A Novel Heparin-dependent Inhibitor of Activated Protein CThat Potentiates Consumptive Coagulopathy in Russell’sViper Envenomation*
Received for publication, November 11, 2011, and in revised form, February 17, 2012 Published, JBC Papers in Press, March 13, 2012, DOI 10.1074/jbc.M111.323063
An-Chun Cheng‡, Hua-Lin Wu§¶, Guey-Yueh Shi§¶, and Inn-Ho Tsai‡�1
From the ‡Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan, the §Department of Biochemistry andMolecular Biology, College of Medicine, National Cheng Kung University, Tainan 70101, Taiwan, the ¶Center for Bioscience andBiotechnology, National Cheng Kung University, Tainan 70101, Taiwan, and the �Institute of Biochemical Sciences, NationalTaiwan University, Taipei 10617, Taiwan
Background: The reason behind the development of consumptive coagulopathies in Russell’s viper bite patients is stillelusive.Results: An inhibitor of activated protein C (a major physiological anticoagulant) synergizes with the venom’s procoagulatingenzymes.Conclusion: Down-regulation of activated protein C in Russell’s viper envenomation is associated with consumptivecoagulation.Significance: The discovery provides new insights into the pathogenesis of Russell’s viper venom-induced coagulopathies.
The activation of coagulation factors V and X by Russell’sviper venom (RVV) has been implicated in the development ofconsumptive coagulopathies in severely envenomed patients.However, factor Va is prone to inactivation by activated proteinC (APC), an important serine protease that negatively regulatesblood coagulation. It is therefore hypothesized that APCmay bedown-regulated by some of the venom components. In thisstudy, we managed to isolate a potent Kunitz-type APC inhibi-tor, named DrKIn-I. Using chromogenic substrate, DrKIn-Idose-dependently inhibited the activity of APC. Heparin poten-tiated the inhibition and reduced the IC50 ofDrKIn-I by 25-fold.DrKIn-I, together with heparin, also protected factor Va fromAPC-mediated inactivation. Using surface plasmon resonance,DrKIn-I exhibited fast binding kinetics with APC (associationrate constant � 1.7 � 107 M�1 s�1). Direct binding assays andkinetic studies revealed that this inhibition (Ki � 53pM) is due tothe tight binding interactions of DrKIn-I with both heparin andAPC.DrKIn-I also effectively reversed the anticoagulant activityof APC and completely restored the thrombin generation inAPC-containing plasma. Furthermore, although the injection ofeither DrKIn-I or RVV-X (the venom factor X-activator) intoICR mice did not significantly deplete the plasma fibrinogenconcentration, co-administration of DrKIn-I with RVV-Xresulted in complete fibrinogen consumption and the deposi-tion of fibrin thrombi in the glomerular capillaries. Our resultsprovide new insights into the pathogenesis of RVV-inducedcoagulopathies and indicate that DrKIn-I is a novel APC inhib-itor that is associated with potentially fatal thrombotic compli-cations in Russell’s viper envenomation.
Envenomation by Russell’s vipers has long been a serioushealth threat in Pakistan, India, Bangladesh, Sri Lanka, andmany parts of Southeast Asia (1, 2). The most striking featuresof these envenomed patients are spontaneous bleeding andincoagulable blood, which result from disseminated intra-vascular coagulation (DIC)2 or consumptive coagulopathyinduced by procoagulant factors in Russell’s viper venom(RVV). Thrombocytopenia and hypofibrinogenemia are there-fore evident in systemically envenomed patients (3, 4).Despite the fact that the procoagulant nature of RVV has
been recognized for decades, our knowledge of the responsiblecomponents in this venom still lingers on factor X-activatingenzyme (RVV-X) and factor V-activating enzyme (RVV-V) (3).The activated factors X (FXa) and V (FVa), in the presence ofcalcium and phospholipids, form a prothrombinase complexthat catalyzes the conversion of prothrombin to �-thrombin(5). It is, however, difficult for coagulations to spread uncon-trollably because there are physiological anticoagulant mecha-nisms that oppose thewidespread formation of�-thrombin (6).Furthermore, animal studies have shown that although RVV-Xis strongly procoagulant in vitro, it failed to effectively decreasethe plasma fibrinogen concentrations in vivo (7). It is, therefore,unlikely that RVV-X and RVV-V are solely responsible for thecoagulopathies seen in Russell’s viper envenomed patients.Based on the severity of bleeding disorders seen in thesepatients, we hypothesized that RVV may contain proteins thatinterfere with the negative regulations of blood coagulation.
* This work was supported by grants from Academia Sinica and from theNational Science Council (NSC 99-2311-B-001-016-MY3).
1 To whom correspondence should be addressed: Institute of BiologicalChemistry, Academia Sinica, No. 128 Academia Road Section 2, Nan-Kang,Taipei 11529, Taiwan. Tel.: 886-2-23665521; Fax: 886-2-23635038; E-mail:[email protected].
2 The abbreviations used are: DIC, disseminated intravascular coagulation;RVV, Russell’s viper venom; FXa, factor Xa; FVa, factor Va; PC, protein C; APC,activated protein C; FVIIIa, factor VIIIa; BPTI, bovine pancreatic trypsininhibitor; DrKIn-I, D. russelii Kunitz inhibitor-I; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DOPS, 1,2-dioleoyl-sn-glycero-3-phosphoser-ine; SPR, surface plasmon resonance; FPLC, fast protein liquid chromatog-raphy; HPLC, high performance liquid chromatography; APTT, activatedpartial thromboplastin time.
