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Physicochemical and biological properties of canine-prothrombin and thrombin
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printed
RESEARCH
in Great Rrirain
?H'iSICOCtiE?lICAL ;\kD BIOLXICAL ?!?OPEB;IE; OF C.I?1'I~E-PP,OTHP,OYGIN A-1ND THRMBI?;
T. Takeuchi and Y. Takeda Department of Medicine. University of Colorado School of Medicine, Denver, Colorak
(Received 21.9.15)?';; in revised form 1.2.1978. Accepted by Editor H.L. Sossel)
.ABSTP,.ACT Canine prothrombin was puriEied by a combination of BaSOi adsorption, elution of adsorbed prothrombin in 0.25 Y sodiin citrate, concentration of the eluate by (NH412SO4 fractionation at 80 % saturation and DEAE cellulose chromatography. The prothrombin was pure by immunoelectrophoresis but some trace impurities were visible by SDS polyacrylamide gel electrophoresis(SDS PAGE). The molecular weight was 51,300 + %O(SD) by SDS PAGE, the extinction coefficient was 13.8 2 0.4(SD) for 10 mg per ml, and the specific activity was 1,200 + 50 (SD) NIH units per mg prothrcmbin. Then, the activation tehavior of prothrombin by activated Factor X in the presence of Factor V, phospholipids and Cai+ was studied, and the molecular weight of each product was determined by SDS PACE. The molecular weights for P2 (intermediate 11, Fa(fragment l), P3cintermediate 2) and Fbcfragment 2) were 60,000, 28,540, 40,400 and 21,240, respectively. Kqxt, thronbin was separated by the use of QAE-Sephadex h-50 chromatography. The molecular weight averaged 38,111 + SOO(SD), the extinction coefficient was 16.0 +_ 0.2(SD) for 10 mg-per ml and the specific activity was 2,000 + 65(SD) NIH units per mg thrombin. Comparison of these results with previous studies of bovine material(Biochen. 11: 4502, 1972; J. Biol. Chem. 249: 594, 1974) showed that the results were generally similar. One peculiar finding with the canine material was that P2 appeared to consist of two polypeptide subunits. Further studies are required to elucidate its si‘gnificance.
IZiTRODUCTIOW A nmber of physicochemical and biological studies of prothrombin and thrombin in man and animals have been reportedcl-191, but very few such studies of canine prothrcznbin and thrombin are available(2, 3). We therefore purified canine proof the physicochemical thrornbin, studied its activation and characterized scr?,e and biological properties of canine prothrcmbin and thrcmbin. 635
vo1.12,so.4
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Purification of canine prothrcmbin. BaSO4 suspension was prepared as described elsewhere(20). About 3 liter of fresh oxalated canine plasma ijasmixed with prepared BaS04 suspension at a ratio of 300 ng BaSO4 per ml plasma. The mixture was stirred gently on a magnetic stirrer for 90 min to adsorb prothrcmbin, and centrifuged at 3,000 rpm for 10 min. The supernate was discarded and the precipitate was washed twice with about 500 ml of i7 ti XaCl in 1 mti sodiun citrate. The washed BaSO4 was then suspended in about 900 ml of 0.25 N sodium citrate and gently stirred on a magnetic stirrer for 90 min to elute adsorbed prothrombin. The mixture was centrifuged at 3,000 rpm for 15 min. The precipitate was discarded. The supernate was then 30 % saturated with (5:i4)2SO4and centrifuged at 5,000 rpm for 15 min. The precipitate was discarded, and the supernate was 80 % saturated with (NH4)2S04, and centrifuged at 15,000 rpm for 15 min. The liquid portion was removed by suction, and the white packed cake on the surface was removed and placed in a dialyzing membrane and dialyzed for 7 hr against 0.025 M citrate buffer(pH 6.0) with several changes of the buffer, The dialyzed material was then chromatographed by the use of DEAE cellulose colmn(2.5 cm x 60 cm in size)(0.89 mEq per g, Schleicher E,Schuell, Inc., Keene, Xew Hampshire). First, the applied material was eluted by the use of 0.025 M citrate buffer(pH 6.0) until OD of the effluent at 280 nm reached 0.02 and then by the use of 0.1 M NaCl in 0.025 M citrate buffer(pH 6.0). Each fraction was measured with respect to its OD at 280 MI and to its prothrombin activity by a modified two-stage prothrombin assay(21) as described later(Fig. 1). The active fractions were collected and the volLane was reduced to about 5 ml by pervaporation or by (NH4)2S04 fractionation at 80 % saturation. The prothrmnbin was dialyzed for 7 hr against 0.1 M NaCl in 0.025 M citrate buffer (pH 6.01, and preserved at -37 degrees C in small aliquots.
.08 9 .04 d (*
20
40
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rr.etion 80
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No. LZO
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FIG. 1 Purification of canine prothrcmbin by DEAE cellulose chromatography: Fractions between the two arrows were collected and further processed. The fraction volume was about 9 ml and the flow rate was about 100 ml per hr. The highest prothrombin activity was set to 100 %. The column size was 2.5 cm x 50 cm. 0.1 M NaCl in 0.025 N citrate buffer(pH 6.0) was used.
