Coagulation indicators and their association with apoptosis ,ammatory indicators and Sharp scores of rheumatoid arthritis

Recent studies have shown that coagulations indicators hava a strong correlation with apoptosis ,ammatory indexes.In the present study, we measured coagulation indicators such PAF,PGI2 and TXB2 in patients with RA and investigated their association with apoptosis,ammatory indicators and Sharp scores of rheumatoid arthritis (RA). A total of 90 patients diagnosed with rheumatoid were enrolled for comparisons. The clinical and laboratory data of were retrospectively reviewed. The X-ray ndings of both were scored according to Sharp's In addition, the of patients with apoptosis indicator,coagulation indicator and ammatory indicator. Explored the relationship between coagulation indiccators and the above indicators.


Abstract Background
Recent studies have shown that coagulations indicators hava a strong correlation with apoptosis , ammatory indexes.In the present study, we measured coagulation indicators such PAF,PGI2 and TXB2 in patients with RA and investigated their association with apoptosis, ammatory indicators and Sharp scores of rheumatoid arthritis (RA).

Methods
A total of 90 patients diagnosed with rheumatoid arthritis were enrolled for comparisons. The clinical and laboratory data of patients were retrospectively reviewed. The X-ray ndings of both hands were scored according to Sharp's criteria. In addition, the statistics of patients with apoptosis indicator,coagulation indicator and ammatory indicator. Explored the relationship between coagulation indiccators and the above indicators.

Results
In this study,there was a strong correlation between the abnormal increase of apoptosis parameters,immune in ammation indicators,Sharp Score and the increase of coagulation indicator in patients with rheumatoid arthritis, and the increase of Sharp Scores,Caspase-3,FasL indexes or the decrease of C-reactive protein (CRP)could be risk factors for the development of RA. The higher the coagulation indicator, the higher the Sharp score,and the high parameters of hypercoagulable state RA.

Conclusions
Higher coagulation indicators are associated with RA disease activity, as well as apoptosis , ammatory indicators and decreased physical activity. Moreover, coagulation indicator are associated with Sharp scores.Coagulation indicator are important for the diagnosis of rheumatoid arthritis and are potential markers of activity and hypercoagulable state in RA patients.

Background
Rheumatoid arthritis (RA) is chronic in ammatory auto-immune disease characterized by persistent synovitis,systemic in ammation, production of autoantibodies,and bone destruction of joints. RA is more frequent among women than men (3:1) and its prevalence is 0.5-1.0% in the adult population [1,2] . A large number of clinical studies have shown that coagulations indicators hava a strong correlation with apoptosis , ammatory indexes ,PAF,PGI2 and and TXB2 have been widely con rmed to be associated with immune and apoptosis in RA,PAF is closely related to synovial in ammation and neovascularization in RA [3,4] . Recent studies have found that TxB2/PGI2 pathway can be involved in the pathological changes of angiogenesis of synovial tissue [5] .
Erosive joint damage and bone destruction are the most common manifestation of RA, which might induce ankylosis, malformation, even loss of normal joint function. Current goals of treatment in RA include achieving disease remission, reducing functional disability as well as minimizing pain. Erosions are the hallmark of bone destruction in RA. Radiographic grading of hands is the most commonly used method for the evaluation of different levels of bone erosion in clinical practice. Sharp score have also been used to measure morphological parameters that quantify the bone erosion and bone destruction, giving useful information for early detection and early treatment of RA. Therefore,we aimed to assess the coagulation index and its associations with apoptosis , ammatory indicators and sharp score in RA and to nd a common potential cause of the coagulation status in RA patients.

