Increased level of fibrinogen chains in the proteome of blood platelets in secondary progressive multiple sclerosis patients

Abstract Epidemiological studies indicate a high risk of stroke, heart failure and myocardial infarction in patients with multiple sclerosis, especially in its secondary progressive (SPMS) phase. Some ischaemic events are directly associated with abnormal platelet functions and their prothrombotic activity. Recent reports, including this study, confirm the increased activation of circulating platelets in SPMS, and also show increased platelet reactivity, among other responses, as well as strong aggregation. In this current study, we conducted a comparative analysis of the platelet proteome in SPMS patients and in healthy controls, to demonstrate the quantitative and qualitative differences likely to affect functional changes observed in SPMS. During densitometry evaluation of 2‐D fluorescence difference gel electrophoresis, we observed differences between the electrophoretic patterns of SPMS platelets and the control samples. To determine a detailed characterisation of the proteome changes in the SPMS patients’ blood platelets, in the next stage, we performed mass spectrometry of selected spots and indicated the increased presence of four proteins (fibrinogen, α‐2 macroglobulin, septin‐14 and tubulin β‐1 chain). The most important of these is the increased amount of prothrombotic protein, fibrinogen, which seems to confirm the accuracy of the imaging and potentially explains the increased risk of platelet‐origin thrombotic events. This study provides new knowledge of the potential existence of the molecular mechanisms responsible for the acceleration of the platelet pro‐coagulant function in SPMS. This can help to identify new targets for therapy, which can then be used not only in the second stage of the disease.

symptoms. 1 On pathological and clinical grounds, MS is a heterogeneous disease, and therefore different pathophysiological pathways can be activated in different MS patients, to initiate and perpetuate the devastating pathologic process. 2 Four main subtypes of the disease are recognized as a result of MS's heterogeneous neurological symptoms. The most common is the first phase of MS, relapsing-remitting (RRMS), in which the disease fluctuates between periods of inflammation/demyelination and remission. Sometimes, the disease starts with a primary progressive stage (PPMS), which is characterized from the beginning by progression without any remission states. After several years of the disease's duration, when the axons have lost their ability to regenerate, in more than 80% of cases RRMS turns into a secondary progressive stage (SPMS), in which the patient suffers irreversible progressive disability. The least widespread of MS subtypes is progressive-relapsing (PRMS). 3 The nature of the pathophysiology of MS is very complex and almost always involves multiple types of cells. It has been well established that various immune cells, such as the T helper lymphocytes (Th1, Th17), B cells and macrophages, are involved in the pathogenesis of MS. 4 Blood platelets have also been suggested as contributing to the development of MS. The interaction of blood platelets and immune and endothelial cells is responsible for the disruption of the blood-brain barrier, which leads to infiltration of lymphocytes, and further, to the formation of inflammatory and demyelinating lesions in CNS. 5,6 It has been proved that blood platelets are an important factor in the development of inflammation in the early phase of MS. 7 The major role of blood platelets in the development of inflammatory response comes from the ease with which they activate and adhere to inflamed endothelial cells or protein components, located within the sub-endothelial layer of blood vessel walls, as well as to the tendency of platelets to aggregate or form conglomerates with leucocytes. [8][9][10] Although studies have confirmed blood platelet hyper-activation in RRMS, and have shown the role of these cells in the development of inflammation and autoimmune processes, 11 there is relatively little information on blood platelet functioning in SPMS. However, for the last few years, the role of platelets in haemostatic mechanisms in the development of the SPMS has been the subject of intensive studies by our research team. [12][13][14] The available data, including our previous studies, clearly indicate excessive intravascular activation of these cells and their hyperresponsiveness to the number of physiological activators in SPMS.
Intensified activation is manifested by, amongst other signs, an increase in adhesion properties, the formation of platelet aggregates and changes in the metabolism of blood platelets.
The latest epidemiological studies confirm the high risk of stroke or myocardial infarction in MS (especially in SPMS). That is, ischaemic events are directly associated with irregular platelet functions and their prothrombotic activity. 15,16 An analysis of over 6000 patient deaths in the Danish National Registry of MS patients revealed that death by vascular or cardiac disease was the most frequently listed cause of death outside of MS itself. 17 Our existing findings state that platelet activation is an epiphenomenon consequent to the progression of MS, and probably secondary to endothelial injury, which causes the exposure of platelets to a variety of stimuli. [12][13][14] A study scheduled by our research group will, for the first time, direct the search for the origins of the hyperactivity of platelets towards molecular changes within platelets in SPMS. Our studies now are aimed at understanding the molecular mechanisms of the clearly heightened prothrombotic activity of blood platelets in SPMS, by analysing their proteomes. For this current study, we conducted a comparative analysis of the platelet proteomes of SPMS patients and healthy controls, to demonstrate the quantitative and qualitative differences in the functional changes observed in SPMS.  scores. 21 On the modified Rankin scale (mRS), patients ranged from 2 to 4 (between slight disability and moderately severe disability). The mRS is a clinician-reported measure of global disability, widely applied in measuring the degree of dependence in the daily activities of patients receiving physical therapy. 22 The Beck Depression Inventory (BDI scores) for the patients were 9.6 ± 4.6. BDI evaluation is one of the most widely used psy-

| Blood platelet isolation
The human blood samples were collected into S-Monovette® CPDA 1 (citrate phosphate, dextrose, adenine) tubes. All blood samples (control group and patients') were drawn in the morning (8 am-9 am) in fasting status, and stored using the same protocol. Blood was taken from the median cubital vein lying within the cubital fossa, anterior to the elbow. The blood platelets were isolated by differential centrifugation, as described by Wachowicz and Kustroń. 24 The whole-

