- Email: [email protected]
Anticoagulant effects of autoprothrombin II-A and prothrombin fragment 1
THROMBOW RESEARCH Vol. 13.No. 2. pp. 233-243. 0 Pngamon PressLtd. 1978. PrintedinGreaf Britain.
1Y349-3848/78/0801-0233SO2.WO
ANTICOAGULANT EFFECTS OF AUTOPROTHROMBIN II-A AND PROTHROMBIN FRAGMENT 1 Walter H. Seegers, Richard A. Marlar and Daniel A. Walz Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201, U.S.A.
(Received 1.5.1978: in revised form 6.6.1978. Accepted by Editor K.M. Brinkhous)
ABSTRACT In 25% sodium citrate solution the quantity and rate of thrombin generated was lower with prothrombin than with prethrombin 1, when the molar ratio of substrate to Factor Xa was ten to one respectively. The difference was found to be due to inhibition by prothrombin fragment 1. Similar differences were observed in physiological saline solution. For the activation of prethrombin 1 in 25% sodium citrate solution Km = 714, and Vmax = 0.173 U/min/unit Factor Xa. In physiological saline solution the respective figures were 992 and 0.015. Protein C, activated by thrombin, reduced the activity of purified AC-globulin and inhibited the production of thrombin from prethrombin 1 by Factor Xa on a competitive basis. The maximum activation of Protein C by thrombin required about 60 min when the molar ratio was 75 to 1. Activated Protein C retained its activity for many hours even in the presence of relatively large quantities of thrombin. INTRODUCTION The natural inhibitors of blood coagulation are sufficiently numerous and effective to offer constant resistance to the function of procoagulants. After being derived from their precursors, several serine proteinases, including thrombin and Factor Xa, are inhibited by plasma antithrombin III.
This investigation was supported by Grant HL-03424-21 from the National Heart, Lung and Blood Institute, National Institutes of Health, U.S. Public Health Service, the McGregor Fund, and the Skillman Foundation, Detroit, MI. The expert technical assistance of Emily Poulik is acknowledged with thanks and appreciation. 233
234
AUTOPROTHROMBIN
II-A
v01.13,N0.2
The inhibition is greatly accelerated by heparin and, as a force from the opposite direction, there are apparently conditions under which the inhibition is retarded. Under suitable conditions Factor X6, prothrombin fragment 1, and Factor IX8 are inhibitors. The modification of Factor X by thrombin (1) yields Factor X6 which is an inhibitor under certain conditions (2). A prothrombin fragment inhibits thrombin in the thrombin-fibrinogen reaction but enhances the esterolytic function of thrombin (3). This fragment was identified as prothrombin fragment 2 (4). Partial activation of Factor IX withourified thrombin produces an inhibitor (2). Prothrombin fragment 1 can enhance the generation of thrombin from prethrombin 1 while it can retard the process if the substrate is prothrombin (5). Further observations on the inhibition of thrombin generation by prothrombin fragment 1 are described in this paper. The other subject of this paper relates to autoprothrombin II-A as an inhibitor. This component of the prothrombin complex was discovered in this laboratory (6) in 1960, and our reports on additional studies have appeared since that time (7-16). In two papers published recently it was erroneously stated that the primary function of this protein is unknown (17,18). It is an inhibitor of blood coagulation, it can modify platelet aggregation, and possibly influences the fibrinolytic process (6-16). The two-chain protein was known to be derived from a precursor in prothrombin complex preparations by activation with thrombin, but the parent protein remained unidentified. Then a fifth vitamin K-dependent protein was isolated from a prothrombin complex preparation (19). Promptly it was found to be the precursor of autoprothrombin II-A (20). The conversion of Protein C to autoprothrombin II-A involves the removal of a peptide from the NH;-terminal end of the heavy chain (18). This peptide has 14 amino acid residues. MATERIALS AND METHODS Purified bovine prothrombin, prothrombin fragment 1, prethrombin 1, Protein C, autoprothrombin II-A which is also called active Protein C, thrombin, and Factor Xa were prepared by methods developed in this laboratory and described (2,4,9,15). All preparations had the high specific activity recorded in the original papers. Each preparation was highly purified as judged by specific activity, polyacrylamide el electrophoresis, and immunological techniques. Factor V (AC-globulin3 was also prepared and assayed by methods developed in this laboratory (21 as well as the assays for prothrombin (15), prethrombin 1 (22), thrombin (151, and Factor X and Xa (23). It has repeatedly been found that prothrombin fragment 1 does not modify the thrombin assay. In the assay for prethrombin 1 one uses the same technique as the two stage assay for prothrombin except that purified AC-globulin and Factor Xa are added to the activation mixture. The prethrombin assays (Table 1) invariably gives the maximum yield of thrombin that can be obtained by any other known means such as use of trypsin, Echis carinatus snake venom, etc. Treating prothrombin complex with thrombin reduces the thrombin yield in the two stage analysis (15). When oral anticoagulants were given, the relative quantity of thrombin generated in the prethrombin assay increased as the drug became effective (24).
GCTOPROTHRO?lBIS 11-d
Vo1.17,No.2
235
TABLE 1 Two Stage Assay and Prethrombin Assay Results with Various Products Product Purified Prothrombin (2)
Two-stage
Prethrombin
2 to 10
10,000
Purified Prethrombin 1
none
12,000
Prothrombin Complex
8,800
Above + Thrombin
Reduced***
*Coumadin Dog Plasma (24)
3 to 5
Dog Plasma**
220
8,800 8,800 9 to 20 270
*Five days after giving Coumadln on a daily basis. **Varies strikingly from dog to dog. ***Less than 8,800 and varies from one experiment to the next. RESULTS THROMBIN GENERATION IN 25%SODIUM CITRATE SOLUTION Perspective: When it was first observed that thrombin generates from a prothrombin complex preparation in 25% sodium citrate solution, it was surprising and self evident that calcium ions are not necessary for thrombin formation (25). Later, generation of thrombin was demonstated in mixtures consisting of purified prethrombin 1 and purified Factor Xa (14). The former does not contain the gaannacarboxyglutamic acid residues found in prothrombin (26-28). Since these residues bind calcium ions and are found in prothrombin fragment 1, it remained to be determined whether this fragment is of any consequence in the absence of calcium ions. Prothrombin or Prethrombin 1: First, equal molar concentrations of prothrombin and prethrombin 1 were separately activated with equal amounts of Factor Xa. Under comparable conditions (Fig. l), the generation of thrombin was far more rapid from prethrombin 1 than from prothrombin. By adding purified prothrombin fragment 1 to the prethrombin 1 activation mixture, at the level of 2 mg/ml, it served as an inhibitor of thrombin formation (Fig. 1). Furthermore, preincubation of prothrombin with thrombin, in the proportions of 10 units to 1 respectively, produced a mixture which generated thrombin more rapidly than from the native prothrombin, but not as rapidly as from prethrombin 1. We can consider a comparison of the quantity of prothrombin fragment 1 produced in the one experiment with the purified prothrombin fragment 1 added in the other. If the digestion of prothrombin with thrombin removed all of the prothrombin fragment 1, its concentration was 0.08 mg/ml in the reaction mixture. This amounts to about 4% of the purified prothrombin fragment 1 added to the prethrombin 1 activation mixture. We do not know, however, whether our purification procedures decreased the inhibitory function of prothrombin fragment 1 or not. It seems certain that prothrombin fragment 1 functioned as an inhibitor, and accounts for the relatively slow generation of thrombin from prothrombin as compared with prethrombin 1.
