Neutrophil-Derived Protein S100A8/A9 Alters the Platelet Proteome in Acute Myocardial Infarction and Is Associated With Changes in Platelet Reactivity

Objective: Platelets are central to acute myocardial infarction (MI). How the platelet proteome is altered during MI is unknown. We sought to describe changes in the platelet proteome during MI and identify corresponding functional consequences. Approach and Results: Platelets from patients experiencing ST-segment–elevation MI (STEMI) before and 3 days after treatment (n=30) and matched patients with severe stable coronary artery disease before and 3 days after coronary artery bypass grafting (n=25) underwent quantitative proteomic analysis. Elevations in the proteins S100A8 and S100A9 were detected at the time of STEMI compared with stable coronary artery disease (S100A8: FC, 2.00; false discovery rate, 0.05; S100A9: FC, 2.28; false discovery rate, 0.005). During STEMI, only S100A8 mRNA and protein levels were correlated in platelets ( R =0.46, P =0.012). To determine whether de novo protein synthesis occurs, activated platelets were incubated with 13C-labeled amino acids for 24 hours and analyzed by mass spectrometry. No incorporation was confidently detected. Platelet S100A8 and S100A9 was strongly correlated with neutrophil abundance at the time of STEMI. When isolated platelets and neutrophils were coincubated under quiescent and activated conditions, release of S100A8 from neutrophils resulted in uptake of S100A8 by platelets. Neutrophils released S100A8/A9 as free heterodimer, rather than in vesicles or extracellular traps. In the community-based Bruneck study (n=338), plasma S100A8/A9 was inversely associated with platelet reactivity—an effect abrogated by aspirin. Conclusions: Leukocyte-to-platelet protein transfer may occur in a thromboinflammatory environment such as STEMI. Plasma S100A8/A9 was negatively associated with platelet reactivity. These findings highlight neutrophils as potential modifiers for thrombotic therapies in coronary artery disease.

Effects of Cancer Presence and Therapy on the Platelet Proteome

Platelets are involved in tumor angiogenesis and cancer progression. Previous studies indicated that cancer could affect platelet content. In the current study, we investigated whether cancer-associated proteins can be discerned in the platelets of cancer patients, and whether antitumor treatment may affect the platelet proteome. Platelets were isolated from nine patients with different cancer types and ten healthy volunteers. From three patients, platelets were isolated before and after the start of antitumor treatment. Mass spectrometry-based proteomics of gel-fractionated platelet proteins were used to compare patients versus controls and before and after treatment initiation. A total of 4059 proteins were detected, of which 50 were significantly more abundant in patients, and 36 more in healthy volunteers. Eight of these proteins overlapped with our previous cancer platelet proteomics study. From these data, we selected potential biomarkers of cancer including six upregulated proteins (RNF213, CTSG, PGLYRP1, RPL8, S100A8, S100A9) and two downregulated proteins (GPX1, TNS1). Antitumor treatment resulted in increased levels of 432 proteins and decreased levels of 189 proteins. In conclusion, the platelet proteome may be affected in cancer patients and platelets are a potential source of cancer biomarkers. In addition, we found in a small group of patients that anticancer treatment significantly changes the platelet proteome.

Alterations of the Platelet Proteome in Lung Cancer: Accelerated F13A1 and ER Processing as New Actors in Hypercoagulability

In order to comprehensively expose cancer-related biochemical changes, we compared the platelet proteome of two types of cancer with a high risk of thrombosis (22 patients with brain cancer, 19 with lung cancer) to 41 matched healthy controls using unbiased two-dimensional differential in-gel electrophoresis. The examined platelet proteome was unchanged in patients with brain cancer, but considerably affected in lung cancer with 15 significantly altered proteins. Amongst these, the endoplasmic reticulum (ER) proteins calreticulin (CALR), endoplasmic reticulum chaperone BiP (HSPA5) and protein disulfide-isomerase (P4HB) were significantly elevated. Accelerated conversion of the fibrin stabilising factor XIII was detected in platelets of patients with lung cancer by elevated levels of a F13A1 55 kDa fragment. A significant correlation of this F13A1 cleavage product with plasma levels of the plasmin–α-2-antiplasmin complex and D-dimer suggests its enhanced degradation by the fibrinolytic system. Protein association network analysis showed that lung cancer-related proteins were involved in platelet degranulation and upregulated ER protein processing. As a possible outcome, plasma FVIII, an immediate end product for ER-mediated glycosylation, correlated significantly with the ER-executing chaperones CALR and HSPA5. These new data on the differential behaviour of platelets in various cancers revealed F13A1 and ER chaperones as potential novel diagnostic and therapeutic targets in lung cancer patients.

