Atanur Ersayin

Aline Thomas

Landry Seyve

Nicole Thielens

Mathieu Castellan

Raphaël Marlu

Benoît Polack

Marie-Claire Dagher
email: marie-claire.dagher@univ-grenoble-alpes.fr

Catalytically inactive Gla-domainless factor Xa binds to TFPI and restores ex vivo coagulation in hemophilia plasma

New hemophilia treatments aim at releasing the inhibition of tissue factor pathway inhibitor (TFPI) on the extrinsic tenase. Our team has demonstrated that Gla-domainless factor Xa (GD-FXa) can restore coagulation in FVIII-or FIX-deficient plasmas and is unable to trigger coagulation per se. In parallel, a catalytically inactive version of GD-FXa, Andexanet alfa, was proposed as antidote to direct oral anticoagulants targeting FXa. Here we show by molecular modeling that GD-FXa and inactive S195A GD-FXa have similar binding properties to TFPI Kunitz 2 domain. The binding parameters between TFPI and inactive S195A GD-FXa, measured using surface plasmon resonance, were similar to those of plasma derived GD-FXa. As previously found for GD-FXa, inactive GD-FXa was able to restore thrombin generation in FVIII-and FIX-deficient plasmas as well as in plasmas from hemophiliacs. We therefore propose to use catalytically inactive GD-FXa in hemophilia.

The problem of inhibitors to substitution factors used to treat hemophilia makes the search for new treatments highly desirable (1). Releasing TFPI inhibition towards the extrinsic tenase is a strategy of choice, with several candidates to this aim (2,3) in clinical trials. Indeed, when the intrinsic tenase pathway leading to factor Xa (FXa) activation is blocked due to FVIII or FIX deficiency, a small amount of FXa can be generated by the extrinsic tenase (FVIIa/tissue factor) but it is immediately neutralized by binding of TFPI through its Kunitz 2 (K2) domain while the Kunitz 1 (K1) domain of TFPI subsequently binds to FVIIa inhibiting the tenase (4,5). Our team has postulated an original anti-TFPI strategy, Gladomainless factor Xa (GD-FXa), deprived of its membrane binding domain and validated the proof of concept of this approach. Indeed GD-FXa reconstituted thrombin generation in plasmas from hemophiliacs (6,7). During its production as recombinant protein, GD-FXa was very unstable due to auto-proteolysis [START_REF] Mertens | Pathways in the activation of human coagulation factor X[END_REF]. Simultaneously, a catalytically inactive version of GD-FXa, Andexanet alfa, was developed and tested as antidote to direct oral anticoagulants, targeting FXa (9). This S195A GD-FXa molecule has currently passed the phase III procedure and is under new drug approval by FDA. During the phase III study, thrombotic complications were observed and the hypothesis of an interaction with TFPI was raised [START_REF] Connolly | Andexanet Alfa for Acute Major Bleeding Associated with Factor Xa Inhibitors[END_REF][START_REF] Mar | 176 Andexanet Alfa: An Investigational Universal Antidote for Reversal of Anticoagulation of Factor Xa Inhibitors in Healthy Human Volunteers[END_REF]. Because of stability problems encountered with wild-type GD-FXa, the inactive S195A GD-FXa mutant appeared easier to handle for biological experiments. This led us to study the binding properties of inactive GD-Xa to TFPI both in silico and ex vivo.

We used molecular modeling and molecular dynamics to characterize the binding interface between TFPI K2 domain and either the wild type or S195A catalytic domains of FXa. Both models were built and submitted to molecular dynamics (see Supplementary material). The simulations showed that the S195A mutation did not significantly modify the molecular interface with the K2 domain both structurally and energetically.

For recombinant protein expression, a drosophila S2 expression system was chosen and GD-FXa was produced as a single polypeptide in which the heavy and the light chains were separated by a double furin cleavage site [START_REF] Thomas | Gla-domainless factor X[END_REF]. Wild-type GD-FXa could not be purified due to auto-proteolysis. For control experiments, we used plasma derived GD-FXa that was more stable. In contrast, the S195A mutant was expressed at a higher level in the supernatant of S2 cells and was purified to near homogeneity. SDS-PAGE analysis showed that the protein was 95% pure and mass spectrometry showed that the protein was properly processed at the furin cleavage site (see Supplementary Figure 3). Recombinant TFPI alpha was produced in the same system.

