Forty-one patients with cancer (lung and brain) enrolled in framework of the Vienna Cancer and Thrombosis Study (CATS) were included in this analysis. CATS is a prospective, observational, single-centre cohort study started in October 2003 at the Medical University of Vienna. Patients included in the present study were recruited between 2014–2017. All detailed inclusion and exclusion criteria were previously described (20, 21). Briefly, adult patients (≥ 18 years) with newly diagnosed cancer or cancer progression after partial of complete remission were enrolled in the study. Patients who were not treated with anticoagulant drugs in the past three months, did not undergo chemotherapy in the last 3 months or receive radiotherapy or surgery within the past 2 weeks, were informed about the study design and gave written consent. At study inclusion, patient history was documented with a structured interview and furthermore, patients enrolled in the CATS study were monitored with a structured questionnaire on a three-month basis for occurrence of VTE and death. For this study and its hypothesis generating approach we used a case control design and included a matched healthy control cohort. Follow ups were made after 3 and 6 months and two years.
Forty-one age and sex matched healthy volunteers were included after giving written informed consent. Venous blood was drawn from cancer patients and healthy controls for platelet proteome investigations, routine and coagulation parameter analysis as well as for further experimental approaches. Data on the study populations can be found in Table 1.
This study was approved by the Ethics Committee of the Medical University of Vienna in accordance with the Declaration of Helsinki (EC no. 126/2003 and 039/2006)
Blood collection, washed platelet and plasma isolation
For platelet isolation, blood was drawn from an antecubital vein into 3.5 mL vacuum tubes containing CTAD (0.129 mM trisodium citrate, 15 mM theophylline, 3.7 mM adenosine, 0.198 mM dipyridamole; Greiner Bio-One, Kremsmünster, Austria) as anticoagulant. The first tube drawn was discarded to avoid any contaminations. Immediately following blood draw, CTAD tubes was centrifuged at 120 xg for 20 min without brake at room temperature. Obtained platelet rich plasma (PRP) was carefully transferred into a fresh tube containing prostacyclin I2 (0.8 µM, PGI2; Sigma-Aldrich, St. Louis, MO, USA) to prevent platelet aggregation and degranulation during the following washing steps. PRP was pelleted by centrifugation for 3 min at 3000 xg at room temperature and the protein pellets were washed twice in phosphate-buffered saline (w/o: Ca2+ and Mg2+) containing PGI2 (0.8 µM). Before the last centrifugation step, platelet count was determined with a Sysmex XN-350 hematocytometer (Sysmex, Kobe, Japan). After the last centrifugation step the supernatant was completely taken off and the platelet pellet was snap-frozen in liquid nitrogen and stored at -80°C until processing.
For preparation of plasma blood was drawn into 3.5 mL vacuum tubes containing sodium citrate (0.129 mM citrate; Greiner Bio-One, Kremsmünster, Austria). The blood was centrifuged at 2500 xg for 15 min at 15°C to separate the cellular fraction and the plasma supernatant was stored at -80°C.
Platelet preparation for 2D-DIGE analysis
The frozen platelet protein pellets were resolubilized in urea-sample buffer (7 M urea, 2 M thiourea, 4% CHAPS, 20 mM Tris-HCl pH 8.68) and incubated for 2 h at 4°C under agitation (800 rpm). Protein quantitation of individual samples was done in triplicate with a Coomassie brilliant blue protein assay kit (Pierce, Thermo Scientific, Rockford, IL, USA). The internal standard (IS) was made by pooling the same protein amounts from each platelet samples of all included study participants. Platelet protein samples and IS were aliquoted and stored at -80°C.