The protein C (PC) pathway, which becomes activated by thethrombin-thrombomodulin complex, represents a major phys-iological anticoagulant component in which the activated pro-tein C (APC) functions by proteolytically inactivating activatedcofactors V (FVa) and VIII (FVIIIa) (8). Although there are sev-eral other physiological anticoagulants such as antithrombinIII, heparin cofactor II and tissue factor pathway inhibitor thateither inhibit thrombin directly or prevent the activation ofprothrombin (9–11), APC remains the only serine proteasethat is involved in anticoagulation. Since Viperidae snake ven-oms are rich in serine protease inhibitors belonging to theKunitz/bovine pancreatic trypsin inhibitor (BPTI) family (12),it is tempting to speculate that some members of this littleunderstood protein family in RVV might target APC to pro-mote the extensive coagulations seen in severely envenomedpatients.In this study, we describe the isolation and kinetic character-
ization of a Kunitz-type protease inhibitor named DrKIn-I(Daboia russelii Kunitz Inhibitor-I) that possesses stronginhibitory activity against APC in the presence of heparin, inboth purified system and in plasma. We demonstrate that itbinds tightly to both APC and heparin.Moreover, we show thatthe presence of this inhibitor greatly exacerbates coagulationand hypofibrinogenemia induced by RVV-X in mice. Thesefindings may necessitate the use of APC or protein C concen-trates in Russell’s viper bite patients, and should offer insightsinto better treatments for these patients.
EXPERIMENTAL PROCEDURES
Materials—Lyophilized venom of Daboia russelii russelii(Pakistan) was purchased from Latoxan. Purified human acti-vated protein C, protein S, factor XIIa (FXIIa), factor XIa(FXIa), factor Xa (FXa), factor IXa (FIXa), factor VIIa (FVIIa),factor Va (FVa), thrombin, plasma kallikrein, and plasmin wereobtained from Hematologic Technologies. Trypsin and tissueplasminogen activator (tPA) were from Merck Chemicals.Urokinase plasminogen activator (uPA) was a kind gift fromPolyamine Corp. Synthetic chromogenic substrates Spec-trozyme PCa, Spectrozyme tPA, and Spectrozyme FIXa werepurchased from American Diagnostica, while S-2222, S-2302,S-2366, S-2288, and S-2251 were from Chromogenix. T-1637was from Sigma-Aldrich. RVV-X was prepared from our labo-ratory according to the method provided by Chen et al. (13).Unfractionated heparin and heparan sulfate were from Sigma-Aldrich, while heparan sulfate dimers, tetramers, hexamers andoctamers were gifts from Dr. Hung Shang-Cheng (GenomicsResearch Center, Academia Sinica, Taiwan). Synthetic phos-pholipids 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)and 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS) werebought from Avanti Polar Lipids. Normal coagulation controlplasma and antithrombin/heparin cofactor II immune-de-pleted plasma were from HYPHEN Biomed and EnzymeResearch Laboratories, respectively. The CM5 sensor chip forsurface plasmon resonance (SPR) analysis was purchased fromGEHealthcare. ICRmice were purchased from BioLASCO andhoused in a pathogen free environment.All animal experimentswere approved by the Academia Sinica Institutional AnimalCare and Utilization Committee.
Purification of Kunitz-type Protease Inhibitors—LyophilizedDaboia russelii russelii crude venom was dissolved in 0.1 M
ammonium acetate (pH 6.5) and loaded onto a SuperdexTM 7510/300 GL column (GE Healthcare) connected to an AKTAFPLC system (GE Healthcare). The proteins were eluted at aflow rate of 1.0 ml/min and collected in volumes of 0.5 ml. Thefractions were analyzed by SDS-PAGE, and those that con-tained proteins in the approximate range of 5 to 10 kDa werepooled together and lyophilized. The proteins were furtherpurified by reversed-phaseHPLC (Waters 600HPLCpumpandcontroller) on a Vydak C-18 (10 �m, 250 � 4.6 mm) column.Elution was carried out with a linear gradient of 20–50% ace-tonitrile in 0.07% w/w trifluoroacetic acid over a period of 60min. The purity of each protein was assessed by SDS-PAGE,and the protein concentrations determined by BCA ProteinAssay kit (Pierce Biotechnology). The molecular weightswere determined by Q-TOF Ultima MALDI instrument(Micromass).Cloning of Kunitz-type Protease Inhibitors—Daboia russelii
formosensis cDNAs prepared from the venom gland mRNAwere amplified using the previously described specific primersfor Kunitz-type protease inhibitors (14). The sense primerwas 5�-CCAGACGGCTCCATCATG-3� while the antisenseprimer was 5�-AAAAGGAATRATCCAGG-3�. The conditionsfor PCR were as follows: denaturation at 92 °C for 1 min,annealing at 60 °C for 1 min, and extension at 72 °C for 1 min(35 cycles). The PCR fragments were inserted into thepGEM-T easy vector (Promega Biotech) and transformedinto JM109 Escherichia coli competent cells. The sequencesof plasmid DNAs from the transformed colonies wereobtained using the DNA-Sequencing System (Model 373A,PE-Applied Biosystems).In Vitro Assays for the Inhibition of APC by DrKIn-I—All
inhibition assays were performed in 96-wells microtiter platesin 25mMTris-HCl (pH 7.4), 150mMNaCl, 2.5mMCaCl2, and 5mg/ml BSA. For comparison between DrKIn-I and DrKIn-II,the amidolytic activity of 10 nM APC, with or without 0.1units/ml heparin, was assayed in the presence or absence ofequal molar concentrations of DrKIn-I or DrKIn-II. Immedi-ately after the addition of APC, Spectrozyme PCa was addedand the rates of p-nitroaniline release were monitored at 405nm for 10 min at 37 °C. For dose response curves, APC wasmixed with different concentrations of DrKIn-I in the presenceor absence of heparin. The final concentrations were as follows:APC (10 nM), heparin (0.1 U/ml) and DrKIn-I (0–100 nM in thepresence of heparin and 0–12800nM in the absence of heparin).Dose-response curves were fitted using GraphPad Prism(GraphPad Software). In other inhibition experiments, varyingconcentrations of heparin (0–1000mU/ml) or different lengthsof heparan sulfate chains (10 �g/ml) were added to equimolarconcentrations of APC and DrKIn-I (20 nM each). In all theinhibition experiments, Spectrozyme PCa was added to a finalconcentration of 0.2 mM. Changes in absorbance were mea-sured using SpectraMax M2e Microplate Reader (MolecularDevices).In assays involving FVa, 20 nM purified FVa was incubated at
