Factor Xa Activation of Factor V Is of Paramount Importance in Initiating the Coagulation SystemClinical Perspective
Lessons From a Tick Salivary Protein
Background—Generation of active procoagulant cofactor factor Va (FVa) and its subsequent association with the enzyme activated factor X (FXa) to form the prothrombinase complex is a pivotal initial event in blood coagulation and has been the subject of investigative effort, speculation, and controversy. The current paradigm assumes that FV activation is initiated by limited proteolysis by traces of (meizo) thrombin.
Methods and Results—Recombinant tick salivary protein TIX-5 was produced and anticoagulant properties were studied with the use of plasma, whole blood, and purified systems. Here, we report that TIX-5 specifically inhibits FXa-mediated FV activation involving the B domain of FV and show that FXa activation of FV is pivotal for plasma and blood clotting. Accordingly, tick feeding is impaired on TIX-5 immune rabbits, displaying the in vivo importance of TIX-5.
Conclusions—Our data elucidate a unique molecular mechanism by which ticks inhibit the host’s coagulation system. From our data, we propose a revised blood coagulation scheme in which direct FXa-mediated FV activation occurs in the initiation phase during which thrombin-mediated FV activation is restrained by fibrinogen and inhibitors.
Ixodes ticks feed for several days on a vertebrate host to obtain a blood meal and potentially to transmit various pathogens, including the causative agent of Lyme borreliosis Borrelia burgdorferi. Ticks tear their way into the dermis and damage small blood vessels after embedding their mouthparts in the host’s skin,1 which will initiate blood coagulation. To counteract host defense mechanisms, ticks introduce saliva at the bite site that contains a variety of proteins with immunosuppressive and anticoagulant properties.2
Editorial see p 203
Clinical Perspective on p 266
Coagulation can be activated via either the contact activation (or intrinsic) pathway or by the tissue factor (TF; or extrinsic) pathway.3,4 The contact and TF pathway converge at the common pathway, which starts at the level of factor X (FX). Activated FX (FXa) forms the prothrombinase complex together with factor Va (FVa) on a phospholipid membrane surface, leading to thrombin generation.5 Thrombin catalyzes several coagulation-related reactions and converts soluble fibrinogen to fibrin, which forms a solid blood clot together with erythrocytes and platelets.6 Activation of the coagulation system can be divided into 2 phases: the initiation phase and propagation phase. The initiation phase, with concurrent FV and FVIII activation and only limited factor IX (FIX) and FX activation,7 is characterized by low rates of thrombin generation; the propagation phase is characterized by rapid, quantitative activation of all available prothrombin. During the initiation phase, low concentrations of enzyme activate the first traces of FV necessary to generate the prothrombinase complex. Traces of thrombin or the active prothrombin activation-intermediate meizothrombin are hypothesized to be responsible for initial FV activation under physiological conditions.8–11 However, despite several decades of intensive research on FV activation, the questions of whether FV has some degree of intrinsic activity before activation and whether FXa plays a significant role in FV activation are still unanswered. The latter is complicated by the extremely rapid feed-forward activation of FV by thrombin and the lack of specific inhibitors of either thrombin-dependent FV activation or FXa-dependent FV activation. Present knowledge of the behavior and function of the trace active reactants during the initiation phase of coagulation is based partly on extrapolation of clotting times12 and computational analysis using models constructed on the basis of the kinetics of the isolated reactions in which FXa activation of FV was assumed to be insignificant.13
Here, we characterize the anticoagulant properties of a tick salivary protein previously designated P2314 that dose-dependently postpones activation of the coagulation system by specifically preventing activation of FV through FXa. Hence, the tick salivary protein P23 was renamed TIX-5 (tick inhibitor of factor Xa toward factor V). Using TIX-5, we demonstrate that the activation of FV by FXa is a crucial event in the initiation of thrombin generation.
Ticks and Rabbits
Ixodes scapularis adult ticks were obtained from a tick colony at the Connecticut Agricultural Experiment Station in New Haven, CT. Ticks were maintained at 23°C and 85% relative humidity under a 14-hour light, 10-hour dark photoperiod. For the immunization studies, ≈6-week-old inbred New Zealand White rabbits (Charles River Laboratories) were used. The work reported in this study was fully compliant with and approved by institutional policies pertinent to biosafety and animal care protocols. The protocol for the use of mice and rabbits was reviewed and approved by the Yale Animal Care and Use Committee (protocol 2008-07941; approval date, March 31, 2010, to March 31, 2011).
Purification of Recombinant I scapularis Salivary Protein
Cloning and expression of TIX-5 (previously designated as P23,14 GenBank No. AEE89467) and p19 (as a control tick salivary protein) in the Drosophila Expression System (Invitrogen) and purification of recombinant protein were performed as described previously.14 Briefly, for the purification of recombinant TIX-5 (rTIX-5) and recombinant P19 (rP19), the coding sequences of TIX-5 and p19 were cloned into the pMT/Bip/V5-HisA plasmid in frame with a His tag and a V5 epitope (Invitrogen) and validated by sequencing. Drosophila melanogaster S2 cells were transfected with the plasmids containing TIX-5 or p19 and the blasticidin selection vector pCOBlast by use of the Calcium Phosphate Transfection Kit (Invitrogen). Subsequently, cells expressing TIX-5 or p19 were selected with blasticidin (25 µg/mL) and were grown in large spinner flasks for 3 days. Thereafter, recombinant protein expression was induced in Drosophila cells with copper sulfate at a final concentration of 500 µmol/L for 4 days and centrifuged at 1000g for 15 minutes. The supernatant was filtered with a 0.22-µm filter (Millipore), and rTIX-5 or rP19 was purified from the supernatant by binding to an Ni-NTA Superflow column (Qiagen) and elution with 250 mmol/L imidazole. The eluted fractions were filtered through a 0.22-µm filter and concentrated with a 5-kDa concentrator (Sigma-Aldrich) through centrifugal concentration at 4°C, washed, and dialyzed against PBS. The purity of the purified rTIX-5 and rP19 was checked by SDS-PAGE followed by Coomassie blue staining, and the concentration was determined by a BCA protein assay kit (Thermo Fisher Scientific Inc).