Platelet proteome analysis by two-dimensional fluorescence differential gel electrophoresis (2D-DIGE)
Prior electrophoresis, proteins were labelled with fluorescent cyanine dyes (5 pmol of CyDyes per µg of protein; GE Healthcare, Uppsala, Sweden) according to previous concentrations (16). The IS was always labelled using Cy2, while Cy3 and Cy5 were used alternately for study samples. Briefly, IPG-Dry-Strips (24 cm, pH 4–7, GE Healthcare, Uppsala, Sweden) were rehydrated for 11 h with 450 µL rehydration buffer (7 M urea, 2 M thiourea, 70 mM DTT, 0.5% pH 4–7 ampholyte; Serva, Heidelberg, Germany) mixed with a total of 36 µg (2x 12 µg sample + 1x 12 µg IS) of alternatively Cy-labelled sample. Isoelectric focusing (IEF) was performed on a Protean I12 IEF unit (Biorad) until 30 kVh was reached.
After IEF, the strips were first equilibrated with gentle shaking in 12.5 mL of equilibration buffer 1 (1% DTT, 50 mM Tris-HCl pH 8.68, 6 M Urea, 30% glycerol and 2% SDS) for 20 min followed by incubation in equilibration buffer 2 (2.5% iodoacetamide, 50 mM Tris-HCl pH 8.68, 6 M Urea, 30% glycerol and 2% SDS) for 15 min. Each of the IPG strips was transferred on 11.5% acrylamide gel (26x 20 cm, 1 mm gel) and sealed with low melting agarose sealing solution (375 mM Tris-HCl pH 8.68, 1% SDS, 0.5% agarose). The SDS-PAGE was performed using an Ettan DALTsix electrophoresis chamber (GE Healthcare, Uppsala, Sweden) under the following conditions: 35 V for 1 h, 50 V for 1.5 h and finally 110 V for 16.5 h at 10°C.
2D-DIGE image analysis
For protein spot detection, 2D-DIGE gels were scanned with three different wavelengths of the particular CyDye at a resolution of 100 µm using a Typhoon 9410 Scanner (GE Healthcare, Uppsala, Sweden). Gel images were analysed via the DeCyder™ software (version 7.2, GE Healthcare, Uppsala, Sweden). Spots were matched to a master 2D-DIGE gel (a representative pH 4–7 platelet protein map of the IS images). On average, 500 protein spots were matched manually to the master gel using the DeCyder™ software. Afterwards an automatic spot match was used which achieved an average of 4310 matched spots per gel. The standardised abundance (SA) of every protein spot was calculated by the DeCyder™ software with two normalisation steps. The first step is the in-gel normalisation by dividing each spot with the center volume of the corresponding spot map and a the second one by dividing each normalised spot volumes against the corresponding spot normalised spot value of the IS. (22). Detailed information about the image analysis was published by Winkler et al. (23).
Protein identification via mass spectrometry
For MS-based identifications, 250 µg unlabelled proteins were separated by 2D-DIGE and proteins were visualized by MS-compatible silver staining (24). Protein spots of interest were excised manually from the gels, de-stained, disulfide were reduced as well as derivatized with iodoacetamide and the proteins were tryptically digested. Subsequently, these peptide samples were applied onto a Dionex Ultimate 3000 RSLC nano-HPLC system (Thermo Scientific) and afterward directly subjected to a QqTOF mass spectrometer oTOF compact (Bruker Daltonics) equipped with nano-flow CaptiveSpray ionisation device. Detailed analytical conditions were previously described (25). The protein identification was obtained with database searches against UniProtKB/Swiss-Prot (2020-06) using Mascot v2.7 server.
One and two-dimensional Western blot analysis
For one-dimensional Western blot (1-D WB), 12 µg total platelet protein were mixed with a sample buffer (150 mM Tris-HCl pH 8.68, 7.5% SDS, 37.5% glycerol, bromine phenol blue, 125 mM DTT) to obtain a final volume of 20 µL. Afterward, samples were boiled for 4 min at 95°C and centrifuged for 3 min at 20,000 xg. Thereafter, the samples were separated on a 11.5% SDS gel (50 V, 20 min and 100 V, 150 min) and blotted (75 V, 120 min) on a nitrocellulose membrane (NC; Pall, East Hills, NY, USA) or polyvinylidene difluoride membrane (PVDF; FluoroTrans® W, Pall, East Hills, NY, USA). For protein quantification, total protein on the membrane was stained using a ruthenium-(II)-tris-(bathophenanthroline disulfonate) (RuBPS; dilution 1:100,000 overnight at 4°C; Sigma-Aldrich St. Louis, MI, USA).
