Proteolytic modulation of factor Xa–antithrombin complex enhances fibrinolysis in plasma

https://doi.org/10.1016/j.bbapap.2013.02.007Get rights and content

Abstract

Our previous work showed that purified coagulation factor Xa (FXa) acquires fibrinolysis cofactor activity after plasmin-mediated cleavage. The predominant functional species is a non-covalent heterodimer of 33 and 13 kDa, termed Xa33/13, which has predicted newly exposed C-terminal lysines that are important for tissue plasminogen activator (tPA)-mediated plasminogen activation to plasmin. To provide evidence that this mechanism occurs in a physiological context, here we demonstrated the appearance of Xa33 in clotting plasma by western blot analysis. Since the normal fate of FXa is stable association with antithrombin (AT), an AT western blot was conducted, which revealed a band of ~ 13 kDa higher apparent molecular weight than AT that appeared concurrent to Xa33. Sequencing of purified proteins confirmed the generation of Xa13 covalently bound to AT and Xa33 (Xa33/13-AT) by cleavages at Lys–Met339 and Lys–Asp389. Ligand blots demonstrated 125I-plasminogen binding to the Xa33 subunit of plasmin-generated Xa33/13-AT. Purified XaAT added to plasma that was induced to clot enhanced the rate of tPA-mediated fibrinolysis by ~ 16-fold. Similarly, purified plasminogen activation by tPA was enhanced by ~ 16-fold by XaAT. Plasmin cleaves XaAT and exposes plasminogen binding sites at least 10-fold faster than FXa. Here we demonstrate a novel function for AT, which accelerates the modulation of FXa into the fibrinolytic form, Xa33/13. The consequent exposure of C-terminal lysine binding sites essential for plasminogen activation enhances fibrinolysis. These results are consistent with a model where auxiliary cofactors link coagulation to fibrinolysis by priming the accelerating role of fibrin.

Graphical abstract

Highlights

► The fibrinolytic fragment of clotting factor Xa (Xa), Xa33/13, was found in plasma. ► A source of Xa33/13 in plasma is from the Xa–antithrombin (XaAT) complex. ► Conversion of Xa to Xa33/13 occurs at least 10-fold faster in complex with AT. ► XaAT accelerates plasma clot lysis and plasmin generation by tPA better than FXa. ► A novel function for XaAT was identified that links coagulation to fibrinolysis.

Introduction

The classical fibrinolysis paradigm defines fibrin as the only required physiological cofactor for acceleration of tissue plasminogen activator (tPA)-mediated conversion of plasminogen to plasmin, the direct clot-dissolving protease [1]. In this model, intact fibrin weakly enhances tPA activity to provide the initial low level of plasmin that induces limited cleavage of fibrin. The C-terminal Lys residues that are consequently exposed provide additional binding sites for tPA and plasminogen, which is pivotal to further accelerate plasmin generation. Thus, “primed” fibrin becomes the ultimate tPA cofactor enabling plasmin activity to exceed the anti-fibrinolytic threshold of plasma.

Contrary to the prevailing cofactor model of fibrinolysis, our recent work demonstrated that purified coagulation factor Xa (FXa) undergoes functional modulation by plasmin, to acquire fibrinolytic cofactor activity as an auxiliary to intact fibrin [2]. It does so at concentrations (10 nM) far below those of fibrin (3 μM). The involvement of FXa was significant only when the functional tPA concentration was similar to or less than the activity obtained when physiological concentrations of tPA (75 ρM) and its primary inhibitor, plasminogen activator inhibitor type-1 (PAI-1; 500 ρM), were combined (i.e. < 10 ρM tPA in the absence of PAI-1). At significantly higher tPA activity (i.e. > 50 ρM tPA), the enhancing effects of FXa were negated because of the intrinsic activation of plasminogen by tPA and consequent “priming” of the fibrin overwhelming the early-stage cofactor effects of FXa or intact fibrin. Therefore, to represent the effects of auxiliary tPA cofactors, such as FXa, in fibrinolysis assays, low levels of tPA are required that are consistent with physiological tPA activity in the context of attenuation by its circulating inhibitors.

