THROMBOSIS @Pergamon
RESEARCH 13; 637~6%% Press Ltd.1978. Printed
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IMNJNOLOGICAL PROPERTIES OF C.XGNE PROTHROMBIN, ITS ACTIVATIOW PRODUCTS, PIASNA AND SERLN
T. Takeuchi and Y. Takeda Pepartnent of Medicine, University of Colorado School of Medicine, Denver, Colorado USA (Received 18.5.1978; in revised form 25.7.1.978. Accepted by Editor H.L. Nossel)
ABSTRACT Immunological properties of canine prothrombin(PO), prethrombin 1 (Pl), fragment l(Fl), fragment 2(F2), thrombin(Tl), plasma and serun were studied by the use of immunoelectrophoresis(IEP), crossed immunoelectrophoresis(CIEP) and Ouchterlony analysis. Antibodies against PO and Tl were used. The results showed that PO, Pl, Fl, F2 and Tl all had a variable affinity with anti-PO antibody but that only PO, Pl and Tl had a demonstrable affinity with anti-T1 antibody. Fl and F2 did not show any detectable affinity. Quantitative affinity study by the use of I-125-labeled proteins gave the following average values for % bound I-125-proteins in 3 hr incubation with the antibody against PO or Tl: with anti-PO antibody, 81 + 2 (SD) % for I-125-PO, 79.1 t 2(SD) % for I-125-Pl, 23.5 f 3(SD) % for I-125-Fl, 25.5 f 2(SD) % for I-125-F2 and 25.2 + 3(SD) % for I-125-Tl, and with anti-T1 antibody, 28 + 2(SD) % for I-125-PO, 80.5 + 2(SD) % for I-125-Pl, 1.0 f O.O3(SD) % for I-125-Fl, 1.3 f O.O3(SD) % for I-125-F2, and 81 f 3(SD) % for I-125-Tl. With the use of CIEP and anti-PO antibody, serm was found to contain Fl, F2 and a trace amount of Pl, and plasma contained only PO, but upon activation with tissue thromboplastin, plasma was found to generate Pl, Fl, F2 and Tl with time. These results indicate that a simple, specific and quantitative immunoassay of Tl by the use of anti-T1 antibody or anti-PO antibody may not be possible at least in dogs, but that CIEP by the use of anti-PO antibody might be used as a diagnostic means of in vivo PO activation.
INTRODUCTION A few studies have appeared of the immunological properties of human and bovine prothrombin and their activation products(l-51, but no such studies of canine prothranbin and its activation products are available. We therefore purified canine prothranbin and its activation products, and studied sane of their immunological properties, using the antibodies against canine prothrcmbin and thranbin. One of the objectives was to find a convenient diagnostic means of prothranbin activation. 637
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MATERIALS AND METHODS Purification of canine orothranbin(PO1.orethrunbin l(P11. fragment l(FL). fragment Z(F21 and thronbin(T1).Canine PO was purified as reported previously(6) by a canbination of BaS04 adsorption, elution of adsorbed PO in 0.25 M sodium citrate, concentrationof the eluate by (NH4)2S04 fractionation at 80 % saturation and DEAE cellulose chranatography.Tl was prepared as described elsewhere(61.For preparationof Fl and F2, purified canine PO was first activated with activated canine Factor X(FXa)(7, 8) in the presence of Ca* and phospholipids,and the activation mixture was chromatographedby the use of QAE-SephadexA-SO chranatography(PharmaciaFine Chemicals Corp., Piscataway, N. J,)(6). The column size was 1.6 cm x 40 cm, and a gradient of 0.1 to 0.6 M NaCl in 0.02 M tris-HCl buffer(pH 7.5) was used. The volwne of the mixing chamber was 120 ml. Then, the peaks 5 and 6 in Fig, 2(6) were collected and the volme was reduced by pervaporation.After dialysis against the starting buffer, each was rechromatographedby the use of a smaller QAESephadex col~~nnof 0.9 cm x 24 cm in size. The size of the mixing chamber was 100 ml, and the same gradient was used. The volume of the purified Fl and F2 vas reduced by pervaporation.In the preparation of Pl, canine PO was first activated with 50 NIH units of bovine thrcmbin(ParkeDavis & Co,, Detroit, Michigan) for 2 hr at 37 degrees C. About 6.8 mg of PO was activated. The flow rate was 10 ml per hr and the fraction volune was about 1.4 ml. The effluent of about 56 ml to 70 ml was collected, and the volume of about 14 ml collected was reduced by pervaporation.The purity of the preparedmaterials was tested by the use of polyacrylamidegel electrophoresis(9)and SDS polyacrylamidegel electrophoresis(SDSPAGE)(Y), using 5.6 g % polyacrylamidegels and 5 mm x 10 cm in size. Prethranbin 2(P2) was not studied because of the difficulty in preparation. Production of the antibody against canine PO or Tl. About 0.5 mg each of PO or Tl in Freund,s canplete adjuvant was intramuscularlyinjected into rabbits weekly for a 3 month period and the antibodies were harvested by a heart puncture within 2 weeks after the last injection, Each antibody was absorbed with BaS04-adsorbedcanine plasma and stored in a freezer in small aliquots. Immunoelectrophoresis(IEP), crossed immuncelectrophoresis(CIEP) and Ouchterlony analysis. The buffer used for IEP, CIEP, and Ouchterlonyanalysis was of the following canposition: barbital 0.55 g, sodium barbital 3.5 g, merthiolate 0.1 g, calcium lactate 0.51 g in 1 liter of H20 at pH 8.6. Agarose gel of 1 g per 100 ml of the buffer was used. The tank buffer for IEP and CIEP was I barbital 5.52 g, sodium barbital 35 g, calciLPnlactate 1.52 g and merthiolate 0.4 g in 4 liter of H20 at pH 8.6. The size of the glass slide for IEP was 2.5 cm x 7.5 cm and that for CIEP was 5 cm x 7.5 cm. Electric current of about 2 to 3 ma per slide was applied for IEP for a 2 hour period and also for the first phase of CIEP. For the second-phase of CIEP, about 10 ma per slide was applied for 2 hr. The second-phasegel contained the antibody against PO or Tl. Quantitation of the affinity of PO, Pl, Fl, F2 and Tl with the antibody against PO or Tl. First, PO, Pl, Fl, F2 and Tl were labeled with I-125 at a ratio of about 0.25 atan iodine per molecule of each protein, using the iodine monochloridemethod of McFarlane(l0).Free I-125 in each preparationwas removed by a passage through a Sephadex G-50 colcPnnequilibratedwith 0.1 M NaCl in 0.02 M tris-HCl buffer(pH 7.5) and 0.9 cm x 25 cm in size. The free I-125 remaining in a preparationwas usually less than 1 % of the total radioactivity. The radioactivityof each was 1.525 x lo6 cpn per ml for I-125-PO, 0.878 x 106 cpm per ml for I-125-~1, 1.085 x 106 cpm per ml for I-125-Fl,
