PARTIAL PURIFICATION OF HUMANPROTHROMBINAND PRO-CONVERTIN: THEIR CHARACTERISTICSAND INTERACTION'

By JESSICA H. LEWIS AND JOHN H. FERGUSON

(From the Department of Physiology, University of North Carolina, Chapel Hill, N. C.)

(Submitted for publication April 6, 1953; accepted May 22, 1953)

Independently and almost simultaneously anumber of investigators recognized the fact that atleast two plasma factors are important in the con-version of prothrombin to thrombin in the presenceof tissue thromboplastin and calcium. Variousnames have been applied to these factors and, ata recent Macy Conference on blood clotting (1),most of the investigators agreed that they couldbe grouped into two entities as follows:

Relatively labile factor

Labile factor (Quick [2, 3])Factor V, Proaccelerin (Owren [4, 5])Plasma Ac globulin (Ware, Guest, and Seegers [6, 7])Prothrombin accelerator (Fantl and Nance [8, 9])Plasmatic co-factor of thromboplastin (Honorato, Rojas,

and Ivanovic [10, 11])Plasma prothrombin conversion factor (Stefanini [12])

Without prejudice and for the sake of simplicity,we have chosen to use the terms proposed byOwren, namely, proaccelerin and proconvertin forthe plasma factors, and accelerin and convertinfor the active (serum) factors.

The investigations presented in this paper dealwith a method for the partial separation of pro-thrombin and proconvertin from human plasmaand the study of their interactions.

MATERIALS

Imidazole buffer: pH 7.3 (26).Accelerin (Accelerator Globulin, AcG): Barium car-

bonate treated beef serum prepared according to themethod of Ware and Seegers (29). For the purposes ofthe present study this crude material served as a satis-factofy source of accelerin, free from prothrombin andproconvertin, and retained full potency on storage at - 20°C. for one year or longer. In additional experiments, whichwill be reported separately, we have employed highlypurified human proaccelerin. This material was not usedin the present experiments as, due to its marked storage

1 This investigation was supported in part by a researchgrant from the Division of Research Grants of the Na-tional Institutes of Health, U. S. Public Health Service.

lability, it must be prepared specially for each individualexperiment, and because we wished to study the reactionsof proconvertin and prothrombin in the presence of ac-celerin rather than proaccelerin.

Thromboplastin: Soluplastin, obtained through thecourtesy of Dr. E. W. Blanchard, Schieffelin and Com-pany. Dog lung thromboplastin, freshly prepared fractionIV + VI of perfused dog lung, previously shown to havehigh thromboplastic potency as well as fibrinolysokinaseactivity (30).

Relatively stable factor

SPCAprecursor (Alexander et al. [1, 13-20])Co-factor V, Proconvertin (Owren [1, 21, 22])Factor VII (Koller, Loeliger, and Duckert [23])Prothrombin accelerator (MacMillan [24])Co-thromboplastin (Mann and Hurn [25, 26])

Stable prothrombin conversion factor (Owen, Bollman,and Magath [27, 28])

Fibrinogen: Humanplasma fraction I (Cohn) preparedas a 0.7 per cent solution in 0.85 per cent sodium chloride(pH 7.3) and adsorbed with 50 mg. barium sulfate per ml.Although this material contains a high concentration ofcitrate, treatment with barium sulfate apparently didimprove its stability. The fraction I was supplied throughthe courtesy of the American National Red Cross andSquibb and Company.

METHODS

Plasma fractionation: Many fractionation procedureswere attempted in the separation of prothrombin and pro-convertin. Although the method outlined below does notsupply absolutely pure fractions, it does yield sufficientseparation of prothrombin and proconvertin to allow studyof the properties of these materials. All procedures werecarried out at 40 C. unless otherwise stated. Various at-tempts to further purify the two fractions were made, butrepeated reprecipitation and readsorption and elution frombarium sulfate did not lead to preparations of significantlygreater purity.

Thrombin activation mixtures: All activation mixtures(except controls in Table I) contained 0.5 ml. imidazolebuffer, 0.5 ml. thromboplastin (diluted 1 to 5 with saline),0.2 ml. accelerin (diluted 1 to 16 with saline) and 0.5 ml.0.15 MCaCl2. Prothrombin and proconvertin were addedin 0.1 ml. amounts unless otherwise stated, and in allcases, sufficient 0.85 per cent sodium chloride was added to

915

JESSICA H. LEWIS AND JOHN H. FERGUSON

OXALATEDHUMANPLASMA(100 ml.)

