Singlechain Factor XII Exhibits Activity When Complexed to Polyphosphate

Journal of Thrombosis and Haemostasis, 12: 1513–1522 DOI: 10.1111/jth.12663 ORIGINAL ARTICLE Single-chain factor XII exhibits activity when complexed to polyphosphate R. ENGEL,*1 C. M. BRAIN,* J. PAGET,† A. S. LIONIKIENE† and N . J . M U T C H † *Faculty of Biological Sciences, University of Leeds, Leeds; and †Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK To cite this article: Engel R, Brain CM, Paget J, Lionikiene AS, Mutch NJ. Single-chain factor XII exhibits activity when complexed to polyphosphate. J Thromb Haemost 2014; 12: 1513–22. prekallikrein to their active forms. Conclusions: Autoacti- Summary. Background: The mechanism underpinning fac- vation of FXII by polyP, of the size found in platelets, tor XII autoactivation was originally characterized with proceeds via an active single-chain intermediate. scFXII– non-physiological surfaces, such as dextran sulfate (DS), polyP70 shows activity towards physiological substrates, ellagic acid, and kaolin. Several ‘natural’ anionic activat- and may represent the primary event in initiating contact ing surfaces, such as platelet polyphosphate (polyP), have activation in vivo. now been identified. Objective: To analyze the autoactivation of FXII by polyP of a similar length to that found in platelets (polyP70). Methods and results: PolyP70 showed Keywords: blood coagulation; factor XII; hemostasis; similar efficacy to DS in stimulating autoactivation of polyphosphates; zymogens. FXII, as detected with amidolytic substrate. Western blot- ting revealed different forms of FXII with the two activating surfaces: two-chain aFXIIa was formed with DS, whereas single-chain FXII (scFXII; 80 kDa) was formed Introduction with polyP70. Dissociation of scFXII from polyP70 abro- Activation of the contact pathway occurs upon reciprocal gated amidolytic activity, suggesting reversible exposure proteolytic cleavage of factor XII and prekallikrein (PK) of the active site. Activity of scFXII–polyP70 was 2+ to their active forms, FXIIa and kallikrein, in the pres- enhanced by Zn and was sensitive to NaCl concentra- ence of a negatively charged surface. In turn, FXIIa tion. A bell-shaped concentration response to polyP70 was cleaves FXI that is tethered to an anionic or membrane evident, as is typical of surface-mediated reactions. Reac- surface, generating active FXIa. FXIa initiates a series of – tion of scFXII polyP70 with various concentrations of ordered cleavages that feed into the prothrombinase com- S2302 generated a sigmoidal curve, in contrast to a hyper- plex and ultimately generate thrombin. FXI and PK a bolic curve for FXIIa, from which a Hill coefficient of require a non-enzymatic cofactor, high molecular weight 3.67 was derived, indicative of positive cooperative bind- kininogen (HK), to facilitate binding of these proteins to – ing. scFXII polyP70 was more sensitive to inhibition by the activating surface, whereas FXII directly associates H-D-Pro-Phe-Arg-chloromethylketone and corn trypsin with the surface via the fibronectin domains in the heavy a inhibitor than FXIIa, but inhibition profiles for chain [1–4]. Zinc ions induce conformational changes in – C1-inhibitor were similar. Active scFXII polyP70 was FXII [5–9] and HK [10–12], enhancing the interaction of also able to cleave its physiological targets FXI and these proteins with the anionic surface. The function of FXII as a coagulation factor was con- Correspondence: Nicola J. Mutch, School of Medicine & Dentistry, tested for many years, as a deficiency in humans is not Institute of Medical Sciences, Foresterhill, University of Aberdeen, associated with a bleeding diathesis. It has now been Aberdeen AB25 2ZD, UK. hypothesized, through the use of mouse models, that Tel.: +44 1224 437492. FXII-driven coagulation is not essential for normal hemo- E-mail: [email protected] stasis, but mediates pathophysiological thrombus formation [13,14]. More recently, monoclonal antibodies 1Now at the Department of Immunopathology, Sanquin Research (mAbs) directed against FXII have been shown to inhibit and Landsteiner Laboratory of the AMC, Amsterdam, the Nether- lands. thrombus formation in primate thrombosis models [15]. These studies have redefined the function of FXII in vivo Received 11 March 2014 and highlighted it as a target for novel anticoagulant Manuscript handled by: R. Camire agents that could potentially prevent nascent thrombus Final decision: P. H. Reitsma, 7 July 2014 growth with minimal bleeding complications. In addition © 2014 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. 