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. Author manuscript; available in PMC: 2011 Jun 1.
Published in final edited form as: Thromb Res. 2010 Feb 13;125(6):e269–e274. doi: 10.1016/j.thromres.2010.01.019

Proteomics of Microparticles after Deep Venous Thrombosis

Eduardo Ramacciotti 1, Angela E Hawley 1, Shirley K Wrobleski 1, Daniel D Myers Jr 1,3, John R Strahler 2, Philip C Andrews 2, Kenneth E Guire 4, Peter K Henke 1, Thomas W Wakefield 1
PMCID: PMC2929804  NIHMSID: NIHMS179921  PMID: 20156641

Abstract

Background

Microparticles (MP) are submicron size membrane vesicles released from activated cells that are associated with thrombosis and inflammation. MP present diverse biological expressions that may be linked to a unique subset of proteins derived from their origin cells.

Methods

To identify these proteins, plasma samples were taken from 9 patients with deep venous thrombosis (DVT) documented by duplex ultrasound, 9 with leg pain but negative for DVT by duplex, and 6 healthy controls without a history of thrombosis, for fold variation. MP were extracted from platelet-poor plasma, digested separately with trypsin and tagged using iTRAQ reagents. The digests were subjected to 2-D LC separation followed by MALDI tandem mass spectrometry. Peak lists were generated and searched against all human sequences. For protein identification, a minimum of two peptides at 95% confidence was required. Later, iTRAQ ratios were generated comparing relative protein levels of DVT patients to baseline. The proteomic analysis was performed twice for each blood sample. Proteins were considered elevated or depressed if the iTRAQ ratio (R) deviated by 20% change from normal and a p-value less than 0.05.

Results

Two proteins (Galectin-3 Binding Protein, [Gal3BP], R=1.76 and Alpha-2 macroglobulin [A2M] R=1.57) were differentially expressed on DVT patients. Nine proteins were depleted including fibrinogen beta and gamma chain precursors (R=0.65).

Conclusions

These proteins influence thrombosis through inflammation, cell shedding, inhibition of fibrinolysis and hemostatic plug formation. Further studies are needed to confirm the mechanistic role of these proteins in the pathogenesis of venous thrombosis in humans.

Keywords: Microparticles, Microparticle proteins, Proteomics, Venous Thrombosis

Introduction

Venous thromboembolism (VTE) remains a significant health problem in the United States with an estimated 900,000 cases of deep venous thrombosis and pulmonary embolism yearly1. The pathophysiology of venous thrombosis has traditionally included stasis of the blood, hypercoagulability and changes in the vein wall (Virchow’s triad).

In the 1970’s Gwendylen Stewart suggested a relationship between thrombosis and inflammation2. Recent studies have investigated the role of procoagulant microparticle (MP) formation in the inflammatory component of venous thrombosis3.

MP are small fragments of cell membrane, present in plasma and shed from different cells and elements such as platelets, leukocytes and endothelial cells. Some MP are rich in tissue factor (TF) and facilitate and amplify coagulation in the presence of thrombus. In addition, MP promotes an interface between inflammation and thrombosis. These small structures are fragments of phospholipid membrane released from different elements and cells in activation and apoptosis; in addition, recent evidence suggests MP carry RNA4 and DNA5. MP may concentrate certain proteins, and have been characterized by evaluating platelet-derived and plasma-derived MP obtained from healthy individuals.6, 7 Fibrinogen was enriched on MP in a nonhuman primate model of venous thrombosis8 9.However, there has been no description of MP protein up-regulation or depletion on human MP in patients with venous thrombosis. Therefore, the purpose of this study was to use proteomic evaluation to identify these proteins, helping to understand the role of MP in human venous thrombogenesis.

Methods

Twenty four (24) subjects make up this study, 9 patients with DVT documented by duplex ultrasound, 9 patients with leg pain but negative for DVT by duplex, and 6 healthy controls with no history of venous thrombosis. Controls were used for fold change; comparisons were made between patients with positive versus negative DVT confirmed by duplex scan, adjusted by controls. Three experiments with 8 patients each (3 DVT positive, 3 DVT negative and 2 controls per experiment, with a total of 24 subjects were performed). At the time of diagnosis (patients) or donation (controls), subjects had an 8.5-mL tube of whole blood drawn into 10% acid citrate dextrose (ACD) by butterfly antecubital stick. Platelet poor plasma (PPP) was obtained by centrifuging blood at 1,500xg and room temperature for 25 min, transferring the plasma to another tube and centrifuging it once more for 2 min at 15,000xg, to remove contaminating cells from the plasma. PFP (4-mL) obtained from each subject was stored in 1-mL aliquots at −70°C for 12 and 24 months. We have performed a pilot study in our methods to compare frozen from fresh samples variability. Testing blood from 4 healthy donors, no significant differences regarding plasma total proteins concentrations, total MP, platelet and monocyte derived MP counts between frozen and fresh samples were observed.