Deglycosylation of rTIX-5
Deglycosylation of rTIX-5 with N-glycosidase (PNGase) F (95 U/mg protein; Sigma) was performed in 0.75% TRITON X-100 in PBS for 24 hours at 37°C. As a control, equal amounts of rTIX-5 were incubated in 0.75% TRITON X-100 in PBS for 24 hours at 37°C. Equal amounts of purified recombinant salivary proteins (1 µg) were electrophoresed on 12% SDS-PAGE, and protein was stained with Coomassie blue.
Human Plasma and Coagulation Factors
Human FXa was purchased from Enzyme Research Laboratory. Human FV and FVa were obtained from Hematologic Technologies Inc. FXI-, FIX-, and protein S–deficient plasmas were purchased from Siemens Healthcare Diagnostics. Antithrombin-deficient plasma was purchased from Affinity Biological. TF pathway inhibitor–deficient plasma was purchased from American Diagnostica, and protein C–deficient plasma was obtained from Kordia. Defibrination of normal human pooled plasma was carried out by mixing plasma with 0.4 batroxobin units per 1 mL reptilase (Roche), which was incubated for 10 minutes at 37°C and kept at room temperature for 10 minutes, and the fibrin clot was removed. Platelet-rich plasma was prepared from citrated blood after centrifugation at 200g for 10 minutes at 25°. Recombinant full-length TF pathway inhibitor produced in Escherichia coli was obtained from American Diagnostica Inc. FVIII was obtained from Baxter. Thrombin was kindly provided by Dr W. Kisiel. FIXa was prepared by activation of FIX (Baxter) by FXIa and purified by an anti-FIX column. Stable R155A meizothrombin was prepared fresh and used immediately as previously described.15
Calibrated automated thrombogram was used to assay the generation of thrombin in clotting plasma with a Fluoroskan Ascent microtiter plate–reading fluorometer (Thermo Labsystems) and Thrombinoscope software (Thrombinoscope BV) according to the manufacturer’s instructions and Hemker et al.16 Thrombin generation was initiated by recalcification of citrated pooled human plasma or citrated rabbit plasma (Harlan) in the presence of recombinant human 1 or 5 pmol/L TF (Innovin, Siemens Healthcare Diagnostics), 4 µmol/L phospholipids (Phosphatidylcholine: Phosphatidylserine: Phosphatidylethanolamines, 60%:20%:20%), and 2.5 mmol/L fluorogenic substrate (Z-Gly-Gly-Arg-AMC; Bachem, Bubendorf, Switzerland) with or without rTIX-5. Thrombin formation was followed for 20 to 60 minutes, and measurements were taken at 20-second intervals. Fluorescence intensity was detected at wavelengths of 355 nm (excitation filter) and 460 nm (emission filter). In some cases, coagulation was initiated with a lyophilized silica reagent (Pathromtin SL, 8 times diluted; Siemens Healthcare Diagnostics), FIXa, or FXa. The following parameters were derived: endogenous thrombin potential, the area under the curve representing the total amount of thrombin generated over time; lag time, the time to the beginning of the explosive burst of thrombin generation; peak, the maximal thrombin concentration; and time to peak, the time until the thrombin peak is reached. Experiments were performed in triplicate and repeated 3 times. TF- or FIXa-dependent thrombin generation in a purified system was determined in the presence of the prothrombin complex concentrate Cofact (Sanquin), which was prepared from human plasma and contains the vitamin K–dependent proteins FII, FVII, FIX, FX, protein S, and protein C. Cofact was diluted so that the assay was performed under nearly physiological concentrations of these factors. Assays were performed in the presence of TF pathway inhibitor, FV, phospholipids (PC:PS:PE, 60%:20%:20%), 3 mmol/L CaCl2, and FVIII. Thrombin activation was initiated by the addition of recombinant 1 pmol/L TF or 8 pmol/L FIXa in the presence of rTIX-5 or rP19 as a control. At specific time intervals, aliquots were removed and diluted in 20 mmol/L EDTA, 100 mmol/L NaCl, and 25 mmol/L Tris (pH 7.5) to stop prothrombin activation. Prothrombinase experiments with purified components were performed as follows. The mediators were prepared at 2 times their final concentration in 25 mmol/L HEPES, 150 mmol/L NaCl, 3 mmol/L CaCl2, and 0.3% BSA and prewarmed to 37°C. Equal volumes of mixtures containing either FXa and phospholipids or FV and Cofact as a thrombin-free prothrombin source were mixed at start of the incubation. rTIX-5 or control was added to the FXa-phospholipid mixture immediately before mixing. Final concentrations were 50 pmol/L FXa, 4 μmol/L phospholipid, 5 µg/mL FV or FVa, and Cofact at a concentration of 0.7 μmol/L prothrombin. Samples were taken in time in 20 mmol/L EDTA, 25 mmol/L Tris, and 100 mmol/L NaCl (pH 7.4) to stop prothrombin activation. Thrombin generation was quantified by adding a final concentration of 0.3 mmol/L thrombin chromogenic substrate S2238 (Chromogenix), and substrate hydrolysis was measured kinetically by determination of absorbances at 405 nm with a kinetic microplate reader. All experiments were carried out in triplicate. Prothrombin activation and FV activation were determined in the purified system by Western blotting. At specific time intervals, aliquots were removed and added to SDS sample buffer with or without 2% 2-mercaptoethanol. Samples were electrophoresed on SDS polyacrylamide gels and transferred to polyvinylidene fluoride membranes. The membranes were blocked with PBS containing 5% milk powder, and the immunoblots were probed with either a sheep antihuman prothrombin antibody (Kordia, NL) or the mouse anti-human FV heavy chain monoclonal antibody AHV-5146 (Hematologic Technologies Inc). Immunoreactive bands were visualized with horseradish peroxidase–conjugated anti-sheep or anti-mouse secondary antibodies (Cell Signaling Technology) and the enhanced chemiluminescence Western Blotting Detection System (GE Healthcare).