Methods
Patients and study design A total of 90 RA patients were included in this retrospective study,which were obtained from the First

Data Collection and Measures
All RA patients data were recorded soon after hospitalization,which including the details regarding patients'past medical,personal,and family histories. Besides this,RA-related data (age,gender,smoking history,alcohol consumption history,disease duration,treatment-condition) was collected from medical records. On the second day of hospitalization,patients general condition was monitored via vital signs,Xray radiography of both hands,Sharp scores,blood biochemistry tests,apoptosis index,coagulation index.
X-ray radiography of both hands were performed to assess the extent of joint destruction,bone erosion and joint space narrowing. The Sharp score of each patients was calculated by 2 independent blinded radiologists. Each joint bone erosion was scored as follows: 0 = normal;1 = cell in ltration with no signs of joint erosion; 2 = in ammation with the presence or erosions limited to discrete foci; and 3 = severe and extensive joint erosion with loss of architecture. Each joint stenosis was scored as follows: 0 = normal; 1 = focal or doubtful narrowing; 2 = generalized narrowing < 50%; 3 = generalized narrowing > 50% or subluxation; 4 = complete luxation or bony ankylosis.

Statistical Analysis
Descriptive results were presented as either n (%) or mean (SD). We conducted univariate analyses on important covariates,including age,gender,anti-CCP or RF positivity,PAF,PGI2,TXB2,Sharp score,caspase-3,caspase-8,Fas,FasL,PDL-2,Bax,Bcl-2,ESR,hs-CRP,IGA,IGG,IGM,C3,and C4.Continuous variables with a normal distribution were compared using Student's t-test,while non-normally distributed variables were compared using the Wilcoxon rank sum test. Categorical variables were evaluated using Fisher's exact test. The correlations between Sharp score and coagulation index were analyzed by Spearman's correlation coe cients. In addition,we constructed a binary linear regression model to determine the association of important covariates with Sharp score. Statistical tests were performed in Excel or in GraphPad Prism version 8.0.
The indicators rise was set to "T",while the indicators decline was set to "F". The Aprior module of SPSS Clementine 11.1 software was used to analyze the correlation between Chinese herbal medicines.The most famous association rule is the Apriori algorithm,which aims to nd out the relationship between items in a data set,also known as shopping blue analysis. In our data,each drug was treated as a variable.The formulae were as follows: where X→Y is an association rule,X (left-hand side [LHS]) and Y (right-hand side [RHS]) represent the set of herb items,σ(X) is the frequency of itemset X,X∪Y is the union of itemset X and Y,σ(X∪Y) is the frequency with which itemset X and itemset Y appear together,support(X→Y) is the frequency with which X and Y appear together,and con dence(X→Y) is the probability that itemset Y appears in the presence of X. The lift is the ratio of the probability of itemset Y appearing in the presence of X to the frequency of Y. Support and con dence are often used to eliminate meaningless combinations; lift is the validity of the rules.

Characteristics of the Study Population
The study sample was composed of 90 RA patients,with a median age of 55 years (range 30-85) and of whom 90% were female. The median number of Sharp score was 69.00 (range21-226.5). The median disease duration was 10.0 (range 0.5-40) years (Table 1). To determine whether correlations existed between Coagulation indicators and apoptosis indicators, ammatory indicators ,Sharp score,a Spearman correlation test was performed. Sharp score,TNF-a,,disease duration were all positively correlated with PAF,caspase-3,FasL,RF were all positively correlated with PGI2,IgG were negatively correlated with PGI2,IL-11,FasL were all positively correlated with TXB2.
. (I-J) There was a close positive correlation of the TXB2 with IL-11,FasL. Logistic regression analysis of risk factors of coagulation indicators was carried out. Signi cant differences in coagulation index was found between RA patients with Sharp score (p = 0.000),CRP (p = 0.023),caspase-3 (p = 0.039),FasL(p = 0.003),indicating that Sharp score,CRP were risk factors for PAF,that caspase-3,FasL were risk factors for PGI2,that Sharp score,FasL were risk factors for TXB2,the higher expression of Sharp score,caspase-3 and FasL,the higher expression of coagulation index,the higher expression of CRP,the lower expression of coagulation index (Table 2). Association rules analysis of coagulation index and apoptosis indicators, ammatory indicators ,Sharp score.
Association rules analysis of coagulation indexs and apoptosis indicators, ammatory indicators ,Sharp score can be found in Table 3. Set the minimum support to 80% and the minimum con dence to 80%. Through Aprior module analysis,the correlation between coagulation indexs and apoptosis indicators, ammatory indicators ,Sharp score was obtained,and the degree of lift was more than 1 and P 0.05. Table 4. Association rules analysis of coagulation index and apoptosis indicators, ammatory indicators ,Sharp score.

