Noninvasive Biomarkers for Alcohol-Related Liver Disease—A Proteomic Related Preliminary Report

Abstract

Increased alcohol intake over decades leads to progressive alcohol-related liver disease (ALD) and contributes to increased mortality. It is characterized by reduced platelet count. Platelets have a role in protecting vascular integrity and involved in liver regeneration. Alcohol affects the platelet count and its function. Platelet function is regulated by their proteins, released during pathophysiological conditions. Therefore, platelet proteome plays a vital role during ALD. This preliminary study consists of 10 patients with ALD. It includes the preparation of human platelets for the proteomic approach. We performed liquid chromatography-mass spectrometry for the samples. A total of 536 proteins were identified in patients with ALD of which 31 proteins were mentioned as a candidate based on their clinical significance. The advancement of diagnostic or therapeutic tools based on the application of platelet proteins in ALD is still far off. Platform for platelet and its proteome research may give diagnostic and prognostic insights into ALD. Platelet proteomes could possibly be concluded as therapeutic and potential diagnostic or prognostic markers in ALD.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12291-023-01120-9.

Introduction

Alcohol dependence is associated with the development of various health issues including liver cirrhosis, cancer and cardiovascular diseases. Excessive drinking contributes to increased metabolic rate, whereas alcohol-induced liver damage will decrease the rate of ethanol metabolism. It causes a wide spectrum of hepatic lesions characterized as steatosis, hepatitis, and fibrosis/cirrhosis. Cirrhosis is the 11th most common cause of increased mortality worldwide and two million deaths were reported due to liver disease [1]. Alcohol-related liver disease (ALD) comprises histopathologic changes, vary from fatty liver to alcoholic steatohepatitis, progressive fibrosis, cirrhosis, and finally liver cancer. Alcohol-related liver disease at any point of time can cause acute alcoholic hepatitis further leading to liver failure. The short-term mortality of 40% in patients with alcoholic hepatitis observed after the onset of clinical symptoms [2]. Continuous excessive extracellular matrix (ECM) deposition leading to liver fibrosis in response to chronic liver injury. Chronic liver disease contributes to mortality and accounts for 18.3% in India [3]. The patients with ALD exhibits an elevated inflammatory response due to liver injury. Aberrant immune fibrosis and hepatic regeneration contribute to disrupted liver architecture. Advanced fibrosis resulting in the permanent substitution of ECM components is considered an irreversible stage.

In liver, during diseased condition platelets can interact with various cellular components and that plays a vital role in hemostasis. Platelets have a protective role in vascular integrity, inflammation, liver regeneration, and tumor metastasis. Platelet proteins are associated with pathophysiological processes of liver. The platelet parameters including platelet, plateletcrit, and platelet distribution width are routinely used as a potential indicator of liver fibrosis [4]. Chronic liver disease develops deterioration of production of clotting factors, detoxification, and release of functional proteins. Alcohol affects platelet count and its function. Alcohol inhibits platelet aggregation. Liu et al. demonstrated ethanol induced mitochondria-mediated intrinsic platelet apoptosis resulting reduced levels of circulating platelets and damages in vivo hemostasis [5]. Thrombocytopenia is a typical impairment of chronic liver disease, characterized by reduced hematopoiesis, lower levels of thrombopoietin (TPO), and increased destruction of platelets in the spleen. Thrombocytopenia is observed in ALD patients, which is associated with the progression of the disease [6]. Platelets adhere to the exposed endothelial tissues during inflammation or damage to liver tissue. Upon adhesion the platelets got activated. Ogasawara et al. observed an inverse correlation between platelet count and platelet activation. The study concluded that patients with ALD were shown increased platelet activation that can contribute to occurrence of thrombocytopenia [7]. The liver is a central regulator of numerous circulating platelets via TPO production and clearance of aged platelets. The coexistence of thrombotic events and inflammation in liver parenchyma contribute to a disturbance in platelet proteome [8]. The relationship between platelet proteome and the pathology of ALD still needs to be discussed.

Liver biopsies, serological investigations, and imaging examinations are currently using in the clinical assessment of hepatic lesions. Liver biopsy is still the gold standard for evaluating liver fibrosis/cirrhosis. Due to the invasiveness and risk of developing complications in this procedure, there is a great demand for new, noninvasive, cost-effective, and safe biomarkers to identify the different stages of liver fibrosis. The specific noninvasive biomarker of the liver structure during disease progression is of crucial interest. The study aims to list the platelet proteins of pathophysiological importance in ALD. Some of the novel platelet proteins related to ALD are yet to be established.