Determination of purity, extinction coefficient, specific activity and molecular weight of canine prothrombin. The purity was first determined by the use of SDS PACE(22), using 5.6 .g% gel with or without beta-mercaptoethanol. The size
of the gel was 5 rm~ x 1G CTII. The purity was also :?sced >:ii:mlawlectrophoresis(IEP) as described elsewhere(23), using anti-canine whole serm(Behring Diagnostics, Sommerville, ?i. J.) and an antibody against canine prothrombin. The anti-canine prothrombin antibody was produced in rabbits by a biweekly intramuscular injection of about 0.5 mg prothrombin in Freund's complete adjwant for a 4 month period and harvested by a heart puncture within 2 weeks after the final injection. For determination of extinction coefficient, 3 different preparations were dialyzed against deionized distilled H20 for 2 days and were lyophilized for 15 hr, using a Virtis lyophilizer(Research Equipment, ?;. Y.). The dried material was further dried over CaCl2 in a dessicator for 7 days. Exactly weighed material was then dissolved in 0.025 ?i citrate buffer(pH 5.0) and the extinction coefficient was obtained as descrited(5). The molecular weight was measured by the use of SDS PACS sith and without the use of beta-mercaptoethano1(22), using 5.6 9% gel of 5 ~‘-3 x 10 cm in size, The standards used were phosphorylase-alpha(92,GOO MW), hman transferrinc 77,000 MW), bovine serm albmin(68,OOO MW), ovalbcnin(43,OOO >1i<), chymotrypsinogen A(25,OOO MW) and ribonuclease A(13,700 MW). Prothrcanbin activity was measured as follows by a modified two-sta.ge assay of Ware and Seegers(21): the reaction mixture was made by mixing in a volume ratio of 2, 2, 1 and 1, respectively, of 0.063 M CaC12 in 0.16 M NaCl, 15 g% acacia solution in 0.16 M NaCl, 0.257 M imidazol buffer(pH 7.3) and 0.16 M SaCl. The mixture of Factors V, VII and X was made by mixing in an equal volume ratio Bacto-ac-globulin (Difco Lab., Detroit, Mich.) and hunan serm both 50 times diluted in 0.16 M NaCl. Bacto-thrcxnboplastin was obtained from Difco Lab. and processed according to the manufacturer's instruction. Fibrinogen solution of 1 g clottable protein per dl was made in the mixture of 1 volume of 0.257 M imidazol buffer CpH 7.3) and 9 volwne of 0.16 3 NaCl. In the measurement of prothrombin activity, 0.1 ml of the material appropriately diluted in 0.16 M NaCl was mixed with 2.4 ml of the mixture of Factors V, VII and X, and 1 ml of this was added to 0.2 ml of thrcnnboplastin, 2.0 ml of the reaction mixture and 0.8 ml of 0.16 M NaCl, and incubated at 28 degrees C. A 0.4 ml aliquot of samples was obtained at 2 min intervals and blown into 0.1 ml fibrinogen solution to determine the clotting time. The standard curve was made by measuring the clotting time of 0.1 ml fibrinogen solution mixed with 0.3 ml reaction mixture to which 0.1 ml standard thrombin was added(Sigma Chemical Co., St Louis, Missouri). Prothrombin activation by activated Factor X(FXa) in the presence of Ca++, phospholipids and Factor V. Canine Factor X(FX), FXa and phospholipids were prepared as described elsewhere(20, 23, 24). In order to determine the optimun condition for canine prothrombin activation, effects of varying amounts of FXa and Factor V on prothranbin activation in the presence of fixed amounts of prothrcmbin, phospholipids and Ca* were studied as shown later. Based on the studies, the following was chosen for prothrombin activation: prothrcnnbin was first dialyzed against 0.1 M NaCl in 0.02 M tris-HCl buffer(pH 7.5). 1 ml of prothrunbin(0.6 mg>, 0.04 ml FXa(0.02 me>, 0.05 ml of 0.25 M CaC12, 0.05 ml phospholipids(0.1 mg), 0.04 ml Factor V 10 times diluted in 0.16 M NaCl, and 0.04 ml of 0.1 M NaCl in 0.02 M tris-HCl buffer(pH 7.5) were mixed and incubated at 37 degrees C for 5 hr. Small aliquots of samples were obtained at appropriate intervals in equal volme of 2 g% SDS in 20 ti tris-HCl buffer (pH 8.0) containing 2 mM EDTA(16). Each sample was analyzed by SDS PAGE(22), using 5.6 g% gels of 5 mm x 10 cm in size. When greater amounts of prothrombin were activated for thrombin preparation, FXa was increased proportionately, but others were kept constant. Preparation of canine thranbin. Prothrombin was activated in the manner shown above, and thrcmbin was separated by QAE-Sephadex A-SO chranatography(l6, 17) (Pharmacia Fine Chemicals Corp., Piscataway, N. J.). The colunn size usually
638
CXSI?iiEPROTHROYBI?; SD
THROIIBIS
Vol.12,?jo.4
was 1.6 cm x 40 cm. A gradient of 0.1 ?I to 0.5 ?iNaCl in 0.02 Y tris-HCl buffer (pH 7.5) was *used. The volme of the nixing chamber was 120 ml(Fig. 2). The active fractions bet-en the two arrows were collected, and the VOlme was The extinction coefficient and clottreduced by pervaporation when necessary. ing activity of thrcmbin were determined as described for prothrombin. - Od.t as0 I)-_ tkrorbtn ,c 11.11,
FIG. 2 QAE-Sephadex A-SO chromatography of activation products of canine prothrombin: The fraction volune was about 1 to 2 ml and the flow rate 2-3 about 8 ml per hr. About 2 mg of prothrcmbin was activated. EESULTS Purity, extinction coefficient, specific .‘, .ivity of prothrcmbin and thrombin. A was filled with prothromFig. 3 shows IEP patterns of purified n’ Xhrcmbin. bin, B with anti-prothrombin antibod- , C with canine plasma, D with prothrcmbin, E with anti-canine whole serun rniY.1 with small amount of anti-prothrtibin antibody and F with canine pla?,.l. It is seen that our prothrcmbin appears to be pure. SDS PAGE patterns c’ prothrombin are shown later. The extinction coefficient of prothranbin averaged 13.8 + O.L(SD) for 10 mg per ml, and the specific activity averaged 1,200 + 50(SD) NIH units per mg prothrunbin. The extinction coefficient of thrcmbig averaged 16.0 + 0.2(SD) for 10 mg per ml and the specific activity was 2,000 t 65(SD) NIH units per mg thranbin. The SDS PAGE patterns of thrombin are shown later. A
B c
:.