Discussion
The association between coagulation indicators and RA has been reported in clinical studies.PAF is a kind of endogenous phospholipid with biological activity.The most effective potent lipid medium found so far, PAF is closely related to synovial in ammation and neovascularization in RA.In a hurry,Bioactive PAF is present in synovial uid of chronic arthritis [3] .PAF not only promotes the synthesis and release of interleukin (IL-1), IL-2, IL-6, and tumor necrosis factor (TNF), but also ampli es the in ammatory response and stimulates the proliferation of synovial cells [4] .This study found that PAF was positively correlated with TNF-A and was associated with many in ammatory responses.Deng et al. found that a variety of PAF subtypes and PAF-like lipids can activate in ammasomes,leading to apoptosis of IL-1 cells and il-18 maturation.To participate in RA immune in ammation and apoptosis imbalance,mediated the formation of bone destruction. Platelet -activating factor (PAF) mediates NLRP3 -NEK7 rather disturbingly named in ammasomes induction independently of PAFR. The correlation analysis of this study showed that Sharp score,TNF-A and course of disease were positively correlated with PAF,and SharpScore was a risk factor for increased PAF.TxA2 mainly from platelets, A strong product generated from physiological activity of peanut four dilute acid metabolism, produced by prostaglandin H2 through the thromboxane synthase, it is considered one of the speci c markers of platelet activation in vivo.TxA2 basically has two active: it is a strong blood small Plate aggregation can promote platelet aggregation to form thrombus;2 it is can make blood vessels, and bronchial smooth muscle contraction, the vascular tension has a regulatory effect.The unstable in aqueous solution, 30 seconds to hydrolysis into thromboxane B2 (TxB2). Recent studies have found that TxB2 pathway can be involved in the pathological changes of angiogenesis of synovial tissue [5] .TxA2 and PGI2 are a pair of arachidonic acid derivatives which are mutually resistant and regulated.TxA2 mainly derived from platelets, it has strong vasoconstriction and platelet aggregation. On the contrary, PGI2 mainly dilates blood vessels and inhibits platelet aggregation, which mainly comes from vascular endothelial cells.
This research also found that caspase-3,FasL,RF were all positively correlated with PGI2,IgG were negatively correlated with PGI2,IL-11,FasL were all positively correlated with TXB2.Apoptosis mainly involves FasL pathway,TNF pathway and granulocyte pathway.FasL system.Fas belongs to the tumor necrosis factor receptor (TNFR) superfamily. Activated T cells can overexpress the death receptor Fas ligand (FasL).Fas binds to FasL as a receptor receiving exogenous apoptotic signals,leading to recruitment of FAS-related death determinant (FADD).FADD binds to caspase-8 premolecules in DED clusters to activate apoptosis and activate the enzyme Caspase8.Caspase8 directly activates the apoptotic enzyme Caspase-3.At the same time,activation of Caspase8 can enhance the permeability of mitochondrial membrane and lead to the release of cytochrome C.Cytochrome C complex and monomer of Caspase9 combine to form a apoptosis complex,which activates the intrinsic apoptotic pathway [6] .(2) the TNF.Membrane type TNF -can activate caspase8 by binding to FADD,exerting effects and inducing apoptosis of ne cells.Granulase pathway.Granzyme can directly crack the caspase-3 precursor (procaspase-3) or indirectly lyse pro-caspase-8 to initiate apoptosis. [7] Caspase is involved in all the major apoptosis pathways,including FAS/FasL,TNF -/TNFR [8,9] . The apoptosis signal transduction initiated by granase requires the involvement of Caspase. Caspase-3,8 and 9 play a major role,and the main mechanism is as follows: Activate THE CASpase-activated DEoxyribonuclease (CAD) and cause DNA fragmentation by inactivating the apoptosis inhibitor ICAD [10] .Destroy the cell structure,the nuclear ber layer,destroy the nuclear membrane structure;(3) To disable the function of the regulatory protein,cut the protein modulation RA is a chronic systemic in ammatory autoimmune disease with the synovium as the main target tissue [11] .The prevalence of RA in China was