37 °C with a mixture containing 1 nM or 5 nM APC, 20 nMprotein S, 20 �M DOPC/DOPS (75:25) and 5–250 nM DrKIn-I
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in the presence or absence of 0.1 U/ml heparin. At specific timeintervals, 5 �l aliquots were removed and mixed with 50 �l ofFV-deficient plasma. The residual FVa activities were quanti-fied in a standard prothrombin time-based assay using a cali-bration curved obtained by adding variable amounts of FVa toFV-deficient plasma. All concentrations given were finalconcentrations.Heparin Binding Assay—A 5ml HiTrap Heparin HP column
(GE Healthcare) that had been pre-equilibrated with 20 mM
Tris-HCl buffer (pH 8.0) was loaded with 70 �g of DrKIn-I.After washingwith 5ml of equilibrating buffer, a 50ml gradientfrom 0.0–1.0MNaClwas applied at a flow rate of 5ml/min andthe salt concentration corresponding to the protein peak wasdetermined as a measure of its heparin binding affinity.Surface Plasmon Resonance Analysis—Biacore T200 (GE
Healthcare) was used for analysis. APC or PC dissolved in 10mM acetate buffer (pH 5.0) was immobilized on a CM5 sensorchip to a response unit (RU) of 1000with an amine coupling kit.Associations and dissociations of DrKIn-I were performed in10 mM HEPES (N-2-hydroxyethylpiperazine-N�-2-ethanesul-fonic acid; pH 7.4), 150 mM NaCl, 3 mM EDTA and 0.05% P20with a flow rate of 60 �l/min. The sensor surface was regener-ated with 90 �l of 2 M MgCl2 and the signals obtained weresubtracted by that obtained from the reference channel thathad not been coated with ligands. Binding kinetics were deter-mined by global fitting to 1:1 Langmuir bindingmodel using theBiaevaluation software (GE Healthcare).Kinetic Analysis of APC Inhibition by DrKIn-I—APC was
incubated with heparin and increasing concentrations ofDrKIn-I for 3 min. The initial reaction velocities (mOD405nm/min) were then determined at 37 °C after the addition ofvarying concentrations of Spectrozyme PCa. The final concen-trations were as follows: APC (20 nM), heparin (0.1 units/ml),DrKIn-I (0–80 nM), and Spectrozyme PCa (0.025–0.4 mM).Initial velocities were plotted against inhibitor concentrationsfor each substrate concentration tested and the plots were sub-jected to nonlinear least squares regression using GraphPadPrism software. The inhibition constant (Ki) of DrKIn-I wasdetermined by global fitting to Morrison’s tight binding Equa-tion 1 as shown below (15),
Vs � �Vo/2Et����Ki� � It � Et�2 � 4Ki�Et]
1/2 � �Ki� � It � Et��
(Eq. 1)
whereVs is the steady state velocity in the presence of inhibitor,while Vo is the velocity in the absence of inhibitor. It is the totalinhibitor concentration and Et is the total enzyme concentra-tion. Ki� is the apparent inhibition constant. For competitiveinhibition, Ki� is related to the true inhibition constant (Ki) byEquation 2,
Ki� � Ki/�1 � S/Km� (Eq. 2)
where S is the substrate concentration andKm is theMichaelis-Menten constant for Spectrozyme PCa, which was determinedto be 0.55 mM.Selectivity Profile of DrKIn-I—DrKIn-I, with or without hep-
arin, was screened for its inhibitory activity against trypsin andalso against serine proteases in the coagulation cascade (FXIIa,
FXIa, FXa, FIXa, FVIIa, thrombin, and kallikrein) and in thefibrinolytic system (plasmin, tPA, and uPA). The amidolyticactivities of these proteases were determined in the presence orabsence of equimolar concentrations of DrKIn-I using theirrespective chromogenic substrates.Determination of APC Inhibitory Activity of DrKIn-I in
Plasma—50 �l of normal coagulation control plasma or anti-thrombin III/heparin cofactor II-double immune-depletedplasma, with or without 0.1 units/ml heparin, was exposed toAPC (40 nM) and varying concentrations of DrKIn-I (40–160nM). 50 �l of activated partial thromboplastin reagent(HYPHENBiomed)was added and incubated for 1min at 37 °C.Finally, clotting was initiated by adding 50 �l of 20 mM CaCl2.Coagulation times, which reflect the activities of APC, wererecorded on a coagulometer (Hemostasis Analyzer KC-1;Sigma Diagnostics).Thrombin Generation Assay—The procoagulant function of
DrKIn-I in heparin-containing plasma was assessed by throm-bin generation assay. Briefly, 80 �l of antithrombin/heparincofactor II-deficient plasma containing 4 �M corn trypsininhibitor, 0.1 units/ml heparin and 30�MDOPC/DOPS (75:25)was incubated with 20 �l of 500-fold diluted tissue factor solu-tion (Innovin; Dade Behring) in the presence or absence of APCand/or DrKIn-I (20 nM each). Following incubation for 3min at37 °C, thrombin generation was initiated by the dispensation of20 �l of 2.5 mM fluorogenic substrate (Z-Gly-Gly-Arg-AMC.HCl) dissolved in 0.15 M NaCl, 60 mg/ml BSA, and 100mM CaCl2. Measurements were taken at 1 min intervals on aSpectraMax M2e Microplate Reader using an excitationwavelength of 360 nm and an emission wavelength of 460nm. Results were evaluated using the Technothrombin TGAsoftware (Technoclone).In Vivo Assays—8–10-week-old ICR mice were used in our
experiments. RVV-X and/or DrKIn-I were administered intra-venously at the indicated concentrations. 3 h after injection,plasma was collected and analyzed for fibrinogen concentra-tions. Briefly, 100 �l of 10-fold diluted plasma was mixed with50 �l of 75 NIH units/ml thrombin (Trinity Biotech) and thetime required for clot formation wasmeasured. Fibrinogen lev-els were determined based on a calibration curve prepared froma fibrinogen reference (HYPHEN Biomed).For histopathological examinations, mice were injected
intravenously with 0.005 �g/g RVV-X and/or 0.04 �g/gDrKIn-I. The mice were anesthetized after 3 h with pentobar-bital, and the kidneys were extracted. Fixed and paraffin-em-bedded kidneys were sectioned at 6�mand subjected to hema-toxylin-eosin staining. The slides were scanned by ScanScopeCS System (Aperio Technologies) with a 20�/0.75 Plan Agoobjective, and images were analyzed using the Aperio Image-Scope software (version 9.1.19.1569).