236
AUTOPROTHROMBIN
0123456
060
TIME IN HOURS
“0
120 240 360 TIME
FIG. 1.
II-A
IN
MINUTES
TIME
IN MINUTES
-z 024 s (x /09
6810
All reactions in 25% sodium citrate solution at 37'C.
Prethrombin 1 activated with Factor Xa at a molar ratio 10 and same retarded with prothrombin fragment 1 (2 mg/ml) (open circles). Prothrombin activation under comparable conditions (open squares), and the same prothrombin first activated with thrombin (closed squares) resulting in more rapid thrombin generation. Upper right. Quantity of Factor Xa required to generate maximum amount of thrombin from prethrombin 1. Lower left. With 33 U/ml of Factor Xa activating prethrombin 1, it required 39 ug of autoprothrombin II-A to completely inhibit thrombin generation. Lower right. Inhibition of Factor Xa (33 U/ml) with 30, 22 or 10 vg autoprothrombin II-A at various concentrations of prethrombin 1. Vmax ? ? 0.173 U/min/unit Factor Xa and Km = 714. In physiological saline solutions at pH 7.2, and the same molar proportions of Factor Xa and prethrombin 1 as described above, prethrombin 1 also converted to thrombin more rapidly than prothrombin.
AUTOPROTHROMBIS
337
11-A
INHIBITION OF THROMBIN GENERATION IN 25% SODIUM CITRATE SOLUTION WITH PURIFIED AUTOPROTHROMBIN II-A Quantity of Factor Xa required. With the prethrombin 1 concentration at 1200 U/ml, the minimum requirement for Factor Xa was 33 U/ml (Fig. 1). This represented'a molar ratio of approximately 9 to 1. With smaller amounts of the enzyme the rate of thrombin generation, as well as the yield, in 180 min, was diminished. Early in the reaction there was a lag phase already noted in our earlier work. Presumably this is when prothrombin fragment 2 is removed from the prethrombin 1 molecule. Larger amounts of enzyme accelerated the reaction, but the yield of thrombin was not increased. Alto 9 molar ratio of enzyme to substrate is thus a favorable condition for studying inhibition of the reaction. Active Protein C. To study the inhibition of thrombin formation, we aqain used 1200 U/ml orethrombin 1 and 33 U/ml of Factor Xa (Fiq. 1). Suitable increments of added purified autoprothrombin II-A reduced ihe thrombin yield progressively until practically none formed when the inhibitor concentration was 39 Pg/ml. Expressing numbers in nanomoles per ml, the conditions for complete inhibition were thus as follows: prethrombin 1 = 4.0; Factor Xa = 0.44 and the inhibitor = 0.7. The purpose of the next step was to try and confirm an earlier conclusion (8) which was that autoprothrombin II-A functions as a competitive inhibitor of Factor Xa. For that purpose the enzyme concentration was set at 33 U/ml and the prethrombin 1 substrate concentration, as well as the inhibitor concentration, were systematically varied. By considering the plotted data (Fig. l), given as l/V0 and l/S at the 90 min time interval, it is clear that competitive inhibition was involved. Our calculations gave values for Vmax = 0.173 U/min/unit Factor Xa and Km = 714. The work was repeated with the results: Vmax = 0.182 U/min/unit Factor Xa and Km = 695. Activation of Protein C. To measure the inhibitor strength of autoprothrombin II-A we used the method previously described (2). The reaction mixture consisted of purified prothrombin complex, thromboplastin, purified AC-globulin and calcium chloride. In that mixture, thromboplastin functions in the formation of Factor Xa. The lipoproteins of thromboplastin and Factor V are accessory to Factor Xa in the formation of thrombin. The function of Factor Xa is retarded by the inhibitor. Possibly AC-globulin is also inhibited. The reaction mixture consisted of the following:* Purified prothrombin complex (12,000 U/ml prothrombin). Purified AC-globulin (Factor V) (500 U/ml). . . . . . . Ortho brain thromboplastin (5 mg/ml). . . . . . . . . . Calcium chloride (0.1 M) in imidazole buffer, pH 7.2 . Sample* or control. . . . . . . . . . . . . . . . . . .
. . . . .
0.1 0.1 0.1 0.1 0.6
ml ml ml ml ml
*Protein C before or after activation added to supply at final concentration of 200 ug/ml in this reaction mixture. ACTIVATION OF PROTEIN C WITH THROMBIN Perspective. Beginning in 1960, reports on work in this laboratory included studies on the qeneration of the inhibitor which we called autoprothrombin II-A. From the comparative amino acid analysis data (20) for‘purified Protein C and purified Autoprothrombin II-A it was evident that only a small amount of protein was removed during the activation with thrombin.
238
AUTOPROTHROMBIN
II-A
Vol.l3,No.2
This is now known to be a peptide consisting of 14 amino acid residues cleaved from the amino terminal end of the B chain of the protein (18). In our further work, sutnnarized below, we obtained information on the quantity of thrombin needed for the activation of purified Protein C. The reaction mixture described above was kept at 37Y and repeatedly samples were removed for thrombin assay. In the example illustrated (Fig. 2) Protein C itself, without first being activated with thrombin, inhibited thrombin formation slightly in the test system. This was expected on the basis of some activation of Protein C because of the thrombin developed in the test itself.
FIG. 2. Composition of activation mixture given in text. Note some thrombin production was inhibited by Protein C. The inhibitory function increased progressively as the digestion of Protein C by thrombin occurred before being placed in the test system.
