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Accelerating Biomarker Discovery Through Electronic Health Records, Automated Biobanking, and Proteomics.
Wells QS, Gupta DK, Smith JG, Collins SP, Storrow AB, Ferguson J, Smith ML, Pulley JM, Collier S, Wang X, Roden DM, Gerszten RE, Wang TJ
(2019) J Am Coll Cardiol 73: 2195-2205
MeSH Terms: Academic Medical Centers, Acceleration, Aged, Automation, Biological Specimen Banks, Biomarkers, Cohort Studies, Electronic Health Records, Female, Heart Failure, Humans, Male, Middle Aged, Proportional Hazards Models, Prospective Studies, Proteomics, Reproducibility of Results, Risk Assessment, Sensitivity and Specificity, Thrombospondins
Show Abstract · Added March 3, 2020
BACKGROUND - Circulating biomarkers can facilitate diagnosis and risk stratification for complex conditions such as heart failure (HF). Newer molecular platforms can accelerate biomarker discovery, but they require significant resources for data and sample acquisition.
OBJECTIVES - The purpose of this study was to test a pragmatic biomarker discovery strategy integrating automated clinical biobanking with proteomics.
METHODS - Using the electronic health record, the authors identified patients with and without HF, retrieved their discarded plasma samples, and screened these specimens using a DNA aptamer-based proteomic platform (1,129 proteins). Candidate biomarkers were validated in 3 different prospective cohorts.
RESULTS - In an automated manner, plasma samples from 1,315 patients (31% with HF) were collected. Proteomic analysis of a 96-patient subset identified 9 candidate biomarkers (p < 4.42 × 10). Two proteins, angiopoietin-2 and thrombospondin-2, were associated with HF in 3 separate validation cohorts. In an emergency department-based registry of 852 dyspneic patients, the 2 biomarkers improved discrimination of acute HF compared with a clinical score (p < 0.0001) or clinical score plus B-type natriuretic peptide (p = 0.02). In a community-based cohort (n = 768), both biomarkers predicted incident HF independent of traditional risk factors and N-terminal pro-B-type natriuretic peptide (hazard ratio per SD increment: 1.35 [95% confidence interval: 1.14 to 1.61; p = 0.0007] for angiopoietin-2, and 1.37 [95% confidence interval: 1.06 to 1.79; p = 0.02] for thrombospondin-2). Among 30 advanced HF patients, concentrations of both biomarkers declined (80% to 84%) following cardiac transplant (p < 0.001 for both).
CONCLUSIONS - A novel strategy integrating electronic health records, discarded clinical specimens, and proteomics identified 2 biomarkers that robustly predict HF across diverse clinical settings. This approach could accelerate biomarker discovery for many diseases.
Copyright © 2019 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.
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20 MeSH Terms
Comparison of protein immunoprecipitation-multiple reaction monitoring with ELISA for assay of biomarker candidates in plasma.
Lin D, Alborn WE, Slebos RJ, Liebler DC
(2013) J Proteome Res 12: 5996-6003
MeSH Terms: Amino Acid Sequence, Animals, Antibodies, Antigens, CD, Biomarkers, Tumor, Carcinoma, Cartilage Oligomeric Matrix Protein, Cattle, Colonic Neoplasms, Endoglin, Enzyme-Linked Immunosorbent Assay, GPI-Linked Proteins, Hernia, Humans, Immunoprecipitation, Mass Spectrometry, Matrix Metalloproteinase 9, Molecular Sequence Data, Receptors, Cell Surface, Thrombospondins, Tissue Inhibitor of Metalloproteinase-1
Show Abstract · Added March 7, 2014
Quantitative analysis of protein biomarkers in plasma is typically done by ELISA, but this method is limited by the availability of high-quality antibodies. An alternative approach is protein immunoprecipitation combined with multiple reaction monitoring mass spectrometry (IP-MRM). We compared IP-MRM to ELISA for the analysis of six colon cancer biomarker candidates (metalloproteinase inhibitor 1 (TIMP1), cartilage oligomeric matrix protein (COMP), thrombospondin-2 (THBS2), endoglin (ENG), mesothelin (MSLN) and matrix metalloproteinase-9 (MMP9)) in plasma from colon cancer patients and noncancer controls. Proteins were analyzed by multiplex immunoprecipitation from plasma with the ELISA capture antibodies, further purified by SDS-PAGE, digested and analyzed by stable isotope dilution MRM. IP-MRM provided linear responses (r = 0.978-0.995) between 10 and 640 ng/mL for the target proteins spiked into a "mock plasma" matrix consisting of 60 mg/mL bovine serum albumin. Measurement variation (coefficient of variation at the limit of detection) for IP-MRM assays ranged from 2.3 to 19%, which was similar to variation for ELISAs of the same samples. IP-MRM and ELISA measurements for all target proteins except ENG were highly correlated (r = 0.67-0.97). IP-MRM with high-quality capture antibodies thus provides an effective alternative method to ELISA for protein quantitation in biological fluids.