Fibrinogen and Whole-Blood Clotting Assay
Thrombin generation time was measured spectrophotometrically by the fibrin polymerization method as previously described.17 Thrombin generation was initiated in citrated plasma by the addition of recombinant TF and 17 mmol/L CaCl2, and results were expressed as T 1/2 (time to reach the midpoint of clear to maximal turbid density of the polymerized fibrin measured at 450 nm). Whole-blood coagulation time was assessed by preincubating fresh citrated human blood with various concentrations of rTIX-5 or PBS as a control at 37°C for 15 minutes and then recalcified by mixing with Iscove modified Dulbecco medium containing 12 mmol/L CaCl2. Tubes were incubated at 37°C and tilted every 30 seconds, and clotting times were recorded. Experiments were performed in triplicate.
FXa Chromogenic Assay
A single-stage chromogenic assay of FXa activity was used to assess the FXa inhibitory activity of rTIX-5. Human FXa was diluted to 2 nmol/L in 10 mmol/L HEPES (pH 7.5) containing 0.3% BSA and 150 mmol/L NaCl. rTIX-5 (with a final concentration of 6.5 μmol/L) was incubated with 100 µL FXa for 15 minutes at 37°C. Fifty microliters of 1 mmol/L S2222 (Chromogenix) was added subsequently, and substrate hydrolysis was determined by measuring absorbance at 405 nm over a period of 5 minutes with a kinetic microplate reader. All experiments were carried out in triplicate.
Immunization of Rabbits With Recombinant I Scapularis Proteins
Three rabbits were immunized subcutaneously with 3 doses containing 50 μg rTIX-5 emulsified with complete Freund adjuvant (first dose) and 2 subsequent booster injections emulsified in incomplete Freund adjuvant at 3-week intervals. Control rabbits were immunized with adjuvant and 50 μg ovalbumin or rP19. To demonstrate that the sera from immunized rabbits recognize tick salivary proteins, 2 μg adult I scapularis salivary gland extract prepared as described earlier18 was electrophoresed on an SDS 12% polyacrylamide gel and transferred to polyvinylidene fluoride membranes. Immunoblotting was performed using the same methods as described above except that the immunoblots were probed with 1:250 dilution of rabbit serum and immunoreactive bands were visualized with horseradish peroxidase–conjugated anti-rabbit secondary antibodies (Cell Signaling Technology). Two weeks after the last immunization, rabbits were infested with 15 I scapularis adult couples on the ear of each rabbit kept in place with small socks attached to each ear. Ticks were allowed to feed to repletion until they naturally detached from the host. From 90 hours after attachment, the rabbits were examined twice a day for detached ticks, and tick weights after repletion were recorded.
Surface Plasmon Resonance
Surface plasmon resonance experiments were performed with a BIACORE 2000 (GE Healthcare). rTIX-5 was immobilized on channel 1 of a research-grade CM5 sensor using amine-coupling chemistry according to the standard protocol of the manufacturer. An activated/deactivated surface served as a reference surface. To collect kinetic binding data, FV variants (1 µmol/L) were injected for 3 minutes in 10 mmol/L HEPES (pH 7.4), 150 mmol/L NaCl, 3 mmol/L CaCl2, and 0.005% Tween-20 as flow buffer, followed by flow buffer alone to observe the dissociation. Phospholipids (PC:PS:PE, 60%:20%:20%) were immobilized on an L1 chip according to the manufacturer’s protocol. After stabilization, 100 nmol/L TIX-5 was injected to flow over the surface for 3 minutes. Subsequently, rTIX-5 was replaced by the flow buffer (10 mmol/L HEPES [pH 7.4], 150 mmol/L NaCl, 3 mmol/L CaCl2), and the dissociation was followed. The curves were processed with the BiaEvaluation 4.1 software.
Fluorescence Binding Assay of FV Basic Peptide
The peptide encoding the basic region of the FV B domain (FVBR; residues 951–1008) was expressed as an SUMO fusion using the SUMOpro bacterial expression system (LifeSensors, Malvern, PA). The SUMO-FVBR fusion was purified on HisTrapFF columns (Amersham); the SUMO tag was cleaved with SUMO protease (LifeSensors); and the FV peptide was purified by ion exchange chromatography. For fluorescence anisotropy experiments, FVBR containing an N-terminal Cys was labeled with Oregon Green488 maleimide (OG488-BR) according to the manufacturer’s instructions (Invitrogen).