Methodology

Sampling

The sample was calculated by using OpenEpi, Version 3, ensuring that the 95% of confidence interval (z = 1.96 for 95%) estimation of the mean fibrosis-4 score (FIB-4), γGT and platelet count in alcoholism patients between age of 43 and 55 with alcoholic liver cirrhosis within 5 points of the mean error (E), a sample size 23. we assumed a standard deviation is 2.3, the continuous outcome of single population ALD sample size is n = 10. The standard deviation was derived from the studies related to alcoholism with chronic liver diseases.

$$\text{n}={[\text{Z}\sigma /\text{E}]}^2$$

Patient Selection

This preliminary study consists of 10 patients with ALD. These participants were recruited from the department of medicine, aarupadai veedu medical college and hospital, Puducherry, India. Primarily the study got approved by the institutional ethics committee. Informed consent was obtained from each participant before taking participation in the study. The progression of ALD in the enrolled patients were assessed using fibrosis-4 index and child–pugh score [9]. The participants were asked to answer questionnaires regarding anthropometric measurements and consumption of alcohol and evaluated laboratory and clinical examinations (Table 1 and Fig. 1).

Table 1.

Clinical profile of alcohol-related liver disease patients

Parameters	ALD (n = 10)
Age (Years)	51.2 ± 3.3
Total bilirubin (mg/dL)	4.21 ± 0.7
Albumin (g/dL)	2.79 ± 0.3
AST (U/L)	192.6 ± 63.0
ALT (U/L)	114.8 ± 45.9
ALP (U/L)	66.5 ± 49.4
GGT (U/L)	82.9 ± 16.8
Uric Acid (mg/dL)	4.55 ± 1.2
Total antioxidant capacity (nmol/µL)	1.02 ± 0.06
Total oxidant status (µmol H2O2 Equiv/L)	14.1 ± 1.7
Total leukocyte count (109/Liter)	5.44 ± 3.7
Platelet count (109/Liter)	1.06 ± 0.2
INR (Sec)	2.03 ± 0.1
Child–Pugh score/cirrhosis SEVERITY (Points)	12.3 ± 0.9
METAVIR/fibrosis-4 Score (Points)	9.377 ± 3.4

AST aspartate aminotransferase, ALT alanine aminotransferase, ALP alkaline phosphate, GGT gamma-glutamyl transferase, INR International normalized ratio

Fig. 1.

Preliminary study to identify candidate biomarkers in alcoholic liver disease patient enrollment flow chart

Inclusion Criteria

The Patients in the age group of more than 18 years with a history of alcohol intake of more than 14 years were included. The patients suffering from clinical symptoms such as ascites, encephalopathy, splenomegaly, autoimmune hepatitis, and kidney-related diseases [10] were recruited in the study.

Exclusion Criteria

The patients suffering from cancer, hepatocellular carcinoma, cardiovascular diseases, blood transfusion (thrombolytic therapy) and hepatotoxic drug-treated patients were excluded from the study.

Preparation of Human Platelet

The whole blood of 5 ml was drawn from the enrolled patients in the 8.5 ml acid-citrate-dextrose vacutainer tubes. The collected blood sample was then transferred to 15 ml polypropylene tubes and centrifuged at 1000 rpm for about 15 min with no brakes. The upper layer consists of pooled human platelet-rich plasma, which is transferred to a new 15 ml polypropylene tube and centrifuged at 1700 rpm for about 15 min with no brakes. The platelet pellet was resuspended gently in 1 ml tyrode's solution (139 mM NaCl, 17 mM NaHCO3. mM KCl, 3 mM CaCl2, 12 mM glucose, and 1 mM MgCl2) and transferred to 2 ml microcentrifuge tubes [11]. Tyrode's buffer acts as a balanced salt solution. An aliquot of 10 µl was used in platelet counting using mindray (BC-5130), an auto hematology analyzer. Added lysis buffer (2% NP40, 30 mM HEPES, 2 mM EDTA and 150 mM NaCl) to the resuspended solution.