The plasmin-mediated acquisition of fibrinolysis activity by FXa occurs in the presence of anionic phospholipid and CaCl2, where intact FXa (FXaα) is proteolysed to FXaβ and then to non-covalently associated fragments of 33 kDa and 13 kDa (Xa33/13) (Fig. 1) exposing C-terminal Lys. The latter cleavage inhibits its role in coagulation [3], [4], [5]. Anionic phospholipid-binding localizes coagulation proteins to sites of fibrin formation [6] and is provided mainly by platelets that are stimulated at the site of vascular damage [7]. By exposing C-terminal Lys, FXaβ and Xa33/13 express binding sites for plasminogen approximately 50-fold faster than fibrin [2], explaining how FXa can participate despite the overwhelming concentration of fibrin. Here one of our goals was to extend the fibrinolysis studies that previously used only purified proteins by investigating whether Xa33/13 generation can be detected in plasma.

Following activation of the inactive precursor factor X (FX) in plasma, the predominant fate of FXa is rapid inhibition by the serpin, antithrombin (AT) [8], which presents itself as a substrate to FXa and is cleaved prior to forming a stable 1:1 XaAT complex [9]. Other protease–serpin complexes are known to render the protease highly susceptible to cleavage [10], [11]. Since FXa must undergo proteolytic modulation to express fibrinolytic function, the effect of complex formation with AT on this acquisition of tPA cofactor activity was evaluated in the current study. We now report that the 33 kDa fragment derived from FXa is detected in plasma and that XaAT is likely a predominant source. Furthermore, XaAT enhances tPA-dependent fibrinolysis of clot formed in plasma, which reveals a novel function for this protease–serpin complex.

Section snippets

Reagents and proteins

Tetrasodium EDTA and sodium chloride (Fisher Scientific, Nepean, ON, Canada), calcium chloride (CaCl2, EMD Chemicals, Inc. Gibbstown, NJ, USA), carboxypeptidase inhibitor 2-guanidinoethylmercaptosuccinic acid (GEMSA, VWR Mississauga, ON, Canada), Innovin (Dade Behring, Mississauga, ON, Canada), 4-2-hydroxyethyl-1-piperazineethanesulfonic acid (HEPES), ε-aminocaproic acid (EACA), polyethylene glycol 8000 (PEG), l-α-phosphatidylcholine (PC) and l-α-phosphatidyl-l-serine (PS, Sigma-Aldrich

Xa33 is generated in plasma consistent with XaAT as a source

Having demonstrated previously that purified FXa is converted to the fibrinolytic derivative, Xa33/13, when treated with plasmin [5], conversion in clotting plasma was now investigated. Western blot analysis using a FX-specific mAb that recognizes the 33 kDa fragment of Xa33/13 (Fig. 2A) shows its formation in plasma that was induced to clot with a source of tissue factor, anionic phospholipid and CaCl2 (i.e. Innovin). We have previously reported that due to the presence of the activation

Discussion

AT is a vital physiological anticoagulant that when deficient causes thrombosis. This well-established status in hemostasis is explained at the level of clot formation through irreversible coagulation protease inhibition [13]. The current work describes a novel function for AT that in addition to decreasing the amount of clot, is also involved in fibrinolysis after clot formation has occurred. Here we show that when AT is complexed with FXa (i.e. XaAT), the rate that the FXa moeity is cleaved

Acknowledgements

We are grateful to Rolinda Carter for protein preparation. The use of equipment supported by the Canadian Foundation for Innovation and administrative support provided by the Michael Smith Research Foundation awarded to the University of British Columbia, Centre for Blood Research are acknowledged. This study was funded equally by a Heart and Stroke Foundation of British Columbia Yukon Grant-in-Aid (E.P.) and a federal Canadian Institutes of Health/Canadian Blood Services Partnership Grant

Cited by (7)

View all citing articles on Scopus
View full text