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0.926 x 106 cp;i per ml for I-125-F2 and 0.620 x 106 cpm per ml for 1-125-X. The protein concentration of each I-125-protein solution was approximately determined by its OD at 280 w and the extinction coefficients reported previously for bovine materialscll, 12), which are 14.4 for PO, 16.4 for Pl, 10.5 for Fl, 13.6 for F2 and 19.5 for Tl. The protein concentration was 95 ug per ml for I-125-PO, 57 ug per ml for I-125-Pl, 111 ug per ml for I-125-Fl, 33 ug per ml for I-125-F2, and 59 ug per ml for 1-125-X. Thus, the specific activity was 1.6 x 104 cpm per ug for I-125-PO, 1.5 x 102 cpr? per ug for I125-Pl, 0.9 x 104 cprn per ug for I-125-Fl, 2.8 x 104 cpm per ug for I-125-F2 and 1.1 x 104 cpm per ug for I-125-Tl. The experiments were performed as follows: to 0.5 ml of 0.1 M NaCl in 0.02 M tris-Hirl b*uffer(pH 7.5) was added 0.1 ml of the antibody against PO or Tl and 0.05 ml of each I-125-protein solution, and the mixture was incubated at rooo temperature for a 3 hr period while being gently rotated on a rotator. Then, to each was added 0.2 ml of antirabbit gamma-globulin(Behring Diagnostics, Scmmerville, N. J.> and each was incubated in a refrigerator for about 10 hr. The mixture was then spun at 3,000 rpm for 10 min, the supernate was removed by suction, and the precipitate was washed 3 times with 0.9 g % NaCl. The washed precipitates were then counted and the % bound I-125-protein was determined by dividing the bound cpm with the cpn of each I-125-protein initially added. Finally, ug I-125-protein bound was calculated fran the % bound I-125-protein and the ug I-125-protein initially added. Activation of canine plasma and examination of the activation products by CIEP. For activation of plasma. defibrinated plasma was used. About 1 ml of canine plasma was first defibrinated with 10 NIH units of bovine thrombin (Parke Davis & Co., Detroit, Michigan). To 0.8 ml of defibrinated plasma was added 0.2 ml of 0.25 M CaC12 and 0.2 ml of tissue thrcmboplastin(Difco Lab., Detroit, Michigan), and the mixture was incubated at 37 degrees C for 15 min. A small aliquot of samples was obtained at 0 set, 10 set, 20 set, 30 set, 1 min, 2 min, 5 min and 15 min, and the activation was stopped by the use of DFP and soy bean trypsin inhibitor(Sigma Chemical Co., St Louis, Missouri) at a final concentration of 8 mM DFP per ml and 83 ug trypsin inhibitor per ml of 0.1 M NaCl in 0.02 M tris-HCl buffer(pH 7.5). RESULTS Purity of PO, Pl, Fl, F2 and Tl as examined by PAGE and SDS PAGE. Fig. 1 shows the SDS PAGE patterns of PO, Pl, Fl, F2 and Tl in canparison with those of activated PO(F) which is at 10 min of incubation with FXa as described previously(6). It is seen that PO, Pl, Fl, F2 and Tl appear to be pure, but PO is really contaminated with a trace amount of impurities and Tl is also contaminated with a trace amount of T2. To determine the degree of T2 contamination, I-125-Tl was electrophoresed by SDS PAGE. The gel was then cut into 2 mm slices, and each was counted to determine the I-125 distribution between Tl and T2. The average value in 3 measurements was 93 + 3(SD) % of the total I-125 in Tl and the remainder in T2. The PAGE patterns of the purified materials are not shown here, because they showed less impurities than those by SDS PAGE. IEP, Ouchterlony analysis and CIEP of PO, Pl, Fl, F2, Tl and activated PO by the use of the antibody against PO or Tl. Fig. 2 shows the IEP patterns and Ouchterlony analysis. The wells A, C, E and C were filled with PO. Wells B, D, F, H, I and J were filled with Tl, Pl, Fl, F2, Tl and activated PO, respectively. The central troughs were filled with anti-PO antibody. It is seen that PO, Tl, Pl, Fl and F2 all formed precipitin lines with the anti-PO antibody, and that F2 had the greatest mobility. Well J shows that activated PO contained
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fl, Pl, Fl and F2. PO was activated with FXa in the presence of Ca*, phospholipids and Factor V as described previously(6).Ouchterlonyanalysis on the left is by the use of anti-PO antibody in the central well and that on the right is by the use of anti-T1 antibody. The peripheralwells a, b, c, d, e and f on both sides were filled with PO, Pl, Tl, Fl, F2 and Tl, respectively, It is seen that all the wells on the left formed visible precipitinlines with the antibody, although Tl in well c and f showed less visible precipitinlines. It is also seen on the right that wells a, b, c, and f formed precipitinlines but that Fl and F2 in wells d and e did not form precipitinlines at all with anti-T1 antibody. Fig, 3 shows the IEP patterns by the use of the antibody against Tl or PO. Wells A, C, E, G, I, and R were filled with canine PO.