RESULTS
Purification and Cloning of Kunitz-type Protease Inhibitors—In view of the fact that Kunitz-type protease inhibitors are rel-atively small with a length of only �60 amino acids (12), thecrude venom of Daboia russelii russelii was first separated intoseveral fractions based on their molecular sizes by gel filtration.The fifth fraction (indicated by a horizontal bar in Fig. 1A) was
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then subjected to a second purification step using reversed-phase HPLC. The first two proteins that were eluted (desig-nated DrKIn-I and DrKIn-II) had approximate yields of 1.7%(w/w) and 2.6% (w/w), respectively (Fig. 1B). The masses ofthese two proteins were determined by MALDI-TOF analysis,which gave anm/z signal of 7548.9 Da for DrKIn-I and anm/zsignal of 6940.3 Da for DrKIn-II. The molecular weights ofthese two proteins were exactly identical to that of the twoKunitz-type protease inhibitors cloned from the venom glandof Daboia russelii formosensis (Fig. 1C) (GenBankTM accessionnumbers JN825729 and JN825730), confirming the identity ofthe first two proteins as Kunitz-type protease inhibitors.Exactly identical sequences have also been cloned from thevenom gland of Daboia russelii siamensis (accession numbersA8Y7N4 and A8Y7N5), indicating that these Kunitz-type pro-tease inhibitors may be conserved throughout the D. russeliispecies. Furthermore, the mass obtained for DrKIn-I and itsinability to be sequenced by Edman degradation indicate thatthe N-terminal of DrKIn-I is in the form of a cyclic pyrogluta-matic acid.DrKIn-I Inhibits APC in the Presence of Heparin—The ability
of DrKIn-I and DrKIn-II to inhibit APC amidolytic activity wasassayed with a chromogenic synthetic substrate, SpectrozymePCa. As shown in Fig. 2A, both inhibitors exhibited little inhib-itory activity against APC in the absence of heparin. However,
in the presence of 0.1 units/ml heparin (180 units/mg), DrKIn-Idecreased the activity of APC by 100%, while DrKIn-IIdecreased the activity by only �20%. Of the two Kunitz-typeprotease inhibitors purified, only DrKIn-I showed a potentinhibitory activity against APC. Dose-response curve ofDrKIn-I obtained in the presence of heparin showed that theincrease in inhibition occurred over a very narrow range ofDrKIn-I concentration, as denoted by a large Hill slope of3.64 0.30 (Fig. 2B). This indicates that DrKIn-I is a tightbinding inhibitor of APC in the presence of heparin where theKd is much lower than the enzyme concentration (16). Further-more, complete inhibition was achieved for equimolar concen-trations of APC and DrKIn-I (Fig. 2B). In contrast, the dose-response curve obtained in the absence of heparin was lesssteep, with a Hill slope of 0.85 0.02 (Fig. 2B). The IC50values in the presence and absence of heparinwere 3.5 0.2 nMand 88.9 1.0 nM, respectively.To determine the concentration of heparin required for the
potentiation of APC inhibition, the enzyme-inhibitor mixturewas spiked with varying concentrations of heparin. 0.01units/ml of heparin potentiated the inhibition by more than70% (Fig. 2C), and at 0.1 units/ml, no APC activity was detect-able, suggesting that only low concentrations of heparin arerequired for APC inhibition.Apart from heparin, we also tested the ability of heparan
sulfate to potentiate DrKIn-I-mediated APC inhibition, sinceheparan sulfate is structurally similar to heparin, and is abun-dant as part of proteoglycans on the surface of endothelial cells(17). As shown in Fig. 2D, heparan sulfate can also act as acofactor for APC inhibition. Furthermore, while heparan sul-fate dimers and tetramers enhanced the inhibition by only �10and �25%, respectively, heparan sulfate hexamers enhancedthe inhibition by �80%, suggesting that heparan sulfate chainsshould be at least 6 units long for sufficient potentiation of APCinhibition (Fig. 2D).In addition to using the synthetic tripeptide (Spectrozyme
PCa) as the substrate ofAPC,we also tested the inhibitory activ-ity of DrKIn-I using FVa, APC’s natural substrate. In theabsence of heparin, APC (1 nM) progressively degraded FVa (20nM) over a period of 10min (Fig. 2E). The addition ofDrKIn-I (5nM) alone had relatively no effect on APC activity. However, inthe presence of heparin, DrKIn-I (5 nM) protected 100% of FVafrom inactivation (Fig. 2E). Without heparin, the addition of a50-fold molar excess of DrKIn-I (250 nM) protected only lessthan 20% of FVa from inactivation (Fig. 2F), confirming thatheparin is absolutely essential for DrKIn-I-mediated APC inhi-bition. Regardless of the type of substrate used, heparin alone at0.1 units/ml did not alter the activity of APC (Fig. 2, A and F).Physical Interactions of DrKIn-I with Heparin and APC—Be-
cause it has been reported that APC possesses a heparin-bind-ing site that allows it to physically interact with heparin (18),what remains to be characterized is the binding of DrKIn-I withboth heparin and APC. The binding of DrKIn-I to heparin wasassessed using a heparin-Sepharose column. As expected,DrKIn-I bound to the heparin column with a very high affinity(Fig. 3A). The inhibitor eluted at 0.95 M NaCl, which was threetimes higher than that required for APC elution (19).