Direct binding of S195A GD-FXa and plasma derived GD-FXa to immobilized TFPI was measured by surface plasmon resonance spectroscopy (SPR). As shown in Figure 1, S195A GD-FXa displayed association and dissociation curves that could be fitted to a 1:1 Langmuir binding model. The K D was 1.49 ± 0.09 nM for plasma derived GD-FXa and 1.99 ± 0.23 nM for S195A GD-FXa. Thus, both proteins were able to bind similarly with high affinity to immobilized TFPI.

We then assessed the effect of inactive GD-FXa in thrombin generation assays in Factor VIII immunodepleted plasma using Hemker's method [START_REF] Hemker | Thrombin generation, a function test of the haemostatic-thrombotic system[END_REF]. Thrombin generation induced by S195A GD-FXa on factor VIII-deficient plasma showed a dose-dependent effect (Figure 2) that was comparable to that of previously studied plasma derived GD-FXa (6). The same result was found in FIX immune-depleted plasma. In addition, we tested plasmas from four severe hemophilia A patients and three severe hemophilia B patients. We checked that the same results could be obtained with different batches of S195A GD-FXa. Parameters of thrombin generation are summarized in Table 1. In all cases, the effect of S195A GD-FXa did not increase at concentrations above 40 nM and the values were comparable to the relevant positive controls. In the case of hemophilia A with inhibitors, as expected Factor VIII did not restore thrombin generation unless in 10-fold excess, but S195A GD-FXa did perfectly well.

We controlled that the protein could not replace FXa in FX-deficient plasma and that S195A GD-FXa induced thrombin generation was dependent on tissue factor. Thus inactive GD-FXa behaves similarly as wild-type GD-FXa and could be suitable for treatment of hemophilia A and B even in the presence of inhibitors, as expected.

We have previously proposed GD-FXa as a bait to TFPI and provided the proof of concept of this approach using plasma derived GD-FXa protein (6). Production of GD-FXa as recombinant protein was not successful. In contrast the catalytically inactive mutant was successfully produced and used as antidote to direct oral anticoagulants [START_REF] Mertens | Pathways in the activation of human coagulation factor X[END_REF], but a general view was that a functional active site was required for TFPI binding. Indeed Broze had shown that diisopropylfluorophosphate-treated FXa lost its ability to bind to TFPI [START_REF] Broze | The rediscovery and isolation of TFPI[END_REF]. However, the reported procoagulant effects of Andexanet alfa during its phase III trial suggested some TFPI binding capacity [START_REF] Connolly | Andexanet Alfa for Acute Major Bleeding Associated with Factor Xa Inhibitors[END_REF]. Although a catalytically inactive molecule was expected to be totally safe, neutralization of TFPI by Andexanet alfa may have contributed to some of the reported thrombotic events.

We performed molecular dynamics to show that the S195A mutation does not significantly modify the binding interface of the serine protease domain with the K2 domain of TFPI, at both structural and energetic levels, and that the theoretical affinity is unchanged. We then set-up expression systems and produced the protein S195A GD-FXa as a single chain in drosophila S2 cell line with the activation peptide replaced by a double furin cleavage site. The S195A GD-FXa was properly processed by furin, efficiently secreted in the cell culture medium and could be easily purified. Moreover, SPR experiments showed that inactive GD-FXa was able to bind to TFPI with affinity constants similar to those of the plasma derived GD-FXa protein, and that it could reconstitute thrombin generation in factor VIII-and IX-deficient plasmas and in plasmas from hemophiliacs as well.

We thus propose a new application for a known product, Andexanet alpha or S195A GD-FXa as a novel anti-hemophilia treatment [START_REF] Dagher | Leurre GD-Xa mutant[END_REF]. Having no catalytic activity, this protein cannot substitute for FXa even at high doses, and is therefore perfectly secure in the context of hemophilia. This potential new hemophilia treatment now requires testing in animal models. The choice of the animal model is crucial as the mouse TFPI sequence has an insertion of six amino acids in the basic C-terminal region involved in prothrombinase inhibition and thus could behave differently from human TFPI (4). A rabbit model of hemophilia would be ideal to prove the efficiency and safety of S195A GD-FXa as the rabbit TFPI sequence differs from that of human TFPI by only one amino acid in the C-terminal basic region. Various sources of plasma were used as indicated and dose-effect curves were performed with S195A GD-FXa. Thrombin generation assays were performed in triplicates according to Hemker [START_REF] Hemker | Thrombin generation, a function test of the haemostatic-thrombotic system[END_REF]. The thrombin generation parameters (mean values) are indicated. Values obtained from normal pooled plasmas are given for reference.