Add 10 Gm. BaSO4Mix 10 min. at 250 C.Centrifuge 15 min. at 5000 RPM

Ba;OfWash 3x salineWash lx dist. waterElute with 10 ml. 0.2M

sodium citrate (250 C.)Repeat elutionCombine eluates

ADSORBEDPLASMA(Reserve for preparation offibrinogen, AHG, Proaccelerin)

|BaSO4 ELUATE

DialyspH to

Centrif,

PRECIPITATEWash with 0.001 M

phosphate buffer, pH 5.5Dissolve 10 ml. saline, pH 7.3

PROTHROMBIN1

e vs. 0.0005 Mphosphate buffer pH. 7.35.5 with 1%acetic acidfuge 15 min. at 5000 RPM

SUPERATANTConcentrate to 10 ml.*Adjust salt to 0.85%pH to 7.3

PROCONVERTIN

* Concentration may be accomplished by evaporation from a dialysis bag, evaporation in vacuo, dialysis againstconcentrated PVP, or lyophilization. The first method was usually employed.

bring the total volume to 5 ml. Employing the BaSO4Eluate (previously dialyzed to remove sodium citrate),which contains both prothrombin and proconvertin, theabove concentrations of CaCl2, thromboplastin, and ac-

celerin were found to be optimal.Activation and clotting were carried out at 25° C.

Experiments showed that a greater yield of thrombin andmore reproducible results were obtained when activationwas carried out in siliconed tubes, which prevent theadsorption of the formed thrombin onto glass. At inter-vals, 0.2 ml. samples of the activation mixtures, in siliconedtubes, were removed with siliconed pipettes and added to0.2 ml. fibrinogen, in glass tubes, and the clotting timesobtained. The concentration of each lot of prothrombinwas adjusted so that approximately a 10 second clottingtime was obtained after complete activation under optimalconditions.

Thrombin "unitage": For simplification in interpreta-tion of the results we have converted clotting times to"units." The most active clotting mixture, after fullactivation, was arbitrarily chosen as containing 100thrombin "units" (per 0.2 ml.). Precise dilutions of thismixture were prepared in siliconed tubes, employing, as

diluting fluid, the same materials as in the activation mix-ture but without the prothrombin or proconvertin. Clot-ting times for each dilution were obtained in triplicate,and a curve, relating concentration of the thrombin mix-ture and clotting time, constructed. By reference to thiscurve, clotting times could then be converted to thesearbitrary "units."

The results presented below are those of individual ex-

periments, performed in duplicate, each of which is il-lustrative of a number of similar experiments carried outon different lots of prothrombin and proconvertin.

TABLE I

Controls (run 4)*

Thrombin IncubationSoluplastin AcG yield time (250 C.)

Mixtures (dil. 1-5) (dii. 1-16) Prothrombin Proconvertin "units" minutes

A 0.5 0.2 0.1 0.3 100.0 3B 0.5 0.2 0.1 95.0 10C 0.5 0.2 0.3 4.0 3D 0.5 0.1 0.3 5.5 60E 0.2 0.1 0.3 1.0 60

* All activation mixtures contained 0.5 ml. 0.15 MCaC12, 0.5 ml. imidazole buffer, and the reagents listed abovein a total volume of 5 ml.

Ba&O4Discard

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HUMANPROTHROMBINAND PROCONVERTIN

RESULTS

Identification and purity of reagents: In orderto establish the purity of our reagents a numberof control tests were carried out.

Five ml. mixtures containing 0.5 ml. imidazoleand the other ingredients (calcium, thrombo-plastin, accelerin, prothrombin, proconvertin)either singly or in any of the possible combinationswere tested for thrombin formation at intervalsduring a one hour incubation period. Appreciablethrombin was formed only in those mixturesshown in Table I, which is illustrative of a typicalrun. The complete mixture (A) activated rapidly,and this mixture was designated as yielding 100"units" of thrombin. In the absence of procon-vertin (B), 95 "units" of thrombin were formed,but the time of complete activation was consider-ably slower. Whenproconvertin was activated inthe absence- of added prothrombin (C), 4.0 "units"of thrombin were formed. If accelerin or thrombo-plastin were omitted (D,E) there was only slightactivation during a one hour period.