Mass Spectrometric Analysis

Mass spectrometric analysis of the isolated platelets from patients with liver disease was performed using nano liquid chromatography-mass spectrometry (LC–MS)-LTQ-orbitrap discovery (Thermo Scientific) coupled to nano-LC (Agilent 1200). The samples were reconstituted in LC buffer (0.2% formic acid and 2% acetonitrile in water). The generated data of the post-MS run was analyzed using proteome discover software, UniProt, and MASCOT search engines for a human database.

Results

The data of baseline characteristics of all study subjects were presented in table. The mean age of patients with ALD was 51.2 ± 3.3 years. Increased levels of ALT, AST, ALP, GGT and uric acid observed in progression of ALD. Platelet count, total antioxidant capacity and oxidant status were displayed in the table. The progression of ALD is measured by using child–pugh score and METVIR (Table 1).

Identification of Biomarker Candidates

The results of a typical chromatograph that is LC–MS shown in the table. A total of 536 proteins were identified. We listed 31 candidate biomarkers of ALD based with increased clinical significance. Out of 31 proteins, ten proteins were belonging to the family of a serine protease inhibitors, and six proteins were belonging to the thrombin family. The proteins histidine-rich glycoprotein and von willebrand are involved in the binding of endothelial cells. Vitronectin, fibronectin, thrombospondin-1, and vinculin are mainly involved in cell–cell and cell–matrix interaction. SERPINC1 regulates blood coagulation. Clusterin functions as a chaperone and prevents blood plasma protein aggregation. SERPING1 forms a complex with C1r or C1s proteases. Von willebrand maintains hemostasis and promotes platelet adhesion to the sites of vascular cell injury (Table 2).

Table 2.

Platelet proteome as candidate biomarkers identified by LC–MS in ALD

Name of the protein	Alternative name(s)	Gene name	Location	Ensembl ID	pI	Molecular mass (Da)	emPAI	Biological role
Plasma protease C1 inhibitor	CI inhibitor, C1-INH, C1NH,	SERPING1	11q12.1	ENSG00000149131	6.09	55,154.19	1.03	Angioedema
Heparin cofactor 2	HCFII, HCF2, HLS2	SERPIND1	22q11.21	ENSG00000099937	6.41	570,700.61	0.39	Contribute in thrombotic risk, Coagulation, Liver hemostasis
Antithrombin-III	AT3, ATIII, Antitrombin-3	SERPINC1	1q25.1	ENSG00000117601	6.32	52,602.44	1.89	Thrombosis, Coagulation, Angiogenesis
Angiotensinogen	AGT	AGT, SERPINA8	1q42.2	ENSG00000135744	5.68	52,069.94	0.85	Blood pressure regulation
Thyroxine-binding globulin	TBG	SERPINA7	Xq22.3	ENSG00000123561	5.87	46,324.5	0.22	Hormone transport, Hypothyroidism
Corticosteroid-binding globulin	CBG	SERPINA6	14q32.13	ENSG00000170099	5.64	45,140.87	0.36	Hormone transport, Steatohepatitis, Cardiovascular disease
Plasma serine protease inhibitor	PCI, Protein C inhibitor, PAI-3	SERPINA5	14q32.13	ENSG00000188488	9.3	45,674.68	0.28	Thrombosis, Angioedema, Cancer, Coagulation
Kallistatin	PI4, KST, KAL	SERPINA4	14q31-32.1	ENSG00000100665	7.34, 4.6, 5.2	48,541.97	0.34	Alcoholic liver cirrhosis, Renal and Cardiovascular disease
Alpha-1-antichymotrypsin	Alpha-1-ACT, AACT, ACT, Antichymotrypsin	SERPINA3	14q32.13	ENSG00000196136	5.33	47,650.87	1.94	Alcoholic liver cirrhosis, Emphysema, Alzheimer`s disease
Alpha-1-antitrypsin	Alpha-1-antitrypsin, PI,	SERPINA1	14q32.13	ENSG00000197249	5.37	46,736.55	4.5	Liver diseases, Emphysema
Plasminogen	PLG	PLG	6q26	ENSG00000122194	7.04	90,568.99	0.05	Thrombosis
Isoform LMW of Kininogen-1	–	KNG1	3q27.3	ENSG00000113889	6.34	71,957.38	0.47	Coagulation
Fibrinogen beta chain	–	FGB	4q31.3	ENSG00000171564	8.54	55,928.15	4.26	Coagulation, Cardiovascular disease,
Alpha-2-macroglobulin	Transcuprein, CPAMD5	A2M	12p13.31	ENSG00000175899	6.03	163,291	2.06	Immune response
Hemopexin	HX, Beta-1B-glycoprotein	HPX	11p15.4	ENSG00000110169	6.55	51,676.37	1.24	Heme-binding and transporter
Histidine-rich glycoprotein	THPH11, HRGP	HRG	3q27.3	ENSG00000113905	7.09	59,578.32	0.47	Coagulation, angiogenesis
Prothrombin	Factor-II	F2	11p11.2	ENSG00000180210	5.63	70,036.87	0.33	Coagulation
Vitronectin	VN	VTN	17q11.2	ENSG00000109072	5.55	54,305.59	0.29	Thrombosis
Von Willebrand factor	Factor VIII, FVIII	VWF	12p13.31	ENSG00000110799	5.29	309,264.51	0.01	Angiogenesis, Liver disease
Thrombospondin-1	TSP-1	THBS1	15q14	ENSG00000137801	4.71	129,382.67	0.15	Chronic liver disease
Vitamin D-binding protein	DBP, GC-globulin	GC	4q13.3	ENSG00000145321	5.32	52,917.54	0.68	Coagulation, Vitamin D storage and transport
Isoform 2 of Clusterin	CLU2	CLU	8p21.1	ENSG00000120885	5.88	52,494.58	0.49	Not known
Protein AMBP (Fragment)	–	AMBP	9q32	ENSG00000106927	5.95	38,999.48	0.9	Not known
Isoform 1 of Vinculin	–	VCL	10q22.2	ENSG00000035403	5.5	123,799.3	0.17	Not known
Tetranectin	TN, C-type lectin domain family 3 member B, Plasminogen kringle-4-binding protein	CLEC3B	3p21.31	ENSG00000163815	5.52	22,536.81	0.29	Liver and cardiac fibrosis
Isoform 10 of Fibronectin	Fibronectin isoform 10 precusor	FN1	2q35	ENSG00000115414	5.31	272,320.36	0.56	Liver fibrogenesis
Talin-1	–	TLN1	9p13.3	ENSG00000137076	5.77	269,767.06	0.34	Liver diseases
Filamin-A	ABP280	FLNA	Xq28	ENSG00000196924	5.7	280,739.01	0.18	Hepatocellular carcinoma,
Coagulation factor XIII A chain	Fibrin stabilizing factor-A	F13A1	6p25.1	ENSG00000124491	5.69	83,268.28	0.12	Coagulation
Coagulation factor XIII B chain	Fibrin stabilizing factor-B	F13B	1q31.3	ENSG00000143278	6.01	75,510.68	0.06	Thrombosis
Coagulation factor XII	Hageman factor	F12	5q35.3	ENSG00000131187	8.04	67,792.09	0.07	Angiogenesis