FIG. 1 SDS PAGE patterns of purified PO(A), Pi(B), Tl(C), Fl(D), and F2(E): Gel F shows the patterns of activatedPO at 10 min incubation(6).It is seen that PO, Pl, Fl, F2 and TL appear Pure.
FIG. 2 IEP and Ouchterlonyanalysis of PO, Pl, Fl, F2, activated PO. See text for details.
Wells B, D, F, H, J, and L were filled with Tl, Pl, Fl, F2, activatedPO and hunan PO as purified by a previous method(61, respectively.The central troughs in the top 5 slides were filled with anti-T1 antibody and the trough in the bottom slide was filled with anti-PO antibody. It is seen that PO, Tl, and Pl all formed precipitinlines with anti-T1 antibody, but that Fl and F2 in wells F and H did not form precipitinlines at all. In well J, it is seen that activated PO containedTl and Pl and that Fl and F2 were not visible by this method, Well L shows a precipitinline formed between hunan PO and anti-caninePO antibody. Fig, 4 shows the CIEP patterns of PO, Pl, Fl, F2, Tl, canine plasma(F) and serun(G).The second-phasegel in A, 8, C, D, F, and G contained 4.1 % of anti-PO antibody and that in E contained anti-T1 antibody. It is seen that
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Activation of canine plasma with tissue thranboplastin as demonstrated by CIEP. Fig. 5 shows the CIEP patterns of activated plasma. The second-phase gelcontained 4.1 Z of anti-PO antibody, It is seen that PO is rapidly consLned with time, that Pl appears(B) and is consumed rapidly, that Fl and F2 are formed in increasing amounts, and that 11 is finally generated(G, H).
FIG. 5 CIEP patterns of canine plasma activated with tissue thromboplastin: Defibrinated plasma was activated and samples were obtained at 0 set(A), 10 set(B), 20 set(C), 30 set(D), 1 min(E), 2 min(F), 5 min(C>, and 15 min(H). It is Seen that PO in plasma was rapidly consumed, Pl appears(B) and is rapidly conswned, that Fl and F2 appear and that Tl is finally formed(G, 9).
DISCUSSION The results(Figs. 2, 3, 5) showed that anti-PO antibody reacted with PO, Pl, Fl, F2 and Tl. On the other hand, anti-T1 antibody reacted with PO, Pl and Tl, but not with Fl and F2. The quantitative affinity study showed that anti-PO antibody had the greatest affinity for PO and that anti-T1 antibody had the greatest affinity for Tl, but that the affinity for PO vas considerably high. The quantitative results were generally in accord with the qualitative results(Figs. 2, 3, 5). The present finding that anti-PO antibody reacts with Tl to a significant extent(Fig. 2B) is in agreement with that by Shapiro(l) using human material, but in contrast with the results of Seegers et al. who used bovine material(5). In an attempt to obtain an antibody specific to Tl, we absorbed the anti-T1 antibody with purified PO, but the absorbed antibody was too weak to be useful for any immunological studies. Thus, these findings indicate that a simple and specific immunoassay of thrombin by the use of anti Tl antibody or anti-PO antibody nay not be possible at least in dogs. In the
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canine serm contained Fl, F2 and Pl by this method.
FIG. 3 IEP patterns of PO, Pl, Fl, F2, Tl, activatedPO and htwan PO. See text for details.
FIG. 4 CIEP patterns of PO, Pl, Fl, F2, Tl, plasma and serum by the use of the antibody against PO or Tl: The second-phasegels in all except E contained 4.1 % anti-PO antibody and that in E contained anti-T1 antibody. It is seen that F2 had the greatest mobility, that plasma(F) contained PO only, whereas serum contained FL, F2 and 'a trace amount Pl.
Quantitationof the affinity of PO, Pl, Fl, F2 and Tl with the antibody against PO or Tl. The average value for % bound I-125-proteinwith anti-PO antibody in 3 measurementswas 81 f: 2(SD) % for I-125-PO, 79.1 f 2(SD) % for I-125-Pl, 23.5 f 3(SD) % for I-125-Fl, 25.5 f 2(SD) % for I-125-F2 and 25.2 +-3(SD) % for I-125-Tl. The average value for % bound I-125-proteinwith anti-T1 antibody in 3 measurements was 28 C 2(SD) % for I-125-PO, 80.5 f 2 (SD) % for I-125-Pl, 1.0 i O.O3(SD) % for I-125-Fl, 1.3 f O.O3(SD) % for I-125-F2 and 81 f 3(SD) % for I-125-Tl. In terms of the actual amount of I125-proteinbound with the anti-PO antibody during the 3 hr incubationin 3 measurementswas 3.8 f O.l(SD) ug for I-125-PO, 2.3 f O.l($D) ug for I-125Pl, 1.3 + O.l(SD) ug for I-125-Fl, 1.4 f O.O8(SD) ug for I-125-F2 and 0.74 f O.O8(SD> ug for I-125-Tl. With the use of anti-T1 antibody, the average value was 1.33 f O.OS(SD) ug for I-125-PO, 2.29 +-O.O6(SD) ug for I-125-Pl, 0.07 + O.O02(SD) ug for I-125-Fl, 0.07 f O.Ol(SD> ug for I-125-F2 and 2.39 f O.O2(SD) ug for I-125-Tl.
