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Plasma fibrin clot proteomics in healthy subjects: Relation to clot permeability and lysis time
Journal of Proteomics 208 (2019) 103487
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Plasma fibrin clot proteomics in healthy subjects: Relation to clot permeability and lysis time
T
Michał Ząbczyka,b,1, Aneta Stachowiczc,d,1, Joanna Natorskaa,b, Rafał Olszaneckic, ⁎ Jacek R. Wiśniewskid, Anetta Undasa,b, a
Institute of Cardiology, Jagiellonian University Medical College, Krakow, Poland Krakow Center for Medical Research and Technology, John Paul II Hospital, Krakow, Poland c Chair of Pharmacology, Jagiellonian University Medical College, Krakow, Poland d Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany b
A R T I C LE I N FO
A B S T R A C T
Keywords: Fibrin Clot Proteomics Clot properties
Background: Little is known about fibrin clot composition in relation to its structure and lysability. We investigated plasma clots protein composition and its associations with clot properties. Methods: We studied 20 healthy subjects aged 31–49 years in whom plasma fibrin clot permeability (Ks) and clot lysis time (CLT) were determined. A proteomic analysis of plasma fibrin clots was based on quantitative liquid chromatography-mass spectrometry. Results: Among 494 clot-bound proteins identified in all clots, the highest concentrations were for fibrinogen chains (about 64% of the clot mass) and fibronectin (13%). α2-antiplasmin (2.7%), factor XIIIA (1.2%), complement component C3 (1.2%), and histidine-rich glycoprotein (HRG, 0.61%) were present at relatively high concentrations. Proteins present in concentrations < 0.5% included (pro)thrombin, plasminogen, apolipoproteins, or platelet factor 4 (PF4). Fibrinogen-α and -γ chains were associated with age, while body-mass index with clot-bound apolipoproteins (all p < .05). Ks correlated with fibrinogen-γ and PF4 amounts within plasma clots. CLT was associated with fibrinogen-α and -γ, PF4, and HRG (all p < .05). Conclusions: This study is the first to show associations of two key measures of clot properties with protein content within plasma clots, suggesting that looser fibrin clots with enhanced lysability contain less fibrinogen-γ chain, platelet-derived PF4, and HRG. Significance: Our study for the first time suggests that more permeable fibrin clots with enhanced lysability contain less fibrinogen-γ chain, platelet-derived factor 4, and histidine-rich glycoprotein, which is related to accelerated clot lysis. The current findings might have functional consequences regarding clot structure, stability, and propagation of thrombin generation, and detailed proteomic analysis of clots in various disorders opens new perspective for coagulation and fibrin research.
1. Introduction There is growing evidence that plasma clot properties are involved in the pathogenesis of venous and arterial thromboembolic events [1,2]. The prothrombotic fibrin clot phenotype, tested mainly in plasma-based assays, including denser fibrin networks relatively resistant to lysis, has been shown as a prognostic marker in patients with venous thromboembolism (VTE) [3–5], coronary artery disease [6] or ischemic stroke [7]. Clot properties are modulated by environmental and genetic factors [8,9]. A number of circulating blood proteins and those derived from cellular blood components or endothelial cells may
affect fibrin clot characteristics [9]. Despite multiple functional assays used to assess fibrin clot properties, the plasma clot proteome has not yet been extensively studied. In plasma clots formed from healthy subjects Talens et al. identified 18 proteins related to blood coagulation, fibrinolysis, lipid metabolism, immune system, and protease inhibition, using 2D gel electrophoresis, mass spectrometry, and Western blot analysis [10]. It has been demonstrated that fibrin network binds in a covalent and non-covalent manner plasma proteins, including albumin, α2-macroglobulin, α2-antiplasmin, apolipoprotein A-IV, A-I, J, and E, along with plasminogen, factor (F)XIII, thrombin, vitronectin, and fibronectin [10]. Moreover,
⁎
Corresponding author at: Institute of Cardiology, Jagiellonian University Medical College, 80 Pradnicka St., 31-202 Cracow, Poland. E-mail address: [email protected] (A. Undas). 1 Authors equally contributed. https://doi.org/10.1016/j.jprot.2019.103487 Received 27 June 2019; Received in revised form 2 August 2019; Accepted 10 August 2019 Available online 16 August 2019 1874-3919/ © 2019 Elsevier B.V. All rights reserved.
Journal of Proteomics 208 (2019) 103487
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were assayed by routine laboratory techniques. Fibrinogen was determined using the Clauss method. High-sensitivity C-reactive protein (CRP) was determined using immunoturbidimetry (Siemens, Marburg, Germany).
actin derived from a small number of platelets present in platelet-poor plasma has been identified within clots [10]. We have shown differences in the protein composition of clots prepared from plasma of patients with an acute phase of myocardial infarction and 2 months later [11]. Post-myocardial infarction clots displayed more heterogeneous protein composition with a variety of molecules incorporated, including haptoglobin or calmodulin [11]. The application of proteomic technologies represents a novel approach to analyze plasma clot-bound proteins giving a unique opportunity to identify and quantify a large number of unique peptides [12,13]. In plasma fibrin clots of patients with antiphospholipid syndrome (APS), compared with VTE subjects and controls decreased amounts of (pro)thrombin, antithrombin III, apolipoprotein A-I, and histidine-rich glycoprotein (HRG) were identified [13]. There were no differences in plasma levels of antithrombin or prothrombin, but lower plasma HRG and apolipoprotein A-I were found [13]. The most differentially changed proteins within the clots of APS patients were bone marrow proteoglycan, complement C5-C9, immunoglobulins, apolipoprotein B-100 or platelet-derived proteins [13]. To our knowledge, there have been no reports assessing correlations between plasma clot properties and protein composition using proteomic analysis. This study investigated the quantitative protein composition of clots prepared ex vivo from citrated plasma of healthy subjects and assessed potential associations of clot-bound protein content with plasma clot permeability and lysis time.
2.1. Plasma clot proteomics Plasma clot proteomics was performed as previously described [12]. Briefly, to 100 μL of citrate plasma was added 20 mmol/L calcium chloride and 1 U/mL human thrombin (Merck, Kenilworth, NJ, USA). This mixture was placed into tubes which after 120 min of incubation were connected to a reservoir of a buffer (0.05 mol/L Tris HCl, 0.1 mol/ L NaCl, pH 7.5) to rinse a fibrin gel for 1 h to remove plasma proteins and heme catabolism products that had not been incorporated into the formed clots [14]. The clots were lysed in a buffer consisting of 0.1 M Tris-HCl, pH 8.0, 1% sodium dodecyl sulfate and 50 mM dithiothreitol at 96 °C for 10 min. Label-free quantitative proteomics of fibrin clots prepared ex vivo from citrated plasma was performed as previously described [12]. Briefly, lysates were processed by MED FASP method using endoproteinase LysC, trypsin, and chymotrypsin for consecutive protein digestion [15]. Protein and peptide concentrations were determined using WF-assay [16]. Aliquots containing 1 μg peptide mixture were analyzed using a QExactive HF mass spectrometer (ThermoFisher Scientific, USA). The mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier: PXD008434. The spectra were searched using Andromeda search engine built-in MaxQuant software. The maximum false peptide and protein discovery rate was specified as 0.01 [12]. Specific protein concentrations were calculated by the ‘Total Protein Approach’ (TPA) using a raw protein intensities from the MaxQuant output [17].
2. Methods We enrolled 20 medication-free apparently healthy subjects including members of the hospital staff and their families. Their characteristics are shown in Table 1. Subjects were eligible if they had no history of thromboembolic events (vein thrombosis, pulmonary embolism, MI, stroke), no signs of acute infection, or recent trauma or surgery. Pregnant women or those who delivered within previous 12 months were excluded. This study was conducted according to the principles expressed in the Declaration of Helsinki and was approved by the Bioethics Committee. All patients gave written informed consent. Fasting blood samples were drawn from an antecubital vein with minimal stasis using atraumatic venipuncture at 8 to 10 AM. Blood samples (vol/vol, 9:1 of 3.2% trisodium citrate) were spun at 2000g for 10 min, and the supernatants were aliquoted and stored at −80 °C for fibrin analysis. Blood cell count, lipid profiles (measured directly by homogenous enzymatic colorimetric method), glucose, and creatinine
2.2. Fibrin clot properties Plasma fibrin clot permeability was measured as described previously [3]. Briefly, 20 mM calcium chloride and 1 U/mL human thrombin (Merck, Kenilworth, NJ, USA) were added to citrated plasma. Tubes containing the clots were connected to a reservoir of a Trisbuffered saline. Its volume flowing through the gels was measured within 60 min. A permeation coefficient (Ks), which indicates the average size of pores formed in the fibrin network with low values indicating tightly packed fibrin structure, was calculated from the equation: Ks = Q × L × η/t × A × Δp, where Q is the flow rate in time t, L is the length of a fibrin gel, η is the viscosity of liquid (in poise), t is percolating time, A is the cross-sectional area (in cm2), and Δp is a differential pressure (in dyne/cm2). Clot lysis time (CLT) was measured as described [18]. Briefly, citrated plasma was mixed with 15 mM calcium chloride, human thrombin (Merck, Kenilworth, NJ, USA) at a final concentration of 0.5 U/mL, 10 μM phospholipid vesicles and 18 ng/mL recombinant t-PA (Boehringer Ingelheim, Ingelheim, Germany). The mixture was transferred to a microtitre plate and its turbidity was measured at 405 nm at 37 °C. CLT was defined as the time from the midpoint of the clear-tomaximum-turbid transition, which represents clot formation, to the midpoint of the maximum-turbid-to-clear transition. Intra-assay and inter-assay coefficients of variation were 6–8% for the two fibrin variables. All assays were performed in triplicates by the experienced investigator unaware of the results of proteomics.