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21 MeSH Terms
Angiogenesis and antiangiogenic therapy of colon cancer liver metastasis.
Stoeltzing O, Liu W, Reinmuth N, Parikh A, Ahmad SA, Jung YD, Fan F, Ellis LM
(2003) Ann Surg Oncol 10: 722-33
MeSH Terms: Angiogenesis Inhibitors, Animals, Colonic Neoplasms, Cyclohexanes, Endothelial Growth Factors, Humans, Liver Neoplasms, Neovascularization, Pathologic, O-(Chloroacetylcarbamoyl)fumagillol, Sesquiterpenes, Spiro Compounds, Thrombospondins, Vascular Endothelial Growth Factor A, Wound Healing
Show Abstract · Added March 5, 2014
The fact that tumor growth and metastatic spread relies on angiogenesis has been widely proven and accepted. The understanding of cancer biology and metastasis formation has led to the development of new therapeutic approaches that target tumor biology. The survival and establishment of metastatic lesions depend on a shift in the normal balance of proangiogenic and antiangiogenic factors that favor angiogenesis. Colorectal cancer is one of the leading cancer deaths worldwide. Angiogenesis has been associated with colon cancer progression and metastatic spread, thereby significantly affecting patient survival. New experimental approaches that inhibit angiogenic processes have demonstrated promising antineoplastic effects on metastatic colorectal cancer and are partially being investigated in clinical trials. This review focuses on angiogenesis in colorectal cancer metastasis formation as a target for antiangiogenic therapy, describing the experience from experimental studies and current clinical trials.
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14 MeSH Terms
The tumor microenvironment: a potential arbitrator of the tumor suppressive and promoting actions of TGFbeta.
Dumont N, Arteaga CL
(2002) Differentiation 70: 574-82
MeSH Terms: Animals, Cell Movement, Endopeptidases, Extracellular Matrix, Humans, Mice, Neoplasms, Neoplastic Processes, Neovascularization, Pathologic, Receptors, Transforming Growth Factor beta, Signal Transduction, Thrombospondins, Transforming Growth Factor beta
Show Abstract · Added March 5, 2014
Transforming growth factor beta (TGFbeta) members are secreted in biologically inactive complexes that must be activated in order to enable binding to their cell surface receptors. Interestingly, many of the proteins that can activate TGFbeta have been implicated in either suppressing or promoting tumorigenesis. Included among these are matrix proteins (thrombospondin-1), receptors (integrins alphanubeta6 and alphanubeta8) and proteases (matrix metalloproteases and plasmin). These proteins cannot only activate TGFbeta, but can also modulate cell responsiveness to TGFbeta. In this section, we review data highlighting the complexity and bidirectionality of TGFbeta matrix interactions within the tumor microenvironment, and propose that these dynamic interactions are a critical spatial and temporal determinant of the effects of TGFbeta on tumorigenesis.
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13 MeSH Terms
Isolation of the fibrinogen-binding region of platelet thrombospondin.
Dixit VM, Grant GA, Frazier WA, Santoro SA
(1984) Biochem Biophys Res Commun 119: 1075-81
MeSH Terms: Blood Platelets, Calcium, Fibrinogen, Glycoproteins, Humans, Magnesium, Peptide Fragments, Protein Binding, Thermolysin, Thrombospondins
Show Abstract · Added March 5, 2014
Purified platelet thrombospondin binds to immobilized fibrinogen if both Ca++ and Mg++ are present. Digestion of the purified molecule with thermolysin results in a limited number of discrete proteolytic fragments. When such digests are subjected to affinity chromatography on immobilized fibrinogen, only the fragments with Mr of 120,000 and 140,000 are specifically bound and subsequently eluted by the addition of EDTA to the column buffer. Examination by SDS-PAGE under both reducing and nonreducing conditions reveals that the fibrinogen-binding domain is derived from the region of the thrombospondin molecule containing the interchain disulfide bonds. The requirement for Ca++ and Mg++ for optimal binding to fibrinogen is also manifest by the Mr 120,000/140,000 thermolytic fragments.
1 Communities
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10 MeSH Terms
Localization of the hemagglutinating activity of platelet thrombospondin to a 140 000-dalton thermolytic fragment.