For proteomic evaluation, PPP was thawed and 400μL was spun down in 1-mL of HEPES buffer [10 mM Hepes/5 mM KCl/1 mM MgCl2/136 mM NaCl2 (pH 7.4)] for 2 h at 4°C, 200,000 × g. Supernatant was removed and MP pellet was resuspended in 400-μl of 0.25M KBr and incubated on ice for 20 min. Samples were then spun down for 2 h at 4°C, 200,000 g. Supernatant was removed and pellet air dried before being resuspended in 250-μl of 1X PBS. The previous steps involving suspension in KBr followed by centrifugation were performed in all experiments to remove soluble serum proteins. Protein concentration was determined using a standard colorimetric BCA total protein assay (Pierce, Rockford, IL).

Protein Isobaric Labeling with iTRAQ Reagents

MP from 200 μL PPP were suspended in 20-μL 0.5M triethylammonium bicarbonate (TEAB) and 0.1% sodium dodecyl sulfate (SDS). For four-plex isobaric labeling, separate aliquots of proteins were treated in parallel, essentially as described by Ross et al. Stock reagents and buffer (TEAB, SDS, Tris (2-carboxyethyl)phosphinea (TCEP), S-methyl methanethiosulfonate (MMTS), and the four isobaric tagging reagents) were obtained in kit form (Applied Biosystems, Foster City, CA). Protein (51.5 μg) was reduced with 2.5 mM TCEP (60°C for 1 h) and cysteine residues blocked with 10 mM MMTS (room temperature for 15 min). Protein was digested with trypsin (porcine modified, Promega; 1:20, w/w) for 20 h. Isobaric tagging iTRAQ reagent (1 unit in 70-μL ethanol) was added directly to the protein digest (70% ethanol final) and the mixture incubated at room temperature for 1 h. The reaction was quenched by addition of 9 volumes 0.1% trifluoroacetic acid (TFA) in water. The reaction mixtures were combined and stored at 4°C.

SCX Peptide Fractionation

For the first dimension of the two-dimension chromatographic separation, an aliquot of the four-plex peptide mixture (200 μg) was applied to a sulfoethyl aspartamide SCX spin column (SEM HIL-SCX, PolyLC, The Nest Group, Inc. Southboro, MA) equilibrated with 10 mM KH2 phosphate, pH 4.5, 20% CH3CN. Excess reagent was washed from the column with 800-μl equilibration buffer. Peptides were eluted using 50 μl volumes of KCl in a stepwise gradient from 25–500 mM KCl in equilibration buffer (25, 50, 75, 100, 150, 225, 350, and 500 mM KCl). Fractions were dried in a vacuum centrifuge.

Reversed-Phase Liquid Chromatography

For the second dimension separation, peptides in SCX fractions were separated by C18 nano LC using an 1100 Series nano HPLC equipped with μWPS autosampler, 2/10 microvalve, MWD UV detector (214 nm) and Micro-FC fraction collector/spotter (Agilent). Each SCX salt step was reconstituted with 43-μl 0.1% TFA, v/v in water and 40-μl injected. Sample was injected onto a C18 cartridge (Zorbax300SB, 5 μm, 5 × 0.3 mm; Agilent), desalted with solvent C (CH3CN:H2O:TFA, 5:95:0.1) at 20-μL/min for 9 min with the effluent discarded. The enrichment cartridge was placed ahead of a C18 column (Zorbax300SB, 3.5 μm, 150 × 0.1 mm; Agilent) equilibrated with solvent A (H2O:TFA, 99.9:0.1). Peptides were eluted with a gradient of solvent B (CH3CN:H2O:TFA, 90:10:0.1) from 6.5% B to 50% B over 90 min at a flow rate of 0.4 μl/min. Column effluent was mixed (micro Tee, Agilent) with matrix (2 mg/ml α-cyano 4-hydroxy cinnamic acid (CHCA in CH3OH: isopropanol: CH3CN:H2O: acetic acid (12:33.3:52:36:0.7) containing 10 mM ammonium phosphate) delivered with a PHD2000 infusion pump (Harvard Apparatus) at 0.9 μl/min. Fractions were spotted at 24 s intervals (192 fractions/LC run) onto large format stainless steel MALDI targets (Applied Biosystems).

Mass Spectrometry

Mass spectra were acquired on an Applied Biosystems 4800 MALDI TOF/TOF Analyzer (TOF/TOF). MS spectra from 800–3500 Da were acquired for each fraction from 750 laser shots of a 200 Hz YAG laser operated in the 3rd harmonic (355 nm). The TOF/TOF was operated in positive ion reflectron mode. Seven point Gaussian smoothing was applied to spectra and a S/N of 30 filter applied for peak picking. Calibration was done using default mode. Plate calibrants were glufib (m/z 1714.787), ACTH (m/z 2753.419), angiotensin (m/z 1440.790), bradykinin (m/z 1048.578), and ACTH 1–17 (m/z 2107.197). The twelve most intense peaks in each MS spectrum were selected for MS/MS analysis. MSMS spectra were acquired from 1500-400 laser shots using “quality dependent” mode (6 peaks at S/N 60); fragment peak picking used S/N 40. MSMS calibration was done with fragments of glufib (m/z 430.242, 684.346, 1056.475 and 1441.634). Fragmentation of the labeled peptides was induced by the use of atmosphere as a collision gas with a pressure of approximately 6 ×10−7 torr and collision energy of 2kV.

