Elsevier

Thrombosis Research

Volume 132, Issue 6, December 2013, Pages 729-734
Thrombosis Research

Regular article
Chronic thromboembolic pulmonary hypertension-associated dysfibrinogenemias exhibit disorganized fibrin structure

https://doi.org/10.1016/j.thromres.2013.09.024Get rights and content

Abstract

Introduction

Mechanisms contributing to the pathogenesis of chronic thromboembolic pulmonary hypertension (CTEPH) are poorly understood. This disorder is characterized by incomplete resolution of pulmonary perfusion defects resulting from acute venous thromboembolism. We previously identified several dysfibrinogenemias in some patients with CTEPH. The purpose of this study was to determine whether fibrin clot architecture might be implicated in the thrombolytic resistance in patients with these CTEPH-associated dysfibrinogenemias.

Materials and Methods

Purified fibrinogen from patients and healthy controls was clotted with thrombin in the presence of calcium. Clot turbidity, porosity, and susceptibility to fibrinolysis were evaluated by spectrophotometric and permeation analyses. Fibrin network structure was assessed by laser-scanning confocal microscopy.

Results

Compared to normal fibrinogen, CTEPH-associated dysfibrinogenemias exhibited low clot turbidity, decreased porosity, and fibrinolytic resistance. In addition, the dysfibrinogenemias exhibited a more disorganized fibrin network structure characterized by thinner fibers, greater network dispersal and more extensive fiber branching.

Conclusions

Abnormal clot architecture and fibrinolytic resistance may contribute to incomplete clot resolution following acute venous thromboembolism in patients with CTEPH-associated dyfibrinogenemia.

Introduction

After acute venous thromboembolism, thrombi either resolve completely or are replaced by chronic intravascular scars, which may have various clinical effects. In the case of acute pulmonary emboli, residual lung perfusion defects are common [1], [2], [3], [4], [5], [6] and probably represent minor degrees of intravascular scarring. However, more extensive scars within the pulmonary arteries may result in chronic thromboembolic pulmonary hypertension (CTEPH), a life-threatening disease [7]. Likewise, residual intraluminal venous thickening is common after acute deep vein thrombosis [8], whereas extensive scarring within the deep veins produces chronic thrombotic venous disease (CTVD), a cause of post-thrombotic syndrome [9].

The factors that contribute to poor resolution of venous thromboemboli and the intravascular scar formation that leads to CTEPH and CTVD are not fully understood. Polymerized fibrin is a major component of acute venous thromboemboli, and resistance to lysis is characteristic of some abnormally formed fibrin clots [10]. We previously reported an unusually high prevalence of dysfibrinogenemias among CTEPH patients [11], which may contribute to a relative resistance to plasmin-mediated fibrinolysis in some cases [12].

We undertook the current series of experiments to determine if CTEPH-associated dysfibrinogenemias lead to abnormal fibrin network formation. In particular, we are interested in alterations that might be implicated in delayed fibrinolysis, abnormal cellular responses to thrombi and subsequent remodeling of thrombotic material into scar tissue.

Section snippets

Materials

Human α-thrombin and glu-plasminogen were purchased from Enzyme Research Laboratories (South Bend, IN). Recombinant human tissue-type plasminogen activator (tPA), 2-chain form, was obtained from Burroughs Wellcome (Research Triangle Park, NC). Human fibrinogen conjugated with Alexa Fluor 488 (9 moles dye/mole protein) was purchased from Molecular Probes (Grand Island, NY). Glass bottom 96-well plates (No. 1.5, uncoated) were obtained from MatTek Corporation (Ashland, MA).

Subjects

The cohort of patients

Fibrinogen Regions Affected by Dysfibrinogenemias

The five dysfibrinogenemias previously reported in CTEPH patients [11] are listed in Table 1. Each occurred in a different patient and all result from heterozygous missense mutations in one or more of the fibrinogen genes. Two patients had compound mutations. None of these mutations were found in a group of 20 healthy controls without a history of venous thromboembolism [11]. The patient with Bβ 235 Pro/Leu dysfibrinogenemia was also heterozygous for the common Bβ 448 Arg/Lys polymorphism, and

Discussion

We observed that fibrin clots derived from patients with CTEPH-associated dysfibrinogenemias have disorganized polymeric structures that may be implicated in delayed thrombolysis and replacement of thrombi by connective tissue. The abnormal fibrin network structures resemble experimentally produced “fine” fibrin clots [20], composed of “thin” fibrin fibers, which may have slower lysis rates than clots with thicker fibers [10], [21], [22], [23]. In keeping with this, we have previously reported

Conflict of Interest Statement

All authors declare no financial or personal conflict of interest.

Acknowledgements

The authors wish to acknowledge the support and inspiration of Dr. Virgil Woods throughout this and other projects. This work was supported by National Institutes of Health grants R01 HL095089 and T32 HL098062. Laser scanning confocal microscopy was performed at the UCSD School of Medicine Light Microscopy Facility, which is supported, in part, by grant P30 NS047101 from the National Institutes of Health.

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