Haverstick DM, Dixit VM, Grant GA, Frazier WA, Santoro SA
(1984) Biochemistry 23: 5597-603
MeSH Terms: Blood Platelets, Calcium, Dithiothreitol, Erythrocytes, Glycoproteins, Hemagglutination, Heparin, Humans, Magnesium, Molecular Weight, Peptide Fragments, Thermolysin, Thrombospondins
Show Abstract · Added March 5, 2014
The platelet protein thrombospondin (TSP) which is secreted from alpha-granules upon platelet activation agglutinates trypsinized, glutaraldehyde-fixed human erythrocytes. Optimal conditions for the hemagglutinating activity require that both Ca2+ and Mg2+ be present in final concentrations of 2 mM. In the presence of dithiothreitol (i.e., reduction of disulfide bonds), the lectin-like activity decreases in a manner proportional to the extent of reduction of the molecule from its native trimeric configuration into its Mr 180 000 subunits. Proteolysis of purified TSP with thermolysin, which produces discrete domains with the capacity to bind fibrinogen and heparin, also diminishes, but does not abolish, the hemagglutinating activity. Fibrinogen was without effect on hemagglutinating activity while heparin was found to be a potent inhibitor. Other proteoglycans such as hyaluronic acid, chondroitin sulfate, keratan sulfate, dermatan sulfate, and heparan sulfate had no effect. That portion of the TSP molecule apparently responsible for the hemagglutinating activity was identified by incubating a thermolytic digest of TSP with red blood cells and then determining which fragment was bound to the cell surface. The binding site resides within a peptide fragment of 140 000 daltons but is absent from an Mr 120 000 fragment derived from the Mr 140 000 fragment. Under the conditions for optimal expression of hemagglutinating activity (i.e., 2 mM MgCl2 and 2 mM CaCl2), this Mr 140 000 fragment was also shown to have heparin binding activity.
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13 MeSH Terms
Isolation and characterization of a heparin-binding domain from the amino terminus of platelet thrombospondin.
Dixit VM, Grant GA, Santoro SA, Frazier WA
(1984) J Biol Chem 259: 10100-5
MeSH Terms: Amino Acid Sequence, Amino Acids, Binding Sites, Blood Platelets, Fibrinolysin, Glycoproteins, Heparin, Humans, Molecular Weight, Peptide Fragments, Protein Binding, Thermolysin, Thrombospondins
Show Abstract · Added March 5, 2014
Calcium-replete thrombospondin has been purified from outdated platelets using heparin-Sepharose affinity chromatography, gelatin-Sepharose to remove fibronectin, and gel filtration to eliminate low-molecular-weight heparin-binding proteins. Edman degradation of six different preparations revealed the amino-terminal sequence of thrombospondin (TSP) to be Asn-Arg-Ile-Pro-Glu-Ser-Gly-Gly-Asp-Asn-Ser-Val-Phe-. This sequence was obtained in initial yields as high as 85%, indicating that no blocked chains are present. Cleavage of calcium-replete TSP with thermolysin or plasmin results in the production of relatively stable fragments. Chromatography of these digests on heparin-Sepharose followed by elution with 0.6 M NaCl affords purification of an Mr 25,000 fragment from the thermolysin digest and an Mr 35,000 fragment from the plasmin digest. The binding of these fragments to heparin-Sepharose does not require divalent metal ions. Neither fragment is disulfide-bonded to other fragments present in the digests. The heparin-binding domains from both digests have similar amino acid compositions and their tryptic peptide maps on high performance liquid chromatography are identical with the exception of one peptide unique to each fragment. Automated Edman degradation in a vapor-phase sequenator of the thermolytic heparin-binding domain electroeluted from sodium dodecyl sulfate-gels indicates that the heparin-binding domain resides at the amino terminus of the Mr 180,000 TSP peptide chain.
1 Communities
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13 MeSH Terms
Effects of anti-thrombospondin monoclonal antibodies on the agglutination of erythrocytes and fixed, activated platelets by purified thrombospondin.
Dixit VM, Haverstick DM, O'Rourke KM, Hennessy SW, Grant GA, Santoro SA, Frazier WA
(1985) Biochemistry 24: 4270-5
MeSH Terms: Antibodies, Monoclonal, Antigen-Antibody Complex, Blood Platelets, Erythrocytes, Glycoproteins, Glycosaminoglycans, Hemagglutination, Humans, Kinetics, Platelet Aggregation, Selenomethionine, Thrombospondins
Show Abstract · Added March 5, 2014
A monoclonal antibody (Mab) has been raised against native thrombospondin (TSP), the endogenous lectin of human platelets, that inhibits the hemagglutination of trypsinized, glutaraldehyde-fixed human erythrocytes by purified TSP. This Mab, designated A2.5, also inhibits the agglutination of fixed, activated platelets by TSP. Mab A2.5 immunoprecipitates a 25-kilodalton (kDa) peptide from chymotryptic digests of TSP that is not disulfide bonded to any other region of the TSP molecule. This fragment represents the previously characterized heparin binding domain of TSP [Dixit, V.M., Grant, G.A., Santoro, S.A., & Frazier, W.A. (1984) J. Biol. Chem. 259, 10100-10105]. In agreement with this assignment, heparin inhibits the binding of Mab A2.5 to TSP. Another Mab, designated C6.7, also blocks TSP-mediated hemagglutination, yet has no effect on the agglutination of fixed, activated platelets by TSP. This Mab has been shown to inhibit the thrombin-stimulated aggregation of live platelets and to immunoprecipitate an 18-kDa fragment from chymotryptic digests, which is distinct from the heparin binding domain [Dixit, V.M., Haverstick, D.M., O'Rourke, K.M., Hennessy, S.W., Grant, G.A., Santoro, S.A., & Frazier, W.A. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 3472-3476].