“0
10 TIME
20
30
“60
IN MINUTES
A combination of Protein C with thrombin in the respective molar ratio of 75 to 1, at pH 7.2 and 37°C fulfilled conditions for the progressive development of autoprothrombin II-A activity over a period of 60 min (Fig. 2). In additional experiments, higher concentrations of thrombin were used, including a molar ratio of 18 to 1. A little more anticoagulant activity developed, and more rapidly in accordance with expectations. There was, however, no loss of inhibitor activity with prolonged standing of purified Protein C with purified thrombin. Thrombin thus activates Protein C but does not destroy the activation product under the conditions of our experiment.
DISCUSSION Autoprothrombin II-A is a competitive inhibitor of Factor Xa. This was previously found in our laboratory. It was also recognized that AC-globulin might be the target of inhibition because purified AC-globulin reversed the inhibition (11). We are able to confirm that AC-globulin has its activity The inhibitor thus inactivates AC-globulin reduced by autoprothrombin II-A. (11,18), and inhibits Factor Xa. Which effect dominates in physiological coagulation remains to be determined. However, the initial function of thrombin is to make AC-globulin more active, and it is not likely that an indirect effect on AC-globulin is produced through the generation of autoprothrombin II-A by thrombin. Whether autoprothrombin II-A might make Acglobulin more active before the reverse occurs remains to be determined.
v01.13,N0.2
AUTOPROTHRO?fBIN 11-A
PROTHROMBIN Prothrombin
239
COMPLEX -
FactorX Factor X 13 Factor Xa f3 Factor IX Factor Ix B Factor lXaf3 Factor IZII Inactive JZII Protein C FIG. 3. Molecular lengths drawn roughly to scale. Interchain disulfide bonds indicated. In prothrombin the NHZ-terminal end starts with prothrombin fragment 1, followed by prothrombin fragment 2. Both of these are deleted during conversion to thrombin. The sequences indicated for Factor X are well documented (2,33). The structure indicated for IXaB remains to be established. Factor IXB converts to Factor IXa8 under the same conditions that Factor IX changes to Factor IXa. In the case of inactive Factor VII, the structure indicated is tentative. With Protein C, the removal of a 14 amino acid residue peptide from the NHZ-terminal end of the heavy chain seems to be the only change. Further work is required to determine the type of competitive inhibition produced by autoprothrombin II-A. At lower concentrations of the inhibitor than shown (Fig. 1) practically no inhibition of the reaction occurred. Quite possibly the inhibitor binds to the substrate. In our analysis of the data we plotted reaction velocity versus inhibitor concentration, and found a relationship in which there was no drop in velocity until the inhibitor concentration was 0.075 mM. Thereafter, the drop was rapid until the inhibitor concentration was about 4 mM. Such a si moidal curve is expected in rare cases of substrate depletion kinetics (34,3z ).
240
AUTOPROTHROMEXN
II-A
Vo1.13,No.2
Thrombin has an effect on each one of the well known components of the It was already shown in prothrombin complex. Consider each one (Fig. 3). 1939 that thrombin modifies prothrombin (29). Now we know that the digestion products are prothrombin fragment 1 and prethrombin 1. The former is an inhibitor under suitable conditions. In the case of another protein, thrombin removes a peptide from the COOH-terminal end of the heavy chain of Factor X (18). The resulting Factor XB can be an inhibitor (2). Similarily, thrombin cleaves a peptide from the COOH-terminal end of Factor IX and the Factor IXB is an inhibitor (30). In the case of Factor VII, thrombin produces activation followed by inactivation (31). Protein C, as elaborated on in this paper, is converted directly to an inhibitor by thrombin (6) due to the removal of a peptide from the NHz-terminal end of the heavy chain (18). One can obtain a partial view of the molecular modifications produced by thrombin in each of five members of the prothrombin complex by organizing the information now available (Fig. 3). Included on the chart is a diagram of the probable structures of thrombin, Factor Xa, Factor IXa, Factor VIIa, and autoprothrombin II-A. In all cases except thrombin, the NHz-terminal end of the plasma protein remains after activation, and as a consequence, the y-carboxyglutamic amino acid residues remain with the active enzyme. Whether the inhibitors produced by thrombin are primarily curiosities of the research laboratory remains to be determined. Each one functions as a negative feedback component and all of them together could be of significance. We need to sort out the physiologically significant members from those that might not qualify for a function. Account must be taken of the relative impact of antithrombin III, az-macroglobulin, aI-antitrypsin and other inhibitors. This also requires a return to considering the whole blood as found in our bodily functioning. Currently there is insufficient information for drawing comprehensive conclusions. It does, however, seem likely that autoprothrombin II-A is an important inhibitor, which additionally has other functions such as platelet aggregation (32), and possibly a synergistic effect on fibrinolysis (12). Atypical prothrombin molecules have been isolated from the blood of steers treated with Oicumarol (36). The modified variants represent precursors along the chain of prothrombin synthesis (37). Presumably the abnormal molecules contain fewer y-carboxyglutamic acid residues than normal prothrombin (26-28). It will then be interesting to study the activation characteristics of purified preparations. One possibility is that thrombin generation will be similar to that with prethrombin 1. On the other hand, the modified prothrombin fragment 1 of the atypical molecules might retain inhibitory functions and contribute to the retardation of thrombin generation as with the normal prothrombin. REFERENCES 1.
SEEGERS, W.H. Blood clotting mechanisms: Rev. Physiol. 3l_, 269-294, 1969.
Three basic reactions.
Annu.
2.
Improved procedures for the NOVOA, E., SEEGERS, W.H. AND HASSOUNA, H.I. purification of selected vitamin K-dependent proteins. Prep. Biochem. 6, 307-338, 1976.
v01.13,N0.2
AUTOPROTHROMBIN
II-A
241
3.
HELDEBRANT, C. and MANN, K. The activation of prothrombin I. Isolation and preliminary characterization of intermediates. J. Biol. Chem. 248, 3642-3652, 1973.
4.
SEEGERS, W.H., WALZ, D.A., REUTERBY, J. and MCCOY, L.E. Isolation and some properties of thrombin-E and other prothrombin derivatives. Thront,. Res. 4_, 829-860, 1974.
5.
SEEGERS, W.H., NOVOA, E., WALZ, D.A., ANOARY, T.J. and HASSOUNA, H.I. Effects of prothrombin fragments on thrombin, on thrombin formation, and separation from Ac-globulin (Factor V). Thromb. Res. &, 83-97, 1976.
6.
MAMMEN, E.F., THOMAS, W.R. and SEEGERS, W.H. Activation of purified prothrombin to autoprothrombin I or autoprothrombin II (platelet cofactor II) or autoprothrombin II-A. Thromb. Diath. Haemorrh. 5, 218 249, 1960.
