous brachial artery imaging was recorded for up to 180 seconds after cuff release, and the highest 5-second average diameter was used as the peak diameter. FMD was calculated as the percentage change from baseline to peak brachial artery diameter. To assess intra-observer reproducibility, FMD measurements from 10 randomly selected participants were reanalyzed by the same observer in a blinded manner. Agreement between repeated measurements was evaluated using the intraclass correlation coefficient (ICC) based on a two-way mixed-effects model with absolute agreement. Blood nitrite and nitrate We collected venous blood via catheter using heparin (143 units/10 mL) as an anticoagulant. The whole blood samples were rapidly mixed with nitrite-stabilizing solution (composed of 0.8 M ferricyanide, 10 mM N-ethylmaleimide, and 1% NP-40 in a 4:1 v/v ratio of sample to stabilizing solution) [ 12 ], and stored at −80 °C. Nitrite (NO 2 - ) was measured using a tri-iodide-based chemiluminescence NO analyzer (CLD88; Eco Medics AG, Duernten, Switzerland). Plasma nitrate (NO 3 - ) was measured by vanadium (III)-based chemiluminescence [ 13 ]. Fractional exhaled NO (FE NO ) Exhaled breath samples were obtained at baseline from all participants utilizing an offline FE NO collection apparatus linked to a reusable Mylar container (ECO MEDICS AG, Duernten, Switzerland). The Mylar container is impermeable and nonreactive to NO, thereby preserving sample integrity. NO concentrations were determined by interfacing the container with the inlet of a chemiluminescence NO analyzer, and findings were reported in parts per billion. Platelet aggregometry and flow cytometry Venous blood was collected from the median cubital vein of fasting participants (10–12 fasting hours) using a 21-gauge winged needle into tubes containing 3.8% sodium citrate (9:1, blood-to-anticoagulant ratio) Platelet-rich plasma (PRP) was prepared by centrifugation at 200 × g for 10 min at 25°C, and platelet-poor plasma (PPP) was obtained by further centrifugation at 5,000 × g for 10 min. Platelet aggregation was performed within 2 hours of blood collection using a Chrono-Log aggregometer (Model 540 VS, Chrono-Log Corp., Havertown, PA, USA). ADP is a weak platelet agonist released from platelet granules to amplify platelet activation. TRAP-6 stimulates platelets by activating thrombin receptor (protease-activated receptor 1), representing the initial trigger of platelet activation. PPP was used to calibrate 100% light transmission, and PRP was preincubated at 37°C before stimulation with ADP (2 and 10 μM) or TRAP-6 (3 and 9 μM). Agonist concentrations were selected based on our preliminary dose–response experiments in healthy volunteers, yielding 50% effective concentration (EC₅₀) values of 2.67 ± 0.38 μM for ADP and 8.00 ± 1.24 μM for TRAP-6 ( S1 Fig ). Therefore, ADP at 2 μM and TRAP-6 at 9 μM were selected as submaximal concentrations to maximize sensitivity for detecting differences in platelet reactivity. For flow cytometry, freshly prepared PRP was diluted 1:10 and stained with FITC-conjugated PAC-1 (activated GPIIb/IIIa), PE-conjugated anti-CD62P (P-selectin), and PE-Cy5-conjugated anti-CD42b (platelet marker). Samples were incubated with phosphate-buffered saline or ADP (0.5 or 1 μM) for 15 min at room temperature in the dark, fixed with 1% paraformaldehyde for an additional 15 min, and analyzed using a BD Accuri C6 Plus flow cytometer (BD Biosciences, San Jose, CA, USA). Platelets were identified by forward- and side-scatter characteristics and CD42b expression. Activated platelets were quantified as the percentages of PAC-1-positive and CD62P-positive events among 10,000 CD42b-positive platelets. Transthoracic echocardiography Standard 2D transthoracic echocardiography was performed for all patients according to the American Society of Echocardiography Guidelines [ 14 – 16 ]. All examinations were conducted by a single experienced observer. Patients were in the left lateral decubitus position after a 10-minute rest. Imaging was acquired using an Affiniti 70C ultrasound scanner (Philips Healthcare, Amsterdam, The Netherlands) with an S5-1 broadband sector phased-array transducer. Grayscale recordings were optimized to achieve a mean frame rate ≥50 frames/s. The estimated systolic pulmonary artery pressure (sPAP) was reported as the estimated right ventricular systolic pressure (eRVSP), calculated using the modified Bernoulli equation: eRVSP = 4 × (TRV max )² + RAP [ 14 ]. The right atrial pressure (RAP) was estimated based on the diameter and collapsibility of the inferior vena cava. Diastolic pulmonary artery pressure (PADP) was calculated as PADP = 4(end-diastolic pulmonary regurgitation velocity) 2 + RAP. The mPAP was estimated as . TAPSE (tricuspid annular plane systolic excursion) to sPAP ratio was calculated as an additional marker of right ventricular (RV)–pulmonary arterial (PA) coupling. TAPSE reflects longitudinal right ventricular systolic shortening, while sPAP represents right ventricular afterload. A lower TAPSE/sPAP ratio indicates impaired RV–PA coupling. Sodium nitrite inhalation As inhaled sodium nitrite (15 and 40 mg) can decrease pulmonary pressure in thalassemia patients with PH [ 17 , 18 ], we tested the effects on pulmonary pressure of inhaled sodium nitrite in two patients as case report. The maximum tolerated dose in healthy subjects is 90 mg [ 19 ]. Sterile sodium nitrite solution for intravenous injection, manufactured by Queen Saovabha Memorial Institute, The Thai Red Cross Society, Thailand, was diluted with saline and administered via a Beurer IH 25/1 nebulizer (Beurer Medical, Ulm, Germany). Normal saline was used as a control. Patient 1 with confirmed SSc-PH received 15 mg of sodium nitrite inhalation, and patient 2 with an intermediate PH probability received incremental doses of 15 mg followed by 30 mg. Each patient first received 15 minutes of saline nebulization, followed by a 15-minute rest, and then 15 minutes of sodium nitrite nebulization, with a 15-minute interval between doses. During inhalation, TRV max was measured by echocardiography every one minute. Systolic blood pressure, diastolic blood pressure and heart rate were assessed using a non-invasive automated sphygmomanometer at baseline, every 5 minutes during inhalation, and at various time points after inhalation cessation. Methemoglobin levels were measured at baseline and 15 minutes after the end of inhalation. Blood nitrite and nitrate levels were measured at baseline, immediately after the end of saline and nitrite inhalation, and 15 minutes and 30 minutes after inhalation. Statistical analysis Statistical analyses were performed using GraphPad Prism® version 10.0 (GraphPad Software, San Diego, CA, USA) and IBM SPSS Statistics for Windows, version 26.0 (IBM Corp., Armonk, NY, USA). Group comparisons were conducted by GraphPad Prism, and correlation analyses were performed in SPSS. Figures were generated by GraphPad Prism. Normality was assessed with the Shapiro–Wilk test. Continuous variables are presented as means ± SD or medians (interquartile range), as appropriate, and categorical variables as frequencies and percentages. Comparisons among groups were performed using one-way analysis of variance with Tukey’s post hoc test or the Kruskal–Wallis test with Dunn’s post hoc test, as appropriate. Categorical variables were compared using a chi-squared test. Correlations were assessed with Spearman’s rank correlation coefficient. Statistical significance was defined as a two-tailed p -value 3.4 m/s or TRV max of 2.9 to 3.4 m/s with at least two other echocardiographic signs of PH) and subsequently had undergone RHC. The diagnosis of PH was finally made by RHC in those patients who were then defined as SSc-PH. Their invasive hemodynamic parameters included mean pulmonary arterial pressure of 27.6 ± 7.9 mmHg, pulmonary arterial wedge pressure of 6.5 (5.0–11.0) mmHg, and pulmonary vascular resistance of 4.5 ± 2.3 Wood units. These parameters were consistent with pre-capillary pulmonary arterial hypertension. SSc-PH patients had a longer disease duration than those with low PH probability ( Table 2 ). Download: PNG larger image TIFF original image Table 2. Clinical characteristics, pulmonary function test, and functional capacity of patients with systemic sclero
## Related Clinical Research