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search for a diagnosticmethod of PO activation,we tried IEP and CIEP. The results(Figs.2H, 4D) showed that F2 had the greatest mobility and it seemed that F2 might be quantitativelyseparated. If this can be done, an appropriate quantitative immunoassayof F2 seems feasible. It would have a same significance as thrombin assay, since fresh plasma does not contain FZ(Fig. 4F) until it becomes activated(Fig.5). This finding is not in accord with the report that bovine plasma contains PO, Pl, Fl, F2 as well as ?2 and/ or Tl(5). Figs. 25 and 5 also imply that IEP and CIEP might be used as a means of detecting PO activation in vivo. IEP requires about 2 hr and CIEP about 4 to 5 hr, so that the time required would not be excessive for then to be clinically useful, but it remains to be seen if they are sensitive enough to detect a mild degree of PO activation in vivo. In our previous study of canine PO activation(6),we noticed the emergence of fragment 1.2(13, 14), but in Figs. 25 and 5, it is seen that we did not detect it. The anti-PO antibody has considerableaffinity with Fl or F2. Therefore, it seems that it should have at least enough affinity with fragment 1.2 for its detection. Thus, the fact that we did not detect its presence seems to be due to the very transient nature of fragment l-2(13, 14). Also, in Fig. 5, the presence of Tl is not apparent despite the presence of Fl and F2 except in Fig. 5G and 5H. This is probably due to the low affinity of anti-PO antibody with Tl. Another point which needs mention is about T2 contamination of our Tl preparation.In Fig. lC, T2 is only faintly visible, but its presence was a very consistent finding in our previous(6)and present studies. We did not separate it fro-aTl to study its property. Therefore, we are uncertain of its significance,but the suggestion that T2 is a degradation product of Tl(13) seems very plausible to us. The finding that human PO has a significantaffinity for anti-caninePO antibody(Fig. 3L) indicates that PO is not a species-specificprotein and is in agreement with that by Waltz et al.(Z). ACKNOWLEDGMENT This work was supported by Research Grant HL-11686 from the National Heart, Lung and Blood Institute,Bethesda, Maryland. REFERENCES 1.
SHAPIRO, S. S. Hunan prothrombin activation. Science 162, 127, 1968.
2.
WALE, D. A., SEEGERS, W. H., HASSOUNA, H. I., AND REUTERBY, J. Hunan and bovine prothrcmbinsimilarities.Thrcmbos. Res. 4, 875, 1974.
3.
TASWELL, C., MCDUFFIE, F. C., AND MANN, K. G. Immunologicalrelationships of the intermediatesof prothrombinactivation. Immunochem.12, 339, 1975.
4.
SHUMAN, M. A., AND MAJERUS, P. W. The measurement of thranbin in clotting blood by radioimmunoassay.J. Clin. Invest. 58, 1249, 1976.
5.
SEEGERS, W. H., HASSOUNA, H. I., AND NOVOA, E. Immunologicalaspects of sane vitamin-K-dependentfactors and preparation of depleted plasmas. Thranbos. Res. 11, 633, 1977.
6.
TAKEUCHI, T., AND TAKEDA, Y. Physicochemicaland biological properties of canine prothrombinand thranbin. Thranbos. Res. 12, 635, 1978.
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7.
ARAI, H., AND TAKEDA, Y. Properties and radioimmunoassayof canine Factor X. Thranbos. Res. 11, 57, 197i.
8.
AR-U, H., AND TAKEDA, Y. Physicochemicaland immunologicalproperties of canine Factor X and activated Factor X. Thrombos. Res. 11, 357, 1975.
9.
FAIRBANKS, G., STECK, T. L., AND WALL&H, D. F. H. Electrophoreticanalysis of the major polypeptidesof the hunan erythrocytemembrane. Biochem. 10, 2606, 1971.
10. MCFARLANE, A. S. Efficient tracer labelling of proteins with iodine. Nature(London).182, 53, 1958. 11. BAJAJ, S. P., BDTOKOWSKI, R. J., AND MANN, K. G. Prothrombin fragments. Ca2+ binding and activation kinetics. J. Biol. Chem. 250, 2150, 1975. 12. OWEN, W. G., ESMON, C. T., AND JACKSON, C. M. The conversion of prothranbin to thrunbin.J. Biol. Chem. 249, 594, 1974. 13. STENN, K. S., AND BLODT, E. R. Mechanism of bovine prothrombinactivation by an insoluble preparationof bovine Factor Xa(Thrambokinase). Biochem. 11, 4502, 1972. 14. ESMON, C. Ti, OWEN, W. C., AND JACKSON, C. M. The conversion of prothrcmbin to thranbin. II. Differentiationbetween thrcmbin- and Factor Xa-catalyzedproteolyses.J. Biol. Chem. 249, 606, 1974.