Table 1 Baseline characteristics of healthy controls. Variable
Healthy controls (n = 20)
Age, years Male, n (%) Body mass index, kg/m2 Current smoking, n (%) White blood cell count, 103/μL Red blood cell count, 106/μL Platelet count, 103/μL Fibrinogen, g/L Glucose, mmol/L Creatinine, μmol/L Total cholesterol, mmol/L Low density lipoprotein cholesterol, mmol/L High density lipoprotein cholesterol, mmol/L Triglycerides, mmol/L High sensitivity C-reactive protein, mg/L Antithrombin, % Clot permeability (Ks), ×10−9 cm2 Clot lysis time (CLT), min
40 (31–49) 5 (25) 23.7 (21.1–25.7) 5 (25) 5.87 (4.99–7.53) 4.56 (4.39–4.79) 239 (211–290) 2.72 (2.34–3.05) 4.9 (4.6–5.1) 68 (60–85) 5.2 (4.6–5.9) 3.1 (2.7–3.6) 1.8 (1.6–2.0) 0.97 (0.7–1.20) 0.84 (0.59–2.11) 104 (94–112) 8.01 (6.47–9.93) 91 (82–109)
2.3. Statistical analysis Variables are presented as numbers (percentages), mean ± standard deviation (SD) or median and interquartile range (IQR) as appropriate. Normal distribution was assessed by Shapiro-Wilk test. The Pearson or Spearman rank correlation coefficients were calculated 2
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(a), kininogen-1, platelet factor 4 (PF4), and FV (Table 2). Of note, 17 (85%) clots showed detectable CRP, but in amounts below 0.001% of the clot mass, with no relation to plasma hsCRP concentrations. Age was positively associated with fibrinogen alpha and gamma chains within clots (r = 0.50, p = .024 and r = 0.54, p = .015, respectively), with no association of fibrinogen beta chain or plasma fibrinogen. Body-mass index (BMI) was positively associated with clotbound apolipoprotein A-I (r = 0.54, p = .018) and inversely with apolipoprotein(a) (r = −0.46, p = .049). A positive correlation between plasma low-density lipoprotein cholesterol (LDL-C) levels and both clot-bound apolipoprotein A-IV (r = 0.55, p = .012) and apolipoprotein B-100 (r = 0.46, p = .043) were found. Plasma high-density lipoprotein cholesterol levels correlated with clot-bound apolipoprotein (a) (r = 0.53, p = .017) and apolipoprotein E (r = 0.64, p = .0025). Similarly, total plasma cholesterol was associated with clot-bound apolipoprotein B-100 (r = 0.60, p-0.0053), apolipoprotein(a) (r = 0.50, p = .042), and apolipoprotein A-IV (r = 0.59, p = .0064). Clot-bound plasminogen (r = −0.62, p = .0034) and vinculin (r = −0.42, p = .032) correlated inversely with fibrinogen alpha and gamma chains, respectively. A positive correlation between FXIII A chain and fibronectin was observed within clots (r = 0.62, p = .0046). Clot-bound CRP was not associated with the content of any proteins detected within clots, except C1q (r = 0.51, p = .030).
Table 2 Quantitative analysis of selected clot-bound proteins of healthy subjects. Protein
Concentration (mg/g total protein)
Fibrinogen alpha chain Fibrinogen beta chain Fibrinogen gamma chain Fibronectin Alpha-2-macroglobulin Alpha-2-antiplasmin Serum albumin Complement C3 Coagulation factor XIII A chain Keratin, type II cytoskeletal 1 Extracellular matrix protein 1 Keratin, type I cytoskeletal 9 Histidine-rich glycoprotein Complement C4-B Apolipoprotein B-100 Apolipoprotein A-I Apolipoprotein A-IV Plasminogen/plasmin (Pro)thrombin Keratin, type I cytoskeletal 14 Keratin, type II cytoskeletal 5 von Willebrand factor Antithrombin-III Vitronectin Apolipoprotein E Kininogen-1 Apolipoprotein(a) Complement C5 Complement C4-A Complement factor H Platelet factor 4 Thrombospondin-1 Complement component C9 Vinculin Coagulation factor V Beta-2-glycoprotein 1
280 ± 12 210 ± 17 150 ± 11 130 ± 15 27 ± 7 22 ± 3 16 ± 9 12 ± 2.4 12 ± 2 9.3 ± 2 7.2 ± 2 6.5 ± 1 6.1 ± 1 4.1 ± 1.7 3.6 ± 3 2.6 ± 0.8 2.4 ± 1 2 ± 0.7 1.6 ± 0.5 1.5 ± 0.3 1 ± 0.2 0.99 ± 0.5 0.79 ± 0.1 0.78 ± 0.2 0.76 ± 0.4 0.34 ± 0.1 0.30 ± 0.3 0.22 ± 0.1 0.21 ± 0.2 0.14 ± 0.1 0.12 ± 0.1 0.088 ± 0.01 0.068 ± 0.02 0.057 ± 0.02 0.051 ± 0.03 0.039 ± 0.02
3.1. Fibrin clot properties In healthy controls Ks values were 8.0 ± 2.9 × 10−9 cm2. Ks correlated with age (r = −0.50, p = .024, Fig. 2A), BMI (r = −0.44, p = .048), plasma fibrinogen (r = −0.56, p = .01, Fig. 2B), total cholesterol (r = −0.48, p = .034), and LDL-C (r = −0.47, p = .038). Regarding proteins detected within plasma clots, Ks showed inverse associations with solely fibrinogen gamma chain (r = −0.54, p = .016, Fig. 2C) and PF4 (r = −0.46, p = .040, Fig. 2D). Of note, clot-bound HRG tended to correlate with Ks (r = −0.40, p = .07). A median CLT was 91 [82–109] min in the studied group. CLT correlated with age (r = 0.51, p = .025, Fig. 3A), plasma fibrinogen (r = 0.51, p = .027, Fig. 3B), total cholesterol (r = 0.70, p = .013) and LDL-C (r = 0.69, p = .015), but not with BMI. CLT was associated with the fibrinogen alpha and gamma chains (r = 0.52, p = .027 and r = 0.46, p = .046, Fig. 3C, D, respectively) and PF4 (r = 0.54, p = .016, Fig. 3E) within clots. Interestingly, CLT showed a strong, positive association with clot-bound HRG (r = 0.65, p = .0028, Fig. 3F). A borderline association between CLT and plasmin (r = −0.38, p = .08) was observed. There were no associations of Ks or CLT with clot-bound lipoproteins, antiplasmin, thrombin, antithrombin, and other proteins identified within the clots.
to test the association between 2 variables with a normal or non-normal distribution, respectively. Differences between 2 groups were compared using the Student's test for normally distributed continuous variables and for non-normally distributed continuous variables the MannWhitney U test was used. A two-sided p-value < .05 was considered statistically significant.
3. Results Proteomic analysis revealed a total of 494 proteins (available at ProteomeXchange Consortium, no. PXD008434) repeatedly identified in the plasma fibrin clots from all of 20 subjects. Among 494 clot-bound proteins we identified blood coagulation factors, lipoprotein metabolism proteins, fibrinolysis inhibitors and activators, proteins involved in immune responses, platelet-derived factors (Table 2), and many others. The highest average concentrations were found for the three fibrinogen chains (about 64% of the total clot protein, Fig. 1) and fibronectin (13%) (Table 2). Interestingly, not only proteins associated with coagulation and fibrinolysis, including α2-antiplasmin (α2AP, 2.3%) and FXIII A chain (1.2%), but also other proteins, including α2-macroglobulin (2.7%), albumin (1.6%), complement component C3 (1.2%), extracellular matrix protein 1 (0.72%), and HRG (0.61%) were present within the clot at relatively high concentrations (Table 2). Plasma albumin levels correlated with the clot-bound albumin (r = 0.43, p = .048). Other proteins incorporated within the fibrin network such as (pro)thrombin, plasminogen, von Willebrand factor, complement C4B, and apolipoproteins A and B-100 were detected at lower concentrations within the clots (ranged from 0.1 to 0.5% of the total clot protein mass). Proteins present at concentrations below 0.1% of the clot mass were antithrombin, vitronectin, apolipoprotein E, apolipoprotein
4. Discussion To our knowledge, this study is the first to show quantitative protein composition of fibrin clots generated from plasma of healthy subjects and its associations with two key measures of clot structure and function, i.e. clot permeability and lysis time. We demonstrated almost 500 proteins incorporated into the plasma fibrin clot, showing similar clot composition as obtained for VTE and APS patients [12,13]. Of note, the present study represents an approach able to quantify clot-bound proteins as the percentage of total clot-mass. In healthy subjects we observed associations between fibrin clot properties and the two clot components i.e. fibrinogen chains and PF4 using proteomic analysis. We failed to find any associations of lipidrelated parameters, inflammatory markers or fibrinolysis modulators with clot properties. This preliminary study suggests that density and lysability of plasma fibrin clots show associations with platelet activation in healthy subjects and possibly others in patients with diseases associated with prothrombotic states. Fibrin constitutes a major protein component of intravascular 3
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Fig. 1. Main clot protein composition in healthy subjects.