1514 R. Engel et al to its role in coagulation, FXII is likely to function in sample buffer, reducing agent and Mops running buffer innate immunity [16], and indeed several lines of evidence were from Life Technologies (Paisley, UK). Pierce spin indicate that these processes are inextricably linked [17– cups with cellulose acetate filters were from Fisher-Scienti- 19]. A second question concerning the function of FXII fic, Loughborough, UK. Unless otherwise stated, the buffer in vivo was the absence of a suitable charged surface to used throughout was 50 mM Tris-HCl (pH 7.4) and stimulate activation. Several potential ‘natural’ activators, 100 mM NaCl. Polymethacrylate beads (Sepabeads EC- including platelet polyphosphate (polyP) [20,21], micro- HA) were a kind gift from Residion SRL (Milan, Italy). particles derived from platelets and erythrocytes [22], Experiments were performed with either polyP65 or polyP70 RNA [23], misfolded proteins [24], collagen [25] and mast (a kind gift from BK Giulini). Comparable results were cell heparin [26], have now been identified. PolyP is an obtained with both preparations of polyP, which, for sim- anionic polymer of phosphate residues that is secreted plicity, will be described as polyP70 throughout the article. from the dense granules of human platelets upon activa- The concentration of polyP70 is expressed in terms of tion by agonists such as thrombin, ADP, and collagen monomer concentration throughout the article [20,33]. [21,27]. Platelet polyP is of a defined size of 60–100 phosphate units in length [21], unlike the extremely long poly- Chromogenic assays mers found in bacteria [28]. Platelet polyP has been found to stimulate FXII-driven procoagulant activity in vitro FXII autoactivation FXII (50 nM) was added to 96-well [20,29] and in vivo [21] and to upregulate bradykinin-dri- microtiter plates (Greiner, Stonehouse, UK) alone or in the À1 ven inflammation [21]. presence of either polyP70 (70 lM) or DS (1.5 lgmL ) Binding of FXII to an anionic surface induces autoacti- with or without 10 lM ZnCl and with or without 1 mM vation, via cleavage at Arg353–Val354. This generates the EDTA. This assay was performed in 50 mM Tris-HCl active protease aFXIIa, an 80-kDa enzyme consisting of (pH 7.4) buffer containing either 100 mM or 140 mM NaCl. a heavy and a light chain linked by a disulfide bond. Fur- The chromogenic substrate S2302 (0.5 mM) was added, and ther proteolytic cleavage at Arg334–Asn335 generates generation of activity was monitored at 405 nm every 30 s bFXIIa, a solution-phase derivative largely composed of the for 2 h at 37 °C in an ELx 808 plate reader (Bio-Tek, protease domain. bFXIIa cleaves PK, but has negligible activ- Potton, UK). Experiments were performed over a range of ity towards FXI, whether surface-bound or in solution [30]. polyP70 (0–2 mM), NaCl (0–1 M) and ZnCl2 (0–100 lM) The mechanism of FXII autoactivation was characterized concentrations. A discontinuous assay for FXII autoacti- prior to the identification of physiological surfaces that were vation was performed by incubating FXII (50 nM) alone, À1 able to support this reaction [5,31,32]. The aim of this study with polyP70 (70 lM) or with DS (1.5 lgmL )at37°C. was to define the ability of polyP, of similar chain length to At various time points (0–120 min), the reaction was that found in platelets, to stimulate autoactivation of FXII stopped by the addition of 1 M NaCl, and FXII activity under physiological conditions. We show that autoactivation was quantified with S2302 (0.5 mM) as described above. of FXII on a ‘natural’ activating surface proceeds via an Gradients from the initial linear sections of absorbance vs. active single-chain intermediate that is capable of cleaving time graphs were calculated and plotted against the inhibi- both synthetic and physiological targets. tor, polyP70, NaCl or ZnCl2 concentrations with GRAPH- PAD PRISM 5.03 (La Jolla, CA, USA). Data were analyzed by linear regression, and accurate line-fitting was estab- Materials and methods lished by analysis of residual data. For western blot analysis of FXII autoactivation, FXII Materials (50 nM) was incubated alone or in the presence of 70 lM À1 FXII, aFXIIa, FXI, PK, kallikrein, horseradish peroxidase polyP70 or 1.5 lgmL DS with or without 10 lM ZnCl2 (HRP)-conjugated polyclonal antibody against FXII, for 60 min at 37 °C. Reducing sample buffer was added, HRP-conjugated antibody against FXI and HRP-conju- and samples were boiled for 10 min prior to being gated antibody against PK were from Enzyme Research resolved on 4–12% NuPAGE gels and transferred to a Laboratories (Swansea, UK). PolyP of average chain poly(vinylidene difluoride) membrane. FXII was detected length 65 (polyP65) and C1-inhibitor (C1-Inh) were from with a polyclonal antibody conjugated to HRP (Enzyme Sigma-Aldrich (Irvine, Scotland). Dextran sulfate (DS) Research Laboratories). A commercial preparation of with an average Mr of 500 000 and EDTA were from aFXIIa (50 nM) was included in the western blot as a Fisher Scientific (Loughborough, UK). L-Pyr-Pro-Arg- positive control.

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