ns: no signal; np: no peak; + indicates the relevant positive control either recombinant FVIII (1U/ml, Octocog alpha, Bayer) in hemophilia A and FVIII-deficient plasmas, or plasma derived FIX (1U/ml, Cryopep) in hemophilia B and FIX-deficient plasmas, or plasma derived FX (1U/ml, Cryopep) in FXdeficient plasma. For Hemophilia A plasma with 50 Bethesda units (BU) inhibitor, values from a positive control with 10U/ml of recombinant FVIII are indicated in parentheses. The GD-FXa samples were injected at the indicated concentrations over immobilized TFPI (1300 RU) in 18 mM Hepes, 135 mM NaCl, 2.5 mM CaCl 2 , 0.005% surfactant P20, pH 7.35. Binding is expressed in resonance units (RU). Fits are shown as red lines and were obtained by global fitting of the data using a 1:1 Langmuir binding model. The K D was calculated from 3 independent experiments.

Molecular Dynamics

To characterize the effect of the S195A mutation on the molecular interactions of FXa with the K2 domain of TFPI, molecular dynamics calculations of the wild-type FXa/K2 and of the S195A FXa/K2 complexes were performed. These simulations were done with the CHARMM software (1, 2) using the CHARMM-36 parameters and topology sets including the CMAP correction (3); the total charge of the system was null so that no counter-ion was added.

The systems were fully solvated in a TIP3P (4) water box of 16 Å edge larger than the protein dimensions. The molecular systems totalize about 35000 atoms including about 4580 protein atoms.

Periodic boundary conditions were used to model the solvent. A minimization procedure was performed with decreasing harmonic forces on all the atoms, totalizing 3000 steps of steepest descent followed by 5000 steps of ABNR minimization without constraints. The structures were then subjected to a CPT molecular dynamics (5) (1 atm, 298 K). The molecular systems were annealed from 48 K to 298 K over a period of 20 ps and maintained at that temperature with velocity scaling. The non-bonding interactions considered up to a cutoff of 14 Å, and smoothly zeroed between 12 Å and 14 Å using a shifting potential. Covalent bonds involving hydrogens were kept fixed using the SHAKE algorithm (6), thus allowing an integration time step of 2 fs. The systems were equilibrated for 10 ns and the production runs lasted 100 ns. The trend of the overall potential energy, root mean square deviations (RMSD) and gyration radius, indicate that the systems are well stabilized after 10 ns.

the very close neighborhood of K2 is conserved, except residue S190 Trypsin which is an alanine in FXa.

Thus, the hydrogen bond of R15(NH1) K2 with the side-chain of residue 190 disappears whether the hydrogen bond between R15(NH1) K2 and the G219 FXa backbone oxygen is maintained. A hydrogen bond between the carboxamide nitrogen of Q192 FXa and the C14(O) K2 appears after 50 ns. The S214(O) FXa entertains a hydrogen bond with D102(OD2) FXa which replaced after 50 ns by a hydrogen bond with W215(NE) FXa . The distribution of the residual interaction energies between FXa residues and the K2 chain (see Supplementary Figure 2) shows that the mutation of S195 into an alanine does not perturb the interface between both proteins.

Estimation of free energies of binding

Approximate free energies of binding of TFPI-K2 to the catalytic domain of wild-type and S195A FXa were calculated using the MMPBSA solvent continuum approximation (8) as follows.

The binding free energy can be approximated as:

G = E -TS

The internal energy terms can be assumed to be the same in the native complex and in the mutated one, so that E can be taken as E vdw . Furthermore, we can assume that the entropic contribution cancels when one calculates the difference in binding free energies G bind between the wild-type and the mutated complexes. Thus, G = E vdw + G elec + G non polar +  E vdw is the standard Lennard-Jones term calculated with a quasi infinite cutoff (of 120.0 Å) for nonbonded interactions. The total electrostatic contribution to the binding free energy was calculated using the Poisson Boltzman equation, with the PBEQ solver. The protein dielectric constant was then taken as 4 and the solvent one as 80. The nonpolar contribution was estimated as a fraction of the surface accessible area (9), with =0.00542 kcal/mol.Å 2 and  = 0.92. The different energy contributions were calculated for each snapshot every 1 ns along the trajectories and the averages (in kcal/mol) are presented in the following