Peptide identifications were performed using GPS Explorer (v3.6, Applied Biosystems) which acts as a front end for the Mascot search engine (v2.1 MatrixScience, London UK). Each MS/MS spectrum was searched against NCBInr mammals (Feb. 2, 2007). Trypsin specificity with one missed cleavage was selected. S-mercaptomethylcysteine and the N-terminal and lysine iTRAQ labels were selected as fixed modifications. Oxidized methionine was considered as a variable modification. The precursor tolerance and MS/MS fragment tolerances were +/− 0.7 and +/− 0.3 Da, respectively. Individual peptide identifications were grouped into protein identifications using GPS Explorer and assigned a total ion C.I.%. Only the peptides with C.I.% at 95% or above for any MS/MS spectrum were retained for further analysis.

Statistical Analysis

The data were processed as follows. The raw peak areas for each experiment were normalized following the methodology in Keshamouni et al.,10 using a quantile method. Subsequently, the desired ratios of DVT versus controls for each experiment were obtained using the random effects ANOVA model introduced in Keshamouni et al.10 and Jagtap et al.11 This model considers the hierarchical structure in the data, namely that every protein is comprised of several peptides and the expression level of each peptide may be measured by multiple spectra. The outcome of this model is a ratio (R) of the treatment effect (2-day expression level) over the control effect (baseline expression level), along with its standard error, which allows us to obtain the p-value for testing the hypothesis R is in the range (0.8–1.2) –no change, versus the alternative one that R is outside that range. The choice of this range is supported by experience with previous experiments, where such ratios usually do not indicate biological activity. Proteins with p-values smaller than 0.05 that rejected the null hypothesis of no change were retained for further processing.

The p-values were adjusted since multiple experiments (3 experiments with 8 samples each: 3 DVT positives, 3 DVT negatives and 2 controls [total n=24]) were performed and some proteins appeared significant in several of them. A false discovery rate12 approach was used to appropriately adjust the p-values, in order to make the appropriate differential expression calls.

All patients signed the informed consent previously to any study procedure. The protocol was approved by the institution IRB.

Results

Protein identifications were based on two peptides with 95% confidence. Of the 151 proteins identified, 35 proteins displayed a greater than 20% enrichment or depletion on MP, in 2 out of 3 experiments, as listed in table 1. Special significance was given to proteins with their level of expression being regulated appearing in 3 out of 3 experiments. Eleven proteins fit this criterion and are listed in Table 2. Among these proteins, two were enriched in MP from DVT patients; the first class of proteins represented was Galectin-3 binding protein (Gal3BP), iTRAQ ratio R=1.76. The second class, Alpha-2 Macroglobulin (A2M) was also enriched (R=1.52). All 151 proteins identified are shown on table 3.

Table 1.

Enriched and depleted proteins on DVT patients 2 out of 3 experiments.

Enriched proteins 2/3 experiments IPI number* i-TRAQ RATIOS
IgGFc-binding protein precursor IPI00242956.4 2.61
Polymeric immunoglobulin receptor precursor IPI00004573.2 1.77
CD5L antigen-like precursor IPI00025204.1 1.76
Galectin-3-binding protein precursor (Gal3BP) IPI00023673.1 1.76
Alpha-2 macroglobulin (A2M) IPI00478003.1 1.57
13 kDa protein IPI00657670.1 1.53
Immunoglobulin J chain IPI00178926.2 1.27
Clusterin isoform 1 precursor IPI00795633.1 1.25
C4A Complement component IPI00643525.1 1.25
C4B Complement component IPI00418163.3 1.25
C1QA Complement subcomponent subunit A precursor IPI00022392.1 1.28
Depleted proteins 2/3 experiments IPI number* i-TRAQ RATIOS
Serpina1 Isoform 1 of Alpha-1-antitrypsin precursor IPI00553177.1 0.79
Histidine-rich glycoprotein precursor IPI00022371.1 0.74
Isoform 1 of Complement factor H precursor IPI00029739.5 0.73
VWF 309 kDa protein IPI00788786.1 0.70
Ig mu heavy chain disease protein IPI00385264.1 0.70
Hemopexin precursor IPI00022488.1 0.69
F2 Prothrombin precursor (Fragment) IPI00019568.1 0.69
IGHG1 protein IPI00815926.1 0.68
Serum amyloid P-component precursor IPI00022391.1 0.68
doublecortin domain containing 5 IPI00418801.4 0.67
Fibrinogen beta chain precursor IPI00298497.3 0.65
Isoform Gamma-B of Fibrinogen gamma chain precursor IPI00021891.5 0.64
Transferrin variant, Serotransferrin precursor IPI00798430.1 0.63
Isoform 2 of Fibrinogen alpha chain precursor; Isoform 1 of Fibrinogen alpha chain precursor IPI00029717.1; IPI00021885.1 0.62
Extracellular matrix protein 1; Extracellular matrix protein 1 precursor IPI00645849.1; IPI00003351.2 0.62
Afamin precursor IPI00019943.1 0.61
Fibronectin 1 isoform 4 preproprotein IPI00414283.5 0.59
IGHA1 protein IPI00166866.3 0.59
Isoform 1 of Serum albumin precursor IPI00745872.2 0.52
Isoform 3 of Fibronectin precursor; Isoform 1 of Fibronectin precursor IPI00339223.1; IPI00022418.1 0.37
Rheumatoid factor RF-IP16 (Fragment) IPI00827646.1 0.34
Lambda-chain precursor IPI00827875.1 0.97
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*