pI Isoelectric pH, LC–MS Liquid chromatography-mass spectrometry, emPAI Exponentially modified protein abundance index

GO (Gene Ontology)-Term Enrichment and String Analysis

Gene ontology analysis provides a comprehensive resource regarding the functions of genes and gene products. The Fig. 2 represents the list of proteins by their gene names classified based on three categories such as (i) biological process, which consists of cellular response, cell adhesion, coagulation, and secretion subdivisions; (ii) cellular component consists of extracellular and granules as subdivisions and (iii) molecular function includes collagen binding, growth factor binding, heparin-binding, serine-type endopeptidase inhibitor, metal ion binding, and Ca-dependent protein binding. Multiple proteins listed in the table are mainly activating during an inflammatory response. Inflammation is one of the significant risk factors for ALD. The proteins SERPING1, SERPIND1, SERPINC1, SERPINA8, SERPINA7, SERPINA6, SERPINA5, SERPINA4, and SERPINA3 belong to the serpin superfamily and are involved in the negative regulation of endopeptidase activity. As endopeptidases have a similar function as matrix metalloproteases, the balanced action of endopeptidases helps regulate the condition of liver function. The proteins such HRG, von willebrand factor (vWF), SERPING1, SERPIND1, SERPINC1, PLG, and FGB have biological roles in hemostasis. The increased vWF plasma levels were observed in cirrhotic patients in support of the reconstitution of hemostasis. Impairment of platelet activation and the membrane phosphatidyl-serine function leads to possible primary hemostasis and risk of bleeding in progressive ALD. Four proteins are involved in platelet activation as mentioned in figure based on cellular components (Fig. 2a). The proteins are classified mainly into three types such as membrane, granules, and microparticles. These alpha-granule proteins include vWF, THBS1, SERPINA8, SERPINA5, PLG, and FGB. A large portion of the proteins localized in the extracellular exosome, extracellular region, and extracellular space. Most of the proteins involved in degranulation end up in the extracellular space. The molecular function of these proteins is enriched due to heparin binding, serine-type endopeptidase inhibitor activity, and Ca-dependent protein binding (Fig. 2a).