For two-dimensional Western blot (2-D WB) analysis, 36 µg Cy2-labeld platelet proteins were separated by IEF on either a 7 cm pH 3–10 or a 24 cm pH 4–7 IPG strip as described for 2D-DIGE gels, and subsequently transferred onto a NC- or PVDF membrane (75 V, 90 min). Afterward, the membrane was blocked with 5% non-fat dry milk (BioRad, Hercules, CA, USA) in 1x PBS containing 0.3% Tween-20 (PBS-T) over night at 4°C under gentle shaking. On the next day, membranes were washed (3x with PBS-T for 5 min, each). For detection, following primary antibodies were used in the corresponding dilutions by incubation for 2 h at room temperature (180 rpm) in PBS-T containing 3% non-fat dry milk: monoclonal anti-Factor F13A1 (ab1834; Abcam, USA) 1:250, polyclonal anti-CD41/Integrin alpha 2b (ab83961; Abcam, Cambridge, UK) 1:200, monoclonal anti-Integrin beta 3 [EPR2342] (ab119992, Abcam, USA) 1:1000, monoclonal anti-Calreticulin [FMC 75] (ab22683; Abcam, USA) 1:500, polyclonal anti-HSPA5 (ab21685; Abcam Cambridge, UK) 1:250 and monoclonal anti-Talin clone 8D4 (SAB4200694, Merck, Germany) 1:300. After washing (3x with PBS-T for 5 min, each), the membranes were incubated with a DyLight 650 conjugated secondary antibody (Novus Biologicals, Littleton, CO, USA), diluted 1:500 or with a horse-radish peroxidase (HRP)- conjugated secondary antibody, diluted 1:20,000 in PBS-T containing 3% non-fat dry milk for 1.5 h in the dark at room temperature (65 rpm). After washing (3x with PBS-T for 5 min, each), the membranes were incubated for 1.5 h in the dark at room temperature (65 rpm). After washing (2x with PBS-T, 1x with 1x PBS for 5 min, each), the antibody fluorescence signals were detected with a Typhoon FLA 9500 imager (GE Healthcare, Uppsala, Sweden) at a resolution of 100 µm. The HRP signal was detected using an Enhanced Chemiluminescent (FluorChem® HD2, Alpha Innotech, CA USA). The 1-D WB antibody signal of F13A1 were normalized by the RuBPS fluorescence signal from the 40 kDa to 100 kDa bands and quantified with ImageQuant 8.0 (GE Healthcare, Uppsala, Sweden).
Measurement of haemostatic biomarkers in plasma
FXIII activity in citrate plasma was measured by Berichrom Factor XIII chromogenic determination (Siemens Healthcare) according to manufacturers’ instructions. Factor VIII activity was measured on a Sysmex CA 7000 analyzer using factor VIII–deficient plasma (Technoclone) and APTT Actin-FS (Dade Behring). D-dimer levels were measured by a quantitative latex assay (STA-LIAtest D-DI; Diagnostica-Stago, Asnieres, France) on a STAR analyser (Diagnostica-Stago) according to the manufacturer’s instructions. Fibrinogen was routinely measured in platelet poor plasma according to Clauss (STA Fibrinogen; Diagnostica Stago, Asnieres, France; normal range: 180–390 mg/dL). C-reactive protein (CRP) was measured with a fully automated particle enhanced immunonephelometry (N high sensitivity CRP, Dade Behring, Marburg, Germany). For sCD62P, to obtain definitely platelet-free plasma a second centrifugation step (Eppendorf) at 13,400 xg for 2 min was performed. These plasma aliquots were also stored at − 80°C, until the determination of sCD62P plasma levels in series. Soluble CD62P levels were measured using a human sCD62P immunoassay (R&D Systems, Minneapolis, MN) following the manufacturer's instructions.