7.
SEEGERS, W.H. and ULUTIN, O.N. Autoprothrombin II-anticoagulant (autoprothrombin II-A). Thromb. Diath. Haemorrh. 6, 270-281, 1961.
8.
MARCINIAK, E., MURANO, G. and SEEGERS, W.H. Inhibitor of blood clotting derived from prothrombin. Thromb. Diath Haemorrh. 18, 161-166, 1967.
9.
SEEGERS, W.H., MCCOY, L.E., GROBEN, H.D., SAKURAGAWA, N. and AGRAWAL, B.B.L. Purification and some properties of autoprothrombin II-A: An anticoagulant perhaps also related to fibrinolysis. Thromb. Res. l_, 443-460, 1972.
10.
AGRAWAL, B.B.L., MCCOY, L.E., GALBRAITH, W., and SEEGERS, W.H. The carbohydrate content of prothrombin, thrombin, autoprothrombin III Thromb. Diath. Haemorrh. Supple(Factor X) and autoprothrombin II-A. ment 57, 229-239, 1974.
11.
A MURANO, G., SEEGERS, W.H. and ZOLTON, R.P. Autoprothrombin II-A: competitive inhibitor of autoprothrombin C (Factor Xa). A review with additions. Thromb. Diath. Haemorrh. Supplement 57, 305-314, 1974.
12.
ZOLTON, R.P. and SEEGERS, W.H. Autoprothrombin II-A: Thrombin removal and mechanism of induction of fibrinolysis. Thromb. Res. 3_, 23-33, 1973.
13.
SEEGERS, W.H., HEENE, D.L. and MARCINIAK, E. Activation of purified prothrombin in ammonium sulfate solutions: Purification of autoprothrombin C. Thromb. Diath Haemorrh. 15, l-11, 1966.
14.
SEEGERS, W.H. and MARCINIAK, E. Some activation characteristics of the prethrombin subunit of prothrombin. Life Sci. 4, 1721-1726, 1965.
15.
SEEGERS, W.H.
16.
SEEGERS, W.H. Physiology of blood coagulation: Advances, problems and present status. PfliiegersArch. 299, 226-246, 1968.
17.
STENFLO, J. and SUTTIE, J.W. Vitamin K-dependent formation of y-carboxyglutamic acid. Annu. Rev. Biochem. 5, 157-172, 1977.
Prothrombin.
Cambridge:
Harvard University Press.
1962.
AUTOPROTHROMBIN
242
II-A
v01.13,~0.2
18. KISIEL,
W., CANFIELD, W.M., ERICSSON, L.H. and DAVIE, E.W. Anticoagulant properties of bovine plasma Protein C following activation by thrombin. Biochemistry 16, 5824-5831, 1977.
19.
STENFLO, J. A new vitamin K-dependent protein. 355-363, 1976.
J. Biol. Chem. 251,
20.
SEEGERS, W.H., NOVOA, E., HENRY, R.L. and HASSOUNA, H.I. Relationship of "new" vitamin K-dependent Protein C and "old" autoprothrombin II-A. Thromb. Res. 8, 543-552, 1976.
21.
DOMBROSE, F.A., YASUI, T., ROUBAL, Z, ROUBAL, A. and SEEGERS, W.H. AC-globulin (Factor V): Preparation of a practical product. Prep. Biochem. 2, 381-396, 1972.
22.
SEEGERS, W.H. and MARCINIAK, E. Autoprothrombin C in irregular blood clotting. Thromb. Diath. Haemorrh. S, l-21, 1962.
23.
RENO, R.S. and SEEGERS, W.H. Two-stage procedure for the quantitative determination of autoprothrombin III concentration and some applications. Thromb. Diath. Haemorrh. l& 198-210, 1967.
24.
MULLER-BERGHAUS, G. and SEEGERS, W.H. Some effects of purified auto;;$hrombin C in blood clotting. Thromb. Diath. Haemorrh. 16, 707-722,
25.
SEEGERS, W.H. Activation of purified prothrombin. Med. 2, 677-680, 1949.
26.
MAGNUSSON, S., PETERSEN, T.E., SOTTRUP-JENSEN, L. and CLAEYS, H. Complete primary sturcture of prothrombin: Isolation, structure and reactivity of ten carboxylated glutamic acid residues and regulation of prothrombin activation by thrombin. In: Proteases and Biological Control. Cold Spring Harbor Laboratory, 1975, Vol. 2.
27.
StrucSTENFLO, J. Vitamin K and the biosynthesis of prothrombin. II. tural comparison of normal and dicoumarol-induced bovine prothrombin. J. Biol. Chem. 247, 8167-8175, 1972.
28.
NELSESTUEN, G.L. and SUTTIE, J.W. The mode of action of vitamin K. Isolation of a peptide containing the vitamin K-dependent portion Of prothrombin. Proc. Natl. Acad. Sci. USA 70, 3366-3370, 1973.
29.
MERTZ, E.T., SEEGERS, W.H. and SMITH, H.P. Inactivation of prothrombin by purified thrombin solutions. Proc. Sot. Exp. Biol. Med. fl, 657-661, 1939.
30.
MARLAR, R. inhibitor. 1978.
31.
RADCLIFFE, R. and NEMERSON, Y. Activation and control of Factor VII by activated Factor X and thrombin. J. Biol. Chem. 250, 388-395, 1975.
Proc. Sot. Exp. Biol.
Formation and properties of a thrombin generated Factor IX Ph.D. Dissertation, Wayne State Univ., Detroit, Michigan,
v01.13,N0.2
AUTOPROTHROMBIN
243
II-A
32. HERMAN, G.E., SEEGERS, W.H, and HENRY, R.L.
Effects of autoprothrombin II-A on epinephrine induced platelet aggregation of normal and coumadin treated dogs. Thrombos. Haemostas. In press, 1978.
33.
FUJIKAWA, K., LEGAS, M.E. and DAVIE, E.W. Bovine Factor X1 (Stuart Factor). Mechanism of activation by a protein from Russell's viper venom. Biochemistry ll_, 4892-4898, 1972.
34.
REINER, J. Behavior of enzyme systems. New York, NY, 192-194, 1969.
35.
SEGEL, I. 1975.
36.
MALHOTRA, O.P. and CARTER, J.R. Isolation and purification of prothrombin from Oicumarolized steers. J. Biol. Chem. 246, 2665-2671, 1971.
37.
MALHOTRA, O.P. 59-60, 1972.
Enzyme kinetics.
Van Nostrand Reinhold Co.