Steady-state fluorescence anisotropy was measured at 25°C in a PTI QuantaMaster fluorescence spectrophotometer (Photon Technology International) using excitation and emission wavelengths of 480 and 520 nm, respectively, with long-pass filters (KV500, CVI Melles Griot) in the emission beam. Displacement experiments were performed by preincubating 30 nmol/L OG488-BR with 20 nmol/L FV-810 and 50 μmol/L PCPS (75:25 PC:PS) in 20 mmol/L HEPES, 150 mmol/L NaCl, 2 mmol/L CaCl2, and 0.1% (wt/vol) PEG-8000, pH 7.4 (assay buffer). Displacement of OG488-BR from FV-810 was monitored by the decrease in OG488-BR anisotropy throughout the titration of unlabeled FVBR peptide or rTIX-5. Fluorescence anisotropy measurements and data analysis were performed as described.19,20
Recombinant FV Variants
Cloning, expression, and characterization of the recombinant FV variants that contain mutations at the 3 thrombin and FXa cleavage sites have previously been described.21 The FV variants were named according to which amino acid replaced the Arg residue present at each cleavage site,22 that is, wild-type FV (Arg709, Arg1018, and Arg1545), RIQ (Arg709, Ile1018, and Gln1545), QRQ (Gln709, Arg1018, and Gln1545), QIR (Gln709, Ile1018, and Arg1545), and QIQQQ (Gln709, Ile1018, Gln1545, Gln1761, and Gln1765). The FV variant lacking the B domain region (FV des 827–1499) was named FV B-dom Δ. Transient expression and collection of the FV variants were performed as previously described.22 Proteins were secreted and collected in serum-free medium (Optimem Glutamax, Gibco), and protein concentration was determined with an FV ELISA as previously described.22 FV variants with partial deletions of the B domain were expressed and purified as described.23
Determination of Rate of FV Activation by Thrombin and FXa in the Presence of rTIX-5
The effect of rTIX-5 on FV activation by FXa, thrombin, and meizothrombin was determined using a method previously described by Safa et al.24 Briefly, purified human FV (final concentration, 133 nmol/L) was incubated at 37°C with purified human thrombin (final concentration, 1 nmol/L) with purified FXa (final concentration, 10 nmol/L) or R155A meizothrombin (final concentration, 0.2 nmol/L) and 4 µmol/L phospholipid vesicles (PC:PS:PE, 60%:20%:20%) in HBSA buffer containing 150 mmol/L NaCl, 25 mmol/L HEPES (pH 7.5), 3 mmol/L CaCl2, and 0.3% BSA. At specific time intervals, aliquots were removed and diluted in 100 mmol/L NaCl and 20 mmol/L Tris (pH 7.5) to evaluate FV activation immediately in a prothrombin time using FV-deficient plasma. As an alternative approach, FV activation was determined by Western blot assessment as described.25 For this, a final concentration of 20 nmol/L purified human FV was incubated at 37°C with purified human thrombin (final concentration, 1 nmol/L) or with purified FXa (final concentration, 10 nmol/L) in the presence or absence of 4 µmol/L phospholipid vesicles (PC:PS:PE, 60%:20%:20%) in HBSA buffer. At specific time intervals, aliquots were removed and added to SDS sample buffer. Samples were electrophoresed on an SDS 5% polyacrylamide gel and transferred to polyvinylidene fluoride membranes. Immunoblotting was performed using the same methods as described above except that the immunoblots were probed with the mouse anti-human FV heavy chain monoclonal antibody AHV-5146 (Hematologic Technologies). The effect of rTIX-5 on the activation of the various FV variants described above by FXa and thrombin was determined using a method previously described.22 In this assay, the FVa cofactor activity was determined via FXa-catalyzed thrombin generation. Briefly, 250 pmol/L various recombinant FV variants was activated by 5 nmol/L FXa or thrombin in the presence or absence of rTIX-5. At different time points, FV concentrations were measured by prothrombin activation in a reaction mixture with 25 mmol/L HEPES (pH 7.5), 150 mmol/L NaCl, 3 mmol/L CaCl2, 1 μmol/L prothrombin, 0.33 nmol/L FXa, 20 μmol/L phospholipid vesicles (PC:PS, 90%:10%), and 0.5 mg/mL ovalbumin. A final concentration of 1 μmol/L Pefabloc TH was added to the mixture to prevent feedback activation of FV by thrombin. Activation of prothrombin was stopped by dilution in ice-cold EDTA buffer, and generated thrombin was measured using the chromogenic substrate S2238 by measuring absorbance at 405 nm over a period of 5 minutes with a kinetic microplate reader.
Data are expressed as mean±SEM. The significance of the difference between the mean values of the groups was analyzed with the Student t test with Prism 5.0 software (GraphPad Software). For comparisons between multiple groups, results were analyzed by the Kruskal-Wallis test with the Dunn posttest for multiple comparisons or 2-way ANOVA when appropriate. If overall significant by ANOVA, differences were analyzed by Bonferroni posttests. Values of P≤0.05 were considered statistically significant.