It seems probable that a pure prothrombinpreparation would not activate in the absence ofproconvertin. In one experiment, the possibilitythat proconvertin might be present as a contami-nant of thromboplastin was explored. Variousthromboplastin preparations (rabbit brain, dogbrain, beef lung, dog lung, cephalin) were substi-tuted for soluplastin, but in each case some activa-tion of the prothrombin occurred.

All of our prothrombin preparations have ac-tivated in the absence of added proconvertin, yield-

90

60-

z 50

0

It-

r0ox4Eut

S 4 6 6 7 lo- MINUTES AT 25° C.

FIG. 1. THROMBINFORMATIONFROMHUMANBaSO.ELUATE, PROTHROMBIN, AND PROCONVERTIN

70)z Z60 1

z> zI0-

20

>61i s X b F t § § l oMINUTES AT 25 Ca

FIG. 2. EFFECT OF VARYING PROTHROMBINCONCEN-ATION

The prothrombin solution was diluted 1 to 10 and em-ployed in 1.0, 0.75, 0.5 and 0.25 ml. amounts. All testscontained 0.2 ml. proconvertin.

ing from 70 to 100 per cent of the expected quan-tity of thrombin. Wehave roughly estimated thequantity of contaminating proconvertin from theincubation time necessary to reach maximal activa-tion. This has varied from 10 to 30 minutes withdifferent lots of prothrombin. The quantity ofprothrombin contaminating a proconvertin prep-aration can be readily assayed, as it is rapidlyconverted to thrombin. In different fractionationruns the proconvertin preparations have formedfrom 0.5 to 6 "units" of thrombin.

In Figure 1, the results of activation of theBaSO4 Eluate and its two fractions, "prothrom-bin" and "proconvertin," are compared. TheBaSO, Eluate activated rapidly, while the pro-thrombin activated much more slowly, but yieldedalmost as much thrombin. The proconvertin alsoactivated rapidly, but the thrombin yield was low.When the prothrombin and proconvertin fractionswere mixed, the activation curve closely resembledthat of the initial BaSO4 Eluate. The most rapidactivation was observed in a test in which con-vertin, formed by pre-incubation of the procon-vertin with calcium and thromboplastin, was addedto the prothrombin (see below).

Effects of varying prothrombin concentration:In this experiment (Figure 2) prothrombin invarying amounts was activated in the presence ofa standard concentration of proconvertin. Thethrombin yields, allowing for that formed from theproconvertin, were found to be proportional to the

917

JESSICA H. LEWIS AND JOHN H. FERGUSON

70-

z 0

2400~~~~~~

MINUTES AT 25 C.

FIG. 3. EFFECTS OF VARYING PROCONVERTINCONCE:N-TRATION

added prothrombin concentrations. The procon-vertin alone (curve 0) yielded 6 "units" of throm-bin. If these 6 "units" were subtracted from theobserved thrombin "units," they became propor-tional to the initial prothrombin concentrations:84, 64, 44, 21:: :1.0, 0.75, 0.5, 0.25.

The same concentrations of prothrombin wereactivated in the absence of added proconvertin(not illustrated). The activation rates were veryslow, the maximal thrombin yield (between 70 to80 per cent of that expected) being obtained after15 to 30) minutes incubation.

Effects of varying proconvertin concentration:In this experiment the prothrombin concentrationwas maintained constant, and the proconvertinconcentration varied. The results (Figure 3)clearly show that the rate of activation of pro-thrombin (in the presence of standard concentra-tions of calcium, tissue thromboplastin, and ac-celerin) is dependent upon the concentration ofproconvertin. The small differences in finalthrombin yields may be accouinted for by the vary-ing amounts of prothrombin present as a contami-nant of the proconvertin.