thrombi and in vitro prepared fibrin clots [8]. Fibrin networks composed of thin, highly compressed fibrin fibers are less permeable, more rigid, and less susceptible to lysis [9]. Fibrinogen concentrations explain up to 18% of the variation in Ks [8], indicating that other factors have to impact this key parameter. Interestingly, we found associations of age and fibrin clot properties with the content of fibrinogen alpha and gamma, but not beta chain, within plasma clots. This finding is surprising because we suspected similar associations of fibrin functional measures with the 3 fibrinogen chains forming the fibrinogen molecule and subsequently fibrin. First, we cannot exclude impaired digestion of the fibrinogen beta chain abolishing associations observed for the 2 remaining chains with Ks. It might be hypothesized that using the current proteomic analysis the fibrinogen gamma chain is the best marker of the fibrinogen content within plasma fibrin clots, which should be recommended while studying interactions among proteins and functional clot tests. In the current study we were not able to distinguish between fibrinogen gamma chain and its gamma' variant. Approximately 8%–15% of total fibrinogen in healthy individuals is composed of gamma/gamma' fibrinogen, which arises after polyadenylation leading to 20-amino acid extension (γ'408–427) substituting the 4 gamma chain amino acids (γA408–411) [19]. The presence of the gamma' chain leads to formation of fibrin clots with smaller pores and thinner fibers, which may be associated with the prothrombotic phenotype [19]. Given the observation that mutations affecting fibrin function, i.e. dysfibrinogenemias are common in the FGG gene [20], it is likely that proteomics of plasma fibrin clots in carriers of genetic abnormalities in FGG may yield new insights into the functional clot alterations, including clot lysability, reported in purified systems. Moreover, it should be mentioned that posttranslational modifications not only of the fibrinogen, but also of plasminogen or antiplasmin molecules, including oxidation, glycation, glycosylation or carbonylation increase with age and may contribute to altered fibrin clot properties [8]. New protein components documented in plasma clots by us and associated with clot properties in healthy individuals were HRG and
PF4. This observation might have potential clinical implications in various diseases linked with thrombotic tendency including platelet activation. PF4, a platelet-specific protein released from alpha granules upon platelet activation, has been previously shown to be associated with the prothrombotic plasma fibrin clot phenotype [21–23]. PF4 might associate with fibrin to impede the lateral association of protofibrils, decreasing porosity of the fibrin network [21]. In vitro, saturating amounts of PF4 (two fibrin molecules per one PF4 monomer) reduced fibrin porosity up to 4.4-fold and modified viscoelastic properties of fibrin network [21], while in patients with essential thrombocythemia and atrial fibrillation plasma PF4 levels correlated inversely with Ks [22,23]. This study supports the view that plateletderived substances, including PF4, are present in plasma clots not only in patients with large platelet counts or following acute thrombotic condition and may affect the fibrin network structure reflected by the pore size. The fact that the association of clot function with PF4 was demonstrated in young healthy subjects free of history of thromboembolism, a role of PF4 appears to be stronger than documented in the literature so far. Unexpectedly, we found a strong positive association of CLT with HRG, suggesting that under physiological conditions this abundant protein may modulate plasmin-mediated clot lysis. HRG is known to be involved in hemostasis displaying both anticoagulant and antifibrinolytic properties [24]. HRG deficiency has been associated with shortened plasma clotting time in human plasma [24] and with arterial thrombosis in HRG-knockout mice [25]. It has been shown in vitro using purified proteins that HRG retarded plasminogen activation by tissue plasminogen activator and interfered plasminogen binding to fibrin [26]. This antifibrinolytic effect of HRG has been proved in a functional test of clot lysis in the present study, which highlights an underestimated role of this protein in fibrin clot susceptibility to lysis. Further studies on HRG as a modulator of clot lysability in health and disease states are warranted. From the methodological point of view, this report provides additional evidence for the superiority of the current proteomic analysis 4
Journal of Proteomics 208 (2019) 103487
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Fig. 2. Associations of plasma fibrin clot permeability (Ks) with age (A), plasma fibrinogen (B), clot-bound fibrinogen gamma chain (C) and platelet factor 4 (D) in 20 healthy individuals.
compared with that used by Talens et al. [10]. who detected in fibrin clots of healthy subjects a relatively small number of proteins [10]. Our method corroborated the presence of multiple other proteins, undetectable by Talens et al. [10] most likely due to limitations of their approach, which was unable to identify low amounts of proteins. We believe that our technique could be recommended to assess protein composition of fibrin clots in various disease states. Interestingly, we were able to detect CRP within most plasma clots, which provides the first proteomic evidence of clot-bound CRP. These amounts of CRP showed no associations with key fibrin characteristics in our group, although it has been shown that both Ks and CLT in patients with chronic obstructive pulmonary disease correlated with plasma CRP levels [27]. A similar observation was made in patients with advanced coronary artery disease [28]. Salonen et al. [29] have shown that CRP binds to various proteins including fibrin(ogen) and thus can modify fibrin formation. Our findings suggest that the impact of CRP on clot properties could be observed in systemic inflammatory states but not in young healthy subjects even if CRP is bound to plasma fibrin clots as evidenced by us. This issue deserves further investigation. The abundance of various lipoproteins in fibrin clots of healthy subjects was identified in this study. We did observe associations of clot-bound lipoproteins with lipid profile in circulating blood. Previous studies have shown that plasma lipoprotein(a) [28,30] as well as high density lipoprotein cholesterol levels [31,32] had an impact on fibrin clot properties. In healthy subjects we did not find any associations between clot properties with clot-bound lipoproteins. Since it has been shown that statins as potent cholesterol-lowering agents improve fibrin
characteristics [6,33,34], our current study suggests that lipid-lowering effects of statins or other agents may at least in part explain antithrombotic effects via altering amounts of clot bound lipoproteins. Protein composition of plasma clots from statin-treated patients however remains to be established. This study has several limitations. First, the sample size was relatively limited. Second, fibrinogen consists of three non-identical pairs of disulfide-bonded chains with different molecular mass (95 kDa for alpha, 55.9 kDa for beta, and 51.5 kDa for gamma chain). To quantify clot-bound proteins each protein concentration was adjusted for a respective molecular weight giving the actual protein percentage of total clot-mass, which might influence data analysis. Third, detailed protein composition of clots made from human plasma have not been compared with protein composition of clots generated from isolated fibrinogen. However, the aim of this analysis was to seek for associations between plasma-based clot assays widely used in clinical settings and protein composition. Moreover, we did not assess fibrin formation since we focused on the two key parameters shown to correlate with clinically relevant outcomes and may have a predictive value for instance in VTE [3]. Finally, since it has been established that the presence of fibrinogen gamma’ chain unfavorably affects the fibrin clot structure [35] it also might influence fibrin clot proteomics. However, in our analysis we were not able to identify clot-bound fibrinogen gamma’ chain, probably due to predominance of the isoform gamma-A splice variant.
5
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Fig. 3. Associations of clot lysis time with age (A), plasma fibrinogen (B), clot-bound fibrinogen alpha chain (C), gamma chain (D), platelet factor 4 (E), and histidinerich glycoprotein (F) in 20 healthy individuals.
5. Conclusions
References
In conclusion, we demonstrated association of plasma fibrin structure and lysability with proteins forming clots, including PF4 and HRG. Further studies are needed to characterize the impact of various diseases as well as genetic and environmental factors, including medications, on clot proteomics.