IPI = international protein index

Table 2.

Enriched and depleted proteins on DVT patients 3 out of 3 experiments and their function on coagulation and inflammation.

Enriched proteins 3/3 experiments IPI number* i-TRAQ RATIOS
Galectin-3-binding protein precursor (Gal3BP) IPI00023673.1 1.76
Alpha-2 macroglobulin (A2M) IPI00478003.1 1.50
Depleted proteins 3/3 experiments IPI number* iTRAQ RATIOS
Serpina1 Isoform 1 of Alpha-1-antitrypsin precursor IPI00553177.1 0.79
Histidine-rich glycoprotein precursor IPI00022371.1 0.76
Hemopexin precursor IPI00022488.1 0.69
Fibrinogen beta chain precursor IPI00298497.3 0.65
Isoform Gamma-B of Fibrinogen gamma chain precursor IPI00021891.5 0.64
Transferrin variant; Serotransferrin precursor IPI00798430.1 0.63
Fibronectin 1 isoform 4 preproprotein IPI00414283.5 0.59
Isoform 1 of Serum albumin precursor IPI00745872.2 0.52
Rheumatoid factor RF-IP16 (Fragment) IPI00827646.1 0.34
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Table 3.

All MP protein identified on the proteomics analysis

Protein IPI
1. 13 kDa protein; APOC3 Apolipoprotein C-III precursor IPI:IPI00657670.1; IPI:IPI00021857.1
2. Alpha-2-macroglobulin (A2M) IPI:IPI00478003.1
3. Anti-(ED-B) scFV (Fragment); - Anti-(ED-B) scFV (Fragment) IPI:IPI00869118.1; IPI:IPI00828062.1
4. Anti-folate binding protein (Fragment) IPI:IPI00828190.1
5. FLJ27059 fis, clone SPL00869 IPI:IPI00442529.1
6. HRV Fab 026-VL (Fragment) IPI:IPI00827510.1
7. Ig heavy chain V-II region SESS precursor IPI:IPI00385557.1
8. Ig heavy chain V-III region GA IPI:IPI00382483.1
9. Ig kappa chain V-I region AG IPI:IPI00387022.1
10. Ig kappa chain V-I region Ni IPI:IPI00387106.1
11. Ig kappa chain V-II region MIL IPI:IPI00387110.1
12. Ig kappa chain V-III region B6 IPI:IPI00387113.1
13. Ig kappa chain V-III region NG9 precursor (Fragment) IPI:IPI00387116.1
14. Ig kappa chain V-III region VH precursor IPI:IPI00829834.1
15. Ig kappa chain V-IV region B17 precursor IPI:IPI00386133.1
16. Ig mu heavy chain disease protein IPI:IPI00385264.1
17. IgG VH protein precursor (Fragment) IPI:IPI00383629.1
18. IGHV4-31 protein IPI:IPI00784810.1
19. Immunglobulin heavy chain variable region IPI:IPI00477804.3
20. Immunoglobulin heavy variable 4-31 IPI:IPI00785084.1
21. Lambda-chain precursor IPI:IPI00827875.1
22. Myosin-reactive immunoglobulin kappa chain variable region (Fragment) IPI:IPI00384402.1
23. Rheumatoid factor light chain variable region precursor (Fragment) IPI:IPI00827486.1
24. Rheumatoid factor RF-IP16 (Fragment) IPI:IPI00827646.1
25. Transthyretin; TTR 13 kDa protein; TTR Transthyretin precursor IPI:IPI00855916.1; IPI:IPI00646384.1; IPI:IPI00022432.1
26. V<gamma>1 protein (Fragment) IPI:IPI00816409.1
27. Variable immnoglobulin anti-estradiol heavy chain (Fragment) IPI:IPI00827581.1
28. Alpha 1B-glycoprotein; A1BG 41 kDa protein; A1BG Alpha-1B-glycoprotein precursor IPI:IPI00745089.2; IPI:IPI00644018.1; IPI:IPI00022895.7
29. Afamin precursor IPI:IPI00019943.1
30. Isoform 1 of Serum albumin precursor IPI:IPI00745872.2
31. AMBP protein precursor IPI:IPI00022426.1
32. Serum amyloid P-component precursor IPI:IPI00022391.1
33. Apolipoprotein A-I precursor IPI:IPI00021841.1