FIGURE 1. Purification of Kunitz-type protease inhibitors. A, 20 mg ofDaboia russelii russelii crude venom was dissolved in 0.1 M ammonium acetatebuffer (pH 6.5) and fractionated by gel filtration using FPLC. The fractionscontaining Kunitz-type protease inhibitors (indicated by a horizontal bar)were pooled together and lyophilized. B, subsequent fractionation usingreversed-phase HPLC. A linear gradient of 20 –50% acetonitrile was appliedover a period of 60 min. The protein peaks corresponding to DrKIn-I andDrKIn-II were indicated. C, amino acid sequences of DrKIn-I and DrKIn-II. Non-identical amino acids are denoted in bold letters.
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Next, we investigated the physical interactions betweenDrKIn-I and APC using surface plasmon resonance. DrKIn-Iconcentrations between 0.78 and 6.25 nMwere flowed across anAPC-coated CM5 sensor chip. DrKIn-I bound to immobilizedAPC with a Kd of �2.6 2.3 nM (Fig. 3B). The association rateconstant was determined to be 1.3 0.8 � 107 M1 s1, whichapproached the diffusion limit of 106�108 M1 s1 in aqueoussolution (20, 21), while the dissociation rate constant was foundto be 3.4 2.2 � 102 s1. Interestingly, no binding wasobserved between DrKIn-I and the immobilized protein Czymogen (Fig. 3B, inset).
Determination of the Inhibition Constant of DrKIn-I—Al-though DrKIn-I binds to APC in the absence of heparin, itseffect on APC-mediated FVa degradation was negligible. Theinhibition constant (Ki) of DrKIn-I was therefore determinedonly in the presence of heparin. By fitting the inhibition curvesglobally to Morrison’s competitive tight binding equation,DrKIn-I was found to inhibit APC with a Ki of 53 39 pM (Fig.4). Although the plot of fractional velocity against inhibitorconcentration showed overlapping inhibition curves for all thesubstrate concentrations tested (0.025–0.4 mM) (Fig. 4 inset),addition of a very high substrate concentration (3.3mM) dimin-
FIGURE 2. Inhibition of APC by DrKIn-I and DrKIn-II in the absence and presence of heparin. A, ability of DrKIn-I and DrKIn-II (10 nM each) to inhibit theamidolytic activity of APC (10 nM) was compared using Spectrozyme PCa (0.2 mM) in the absence and presence of 0.1 units/ml heparin. B, dose-response curvesof APC (10 nM) inhibition by DrKIn-I in the absence (E) and presence (F) of 0.1 units/ml heparin. C, to determine the minimum concentration of heparin requiredfor the potentiation APC inhibition, the activity of APC (20 nM) was measured after the addition of DrKIn-I (20 nM) and varying concentrations of heparin (0 –1000mU/ml). D, to assess the ability of heparan sulfate and the minimum length of heparan sulfate required to potentiate DrKIn-I-mediated APC inhibition, theamidolytic activity of APC was determined in the presence of DrKIn-I (20 nM) and different lengths of heparan sulfate chains (10 �g/ml each). E, time course ofFVa inactivation by APC (1 nM) was assessed in the absence (F) or presence (f) of 5 nM DrKIn-I, or in the presence of 5 nM DrKIn-I supplemented with 0.1 units/mlheparin (Œ), as described under “Experimental Procedures.” F, effect of DrKIn-I (0 –250 nM) and/or heparin (0.1 units/ml) on APC-mediated FVa inactivation wasdetermined after 10 min of incubation at 37 °C with APC (5 nM). Results shown are means S.D. of three experiments.
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ished the APC-inhibitory activity of DrKIn-I (data not shown),indicating that the inhibition is truly competitive in nature. Thelack of substrate concentration effect therefore suggests that atlower substrate concentrations that are more experimentallyfeasible, the substrate is unable to effectively compete with theinhibitor.
Selectivity Profile of DrKIn-I—The inhibitory activity ofDrKIn-I, in the presence or absence of heparin, was screenedagainst the classic serine protease trypsin and also against ser-ine proteases in the coagulation and fibrinolytic systems. Apartfrom APC, DrKIn-I at the same molar concentration as theenzyme active site also significantly inhibited the activities oftrypsin (�45% inhibition), FXIa (�40% inhibition), and plas-min (�20% inhibition in the absence of heparin, and �70%inhibition in the presence of heparin) (Fig. 5). Notably, amongall the serine proteases tested, only APC showed 100% inhibi-tion by DrKIn-I in the presence of heparin.To compare the potencies of DrKIn-I against FXIa, plasmin
and APC, the Ki for FXIa and plasmin inhibition were alsodetermined. Using chromogenic substrates, the Ki values forFXIa and plasmin inhibition in the presence of heparin werefound to be 1.33 0.08 nM and 1.56 0.09 nM, respectively(data not shown). These values were at least 25-fold higher thanthat for APC inhibition (�53 pM), supporting our hypothesisthat APC is the preferential target of DrKIn-I.DrKIn-I Nullifies the Effect of APC in Heparin-containing
Plasma—DrKIn-I-mediated APC inhibition in human plasmawas demonstrated using the conventional activated partialthromboplastin time (APTT)-based clotting assay. In theabsence of heparin, APC (40 nM) prolonged the clotting time ofnormal plasma by �7-fold (Fig. 6A). Although DrKIn-I attenu-ated the prolongation, its effect was relatively small. However,when exogenous heparin was added, the clotting time waseffectively shortened (Fig. 6A). To exclude the synergistic effectof antithrombin and heparin cofactor II on heparin-inducedprolongation of clotting time, we also tested theAPC inhibitoryactivity of DrKIn-I in antithrombin/heparin cofactor II-defi-cient plasma. Similar to the results obtained with normalplasma,DrKIn-I restored the clotting time to that of the controllevel only when heparin was added (Fig. 6B).The procoagulant nature of DrKIn-I in the presence of hep-
arin was further confirmed by thrombin generation assay. As
FIGURE 3. Physical interactions of DrKIn-I with heparin and APC. A, 70 �gof DrKIn-I in 0.1 ml equilibrating buffer (20 mM Tris-HCl, pH 8.0) was applied toa 5 ml HiTrap Heparin column and eluted with a 50 ml gradient from 0.0 –1.0M NaCl. The salt concentration corresponding to the elution peak was deter-mined to be 0.95 M. B, binding of DrKIn-I (0.78, 1.56, 2.34, 3.13, 4.68, and 6.25nM) to immobilized APC was assessed in buffer containing 10 mM HEPES, 0.15M NaCl, 3 mM EDTA, and 0.05% P20 at a flow rate of 60 �l/min at 25 °C. Theassociation and dissociation rate constants, determined by global fitting to1:1 Langmuir binding model, were 1.3 0.8 � 107
M1 s1 and 3.4 2.2 �
102 s1, respectively. The thin lines represent the global fit to the responsedata. No binding was observed when DrKIn-I (31.25, 62.5, 125 nM) was flowedacross immobilized PC (inset). Results shown are means S.D. of three exper-iments. Representative binding traces are shown.