‘0 -
- i
-- _.
_._t
.-
-..
A. ;
___.
7 -~
FIG.
3
with prothrornbin, B with ID patterns of purified prothrunbin: A was filled anti-prothranbin antibody, C with canine plasma, D with prothranbin, E with anti-canine whole serun, and F with canine plasma. It is seen that our prothranbin appears to be pure.
Effects of F?(a and Factor V on prothrcmbin activation in the presence of Cal and phospholipids. Fig. 4 shows the effects of var:;ing amounts of FXa on prothrcmbin activation in the presence of fixed amounts of orothrornbin,Ca++ an? phospholipids. The volume and the amount of prothrombin iiere fixed to 1 ml and 0.6 mg, those of phospholipids were 0.05 ml and 0.1 ng, and 0.05 ml of 0.25 Y CaC12 was used. The total volt_xae of the incubation mixture was fixed to 1.22 ml using 0.1 M MaCl in 0.02 ?i tris-HCl buffer(pH 7.5). X shows the activation pattern with 0.06 ng of FXa, B is that with 0.03 ng of FXa, C is that with 0.02 mg of FXa and D is that with 0.015 mg of FXa. It is seen that the degree of prothrombin activation depends on the a;nount of FXa. Fig. 5 shows the effects of varying amounts of Factor V on prothrombin activation in the presence of fixed amounts of prothrcmbin, FXa, phospholipids and Ca++. The volume and amount of prothrombin were fixed to 1 ml and 0.6 mg, those of FXa were 0.04 ml and 0.02 mg, those of phospholipids were 0.05 ml and 0.1 mg, and 0.05 ml of 0.25 M CaC12 was used. The total vo1lmii.e of the activation mixture was fixed to 1.22 ml with 0.1 M NaCl in 0.02 ?l tris-Kl bluffer(pH 7.5). A shows the activation pattern when 0.08 ml of Factor V 10 times diluted in 0.1 M SaCl in 0.02 M tris-HC1 buffer(pH 7.5) was used, B is that with 0.04 ml of Factor V and C is that with 0.02 ml of Factor V. D shows the activation of prothrombin when Factor V was not added initially but 0.04 ml of Factor V Kas added at 60 min of incubation. It is obvious that prothrcmbin activation greatly depends on Factor V. Although not shown, our purified prothrcmbin did not generate detectable thrombin activity after 7 day incubation at 37 degrees C in 25 g% sodium citrate solution. Ihl._b," .Sli.,l, NIH" 2009.'=, .
thl0mt.i” .ct..,t, ” NW” P.‘sr.I 180 140
.
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Effects of varying amounts of FXa on prothrcmbin activation in the presence of fixed amounts of prothranbin, phospholipids and Ca++: the incubation temperature was 37 degrees C. A is the activation pattern with 0.06 mg FXa, B is that with 0.03 mg FXa, C is that with 0.02 mg FXa and D is that with 0.015 mg of FXa per 1.22 ml. The amount of prothrombin was 0.6 mg per 1.22 ml. See text for details.
. 220 . . . 200 . .
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*A
.
.B
180 * 180 *.
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-D
Effects of varying amounts of Factor V on prothrcmbin activation in the presence of fixed amounts of prothrombin, FXa, Ca* and phospholipids: A shows the activation pattern with 0.08 cl of Factor V, B is that with 0.04 ml and C is that with 0.02 ml. D shows the activation when Factor V was not added initially, but 0.04 ml Factor V was added at 60 min incubation at 37 degrees C.
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Products of prothrornbinactivation r;ith ti!!e. Fig. 6 shous the acti;*ationpro-trated b:;SDS ?ri;E without 'beta-mercaducts of prothrombin with t&.e as demon3 ptoethanol. The activation condition uas the saiae as in Fig. SB. The gels A to J represent the SDS PAGE patterns at 0, 5, 10, 15, 30, 60, 90, 120, 180 and 300 nin of incubation at 37 degrees C. Various products are 1a:belled on vertical line according to Stenn and Blout(12) as Pl(prothrombin), P2, Fx, Tl, P3, Fa, T2, and Fb. Gel K shows the thrombin separated by QAE-Sephadex A-50 chraaatography(Fig. 2) and gel L shows the pattern of Fa and Fb obtained from peak 5 of Fig. 2. The SDS PAGE pattern of peak 6 in Fig. 2 was similar to that in-gel L except that the amount of Fb appeared to be greater than Fa. The peak 1 in Fig. 2 was found to icecontaninant proteins due to the use of iTpure Factor V. The peaks 3 and 4 in Fig. 2 were not analyzed. Fig. 7 shows SDS PXE patterns of the same samples as in Fig. 5 except that they were reduced by beta-mercaptoethanol. The products are designated according to Stenn and Blo-t(l2) as Pl(prothrombin), ~2, Fx, P3, Tlb, Fa, T2b, Fb, T2a and Tla. Gel K shows the patterns of various standards as given earlier. The molecular weights of the products obtained by SDS PAGE of reduced and non-reduced samples are given in Table 1. It is seen in Fig. G that P2 appears to be a doublet and in Fig. 7 that P2 is split into two components. This was a very consistent finding with canine material. Fig. 8 shows SDS PACE patterns of prothronbin(A), prothrombin activated with thrombin only for 2 hr at 37 degrees C(B) and prothror.;bin activated with FXa in the presence of Factor V, phospholipids and Ca++ for 2 min as in Fig. 58(C). It is seen that thrombin generates only P2 and Fa from prothrwnbin.