RESEARCH 13; 637~6%% Press Ltd.1978. Printed
in Great Britain 00~~-38~8~7~/lOOl-O~37
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IMNJNOLOGICAL PROPERTIES OF C.XGNE PROTHROMBIN, ITS ACTIVATIOW PRODUCTS, PIASNA AND SERLN
T. Takeuchi and Y. Takeda Pepartnent of Medicine, University of Colorado School of Medicine, Denver, Colorado USA (Received 18.5.1978; in revised form 25.7.1.978. Accepted by Editor H.L. Nossel)
ABSTRACT Immunological properties of canine prothrombin(PO), prethrombin 1 (Pl), fragment l(Fl), fragment 2(F2), thrombin(Tl), plasma and serun were studied by the use of immunoelectrophoresis(IEP), crossed immunoelectrophoresis(CIEP) and Ouchterlony analysis. Antibodies against PO and Tl were used. The results showed that PO, Pl, Fl, F2 and Tl all had a variable affinity with anti-PO antibody but that only PO, Pl and Tl had a demonstrable affinity with anti-T1 antibody. Fl and F2 did not show any detectable affinity. Quantitative affinity study by the use of I-125-labeled proteins gave the following average values for % bound I-125-proteins in 3 hr incubation with the antibody against PO or Tl: with anti-PO antibody, 81 + 2 (SD) % for I-125-PO, 79.1 t 2(SD) % for I-125-Pl, 23.5 f 3(SD) % for I-125-Fl, 25.5 f 2(SD) % for I-125-F2 and 25.2 + 3(SD) % for I-125-Tl, and with anti-T1 antibody, 28 + 2(SD) % for I-125-PO, 80.5 + 2(SD) % for I-125-Pl, 1.0 f O.O3(SD) % for I-125-Fl, 1.3 f O.O3(SD) % for I-125-F2, and 81 f 3(SD) % for I-125-Tl. With the use of CIEP and anti-PO antibody, serm was found to contain Fl, F2 and a trace amount of Pl, and plasma contained only PO, but upon activation with tissue thromboplastin, plasma was found to generate Pl, Fl, F2 and Tl with time. These results indicate that a simple, specific and quantitative immunoassay of Tl by the use of anti-T1 antibody or anti-PO antibody may not be possible at least in dogs, but that CIEP by the use of anti-PO antibody might be used as a diagnostic means of in vivo PO activation.
INTRODUCTION A few studies have appeared of the immunological properties of human and bovine prothrombin and their activation products(l-51, but no such studies of canine prothranbin and its activation products are available. We therefore purified canine prothranbin and its activation products, and studied sane of their immunological properties, using the antibodies against canine prothrcmbin and thranbin. One of the objectives was to find a convenient diagnostic means of prothranbin activation. 637
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MATERIALS AND METHODS Purification of canine orothranbin(PO1.orethrunbin l(P11. fragment l(FL). fragment Z(F21 and thronbin(T1).Canine PO was purified as reported previously(6) by a canbination of BaS04 adsorption, elution of adsorbed PO in 0.25 M sodium citrate, concentrationof the eluate by (NH4)2S04 fractionation at 80 % saturation and DEAE cellulose chranatography.Tl was prepared as described elsewhere(61.For preparationof Fl and F2, purified canine PO was first activated with activated canine Factor X(FXa)(7, 8) in the presence of Ca* and phospholipids,and the activation mixture was chromatographedby the use of QAE-SephadexA-SO chranatography(PharmaciaFine Chemicals Corp., Piscataway, N. J,)(6). The column size was 1.6 cm x 40 cm, and a gradient of 0.1 to 0.6 M NaCl in 0.02 M tris-HCl buffer(pH 7.5) was used. The volwne of the mixing chamber was 120 ml. Then, the peaks 5 and 6 in Fig, 2(6) were collected and the volme was reduced by pervaporation.After dialysis against the starting buffer, each was rechromatographedby the use of a smaller QAESephadex col~~nnof 0.9 cm x 24 cm in size. The size of the mixing chamber was 100 ml, and the same gradient was used. The volume of the purified Fl and F2 vas reduced by pervaporation.In the preparation of Pl, canine PO was first activated with 50 NIH units of bovine thrcmbin(ParkeDavis & Co,, Detroit, Michigan) for 2 hr at 37 degrees C. About 6.8 mg of PO was activated. The flow rate was 10 ml per hr and the fraction volune was about 1.4 ml. The effluent of about 56 ml to 70 ml was collected, and the volume of about 14 ml collected was reduced by pervaporation.The purity of the preparedmaterials was tested by the use of polyacrylamidegel electrophoresis(9)and SDS polyacrylamidegel electrophoresis(SDSPAGE)(Y), using 5.6 g % polyacrylamidegels and 5 mm x 10 cm in size. Prethranbin 2(P2) was not studied because of the difficulty in preparation. Production of the antibody against canine PO or Tl. About 0.5 mg each of PO or Tl in Freund,s canplete adjuvant was intramuscularlyinjected into rabbits weekly for a 3 month period and the antibodies were harvested by a heart puncture within 2 weeks after the last injection, Each antibody was absorbed with BaS04-adsorbedcanine plasma and stored in a freezer in small aliquots. Immunoelectrophoresis(IEP), crossed immuncelectrophoresis(CIEP) and Ouchterlony analysis. The buffer used for IEP, CIEP, and Ouchterlonyanalysis was of the following canposition: barbital 0.55 g, sodium barbital 3.5 g, merthiolate 0.1 g, calcium lactate 0.51 g in 1 liter of H20 at pH 8.6. Agarose gel of 1 g per 100 ml of the buffer was used. The tank buffer for IEP and CIEP was I barbital 5.52 g, sodium barbital 35 g, calciLPnlactate 1.52 g and merthiolate 0.4 g in 4 liter of H20 at pH 8.6. The size of the glass slide for IEP was 2.5 cm x 7.5 cm and that for CIEP was 5 cm x 7.5 cm. Electric current of about 2 to 3 ma per slide was applied for IEP for a 2 hour period and also for the first phase of CIEP. For the second-phase of CIEP, about 10 ma per slide was applied for 2 hr. The second-phasegel contained the antibody against PO or Tl. Quantitation of the affinity of PO, Pl, Fl, F2 and Tl with the antibody against PO or Tl. First, PO, Pl, Fl, F2 and Tl were labeled with I-125 at a ratio of about 0.25 atan iodine per molecule of each protein, using the iodine monochloridemethod of McFarlane(l0).Free I-125 in each preparationwas removed by a passage through a Sephadex G-50 colcPnnequilibratedwith 0.1 M NaCl in 0.02 M tris-HCl buffer(pH 7.5) and 0.9 cm x 25 cm in size. The free I-125 remaining in a preparationwas usually less than 1 % of the total radioactivity. The radioactivityof each was 1.525 x lo6 cpn per ml for I-125-PO, 0.878 x 106 cpm per ml for I-125-~1, 1.085 x 106 cpm per ml for I-125-Fl,