Figure 1 :

 1 Figure 1: Representative kinetic analysis of the interaction of S195A GD-FXa with immobilized TFPI.

Figure 2 :

 2 Figure 2: Correction of thrombin generation by S195A GD-FXa in FVIII-immunodepleted plasma

Table 1 :

 1 Parameters of thrombin generation assay after supplementation of hemophilia A and B plasmas and immuno-depleted plasmas by S195A GD-FXa

			Endogenous thrombin		Peak height			Lag time			Time to peak
			potential (nM.min)		(nM)			(min)				(min)
								S195A GD-FXa concentration (nM)
	Plasma	0	20	40	+	0	20	40	+	0	20	40	+	0	20	40	+
		P1A	186	1227	1281	760	27	68	69	59	5.3	4.1	4.1	7.6	5	15	16	14
		P2A	305	1543	1613	1578	15	107	113	185	5.2	4.2	4.3	5.8	18	13	13	10
	Hemo
		P3A	618	1498	1473	1530	28	100	100	249	3.5	3.3	3.3	4.0	16	14	14	7.4
	philia
	A	P4A				148				6.4				4.2				20
		inhibitor	176	803	815		7.3	46	47		4.0	3.7	3.7		19	11	10
						(400)				(28)				(5)			(13)
		positive
		P1B	448	1187	1217	1301	20.4	88	103	248	4.7	4.0	4.0	4.0	15	12	11	7.0
	Hemo
		P2B	397	1281	1327	1375	19	115	120	215	5.1	4.4	4.3	4.5	14	7.8	11	11
	philia
		P3B	320	1450	1456	1498	11	90	93	203	5.5	4.7	4.7	5.2	19	8.9	12	12
	B
		P4B	429	1177	1197	1215	22	212	110	114	5.1	4.3	4.2	4.5	13	7.6	10	10
	FIX-deficient	740	1523	1613	1663	37	120	149	277	3.6	3.3	3.6	4.2	12	11	10	7.4
	FX-deficient	ns	ns	ns	976	np	np	np	62	np	np	np	4.0	np	np	np	11
	Normal pool	634	-	-	-	54	-	-	-	6.6	-	-	-	12	-	-	-

  table.

	System	E vdw	G elec	G non polar	G
	Wild	-14.47 ± 1.73 -15.52 ±2.49 -9.24 ± 0.85	-39.24 ± 2.25
	SA195	-14.95 ±1.07 -16.79 ±2.16 -9.38 ±0.47	-41.14 ± 2.39

Acknowledgements

The simulations were performed using the Froggy platform of the CIMENT infrastructure, which is supported by the Rhône-Alpes region (GRANT CPER07_13 CIRA) and the Equip@Meso project (reference ANR-10-EQPX-29-01) of the program Investissements d'Avenir supervised by the Agence Nationale pour la Recherche. The authors would like to thank Pierre Girard and Antoine Fortuné for technical support.

This work used the platforms of the Grenoble Instruct-ERIC Centre (ISBG; UMS 3518 CNRS-CEA-UJF-EMBL) with support from FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB). We thank Isabelle Bally and Jean-Baptiste Reiser for assistance and access to the SPR facility. We thank Elisabetta Boeri Erba and Luca Signor for the technical support to acquire MS spectra. We are indebted to Xavier Brazzoloto who provided the drosophila expression system. We gratefully acknowledge our colleagues of LFB SA Toufik Abache, Alexandre Fontayne and Jean-Luc Plantier for fruitful discussions.

Funding

The project was funded by the Agence Nationale pour la Recherche 13-RPIB-0011 Author's contributions: AE, AT, LS, NT, MC performed and analyzed the experiments AT, RM, NT, BP, MCD designed and analyzed the experiments MCD supervised the experiments and wrote the manuscript

Even though there is a rough approximation for the calculation of the free energies of binding by neglecting the solvation entropy, the G values indicate that the S195A mutation does not perturb the affinity of TFPI-K2 for FXa.