34. Apolipoprotein A-II precursor IPI:IPI00021854.1
35. Apolipoprotein E precursor IPI:IPI00021842.1
36. apolipoprotein L1 isoform a precursor IPI:IPI00852826.1
37. Isoform 1 of Apolipoprotein-L1 precursor; APOL1 Isoform 2 of Apolipoprotein-L1 precursor IPI:IPI00514475.4; IPI:IPI00186903.3
38. C10orf79 Isoform 1 of WD repeat-containing protein C10orf79; C10orf79 hypothetical protein LOC80217 IPI:IPI00478993.4; IPI:IPI00329556.6
39. C1QA Complement C1q subcomponent subunit A precursor IPI:IPI00022392.1
40. C1QB Complement component 1, q subcomponent, B chain (Fragment); C1QB complement component 1, q subcomponent, B chain precursor IPI:IPI00643948.2; IPI:IPI00477992.1
41. Complement C1q subcomponent subunit C precursor IPI:IPI00022394.2
42. C1R;C17orf13;RP11-114H20.1;LOC442122;ACYP1 Complement C1r subcomponent precursor IPI:IPI00296165.5
43. Complement C1s subcomponent precursor IPI:IPI00017696.1
44. Complement C2 precursor (Fragment) IPI:IPI00303963.1
45. Complement C3 precursor (Fragment) IPI:IPI00783987.2
46. Complement component 4A IPI:IPI00643525.1
47. C4B C4B1 IPI:IPI00418163.3
48. C4BPA C4b-binding protein alpha chain precursor IPI:IPI00021727.1
49. C4BPB Isoform 2 of C4b-binding protein beta chain precursor; C4BPB Isoform 1 of C4b- binding protein beta chain precursor IPI:IPI00555752.2; IPI:IPI00025862.2
50. Complement C5 precursor IPI:IPI00032291.1
51. Complement component C6 precursor IPI:IPI00009920.2
52. Complement component C8 alpha chain precursor IPI:IPI00011252.1
53. Complement component C8 beta chain precursor IPI:IPI00294395.1
54. Complement component 8, gamma polypeptide; C8G Complement component C8 gamma chain precursor IPI:IPI00513935.1; IPI:IPI00011261.2
55. Complement component C9 precursor IPI:IPI00022395.1
56. CD5 antigen-like precursor IPI:IPI00025204.1
57. B-factor, properdin; CFB Isoform 1 of Complement factor B precursor (Fragment) IPI:IPI00639937.1; IPI:IPI00019591.1
58. Isoform 1 of Complement factor H precursor IPI:IPI00029739.5
59. CLASP1 protein; Isoform 3 of CLIP-associating protein 1; Isoform 1 of CLIP-associating protein 1 IPI:IPI00828013.2; IPI:IPI00744551.2; IPI:IPI00396279.3
60. Clusterin isoform 1; CLU Clusterin precursor IPI:IPI00795633.1; IPI:IPI00400826.1; IPI:IPI00291262.3
61. Ceruloplasmin precursor IPI:IPI00017601.1
62. Doublecortin domain containing 5 IPI:IPI00418801.4
63. DENND1A 111 kDa protein; DENND1A Isoform 1 of DENN domain-containing protein 1A IPI:IPI00873295.1; IPI:IPI00170641.3
64. Dystrophin Dp427p1 isoform; DMD Isoform 3 of Dystrophin; DMD Isoform 4 of Dystrophin IPI:IPI00472316.1; IPI:IPI00304639.2; IPI:IPI00006091.2
65. DNAJC11 DnaJ (Hsp40) homolog, subfamily C, member 11 IPI:IPI00477887.1
66. Extracellular matrix protein 1; ECM1 Extracellular matrix protein 1 precursor IPI:IPI00645849.1; IPI:IPI00003351.2
67. Translation initiation factor eIF-2B subunit beta IPI:IPI00028083.1
68. Coagulation factor XII precursor IPI:IPI00019581.1
69. A1 Coagulation factor XIII A chain precursor IPI:IPI00297550.8
70. Coagulation factor XIII B chain precursor IPI:IPI00007240.2
71. F2 Prothrombin precursor (Fragment) IPI:IPI00019568.1
72. FAM135B hypothetical protein LOC51059 IPI:IPI00852662.1