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0.926 x 106 cp;i per ml for I-125-F2 and 0.620 x 106 cpm per ml for 1-125-X. The protein concentration of each I-125-protein solution was approximately determined by its OD at 280 w and the extinction coefficients reported previously for bovine materialscll, 12), which are 14.4 for PO, 16.4 for Pl, 10.5 for Fl, 13.6 for F2 and 19.5 for Tl. The protein concentration was 95 ug per ml for I-125-PO, 57 ug per ml for I-125-Pl, 111 ug per ml for I-125-Fl, 33 ug per ml for I-125-F2, and 59 ug per ml for 1-125-X. Thus, the specific activity was 1.6 x 104 cpm per ug for I-125-PO, 1.5 x 102 cpr? per ug for I125-Pl, 0.9 x 104 cprn per ug for I-125-Fl, 2.8 x 104 cpm per ug for I-125-F2 and 1.1 x 104 cpm per ug for I-125-Tl. The experiments were performed as follows: to 0.5 ml of 0.1 M NaCl in 0.02 M tris-Hirl b*uffer(pH 7.5) was added 0.1 ml of the antibody against PO or Tl and 0.05 ml of each I-125-protein solution, and the mixture was incubated at rooo temperature for a 3 hr period while being gently rotated on a rotator. Then, to each was added 0.2 ml of antirabbit gamma-globulin(Behring Diagnostics, Scmmerville, N. J.> and each was incubated in a refrigerator for about 10 hr. The mixture was then spun at 3,000 rpm for 10 min, the supernate was removed by suction, and the precipitate was washed 3 times with 0.9 g % NaCl. The washed precipitates were then counted and the % bound I-125-protein was determined by dividing the bound cpm with the cpn of each I-125-protein initially added. Finally, ug I-125-protein bound was calculated fran the % bound I-125-protein and the ug I-125-protein initially added. Activation of canine plasma and examination of the activation products by CIEP. For activation of plasma. defibrinated plasma was used. About 1 ml of canine plasma was first defibrinated with 10 NIH units of bovine thrombin (Parke Davis & Co., Detroit, Michigan). To 0.8 ml of defibrinated plasma was added 0.2 ml of 0.25 M CaC12 and 0.2 ml of tissue thrcmboplastin(Difco Lab., Detroit, Michigan), and the mixture was incubated at 37 degrees C for 15 min. A small aliquot of samples was obtained at 0 set, 10 set, 20 set, 30 set, 1 min, 2 min, 5 min and 15 min, and the activation was stopped by the use of DFP and soy bean trypsin inhibitor(Sigma Chemical Co., St Louis, Missouri) at a final concentration of 8 mM DFP per ml and 83 ug trypsin inhibitor per ml of 0.1 M NaCl in 0.02 M tris-HCl buffer(pH 7.5). RESULTS Purity of PO, Pl, Fl, F2 and Tl as examined by PAGE and SDS PAGE. Fig. 1 shows the SDS PAGE patterns of PO, Pl, Fl, F2 and Tl in canparison with those of activated PO(F) which is at 10 min of incubation with FXa as described previously(6). It is seen that PO, Pl, Fl, F2 and Tl appear to be pure, but PO is really contaminated with a trace amount of impurities and Tl is also contaminated with a trace amount of T2. To determine the degree of T2 contamination, I-125-Tl was electrophoresed by SDS PAGE. The gel was then cut into 2 mm slices, and each was counted to determine the I-125 distribution between Tl and T2. The average value in 3 measurements was 93 + 3(SD) % of the total I-125 in Tl and the remainder in T2. The PAGE patterns of the purified materials are not shown here, because they showed less impurities than those by SDS PAGE. IEP, Ouchterlony analysis and CIEP of PO, Pl, Fl, F2, Tl and activated PO by the use of the antibody against PO or Tl. Fig. 2 shows the IEP patterns and Ouchterlony analysis. The wells A, C, E and C were filled with PO. Wells B, D, F, H, I and J were filled with Tl, Pl, Fl, F2, Tl and activated PO, respectively. The central troughs were filled with anti-PO antibody. It is seen that PO, Tl, Pl, Fl and F2 all formed precipitin lines with the anti-PO antibody, and that F2 had the greatest mobility. Well J shows that activated PO contained
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fl, Pl, Fl and F2. PO was activated with FXa in the presence of Ca*, phospholipids and Factor V as described previously(6).Ouchterlonyanalysis on the left is by the use of anti-PO antibody in the central well and that on the right is by the use of anti-T1 antibody. The peripheralwells a, b, c, d, e and f on both sides were filled with PO, Pl, Tl, Fl, F2 and Tl, respectively, It is seen that all the wells on the left formed visible precipitinlines with the antibody, although Tl in well c and f showed less visible precipitinlines. It is also seen on the right that wells a, b, c, and f formed precipitinlines but that Fl and F2 in wells d and e did not form precipitinlines at all with anti-T1 antibody. Fig, 3 shows the IEP patterns by the use of the antibody against Tl or PO. Wells A, C, E, G, I, and R were filled with canine PO.