| RE SULTS
To make a detailed assessment of the changes in the proteome of blood platelets in patients with SPMS, we performed two-step analysis of the clinical material using 2-D fluorescence difference gel electrophoresis (2D-DIGE) and MALDI-TOF/TOF mass spectrometry-based proteomics. In the first step of our research, we performed a two-dimensional, electrophoretic comparative separation of the blood platelet proteins obtained from the SPMS patients, and the healthy control group. During densitometric evaluation of the obtained digital images of protein maps, we observed differences between the electrophoretic patterns of SPMS platelets and the control samples ( Figure 1). In gels with SPMS platelet proteins, additional groups of peptides were present. Next, we performed spotmatching in gels (indicating the control platelets as a gel reference), to determine the peptide expression coefficient. The pixel volume of each spot was calculated, normalized and compared between the two groups using the Student's t test. For all the selected spots, the P-values between the SPMS and control groups were <0.05. Of all the peptides identified in the platelet proteome, nine spots (P1-P9 highlighted in Figure 1A) had significant up-regulation in expression, relative to the control. The greatest increase of expression (coefficient = 6.2 ± 1.6) was obtained for spot P9, while spots P5-P8 had expression coefficient values ranging from 4.2 to 3.4. Expression coefficients for peptides P1-P4 were in the range of 1.8-1.4. Detailed values for each spot are presented in Table 1.
For detailed characterization of the proteome changes in the blood platelets of the SPMS patients, in the next stage of our research we performed mass spectrometry analysis of the P1-P9 spots. The differentially expressed protein spots were excised from the preparative gels, subjected to trypsin digestion, then sequenced using MALDI-TOF/TOF spectrometry. All spots were successfully identified as known proteins in the SWISS-PROT database. The results of the mass spectrometry analysis for the identified spots are listed in Table 1. Peptides P1 and P2 were characterized as a fibrinogen beta chain, P3 was α-2 macroglobulin, P4 was septin-14, spots P5-P8 were derived from the fibrinogen gamma chain, and P9 from the tubulin β-1 chain ( Table 2).

| D ISCUSS I ON
Blood platelets are a crucial element of the coagulation process and are involved in various pathologies, such as atherosclerosis and thrombosis. Due to the large number of specific membrane receptors, blood platelets are highly reactive cells, readily activated by many physiological and un-physiological agonists. 26,27 In the human body, about 1 × 10 11 platelets are created every day as a result of complex processes of differentiation, maturation and fragmentation of megakaryocytes. 28,29 It has long been known that platelet proteins can have different origins-some are synthesized in megakaryocytes, and some derive directly from blood plasma. 30 However, studies completed in recent years have shown that un-nucleated platelets are able to succeed protein synthesis. About 5000 mRNA transcripts have been detected in blood platelets, which represent half of all transcripts located in megakaryocytes. 31 Additional blood platelets contain very stable mRNA transcripts with a long lifespan, correlated with platelet lifetime. This is very important, as F I G U R E 1 High-resolution 2D-DIGE electrophoretic patterns of blood platelet proteome obtained from SPMS patients (A), and the control group (B). Proteins were separated using isoelectric focusing (pH range 3-10, 7 cm), and 12.5% SDS-PAGE gels. This is a representative image of the 2D analysis. Spots highlighted as P1-P9 have increased expression in SPMS platelets in comparison to the control group, and were selected for mass spectrometry analysis the absence of a nucleus in these cells means they are unable to restore their mRNA pool. 32 Proteomics can be a helpful tool in the search for biomarkers, in the diagnosis of rare platelet disorders or in the dissection of molecular mechanisms underlying a specific condition. Proteomic blood platelet analysis is probably the best tool, and an invaluable one, for characterizing the fundamental processes that affect platelet homeostasis, thus determining the roles of platelets in health and disease. 33 Studies performed in the last few years have demonstrated that changes in blood platelet proteomes are associated with the risk of thrombotic events. The first studies that can be said to have initiated the field of platelet proteomics in clinical research were performed in 2008 by Arias-Salgado et al. 34 In that paper, the authors described blood platelets obtained from patients with arterial thrombosis having three decreased protein spots, and five increased protein spots, involved in cytoskeletal organization. Subsequent  This research group identified six differentially expressed proteins: two involved in energy metabolism, three associated with the cytoskeleton and one involved in protein degradation.
Our two-dimensional differential gel electrophoresis and mass spectrometry-based proteomic study indicated the increased pres- GPIIb-IIIa on agonist-stimulated platelets with a dissociation constant (Kd) of approximately 100 nM, which is nearly 100 times less than the concentration of fibrinogen in plasma. 39 Blood platelets contain three types of granules in their structure: dense granules, lambda granules and alpha granules. Activated platelets secrete the contents of these granules through their canalicular systems to the exterior environment. Fibrinogen is a part of the alpha granules' content, and is realized during activation. 26 These released molecules can play a major role in the formation of blood platelet aggregates. A study performed by Ang et al 40 suggested that the plasma fibrinogen level increases blood platelet reactivity, and is a significant predictor of coronary ischaemic events.
There is relatively little information about blood platelets' functioning in MS However, the available data, including from our own earlier studies, 12,14 clearly indicate that SPMS platelets are more sensitive to agonists and that their response is significantly stronger than that of platelets obtained from healthy patients. The mechanisms of increased platelet activation in MS also remain unknown. Our comparative analysis of the platelet proteome presented in this study demonstrated significant differences in the most important prothrombotic protein-fibrinogen, which seems to confirm the accuracy of, and potentially explain, our previous observations. Understanding

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors confirm no conflicts of interest.