[1] A.S. Wolberg, R.A. Campbell, Thrombin generation, fibrin clot formation and hemostasis, Transfus. Apher. Sci. 38 (2008) 15–23. [2] R.A. Ariëns, Fibrin(ogen) and thrombotic disease, J. Thromb. Haemost. 11 Suppl 1 (2013) 294–305. [3] A. Undas, K. Zawilska, M. Ciesla-Dul, A. Lehmann-Kopydłowska, A. Skubiszak, K. Ciepłuch, W. Tracz, Altered fibrin clot structure/function in patients with idiopathic venous thromboembolism and in their relatives, Blood 114 (2009) 4272–4278. [4] J. Siudut, M. Grela, E. Wypasek, K. Plens, A. Undas, Reduced plasma fibrin clot permeability and susceptibility to lysis are associated with increased risk of postthrombotic syndrome, J. Thromb. Haemost. 14 (2016) 784–793. [5] M. Zabczyk, K. Plens, W. Wojtowicz, A. Undas, Prothrombotic fibrin clot phenotype is associated with recurrent pulmonary embolism after discontinuation of anticoagulant therapy, Arterioscler. Thromb. Vasc. Biol. 37 (2017) 365–373. [6] A. Undas, M. Celinska-Löwenhoff, T. Löwenhoff, A. Szczeklik, Statins, fenofibrate, and quinapril increase clot permeability and enhance fibrinolysis in patients with coronary artery disease, J. Thromb. Haemost. 4 (2006) 1029–1036. [7] A. Undas, P. Podolec, K. Zawilska, M. Pieculewicz, I. Jedliński, E. Stepień, E. Konarska-Kuszewska, P. Weglarz, M. Duszyńska, E. Hanschke, T. Przewlocki, W. Tracz, Altered fibrin clot structure/function in patients with cryptogenic ischemic stroke, Stroke 40 (2009) 1499–1501. [8] A. Undas, R.A. Ariëns, Fibrin clot structure and function: a role in the pathophysiology of arterial and venous thromboembolic diseases, Arterioscler. Thromb. Vasc. Biol. 31 (2011) e88–e99. [9] M. Ząbczyk, A. Undas, Plasma fibrin clot structure and thromboembolism: clinical implications, Pol. Arch. Intern. Med. 127 (2017) 873–881. [10] S. Talens, F.W. Leebeek, J.A. Demmers, D.C. Rijken, Identification of fibrin clotbound plasma proteins, PLoS One 7 (2012) e41966. [11] M. Suski, J. Siudut, M. Ząbczyk, R. Korbut, R. Olszanecki, A. Undas, Shotgun analysis of plasma fibrin clot-bound proteins in patients with acute myocardial infarction, Thromb. Res. 135 (2015) 754–759. [12] A. Stachowicz, J. Siudut, M. Suski, R. Olszanecki, R. Korbut, A. Undas, J.R. Wiśniewski, Optimization of quantitative proteomic analysis of clots generated from plasma of patients with venous thromboembolism, Clin. Proteomics 14 (2017) 38. [13] A. Stachowicz, M. Zabczyk, J. Natorska, M. Suski, R. Olszanecki, R. Korbut, J.R. Wiśniewski, A. Undas, Differences in plasma fibrin clot composition in patients with thrombotic antiphospholipid syndrome compared with venous thromboembolism, Sci. Rep. 8 (2018) 17301. [14] M. Ząbczyk, A. Piłat, M. Awsiuk, A. Undas, An automated method for fibrin clot permeability assessment, Blood Coagul. Fibrinolysis 26 (2015) 104–109. [15] J.R. Wiśniewski, M. Mann, Consecutive proteolytic digestion in an enzyme reactor increases depth of proteomic and phosphoproteomic analysis, Anal. Chem. 84
Authors' contributions AU, RO, and JRW were responsible for the conception and design of the study. AS was responsible for analyses of the samples. MZ, AS, JN, AU and JRW were responsible for the interpretation of the data. MZ and JN drafted the article. All authors revised the paper critically for important intellectual content and gave final approval of the version to be published. All authors read and approved the final manuscript. Funding sources This work was supported by the Max-Planck Society for the Advancement of Science and by the German Research Foundation (DFG/Gottfried Wilhelm Leibniz Prize) and the Jagiellonian University Medical College (K/ZDS/007717, to A.U.). Availability of data and materials The mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier: PXD006814. Declaration of Competing Interest The authors declare that they have no competing interests. 6
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(2012) 2631–2637. [16] J.R. Wiśniewski, F.Z. Gaugaz, Fast and sensitive total protein and peptide assays for proteomic analysis, Anal. Chem. 87 (2015) 4110–4116. [17] J.R. Wiśniewski, Label-free and standard-free absolute quantitative proteomics using the "total protein" and "proteomic ruler" approaches, Methods Enzymol. 585 (2017) 49–60. [18] M. Pieters, H. Philippou, A. Undas, Z. de Lange, D.C. Rijken, N.J. Mutch, Subcommittee on Factor XIII and Fibrinogen, and the Subcommittee on Fibrinolysis. An international study on the feasibility of a standardized combined plasma clot turbidity and lysis assay: communication from the SSC of the ISTH, J. Thromb. Haemost. 16 (2018) 1007–1012. [19] S. Uitte de Willige, K.F. Standeven, H. Philippou, R.A. Ariëns, The pleiotropic role of the fibrinogen gamma' chain in hemostasis, Blood 114 (2009) 3994–4001. [20] S. Uitte de Willige, M.C. de Visser, J.J. Houwing-Duistermaat, F.R. Rosendaal, H.L. Vos, R.M. Bertina, Genetic variation in the fibrinogen gamma gene increases the risk for deep venous thrombosis by reducing plasma fibrinogen gamma' levels, Blood 106 (2005) 4176–4183. [21] A.A. Amelot, M. Tagzirt, G. Ducouret, R.L. Kuen, B.F. Le Bonniec, Platelet factor 4 (CXCL4) seals blood clots by altering the structure of fibrin, J. Biol. Chem. 282 (2007) 710–720. [22] R. Małecki, M. Gacka, M. Kuliszkiewicz-Janus, U. Jakobsche-Policht, J. Kwiatkowski, R. Adamiec, A. Undas, Altered plasma fibrin clot properties in essential thrombocythemia, Platelets 27 (2016) 110–116. [23] L. Drabik, P. Wołkow, A. Undas, Denser plasma clot formation and impaired fibrinolysis in paroxysmal and persistent atrial fibrillation while on sinus rhythm: association with thrombin generation, endothelial injury and platelet activation, Thromb. Res. 136 (2015) 408–414. [24] T.T. Vu, B.A. Leslie, A.R. Stafford, J. Zhou, J.C. Fredenburgh, J.I. Weitz, Histidinerich glycoprotein binds DNA and RNA and attenuates their capacity to activate the intrinsic coagulation pathway, Thromb. Haemost. 115 (2016) 89–98. [25] T.T. Vu, J. Zhou, B.A. Leslie, A.R. Stafford, J.C. Fredenburgh, R. Ni, S. Qiao, N. Vaezzadeh, W. Jahnen-Dechent, B.P. Monia, P.L. Gross, J.I. Weitz, Arterial thrombosis is accelerated in mice deficient in histidine-rich glycoprotein, Blood 125
(2015) 2712–2719. [26] H.R. Lijnen, M. Hoylaerts, D. Collen, Isolation and characterization of a human plasma protein with affinity for the lysine binding sites in plasminogen. Role in the regulation of fibrinolysis and identification as histidine-rich glycoprotein, J. Biol. Chem. 255 (1980) 10214–10222. [27] A. Undas, P. Kaczmarek, K. Sladek, E. Stepien, W. Skucha, M. Rzeszutko, I. Gorkiewicz-Kot, W. Tracz, Fibrin clot properties are altered in patients with chronic obstructive pulmonary disease: beneficial effects of simvastatin treatment, Thromb. Haemost. 102 (2009) 1176–1182. [28] A. Undas, D. Plicner, E. Stepien, R. Drwila, J. Sadowski, Altered fibrin clot structure in patients with advanced coronary artery disease: a role of C-reactive protein, lipoprotein(a), and homocysteine, J. Thromb. Haemost. 5 (2007) 1988–1990. [29] E.M. Salonen, T. Vartio, K. Hedman, A. Vaheri, Binding of fibronectin by the acute phase C-reactive protein, J. Biol. Chem. 259 (1984) 1496–1501. [30] A. Undas, E. Stepien, W. Tracz, A. Szczeklik, Lipoprotein(a) as a modifier of fibrin clot permeability and susceptibility to lysis, J. Thromb. Haemost. 4 (2006) 973–975. [31] M. Ząbczyk, Ł. Hońdo, M. Krzek, A. Undas, High-density cholesterol and apolipoprotein AI as modifiers of plasma fibrin clot properties in apparently healthy individuals, Blood Coagul. Fibrinolysis 24 (2013) 50–54. [32] R.C. Kotzé, R.A. Ariëns, Z. de Lange, M. Pieters, CVD risk factors are related to plasma fibrin clot properties independent of total and or γ' fibrinogen concentration, Thromb. Res. 134 (2014) 963–969. [33] J.H. Griffin, K. Kojima, C.L. Banka, L.K. Curtiss, J.A. Fernandez, High-density lipoprotein enhancement of anticoagulant activities of plasma protein S and activated protein C, J. Clin. Invest. 103 (1999) 219–227. [34] C. Oslakovic, M.J. Krisinger, A. Andersson, M. Jauhiainen, C. Ehnholm, B. Dahlbäck, Anionic phospholipids lose their procoagulant properties when incorporated into high density lipoproteins, J. Biol. Chem. 284 (2009) 5896–5904. [35] P. Allan, S. Uitte de Willige, R.H. Abou-Saleh, S.D. Connell, R.A. Ariëns, Evidence that fibrinogen γ' directly interferes with protofibril growth: implications for fibrin structure and clot stiffness, J. Thromb. Haemost. 10 (2012) 1072–1080.