73. FAM75A7;FAM75A1 Family with sequence similarity 75, member A1 IPI:IPI00845457.1
74. IgGFc-binding protein precursor IPI:IPI00242956.4
75. Isoform 1 of Ficolin-3 precursor IPI:IPI00293925.2
76. Isoform 2 of Ficolin-3 precursor IPI:IPI00419744.4
77. Isoform 1 of Fibrinogen alpha chain precursor IPI:IPI00021885.1
78. Isoform 2 of Fibrinogen alpha chain precursor; FGA Isoform 1 of Fibrinogen alpha chain precursor IPI:IPI00029717.1; IPI:IPI00021885.1
79. Fibrinogen beta chain precursor IPI:IPI00298497.3
80. Isoform Gamma-B of Fibrinogen gamma chain precursor IPI:IPI00021891.5
81. fibronectin 1 isoform 4 preproprotein IPI:IPI00414283.5
82. Isoform 1 of Fibronectin precursor IPI:IPI00022418.1
83. Isoform 3 of Fibronectin precursor; FN1 Isoform 1 of Fibronectin precursor IPI:IPI00339223.1; IPI:IPI00022418.1
84. Isoform 2 of GRIP and coiled-coil domain-containing protein 2 IPI:IPI00333197.4
85. Glutamate [NMDA] receptor subunit epsilon-1 precursor IPI:IPI00029768.1
86. Alpha 2 globin variant (Fragment); HBA2;HBA1 Hemoglobin subunit alpha IPI:IPI00853068.1; IPI:IPI00410714.5
87. Hemoglobin subunit beta IPI:IPI00654755.3
88. Hemoglobin subunit delta IPI:IPI00473011.3
89. Huntingtin IPI:IPI00002335.1
90. Haptoglobin precursor IPI:IPI00641737.1
91. Isoform 2 of Haptoglobin-related protein precursor; HPR Isoform 1 of Haptoglobin-related protein precursor IPI:IPI00607707.1; IPI:IPI00477597.1
92. Hemopexin precursor IPI:IPI00022488.1
93. Histidine-rich glycoprotein precursor IPI:IPI00022371.1
94. Isoform 2 of Isocitrate dehydrogenase [NAD] subunit alpha, mitochondrial precursor; IDH3A Isoform 1 of Isocitrate dehydrogenase [NAD] subunit alpha, mitochondrial precursor IPI:IPI00792971.2; IPI:IPI00030702.1
95. Insulin-like growth factor-binding protein complex acid labile chain precursor IPI:IPI00020996.3
96. FLJ14473 fis, clone MAMMA1001080, highly similar to Homo sapiens SNC73 protein (SNC73) mRNA IPI:IPI00386879.1
97. IGHA1 protein IPI:IPI00166866.3
98. IGHA2 protein IPI:IPI00783993.1
99. IGHD protein IPI:IPI00418422.3
100. IGHG1 protein IPI:IPI00815926.1
101. IGHG3 protein IPI:IPI00472345.1
102. Ig gamma-4 chain C region; IGHG4 protein IPI:IPI00829814.1; IPI:IPI00550640.2
103. IGHM protein IPI:IPI00472610.2
104. immunoglobulin J chain IPI:IPI00178926.2
105. IGKC protein IPI:IPI00430847.1
106. Ig kappa chain V-II region GM607 precursor (Fragment) IPI:IPI00743389.1
107. IGKV2-24 protein IPI:IPI00440577.3
108. Similar to Ig kappa chain V-III region VG precursor IPI:IPI00784430.3
109. Myosin-reactive immunoglobulin light chain variable region IPI:IPI00549330.4
110. IGL@ protein IPI:IPI00829640.1
111. IGLV2-14 protein IPI:IPI00816555.1
112. IGLV3-21 protein IPI:IPI00815938.1
113. V2-19 protein; IGLV3-16 V2-11 protein IPI:IPI00552852.2; IPI:IPI00552771.2
114. Zinc finger protein Pegasus IPI:IPI00154612.3
115. Inter-alpha-trypsin inhibitor heavy chain H1 precursor IPI:IPI00292530.1
116. Inter-alpha (Globulin) inhibitor H2; ITIH2 Inter-alpha-trypsin inhibitor heavy chain H2 precursor IPI:IPI00645038.1; IPI:IPI00305461.2
117. Isoform 1 of Kinesin-like protein KIF21B; KIF21B Isoform 2 of Kinesin-like protein KIF21B IPI:IPI00872997.1; IPI:IPI00644778.2