John Wiley and Sons, New York, NY, 203-206,
Atypical prothrombins induced by Dicumarol.
Nature 239,
1Y349-3848/78/0801-0233SO2.WO
ANTICOAGULANT EFFECTS OF AUTOPROTHROMBIN II-A AND PROTHROMBIN FRAGMENT 1 Walter H. Seegers, Richard A. Marlar and Daniel A. Walz Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan 48201, U.S.A.
(Received 1.5.1978: in revised form 6.6.1978. Accepted by Editor K.M. Brinkhous)
ABSTRACT In 25% sodium citrate solution the quantity and rate of thrombin generated was lower with prothrombin than with prethrombin 1, when the molar ratio of substrate to Factor Xa was ten to one respectively. The difference was found to be due to inhibition by prothrombin fragment 1. Similar differences were observed in physiological saline solution. For the activation of prethrombin 1 in 25% sodium citrate solution Km = 714, and Vmax = 0.173 U/min/unit Factor Xa. In physiological saline solution the respective figures were 992 and 0.015. Protein C, activated by thrombin, reduced the activity of purified AC-globulin and inhibited the production of thrombin from prethrombin 1 by Factor Xa on a competitive basis. The maximum activation of Protein C by thrombin required about 60 min when the molar ratio was 75 to 1. Activated Protein C retained its activity for many hours even in the presence of relatively large quantities of thrombin. INTRODUCTION The natural inhibitors of blood coagulation are sufficiently numerous and effective to offer constant resistance to the function of procoagulants. After being derived from their precursors, several serine proteinases, including thrombin and Factor Xa, are inhibited by plasma antithrombin III.
This investigation was supported by Grant HL-03424-21 from the National Heart, Lung and Blood Institute, National Institutes of Health, U.S. Public Health Service, the McGregor Fund, and the Skillman Foundation, Detroit, MI. The expert technical assistance of Emily Poulik is acknowledged with thanks and appreciation. 233
234
AUTOPROTHROMBIN
II-A
v01.13,N0.2
The inhibition is greatly accelerated by heparin and, as a force from the opposite direction, there are apparently conditions under which the inhibition is retarded. Under suitable conditions Factor X6, prothrombin fragment 1, and Factor IX8 are inhibitors. The modification of Factor X by thrombin (1) yields Factor X6 which is an inhibitor under certain conditions (2). A prothrombin fragment inhibits thrombin in the thrombin-fibrinogen reaction but enhances the esterolytic function of thrombin (3). This fragment was identified as prothrombin fragment 2 (4). Partial activation of Factor IX withourified thrombin produces an inhibitor (2). Prothrombin fragment 1 can enhance the generation of thrombin from prethrombin 1 while it can retard the process if the substrate is prothrombin (5). Further observations on the inhibition of thrombin generation by prothrombin fragment 1 are described in this paper. The other subject of this paper relates to autoprothrombin II-A as an inhibitor. This component of the prothrombin complex was discovered in this laboratory (6) in 1960, and our reports on additional studies have appeared since that time (7-16). In two papers published recently it was erroneously stated that the primary function of this protein is unknown (17,18). It is an inhibitor of blood coagulation, it can modify platelet aggregation, and possibly influences the fibrinolytic process (6-16). The two-chain protein was known to be derived from a precursor in prothrombin complex preparations by activation with thrombin, but the parent protein remained unidentified. Then a fifth vitamin K-dependent protein was isolated from a prothrombin complex preparation (19). Promptly it was found to be the precursor of autoprothrombin II-A (20). The conversion of Protein C to autoprothrombin II-A involves the removal of a peptide from the NH;-terminal end of the heavy chain (18). This peptide has 14 amino acid residues. MATERIALS AND METHODS Purified bovine prothrombin, prothrombin fragment 1, prethrombin 1, Protein C, autoprothrombin II-A which is also called active Protein C, thrombin, and Factor Xa were prepared by methods developed in this laboratory and described (2,4,9,15). All preparations had the high specific activity recorded in the original papers. Each preparation was highly purified as judged by specific activity, polyacrylamide el electrophoresis, and immunological techniques. Factor V (AC-globulin3 was also prepared and assayed by methods developed in this laboratory (21 as well as the assays for prothrombin (15), prethrombin 1 (22), thrombin (151, and Factor X and Xa (23). It has repeatedly been found that prothrombin fragment 1 does not modify the thrombin assay. In the assay for prethrombin 1 one uses the same technique as the two stage assay for prothrombin except that purified AC-globulin and Factor Xa are added to the activation mixture. The prethrombin assays (Table 1) invariably gives the maximum yield of thrombin that can be obtained by any other known means such as use of trypsin, Echis carinatus snake venom, etc. Treating prothrombin complex with thrombin reduces the thrombin yield in the two stage analysis (15). When oral anticoagulants were given, the relative quantity of thrombin generated in the prethrombin assay increased as the drug became effective (24).
GCTOPROTHRO?lBIS 11-d
Vo1.17,No.2
235
TABLE 1 Two Stage Assay and Prethrombin Assay Results with Various Products Product Purified Prothrombin (2)
Two-stage
Prethrombin
2 to 10
10,000
Purified Prethrombin 1
none
12,000
Prothrombin Complex
8,800
Above + Thrombin
Reduced***
*Coumadin Dog Plasma (24)
3 to 5
Dog Plasma**
220
8,800 8,800 9 to 20 270
*Five days after giving Coumadln on a daily basis. **Varies strikingly from dog to dog. ***Less than 8,800 and varies from one experiment to the next. RESULTS THROMBIN GENERATION IN 25%SODIUM CITRATE SOLUTION Perspective: When it was first observed that thrombin generates from a prothrombin complex preparation in 25% sodium citrate solution, it was surprising and self evident that calcium ions are not necessary for thrombin formation (25). Later, generation of thrombin was demonstated in mixtures consisting of purified prethrombin 1 and purified Factor Xa (14). The former does not contain the gaannacarboxyglutamic acid residues found in prothrombin (26-28). Since these residues bind calcium ions and are found in prothrombin fragment 1, it remained to be determined whether this fragment is of any consequence in the absence of calcium ions. Prothrombin or Prethrombin 1: First, equal molar concentrations of prothrombin and prethrombin 1 were separately activated with equal amounts of Factor Xa. Under comparable conditions (Fig. l), the generation of thrombin was far more rapid from prethrombin 1 than from prothrombin. By adding purified prothrombin fragment 1 to the prethrombin 1 activation mixture, at the level of 2 mg/ml, it served as an inhibitor of thrombin formation (Fig. 1). Furthermore, preincubation of prothrombin with thrombin, in the proportions of 10 units to 1 respectively, produced a mixture which generated thrombin more rapidly than from the native prothrombin, but not as rapidly as from prethrombin 1. We can consider a comparison of the quantity of prothrombin fragment 1 produced in the one experiment with the purified prothrombin fragment 1 added in the other. If the digestion of prothrombin with thrombin removed all of the prothrombin fragment 1, its concentration was 0.08 mg/ml in the reaction mixture. This amounts to about 4% of the purified prothrombin fragment 1 added to the prethrombin 1 activation mixture. We do not know, however, whether our purification procedures decreased the inhibitory function of prothrombin fragment 1 or not. It seems certain that prothrombin fragment 1 functioned as an inhibitor, and accounts for the relatively slow generation of thrombin from prothrombin as compared with prethrombin 1.