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Journal of Proteomics journal homepage: www.elsevier.com/locate/jprot
Plasma fibrin clot proteomics in healthy subjects: Relation to clot permeability and lysis time
T
Michał Ząbczyka,b,1, Aneta Stachowiczc,d,1, Joanna Natorskaa,b, Rafał Olszaneckic, ⁎ Jacek R. Wiśniewskid, Anetta Undasa,b, a
Institute of Cardiology, Jagiellonian University Medical College, Krakow, Poland Krakow Center for Medical Research and Technology, John Paul II Hospital, Krakow, Poland c Chair of Pharmacology, Jagiellonian University Medical College, Krakow, Poland d Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany b
A R T I C LE I N FO
A B S T R A C T
Keywords: Fibrin Clot Proteomics Clot properties
Background: Little is known about fibrin clot composition in relation to its structure and lysability. We investigated plasma clots protein composition and its associations with clot properties. Methods: We studied 20 healthy subjects aged 31–49 years in whom plasma fibrin clot permeability (Ks) and clot lysis time (CLT) were determined. A proteomic analysis of plasma fibrin clots was based on quantitative liquid chromatography-mass spectrometry. Results: Among 494 clot-bound proteins identified in all clots, the highest concentrations were for fibrinogen chains (about 64% of the clot mass) and fibronectin (13%). α2-antiplasmin (2.7%), factor XIIIA (1.2%), complement component C3 (1.2%), and histidine-rich glycoprotein (HRG, 0.61%) were present at relatively high concentrations. Proteins present in concentrations < 0.5% included (pro)thrombin, plasminogen, apolipoproteins, or platelet factor 4 (PF4). Fibrinogen-α and -γ chains were associated with age, while body-mass index with clot-bound apolipoproteins (all p < .05). Ks correlated with fibrinogen-γ and PF4 amounts within plasma clots. CLT was associated with fibrinogen-α and -γ, PF4, and HRG (all p < .05). Conclusions: This study is the first to show associations of two key measures of clot properties with protein content within plasma clots, suggesting that looser fibrin clots with enhanced lysability contain less fibrinogen-γ chain, platelet-derived PF4, and HRG. Significance: Our study for the first time suggests that more permeable fibrin clots with enhanced lysability contain less fibrinogen-γ chain, platelet-derived factor 4, and histidine-rich glycoprotein, which is related to accelerated clot lysis. The current findings might have functional consequences regarding clot structure, stability, and propagation of thrombin generation, and detailed proteomic analysis of clots in various disorders opens new perspective for coagulation and fibrin research.
1. Introduction There is growing evidence that plasma clot properties are involved in the pathogenesis of venous and arterial thromboembolic events [1,2]. The prothrombotic fibrin clot phenotype, tested mainly in plasma-based assays, including denser fibrin networks relatively resistant to lysis, has been shown as a prognostic marker in patients with venous thromboembolism (VTE) [3–5], coronary artery disease [6] or ischemic stroke [7]. Clot properties are modulated by environmental and genetic factors [8,9]. A number of circulating blood proteins and those derived from cellular blood components or endothelial cells may
affect fibrin clot characteristics [9]. Despite multiple functional assays used to assess fibrin clot properties, the plasma clot proteome has not yet been extensively studied. In plasma clots formed from healthy subjects Talens et al. identified 18 proteins related to blood coagulation, fibrinolysis, lipid metabolism, immune system, and protease inhibition, using 2D gel electrophoresis, mass spectrometry, and Western blot analysis [10]. It has been demonstrated that fibrin network binds in a covalent and non-covalent manner plasma proteins, including albumin, α2-macroglobulin, α2-antiplasmin, apolipoprotein A-IV, A-I, J, and E, along with plasminogen, factor (F)XIII, thrombin, vitronectin, and fibronectin [10]. Moreover,
⁎
Corresponding author at: Institute of Cardiology, Jagiellonian University Medical College, 80 Pradnicka St., 31-202 Cracow, Poland. E-mail address: [email protected] (A. Undas). 1 Authors equally contributed. https://doi.org/10.1016/j.jprot.2019.103487 Received 27 June 2019; Received in revised form 2 August 2019; Accepted 10 August 2019 Available online 16 August 2019 1874-3919/ © 2019 Elsevier B.V. All rights reserved.
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were assayed by routine laboratory techniques. Fibrinogen was determined using the Clauss method. High-sensitivity C-reactive protein (CRP) was determined using immunoturbidimetry (Siemens, Marburg, Germany).
actin derived from a small number of platelets present in platelet-poor plasma has been identified within clots [10]. We have shown differences in the protein composition of clots prepared from plasma of patients with an acute phase of myocardial infarction and 2 months later [11]. Post-myocardial infarction clots displayed more heterogeneous protein composition with a variety of molecules incorporated, including haptoglobin or calmodulin [11]. The application of proteomic technologies represents a novel approach to analyze plasma clot-bound proteins giving a unique opportunity to identify and quantify a large number of unique peptides [12,13]. In plasma fibrin clots of patients with antiphospholipid syndrome (APS), compared with VTE subjects and controls decreased amounts of (pro)thrombin, antithrombin III, apolipoprotein A-I, and histidine-rich glycoprotein (HRG) were identified [13]. There were no differences in plasma levels of antithrombin or prothrombin, but lower plasma HRG and apolipoprotein A-I were found [13]. The most differentially changed proteins within the clots of APS patients were bone marrow proteoglycan, complement C5-C9, immunoglobulins, apolipoprotein B-100 or platelet-derived proteins [13]. To our knowledge, there have been no reports assessing correlations between plasma clot properties and protein composition using proteomic analysis. This study investigated the quantitative protein composition of clots prepared ex vivo from citrated plasma of healthy subjects and assessed potential associations of clot-bound protein content with plasma clot permeability and lysis time.
2.1. Plasma clot proteomics Plasma clot proteomics was performed as previously described [12]. Briefly, to 100 μL of citrate plasma was added 20 mmol/L calcium chloride and 1 U/mL human thrombin (Merck, Kenilworth, NJ, USA). This mixture was placed into tubes which after 120 min of incubation were connected to a reservoir of a buffer (0.05 mol/L Tris HCl, 0.1 mol/ L NaCl, pH 7.5) to rinse a fibrin gel for 1 h to remove plasma proteins and heme catabolism products that had not been incorporated into the formed clots [14]. The clots were lysed in a buffer consisting of 0.1 M Tris-HCl, pH 8.0, 1% sodium dodecyl sulfate and 50 mM dithiothreitol at 96 °C for 10 min. Label-free quantitative proteomics of fibrin clots prepared ex vivo from citrated plasma was performed as previously described [12]. Briefly, lysates were processed by MED FASP method using endoproteinase LysC, trypsin, and chymotrypsin for consecutive protein digestion [15]. Protein and peptide concentrations were determined using WF-assay [16]. Aliquots containing 1 μg peptide mixture were analyzed using a QExactive HF mass spectrometer (ThermoFisher Scientific, USA). The mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier: PXD008434. The spectra were searched using Andromeda search engine built-in MaxQuant software. The maximum false peptide and protein discovery rate was specified as 0.01 [12]. Specific protein concentrations were calculated by the ‘Total Protein Approach’ (TPA) using a raw protein intensities from the MaxQuant output [17].
2. Methods We enrolled 20 medication-free apparently healthy subjects including members of the hospital staff and their families. Their characteristics are shown in Table 1. Subjects were eligible if they had no history of thromboembolic events (vein thrombosis, pulmonary embolism, MI, stroke), no signs of acute infection, or recent trauma or surgery. Pregnant women or those who delivered within previous 12 months were excluded. This study was conducted according to the principles expressed in the Declaration of Helsinki and was approved by the Bioethics Committee. All patients gave written informed consent. Fasting blood samples were drawn from an antecubital vein with minimal stasis using atraumatic venipuncture at 8 to 10 AM. Blood samples (vol/vol, 9:1 of 3.2% trisodium citrate) were spun at 2000g for 10 min, and the supernatants were aliquoted and stored at −80 °C for fibrin analysis. Blood cell count, lipid profiles (measured directly by homogenous enzymatic colorimetric method), glucose, and creatinine
2.2. Fibrin clot properties Plasma fibrin clot permeability was measured as described previously [3]. Briefly, 20 mM calcium chloride and 1 U/mL human thrombin (Merck, Kenilworth, NJ, USA) were added to citrated plasma. Tubes containing the clots were connected to a reservoir of a Trisbuffered saline. Its volume flowing through the gels was measured within 60 min. A permeation coefficient (Ks), which indicates the average size of pores formed in the fibrin network with low values indicating tightly packed fibrin structure, was calculated from the equation: Ks = Q × L × η/t × A × Δp, where Q is the flow rate in time t, L is the length of a fibrin gel, η is the viscosity of liquid (in poise), t is percolating time, A is the cross-sectional area (in cm2), and Δp is a differential pressure (in dyne/cm2). Clot lysis time (CLT) was measured as described [18]. Briefly, citrated plasma was mixed with 15 mM calcium chloride, human thrombin (Merck, Kenilworth, NJ, USA) at a final concentration of 0.5 U/mL, 10 μM phospholipid vesicles and 18 ng/mL recombinant t-PA (Boehringer Ingelheim, Ingelheim, Germany). The mixture was transferred to a microtitre plate and its turbidity was measured at 405 nm at 37 °C. CLT was defined as the time from the midpoint of the clear-tomaximum-turbid transition, which represents clot formation, to the midpoint of the maximum-turbid-to-clear transition. Intra-assay and inter-assay coefficients of variation were 6–8% for the two fibrin variables. All assays were performed in triplicates by the experienced investigator unaware of the results of proteomics.