Activation of proconvertin: In even the highestconcentration of proconbertin employed in the ex-periment illustrated in Figure 3, there was aninitial lag in thrombin formation during the firstminute of incubation. Pre-incubation of the proconvertin with thromboplastin and calcium, as firstsuggested by Mann (25) in his studies on cothromboplastin, eliminates this lag (see Figure 1).Experiments not illustrated showed that the adc-dition of accelerin during the pre-incubation was

not essential to this conversion of proconvertin toconvertin. Pre-incubation of the proconvertinwith either calcium or thromboplastin alone didnot cause the formation of convertin. Additionalexperiments also showed that the addition ofthrombin to the usual proconvertin-prothrombinmixture did not eliminate the initial lag in throm-bin formation.

Rate of activation of proconvertin: In a seriesof identical mixtures, the proconvertin was in-cubated with calcium and thromboplastin for vary-ing lengths of time before addition to the pro-thrombin and accelerin. The results are illustratedin Figure 4. After 10 minutes pre-incubation ofproconvertin, the initial lag in thrombin formationhad been eliminated. Longer pre-incubation didnot increase the convertin activity.

Effects of varying convertin concentration: Inthis experiment, various amounts of proconvertinwere pre-incubated for 10 minutes with calciumand thromboplastin before addition to prothrom-bin and accelerin. The results, illustrated in Fig-ure 5, show that the rapidity of activation of pro-thrombin was dependent upon the convertinconcentration.

Action of thromboplastin: The experiments pre-sented above showed that calcium and thrombo-plastin convert proconvertin to convertin. Weattempted to determine whether calcium andthromboplastin were also important in the con-version of prothrombin to thrombin in the presenceof convertin and accelerin. In order to do this,proconvertin was incubated with calcium andthromboplastin and then attempts were made toremove these activators. Although a number of

370

60

z 0

I0-

FIG. 4. ACTIVATION OF PROCONVERTIN

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HUMANPROTHROMBINAND PROCONVERTIN

procedures for the removal of calcium (passagethrough the ion-exchange resin, Dowex 50, oxala-tion, citration, and dialysis) were tried, no con-vertin activity could be demonstrated after re-moval of the calcium.

On the other hand, substituting freshly prepareddog lung thromboplastin for soluplastin, it waspossible to show full convertin activity after re-moval of most of the thromboplastin by high speedcentrifugation. The results are shown in Figure 6.In the first test (A), calcium, dog lung thrombo-plastin, and proconvertin were incubated for 10minutes and then added to prothrombin and ac-celerin. In the next test (B) proconvertin wasagain incubated with thromboplastin and calcium,and the mixture was centrifuged at 20,000 RPMfor 20 minutes before adding to the prothrombinand accelerin. In test (C) the same procedureof incubation and centrifugation was followed asfor test (B), but fresh thromboplastin was addedalong with the prothrombin and accelerin in thefinal activation mixture. In the last test (D), thecalcium and thromboplastin were incubated, with-out proconvertin, and centrifuged before adding tothe prothrombin, proconvertin, and accelerin.This test shows that a trace of thromboplastin re-mained after the centrifugation, as some thrombinwas formed. In the absence of thromboplastin, noactivation was observed. The similarity betweencurves (A) and (C) and their striking differencefrom curve (B) suggest that convertin is presentafter the removal of thromboplastin, but that it canfully activate the prothrombin only in the presence

80

I0-

60450-x

FIG. 5. EFFECTS OF VARYING CONVERTIN CONCENTRA-TRATION

a

-7-

Z 60 Hz250

240M 0

20

106

MINUTES AT 25 C

FIG. 6. ACrION OF THROMBOPLASTIN(SEz TEXT)

of adequate thromboplastin, as well as accelerinand calcium.

Stability of prothrombin and proconvertin: Inorder to test the stability of prothrombin and pro-convertin, samples were heated to various temper-atures, while control samples were kept at 40 C.In Table II the residual activity, after treatment, isrecorded as percentage of that in the controlsamples. Proconvertin activity was estimated bycomparison to a series of activation curves inwhich the concentration of the control proconvertinwas varied. The results suggest that both frac-tions are relatively heat labile.