118. Isoform HMW of Kininogen-1 precursor IPI:IPI00032328.2
119. Keratin, type I cytoskeletal 9 IPI:IPI00019359.3
120. Laminin subunit gamma-1 precursor IPI:IPI00298281.3
121. Galectin-3-binding protein IPI:IPI00023673.1
122. PTD016 protein; LOC51136 CDNA FLJ25783 fis, clone TST06726 IPI:IPI00297702.4; IPI:IPI00007646.5
123. LOC646903 protein IPI:IPI00061475.4
124. LOC729628 similar to Hornerin IPI:IPI00787795.1
125. Isoform Long of Alpha-mannosidase IIx IPI:IPI00027703.1
126. LOC730410;HLA-C;HLA-B MHC class I antigen IPI:IPI00816057.1
127. Alpha-1-acid glycoprotein 1 precursor IPI:IPI00022429.3
128. Alpha-1-acid glycoprotein 2 precursor IPI:IPI00020091.1
129. Isoform 2 of Prolyl 4-hydroxylase subunit alpha-1 precursor; Isoform 1 of Prolyl 4-hydroxylase subunit alpha-1 precursor IPI:IPI00218682.1; IPI:IPI00009923.1
130. pecanex-like 3; PCNXL3 similar to pecanex-like 3 isoform 5 similar to pecanex-like 3 isoform 2 IPI:IPI00869136.1; IPI:IPI00787937.1; IPI:IPI00738957.2
131. Isoform 2 of N-acetylmuramoyl-L-alanine amidase precursor; Isoform 1 of N-acetylmuramoyl- L-alanine amidase precursor IPI:IPI00394992.1; IPI:IPI00163207.1
132. Polymeric immunoglobulin receptor precursor IPI:IPI00004573.2
133. Plasminogen precursor IPI:IPI00019580.1
134. PROS1 80 kDa protein IPI:IPI00873445.1
135. Pregnancy zone protein precursor IPI:IPI00025426.1
136. Reticulocalbin-1 precursor IPI:IPI00015842.1
137. Serum amyloid A protein precursor IPI:IPI00552578.2
138. SERPINA1 Isoform 1 of Alpha-1-antitrypsin precursor IPI:IPI00553177.1
139. SERPINA3 Alpha-1-antichymotrypsin precursor IPI:IPI00550991.3
140. SERPINA3 Isoform 1 of Alpha-1-antichymotrypsin precursor; SERPINA3 Alpha-1- antichymotrypsin precursor IPI:IPI00847635.1; IPI:IPI00550991.3
141. SERPINC1 protein IPI:IPI00844156.2
142. SERPIND1 Heparin cofactor 2 precursor IPI:IPI00292950.4
143. Isoform 2 of Sodium- and chloride-dependent transporter XTRP3; Solute carrier family 6, member 20 isoform 1 IPI:IPI00216628.1; IPI:IPI00152803.4
144. TF Transferrin variant (Fragment); TF Serotransferrin precursor IPI:IPI00798430.1; IPI:IPI00022463.1
145. TGFB1-induced anti-apoptotic factor 1 IPI:IPI00294690.2
146. Isoform 8 of Titin IPI:IPI00759542.1
147. Vitronectin precursor IPI:IPI00298971.1
148. VWF 309 kDa protein IPI:IPI00788786.1
149. VWF von Willebrand factor precursor IPI:IPI00023014.1
150. H14 nuclear protein UKp68 isoform 4 IPI:IPI00168449.1
151. ZNF148 78 kDa protein; SLC12A8 Isoform 2 of Solute carrier family 12 member 8; SLC12A8 Isoform 1 of Solute carrier family 12 member 8; ZNF148 82 kDa protein IPI:IPI00872544.1; IPI:IPI00867607.1; IPI:IPI00855723.2; IPI:IPI00747011.1
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Nine proteins were depleted in MP of DVT patients; among them, fibrinogen beta chain precursor (R=0.65) and fibrinogen gamma chain precursor, (R=0.64). Other polypeptides, not related to thrombosis or inflammation, were also depleted as shown in table 2. All of these proteins were depleted on DVT patients compared to negatives as indicated by iTRAQ ratios of less than 0.8. Complementary information (DVT positive and DVT negative distribution, D-dimer levels, MP count) is shown on table 4.