Table 1 Baseline characteristics of healthy controls. Variable
Healthy controls (n = 20)
Age, years Male, n (%) Body mass index, kg/m2 Current smoking, n (%) White blood cell count, 103/μL Red blood cell count, 106/μL Platelet count, 103/μL Fibrinogen, g/L Glucose, mmol/L Creatinine, μmol/L Total cholesterol, mmol/L Low density lipoprotein cholesterol, mmol/L High density lipoprotein cholesterol, mmol/L Triglycerides, mmol/L High sensitivity C-reactive protein, mg/L Antithrombin, % Clot permeability (Ks), ×10−9 cm2 Clot lysis time (CLT), min
40 (31–49) 5 (25) 23.7 (21.1–25.7) 5 (25) 5.87 (4.99–7.53) 4.56 (4.39–4.79) 239 (211–290) 2.72 (2.34–3.05) 4.9 (4.6–5.1) 68 (60–85) 5.2 (4.6–5.9) 3.1 (2.7–3.6) 1.8 (1.6–2.0) 0.97 (0.7–1.20) 0.84 (0.59–2.11) 104 (94–112) 8.01 (6.47–9.93) 91 (82–109)
2.3. Statistical analysis Variables are presented as numbers (percentages), mean ± standard deviation (SD) or median and interquartile range (IQR) as appropriate. Normal distribution was assessed by Shapiro-Wilk test. The Pearson or Spearman rank correlation coefficients were calculated 2
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(a), kininogen-1, platelet factor 4 (PF4), and FV (Table 2). Of note, 17 (85%) clots showed detectable CRP, but in amounts below 0.001% of the clot mass, with no relation to plasma hsCRP concentrations. Age was positively associated with fibrinogen alpha and gamma chains within clots (r = 0.50, p = .024 and r = 0.54, p = .015, respectively), with no association of fibrinogen beta chain or plasma fibrinogen. Body-mass index (BMI) was positively associated with clotbound apolipoprotein A-I (r = 0.54, p = .018) and inversely with apolipoprotein(a) (r = −0.46, p = .049). A positive correlation between plasma low-density lipoprotein cholesterol (LDL-C) levels and both clot-bound apolipoprotein A-IV (r = 0.55, p = .012) and apolipoprotein B-100 (r = 0.46, p = .043) were found. Plasma high-density lipoprotein cholesterol levels correlated with clot-bound apolipoprotein (a) (r = 0.53, p = .017) and apolipoprotein E (r = 0.64, p = .0025). Similarly, total plasma cholesterol was associated with clot-bound apolipoprotein B-100 (r = 0.60, p-0.0053), apolipoprotein(a) (r = 0.50, p = .042), and apolipoprotein A-IV (r = 0.59, p = .0064). Clot-bound plasminogen (r = −0.62, p = .0034) and vinculin (r = −0.42, p = .032) correlated inversely with fibrinogen alpha and gamma chains, respectively. A positive correlation between FXIII A chain and fibronectin was observed within clots (r = 0.62, p = .0046). Clot-bound CRP was not associated with the content of any proteins detected within clots, except C1q (r = 0.51, p = .030).
Table 2 Quantitative analysis of selected clot-bound proteins of healthy subjects. Protein
Concentration (mg/g total protein)
Fibrinogen alpha chain Fibrinogen beta chain Fibrinogen gamma chain Fibronectin Alpha-2-macroglobulin Alpha-2-antiplasmin Serum albumin Complement C3 Coagulation factor XIII A chain Keratin, type II cytoskeletal 1 Extracellular matrix protein 1 Keratin, type I cytoskeletal 9 Histidine-rich glycoprotein Complement C4-B Apolipoprotein B-100 Apolipoprotein A-I Apolipoprotein A-IV Plasminogen/plasmin (Pro)thrombin Keratin, type I cytoskeletal 14 Keratin, type II cytoskeletal 5 von Willebrand factor Antithrombin-III Vitronectin Apolipoprotein E Kininogen-1 Apolipoprotein(a) Complement C5 Complement C4-A Complement factor H Platelet factor 4 Thrombospondin-1 Complement component C9 Vinculin Coagulation factor V Beta-2-glycoprotein 1
280 ± 12 210 ± 17 150 ± 11 130 ± 15 27 ± 7 22 ± 3 16 ± 9 12 ± 2.4 12 ± 2 9.3 ± 2 7.2 ± 2 6.5 ± 1 6.1 ± 1 4.1 ± 1.7 3.6 ± 3 2.6 ± 0.8 2.4 ± 1 2 ± 0.7 1.6 ± 0.5 1.5 ± 0.3 1 ± 0.2 0.99 ± 0.5 0.79 ± 0.1 0.78 ± 0.2 0.76 ± 0.4 0.34 ± 0.1 0.30 ± 0.3 0.22 ± 0.1 0.21 ± 0.2 0.14 ± 0.1 0.12 ± 0.1 0.088 ± 0.01 0.068 ± 0.02 0.057 ± 0.02 0.051 ± 0.03 0.039 ± 0.02
3.1. Fibrin clot properties In healthy controls Ks values were 8.0 ± 2.9 × 10−9 cm2. Ks correlated with age (r = −0.50, p = .024, Fig. 2A), BMI (r = −0.44, p = .048), plasma fibrinogen (r = −0.56, p = .01, Fig. 2B), total cholesterol (r = −0.48, p = .034), and LDL-C (r = −0.47, p = .038). Regarding proteins detected within plasma clots, Ks showed inverse associations with solely fibrinogen gamma chain (r = −0.54, p = .016, Fig. 2C) and PF4 (r = −0.46, p = .040, Fig. 2D). Of note, clot-bound HRG tended to correlate with Ks (r = −0.40, p = .07). A median CLT was 91 [82–109] min in the studied group. CLT correlated with age (r = 0.51, p = .025, Fig. 3A), plasma fibrinogen (r = 0.51, p = .027, Fig. 3B), total cholesterol (r = 0.70, p = .013) and LDL-C (r = 0.69, p = .015), but not with BMI. CLT was associated with the fibrinogen alpha and gamma chains (r = 0.52, p = .027 and r = 0.46, p = .046, Fig. 3C, D, respectively) and PF4 (r = 0.54, p = .016, Fig. 3E) within clots. Interestingly, CLT showed a strong, positive association with clot-bound HRG (r = 0.65, p = .0028, Fig. 3F). A borderline association between CLT and plasmin (r = −0.38, p = .08) was observed. There were no associations of Ks or CLT with clot-bound lipoproteins, antiplasmin, thrombin, antithrombin, and other proteins identified within the clots.
to test the association between 2 variables with a normal or non-normal distribution, respectively. Differences between 2 groups were compared using the Student's test for normally distributed continuous variables and for non-normally distributed continuous variables the MannWhitney U test was used. A two-sided p-value < .05 was considered statistically significant.
3. Results Proteomic analysis revealed a total of 494 proteins (available at ProteomeXchange Consortium, no. PXD008434) repeatedly identified in the plasma fibrin clots from all of 20 subjects. Among 494 clot-bound proteins we identified blood coagulation factors, lipoprotein metabolism proteins, fibrinolysis inhibitors and activators, proteins involved in immune responses, platelet-derived factors (Table 2), and many others. The highest average concentrations were found for the three fibrinogen chains (about 64% of the total clot protein, Fig. 1) and fibronectin (13%) (Table 2). Interestingly, not only proteins associated with coagulation and fibrinolysis, including α2-antiplasmin (α2AP, 2.3%) and FXIII A chain (1.2%), but also other proteins, including α2-macroglobulin (2.7%), albumin (1.6%), complement component C3 (1.2%), extracellular matrix protein 1 (0.72%), and HRG (0.61%) were present within the clot at relatively high concentrations (Table 2). Plasma albumin levels correlated with the clot-bound albumin (r = 0.43, p = .048). Other proteins incorporated within the fibrin network such as (pro)thrombin, plasminogen, von Willebrand factor, complement C4B, and apolipoproteins A and B-100 were detected at lower concentrations within the clots (ranged from 0.1 to 0.5% of the total clot protein mass). Proteins present at concentrations below 0.1% of the clot mass were antithrombin, vitronectin, apolipoprotein E, apolipoprotein
4. Discussion To our knowledge, this study is the first to show quantitative protein composition of fibrin clots generated from plasma of healthy subjects and its associations with two key measures of clot structure and function, i.e. clot permeability and lysis time. We demonstrated almost 500 proteins incorporated into the plasma fibrin clot, showing similar clot composition as obtained for VTE and APS patients [12,13]. Of note, the present study represents an approach able to quantify clot-bound proteins as the percentage of total clot-mass. In healthy subjects we observed associations between fibrin clot properties and the two clot components i.e. fibrinogen chains and PF4 using proteomic analysis. We failed to find any associations of lipidrelated parameters, inflammatory markers or fibrinolysis modulators with clot properties. This preliminary study suggests that density and lysability of plasma fibrin clots show associations with platelet activation in healthy subjects and possibly others in patients with diseases associated with prothrombotic states. Fibrin constitutes a major protein component of intravascular 3
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Fig. 1. Main clot protein composition in healthy subjects.