1 Communities
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12 MeSH Terms
Characterization of the platelet agglutinating activity of thrombospondin.
Haverstick DM, Dixit VM, Grant GA, Frazier WA, Santoro SA
(1985) Biochemistry 24: 3128-34
MeSH Terms: Blood Platelets, Calcium, Cations, Divalent, Electrophoresis, Polyacrylamide Gel, Glycoproteins, Humans, Kinetics, Magnesium, Molecular Weight, Peptide Fragments, Platelet Aggregation, Thermolysin, Thrombospondins
Show Abstract · Added March 5, 2014
Thrombospondin (TSP) is a glycoprotein secreted from the alpha-granules of platelets upon activation. In the presence of divalent cations, the secreted protein binds to the surface of the activated platelets and is responsible for the endogenous lectin-like activity associated with activated platelets. Platelets fixed with formaldehyde following activation by thrombin are agglutinated by exogenously added TSP. Fixed, nonactivated platelets are not agglutinated. The platelet agglutinating activity of TSP is optimally expressed in the presence of 2 mM each of Mg2+ and Ca2+. Reduction of the disulfide bonds within the TSP molecule inhibits its platelet agglutinating activity. TSP bound to the surface of fixed, activated platelets can be eluted by the addition of disodium ethylenediaminetetraacetate. This approach was exploited to identify the region of the TSP molecule containing the platelet binding site. The binding site resides within a thermolytic fragment of TSP with Mr 140 000 but is not present in the Mr 120 000 fragment derived from the polypeptide of Mr 140 000. Since both the Mr 140 000 and 120 000 fragments contain fibrinogen binding sites, this finding suggests that the binding of TSP to the platelet surface requires interaction with other platelet surface components in addition to fibrinogen. The observation that fibrinogen only partially inhibits the TSP-mediated agglutination of fixed, activated platelets is consistent with this interpretation.
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13 MeSH Terms
The platelet glycoprotein thrombospondin binds specifically to sulfated glycolipids.
Roberts DD, Haverstick DM, Dixit VM, Frazier WA, Santoro SA, Ginsburg V
(1985) J Biol Chem 260: 9405-11
MeSH Terms: Animals, Blood Platelets, Cations, Divalent, Edetic Acid, Glycoproteins, Humans, Hydrogen-Ion Concentration, Kinetics, Phospholipids, Protein Binding, Sheep, Sulfoglycosphingolipids, Thrombospondins
Show Abstract · Added March 5, 2014
The human platelet glycoprotein thrombospondin (TSP) binds specifically and with high affinity to sulfatides (galactosylceramide-I3-sulfate). Binding of 125I-TSP to lipids from sheep and human erythrocytes and human platelets resolved on thin layer chromatograms indicates that sulfatides are the only lipids in the membrane which bind TSP. Binding to less than 2 ng of sulfatide could be detected. TSP failed to bind to other purified lipids including cholesterol 3-sulfate, phospholipids, neutral glycolipids, and gangliosides. Binding of 125I-TSP was inhibited by unlabeled TSP, by low pH, and by reduction of intersubunit disulfide bonds with dithiothreitol. A monoclonal antibody against TSP (A2.5), which inhibits hemagglutination and agglutination of fixed activated platelets by TSP, strongly inhibited TSP binding to sulfatides. A second monoclonal antibody (C6.7), which inhibits hemagglutination and aggregation of thrombin-activated live platelets, weakly inhibited sulfatide binding. Binding was inhibited by high ionic strength and by some monosaccharide sulfates including methyl-alpha-D-GlcNAc-3-sulfate. Neutral sugars did not inhibit. Fucoidan, a sulfated fucan, strongly inhibited binding with 50% inhibition at 0.3 micrograms/ml fucoidan. Other sulfated polysaccharides including heparin and dextran sulfates were good inhibitors, whereas hyaluronic acid and keratan sulfate were very weak.
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13 MeSH Terms