Both Contact- and TF-Mediated Activation of Coagulation Is Inhibited by rTIX-5 in a Dose-Dependent Manner
I scapularis protein TIX-5 was recently identified as an antigenic salivary protein after a yeast surface display screen with I scapularis immune rabbit serum (designated P23) and showed anticoagulant activity by significant prolongation of the lag time of thrombin formation in human plasma after the initiation of coagulation with 1 pmol/L TF.14 We now show that rTIX-5 dramatically prolonged lag time and time to peak thrombin generation initiated in human plasma through the contact activation route (Figure 1A) and by 1 and 5 pmol/L TF (Figure 1B and 1C), with more subtle effects on the total amount of thrombin formed (endogenous thrombin potential). rTIX-5 retained its anticoagulant properties after initiation of the TF coagulation pathway in both FVIII- (Figure 1D) and FXI- (Figure 1E) deficient plasma, which indicates that TIX-5 does not exert its inhibitory action through interference with FVIII activity or generation of thrombin through feedback activation via FXI.26 rTIX-5 also showed a prolonged lag time and decreased thrombin formation in platelet-rich plasma when coagulation was initiated with 1 pmol/L TF (Figure 1F). The effect of rTIX-5 was dose dependent (Figure 1G) and was confirmed in a fibrinogen-dependent plasma clotting assay (Figure I in the online-only Data Supplement). On degradation by proteinase K, rTIX-5 lost its anticoagulant properties (Figure II in the online-only Data Supplement). Spontaneous clotting of human whole blood was also dose-dependently extended by rTIX-5 (Figure 1H), underscoring its efficacy in a more relevant system.
Immunization With rTIX-5 Impairs Adult Tick Feeding on Rabbits
To elucidate the biological importance of TIX-5 in tick feeding, rabbits were immunized with rTIX-5. Immunization induced an antibody response that recognized native TIX-5 in salivary gland extract of I scapularis adults (Figure 2A). Immune serum antibodies detected multiple bands in the range of 23 to 30 kDa, representing differentially glycosylated forms of TIX-5 (Figure 2A and Figure III in the online-only Data Supplement). Serum from ovalbumin-immunized rabbits showed no reaction with proteins in salivary gland extract. Antisera to a tick salivary control protein rP19 recognized native P19 but not native TIX-5 (Figure 2A). Adult tick engorgement weights after spontaneous detachment were significantly lower after feeding on rTIX-5–immune rabbits compared with rP19-immune control rabbits (Figure 2B). In line, rTIX-5 also inhibited TF- or contact activation pathway–initiated thrombin generation in rabbit plasma (Figure 2C and 2D).
rTIX-5 Inhibits FXa-Driven Thrombin Generation Independently of the Active Site and Phospholipids
Because rTIX-5 inhibited the coagulation system initiated via either the contact activation or TF pathway, we assessed whether rTIX-5 inhibited the common pathway of coagulation. When thrombin formation was initiated in plasma by 30 pmol/L FXa and various concentrations of phospholipids, rTIX-5 inhibited thrombin generation (Figure 3A). To assess whether rTIX-5 inhibited FXa generation through feedback activation, we initiated coagulation with 30 pmol/L FXa in FX-deficient plasma and demonstrated that rTIX-5 retained its anticoagulant properties (Figure 3B). Tick saliva contains active site inhibitors of FXa, including Salp14.27 However, rTIX-5 did not show a direct effect on FXa in a chromogenic assay (Figure 3C), indicating that rTIX-5 is not an active site inhibitor of FXa. Because the effect of rTIX-5 was more evident in the presence of lower concentrations of phospholipids (Figure 3A), we assessed whether rTIX-5 was able to neutralize phospholipids. In the presence of high concentrations of phospholipids (20 µmol/L), rTIX-5 still inhibited TF-initiated thrombin generation (Figure 3D), indicating that rTIX-5 does not simply neutralize phospholipids.
Inhibitors of the Initiation Phase of Coagulation Reinforce the Anticoagulant Effect of rTIX-5
We assessed whether physiological inhibitors of the human coagulation system influenced the anticoagulant properties of rTIX-5. Fibrinogen, also referred to as antithrombin I, reduces thrombin generation by binding thrombin with high affinity.28 Defibrination of normal human plasma resulted in a clear reduction of the lag time when coagulation was triggered with 1 or 5 pmol/L TF (Figure 4A), in line with a study performed by de Bosch et al.29 When coagulation was triggered with 1 pmol/L TF, rTIX-5 prolonged the lag time by 1.5 minutes in the absence of and 5.7 minutes in the presence of fibrinogen (Figure 4A). Activation of coagulation by 5 pmol/L TF with rTIX-5 resulted in a prolongation of lag time by 2.0 and 3.2 minutes in the absence and presence of fibrinogen, respectively (Figure 4A). Thus, the inhibitory effect of rTIX-5 is greatly reduced in fibrinogen-depleted plasma. In line with this observation, the absence of other physiological inhibitors of the initiation phase, for example, antithrombin, TF pathway inhibitor, and protein S, resulted in a reduced anticoagulant effect of rTIX-5 (Figure IV in the online-only Data Supplement). The absence of protein C, which is not an inhibitor of the initiation phase, did not influence the effect of rTIX-5 (Figure IV in the online-only Data Supplement).
Preactivation of Prothrombin and FV Bypasses the Anticoagulant Effect of rTIX-5
Because the absence of thrombin inhibitors or thrombin generation inhibitors dampened the anticoagulant activity of rTIX-5, we further investigated whether rTIX-5 was able to inhibit coagulation in the presence of preactivated prothrombin. In the presence of 3 nmol/L thrombin, rTIX-5 was not able to inhibit further thrombin generation via the feedback loop through FXI activation (Figure 4B). Next, we studied the anticoagulant effect of rTIX-5 in the presence of preactivated FV. Interestingly, rTIX-5 significantly inhibited coagulation when 20 nmol/L FV was added to FV-deficient plasma, whereas this effect was abrogated in the presence of 20 nmol/L FVa (Figure 4C), which suggested that rTIX-5 postpones the activation of FV, leading to reduced activation of prothrombin. Human plasma contains numerous factors or proteins that could be involved in the inhibitory effect of rTIX-5 on FV activation. We therefore investigated the anticoagulant effect of rTIX-5 on thrombin generation in a partially purified system containing the vitamin K–dependent coagulation factors (Cofact) supplemented with FV, FVIII, TF pathway inhibitor, phospholipids, and CaCl2. In this system, thrombin generation was inhibited by rTIX-5 after initiation with 1 pmol/L TF (Figure 4D) and when initiated with 8 pmol/L FIXa (Figure V in the online-only Data Supplement). Thus, by using this system of purified coagulation factors, we showed by Western blotting that thrombin generation (Figure 4E) and FV activation (Figure 4F) were postponed in the presence of rTIX-5. In line with these data, thrombin generation was inhibited by rTIX-5 in a prothrombinase experiment with procofactor FV, whereas rTIX-5 did not inhibit prothrombinase activity started with preformed FVa (Figure 4G). These data prompted us to study whether rTIX-5 inhibits coagulation activation by interference with FV activation.