about 0.32-0.36%,and its pathological features were synovial cell proliferation,in ammatory cell in ltration,synovial pannus formation,and invasion and destruction of cartilage and bone tissue [12] .Repeated joint in ammation,resulting in the destruction of joint structure,deformity,loss of function [13] .The etiology of this disease is not yet clear. Currently,there are mainly molecular simulation theory,IgG glycation defect theory,MHCII molecule overexpression,and abnormal apoptosis [14] to explain the pathogenesis of RA.In recent years,studies have suggested that the apoptosis of RA synovial cells, broblasts,lymphocytes,and chondrocytes is abnormal,and synovial hyperplasia is a manifestation of abnormal apoptosis [15] .The mechanism of APOPTOSIS in RA cells and its correlation with in ammation are mainly involved in FasL pathway,TNF pathway and granulase pathway.FasL system.Fas belongs to the tumor necrosis factor receptor (TNFR) superfamily [16] . Activated T cells can overexpress the death receptor Fas ligand (FasL).Fas binds to FasL as a receptor receiving exogenous apoptotic signals,leading to recruitment of FAS-related death determinant (FADD) [17] .FADD binds to caspase-8 premolecules in DED clusters to activate apoptosis and activate the enzyme Caspase8.Caspase8 directly activates the apoptotic enzyme Caspase-3 [18] .At the same time,activation of Caspase8 can enhance the permeability of mitochondrial membrane and lead to the release of cytochrome C.Cytochrome C complex and monomer of Caspase9 combine to form a apoptosis complex,which activates the intrinsic apoptotic pathway [19] .(2) the TNF.Membrane type TNF -can activate caspase8 by binding to FADD,exerting effects and inducing apoptosis of ne cells.Granulase pathway.Granzyme can directly crack the caspase-3 precursor (pro-caspase-3) or indirectly lyse procaspase-8 to initiate apoptosis.Caspase is involved in all the major apoptosis pathways,including FAS/FasL,TNF -/TNFR [20] . The apoptosis signal transduction initiated by granase requires the involvement of Caspase. Caspase-3,8 and 9 play a major role,and the main mechanism is as follows: Activate THE caspase-activated deoxyribonuclease (CAD) and cause DNA fragmentation by inactivating the apoptosis inhibitor ICAD.Destroy the cell structure,the nuclear ber layer,destroy the nuclear membrane structure;Make the regulatory protein lose its function,cut the regulatory domain and catalytic domain of the protein,and inactivate the protein [21] .
The association rules in this paper suggest that the increase of parameters of high coagulation state is strongly correlated with the increase of caspase-3,TNF-a,IL-11 and FasL,with the support degree greater than 80%,con dence degree greater than 89%,and improvement degree greater than 1. The Logistic regression analysis of the coagulation parameters and immune indicators showed that the coagulation index was strongly associated with the activity of RA and involved in disease progression as a risk factor.The index of coagulation can be used as an important clinical index to predict bone damage and affect bone erosion.

Conclusions
Higher coagulation indicators are associated with RA disease activity, as well as apoptosis , ammatory indicators and decreased physical activity. Moreover, coagulation indicator are associated with Sharp scores.Coagulation indicator are important for the diagnosis of rheumatoid arthritis and are potential markers of activity and hypercoagulable state in RA patients. Before participating in the study,the patients lled in a written informed consent forms. A written informed consent was obtained from all the study participants.(or their parent or legal guardian in the case of children under 16).

Consent to publish
The research has obtained the informed consent for the publication from that person (or their parent or legal guardian in the case of children under 18) about he details relating to an individual person.

Availability of data and materials
The raw data supporting the conclusions of this article will be made available by the authors,without undue reservation to any quali ed researcher.

Figure 1
Spearman correlation analysis of coagulation indicators and apoptosis index, ammatory indicators,Sharp score.