DISCUSSION

These investigations show that human pro-thrombin and proconvertin can be partially sepa-rated from oxalated blood plasma by a simplefractionation procedure. Koller, Loeliger, andDuckert (23) and Owren (1, 22) have accom-plished a similar separation of these factors frombovine blood by using a differential adsorptionmethod. However, they state that this technicdoes not work on human blood. The excellentstudies of Alexander and his co-workers (1, 13-20) have shown that human serum contains, inaddition to accelerin, another factor, which theyhave called SPCA. Apparently SPCA is verysimilar to proconvertin and may prove to be amixture of proconvertin and convertin. Ourstudies confirm many of the findings of theseworkers.

Activation of our human plasma fractions in thepresence of standard concentrations of calcium,

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JESSICA H. LEWIS AND JOHN H. FERGUSON

TABLE II

Stability of prothrombin and proconvertin

Residual activity

Temperature Prothrombin Proconvertin

37° C. for 4 hours 82 100S6° C. for 5 mins. 30 3465°C.for3mins. 14 28

thromboplastin, and accelerin gave final thrombinyields which were quantitatively dependent uponthe initial concentration of prothrombin, but notof proconvertin. On the other hand, rate ofthrombin formation was dependent upon procon-vertin concentration and, in the presence of afixed amount of prothrombin, the quantity ofthrombin measured in early stages of activationwas almost directly proportional to the proconver-tin concentration. This strongly suggests that theordinary one-stage prothrombin time (Quick test),in the presence of normal proaccelerin, largely re-flects proconvertin, rather than prothrombin con-centration.

Our findings that calcium and thromboplastin(but not accelerin) activate proconvertin to con-vertin extend the data of Mann and Hurn (25,26), Landwehr and Alexander (18), and Owren(1), which previously pointed to some non-prothrombin plasma factor being activated bycalcium and thromboplastin. In our cited isolatedsystems, complete activation of proconvertin bycalcium and thromboplastin required about tenminutes, but other experiments, not illustrated,indicate that the rate of convertin formation isvery dependent upon the potency of the thrombo-plastin. This observation might explain the factthat fractions, which we have prepared from spon-taneously clotted serum, have contained primarilyproconvertin.

During spontaneous plasma coagulation, throm-bin formation is very slow during the first fewminutes. This "lag phase" can be partially elimi-nated by introducing tissue thromboplastin, whichby-passes the physiological action of a number ofplasma substances and platelets. In our isolatedsystem, we are able to reduce still further this"lag phase" by employing accelerin instead of pro-accelerin. Complete elimination of the "lag phase"is accomplished by substituting convertin for pro-convertin.

Additional experiments showed that convertinwas unable to form thrombin from prothrombinunless calcium, thromboplastin, and accelerin werealso present.

CONCLUSIONS

1. The final thrombin yield, in the presence ofstandard amounts of calcium, thromboplastin, ac-celerin, and proconvertin, is dependent upon theinitial prothrombin concentration.

2. The rate of thrombin formation, in the pres-ence of standard amounts of calcium, thrombo-plastin, accelerin, and prothrombin, is dependentupon the proconvertin concentration.

3. Proconvertin is activated to convertin bycalcium and thromboplastin. The presence of ac-celerin is not necessary.

4. The "lag phase" in thrombin formition in amixture containing prothrombin, thromboplastin,calcium, accelerin, and proconvertin can be elimi-nated by pre-incubation of the proconvertin, cal-cium, and thromboplastin.

REFERENCES

1. Blood Clotting and Allied Problems, Transactionsof Fifth Conference, The Josiah Macy, Jr. Founda-tion, Joseph E. Flynn, Ed., New York, 1952.

2. Quick, A. J., On the constitution of prothrombin.Am. J. Physiol., 1943, 140, 212.

3. Quick, A. J., Components of the prothrombin complex.Am. J. Physiol., 1947, 151, 63.

4. Owren, P. A., Nye undersokelser over blodets koagu-lasjon. Det, Norske Videnskapsakademi I Oslo.Arbok., 1944, p. 21.

5. Owren, P. A., The coagulation of the blood; investi-gations on a new clotting factor. Acta med.Scandinav., supp., 1947, 194, 1.

6. Ware, A. G., Guest, M. M., and Seegers, W. H., Afactor in plasma which accelerates the activationof prothrombin. J. Biol. Chem., 1947, 169, 231.

7. Ware, A. G., and Seegers, W. H., Plasma acceleratorglobulin; partial purification, quantitative determi-nation and properties. J. Biol. Chem., 1948, 172,699.

