Elsevier

Journal of Proteomics

Volume 229, 30 October 2020, 103946
Journal of Proteomics

Plasma fibrin clot proteomics in patients with acute pulmonary embolism: Association with clot properties

https://doi.org/10.1016/j.jprot.2020.103946Get rights and content

Highlights

  • Fibrin clot proteome differs between pulmonary embolism patients and healthy controls.

  • In pulmonary embolism, clots proteome is associated with fibrin clot properties.

  • Proteins unrelated to coagulation modulate fibrin phenotype in acute thrombosis.

Abstract

It has been reported that 476 proteins can be detected in plasma fibrin clots from patients with venous thromboembolism. Plasma fibrin clots proteomic composition in relation to their properties has not been studied in acute pulmonary embolism (PE). Clots generated from plasma of 20 PE patients and 20 healthy controls were assessed using mass spectrometry, clot permeability (Ks), and clot lysis time (CLT). The proteomic composition of plasma fibrin clots from acute PE patients differed from that of control subjects in regard to 198 clot-bound proteins. In the acute PE group, we observed increased clot-bound fibrinogen, apolipoprotein B-100, platelet glycoprotein Ib, lipopolysaccharide-binding protein, and histones H3 + 4 and reduced fibronectin, α2-antiplasmin, α2-macroglobulin, factor (F)XIII, histidine-rich glycoprotein, antithrombin, von Willebrand Factor, plasminogen, and prothrombin. Among PE patients, low Ks (≤3.83 × 10−9 cm2) was associated with increased clot-bound C-reactive protein, kininogen-1, protein S, β-2-microglobulin, and thromboxane-A synthase when compared with patients having Ks > 3.83 × 10−9 cm2. Ks correlated inversely with FIX and FV, thrombin-activatable fibrinolysis inhibitor, complement C1s, C7, C8, and apolipoprotein A-I. The specific protein composition in plasma fibrin clots from acute PE patients is associated with denser clot formation. Several proteins unrelated to the coagulation system can modulate fibrin phenotype in acute thrombotic states.

Significance

Our study significantly advances the field of thrombosis and hemostasis. The plasma fibrin clot proteomics findings fill the gap of knowledge about the presence and the role of other proteins to the plasma fibrin clot in the acute phase of pulmonary embolism, aside fibrinogen, which is the main component of fibrin. The reported methodology, which involves the sample preparation using Multienzyme Digestion-Filter Aided Sample Preparation (MED FASP), data acquisition with the Quadrupole-Orbitrap mass spectrometer, and data analysis using the advanced tools such as MaxQuant, Total Protein Approach and Perseus, allows to gain not only the qualitative, but also the quantitative insights into the microworld of proteins entangled among the fibrin network. By comparing the clots formed from plasma of patients with acute pulmonary embolism with the clots from healthy control, we provide the specific protein composition associated with unfavorable clot properties observed in this disease. Moreover, our findings emphasize that several proteins unrelated to the coagulation system, can modulate fibrin phenotype in acute thrombotic states.

Introduction

Prothrombotic fibrin clot properties, including reduced clot porosity and susceptibility to lysis, are known to be associated with venous and arterial thrombotic events. Fibrin clot properties can be assessed using several methods, including clot permeability, compaction, and turbidity to determine clot absorbance and clot lysis time [1]. Clots generated from the plasma of patients who were evaluated 7 months after a diagnosis of venous thromboembolism (VTE) (34% of these patients had pulmonary embolism [PE]) were characterized by lower clot permeability, lower compaction, higher maximum absorbance, and prolonged clot lysis time than VTE-free relatives and controls [2]. Reduced clot permeability and prolonged clot lysis time have been shown to be good predictors of recurrent deep vein thrombosis (DVT) and PE [[3], [4], [5]]. However, clot structure and function, including clot formation, lysis, and viscoelastic properties assessed in vitro, differed patients with acute DVT from those with PE [2,6]. Analysis of thrombotic material removed from pulmonary arteries of acute PE patients showed that fibrin is the main component of the thrombus [7,8]. When plasma clot lysability in the presence of tissue plasminogen activator is assessed, the impact of fibrinogen concentration is negligible. The key contributors to clot lysis time variation in venous thrombosis patients include plasminogen activator inhibitor 1 (PAI-1), thrombin activatable fibrinolysis inhibitor (TAFI), prothrombin, and α2-antiplasmin levels [9]. Although there have been multiple studies on plasma fibrin clot properties in acute PE [[4], [5], [6]], a comparison between plasma clot composition of acute PE patients and those of healthy subjects has not been investigated. In healthy subjects, the quantitative protein composition of fibrin clots generated from plasma has been reported to associate with clot permeability and lysis time, the two key measures of clot structure and function [10]. Using conventional biochemical assays, it has been shown that in acute PE patients, plasma levels of factor XIIIa (FXIIIa) and soluble thrombomodulin were lower, whereas plasma levels of von Willebrand Factor (vWF) antigen and fibrinogen were higher when compared with results after 7 months follow-up [11]. Using a mass spectrometer (MS)-based high-throughput approach, as many as 476 proteins incorporated into plasma clots can be quantified in a single MS run [12].

To the best of our knowledge, there have been no reports on proteomic analyses comparing the clot composition between acute PE patients and healthy subjects. Therefore, we investigated whether clot-bound protein content could be associated with prothrombotic or unfavorable clot characteristics, as evidenced by low plasma clot permeability and prolonged clot lysis time among acute PE patients.

Section snippets

Study design

We evaluated 20 patients admitted due to acute PE and 20 sex-matched apparently healthy controls. The diagnosis of PE was based on the presence of typical symptoms and positive results of high resolution spiral computed tomography, according to current guidelines [13]. The diagnosis of lower or upper limb DVT required a positive finding on color duplex sonography. An iliac/caval DVT was defined as abnormal duplex flow patterns typical of thrombosis or an intraluminal filling defect on contrast

Acute PE vs. healthy control subjects

The baseline characteristics of acute PE patients and healthy controls are presented in Table 1. None of the PE patients or controls declared current smoking. Healthy subjects were medication-free. Prior to study enrolment, medications used by acute PE patients were angiotensin converting enzyme inhibitor (n = 9, 45%), β-blocker (n = 8, 40%), aspirin (n = 7, 35%), statin (n = 6, 20%), proton pump inhibitor (n = 5, 25%), angiotensin receptor blocker (n = 1, 5%), and calcium channel blocker

Discussion

Our study is the first to show that in acute PE patients, the proteome of fibrin clots differs largely from healthy controls. Moreover, we have observed that fibrin clot protein composition is associated with clot permeability as reflected by Ks, a key measure of fibrin network density. In acute PE, this parameter correlated with coagulation factor IX and V, carboxypeptidase B2 (TAFI), complement C1s, C7, C8 α chain, and apolipoprotein A-I, which suggests the impact of proteins unrelated to the

Conclusions

Our study is the first to demonstrate differences between the protein composition of fibrin clots generated from plasma of acute PE patients and healthy controls, suggesting that clot properties might be modulated by diverse proteins, including complement components and apolipoproteins. We have extended our previous observations [10] on the relationship between fibrin clot protein content and fibrin clot properties in the apparently healthy subjects, by showing that in acute PE, decreased clot

Sources of funding

This work was supported by the Polish National Science Centre (UMO-2015/B/NZ5/02215 to A.U.).

Disclosures

None.

Acknowledgements

None.

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