Anti-Platelet Properties of Phenolic and Nonpolar FractionsIsolated from Various Organs of Elaeagnus rhamnoides (L.) A. Nelson in Whole Blood

Sea buckthorn (Elaeagnus rhamnoides (L.) A. Nelson) is a shrub growing in coastal areas. Its organs contain a range of bioactive substances including vitamins, fatty acids, various micro and macro elements, as well as phenolic compounds. Numerous studies of sea buckthorn have found it to have anticancer, anti-ulcer, hepatoprotective, antibacterial, and antiviral properties. Some studies suggest that it also affects the hemostasis system. The aim of the study was to determine the effect of six polyphenols rich and triterpenic acids rich fractions (A–F), taken from various organs of sea buckthorn, on the activation of blood platelets using whole blood, and to assess the effect of the tested fractions on platelet proteins: fraction A (polyphenols rich fraction from fruits), fraction B (triterpenic acids rich fraction from fruits), fraction C (polyphenols rich fraction from leaves), fraction D (triterpenic acids rich fraction from leaves), fraction E (polyphenols rich fraction from twigs), and fraction F (triterpenic acids rich fraction from twigs). Hemostasis parameters were determined using flow cytometry and T-TAS (Total Thrombus-formation Analysis System). Additionally, electrophoresis was performed under reducing and non-reducing conditions. Although all tested fractions inhibit platelet activation, the greatest anti-platelet activity was demonstrated by fraction A, which was rich in flavonol glycosides. In addition, none of the tested fractions (A–F) caused any changes in the platelet proteome, and their anti-platelet potential is not dependent on the P2Y12 receptor.

Accelerated endoplasmic reticulum and coagulation factor processing in platelets are linked with the hypercoagulable state of patients with lung cancer

Background The risk of venous thromboembolism in cancer is nine times higher than in the general population and the second leading cause of death in these patients. Platelets play a key role in tumour growth, metastasis, and cancer-associated thrombosis. Despite this widely observed functional role of platelets in the prothrombotic state of certain cancers, the underlying molecular mechanisms are largely unknown. Methods In order to comprehensively expose cancer-related biochemical changes, we compared the platelet proteome of two types of cancer with a high risk of thrombosis (22 patients with brain cancer, 19 with lung cancer) to 41 matched healthy controls using unbiased two-dimensional differential in-gel electrophoresis. Cancer-related platelet proteins were statistically characterised by adjusted one-way ANOVA and planned comparisons post hoc tests. Significantly changed platelet proteins were identified by mass spectrometry. Afterward, selected cancer-associated platelet proteins were validated by 1-D and 2-D Western blots and/or functional assessments were made by association analysis with central haemostatic plasma parameters. Results The examined platelet proteome was unchanged in patients with brain cancer, but considerably affected in lung cancer with 15 significantly altered proteins. Amongst these, the endoplasmic reticulum (ER) proteins CALR, HSPA5 and P4HB were significantly elevated. Accelerated conversion of the fibrin stabilising factor has been detected in platelets of patients with lung cancer by elevated levels of a F13A1 55 kDa fragment. A significant correlation of this F13A1 cleavage product with plasma D-dimer levels suggests an enhanced degradation in platelets by the fibrinolytic system. Functional protein association network analysis showed that lung cancer-related protein profiles were involved in platelet degranulation and upregulated ER protein processing. The latter is important in the glycosylation of coagulation factors. As a possible outcome, plasma FVIII, an immediate end product for ER-mediated glycosylation, correlated significantly with the ER-executing chaperones CALR and HSPA5. In addition, several lung cancer-related platelet proteins such as decreased levels of ITGA2B are significantly linked to the plasmatic haemostasis factors D-dimer and fibrinogen. Conclusions These new data on the differential behaviour of platelets in various cancers revealed F13A1 and ER chaperones as potential novel diagnostic and therapeutic targets in lung cancer patients.

Uncomplicated Plasmodium vivax malaria: Mapping the proteome from circulating platelets

Background: Thrombocytopenia is frequent in uncomplicated Plasmodium vivax malaria. Contribution of platelets to pathogenesis is unknown and poorly understood. Our study explores the platelet proteome from uncomplicated P. vivax malaria patients to fingerprint molecular pathways in relation to platelet function. Also, plasma levels of platelet activation (Platelet factor 4 – PF4/CXCL4) and endothelial activation (Von Willebrand factor – VWf) markers, in conjunction with some in vitro interactions between platelets and P. vivax infected erythrocytes ( Pv -IEs) were measured to explore platelet responses during infection and their effect on parasite development. Methods: This study was performed in a cohort of 48 patients and 25 healthy controls. Platelets were purified from a subgroup of 5 patients and 5 healthy controls to be analyzed by LC-MS/MS. In all participants enrolled in this study, PF4/CXCL4 and VWf plasma levels were measured. Finally, a subsample of 10 P. vivax isolates were co-cultured with platelets to measure P v- IE schizonts inhibition as well as platelet activation due to their interaction. Results: In total 28 out of 215 proteins were significantly abundant in the proteomes from patients. The most significantly decreased protein was PF4/CXCL4 followed by other proteins related to platelet activation, cytoskeletal remodeling, and adhesion to endothelial cells. In contrast, acute phase proteins including SERPINs and Amyloid Serum A 1 (SAA1) were increased. High VWf plasma levels in patients suggested endothelial activation. Interestingly, PF4/CXCL4 plasma levels were similar between patients and controls, but high levels of this protein were found in co-cultures, and platelets inhibited Pv -IEs development to schizonts. Conclusions: Platelet proteome from patients with uncomplicated P. vivax malaria suggests platelet degranulation, platelet activation, cytoskeletal remodeling, and adhesion to endothelial cells. According to the evidenced endothelial activation our study plus the suggested specific localization of PF4/CXCL4 during P. vivax infection due to the normal levels in plasma, and the inhibition of Pv -IE schizonts development; our study suggest that platelets are active players during the response to P . vi vax infection. Future studies are needed to further investigate the molecular pathways of interaction between altered platelet proteins and host response; which could affect parasite control as well as disease progression.