Fig. 2.

a GO term enrichment analysis of biological processes, cellular components and molecular functions; b STRING analysis

String analysis was employed to monitor possible connectivity networks between candidate proteins identified in this study. The functional association is the primary interaction unit of string analysis, i.e., a link between proteins contribute to a specific biological function [12]. The interaction cluster consists of three different colors red, blue, and green. A group of blue sets consists of FLNA, SERPINA1, CLU, THBS1, TLN1, and VCL. No binding partners were identified for GC, SERPINA4, GIG25, SERPINA7, and HPX. A group of green clusters consists of AMBP, CLEC3B, F12, F13A1, F13B, GIG25, HPX, HRG, KNG1, PLG, SERPINA6, and VTN. A group of red clusters consists of A2M, AGT, F2, FGB, SERPINA5, SERPINC1, SERPIND1, SERPING1, and vWF (Fig. 2b).

Discussion

Chronic alcohol consumption is a major health concern with vast social, economic, and clinical consequences, with a mortality rate of 3.3 million in 2012 [13]. Increased alcohol intake over decades leads to the deterioration of most of the organs in the human body. The liver sustains an increased degree of tissue injury due to alcohol intake, whereas liver is the primary site of ethanol metabolism. Acute alcohol intoxication damages hemostasis [14]. Hyperfibrinolysis enhanced intravascular coagulation, either quantitative or qualitative platelet defects, and reduced synthesis of clotting factors are well observed in chronic liver diseases [15]. The liver parenchymal cell produces most of the coagulation factors except vWf. Consumption of alcohol contributes to variation in plasma levels of both vWF and ADAMTS13 [16]. The liver mainly produces ADAMTS13, which cleaves vWF, thereby preventing formation of microthrombi. It has a functional role in the pathogenesis of the liver disease. Our study found this protein in platelet of ALD patients. Increased vWF and deceased ADAMTS13 might lead to thrombotic risk, whereas ADAMTS13 is positively associated with gamma-glutamyl transferase (GGT) [17]. GGT is a liver enzyme that can predict ALD progression. Hence the lower levels of ADMTS13 can predict progression of ALD.

The majority of these platelet proteins are localized in alpha granules. The proteins released from the alpha-granules contributed to angiogenesis, inflammation, and coagulation. Proteins present in the alpha-granule membrane are associated with the platelet membrane and aid in a fusion of alpha-granules with the plasma membrane. These transmembrane proteins significantly regulate platelet function in ALD. Platelets also contain dense granules and lysosomes secreted upon platelet activation. Rijkers et al. identified 2501 proteins of which 21 proteins were significantly changed in stored platelets [18]. Even though the platelet functionality is reduced in stored condition, changes in proteins level appear to be relatively minor but their ability to respond to agonists in a coordinated manner is greatly reduced. We identified data set of 536 accurately quantified platelet proteins of patients with ALD. In that identified platelet proteins, most of the proteins belong to the serpin superfamily. Lower levels of serpins in plasma have been associated with an increased risk of thrombosis [19]. Anticoagulant serpins act as negative regulators of coagulation. Serpins inhibiting target coagulation proteases by forming irreversible complexes. Present study found CLEC3B and SERPINA4 in platelet dense granule lumen. Whereas SERPINA4 is multifunctional and expressed in liver. The concentration of SERPINA4 depends on the degree of severity of ALD. Antithrombin and SERPINA4 was involved in inhibition of plasma kallikrein. Kallikrein-kinin system has an important role in coagulation, inflammation, and tissue injury and its regulatory activity in liver injury is yet to be defined. C1-inhibitor is an anticoagulant serpin. C11NH is a C1 inhibitor that protects from alcohol-induced liver disease. In contrast, C11NH is encoded by SERPING1. A study on ethanol-fed mice showed that C11NH levels are associated with reduced liver injury [20].