thrombi and in vitro prepared fibrin clots [8]. Fibrin networks composed of thin, highly compressed fibrin fibers are less permeable, more rigid, and less susceptible to lysis [9]. Fibrinogen concentrations explain up to 18% of the variation in Ks [8], indicating that other factors have to impact this key parameter. Interestingly, we found associations of age and fibrin clot properties with the content of fibrinogen alpha and gamma, but not beta chain, within plasma clots. This finding is surprising because we suspected similar associations of fibrin functional measures with the 3 fibrinogen chains forming the fibrinogen molecule and subsequently fibrin. First, we cannot exclude impaired digestion of the fibrinogen beta chain abolishing associations observed for the 2 remaining chains with Ks. It might be hypothesized that using the current proteomic analysis the fibrinogen gamma chain is the best marker of the fibrinogen content within plasma fibrin clots, which should be recommended while studying interactions among proteins and functional clot tests. In the current study we were not able to distinguish between fibrinogen gamma chain and its gamma' variant. Approximately 8%–15% of total fibrinogen in healthy individuals is composed of gamma/gamma' fibrinogen, which arises after polyadenylation leading to 20-amino acid extension (γ'408–427) substituting the 4 gamma chain amino acids (γA408–411) [19]. The presence of the gamma' chain leads to formation of fibrin clots with smaller pores and thinner fibers, which may be associated with the prothrombotic phenotype [19]. Given the observation that mutations affecting fibrin function, i.e. dysfibrinogenemias are common in the FGG gene [20], it is likely that proteomics of plasma fibrin clots in carriers of genetic abnormalities in FGG may yield new insights into the functional clot alterations, including clot lysability, reported in purified systems. Moreover, it should be mentioned that posttranslational modifications not only of the fibrinogen, but also of plasminogen or antiplasmin molecules, including oxidation, glycation, glycosylation or carbonylation increase with age and may contribute to altered fibrin clot properties [8]. New protein components documented in plasma clots by us and associated with clot properties in healthy individuals were HRG and
PF4. This observation might have potential clinical implications in various diseases linked with thrombotic tendency including platelet activation. PF4, a platelet-specific protein released from alpha granules upon platelet activation, has been previously shown to be associated with the prothrombotic plasma fibrin clot phenotype [21–23]. PF4 might associate with fibrin to impede the lateral association of protofibrils, decreasing porosity of the fibrin network [21]. In vitro, saturating amounts of PF4 (two fibrin molecules per one PF4 monomer) reduced fibrin porosity up to 4.4-fold and modified viscoelastic properties of fibrin network [21], while in patients with essential thrombocythemia and atrial fibrillation plasma PF4 levels correlated inversely with Ks [22,23]. This study supports the view that plateletderived substances, including PF4, are present in plasma clots not only in patients with large platelet counts or following acute thrombotic condition and may affect the fibrin network structure reflected by the pore size. The fact that the association of clot function with PF4 was demonstrated in young healthy subjects free of history of thromboembolism, a role of PF4 appears to be stronger than documented in the literature so far. Unexpectedly, we found a strong positive association of CLT with HRG, suggesting that under physiological conditions this abundant protein may modulate plasmin-mediated clot lysis. HRG is known to be involved in hemostasis displaying both anticoagulant and antifibrinolytic properties [24]. HRG deficiency has been associated with shortened plasma clotting time in human plasma [24] and with arterial thrombosis in HRG-knockout mice [25]. It has been shown in vitro using purified proteins that HRG retarded plasminogen activation by tissue plasminogen activator and interfered plasminogen binding to fibrin [26]. This antifibrinolytic effect of HRG has been proved in a functional test of clot lysis in the present study, which highlights an underestimated role of this protein in fibrin clot susceptibility to lysis. Further studies on HRG as a modulator of clot lysability in health and disease states are warranted. From the methodological point of view, this report provides additional evidence for the superiority of the current proteomic analysis 4
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Fig. 2. Associations of plasma fibrin clot permeability (Ks) with age (A), plasma fibrinogen (B), clot-bound fibrinogen gamma chain (C) and platelet factor 4 (D) in 20 healthy individuals.
compared with that used by Talens et al. [10]. who detected in fibrin clots of healthy subjects a relatively small number of proteins [10]. Our method corroborated the presence of multiple other proteins, undetectable by Talens et al. [10] most likely due to limitations of their approach, which was unable to identify low amounts of proteins. We believe that our technique could be recommended to assess protein composition of fibrin clots in various disease states. Interestingly, we were able to detect CRP within most plasma clots, which provides the first proteomic evidence of clot-bound CRP. These amounts of CRP showed no associations with key fibrin characteristics in our group, although it has been shown that both Ks and CLT in patients with chronic obstructive pulmonary disease correlated with plasma CRP levels [27]. A similar observation was made in patients with advanced coronary artery disease [28]. Salonen et al. [29] have shown that CRP binds to various proteins including fibrin(ogen) and thus can modify fibrin formation. Our findings suggest that the impact of CRP on clot properties could be observed in systemic inflammatory states but not in young healthy subjects even if CRP is bound to plasma fibrin clots as evidenced by us. This issue deserves further investigation. The abundance of various lipoproteins in fibrin clots of healthy subjects was identified in this study. We did observe associations of clot-bound lipoproteins with lipid profile in circulating blood. Previous studies have shown that plasma lipoprotein(a) [28,30] as well as high density lipoprotein cholesterol levels [31,32] had an impact on fibrin clot properties. In healthy subjects we did not find any associations between clot properties with clot-bound lipoproteins. Since it has been shown that statins as potent cholesterol-lowering agents improve fibrin
characteristics [6,33,34], our current study suggests that lipid-lowering effects of statins or other agents may at least in part explain antithrombotic effects via altering amounts of clot bound lipoproteins. Protein composition of plasma clots from statin-treated patients however remains to be established. This study has several limitations. First, the sample size was relatively limited. Second, fibrinogen consists of three non-identical pairs of disulfide-bonded chains with different molecular mass (95 kDa for alpha, 55.9 kDa for beta, and 51.5 kDa for gamma chain). To quantify clot-bound proteins each protein concentration was adjusted for a respective molecular weight giving the actual protein percentage of total clot-mass, which might influence data analysis. Third, detailed protein composition of clots made from human plasma have not been compared with protein composition of clots generated from isolated fibrinogen. However, the aim of this analysis was to seek for associations between plasma-based clot assays widely used in clinical settings and protein composition. Moreover, we did not assess fibrin formation since we focused on the two key parameters shown to correlate with clinically relevant outcomes and may have a predictive value for instance in VTE [3]. Finally, since it has been established that the presence of fibrinogen gamma’ chain unfavorably affects the fibrin clot structure [35] it also might influence fibrin clot proteomics. However, in our analysis we were not able to identify clot-bound fibrinogen gamma’ chain, probably due to predominance of the isoform gamma-A splice variant.
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Fig. 3. Associations of clot lysis time with age (A), plasma fibrinogen (B), clot-bound fibrinogen alpha chain (C), gamma chain (D), platelet factor 4 (E), and histidinerich glycoprotein (F) in 20 healthy individuals.
5. Conclusions
References
In conclusion, we demonstrated association of plasma fibrin structure and lysability with proteins forming clots, including PF4 and HRG. Further studies are needed to characterize the impact of various diseases as well as genetic and environmental factors, including medications, on clot proteomics.