FIG. 7 SDS PAGE patterns of activation products of prothrunbin with time: the act!.vationcondition was the same as in Fig. 5B. Samples were not reduced with teta-mercaptoethanol. See text for details.
SDS PAGE patterns of activaproducts of prothrombin with time: Gels A to J represent the patterns of the same samples as in Fig. 6 except that they were reduced. See text for details.
>folecular Ueights of Redsuced and Nonrediiced Activation Products of Prothrabin
Reduced Pl 32
P3 Tl Tlb Tla T2 T2b T2a Fx Fa Fb
Yonreduced
81,300 2 85OtSD) 60,000 t 970(SD) 40,400 +_ ijO
$1,900
2
78OCSD)
62,500 e l,OOO(SD) 35,243 + iOO(SD) 35,111 I SOO(SD)
33,440 k l,OOO(SD) 6,000 2 62O(SD) 27,492
+ 7OO(SD)
19,800 + 1,50O(SD) 9,367 z 650(SD) 2 l,?oo(so> 29,928 +_ 75O(SD) 19,s43 2 l,SOO(SD)
44,300
28,540 + SOO(SD) 21,240 ; l,lOO(SD)
Average value of 3 measurements is
giver1
for each,
FIG. 8 SDS PACE patterns of prothrombin(A), activation products of prothrombin by thrombin at 2 hr incubation(B) and activation products of prothronbin by FXa in the presence of phospholipids, Ca* and Factor V at 2 min incubation(C): 0.6 ng of prothrombin was activated with 1 NIH unit of thrombin at 37 degrees C(B). See text for further details.
Our purification method for canine prothrmbic is no more than a modification of previo!zsmethods(l-7, 10-12, 15-19>, bl;t it is simple and rapid and a preparation can %tecompleted within 60 hr with a recovery of abo-t 17 7:. The prepared prothrombin appeared to be pure by imzzun.oelectrophoresis(Fig. 3) but there were trace contaminants visible by SDS ?.iGE(Figs. 6-8). The molecullar weight of 81,300 i 85O(SD) for canine prothr:mbin(Table 1) is similar to 83,000 +_ 6,OOO(SD)(l2? but appears somewhat larger than 72,000 to 74,000(16), 72,000 2 5,000(SD)(l3) and 68,900(l) reported for bovine prothrcmbin. Rosenberg et al.(lY) reported 67,800 and Lanchantin and his associates(S) published 65,000 to 70,000 for hman prothrombin. However, we were unabie to find data on canine prothrcmbin. Some of these differences at least appear to be due to the difference in the methodology. The specific activity of 1,200 + 5O(SD) ?JIH units per mg canine prothrcmbin in the present study is to 1,250 f lOO(SD>(lS>, 1,200 to 1,400(12) and 1,100 to 1,300 NIH units per mg(lO) but appears less than 2,400 to 3,000 Iowa units par mg(25) reported for bovine prothrombin. Lanchantin et al.{71 reported 2,444 f- 276(SD) NIH units per mg for human prothrombin which is nuch higher than our value. ?+oore and his coworkers(3) publis‘ned a value of 2,400 to 3,000 Ioua units per mg for canine prothrombin, which appears somewhat greater than our value. The molecular weights for activation products of canine prot‘nrcmbin(Table 1) were more or less similar to those of bovine prothrombin reported by Stenn and Blout(l2) and Owen et a1.(16). Studies by these investigators(l2, 16) and others(l4, 26) led to the concept excluding the details that thrombin cleaves prothranbin(P1) to PZ(intermediate 1) and Faifragment l), that FXa breaks down P2 to P3(intermediate 2) and Fb(fragment 2) and that FXa generates Tl(thrcmbin) from P3. It is also stated that Tl is degraded to T2 with time, that Tlb and Tla are the subunits of Tl, and that T2b and T2a are the subunits of T2(12). Our studies of prothrcmbin activation with thrombin(Fig. 8B) certainly formed P2 and Fa only, but we did not purify P2 or P3 and study their behavior of activation with FXa. However, the SKI of the molecular weights of P2 and Fa is roughly equal to that of Pl, the sm of those of P3 and Fb is about equal to that of P2, and the slpns of those of Tlb and Tla and of T2a and T2b approximate that of Tl and of T2, respectively(Table 1). Thus, our results generally appear to support their findings(l2, 16). Only peculiar finding with canine prothrombin was that P2 appeared to consist of two polypeptide units joined by a disulphide bond(Figs. 6-8). We are uncertain of its significance at this time. The SDS PAGE pattern of our thrombin preparation is shown in Fig. 6K, in which both Tl and T2 are present. We were unable to obtain a single protein with thronbin activity. Therefore, our thrcmbin preparations are a mixture of Tl and T2. The molecular weight of 38,100 +_ SOO(SD> for canine.Tl is similar to 37,100 (121, 37,000(16) and 39,000(9) reported for bovine thrombin and 36,000 for himan thrcmbin(l9), but the specific activity of 2,000 f 65(SD) NIH units per mg canine thrombin in the present study appears somewhat less than 2,400(16) and 2,700 NIH units per mg(9) reported for bovine thrcmbin and much less than 9,800 to 10,600 Iowa units per mg dry weight reported for hunan thrombin(25). We were again unable to find the data on canine thrcmbin. Thus, the properties of canine prothrombin and thrombin appear to be generally similar to bovine material except the peculiarity of P2 as discussed above. Siiiiar
ACKNOWLEDCMENT This work was supported by Research Grant HI.-116% from the National Heart, Lung and Blood Institute, Bethesda, Maryland.