236
AUTOPROTHROMBIN
0123456
060
TIME IN HOURS
“0
120 240 360 TIME
FIG. 1.
II-A
IN
MINUTES
TIME
IN MINUTES
-z 024 s (x /09
6810
All reactions in 25% sodium citrate solution at 37'C.
Prethrombin 1 activated with Factor Xa at a molar ratio 10 and same retarded with prothrombin fragment 1 (2 mg/ml) (open circles). Prothrombin activation under comparable conditions (open squares), and the same prothrombin first activated with thrombin (closed squares) resulting in more rapid thrombin generation. Upper right. Quantity of Factor Xa required to generate maximum amount of thrombin from prethrombin 1. Lower left. With 33 U/ml of Factor Xa activating prethrombin 1, it required 39 ug of autoprothrombin II-A to completely inhibit thrombin generation. Lower right. Inhibition of Factor Xa (33 U/ml) with 30, 22 or 10 vg autoprothrombin II-A at various concentrations of prethrombin 1. Vmax ? ? 0.173 U/min/unit Factor Xa and Km = 714. In physiological saline solutions at pH 7.2, and the same molar proportions of Factor Xa and prethrombin 1 as described above, prethrombin 1 also converted to thrombin more rapidly than prothrombin.
AUTOPROTHROMBIS
337
11-A
INHIBITION OF THROMBIN GENERATION IN 25% SODIUM CITRATE SOLUTION WITH PURIFIED AUTOPROTHROMBIN II-A Quantity of Factor Xa required. With the prethrombin 1 concentration at 1200 U/ml, the minimum requirement for Factor Xa was 33 U/ml (Fig. 1). This represented'a molar ratio of approximately 9 to 1. With smaller amounts of the enzyme the rate of thrombin generation, as well as the yield, in 180 min, was diminished. Early in the reaction there was a lag phase already noted in our earlier work. Presumably this is when prothrombin fragment 2 is removed from the prethrombin 1 molecule. Larger amounts of enzyme accelerated the reaction, but the yield of thrombin was not increased. Alto 9 molar ratio of enzyme to substrate is thus a favorable condition for studying inhibition of the reaction. Active Protein C. To study the inhibition of thrombin formation, we aqain used 1200 U/ml orethrombin 1 and 33 U/ml of Factor Xa (Fiq. 1). Suitable increments of added purified autoprothrombin II-A reduced ihe thrombin yield progressively until practically none formed when the inhibitor concentration was 39 Pg/ml. Expressing numbers in nanomoles per ml, the conditions for complete inhibition were thus as follows: prethrombin 1 = 4.0; Factor Xa = 0.44 and the inhibitor = 0.7. The purpose of the next step was to try and confirm an earlier conclusion (8) which was that autoprothrombin II-A functions as a competitive inhibitor of Factor Xa. For that purpose the enzyme concentration was set at 33 U/ml and the prethrombin 1 substrate concentration, as well as the inhibitor concentration, were systematically varied. By considering the plotted data (Fig. l), given as l/V0 and l/S at the 90 min time interval, it is clear that competitive inhibition was involved. Our calculations gave values for Vmax = 0.173 U/min/unit Factor Xa and Km = 714. The work was repeated with the results: Vmax = 0.182 U/min/unit Factor Xa and Km = 695. Activation of Protein C. To measure the inhibitor strength of autoprothrombin II-A we used the method previously described (2). The reaction mixture consisted of purified prothrombin complex, thromboplastin, purified AC-globulin and calcium chloride. In that mixture, thromboplastin functions in the formation of Factor Xa. The lipoproteins of thromboplastin and Factor V are accessory to Factor Xa in the formation of thrombin. The function of Factor Xa is retarded by the inhibitor. Possibly AC-globulin is also inhibited. The reaction mixture consisted of the following:* Purified prothrombin complex (12,000 U/ml prothrombin). Purified AC-globulin (Factor V) (500 U/ml). . . . . . . Ortho brain thromboplastin (5 mg/ml). . . . . . . . . . Calcium chloride (0.1 M) in imidazole buffer, pH 7.2 . Sample* or control. . . . . . . . . . . . . . . . . . .
. . . . .
0.1 0.1 0.1 0.1 0.6
ml ml ml ml ml
*Protein C before or after activation added to supply at final concentration of 200 ug/ml in this reaction mixture. ACTIVATION OF PROTEIN C WITH THROMBIN Perspective. Beginning in 1960, reports on work in this laboratory included studies on the qeneration of the inhibitor which we called autoprothrombin II-A. From the comparative amino acid analysis data (20) for‘purified Protein C and purified Autoprothrombin II-A it was evident that only a small amount of protein was removed during the activation with thrombin.
238
AUTOPROTHROMBIN
II-A
Vol.l3,No.2
This is now known to be a peptide consisting of 14 amino acid residues cleaved from the amino terminal end of the B chain of the protein (18). In our further work, sutnnarized below, we obtained information on the quantity of thrombin needed for the activation of purified Protein C. The reaction mixture described above was kept at 37Y and repeatedly samples were removed for thrombin assay. In the example illustrated (Fig. 2) Protein C itself, without first being activated with thrombin, inhibited thrombin formation slightly in the test system. This was expected on the basis of some activation of Protein C because of the thrombin developed in the test itself.
FIG. 2. Composition of activation mixture given in text. Note some thrombin production was inhibited by Protein C. The inhibitory function increased progressively as the digestion of Protein C by thrombin occurred before being placed in the test system.