Table 4.

DVT and negative patients, D-dimer levels and MP counts.

ID numbers Initials D-dimer levels (ng/ml) Total MP number Observations
DVT patients
5038-003 MB 0.82 9667.5
5038-006 KZ 1.20 2977.5
5038-007 MJ 5.05 15765
5038-009 BS 6.00 8920
5038-012 GT 6.90 2032.5
5038-013 RW 0.97 16237.5
5038-015 RT 3.17 36592.5
5038-017 LH 1.42 182125 2.96 (DVT positive D-dimer average level)
5038-019 VH 1.10 105945 42,251 (MP average count on DVT positive patients)
DVT Negative patients
2460-002 BP 0.41 40435
2460-004 YSH 0.32 110240
2460-005 EA 2.36 57980
2460-006 AR 5.68 59620
2460-007 DCP 0.42 31580
2460-008 DB 0.26 25815
2460-009 AD 7.44
2460-200 TB 0.58 52155 2.07 (DVT negative D-dimer average level)
2460-201 MC 1.19 18250 49,509 (MP average count on DVT negative patients)
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Discussion

Thrombosis and inflammation are inter-related processes. At the time of thrombus formation, MP are recruited to the thrombus site, playing a role in thrombus modulation.

MP are submicron vesicles shed from plasma membranes, in response to cell activation, injury and/or apoptosis. Recent investigations suggest that MP, which are prothrombotic in part by virtue of carrying TF,13–16 are important in early venous thrombogenesis.17 MP are recruited to the area of thrombosis,18 where they amplify coagulation via TF and factor VIIa.13, 17–19 Co-localization of fibrin, platelets, and leukocytes in the developing thrombus 13, 20 and the leukocyte-platelet interactions generating TF 21 support the central role of inflammation in thrombogenesis 22, with MP interacting both in coagulation and inflammation processes. MP are also found in healthy individuals 23, as well as pathological conditions such as severe sepsis, where their reduction was found to correlate with organ dysfunction and mortality.24 The current study evaluated MP proteins with proteomics analysis in human venous thrombosis. Since MP present diverse biological expressions that may be linked to a unique subset of proteins, derived from their origin cells, the assumption is that those proteins are crucial for influencing thrombogenesis and are regulated during the course of venous thrombosis. Thus, identifying and understanding the nature of these proteins can serve to provide valuable insight into the mechanistic role of MP in human venous thrombogenesis.

Enriched Proteins: Gal3BP andA2M

Gal3BP precursor is a polypeptide from the lectin family that promotes integrin-mediated cell adhesion25. In addition, it plays a key role in human platelet activation and function, synergizes with ADP or thrombin to induce platelet aggregation and ATP release, induces up-regulation of P-selectin, induces GPIIIa expression, promotes shedding of MPs, and favors the generation of leukocyte-platelet aggregates26. Those effects of Gal3BP may play an important role in thrombosis and inflammation, potentially amplifying the thrombus progression. Gal3BP was enriched in MP of all DVT patients, leading to the idea that this protein is over-expressed during thrombus formation process. Previous studies have demonstrated that P-selectin inhibition decreases thrombogenesis27. An inhibition of Gal3BP would promote an upstream inhibition of P-selectin and even a decrease in MP generation, decreasing thrombus propagation.

On the other hand, A2M plays an important role in thrombogenesis and fibrinolysis. It functions as an inhibitor of fibrinolysis by inhibiting plasmin and kallikrein, and as an inhibitor of coagulation by inhibiting thrombin. In all DVT patients A2M was enriched. MP, by virtue of having this inhibitor, may play a role in thrombus formation (by its inhibition of plasmin) facilitating thrombus progression.

Proteins Depleted in MP of DVT Patients

Nine proteins were observed to be depleted in MP from DVT patients compared to controls. Seven of them, Serpina1 Isoform 1 of Alpha-1-antitrypsin precursor, Histidine-rich glycoprotein precursor, Hemopexin precursor, Transferrin variant serotransferrin precursor, Fibrinogen, Serum albumin precursor and Rheumatoid factor RF-IP16, play no known major role in inflammation or thrombosis.

Fibrinogen is a six-chain protein precursor to the clot structural protein, fibrin and dimer of α, β, and γ polypeptides.28,29 Proteomic analysis detected 2 of these polypeptides (Fibrinogen beta chain precursor and Isoform Gamma-B of Fibrinogen gamma chain precursor) on MP, both depleted on MP from DVT patients. The presence of fibrinogen suggests a number of potential mechanisms regarding the role of MP in the thrombotic process. Interestingly, in our previous study of experimental DVT in primates, fibrinogen was enriched in early stages of thrombosis (day 2)8. In spite of having all samples collected before the initiation of DVT treatment, the time point of blood collection in human samples cannot be precise, as it was in our controlled experimental primate model, where samples were collected precisely 48 hours after thrombus initiation. Many patients do not present immediately upon the development of leg symptoms and we thus have no way to know the precise timing of thrombus initiation. The down-regulation of fibrinogen in this study may be due to its consumption (fibrinogen taken into the thrombus and consumed).

Unexpectedly, a number of proteins such as tissue factor (TF), P-selectin, and P-selectin glycoprotein ligand-1 (PSGL-1) were not observed on MP using proteomics analysis, in both experiments (this human DVT MP analysis and our previous experimental primate DVT model study8). This is interesting since it was reported that the amount of TF activity on MP correlate with the presence or absence of experimentally induced venous thrombosis15, 16, 30, 31 and P-selectin and PSGL-1 has been found critical in venous thrombogenesis. Potential reasons for this absence include the saturation of these proteins by their receptors, or glycosylation. Highly glycosylated proteins might not be detected by proteomic analysis, and PSGL-1 as a receptor for P-selectin, a member of lectin family, is glycosylated. A more likely explanation for their absence is low levels of these proteins in the MP preparation, present on MP but not detected by proteomics analysis.

Additionally, TF has been reported on MP by ELISA methods30, but the majority of recent studies addressing MP Proteomics also had not detected TF.

Conclusions

In this study, complementary to our previous proteome analysis of MP in a large animal model of venous thrombosis, a diverse group of proteins associated with circulating MP in human patients presenting venous thrombosis were characterized. We can speculate that these proteins may influence thrombosis through inhibition of inflammation and MP generation (Gal3BP), coagulation cascade and fibrinolysis (A2M) and hemostatic plug formation (fibrinogen). New direct experimental studies evaluating the role of these proteins in thrombogenesis are warranted. Proteomic data from this investigation will be used to design upcoming studies aimed at identifying novel biomarkers and future target therapies for venous thrombosis.

Acknowledgments

Supported by NIH grant 070766 (TWW)

Footnotes

Conflict of interest

None.

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