643
REFEEE?;CES
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printed
RESEARCH
in Great Rrirain
?H'iSICOCtiE?lICAL ;\kD BIOLXICAL ?!?OPEB;IE; OF C.I?1'I~E-PP,OTHP,OYGIN A-1ND THRMBI?;
T. Takeuchi and Y. Takeda Department of Medicine. University of Colorado School of Medicine, Denver, Colorak
(Received 21.9.15)?';; in revised form 1.2.1978. Accepted by Editor H.L. Sossel)
.ABSTP,.ACT Canine prothrombin was puriEied by a combination of BaSOi adsorption, elution of adsorbed prothrombin in 0.25 Y sodiin citrate, concentration of the eluate by (NH412SO4 fractionation at 80 % saturation and DEAE cellulose chromatography. The prothrombin was pure by immunoelectrophoresis but some trace impurities were visible by SDS polyacrylamide gel electrophoresis(SDS PAGE). The molecular weight was 51,300 + %O(SD) by SDS PAGE, the extinction coefficient was 13.8 2 0.4(SD) for 10 mg per ml, and the specific activity was 1,200 + 50 (SD) NIH units per mg prothrcmbin. Then, the activation tehavior of prothrombin by activated Factor X in the presence of Factor V, phospholipids and Cai+ was studied, and the molecular weight of each product was determined by SDS PACE. The molecular weights for P2 (intermediate 11, Fa(fragment l), P3cintermediate 2) and Fbcfragment 2) were 60,000, 28,540, 40,400 and 21,240, respectively. Kqxt, thronbin was separated by the use of QAE-Sephadex h-50 chromatography. The molecular weight averaged 38,111 + SOO(SD), the extinction coefficient was 16.0 +_ 0.2(SD) for 10 mg-per ml and the specific activity was 2,000 + 65(SD) NIH units per mg thrombin. Comparison of these results with previous studies of bovine material(Biochen. 11: 4502, 1972; J. Biol. Chem. 249: 594, 1974) showed that the results were generally similar. One peculiar finding with the canine material was that P2 appeared to consist of two polypeptide subunits. Further studies are required to elucidate its si‘gnificance.
IZiTRODUCTIOW A nmber of physicochemical and biological studies of prothrombin and thrombin in man and animals have been reportedcl-191, but very few such studies of canine prothrcznbin and thrombin are available(2, 3). We therefore purified canine proof the physicochemical thrornbin, studied its activation and characterized scr?,e and biological properties of canine prothrcmbin and thrcmbin. 635
vo1.12,so.4
636
Purification of canine prothrcmbin. BaSO4 suspension was prepared as described elsewhere(20). About 3 liter of fresh oxalated canine plasma ijasmixed with prepared BaS04 suspension at a ratio of 300 ng BaSO4 per ml plasma. The mixture was stirred gently on a magnetic stirrer for 90 min to adsorb prothrcmbin, and centrifuged at 3,000 rpm for 10 min. The supernate was discarded and the precipitate was washed twice with about 500 ml of i7 ti XaCl in 1 mti sodiun citrate. The washed BaSO4 was then suspended in about 900 ml of 0.25 N sodium citrate and gently stirred on a magnetic stirrer for 90 min to elute adsorbed prothrombin. The mixture was centrifuged at 3,000 rpm for 15 min. The precipitate was discarded. The supernate was then 30 % saturated with (5:i4)2SO4and centrifuged at 5,000 rpm for 15 min. The precipitate was discarded, and the supernate was 80 % saturated with (NH4)2S04, and centrifuged at 15,000 rpm for 15 min. The liquid portion was removed by suction, and the white packed cake on the surface was removed and placed in a dialyzing membrane and dialyzed for 7 hr against 0.025 M citrate buffer(pH 6.0) with several changes of the buffer, The dialyzed material was then chromatographed by the use of DEAE cellulose colmn(2.5 cm x 60 cm in size)(0.89 mEq per g, Schleicher E,Schuell, Inc., Keene, Xew Hampshire). First, the applied material was eluted by the use of 0.025 M citrate buffer(pH 6.0) until OD of the effluent at 280 nm reached 0.02 and then by the use of 0.1 M NaCl in 0.025 M citrate buffer(pH 6.0). Each fraction was measured with respect to its OD at 280 MI and to its prothrombin activity by a modified two-stage prothrombin assay(21) as described later(Fig. 1). The active fractions were collected and the volLane was reduced to about 5 ml by pervaporation or by (NH4)2S04 fractionation at 80 % saturation. The prothrmnbin was dialyzed for 7 hr against 0.1 M NaCl in 0.025 M citrate buffer (pH 6.01, and preserved at -37 degrees C in small aliquots.
.08 9 .04 d (*
20
40
60
rr.etion 80
100
No. LZO
140
160
180
200
FIG. 1 Purification of canine prothrcmbin by DEAE cellulose chromatography: Fractions between the two arrows were collected and further processed. The fraction volume was about 9 ml and the flow rate was about 100 ml per hr. The highest prothrombin activity was set to 100 %. The column size was 2.5 cm x 50 cm. 0.1 M NaCl in 0.025 N citrate buffer(pH 6.0) was used.