[1] A.S. Wolberg, R.A. Campbell, Thrombin generation, fibrin clot formation and hemostasis, Transfus. Apher. Sci. 38 (2008) 15–23. [2] R.A. Ariëns, Fibrin(ogen) and thrombotic disease, J. Thromb. Haemost. 11 Suppl 1 (2013) 294–305. [3] A. Undas, K. Zawilska, M. Ciesla-Dul, A. Lehmann-Kopydłowska, A. Skubiszak, K. Ciepłuch, W. Tracz, Altered fibrin clot structure/function in patients with idiopathic venous thromboembolism and in their relatives, Blood 114 (2009) 4272–4278. [4] J. Siudut, M. Grela, E. Wypasek, K. Plens, A. Undas, Reduced plasma fibrin clot permeability and susceptibility to lysis are associated with increased risk of postthrombotic syndrome, J. Thromb. Haemost. 14 (2016) 784–793. [5] M. Zabczyk, K. Plens, W. Wojtowicz, A. Undas, Prothrombotic fibrin clot phenotype is associated with recurrent pulmonary embolism after discontinuation of anticoagulant therapy, Arterioscler. Thromb. Vasc. Biol. 37 (2017) 365–373. [6] A. Undas, M. Celinska-Löwenhoff, T. Löwenhoff, A. Szczeklik, Statins, fenofibrate, and quinapril increase clot permeability and enhance fibrinolysis in patients with coronary artery disease, J. Thromb. Haemost. 4 (2006) 1029–1036. [7] A. Undas, P. Podolec, K. Zawilska, M. Pieculewicz, I. Jedliński, E. Stepień, E. Konarska-Kuszewska, P. Weglarz, M. Duszyńska, E. Hanschke, T. Przewlocki, W. Tracz, Altered fibrin clot structure/function in patients with cryptogenic ischemic stroke, Stroke 40 (2009) 1499–1501. [8] A. Undas, R.A. Ariëns, Fibrin clot structure and function: a role in the pathophysiology of arterial and venous thromboembolic diseases, Arterioscler. Thromb. Vasc. Biol. 31 (2011) e88–e99. [9] M. Ząbczyk, A. Undas, Plasma fibrin clot structure and thromboembolism: clinical implications, Pol. Arch. Intern. Med. 127 (2017) 873–881. [10] S. Talens, F.W. Leebeek, J.A. Demmers, D.C. Rijken, Identification of fibrin clotbound plasma proteins, PLoS One 7 (2012) e41966. [11] M. Suski, J. Siudut, M. Ząbczyk, R. Korbut, R. Olszanecki, A. Undas, Shotgun analysis of plasma fibrin clot-bound proteins in patients with acute myocardial infarction, Thromb. Res. 135 (2015) 754–759. [12] A. Stachowicz, J. Siudut, M. Suski, R. Olszanecki, R. Korbut, A. Undas, J.R. Wiśniewski, Optimization of quantitative proteomic analysis of clots generated from plasma of patients with venous thromboembolism, Clin. Proteomics 14 (2017) 38. [13] A. Stachowicz, M. Zabczyk, J. Natorska, M. Suski, R. Olszanecki, R. Korbut, J.R. Wiśniewski, A. Undas, Differences in plasma fibrin clot composition in patients with thrombotic antiphospholipid syndrome compared with venous thromboembolism, Sci. Rep. 8 (2018) 17301. [14] M. Ząbczyk, A. Piłat, M. Awsiuk, A. Undas, An automated method for fibrin clot permeability assessment, Blood Coagul. Fibrinolysis 26 (2015) 104–109. [15] J.R. Wiśniewski, M. Mann, Consecutive proteolytic digestion in an enzyme reactor increases depth of proteomic and phosphoproteomic analysis, Anal. Chem. 84
Authors' contributions AU, RO, and JRW were responsible for the conception and design of the study. AS was responsible for analyses of the samples. MZ, AS, JN, AU and JRW were responsible for the interpretation of the data. MZ and JN drafted the article. All authors revised the paper critically for important intellectual content and gave final approval of the version to be published. All authors read and approved the final manuscript. Funding sources This work was supported by the Max-Planck Society for the Advancement of Science and by the German Research Foundation (DFG/Gottfried Wilhelm Leibniz Prize) and the Jagiellonian University Medical College (K/ZDS/007717, to A.U.). Availability of data and materials The mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier: PXD006814. Declaration of Competing Interest The authors declare that they have no competing interests. 6
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M. Ząbczyk, et al.
(2012) 2631–2637. [16] J.R. Wiśniewski, F.Z. Gaugaz, Fast and sensitive total protein and peptide assays for proteomic analysis, Anal. Chem. 87 (2015) 4110–4116. [17] J.R. Wiśniewski, Label-free and standard-free absolute quantitative proteomics using the "total protein" and "proteomic ruler" approaches, Methods Enzymol. 585 (2017) 49–60. [18] M. Pieters, H. Philippou, A. Undas, Z. de Lange, D.C. Rijken, N.J. Mutch, Subcommittee on Factor XIII and Fibrinogen, and the Subcommittee on Fibrinolysis. An international study on the feasibility of a standardized combined plasma clot turbidity and lysis assay: communication from the SSC of the ISTH, J. Thromb. Haemost. 16 (2018) 1007–1012. [19] S. Uitte de Willige, K.F. Standeven, H. Philippou, R.A. Ariëns, The pleiotropic role of the fibrinogen gamma' chain in hemostasis, Blood 114 (2009) 3994–4001. [20] S. Uitte de Willige, M.C. de Visser, J.J. Houwing-Duistermaat, F.R. Rosendaal, H.L. Vos, R.M. Bertina, Genetic variation in the fibrinogen gamma gene increases the risk for deep venous thrombosis by reducing plasma fibrinogen gamma' levels, Blood 106 (2005) 4176–4183. [21] A.A. Amelot, M. Tagzirt, G. Ducouret, R.L. Kuen, B.F. Le Bonniec, Platelet factor 4 (CXCL4) seals blood clots by altering the structure of fibrin, J. Biol. Chem. 282 (2007) 710–720. [22] R. Małecki, M. Gacka, M. Kuliszkiewicz-Janus, U. Jakobsche-Policht, J. Kwiatkowski, R. Adamiec, A. Undas, Altered plasma fibrin clot properties in essential thrombocythemia, Platelets 27 (2016) 110–116. [23] L. Drabik, P. Wołkow, A. Undas, Denser plasma clot formation and impaired fibrinolysis in paroxysmal and persistent atrial fibrillation while on sinus rhythm: association with thrombin generation, endothelial injury and platelet activation, Thromb. Res. 136 (2015) 408–414. [24] T.T. Vu, B.A. Leslie, A.R. Stafford, J. Zhou, J.C. Fredenburgh, J.I. Weitz, Histidinerich glycoprotein binds DNA and RNA and attenuates their capacity to activate the intrinsic coagulation pathway, Thromb. Haemost. 115 (2016) 89–98. [25] T.T. Vu, J. Zhou, B.A. Leslie, A.R. Stafford, J.C. Fredenburgh, R. Ni, S. Qiao, N. Vaezzadeh, W. Jahnen-Dechent, B.P. Monia, P.L. Gross, J.I. Weitz, Arterial thrombosis is accelerated in mice deficient in histidine-rich glycoprotein, Blood 125
(2015) 2712–2719. [26] H.R. Lijnen, M. Hoylaerts, D. Collen, Isolation and characterization of a human plasma protein with affinity for the lysine binding sites in plasminogen. Role in the regulation of fibrinolysis and identification as histidine-rich glycoprotein, J. Biol. Chem. 255 (1980) 10214–10222. [27] A. Undas, P. Kaczmarek, K. Sladek, E. Stepien, W. Skucha, M. Rzeszutko, I. Gorkiewicz-Kot, W. Tracz, Fibrin clot properties are altered in patients with chronic obstructive pulmonary disease: beneficial effects of simvastatin treatment, Thromb. Haemost. 102 (2009) 1176–1182. [28] A. Undas, D. Plicner, E. Stepien, R. Drwila, J. Sadowski, Altered fibrin clot structure in patients with advanced coronary artery disease: a role of C-reactive protein, lipoprotein(a), and homocysteine, J. Thromb. Haemost. 5 (2007) 1988–1990. [29] E.M. Salonen, T. Vartio, K. Hedman, A. Vaheri, Binding of fibronectin by the acute phase C-reactive protein, J. Biol. Chem. 259 (1984) 1496–1501. [30] A. Undas, E. Stepien, W. Tracz, A. Szczeklik, Lipoprotein(a) as a modifier of fibrin clot permeability and susceptibility to lysis, J. Thromb. Haemost. 4 (2006) 973–975. [31] M. Ząbczyk, Ł. Hońdo, M. Krzek, A. Undas, High-density cholesterol and apolipoprotein AI as modifiers of plasma fibrin clot properties in apparently healthy individuals, Blood Coagul. Fibrinolysis 24 (2013) 50–54. [32] R.C. Kotzé, R.A. Ariëns, Z. de Lange, M. Pieters, CVD risk factors are related to plasma fibrin clot properties independent of total and or γ' fibrinogen concentration, Thromb. Res. 134 (2014) 963–969. [33] J.H. Griffin, K. Kojima, C.L. Banka, L.K. Curtiss, J.A. Fernandez, High-density lipoprotein enhancement of anticoagulant activities of plasma protein S and activated protein C, J. Clin. Invest. 103 (1999) 219–227. [34] C. Oslakovic, M.J. Krisinger, A. Andersson, M. Jauhiainen, C. Ehnholm, B. Dahlbäck, Anionic phospholipids lose their procoagulant properties when incorporated into high density lipoproteins, J. Biol. Chem. 284 (2009) 5896–5904. [35] P. Allan, S. Uitte de Willige, R.H. Abou-Saleh, S.D. Connell, R.A. Ariëns, Evidence that fibrinogen γ' directly interferes with protofibril growth: implications for fibrin structure and clot stiffness, J. Thromb. Haemost. 10 (2012) 1072–1080.
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