FIGURE 4. Kinetic analysis of APC inhibition by DrKIn-I in the presence of0.1 units/ml heparin. Initial velocities of APC (20 nM) were measured in thepresence of increasing concentrations of DrKIn-I (0 – 80 nM) using differentconcentrations of Spectrozyme PCa (F, 0.4 mM; f, 0.2 mM; Œ, 0.1 mM; �, 0.05mM; �, 0.025 mM) as substrate. Solid lines represent best least squares fit toMorrison’s competitive tight binding equation, which gave an inhibition con-stant (Ki) of 53 39 pM. Inset shows the secondary plot of fractional velocity(Vi/V0) versus DrKIn-I concentration, where Vi is the initial velocity in the pres-ence of DrKIn-I and V0 is the initial velocity in the absence of DrKIn-I.
FIGURE 5. Selectivity profile of DrKIn-I. The inhibitory activity of DrKIn-I, inthe presence or absence of heparin (0.1 units/ml), was screened against tryp-sin and also against serine proteases in the coagulation and fibrinolytic sys-tems. In each of these experiments, the molar ratio of protease active site toinhibitor was 1:1. The final concentrations of these proteases and theirrespective substrates were as follows: FXIIa (20 nM)/S-2302 (0.2 mM), FXIa (2.5nM)/S-2366 (0.2 mM), FXa (10 nM)/S-2222 (1.3 mM), FIXa (200 nM)/SpectrozymeFIXa (1.3 mM), FVIIa (100 nM)/S-2288 (1.3 mM), thrombin (5 nM)/T-1637 (0.2mM), kallikrein (5 nM)/S-2302 (0.2 mM), plasmin (20 nM)/S-2251 (0.2 mM), tPA(80 nM)/Spectrozyme tPA (0.2 mM), uPA (100 nM)/S-2288 (1.3 mM), APC (10nM)/Spectrozyme PCa (0.2 mM), and trypsin (5 nM)/S-2222 (0.2 mM). Resultsshown are means S.D. of at least three experiments.
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expected, although DrKIn-I itself did not alter the thrombingeneration profile, it completely restored the generation ofthrombin in APC-containing plasma (Fig. 6C). Combined,these results suggest that DrKIn-I is able to specifically targetAPC in plasma, an environment where dozens of other serineproteases exist.DrKIn-I Aggravates Hypofibrinogenemia and Coagulation
Induced by RVV-X in Mice—Patients severely envenomed byRVV often develop bleeding disorders due to the consumptionof coagulation factors, the most prominent of which is fibrino-gen. We therefore speculated that DrKIn-I, being a potentinhibitor of APC, should potentiate the procoagulant effect ofRVV-X. In line with our hypothesis, DrKIn-I did not decreasethe plasma fibrinogen level when the stimulus (RVV-X) wasexcluded (Fig. 7A). However, DrKIn-I significantly and dose-dependently decreased the level of fibrinogen in mice whenRVV-X (0.02 �g/g) was co-injected with the inhibitor (Fig. 7A).Furthermore, co-injection of RVV-X with higher doses ofDrKIn-I (� 0.08�g/g) led to immediate death (data not shown).
Because renal failure is the most common complication inenvenomed patients (22), a histopathological examination ofthe kidneyswas conducted, and renal glomeruli, the basic bloodfiltration units which determine the overall function of the kid-
FIGURE 6. DrKIn-I nullifies the effect of APC in heparin-containingplasma. Purified APC (40 nM) was added to normal plasma (A) or antithrom-bin III and heparin cofactor II-deficient plasma (B) containing varying concen-trations of DrKIn-I (40, 80, 160 nM) in the presence or absence of heparin (0.1units/ml). After incubation for 1 min at 37 °C, plasma APTT was measured.Clotting times were compared with the control experiments performed in theabsence of DrKIn-I and heparin. Results shown are means S.D. of threeexperiments. C, effect of 40 nM DrKIn-I and/or 40 nM APC on thrombin gener-ation was assessed in the presence of 0.1 units/ml heparin as described in“Experimental Procedures.” Representative thrombin generation curves areshown.
FIGURE 7. DrKIn-I potentiates the procoagulant activity of RVV-X. A, ICRmice were injected intravenously with the indicated concentrations of RVV-Xand/or DrKIn-I. Plasma fibrinogen concentrations were determined 3 h afterinjection. Each error bar represents S.E. about the mean (n � 4). B, histologicalsections of hematoxylin-and-eosin stained kidneys of mice administered withnormal saline (i), 0.04 �g/g DrKIn-I (ii), 0.005 �g/g RVV-X (iii), or 0.04 �g/gDrKIn-I plus 0.005 �g/g RVV-X (iv). Images were obtained using the Scan-Scope CS System. Original magnification � 400.