“0
10 TIME
20
30
“60
IN MINUTES
A combination of Protein C with thrombin in the respective molar ratio of 75 to 1, at pH 7.2 and 37°C fulfilled conditions for the progressive development of autoprothrombin II-A activity over a period of 60 min (Fig. 2). In additional experiments, higher concentrations of thrombin were used, including a molar ratio of 18 to 1. A little more anticoagulant activity developed, and more rapidly in accordance with expectations. There was, however, no loss of inhibitor activity with prolonged standing of purified Protein C with purified thrombin. Thrombin thus activates Protein C but does not destroy the activation product under the conditions of our experiment.
DISCUSSION Autoprothrombin II-A is a competitive inhibitor of Factor Xa. This was previously found in our laboratory. It was also recognized that AC-globulin might be the target of inhibition because purified AC-globulin reversed the inhibition (11). We are able to confirm that AC-globulin has its activity The inhibitor thus inactivates AC-globulin reduced by autoprothrombin II-A. (11,18), and inhibits Factor Xa. Which effect dominates in physiological coagulation remains to be determined. However, the initial function of thrombin is to make AC-globulin more active, and it is not likely that an indirect effect on AC-globulin is produced through the generation of autoprothrombin II-A by thrombin. Whether autoprothrombin II-A might make Acglobulin more active before the reverse occurs remains to be determined.
v01.13,N0.2
AUTOPROTHRO?fBIN 11-A
PROTHROMBIN Prothrombin
239
COMPLEX -
FactorX Factor X 13 Factor Xa f3 Factor IX Factor Ix B Factor lXaf3 Factor IZII Inactive JZII Protein C FIG. 3. Molecular lengths drawn roughly to scale. Interchain disulfide bonds indicated. In prothrombin the NHZ-terminal end starts with prothrombin fragment 1, followed by prothrombin fragment 2. Both of these are deleted during conversion to thrombin. The sequences indicated for Factor X are well documented (2,33). The structure indicated for IXaB remains to be established. Factor IXB converts to Factor IXa8 under the same conditions that Factor IX changes to Factor IXa. In the case of inactive Factor VII, the structure indicated is tentative. With Protein C, the removal of a 14 amino acid residue peptide from the NHZ-terminal end of the heavy chain seems to be the only change. Further work is required to determine the type of competitive inhibition produced by autoprothrombin II-A. At lower concentrations of the inhibitor than shown (Fig. 1) practically no inhibition of the reaction occurred. Quite possibly the inhibitor binds to the substrate. In our analysis of the data we plotted reaction velocity versus inhibitor concentration, and found a relationship in which there was no drop in velocity until the inhibitor concentration was 0.075 mM. Thereafter, the drop was rapid until the inhibitor concentration was about 4 mM. Such a si moidal curve is expected in rare cases of substrate depletion kinetics (34,3z ).
240
AUTOPROTHROMEXN
II-A
Vo1.13,No.2
Thrombin has an effect on each one of the well known components of the It was already shown in prothrombin complex. Consider each one (Fig. 3). 1939 that thrombin modifies prothrombin (29). Now we know that the digestion products are prothrombin fragment 1 and prethrombin 1. The former is an inhibitor under suitable conditions. In the case of another protein, thrombin removes a peptide from the COOH-terminal end of the heavy chain of Factor X (18). The resulting Factor XB can be an inhibitor (2). Similarily, thrombin cleaves a peptide from the COOH-terminal end of Factor IX and the Factor IXB is an inhibitor (30). In the case of Factor VII, thrombin produces activation followed by inactivation (31). Protein C, as elaborated on in this paper, is converted directly to an inhibitor by thrombin (6) due to the removal of a peptide from the NHz-terminal end of the heavy chain (18). One can obtain a partial view of the molecular modifications produced by thrombin in each of five members of the prothrombin complex by organizing the information now available (Fig. 3). Included on the chart is a diagram of the probable structures of thrombin, Factor Xa, Factor IXa, Factor VIIa, and autoprothrombin II-A. In all cases except thrombin, the NHz-terminal end of the plasma protein remains after activation, and as a consequence, the y-carboxyglutamic amino acid residues remain with the active enzyme. Whether the inhibitors produced by thrombin are primarily curiosities of the research laboratory remains to be determined. Each one functions as a negative feedback component and all of them together could be of significance. We need to sort out the physiologically significant members from those that might not qualify for a function. Account must be taken of the relative impact of antithrombin III, az-macroglobulin, aI-antitrypsin and other inhibitors. This also requires a return to considering the whole blood as found in our bodily functioning. Currently there is insufficient information for drawing comprehensive conclusions. It does, however, seem likely that autoprothrombin II-A is an important inhibitor, which additionally has other functions such as platelet aggregation (32), and possibly a synergistic effect on fibrinolysis (12). Atypical prothrombin molecules have been isolated from the blood of steers treated with Oicumarol (36). The modified variants represent precursors along the chain of prothrombin synthesis (37). Presumably the abnormal molecules contain fewer y-carboxyglutamic acid residues than normal prothrombin (26-28). It will then be interesting to study the activation characteristics of purified preparations. One possibility is that thrombin generation will be similar to that with prethrombin 1. On the other hand, the modified prothrombin fragment 1 of the atypical molecules might retain inhibitory functions and contribute to the retardation of thrombin generation as with the normal prothrombin. REFERENCES 1.
SEEGERS, W.H. Blood clotting mechanisms: Rev. Physiol. 3l_, 269-294, 1969.
Three basic reactions.
Annu.
2.
Improved procedures for the NOVOA, E., SEEGERS, W.H. AND HASSOUNA, H.I. purification of selected vitamin K-dependent proteins. Prep. Biochem. 6, 307-338, 1976.
v01.13,N0.2
AUTOPROTHROMBIN
II-A
241
3.
HELDEBRANT, C. and MANN, K. The activation of prothrombin I. Isolation and preliminary characterization of intermediates. J. Biol. Chem. 248, 3642-3652, 1973.
4.
SEEGERS, W.H., WALZ, D.A., REUTERBY, J. and MCCOY, L.E. Isolation and some properties of thrombin-E and other prothrombin derivatives. Thront,. Res. 4_, 829-860, 1974.
5.
SEEGERS, W.H., NOVOA, E., WALZ, D.A., ANOARY, T.J. and HASSOUNA, H.I. Effects of prothrombin fragments on thrombin, on thrombin formation, and separation from Ac-globulin (Factor V). Thromb. Res. &, 83-97, 1976.
6.
MAMMEN, E.F., THOMAS, W.R. and SEEGERS, W.H. Activation of purified prothrombin to autoprothrombin I or autoprothrombin II (platelet cofactor II) or autoprothrombin II-A. Thromb. Diath. Haemorrh. 5, 218 249, 1960.
7.