FIG. 1 SDS PAGE patterns of purified PO(A), Pi(B), Tl(C), Fl(D), and F2(E): Gel F shows the patterns of activatedPO at 10 min incubation(6).It is seen that PO, Pl, Fl, F2 and TL appear Pure.
FIG. 2 IEP and Ouchterlonyanalysis of PO, Pl, Fl, F2, activated PO. See text for details.
Wells B, D, F, H, J, and L were filled with Tl, Pl, Fl, F2, activatedPO and hunan PO as purified by a previous method(61, respectively.The central troughs in the top 5 slides were filled with anti-T1 antibody and the trough in the bottom slide was filled with anti-PO antibody. It is seen that PO, Tl, and Pl all formed precipitinlines with anti-T1 antibody, but that Fl and F2 in wells F and H did not form precipitinlines at all. In well J, it is seen that activated PO containedTl and Pl and that Fl and F2 were not visible by this method, Well L shows a precipitinline formed between hunan PO and anti-caninePO antibody. Fig, 4 shows the CIEP patterns of PO, Pl, Fl, F2, Tl, canine plasma(F) and serun(G).The second-phasegel in A, 8, C, D, F, and G contained 4.1 % of anti-PO antibody and that in E contained anti-T1 antibody. It is seen that
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Activation of canine plasma with tissue thranboplastin as demonstrated by CIEP. Fig. 5 shows the CIEP patterns of activated plasma. The second-phase gelcontained 4.1 Z of anti-PO antibody, It is seen that PO is rapidly consLned with time, that Pl appears(B) and is consumed rapidly, that Fl and F2 are formed in increasing amounts, and that 11 is finally generated(G, H).
FIG. 5 CIEP patterns of canine plasma activated with tissue thromboplastin: Defibrinated plasma was activated and samples were obtained at 0 set(A), 10 set(B), 20 set(C), 30 set(D), 1 min(E), 2 min(F), 5 min(C>, and 15 min(H). It is Seen that PO in plasma was rapidly consumed, Pl appears(B) and is rapidly conswned, that Fl and F2 appear and that Tl is finally formed(G, 9).
DISCUSSION The results(Figs. 2, 3, 5) showed that anti-PO antibody reacted with PO, Pl, Fl, F2 and Tl. On the other hand, anti-T1 antibody reacted with PO, Pl and Tl, but not with Fl and F2. The quantitative affinity study showed that anti-PO antibody had the greatest affinity for PO and that anti-T1 antibody had the greatest affinity for Tl, but that the affinity for PO vas considerably high. The quantitative results were generally in accord with the qualitative results(Figs. 2, 3, 5). The present finding that anti-PO antibody reacts with Tl to a significant extent(Fig. 2B) is in agreement with that by Shapiro(l) using human material, but in contrast with the results of Seegers et al. who used bovine material(5). In an attempt to obtain an antibody specific to Tl, we absorbed the anti-T1 antibody with purified PO, but the absorbed antibody was too weak to be useful for any immunological studies. Thus, these findings indicate that a simple and specific immunoassay of thrombin by the use of anti Tl antibody or anti-PO antibody nay not be possible at least in dogs. In the
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canine serm contained Fl, F2 and Pl by this method.
FIG. 3 IEP patterns of PO, Pl, Fl, F2, Tl, activatedPO and htwan PO. See text for details.
FIG. 4 CIEP patterns of PO, Pl, Fl, F2, Tl, plasma and serum by the use of the antibody against PO or Tl: The second-phasegels in all except E contained 4.1 % anti-PO antibody and that in E contained anti-T1 antibody. It is seen that F2 had the greatest mobility, that plasma(F) contained PO only, whereas serum contained FL, F2 and 'a trace amount Pl.
Quantitationof the affinity of PO, Pl, Fl, F2 and Tl with the antibody against PO or Tl. The average value for % bound I-125-proteinwith anti-PO antibody in 3 measurementswas 81 f: 2(SD) % for I-125-PO, 79.1 f 2(SD) % for I-125-Pl, 23.5 f 3(SD) % for I-125-Fl, 25.5 f 2(SD) % for I-125-F2 and 25.2 +-3(SD) % for I-125-Tl. The average value for % bound I-125-proteinwith anti-T1 antibody in 3 measurements was 28 C 2(SD) % for I-125-PO, 80.5 f 2 (SD) % for I-125-Pl, 1.0 i O.O3(SD) % for I-125-Fl, 1.3 f O.O3(SD) % for I-125-F2 and 81 f 3(SD) % for I-125-Tl. In terms of the actual amount of I125-proteinbound with the anti-PO antibody during the 3 hr incubationin 3 measurementswas 3.8 f O.l(SD) ug for I-125-PO, 2.3 f O.l($D) ug for I-125Pl, 1.3 + O.l(SD) ug for I-125-Fl, 1.4 f O.O8(SD) ug for I-125-F2 and 0.74 f O.O8(SD> ug for I-125-Tl. With the use of anti-T1 antibody, the average value was 1.33 f O.OS(SD) ug for I-125-PO, 2.29 +-O.O6(SD) ug for I-125-Pl, 0.07 + O.O02(SD) ug for I-125-Fl, 0.07 f O.Ol(SD> ug for I-125-F2 and 2.39 f O.O2(SD) ug for I-125-Tl.