Platelets are anuclear cells derived from megakaryocytes. It plays an essential role in hemostasis and secretion of growth factors such as platelet-derived growth factor (PDGF) and hepatocyte growth factor. Michalak et al. noticed positive dependence between PCT, TGF-alpha, and PDGF-AB in alcohol-related liver cirrhosis [4]. Liver disease is associated with abnormality in fibrinolysis. Increased plasmin activity and decreased levels of plasmin inhibitors have been observed in cirrhosis. The concentrations of t-PA, t-PA-PAI-1 complex and PAI-1 antigen were all considerably increased in ALD/cirrhosis [21]. Liver fibrogenesis is initiated by the destruction of hepatocytes and exemplifies the wound-healing process leading to increased deposition of matrix proteins, elastin, collagen, proteoglycans, and glycoproteins. Increased platelet count is directly correlated with the reversal of liver fibrosis. Platelet can exert positive effect on the regeneration of the liver. Alcohol suppresses platelet aggregation via anti-thrombotic activity. Alcohol may alter the levels of blood coagulation factors including fibrinogen.

Thrombocytopenia is the most common complication of chronic liver disease. Thrombocytopenia contributes to the pathogenesis of cirrhosis. Activation of coagulation is essential to limit blood loss that requires balance between pro- and anti-coagulant proteins in the bloodstream. The platelets may be hyporesponsive or dysfunctional due to basal activation and degranulation during circulation [22]. Panova et al. concluded that a platelet-dependent thrombin generation assay could be used as a research tool to investigate the characteristics of platelets related to various clinical conditions [23]. Present findings listed blood coagulation proteins in patients with ALD. Anticoagulant proteins and protease inhibitors together function to inactivate proteases involved in the coagulation system, indicating negative regulation of coagulation by serpins. Inhibitory activity of serpins is enhanced by binding to heparin, a negatively charged glycosaminoglycans (GAGs). Present study listed some of the heparin-binding platelet proteins in patients with ALD. Antithrombin is a 58 kDa protein encoded by the SERPINC1 gene. A study indicated that the platelet count decreased with increased platelet activation. Platelet activation is based on the expression of platelet CD62p. Increased expression of platelet CD62 was observed ethanol-fed mice [24]. Moreover, chronic alcohol intake alone can cause chronic platelet activation. Platelet CD26p is mainly stored in alpha-granules, which is associated with platelet adhesion, inflammatory response, and platelet aggregation. It has been observed that some of the proteins expressed in alpha-granules are involved in platelet adhesion and aggregation during ALD progression. An endothelial injury could release collagen in several intracellular signaling pathways, leading to rapid platelet activation.

Biomarkers are now developing with enormous scientific and clinical value in a complete journey of disease progression. Present study identified a total of 536 proteins of which 31 were expressed in patients with ALD. Interestingly, although the platelet count is significantly reduced in these patients and significantly decreased platelet functionality can be observed during disease progression. Platelet proteomes indicating adhesion, hyperactivation, and aggregation can assert characteristics of disease pathogenesis. Current research revealed that platelet proteomes could possibly be used as potential diagnostic and prognostic markers in ALD. Proteomic applications have open novel signaling pathways and secreted proteins regulating platelet activation and inhibition. Validation of novel platelet proteins from present findings on ALD will broaden the knowledge. It may result in the identification of noninvasive biomarkers for improvement in tailored clinical management. Thus, an attempt to identify various platelet proteins as biomarkers significantly impacts clinical practice.

Conclusion

The advancement of diagnostic or therapeutic tools based on applying the platelet proteome approach in development of ALD is still far off. Hence, a platform for platelet and its proteome research may give diagnostic and prognostic insights into ALD. Present findings identified tetranectin, SERPINA4, and SERPING1 as new potential biomarkers in non-invasive diagnostics of ALD progression. These data demonstrated that involvement of platelet proteome during ALD pathogenesis results in reduced number of circulating platelets and impairment of hemostasis. These findings reveal the new potential biomarker of platelet proteome during pathogenesis of ALD in patients experiencing alcohol intoxication.

Supplementary Information

Below is the link to the electronic supplementary material.

Author Contributions

The authors contributed equally for all its content, writing and reviewing, and/or editing of the manuscript before submission.

Funding

We are not received any fund from internal or external sources for this study.

Declarations

Conflict of interest

The authors declare no conflicts of interest.

Consent Informed

Consent was obtained from all the study participants.

Ethical Approval

All are in accordance with ethical standards.