8. Fantl, P., and Nance, M., Acceleration of thrombinformation by a plasma component. Nature, 1946,158, 708.

9. Fantl, P., and Nance, M., The physiological activa-tion of prothrombin. M. J. Australia, 1948, 1, 128.

10. Honorato, R., The plasmatic cofactor of thrombo-plastin; its adsorption, with prothrombin and fibrin-ogen, by alumina and tricalcium phosphate gels.Am. J. Physiol, 1947, 150, 381.

11. Honorato, R., Rojas, C., and Ivanovic, N., Destruc-

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HUMANPROTHROMBINAND PROCONVERTIN

cion del cofactor plasmatico de la tromboplastina.Consumo de este factor en la coagulation sanguinea.Bol. Soc. biol. Santiago, Chile, 1949, 7, 35.

12. Stefanini, M., Autocatalytic formation of thrombin inblood coagulation. Acta med. Scandinav., 1951,140, 290.

13. Alexander, B., De Vries, A., and Goldstein, R., Aprothrombin activator in serum, and its significancein certain hemorrhagic diseases. J. Clin. Invest.,1948, 27, 523.

14. Alexander, B., De Vries, A., Goldstein, R., and Land-wehr, G., A prothrombin conversion accelerator inserum. Science, 1949, 109, 545.

15. De Vries, A., Alexander, B., and Goldstein, R., Afactor in serum which accelerates the conversion ofprothrombin to thrombin. I. Its determination andsome physiologic and biochemical properties.Blood, 1949, 4, 247.

16. Alexander, B., De Vries, A., and Goldstein, R., Afactor in serum which accelerates the conversionof. prothrombin to thrombin. II. Its evolution withspecial reference to the influence of conditions whichaffect blood coagulation. Blood, 1949, 4, 739.

17. Alexander, B., Goldstein, R., and Landwehr, G., Theprothrombin conversion accelerator of serum(SPCA): its partial purification and its propertiescompared with serum Ac-globulin. J. Clin. Invest.,1950, 29, 881.

18. Landwehr, G., and Alexander, B., Interaction ofSPCA with platelets or thromboplastin in pro-thrombin conversion. Federation Proc., 1951, 10,212.

19. Alexander, B., and Landwehr, G., Stability of humanplasma prothrombin fractions. Federation Proc.,1952, 11, 180.

20. Goldstein, R., Le Bolloch, A. G., and Alexander, B.,SPCA: An essential factor for prothrombin con-version. Federation Proc., 1953, 12, 209.

21. Owren, P. A., The prothrombin activating complexand its clinical significance. Proc. 3rd Internat.Cong., Internat. Soc. Hemat., New York, Gruneand Stratton, 1951, p. 379.

22. Owren, P. A., Proconvertin, the new clotting factor.Scandinav. J. Clin. & Lab. Invest., 1951, 3, 168.

23. Koller, F., Loeliger, A., and Duckert, F., Experimentson a new clotting factor (Factor VII). Actahaemat., 1951, 6, 1.

24. MacMillan, R. L., Observation on the mechanism ofaction of dicoumarol. Science, 1948, 108, 416.

25. Mann, F. D. Co-thromboplastin assay; a means ofstudy of abnormalities of blood coagulation. Am.J. Gin. Path., 1949, 19, 861.

26. Mann, F. D., and Hurn, M., Co-thromboplastin, aprobable factor in coagulation of blood. Am. J.Physiol., 1951, 164, 105.

27. Owen, C. A., Jr., and Bollman, J. L., Prothrombinconversion factor of dicumarol plasma. Proc. Soc.Exper. Biol. & Med., 1948, 67, 231.

28. Owen, C. A., Jr., Magath, T. B., and Bollman, J. L.,Prothrombin conversion factors in blood coagula-tion. Am. J. Physiol., 1951, 166, 1.

29. Ware, A. G., and Seegers, W. H., Two-stage pro-cedure for the quantitative determination of pro-thrombin concentration. Am. J. Clin. Path., 1949,19, 471.

30. Lewis, J. H., and Ferguson, J. H., Studies on a pro-teolytic enzyme system of the blood. II. Fibrinoly-sokinase activators for profibrinolysin. J. Gin.Invest., 1950, 29, 1059.

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