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neys, were examined. In our experiments, injection of DrKIn-I(0.04 �g/g) or RVV-X (0.005 �g/g) alone did not result in thedeposition of fibrin. However, when administered together,fibrin thrombi were observed in at least 30% of the glomerularcapillaries (Fig. 7B). These results agreed well with the fibrino-gen determination experiments, confirming DrKIn-I as a newprocoagulant component in RVV that synergizes with RVV-Xin promoting coagulations in Russell’s viper bite patients.
DISCUSSION
Russell’s vipers are responsible formost of the snakebite inci-dents in many parts of South and Southeast Asia where themajority of the patients often suffer frommild to severe coagu-lopathies, depending on the extent of envenomation (2). It is,therefore, important to fully understand the causes of thesecoagulopathies so that effective treatments could be given. Inthis study, we have demonstrated that besides having RVV-Xand RVV-V (proteases that activate FX and FV, respectively) asthe main procoagulating components in the venom, RVV alsocontains DrKIn-I, a heparin-dependent tight binding inhibitorof APC that effectively potentiates coagulation in RVV-X-stim-ulated mice.DrKIn-I, amember of the snakeKunitz/BPTI family, consists
of 66 amino acids, with three conserved disulfide linkages tostabilize the overall structure (12). DrKIn-I is unique among allthe other Kunitz-type protease inhibitors in that it is extremelybasic (predicted pI � 9.6), with two putative heparin-bindingmotifs in its C-terminal region (49TRKKCRQ55 and60PRKGRP65) (23, 24). The presence of these -XBBBXBX- and-XBBXBX- regions (whereX represents uncharged amino acidsand B represents basic amino acids) probably contributes to thehigh affinity of DrKIn-I toward heparin and allows heparin topotentiate the inhibition of APC by DrKIn-I. This is supportedby our findings that although DrKIn-I and DrKIn-II are highlyidentical, with a percent identity of 71%, DrKIn-II, which lacksthe heparin-binding motifs, showed no affinity toward heparincolumn (data not shown). Consequently, heparin was unable toenhance the inhibition of APC by DrKIn-II.Although the exact mechanism for DrKIn-I-mediated APC
inhibition in the presence of heparin has not been elucidated,the absence of a bell-shaped response curve in the plot of APCactivity versus heparin concentration (Fig. 2C) suggests thatDrKIn-I does not employ the typical template mechanismwhereby both the protease and the inhibitor bind to a heparinmolecule in proximity to each other (25). Furthermore,DrKIn-Ionly requires a length of 6 saccharide units to enhance the inhi-bition of APC by �80% (Fig. 2D). This is in contrast with thetypical template mechanism which requires heparin moleculesto be at least 18 saccharide units long (26). The ability of hepa-ran sulfate hexamers to act as cofactors for APC inhibition alsosuggests a non-template based mechanism, since 6 saccharideunits would be insufficient to bridge both the protease and theinhibitor.The reason for the presence of APC inhibitors in the D. rus-
selii species is not hard to understand, as it is known thatuncomplexed FVa (those that are not bound to FXa or pro-thrombin) are prone to enzymatic degradation by APC (27, 28).It is, therefore, reasonable from the evolutionary point of view
for RVV tonot only activate FV, but also to protect the activatedcofactor (FVa) from APC-mediated inactivation. The presenceof DrKIn-I ensures that there is always a constant supply of FVafor the formation of prothrombinase complexes with FXa.Although APC inactivates both FVa and FVIIIa, we have nottested whether DrKIn-I protects FVIIIa from degradation. Thisis because RVV induces coagulations primarily through thecommon pathway. In addition, factor IX (the protease thatcomplexes with FVIIIa) is rarely activated or consumed inenvenomed patients (3), suggesting that relative to FVa, FVIIIaplays only a minor role in RVV-induced coagulopathies seen inthese patients.Over the years, several plasma APC inhibitors belonging to
the serpin family have been found, including protein C inhibi-tor and �-1-antitrypsin (29, 30). In the absence of heparin, bothserpins inhibit APC slowly, with second order rate constants of2.5 � 103 M1 s1 and 1.0 � 10 M1 s1, respectively. DrKIn-I,however, is the first APC inhibitor discovered that belongs tothe Kunitz/BPTI family. It differs from the serpin-type APCinhibitors in that it is not a slow-binding inhibitor. Using thesynthetic substrate, DrKIn-I inhibited the amidolytic activity ofAPC as soon as it was added to the enzyme. While heparinenhances the second order rate constant of protein C inhibitorby 30–230-fold (29, 31), the binding betweenAPC andDrKIn-Iis intrinsically fast, with an association rate constant of �1.3 �107 M1 s1. These differences suggest that DrKIn-I is the onlyinhibitor discovered that exhibits fast-binding kinetics withAPC. The low Ki of �53 39 pM suggests that DrKIn-I, in thepresence of heparin, may be the most potent APC inhibitorfound to date.The selectivity profile of DrKIn-I suggests that besides APC,
the inhibitor may also target FXIa and plasmin. However ourkinetic analyses indicate that theKi forAPC inhibition is at least25-fold lower than that for FXIa and plasmin inhibition.Althoughhigh concentrations ofDrKIn-Iwould invariably pro-long the APTT clotting time (probably due to the inhibition ofFXIa), our plasma experiments indicate that at concentrationsbelow 160 nM, the effect of DrKIn-I on APTT was negligible(Fig. 6A). Furthermore, we have also performed euglobulin clotlysis assays on both DrKIn-I and aprotinin (a well known plas-min inhibitor) (32) to assess the plasmin inhibitory activity ofDrKIn-I. Whereas 20 nM aprotinin prolonged the euglobulinclot lysis time by �9 h, 20 nM of DrKIn-I failed to prolong theclot lysis time, either in the presence or absence of heparin (datanot shown). At a concentration of 100 nM, aprotinin prolongedthe clot lysis time by more than 16 h, while DrKIn-I only pro-longed the clot lysis time by approximately an hour. Combined,these data support our hypothesis that among all the serineproteases tested, APC is the preferential target of DrKIn-I.In our experiments, we have demonstrated that both heparin