Determination of purity, extinction coefficient, specific activity and molecular weight of canine prothrombin. The purity was first determined by the use of SDS PACE(22), using 5.6 .g% gel with or without beta-mercaptoethanol. The size
of the gel was 5 rm~ x 1G CTII. The purity was also :?sced >:ii:mlawlectrophoresis(IEP) as described elsewhere(23), using anti-canine whole serm(Behring Diagnostics, Sommerville, ?i. J.) and an antibody against canine prothrombin. The anti-canine prothrombin antibody was produced in rabbits by a biweekly intramuscular injection of about 0.5 mg prothrombin in Freund's complete adjwant for a 4 month period and harvested by a heart puncture within 2 weeks after the final injection. For determination of extinction coefficient, 3 different preparations were dialyzed against deionized distilled H20 for 2 days and were lyophilized for 15 hr, using a Virtis lyophilizer(Research Equipment, ?;. Y.). The dried material was further dried over CaCl2 in a dessicator for 7 days. Exactly weighed material was then dissolved in 0.025 ?i citrate buffer(pH 5.0) and the extinction coefficient was obtained as descrited(5). The molecular weight was measured by the use of SDS PACS sith and without the use of beta-mercaptoethano1(22), using 5.6 9% gel of 5 ~‘-3 x 10 cm in size, The standards used were phosphorylase-alpha(92,GOO MW), hman transferrinc 77,000 MW), bovine serm albmin(68,OOO MW), ovalbcnin(43,OOO >1i<), chymotrypsinogen A(25,OOO MW) and ribonuclease A(13,700 MW). Prothrcanbin activity was measured as follows by a modified two-sta.ge assay of Ware and Seegers(21): the reaction mixture was made by mixing in a volume ratio of 2, 2, 1 and 1, respectively, of 0.063 M CaC12 in 0.16 M NaCl, 15 g% acacia solution in 0.16 M NaCl, 0.257 M imidazol buffer(pH 7.3) and 0.16 M SaCl. The mixture of Factors V, VII and X was made by mixing in an equal volume ratio Bacto-ac-globulin (Difco Lab., Detroit, Mich.) and hunan serm both 50 times diluted in 0.16 M NaCl. Bacto-thrcxnboplastin was obtained from Difco Lab. and processed according to the manufacturer's instruction. Fibrinogen solution of 1 g clottable protein per dl was made in the mixture of 1 volume of 0.257 M imidazol buffer CpH 7.3) and 9 volwne of 0.16 3 NaCl. In the measurement of prothrombin activity, 0.1 ml of the material appropriately diluted in 0.16 M NaCl was mixed with 2.4 ml of the mixture of Factors V, VII and X, and 1 ml of this was added to 0.2 ml of thrcnnboplastin, 2.0 ml of the reaction mixture and 0.8 ml of 0.16 M NaCl, and incubated at 28 degrees C. A 0.4 ml aliquot of samples was obtained at 2 min intervals and blown into 0.1 ml fibrinogen solution to determine the clotting time. The standard curve was made by measuring the clotting time of 0.1 ml fibrinogen solution mixed with 0.3 ml reaction mixture to which 0.1 ml standard thrombin was added(Sigma Chemical Co., St Louis, Missouri). Prothrombin activation by activated Factor X(FXa) in the presence of Ca++, phospholipids and Factor V. Canine Factor X(FX), FXa and phospholipids were prepared as described elsewhere(20, 23, 24). In order to determine the optimun condition for canine prothrombin activation, effects of varying amounts of FXa and Factor V on prothranbin activation in the presence of fixed amounts of prothrcmbin, phospholipids and Ca* were studied as shown later. Based on the studies, the following was chosen for prothrombin activation: prothrcnnbin was first dialyzed against 0.1 M NaCl in 0.02 M tris-HCl buffer(pH 7.5). 1 ml of prothrunbin(0.6 mg>, 0.04 ml FXa(0.02 me>, 0.05 ml of 0.25 M CaC12, 0.05 ml phospholipids(0.1 mg), 0.04 ml Factor V 10 times diluted in 0.16 M NaCl, and 0.04 ml of 0.1 M NaCl in 0.02 M tris-HCl buffer(pH 7.5) were mixed and incubated at 37 degrees C for 5 hr. Small aliquots of samples were obtained at appropriate intervals in equal volme of 2 g% SDS in 20 ti tris-HCl buffer (pH 8.0) containing 2 mM EDTA(16). Each sample was analyzed by SDS PAGE(22), using 5.6 g% gels of 5 mm x 10 cm in size. When greater amounts of prothrombin were activated for thrombin preparation, FXa was increased proportionately, but others were kept constant. Preparation of canine thranbin. Prothrombin was activated in the manner shown above, and thrcmbin was separated by QAE-Sephadex A-SO chranatography(l6, 17) (Pharmacia Fine Chemicals Corp., Piscataway, N. J.). The colunn size usually
638
CXSI?iiEPROTHROYBI?; SD
THROIIBIS
Vol.12,?jo.4
was 1.6 cm x 40 cm. A gradient of 0.1 ?I to 0.5 ?iNaCl in 0.02 Y tris-HCl buffer (pH 7.5) was *used. The volme of the nixing chamber was 120 ml(Fig. 2). The active fractions bet-en the two arrows were collected, and the VOlme was The extinction coefficient and clottreduced by pervaporation when necessary. ing activity of thrcmbin were determined as described for prothrombin. - Od.t as0 I)-_ tkrorbtn ,c 11.11,
FIG. 2 QAE-Sephadex A-SO chromatography of activation products of canine prothrombin: The fraction volune was about 1 to 2 ml and the flow rate 2-3 about 8 ml per hr. About 2 mg of prothrcmbin was activated. EESULTS Purity, extinction coefficient, specific .‘, .ivity of prothrcmbin and thrombin. A was filled with prothromFig. 3 shows IEP patterns of purified n’ Xhrcmbin. bin, B with anti-prothrombin antibod- , C with canine plasma, D with prothrcmbin, E with anti-canine whole serun rniY.1 with small amount of anti-prothrtibin antibody and F with canine pla?,.l. It is seen that our prothrcmbin appears to be pure. SDS PAGE patterns c’ prothrombin are shown later. The extinction coefficient of prothranbin averaged 13.8 + O.L(SD) for 10 mg per ml, and the specific activity averaged 1,200 + 50(SD) NIH units per mg prothrunbin. The extinction coefficient of thrcmbig averaged 16.0 + 0.2(SD) for 10 mg per ml and the specific activity was 2,000 t 65(SD) NIH units per mg thranbin. The SDS PAGE patterns of thrombin are shown later. A
B c
:.