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search for a diagnosticmethod of PO activation,we tried IEP and CIEP. The results(Figs.2H, 4D) showed that F2 had the greatest mobility and it seemed that F2 might be quantitativelyseparated. If this can be done, an appropriate quantitative immunoassayof F2 seems feasible. It would have a same significance as thrombin assay, since fresh plasma does not contain FZ(Fig. 4F) until it becomes activated(Fig.5). This finding is not in accord with the report that bovine plasma contains PO, Pl, Fl, F2 as well as ?2 and/ or Tl(5). Figs. 25 and 5 also imply that IEP and CIEP might be used as a means of detecting PO activation in vivo. IEP requires about 2 hr and CIEP about 4 to 5 hr, so that the time required would not be excessive for then to be clinically useful, but it remains to be seen if they are sensitive enough to detect a mild degree of PO activation in vivo. In our previous study of canine PO activation(6),we noticed the emergence of fragment 1.2(13, 14), but in Figs. 25 and 5, it is seen that we did not detect it. The anti-PO antibody has considerableaffinity with Fl or F2. Therefore, it seems that it should have at least enough affinity with fragment 1.2 for its detection. Thus, the fact that we did not detect its presence seems to be due to the very transient nature of fragment l-2(13, 14). Also, in Fig. 5, the presence of Tl is not apparent despite the presence of Fl and F2 except in Fig. 5G and 5H. This is probably due to the low affinity of anti-PO antibody with Tl. Another point which needs mention is about T2 contamination of our Tl preparation.In Fig. lC, T2 is only faintly visible, but its presence was a very consistent finding in our previous(6)and present studies. We did not separate it fro-aTl to study its property. Therefore, we are uncertain of its significance,but the suggestion that T2 is a degradation product of Tl(13) seems very plausible to us. The finding that human PO has a significantaffinity for anti-caninePO antibody(Fig. 3L) indicates that PO is not a species-specificprotein and is in agreement with that by Waltz et al.(Z). ACKNOWLEDGMENT This work was supported by Research Grant HL-11686 from the National Heart, Lung and Blood Institute,Bethesda, Maryland. REFERENCES 1.
SHAPIRO, S. S. Hunan prothrombin activation. Science 162, 127, 1968.
2.
WALE, D. A., SEEGERS, W. H., HASSOUNA, H. I., AND REUTERBY, J. Hunan and bovine prothrcmbinsimilarities.Thrcmbos. Res. 4, 875, 1974.
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TASWELL, C., MCDUFFIE, F. C., AND MANN, K. G. Immunologicalrelationships of the intermediatesof prothrombinactivation. Immunochem.12, 339, 1975.
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SHUMAN, M. A., AND MAJERUS, P. W. The measurement of thranbin in clotting blood by radioimmunoassay.J. Clin. Invest. 58, 1249, 1976.
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SEEGERS, W. H., HASSOUNA, H. I., AND NOVOA, E. Immunologicalaspects of sane vitamin-K-dependentfactors and preparation of depleted plasmas. Thranbos. Res. 11, 633, 1977.
6.
TAKEUCHI, T., AND TAKEDA, Y. Physicochemicaland biological properties of canine prothrombinand thranbin. Thranbos. Res. 12, 635, 1978.
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7.
ARAI, H., AND TAKEDA, Y. Properties and radioimmunoassayof canine Factor X. Thranbos. Res. 11, 57, 197i.
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AR-U, H., AND TAKEDA, Y. Physicochemicaland immunologicalproperties of canine Factor X and activated Factor X. Thrombos. Res. 11, 357, 1975.
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FAIRBANKS, G., STECK, T. L., AND WALL&H, D. F. H. Electrophoreticanalysis of the major polypeptidesof the hunan erythrocytemembrane. Biochem. 10, 2606, 1971.
10. MCFARLANE, A. S. Efficient tracer labelling of proteins with iodine. Nature(London).182, 53, 1958. 11. BAJAJ, S. P., BDTOKOWSKI, R. J., AND MANN, K. G. Prothrombin fragments. Ca2+ binding and activation kinetics. J. Biol. Chem. 250, 2150, 1975. 12. OWEN, W. G., ESMON, C. T., AND JACKSON, C. M. The conversion of prothranbin to thrunbin.J. Biol. Chem. 249, 594, 1974. 13. STENN, K. S., AND BLODT, E. R. Mechanism of bovine prothrombinactivation by an insoluble preparationof bovine Factor Xa(Thrambokinase). Biochem. 11, 4502, 1972. 14. ESMON, C. Ti, OWEN, W. C., AND JACKSON, C. M. The conversion of prothrcmbin to thranbin. II. Differentiationbetween thrcmbin- and Factor Xa-catalyzedproteolyses.J. Biol. Chem. 249, 606, 1974.