rTIX-5 Specifically Inhibits FXa-Mediated FV Activation
To elucidate the mechanism by which rTIX-5 prolonged FV activation, we explored the role of rTIX-5 in the direct activation of FV. Because thrombin and the active prothrombin activation-intermediate meizothrombin have been hypothesized to be the most significant contributors in formation of FVa (reviewed elsewhere9,10), we determined whether FV activation by them was inhibited by rTIX-5. We demonstrate here that rTIX-5 did not affect the activation of FV by 1 nmol/L thrombin (Figure 5A) or by 0.2 nmol/L meizothrombin (Figure 5B) in the presence of phospholipids as evaluated by both Western blot analysis (top) and FVa clot assay (bottom). Strikingly, rTIX-5 abrogated activation of FV by 10 nmol/L FXa-phospholipid (Figure 5C), demonstrated by Western blotting of active fragments of both FV and FVa clot assay. Activation of FV by FXa in the absence of phospholipids was negligible (data not shown). rTIX-5 dose-dependently inhibited FXa-mediated FV activation with a half-maximal inhibitory concentration (IC50) for rTIX-5 of ≈3.2 µmol/L (Figure 5D). It is currently assumed that small amounts of thrombin are generated by FXa when assembled on a membrane surface that subsequently activate sufficient FV to induce prothrombinase activity. Importantly, direct activation of prothrombin by FXa in the absence of FV was not impaired by rTIX-5 (Figure 5E), which corroborates that rTIX-5 is not an FXa active site inhibitor, and does not bind to an exosite of FXa involved in prothrombin activation. Collectively, these data demonstrate that rTIX-5 is a highly specific inhibitor of FXa-mediated FV activation. In line with this, the inhibiting effect of TIX-5 on clot formation was essentially absent in FV-deficient human plasma initiated with 30 nmol/L FXa (Figure 5F). The nominal inhibiting effect of TIX-5 in FV-deficient plasma can be explained by traces of FV, as shown by means of FV neutralizing IgG and activated protein C (Figure 5F).
Inhibitory Action of rTIX-5 on FV Activation is B Domain Dependent
To further pinpoint the anticoagulant effect of TIX-5, we used FV mutants that lack 1, several, or all Arg residues involved in the proteolytic activation of FV or miss the central B domain as described by Segers et al.22 With this method, rTIX-5 also inhibited FXa-catalyzed (Figure 6B) but not thrombin-mediated (Figure 6A) FV activation. FXa activation of the FV mutants that lacked 1 or more activation sites was still inhibited by rTIX-5 (Figure 6C–6F and 6H), whereas TIX-5 specifically failed to inhibit FXa activation of mutants lacking the central B domain (Figure 6G and 6H). TIX-5 did not affect activation of the FV mutants by thrombin (data not shown). In line with this, rTIX-5 significantly inhibited coagulation when recombinant wild-type FV was added to FV-deficient plasma, whereas this effect was abrogated in the presence of the FV mutant lacking the central B domain (Figure 6I). These results indicate that TIX-5 is not simply inhibiting the proteolysis of 1 specific cleavage site but interferes via a B domain–dependent mechanism.
Both the Acidic and Basic Regions of the FV B Domain Support the Inhibitory Effect or TIX-5
It was shown very recently that the B domain, which contains basic and acidic regions (Figure 7A), hinders FXa binding to the high-affinity FXa binding site normally exposed only after proteolysis of the B domain.23 Because TIX-5 also harbors a basic region (Figure 7A), we next assessed whether TIX-5 was able to compete for binding to the acidic region of FV-810, an FV B domain derivative that contains the B domain acidic region but not the basic region. Nevertheless, rTIX-5 did not compete with the FV basic region on the basis of competitive binding experiments (Figure 7B). In line with these results, rTIX-5 inhibited thrombin formation when the various factor B domain variants with the basic region (FV-B152), acidic region (FV-810), or both basic and acidic regions (FV-1033) were added to FV-deficient plasma (Figure 7C). Of note, thrombin formation was less inhibited by rTIX-5 in the presence of FV-B152 or FV-810 compared with plasma-derived FV (wild-type FV) or FV-1033, indicating that both the acidic and basic regions of the FV B domain support the inhibiting effect of rTIX-5 (Figure 7C). rTIX-5 abrogated activation of FV-810 (Figure 7D), FV-B152 (Figure 7E), and FV-1033 (Figure 7F) by 10 nmol/L FXa in the presence of phospholipids, as demonstrated by Western blotting of active fragments of FV. To characterize specific interactions of rTIX-5 with FXa or FV, surface plasmon resonance experiments were performed that revealed that rTIX-5 bound to FV-1033, FV-B152, and to a lesser extent FV-810, but not to FXa, under these conditions (Figure 7G). Binding analysis of TIX-5 to a phospholipid layer showed an additional interaction of TIX-5 with phospholipids (Figure 7H).