‘0 -
- i
-- _.
_._t
.-
-..
A. ;
___.
7 -~
FIG.
3
with prothrornbin, B with ID patterns of purified prothrunbin: A was filled anti-prothranbin antibody, C with canine plasma, D with prothranbin, E with anti-canine whole serun, and F with canine plasma. It is seen that our prothranbin appears to be pure.
Effects of F?(a and Factor V on prothrcmbin activation in the presence of Cal and phospholipids. Fig. 4 shows the effects of var:;ing amounts of FXa on prothrcmbin activation in the presence of fixed amounts of orothrornbin,Ca++ an? phospholipids. The volume and the amount of prothrombin iiere fixed to 1 ml and 0.6 mg, those of phospholipids were 0.05 ml and 0.1 ng, and 0.05 ml of 0.25 Y CaC12 was used. The total volt_xae of the incubation mixture was fixed to 1.22 ml using 0.1 M MaCl in 0.02 ?i tris-HCl buffer(pH 7.5). X shows the activation pattern with 0.06 ng of FXa, B is that with 0.03 ng of FXa, C is that with 0.02 mg of FXa and D is that with 0.015 mg of FXa. It is seen that the degree of prothrombin activation depends on the a;nount of FXa. Fig. 5 shows the effects of varying amounts of Factor V on prothrombin activation in the presence of fixed amounts of prothrcmbin, FXa, phospholipids and Ca++. The volume and amount of prothrombin were fixed to 1 ml and 0.6 mg, those of FXa were 0.04 ml and 0.02 mg, those of phospholipids were 0.05 ml and 0.1 mg, and 0.05 ml of 0.25 M CaC12 was used. The total vo1lmii.e of the activation mixture was fixed to 1.22 ml with 0.1 M NaCl in 0.02 ?l tris-Kl bluffer(pH 7.5). A shows the activation pattern when 0.08 ml of Factor V 10 times diluted in 0.1 M SaCl in 0.02 M tris-HC1 buffer(pH 7.5) was used, B is that with 0.04 ml of Factor V and C is that with 0.02 ml of Factor V. D shows the activation of prothrombin when Factor V was not added initially but 0.04 ml of Factor V Kas added at 60 min of incubation. It is obvious that prothrcmbin activation greatly depends on Factor V. Although not shown, our purified prothrcmbin did not generate detectable thrombin activity after 7 day incubation at 37 degrees C in 25 g% sodium citrate solution. Ihl._b," .Sli.,l, NIH" 2009.'=, .
thl0mt.i” .ct..,t, ” NW” P.‘sr.I 180 140
.
-
Effects of varying amounts of FXa on prothrcmbin activation in the presence of fixed amounts of prothranbin, phospholipids and Ca++: the incubation temperature was 37 degrees C. A is the activation pattern with 0.06 mg FXa, B is that with 0.03 mg FXa, C is that with 0.02 mg FXa and D is that with 0.015 mg of FXa per 1.22 ml. The amount of prothrombin was 0.6 mg per 1.22 ml. See text for details.
. 220 . . . 200 . .
.
*A
.
.B
180 * 180 *.
*-
-D
Effects of varying amounts of Factor V on prothrcmbin activation in the presence of fixed amounts of prothrombin, FXa, Ca* and phospholipids: A shows the activation pattern with 0.08 cl of Factor V, B is that with 0.04 ml and C is that with 0.02 ml. D shows the activation when Factor V was not added initially, but 0.04 ml Factor V was added at 60 min incubation at 37 degrees C.
640
C.%?;ISE PROTHROYBI?;
XSD
THROYBIS
Vol.l',So.4
Products of prothrornbinactivation r;ith ti!!e. Fig. 6 shous the acti;*ationpro-trated b:;SDS ?ri;E without 'beta-mercaducts of prothrombin with t&.e as demon3 ptoethanol. The activation condition uas the saiae as in Fig. SB. The gels A to J represent the SDS PAGE patterns at 0, 5, 10, 15, 30, 60, 90, 120, 180 and 300 nin of incubation at 37 degrees C. Various products are 1a:belled on vertical line according to Stenn and Blout(12) as Pl(prothrombin), P2, Fx, Tl, P3, Fa, T2, and Fb. Gel K shows the thrombin separated by QAE-Sephadex A-50 chraaatography(Fig. 2) and gel L shows the pattern of Fa and Fb obtained from peak 5 of Fig. 2. The SDS PAGE pattern of peak 6 in Fig. 2 was similar to that in-gel L except that the amount of Fb appeared to be greater than Fa. The peak 1 in Fig. 2 was found to icecontaninant proteins due to the use of iTpure Factor V. The peaks 3 and 4 in Fig. 2 were not analyzed. Fig. 7 shows SDS PXE patterns of the same samples as in Fig. 5 except that they were reduced by beta-mercaptoethanol. The products are designated according to Stenn and Blo-t(l2) as Pl(prothrombin), ~2, Fx, P3, Tlb, Fa, T2b, Fb, T2a and Tla. Gel K shows the patterns of various standards as given earlier. The molecular weights of the products obtained by SDS PAGE of reduced and non-reduced samples are given in Table 1. It is seen in Fig. G that P2 appears to be a doublet and in Fig. 7 that P2 is split into two components. This was a very consistent finding with canine material. Fig. 8 shows SDS PACE patterns of prothronbin(A), prothrombin activated with thrombin only for 2 hr at 37 degrees C(B) and prothror.;bin activated with FXa in the presence of Factor V, phospholipids and Ca++ for 2 min as in Fig. 58(C). It is seen that thrombin generates only P2 and Fa from prothrwnbin.