- [Machine learning and deep learning prediction of hypertension and key risk factors in Bangladesh](https://medichelpline.com/clinical-feed/plos-one-12-machine-learning-and-deep-learning-based-prediction-of-hypertension-and.md)
- [Dyslipidemia care in Japan before and after the 2024 fee revision: nationwide claims analysis](https://medichelpline.com/clinical-feed/plos-one-19-real-world-patterns-of-dyslipidemia-care-before-and-after-a-national-fee.md)
- [Antihypertensive Drug Class and Risk of Incident Alzheimer’s Disease and Related Dementias: Replic](https://medichelpline.com/clinical-feed/medrxiv-17-antihypertensive-medication-class-and-incident-alzheimers-disease-and-related.md)
- [GLP-1 Receptor Agonists and Pulmonary Hypertension: Mechanistic Rationale and Evidence Gaps](https://medichelpline.com/clinical-feed/pubmed-42747605.md) (DOI: 10.1007/s00408-026-00939-2)
- [Adherence to Medications, Lifestyle Advice, and Self‑Monitoring for Type 2 Diabetes and Hypertensi](https://medichelpline.com/clinical-feed/plos-medicine-0-adherence-to-medications-lifestyle-advice-and-self-monitoring-for-type-2.md)

## Navigation
- [← Back to Rheumatology Feed](https://medichelpline.com/clinical-feed/rheumatology.md)
- [← All Clinical Specialties](https://medichelpline.com/clinical-feed.md)
## Medical & Regulatory Disclaimer

> [!CAUTION]
> MedicHelpline content is structured for research, educational, and professional discovery purposes. It does not constitute individual medical advice, clinical diagnosis, or treatment recommendations.
> Always verify dosing, contraindications, and regulatory alerts against official product labeling and primary regulatory sources before clinical decision-making.