and heparan sulfates can act as cofactors for APC inhibition. Itis, however, uncertain which of the two plays a more importantrole. Since heparan sulfates are abundant on the surface ofendothelial cells (17), it is speculated that heparan sulfates mayplay a more important role compared with heparin. Althoughthe endogenous heparin level in plasma has been reported to bein the range of 1.0�2.4 �g/ml or 0.1�0.2 units/ml (33, 34),these heparin molecules are already bound with other plasma
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proteins and are therefore unavailable to act as cofactors forAPC inhibition (33). The need to add heparin to our plasmaexperiments (Fig. 6, A and B) also indicates that most of theseheparin molecules are already in a complexed state with otherproteins. However, it has also been documented that mast cellscan degranulate and release heparinwhen stimulatedwith RVV(35), and that these heparin molecules can enhance the activityof RVV-V, causing an increase in the activation of FV by nearly4-fold (36). Furthermore, endogenous heparin levels have beenshown to increase in response to hemorrhagic shock (37).Metzet al. also recently showed that mast cells do degranulate andrelease carboxypeptidase A in response to snake and honeybeevenom exposure (38). These references therefore suggest thatbesides heparan sulfate, heparin molecules secreted by mastcells also play an important role in the pathogenesis of Russell’sviper envenomation.The importance of APC in maintaining the patency of blood
vessels has been well documented (39). It has been suggestedthat APC is particularly important in regulating coagulations inthemicrocirculation, as small blood vessels and capillaries havehigher concentrations of thrombomodulin compared withlarger vessels (39). Homozygous PC deficiency often results inDIC and microvascular thrombosis (purpura fulminans) innewborn infants (40). Furthermore, in a primate sepsis model,it was found that PC depletion resulted in complete fibrinogenconsumption, organ failure, and death when the animals werestimulated with an otherwise nonlethal dose of E. coli (41). Inview of the above references, it is not surprising that DrKIn-Ican induce fibrinogen consumption and the formation of fibrinthrombi in the glomerular capillaries when co-administeredwith low doses of RVV-X (Fig. 7). Consistent with our findings,Rapaport et al. reported that administration of RVV-X aloneinto normal rabbits did not result in significant fibrinogendepletion (7). Although he demonstrated that fibrinogen con-sumption could only be achieved by RVV-X in antithrombin-immunodepleted rabbits, antithrombin was found to be theonly anticoagulant thatwaswithin the normal range inRussell’sviper bite patients (3). Our results therefore help to explain whyseverely envenomed patients often develop bleeding complica-tions and suggest that APC, and not antithrombin, is the majoranticoagulant that is down-regulated in these patients.Histopathologically, fibrin thrombi were observed in the glo-
merular capillaries of mice that received both DrKIn-I andRVV-X. The presence of these glomerular thrombi corrobo-rated with the depletion of fibrinogen, suggesting that thesemice might be experiencing DIC-like symptoms (42). SinceAPC deficiency is associated with microvascular thrombosisand that DIC is always manifested with renal dysfunction (42),it is speculated that the occlusion of glomerular capillariesinduced byDrKIn-I-mediated APC inhibitionmay be the causeof acute renal failure. Although it has been documented thatRVV-induced renal failure may be due to the direct nephro-toxic components in the venom (43), other studies have shownthat intravascular clotting andhemolysismay be themain causeof renal failures in these patients (44, 45). In support of ourhypothesis, it was found that none of the envenomed patientswho did not develop DIC suffered from renal failures (46). Fur-thermore, Gupta et al. demonstrated using a ratmodel of endo-
toxemia that APC could significantly improve the renal bloodflow in rats with acute renal failure (47). These evidences sug-gest that DrKIn-I, through the inhibition of APC, may intensifythe kidney damage induced by the procoagulating enzymes inRVV.The present study stresses on the importance of APC in
RVV-induced coagulopathies because this physiological anti-coagulant is inhibited by DrKIn-I from the same venom withlethal consequences. Fromour results, we can imply that if APCis replenished in Russell’s viper bite patients, the concentra-tions of RVV-X and RVV-V needed for the induction of con-sumptive coagulopathy would be much higher. Since severelyenvenomed patients develop bleeding disorders (fibrinogenconsumption andmicrovascular occlusionwith fibrin thrombi)similar to that seen in patients with severe sepsis and homozy-gous PC deficiency (40, 41), administration of PC concentrate(Ceprotin) or recombinant APC (Drotrecogin �-activated orXigris) before the consumption of coagulation factors shouldbe able to pacify, if not prevent, the development of DIC orDIC-like symptoms in envenomed patients (48, 49). This, inaddition to treatments with antivenom, should reduce fibrindepositions in the glomerular capillaries, and should subse-quently ameliorate the damage done to the kidneys. The dis-covery of a fast and tight binding inhibitor of APC thereforeprovides new insights into the pathogenesis of RVV-inducedcoagulopathies, which may form a basis for the administrationof APC or PC concentrates in Russell’s viper bite patients.
Acknowledgments—We thank Technology Commons, College of LifeScience and the School of Veterinary Medicine, National TaiwanUniversity for giving us access to Biacore T200 and for the preparationand staining of tissue sections. We also thank Ying-Ming Wang andShing Tseng for the cloning of Kunitz inhibitors and for the acquire-ment of pathological data, respectively. In addition, wewould also liketo thank Dr. Sheng-Wei Lin for his technical assistance in bindingkinetics experiments.
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