FIG. 7 SDS PAGE patterns of activation products of prothrunbin with time: the act!.vationcondition was the same as in Fig. 5B. Samples were not reduced with teta-mercaptoethanol. See text for details.
SDS PAGE patterns of activaproducts of prothrombin with time: Gels A to J represent the patterns of the same samples as in Fig. 6 except that they were reduced. See text for details.
>folecular Ueights of Redsuced and Nonrediiced Activation Products of Prothrabin
Reduced Pl 32
P3 Tl Tlb Tla T2 T2b T2a Fx Fa Fb
Yonreduced
81,300 2 85OtSD) 60,000 t 970(SD) 40,400 +_ ijO
$1,900
2
78OCSD)
62,500 e l,OOO(SD) 35,243 + iOO(SD) 35,111 I SOO(SD)
33,440 k l,OOO(SD) 6,000 2 62O(SD) 27,492
+ 7OO(SD)
19,800 + 1,50O(SD) 9,367 z 650(SD) 2 l,?oo(so> 29,928 +_ 75O(SD) 19,s43 2 l,SOO(SD)
44,300
28,540 + SOO(SD) 21,240 ; l,lOO(SD)
Average value of 3 measurements is
giver1
for each,
FIG. 8 SDS PACE patterns of prothrombin(A), activation products of prothrombin by thrombin at 2 hr incubation(B) and activation products of prothronbin by FXa in the presence of phospholipids, Ca* and Factor V at 2 min incubation(C): 0.6 ng of prothrombin was activated with 1 NIH unit of thrombin at 37 degrees C(B). See text for further details.
Our purification method for canine prothrmbic is no more than a modification of previo!zsmethods(l-7, 10-12, 15-19>, bl;t it is simple and rapid and a preparation can %tecompleted within 60 hr with a recovery of abo-t 17 7:. The prepared prothrombin appeared to be pure by imzzun.oelectrophoresis(Fig. 3) but there were trace contaminants visible by SDS ?.iGE(Figs. 6-8). The molecullar weight of 81,300 i 85O(SD) for canine prothr:mbin(Table 1) is similar to 83,000 +_ 6,OOO(SD)(l2? but appears somewhat larger than 72,000 to 74,000(16), 72,000 2 5,000(SD)(l3) and 68,900(l) reported for bovine prothrcmbin. Rosenberg et al.(lY) reported 67,800 and Lanchantin and his associates(S) published 65,000 to 70,000 for hman prothrombin. However, we were unabie to find data on canine prothrcmbin. Some of these differences at least appear to be due to the difference in the methodology. The specific activity of 1,200 + 5O(SD) ?JIH units per mg canine prothrcmbin in the present study is to 1,250 f lOO(SD>(lS>, 1,200 to 1,400(12) and 1,100 to 1,300 NIH units per mg(lO) but appears less than 2,400 to 3,000 Iowa units par mg(25) reported for bovine prothrombin. Lanchantin et al.{71 reported 2,444 f- 276(SD) NIH units per mg for human prothrombin which is nuch higher than our value. ?+oore and his coworkers(3) publis‘ned a value of 2,400 to 3,000 Ioua units per mg for canine prothrombin, which appears somewhat greater than our value. The molecular weights for activation products of canine prot‘nrcmbin(Table 1) were more or less similar to those of bovine prothrombin reported by Stenn and Blout(l2) and Owen et a1.(16). Studies by these investigators(l2, 16) and others(l4, 26) led to the concept excluding the details that thrombin cleaves prothranbin(P1) to PZ(intermediate 1) and Faifragment l), that FXa breaks down P2 to P3(intermediate 2) and Fb(fragment 2) and that FXa generates Tl(thrcmbin) from P3. It is also stated that Tl is degraded to T2 with time, that Tlb and Tla are the subunits of Tl, and that T2b and T2a are the subunits of T2(12). Our studies of prothrcmbin activation with thrombin(Fig. 8B) certainly formed P2 and Fa only, but we did not purify P2 or P3 and study their behavior of activation with FXa. However, the SKI of the molecular weights of P2 and Fa is roughly equal to that of Pl, the sm of those of P3 and Fb is about equal to that of P2, and the slpns of those of Tlb and Tla and of T2a and T2b approximate that of Tl and of T2, respectively(Table 1). Thus, our results generally appear to support their findings(l2, 16). Only peculiar finding with canine prothrombin was that P2 appeared to consist of two polypeptide units joined by a disulphide bond(Figs. 6-8). We are uncertain of its significance at this time. The SDS PAGE pattern of our thrombin preparation is shown in Fig. 6K, in which both Tl and T2 are present. We were unable to obtain a single protein with thronbin activity. Therefore, our thrcmbin preparations are a mixture of Tl and T2. The molecular weight of 38,100 +_ SOO(SD> for canine.Tl is similar to 37,100 (121, 37,000(16) and 39,000(9) reported for bovine thrombin and 36,000 for himan thrcmbin(l9), but the specific activity of 2,000 f 65(SD) NIH units per mg canine thrombin in the present study appears somewhat less than 2,400(16) and 2,700 NIH units per mg(9) reported for bovine thrcmbin and much less than 9,800 to 10,600 Iowa units per mg dry weight reported for hunan thrombin(25). We were again unable to find the data on canine thrcmbin. Thus, the properties of canine prothrombin and thrombin appear to be generally similar to bovine material except the peculiarity of P2 as discussed above. Siiiiar
ACKNOWLEDCMENT This work was supported by Research Grant HI.-116% from the National Heart, Lung and Blood Institute, Bethesda, Maryland.
643
REFEEE?;CES
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Cni\yrsity ?ress, Cambrid.ge, SEEGERS, W. H. Pro'irosbin, ;?'arv;rd ?lassachssetts, 195;.
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