Ersayin et al. Supplementary online material

Supplementary Methods

Production of TFPI alpha and S195A GD-FXa

The optimized cDNA sequences were obtained from Genscript. The proteins were expressed in S2 cells (Drosophila Expression System, Life Technologies). GD-FXa was produced as a single polypeptide chain where the activation peptide was replaced by a linker containing a double furin cleavage site. An HPC4 tag (Protein C derived epitope) was present at the C-terminus. S2 cells were transfected with Freestyle Max Reagent (Life Technologies), and the supernatant was collected at day 6 post transfection. TFPI was purified on a SP-Sepharose column (GE Healthcare) and displayed 95% purity after this step. GD-FXa was very unstable and could not be purified. After cation exchange chromatography, the S195A GD-FXa protein was further purified on an anti-Protein C Affinity matrix (Roche). TFPI activity was checked by inhibition of FXa using the chromogenic substrate PNAPEP-1025 (Cryopep) and by binding to FXa by surface plasmon resonance. For S195A GD-FXa, correct processing at the furin cleavage site was confirmed by mass spectrometry analysis.

Surface plasmon resonance

First, to assess the functionality of TFPI, increasing concentrations of FXa (Cryopep) were injected over TFPI covalently immobilized on CM5 sensorchip (GE Healthcare). The kinetic constants were calculated using the Biaeval software (GE Healthcare) and the ratio of the dissociation and association rate constants yielded a K D of 2.2 nM. Immobilized TFPI was then used to measure the binding constants of plasma derived GD-FXa (Cryopep) or S195A GD-FXa.

Thrombin generation assay

Thrombin generation was assayed in FVIII or FIX immuno-depleted plasmas and in plasmas from hemophilia A and B patients (Cryopep). One hemophilia A plasma with inhibitor (50 BU) was also assayed. Plasma was spiked with S195A GD-FXa or recombinant FVIII (Octocog alpha, Bayer Pharma) or plasma derived FIX (Cryopep) and thrombin generation was triggered by 1 pM tissue factor and 4 µM phospholipids (PPP-reagent low, Stago) in the presence of a fluorogenic substrate (FluCa kit, Diagnostica Stago). All assays were run in triplicates on a CAT fluorimeter (Stago). Thrombin generation was monitored after excitation at 390 nm and emission at 460 nm for 60 min at 37°C. The curves were fitted

Supplementary results

The molecular interface along the dynamics

The hydrogen bond between S195(O) FXa and R15(N) K2 that was present in the starting porcine trypsin-K2 complex (7), is not present in the wild-type FXa-K2 complex as the serine side-chain is hydrogen bonded to surrounding water molecules. As shown in Supplementary Figure 1, the R15(N) K2 is stabilized by two strong and stable hydrogen bonds with respectively G193(N) FXa and S195(N) FXa. The hydrogen bond between G216(N) FXa and I13(O) K2 is conserved throughout the dynamics. The R15 K2 side-chain extends in the specificity pocket with its guanidinium group forming a salt-bridge with the carboxylate group of D189 FXa , fully conserved along the trajectories. When compared to the porcine trypsin-K2 complex (7),

Supplementary figures

Supplementary Figure 1: Model of the interaction between the catalytic domain of FXa and the K2 domain of TFPI.

The catalytic domain of FXa and the K2 domain of TFPI are respectively drawn as cyan and green ribbons. The calcium ion is represented as a purple sphere. A zoom on the active site shows the insertion of R15 K2 within the FXa active site. The main hydrogen bonds stabilizing the interface are shown.

Supplementary Figure 2: Distribution of the residual interaction energies of the catalytic domain of FXa with the K2 domain of TFPI.

The plots are for wild-type GD-FXa (A) and S195A GD-FXa (B) catalytic domains residues.

Supplementary Figure 3A: SDS-PAGE analysis of purified S195A GD-FXa.

The protein was purified from the supernatant of drosophila S2 cells at day 6 post transfection as described in Methods. Under reducing conditions, the light chain that does not contain tryptophan is not visible on Stain Free gels (Biorad, data not shown). But as expected, the revelation with Coomassie Blue confirmed the presence of the light chain. UR: unreduced, HC: heavy chain, LC: light chain, Std: protein molecular weight standards.

Supplementary Figure 3B: Quality control of purified S195A GD-FXa by mass spectrometry.

The sample was analyzed on a 6210 LC-ESI-TOF mass spectrometer (Agilent Technologies). The spectrum presented was obtained after deconvolution of the raw spectrum. The main peak at 41198 Da corresponds to the two chains linked by a disulfide bridge after cleavage by furin between the two chains with elimination of the peptide RKRRKR. The smaller peak 37889.74 may correspond to degradation of the protein.

Supplementary references