Journal of Human Hypertension https://doi.org/10.1038/s41371-021-00555-y ARTICLE Differentially expressed proteins in platelets derived from patients with hypertension 1 1 2 3 Yobana Armenta-Medina ● Ivette Martínez-Vieyra ● Oscar Medina-Contreras ● Claudia G. Benitez-Cardoza ● 3 4 1 Albertana Jiménez-Pineda ● César A. Reyes-López ● Doris Cerecedo Received: 19 February 2021 / Revised: 7 May 2021 / Accepted: 20 May 2021 © The Author(s), under exclusive licence to Springer Nature Limited 2021 Abstract Hypertension (HTN) causes end-organ damage and is a major cause of morbidity and mortality globally. Recent studies suggested blood cells participate in the maintenance of HTN. Platelets—anucleated cell fragments derived from megakaryocytes—exert diverse functions, including their well-characterized role in the formation of hemostatic clots. However, platelets from patients with HTN exhibit altered membrane lipid and protein compositions that impact platelet function and lead to formation of aggregates and vascular obstructions. Here, for the first time, we have identified, by proteomic analyses, the most relevant 11 proteins that show the greatest difference in their expression in platelets derived 1234567890();,: 1234567890();,: from patients with HTN, in comparison with those from normotensive individuals. These proteins are involved in cytoskeletal organization and the coagulation cascade that contributes to platelet activation, release of granule contents, and aggregation, which culminate in thrombus formation. These results have important implications in our understanding of the molecular mechanisms associated with the development of HTN, and in consequence, the development of new strategies to counteract the cardiovascular disorders associated with constitutive activation of platelets in HTN. Introduction rejuvenating our interest in better understanding this condi- tion [1]. Mechanisms such as overactivation of the Hypertension (HTN) is a common, multifactorial disease and renin–angiotensin–aldosterone system and sympathetic ner- the major risk factor for heart attacks, stroke, and kidney vous system, hypertrophy, endothelial dysfunction, and oxi- failure. Furthermore, HTN has been implicated in higher dative stress have been related the pathogenesis of HTN [2]. susceptibility and adverse outcomes of patients infected HTN is characterized by spontaneous platelet aggrega- with the current pandemic caused by SARS-CoV2, thereby tion due to increased platelet activation. Hemodynamic and vascular factors generated by the higher sheer forces in patients with HTN, as well as atherosclerosis, contribute to platelet activation [3]. In addition, the release of higher Supplementary information The online version contains concentrations of catecholamines and increased sensitivity supplementary material available at https://doi.org/10.1038/s41371- of platelets to these patients with HTN also plays key roles 021-00555-y. in platelet activation [4]. * Doris Cerecedo Activated platelets undergo a dramatic change in shape [email protected] from disc shaped to a spherical morphology due to dynamic reorganization of cytoskeletal components, including 1 Laboratorio de Hematobiología, ENMyH, Instituto Politécnico Nacional, Mexico City, Mexico assembly and disassembly of structural proteins. The per- 2 centage of platelets exhibiting active actin polymerization Unidad de Investigación Epidemiológica en Endocrinología y – Nutrición, Hospital Infantil de México Federico Gómez, increases from ~35 40 to 70% during the activation pro- Mexico City, Mexico cess. In turn, activated platelets form filopodia and lamel- 3 Laboratorio de Investigación Bioquímica, ENMyH, Instituto lipodia adhere to each other and release their granule Politécnico Nacional, Mexico City, Mexico contents [5]. 4 Laboratorio de Bioquímica Estructural, ENMyH, Instituto Platelets from patients with HTN are constituently acti- Politécnico Nacional, Mexico City, Mexico vated and exhibit morphological, biophysical, biochemical, Y. Armenta-Medina et al. and functional modifications [6]. Recently, we reported that Table 1 General characteristics of hypertensive patients and controls. platelets from patients with HTN possess numerous long Parameter Hypertensive Controls p value filopodia, exhibit deregulated expression of cytoskeletal proteins, and overexpress the epithelial sodium channel. Age ± SD (years) 55.83 ± 9.26 49 ± 12.56 0.2439 – These changes were associated with altered phospholipid Sex (%male/%female) 38/62 40/60 and cholesterol contents and changes in the microviscosity SBP (mmHg) 149 ± 23 117 ± 5 0.0019 of the plasma membrane [7]. Atomic force microscopy and DBP (mmHg) 89 ± 8 79 ± 4 0.0001 liquid chromatography–tandem mass spectrometry BMI 28 ± 5 27 ± 4 0.0898 ns (LC–MS/MS) confirmed the changes in fluidity and altered Glucose (mg/dl) 105 ± 11.5 89 ± 5 0.0001 lipid contents in platelets from patients with HTN compared Total cholesterol (mg/dl) 185.8 ± 10 191 ± 13 0.1815 ns to platelets from normotensive individuals (NTI). In addi- Triglycerides (mg/dl) 220.4 ± 52.5 140 ± 38 0.0001 tion, transmission electron microscopy revealed changes in MPV (fl) 9.1 ± 0.84 8.7 ± 0.6 0.403 the inner membranous structure of platelets from patients Platelet count (×109) 161 ± 21.9 235 ± 30.3 0.0007 with HTN [8]. Hypertension grades (%) Proteomic analysis provides an important tool to identify Controlled 44.4 – proteins; ~80% of the total protein content of platelets has 1 44.4 – been characterized [9]. The aim of the present study was to 2 11.2 – identify differentially expressed proteins in platelets derived Values are the mean ± SD of ten patients and controls in each group. from patients with HTN compared to platelets from NTI. BMI body mass index, SBP systolic blood pressure, DBP diastolic Using 2D electrophoresis, we resolved whole platelet pro- blood pressure, MPV mean platelet volume, ns non significant. teins from nine patients with HTN and nine NTI. The 11 most importantly differentially expressed electrophoretic entities, between the two groups, were identified using a blood samples in an automatic blood counter (Beckman LC–MS/MS approach and an interactome of the identified Coulter) within 30 min after venipuncture. proteins was built using STRING. This information may HTN grades were defined according to 2013 ESC/ESH help to identify specific therapeutic target proteins to ame- Guidelines for the management of arterial HTN. liorate or avoid thrombotic events, or identify biomarkers of Controlled BP was defined as systolic blood pressure disease progression. (SBP) < 140 mmHg and/or diastolic blood pressure (DBP) < 90 mmHg; grade 1 was defined as SBP 140–159 mmHg and/or DBP 90–99 mmHg; grade 2 was defined as Materials and methods SBP 160–179 mmHg and/or DBP 100–109 mmHg; and grade 3 was defined as SBP ≥ 180 mmHg and/or DBP ≥ Reagents 110 mmHg. All subjects maintained their usual diets; the patients SDS, acrylamide, bis-acrylamide, urea, thiourea, CHAPS, with HTN maintained their treatment, which included ena- TEMED, DTT, Tris, TCA, acetic acid, ethanol, Coomassie lapril, losartan, telmisartan, or amlodipine; no platelet brilliant blue G 250, aluminum sulfate, and orthophosphoric aggregation inhibitors were prescribed. The NTI group had acid were purchased from Sigma Chemical Co. (St. Louis, not received any drug treatment during the previous MO, USA). Immobilized pH gradient strips (pH 3–10) and 10 days. Exclusion criteria included pregnancy, smoking, the 2D electrophoresis reagents and electrophoresis instru- valvular heart disease, type 2 diabetes mellitus, and dysli- ments were obtained from Bio-Rad Laboratories Inc. pidemia. For the 2D gel analysis the patients were grouped (Hercules, CA, USA). regardless the treatment they were receiving. Subjects Platelet sample preparation Human peripheral blood samples were collected from Platelet isolation, protein extraction, and protein preparation patients diagnosed with essential systemic arterial HTN for analysis were performed as previously reported by (n = 9) and NTI (n = 9) after obtaining signed informed García et al. [10]. consent. This project was approved by the ethics com- LC–MS/MS approach analyzes many proteins in a single mittee of the ENMyH Instituto Politécnico Nacional experiment, this methodology is not suitable to assess large (No. 001/2019). Table 1 summarizes the anthropometric number of individuals; the washed platelet suspensions characteristics of the two groups. Mean platelet volume were pooled in groups of three to produce three samples for (MPV) and platelet count were measured from whole patients with HTN and three samples for NTI. Each pooled Differentially expressed proteins in platelets derived from patients with hypertension sample was assessed on three independent gels; therefore, a Irapuato, México. All experiments were performed on a total of nine gels for patients with HTN and nine gels for nanoAcquity nanoflow LC system (Waters Corporation) NTI were obtained and analyzed. coupled to a linear ion trap LTQ Velos mass spectrometer One thousand two hundred and fifty micrograms of total (Thermo Fisher Scientific, Bremen, Germany) equipped protein extracts were solubilized in a rehydration buffer with a nanoelectrospray ion source. Solvent A was 0.1% (8 M urea, 2 M thiourea, CHAPS 2%, 50 mM DTT) sup- formic acid and solvent B was 100% acetonitrile in 0.1% plemented with 1% Bio-LyteTM ampholyte (Bio-Rad)), and formic acid. Protein digestate samples (3 µl) were bound to were loaded onto 17 cm immobilized pH gradient IPG