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Optimization of quantitative proteomic analysis of clots generated from plasma of patients with venous thromboembolism.

ABSTRACT: Background:It is well known that fibrin network binds a large variety of proteins, including inhibitors and activators of fibrinolysis, which may affect clot properties, such as stability and susceptibility to fibrinolysis. Specific plasma clot composition differs between individuals and may change in disease states. However, the plasma clot proteome has not yet been in-depth analyzed, mainly due to technical difficulty related to the presence of a highly abundant protein-fibrinogen and fibrin that forms a plasma clot. Methods:The aim of our study was to optimize quantitative proteomic analysis of fibrin clots prepared ex vivo from citrated plasma of the peripheral blood drawn from patients with prior venous thromboembolism (VTE). We used a multiple enzyme digestion filter aided sample preparation, a multienzyme digestion (MED) FASP method combined with LC-MS/MS analysis performed on a Proxeon Easy-nLC System coupled to the Q Exactive HF mass spectrometer. We also evaluated the impact of peptide fractionation with pipet-tip strong anion exchange (SAX) method on the obtained results. Results:Our proteomic approach revealed 476 proteins repeatedly identified in the plasma fibrin clots from patients with VTE including extracellular vesicle-derived proteins, lipoproteins, fibrinolysis inhibitors, and proteins involved in immune responses. The MED FASP method using three different enzymes: LysC, trypsin and chymotrypsin increased the number of identified peptides and proteins and their sequence coverage as compared to a single step digestion. Peptide fractionation with a pipet-tip strong anion exchange (SAX) protocol increased the depth of proteomic analyses, but also extended the time needed for sample analysis with LC-MS/MS. Conclusions:The MED FASP method combined with a label-free quantification is an excellent proteomic approach for the analysis of fibrin clots prepared ex vivo from citrated plasma of patients with prior VTE.

SUBMITTER: Stachowicz A

PROVIDER: S-EPMC5706328 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Optimization of quantitative proteomic analysis of clots generated from plasma of patients with venous thromboembolism.

Stachowicz Aneta A Siudut Jakub J Suski Maciej M Olszanecki Rafał R Korbut Ryszard R Undas Anetta A Wiśniewski Jacek R JR

Clinical proteomics 20171128

<h4>Background</h4>It is well known that fibrin network binds a large variety of proteins, including inhibitors and activators of fibrinolysis, which may affect clot properties, such as stability and susceptibility to fibrinolysis. Specific plasma clot composition differs between individuals and may change in disease states. However, the plasma clot proteome has not yet been in-depth analyzed, mainly due to technical difficulty related to the presence of a highly abundant protein-fibrinogen and ...[more]

PMID: 29209155

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Project description:Essentials Venous thromboembolism (VTE) recurrence leads to decreased clot elastic modulus in plasma. Recurrent VTE is not linked to changes in clot structure, fiber radius, or factor XIII activity. Other plasma components may play a role in VTE recurrence. Prospective studies should resolve if clot stiffness can be used as predictor for recurrent VTE. SUMMARY: Background Venous thromboembolism (VTE) is associated with a high risk of recurrent events after withdrawal of anticoagulation. Objectives To determine the difference in plasma clot mechanical properties between patients with recurrent VTE (rVTE) and those with non-recurrent VTE (nrVTE). Methods We previously developed a system for determining clot mechanical properties by use of an in-house magnetic tweezers system. This system was used to determine the mechanical properties of clots made from plasma of 11 patients with rVTE and 33 with nrVTE. Plasma was mixed with micrometer-sized beads, and thrombin and calcium were added to induce clotting; the mixture was then placed in small capillary tubes, and clotting was allowed to proceed overnight. Bead displacements upon manipulation with magnetic forces were analyzed to determine clot elastic and viscous moduli. Fibrin clot structure was analyzed with turbidimetry and confocal microscopy. Factor XIII was measured by pentylamine incorporation into fibrin. Results Clots from rVTE patients showed nearly two-fold less elastic and less viscous moduli than clots from nrVTE patients, regardless of male sex, unprovoked events, family history of VTE, fibrinogen concentration, or body mass index. No differences were observed in clot structure, fibrinolysis rates, or FXIII levels. Conclusion Using magnetic tweezers for the first time in patient samples, we found that plasma clots from rVTE patients showed a reduced elastic modulus and a reduced viscous modulus as compared with clots from nrVTE patients. These data indicate a possible role for fibrin clot viscoelastic properties in determining VTE recurrence.

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Optimization of quantitative proteomic analysis of clots generated from plasma of patients with venous thromboembolism.

Publications

Optimization of quantitative proteomic analysis of clots generated from plasma of patients with venous thromboembolism.

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