Fluorogenic F13A1 activity assay from platelet samples
Washed platelets from 42 patient´s samples were lysed by 1% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) and sonicated for 1 min at 4°C. After centrifugation (15,000 xg, 4°C) lysed platelets were transferred to a fresh tube. Protein concentration in the lysate was determined by Coomassie. F13A1 activity in platelets was performed by a fluorogenic FXIIIA enzyme activity kit (Zedira GmbH, #F001, Darmstadt, Germany) essentially according to the manufacturers’ protocol with minor modifications. Measurements were performed in triplicate in 96-well flat bottom black microplates (Thermo Scientific, #137101, Denmark). Ten µL of lysed samples was mixed with 90 µL reagent mix solution (modified peptide, 100 NIH Units thrombin, Tris buffer pH 7.5 containing CaCl2, NaCl, polyethylene glycol (PEG), glycine methyl ester, clot inhibitor peptide, Heparin antagonist (hexadimethrine bromide) and sodium azide), creating a final volume of 100 µL/well. As a control for specificity of enzymatic F13A1 activity, the irreversible transglutaminase inhibitor T101 (50 µM final concentration) was used. The enzyme kinetic were recorded at 37°C using a Varioskan LUX microplate reader (Thermo Fisher Scientific) with excitation at 313 nm and emission at 418 nm in the kinetic mode, absorbance was read every 36 s for 15 min and the change of absorbance between 0 and 15 min were detected. F13A1 activity was determined on the basis of a standard curve constructed with washed human platelets. Microsoft excel was used for further processing of enzyme kinetic data.
Biological pathway analysis
Biological data base analysis was made from the fifteen lung cancer-related platelet proteins. The data source for the protein-protein interaction (PPI) networks was the protein query of the STRING database (26), with the settings (active interaction sources: experiments and databases; score = 0.4; maximal additional interactors = 0). For the functional enrichment, the Gene Ontology Biological Processes and KEEG pathway analysis were used for the PPI networks with a specific colour for each biological process and KEGG pathway. The STRING Version 11.0b was used.
In addition, we applied the NetworkAnalyst platform (27, 28) for further analysis. The differentially expressed proteins were uploaded to the web-platform (www.networkanalyst.ca) and generic protein-protein interaction (PPI) network analysis was performed with the Imex interactome (29) resulting in a first-order network with 722 nodes and 889 edges comprising the 15 lung cancer proteins as seeds. This network was downloaded as graphML-file and imported into the Cytoscape 3.8.2 program. The stringApp was used to “STRINGify” the network, followed by functional enrichment of pathways and gene ontologies, which were sorted according to p-values. Relevant pathways were selected and coloured using the bypass function for node colours.
For statistical analysis, from each 2D-DIGE image only protein spots are included which could be matched by the IS spot map with more than 95% of all 2-D platelet proteome maps of this study. This quality selection limits resulting protein spots to 617 from in average 2720 to the master gel matched spots. One-way and two-way analysis of variance (ANOVA) were calculated for these 617 highly reliable matched spots between the three study groups (healthy controls, patients with lung and brain cancer). Resulting p values of the one-way ANOVA were false discovery rate corrected (FDR) by Benjamini Hochberg for multiple comparisons (30). FDR-corrected one-way ANOVA and unadjusted two-way ANOVA were calculated with the Extended Data Analysis (EDA) module of the DeCyder™ software (version 7.2, GE Healthcare, Uppsala, Sweden). Supervised principal component analysis (PCA) of one-way ANOVA significant spots was also performed by this EDA module to control sample clustering. To assess the effect of mortality on the examined platelet proteom between cancer type groups, the two-way ANOVA main effects “mortality”, “cancer type group” and interaction term “cancer type group ∗ mortality” were included. Significant differences between control group and each cancer group were analysed by planned post-hoc contrasts analysis with unpaired Student´s t-Test in SPSS and corrected for multiple comparison by the online FDR calculator (31). For a direct comparison of different parameters the effect size was calculated by Cohen’s D = (mean1-mean2)/standard deviationpooled). Graphs were created with GraphPad Prism 6 (GraphPad Software, Inc. San Diego California, USA).