SEEGERS, W.H. and ULUTIN, O.N. Autoprothrombin II-anticoagulant (autoprothrombin II-A). Thromb. Diath. Haemorrh. 6, 270-281, 1961.
8.
MARCINIAK, E., MURANO, G. and SEEGERS, W.H. Inhibitor of blood clotting derived from prothrombin. Thromb. Diath Haemorrh. 18, 161-166, 1967.
9.
SEEGERS, W.H., MCCOY, L.E., GROBEN, H.D., SAKURAGAWA, N. and AGRAWAL, B.B.L. Purification and some properties of autoprothrombin II-A: An anticoagulant perhaps also related to fibrinolysis. Thromb. Res. l_, 443-460, 1972.
10.
AGRAWAL, B.B.L., MCCOY, L.E., GALBRAITH, W., and SEEGERS, W.H. The carbohydrate content of prothrombin, thrombin, autoprothrombin III Thromb. Diath. Haemorrh. Supple(Factor X) and autoprothrombin II-A. ment 57, 229-239, 1974.
11.
A MURANO, G., SEEGERS, W.H. and ZOLTON, R.P. Autoprothrombin II-A: competitive inhibitor of autoprothrombin C (Factor Xa). A review with additions. Thromb. Diath. Haemorrh. Supplement 57, 305-314, 1974.
12.
ZOLTON, R.P. and SEEGERS, W.H. Autoprothrombin II-A: Thrombin removal and mechanism of induction of fibrinolysis. Thromb. Res. 3_, 23-33, 1973.
13.
SEEGERS, W.H., HEENE, D.L. and MARCINIAK, E. Activation of purified prothrombin in ammonium sulfate solutions: Purification of autoprothrombin C. Thromb. Diath Haemorrh. 15, l-11, 1966.
14.
SEEGERS, W.H. and MARCINIAK, E. Some activation characteristics of the prethrombin subunit of prothrombin. Life Sci. 4, 1721-1726, 1965.
15.
SEEGERS, W.H.
16.
SEEGERS, W.H. Physiology of blood coagulation: Advances, problems and present status. PfliiegersArch. 299, 226-246, 1968.
17.
STENFLO, J. and SUTTIE, J.W. Vitamin K-dependent formation of y-carboxyglutamic acid. Annu. Rev. Biochem. 5, 157-172, 1977.
Prothrombin.
Cambridge:
Harvard University Press.
1962.
AUTOPROTHROMBIN
242
II-A
v01.13,~0.2
18. KISIEL,
W., CANFIELD, W.M., ERICSSON, L.H. and DAVIE, E.W. Anticoagulant properties of bovine plasma Protein C following activation by thrombin. Biochemistry 16, 5824-5831, 1977.
19.
STENFLO, J. A new vitamin K-dependent protein. 355-363, 1976.
J. Biol. Chem. 251,
20.
SEEGERS, W.H., NOVOA, E., HENRY, R.L. and HASSOUNA, H.I. Relationship of "new" vitamin K-dependent Protein C and "old" autoprothrombin II-A. Thromb. Res. 8, 543-552, 1976.
21.
DOMBROSE, F.A., YASUI, T., ROUBAL, Z, ROUBAL, A. and SEEGERS, W.H. AC-globulin (Factor V): Preparation of a practical product. Prep. Biochem. 2, 381-396, 1972.
22.
SEEGERS, W.H. and MARCINIAK, E. Autoprothrombin C in irregular blood clotting. Thromb. Diath. Haemorrh. S, l-21, 1962.
23.
RENO, R.S. and SEEGERS, W.H. Two-stage procedure for the quantitative determination of autoprothrombin III concentration and some applications. Thromb. Diath. Haemorrh. l& 198-210, 1967.
24.
MULLER-BERGHAUS, G. and SEEGERS, W.H. Some effects of purified auto;;$hrombin C in blood clotting. Thromb. Diath. Haemorrh. 16, 707-722,
25.
SEEGERS, W.H. Activation of purified prothrombin. Med. 2, 677-680, 1949.
26.
MAGNUSSON, S., PETERSEN, T.E., SOTTRUP-JENSEN, L. and CLAEYS, H. Complete primary sturcture of prothrombin: Isolation, structure and reactivity of ten carboxylated glutamic acid residues and regulation of prothrombin activation by thrombin. In: Proteases and Biological Control. Cold Spring Harbor Laboratory, 1975, Vol. 2.
27.
StrucSTENFLO, J. Vitamin K and the biosynthesis of prothrombin. II. tural comparison of normal and dicoumarol-induced bovine prothrombin. J. Biol. Chem. 247, 8167-8175, 1972.
28.
NELSESTUEN, G.L. and SUTTIE, J.W. The mode of action of vitamin K. Isolation of a peptide containing the vitamin K-dependent portion Of prothrombin. Proc. Natl. Acad. Sci. USA 70, 3366-3370, 1973.
29.
MERTZ, E.T., SEEGERS, W.H. and SMITH, H.P. Inactivation of prothrombin by purified thrombin solutions. Proc. Sot. Exp. Biol. Med. fl, 657-661, 1939.
30.
MARLAR, R. inhibitor. 1978.
31.
RADCLIFFE, R. and NEMERSON, Y. Activation and control of Factor VII by activated Factor X and thrombin. J. Biol. Chem. 250, 388-395, 1975.
Proc. Sot. Exp. Biol.
Formation and properties of a thrombin generated Factor IX Ph.D. Dissertation, Wayne State Univ., Detroit, Michigan,
v01.13,N0.2
AUTOPROTHROMBIN
243
II-A
32. HERMAN, G.E., SEEGERS, W.H, and HENRY, R.L.
Effects of autoprothrombin II-A on epinephrine induced platelet aggregation of normal and coumadin treated dogs. Thrombos. Haemostas. In press, 1978.
33.
FUJIKAWA, K., LEGAS, M.E. and DAVIE, E.W. Bovine Factor X1 (Stuart Factor). Mechanism of activation by a protein from Russell's viper venom. Biochemistry ll_, 4892-4898, 1972.
34.
REINER, J. Behavior of enzyme systems. New York, NY, 192-194, 1969.
35.
SEGEL, I. 1975.
36.
MALHOTRA, O.P. and CARTER, J.R. Isolation and purification of prothrombin from Oicumarolized steers. J. Biol. Chem. 246, 2665-2671, 1971.
37.
MALHOTRA, O.P. 59-60, 1972.
Enzyme kinetics.
Van Nostrand Reinhold Co.
John Wiley and Sons, New York, NY, 203-206,
Atypical prothrombins induced by Dicumarol.
Nature 239,