We report here that TIX-5, a major antigen in tick saliva, inhibits blood coagulation by specific inhibition of FXa-dependent FV activation. The mechanism by which TIX-5 specifically inhibits FXa-mediated FV activation involves the central B domain of FV, which contains the initial preferential cleavage site for FXa.11 Most important, for the first time, delineation of the molecular anticoagulant mechanism of TIX-5 provides evidence that FXa is a physiologically relevant activator of FV, challenging the dogma that thrombin or the active prothrombin activation-intermediate meizothrombin are the only physiological activators of FV in the initiation phase of coagulation.9–12,30
It is of crucial importance that enzymes of the coagulation system and their cofactors circulate in an inactive form under physiological conditions and are activated promptly but only when necessary. Traces of activated FV are likely inactivated by activated protein C. Activated protein C is a crucial antithrombotic mechanism, as shown by the association of the FV-Leiden mutation with thrombosis, which renders APC-resistant FVa.31 In the common pathway of coagulation, FX and FV are activated to FXa and FVa, respectively. FVa is the nonenzymatic cofactor of the prothrombinase complex, which accelerates the conversion of prothrombin to thrombin by FXa ≈300 000 times.32,33 The high efficiency of FXa to generate thrombin in the presence of only minute traces of FVa, together with the relative poor activation of FV by FXa and extremely efficient FV activation by (meizo)thrombin, has hampered studies on the contribution of FV activation by FXa in relevant thrombin-generating systems. Activation of FV involves cleavages in the procofactor form that release the central gatekeeper B domain to generate active FVa, a noncovalent complex formed by the N-terminal–derived heavy chain and the C-terminal–derived light chain11 (Figure VI in the online-only Data Supplement). Although thrombin directly cleaves FV at Arg709 and subsequently at Arg1545 to liberate the heavy chain and light chain, respectively, the sequence of events is different for FXa. FXa preferentially first cleaves the FV B domain at Arg1018 and subsequently at position Arg709 and Arg1545.21 Using mutant FV molecules, we were able to show that TIX-5 inhibited FV activation by FXa only when the B-domain was present in the procofactor form. An FV mutant that lacks the B domain was activated by FXa in a manner that is not inhibited by TIX-5, showing that TIX-5 inhibits FXa-catalyzed FV proteolytic activation in a B domain–dependent mechanism. It was shown very recently that the B domain, which contains basic and acidic regions, hinders FXa binding to the high-affinity FXa binding site normally exposed only after proteolysis of the B domain.23 We hypothesize that TIX-5 cooperates with the inhibitory B domain segments to impair FXa-mediated activation of FV. In line with this hypothesis, TIX-5 inhibited FXa-mediated activation of all 4 FV variants with mutated cleavage sites. Thus, TIX-5 does not block the cleavage sites themselves but prevents the accessibility of FXa to activate FV.
Although an obvious basic region is present in TIX-5, binding of TIX-5 to the acidic region of FV did not appear to dominate binding. The Surface Plasmon Resonance experiments revealed rather that the FVBR seems to support a larger part of the direct protein-protein interaction between TIX-5 and FV. Importantly, TIX-5 interacted with and inhibited FV variants that lack either the acidic or basic region in the B domain, indicating that the action of TIX-5 is not caused by a simple 1-site binding event. Thus, the exact molecular mechanism by which TIX-5 inhibits FXa-mediated FV activation remains elusive. Although FV variants that lack either the acidic or basic region in the B domain behave as active cofactors,23 TIX-5 is still able to turn these derivatives into partially active or procofactor proteins. Because this is not the case for FVa or B domain–less FV, these findings emphasize a dependency of parts of the FV B domain in the function of TIX-5 as coagulation inhibitor. In our current view, on the basis of the notion that TIX-5 has some phospholipid-binding properties, we construe that TIX-5 prevents functional low-affinity activating Xa-FV interactions that drive the FXa-mediated FV activation on phospholipid membranes. Importantly, interaction of TIX-5 with the phospholipid surface does not interfere with other phospholipid-dependent reactions, including the activation of prothrombin by FXa and the phospholipid-dependent FV activation by meizothrombin. Additionally, in the presence of excessive amounts of phospholipids (20 µmol/L), TIX-5 still inhibited TF-initiated thrombin generation. Together, these data exclude that TIX-5 is just a phospholipid scavenger and show that TIX-5 is a specific inhibitor of the phospholipid-dependent activation of FV by FXa.
The ability of FXa to activate the FV QIQQQ variant, which lacks all specific FXa activation sites, is puzzling. However, Thorelli et al21 demonstrated that FXa is more promiscuous in cleaving at other sites than the traditional 709, 1018, and 1545 thrombin cleavage sites. Potential other exposed Args are vulnerable to cleavage by FXa in the B domain or Arg residues in the heavy chain and light chain that would generate truncated heavy chain/light chain fragments, forming a partially active FVa, which could explain how FV QIQQQ becomes activated by FXa.
It has been proposed that the α-thrombin responsible for early FV activation is produced directly by FXa on phospholipids during the initial phase of coagulation.9,34 TIX-5 did not affect direct activation of prothrombin to thrombin on phospholipids by FXa, nor did it affect the enzymatic activity of FXa or thrombin toward small chromogenic substrates. Consistent with these observations and characteristic for its mode of action, TIX-5 did not inhibit thrombin generation in plasma in the presence of FVa or started by traces of thrombin and positive feedback by FXI activation, whereas both the contact activation and TF pathway of coagulation were inhibited by TIX-5. Indeed, in a purified system, prothrombinase activity was exclusively inhibited by TIX-5 in the presence of procofactor FV, whereas inhibition of thrombin formation by TIX-5 was abrogated in prothrombinase with preactivated FVa.