Mild hyperlipidemia in mice aggravates platelet responsiveness in thrombus formation and exploration of platelet proteome and lipidome

Hyperlipidemia is a well-established risk factor for cardiovascular diseases. Millions of people worldwide display mildly elevated levels of plasma lipids and cholesterol linked to diet and life-style. While the prothrombotic risk of severe hyperlipidemia has been established, the effects of moderate hyperlipidemia are less clear. Here, we studied platelet activation and arterial thrombus formation in Apoe−/− and Ldlr−/− mice fed a normal chow diet, resulting in mildly increased plasma cholesterol. In blood from both knockout mice, collagen-dependent thrombus and fibrin formation under flow were enhanced. These effects did not increase in severe hyperlipidemic blood from aged mice and upon feeding a high-fat diet (Apoe−/− mice). Bone marrow from wild-type or Ldlr−/− mice was transplanted into irradiated Ldlr−/− recipients. Markedly, thrombus formation was enhanced in blood from chimeric mice, suggesting that the hyperlipidemic environment altered the wild-type platelets, rather than the genetic modification. The platelet proteome revealed high similarity between the three genotypes, without clear indication for a common protein-based gain-of-function. The platelet lipidome revealed an altered lipid profile in mildly hyperlipidemic mice. In conclusion, in Apoe−/− and Ldlr−/− mice, modest elevation in plasma and platelet cholesterol increased platelet responsiveness in thrombus formation and ensuing fibrin formation, resulting in a prothrombotic phenotype.

The Effects of Storage on Platelet Proteome Assessed by Protein Expression Changes and Lysine Modification Changes Using Tmt Isobaric Labeling

Purpose:To provide the first insight into the proteomic dynamics during platelet storage.Experiment design: In this study, based on TMT-labeled LC-MS/MS analysis, combined with antibody-affinity enrichment and purification for acetylated and succinylated peptides, we performed quantification of global proteomics, acetylome and succinylome.Simultaneously, dynamic molecular changes and functional transformation of platelet were also characterized under proper conditions stored for 1, 3, 5, 7 days, respectively.Results:3,100 proteins are quantified from a total of 3,609 proteins identified from platelets. Out of 1,308 acetylated sites identified in 648 proteins, 790 sites in 396 proteinsare quantifiable. There are 1,947 succinylated sites in 959 proteins in which 1,279 sites in 661 proteins are quantifiable.We screened the differential expression changes of global proteins, acetyl- and succinyl- proteins, and systematically interpreted their molecular functions, biological processes, cellular components, pathways and motif characters to fully investigate the molecular dynamics and biological functions of platelets. Conclusions and clinical relevance:This paper is the first systematic exploration of proteomes and modified proteomes of platelet dynamics during storage in the aim to improve our understanding of platelet biology, which may be a valuable reference for further research and clinical application.

Comparative proteomics reveals unexpected quantitative phosphorylation differences linked to platelet activation state

There is a need to assess platelet activation in patients with thrombotic disorders. P-selectin and activated integrin αIIbβ3 are usually quantified by flow cytometry to measure platelet activation. Monitoring changes in vasodilator-stimulated phosphoprotein (VASP) phosphorylation is an established method to determine the platelet-reactivity status. To study disruptions of platelet reactivity more comprehensively, we compared the human non-secretory platelet proteome after in-vitro -activation and –inhibition with their respective untreated controls using unbiased fluorescence two-dimensional differential in-gel electrophoresis. The non-secretory platelet proteome was more severely affected during inhibition than during activation. Strikingly, while VASP reached a 1.3-fold increase in phosphorylation levels in inhibited platelets, other protein kinase A targets showed several-fold stronger inhibition-induced phosphorylation levels, including LIM and SH3 domain protein 1 (6.7-fold), Src kinase-associated phosphoprotein 2 (4.6-fold), and Ras-related protein Rap1b (4.1-fold). Moreover, phosphorylation of integrin-linked protein kinase (ILK) and pleckstrin (PLEK) species was associated with P-selectin surface expression. The discrimination power between activation and inhibition was more pronounced for dephosphorylated ILK (3.79 Cohen’s d effect size) and phosphorylated PLEK (3.77) species than for P-selectin (2.35). These data reveal new insights into the quantitative changes of the platelet reactivity proteome and suggest powerful alternatives to characterise their activation and inactivation potential.