Furthermore, by using TIX-5 as a tool, we uncovered that the importance of FXa-dependent FV activation for thrombin generation in plasma is largely dependent on the presence of fibrinogen. This underscores the notion that fibrinogen, also referred to as antithrombin I, binds and inhibits thrombin,28,35 thereby shifting the role of FXa to a crucial FV activator in plasma. In a reconstituted system with purified coagulation factors, we were able to confirm the inhibitory role of TIX-5 on thrombin generation in the complete absence of fibrinogen, which indicates that the action of TIX-5 is not dependent but is more efficient in the presence of fibrinogen. Similar observations were made in terms of the other physiological coagulation inhibitors that regulate the initiation phase of thrombin generation, including antithrombin-III,25 TF pathway inhibitor, and protein S,36,37 indicating that TIX-5 efficiently exploits the halt on thrombin generation provided by the host coagulation inhibitors. Thus, we observed that rTIX-5 inhibits thrombin generation profoundly when the initiating trigger is low and when thrombin formation and function are counterbalanced by coagulation inhibitors of the host.
The coagulation system is triggered immediately after the tick’s mouth parts penetrate and damage the host’s tissue. To secure their blood meal, ticks have been shown to target several parts of the host coagulation system, among them platelets, thrombin, FXa, and fibrinogen,38 by alternating phases of sucking blood and secreting saliva into the feeding pit, with each phase lasting as long as 5 to 20 minutes.39 Interestingly, nonanticoagulated whole blood was kept in a fluid state for 19.5 minutes by rTIX-5 compared with 5.5 minutes in the absence of rTIX-5. Considering that ticks have a wide repertoire of anticoagulant proteins, the anticoagulant effect of these additional inhibitors is most likely enhanced in the presence of TIX-5 because TIX-5 inhibits the generation of FVa, which stabilizes and protects FXa against inhibition.40 Interestingly, by performing a bioinformatics analysis of TIX-5, we identified, besides several homologs in I scapularis, a partial amino acid sequence coding for an annotated homolog in Ornithodoros coriaceus (Figure VII and Table I in the online-only Data Supplement). Because Ornithodoros is part of a distinct family of ticks, that is, Argasidae,41 it is likely that TIX-5 is a member of a larger protein family in ticks. Antitick vaccines to prevent pathogen transmission are the topic of extensive investigation, and TIX-5 or its homologs could be used, possibly in combination with other tick proteins, in efforts to develop vaccines that render tick immunity or tick resistance.42 In line with this, we show here that adult I scapularis weights were dramatically reduced after feeding on rTIX-5–immunized rabbits, which is a major parameter indicative of tick immunity.43
Our findings show evidence for the facilitation of rapid thrombin generation by direct FXa-mediated FV activation under physiological blood and plasma clotting conditions. This was accomplished by the use of a novel anticoagulant tick protein, TIX-5, that inhibits the coagulation system in a unique way. Thus, we propose a scheme for blood coagulation as shown in Figure 8 in which FXa-dependent FV activation is pivotal during the initiation phase while traces of initial thrombin are captured by physiological thrombin inhibitors. By forming the initial prothrombinase complexes, thrombin generation enters the transition phase, with complete FV activation leading to the explosive propagation phase of thrombin generation. This study, using TIX-5 as a tool to inhibit FXa-mediated activation of FV, has broad implications for the understanding of the initiation phase of coagulation and indicates that inhibition of FV activation by FXa may provide an alternative therapeutic strategy for thrombotic diseases.
Cofact was generously provided by Ruud Zoethout (Sanquin). We thank Han Levels, Rolf Urbanus, and Sukanya Narasimhan for technical support. We thank Nan van Geloven and Arjan Hoogendijk for statistical assistance.
Sources of Funding
Dr Schuijt is supported by a grant from the Onze Lieve Vrouwe Gasthuis research fund. This study was supported by grants 41440, 49200, and 32947 from the National Institutes of Health. Dr Hovius is a recipient of a VENI stipend (91611065) from the Netherlands Organization for Health Research and Development (ZonMw). Dr Fikrig is an investigator of the Howard Hughes Medical Institute. Dr Bunce is supported by grants T32 HL07439 from the National Institutes of Health. Dr Camire is supported by grants R01-HL88010 and P01-HL74124, project 2, from the National Institutes of Health.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/lookup/suppl/doi:10.1161/CIRCULATIONAHA.113.003191/-/DC1.
- Received April 12, 2013.
- Accepted May 10, 2013.
- © 2013 American Heart Association, Inc.
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It is of crucial importance that enzymes of the coagulation system and their cofactors circulate in an inactive form under physiological conditions and are activated promptly but only when necessary. In this study, we demonstrate, using a novel tick-derived coagulation inhibitor, that the activation of factor V by factor Xa is a crucial event in the initiation of thrombin generation. Efforts to introduce novel anticoagulants based on direct factor Xa or thrombin inhibition have provoked discussion about the implications these new strategies may have. In light of our findings, it can be hypothesized that this new class of direct inhibitors have a differential influence on the initial factor V activation. In other words, part of the anticoagulant mechanism of direct factor Xa inhibitors is potentially explained by inhibiting the generation of the first factor Va molecules. In addition, our findings may prompt to a new class of coagulation inhibitors that target this aspect of the initiation phase of coagulation.