ARCHIVES
OF
BIOCHEMISTRY
Rat Prothrombin:
AND
BIOPHYSICS
176,
Purification, G. A. GRANT’
Department
of Biochemistry,
650-662
College
of Agricultural Madison, Received
(1976)
Characterization, AND
J. W. SUTTIE
and Life Sciences, Wisconsin 53706 March
and Activation’
University
of Wisconsin-Madison,
15, 1976
Prothrombin has been purified from rat plasma and its properties compared to prothrombin isolated from other species. The molecular weight, amino acid composition, and amino-terminal sequence of rat prothrombin are similar to human and bovine prothrombin. Rat prothrombin binds to phospholipid in the presence of calcium ions, and calcium-binding measurements indicate that it may bind somewhat more calcium than does bovine prothrombin. The proteolytic cleavage of purified rat prothrombin by Factor X, or thrombin yields the same peptides that are formed from similar proteolysis of bovine prothrombin. Factor V and phospholipid were shown to enhance the rate of Factor X, and calcium ion generation of thrombin from rat prothrombin.
Prothrombin, the plasma zymogen of thrombin, has been purified from human (l-4) and bovine (5-10) plasma and extensively characterized. Prothrombin from these two species is a glycoprotein composed of a single polypeptide chain with an apparent molecular weight of 68,00074,000. The complete amino acid sequence of bovine prothrombin (11, 12) and the sequence of portions of human prothrombin (4, 13) have recently been reported. Thrombin is generated from prothrombin by the action of Factor X, in the presence of Factor V, Ca*+, and phospholipid; and the pathway of this activation has been the subject of intensive investigation in recent years (3, 4, 14-20). Prothrombin and Factor X, contain a number of y-carboxyglutamic acid residues (21-23) in their amino-terminal region, and, during the activation of prothrombin, these proteins are bound to the negatively charged surface by Ca*+ interactions to the y-carboxy-
glutamyl residues (241. These residues arise from a post-translational vitamin Kdependent carboxylation of glutamyl residues in liver microsomal precursors of these proteins (25). We have recently reported (26) the purification and partial characterization of one form of the prothrombin precursor from the rat liver and have additional evidence for the existence of multiple forms of this protein. An understanding of the relationship of these liver precursor proteins to the final carboxylated product depends on a knowledge of the structural and functional properties of rat plasma prothrombin. Since it has been claimed (27, 28) that rat prothrombin differs significantly in some of its structural properties from human and bovine prothrombin, we are therefore reporting a purification and characterization of this protein. In addition, the activation of rat prothrombin by Factor X, and its proteolysis by thrombin have been studied; and the peptides released from these digestions have been compared to similar products obtained from bovine prothrombin digestion.
’ Research supported by the College of Agricultural and Life Sciences, University of WisconsinMadison, and in part by Grant No. AM-14881 from the National Institutes of Health, USPHS, and in part by National Institutes of Health Training Grant No. GM-00236 BCH. ’ Present address: Department of Biological Chemistry, Division of Biology and Biomedical Sciences, Washington University, St. Louis, MO. 63110.
MATERIALS
650 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
AND
METHODS
Assays and measurements. Prothrombin was measured by the two-stage assay of Ware and Seegers as modified by Shapiro and Waugh (29) and by
RAT
651
PROTHROMBIN
activation with&his carinatus venom as previously described (30). Clotting times were converted to thrombin activity (NH-I units) by comparison with a standard curve prepared from NIH standard thrombin (Lot 3B). Cephalin was the source of lipid for all activations done in the presence of phospholipid. Calcium binding was performed by equilibrium dialysis (31). Phospholipid vesicles (dioleoylphosphatidylglycerol:dioleoylphosphatidylcholine, 1:l molar ratio) used for binding studies were prepared as described by Gitel et al. (32).3 Rat prothrombin was incubated with labeled phospholipid vesicles in the presence of either calcium ions or EDTA for 30 min and then applied to a 0.9-cm column of Bio-Gel A0.5m. Polyacrylamide disc gel electrophoresis was performed by the method of Davis (33). Polyacrylamide disc gel electrophoresis in the presence of sodium dodecyl sulfate was performed according to Laemmli (34), and peptide molecular weight was estimated from these gels by comparison to a curve prepared with standard proteins after disulfide bond reduction with P-mercaptoethanol prior to electrophoresis. Molecular weight estimation by sedimentation equilibrium was performed by the meniscus depletion technique on a Spinco Model E ultracentrifuge employing Raleigh interference optics (35). Partial specific volumes were calculated from amino acid and carbohydrate compositions using residue specific volumes (36, 37). Amino acid analyses were performed on a Durrum Model D-500 analyzer using single-column technology. Tryptophan was determined spectrophotometrically by the procedure of Bencze and Schmid (38). Cysteine and methionine were determined as cysteic acid and methionine sulfone alter oxidation with performic acid (39). Serine and threonine were determined by extrapolation to zero time assuming a first-order rate of destruction. Hydrolysis in 6 N HCl was allowed to proceed for 24, 48, and 96 h at 110°C in sealed evacuated tubes. Aminoterminal amino acid analysis was performed using the dansyl method as described by Gray for proteins (40). The dansyl amino acids were identified by thinlayer chromatography on polyamide sheets as described by Hartley (41). Amino-terminal amino acid sequencing was performed (42) on a Beckman Model 890-C sequencer using a protein program employing Quadrol buffer. The phenylthiazolinone derivatives were converted to the free amino acids by acid hydrolysis and identified on a Durrum D-500 analyzer. Hexose analyses were performed by the procedure of DuBoiset al. (43). Sialic acid was determined by the procedure of Warren (44) after hydrolysis of the glycoprotein for 1 h at 80°C in 0.1 N H,SO,, and hexosamines were determined by amino acid analy-
sis after hydrolysis for 4 h at 110°C in 1 N HCl. All hydrolyses were done in sealed evacuated tubes. Reagents. Phenylmethylsulfonyl fluoride, soybean trypsin inhibitor (type II-S), and sodium heparin were purchased from Sigma Chemical Company, St. Louis, MO. Soybean trypsin inhibitor was covalently linked to agarose by the method of Cuatrecasus (46) as previously described (26). BenzamidineHCl was purchased from Aldrich Chemical Company, Milwaukee, Wis. QAE-Sephadex’ was a product of Pharmacia (Uppsala). Bovine Factor X, Factor X-activating enzyme, bovine Factor V, and cephalin were obtained and prepared as previously described (26).3 Bovine prothrombin was prepared as described by Stenflo (45). Ratprothrombin isolation. For the isolation of rat prothrombin, blood was drawn by cardiac puncture from large male donor rats under ether anesthesia into one-tenth volume of 2.85% sodium citrate and centrifuged at 3600g for 16-15 min, and the plasma was immediately frozen. After thawing, the plasma was centrifuged at 8OOOg for 5 min to remove insoluble material and treated with sodium heparin (10 units/ml) and soybean trypsin inhibitor (20 pg/ml). All subsequent steps were performed at 4°C except for the ion-exchange chromatography which was performed at room temperature. Ten milliliters of 1 M BaC12.H,0 was added slowly to each 125 ml of gently stirring plasma, and stirring was continued for 15-30 min. The barium citrate precipitate, with adsorbed prothrombin, was collected by centrifugation and washed three times with 0.9% NaCl, 0.005 M sodium citrate. After the final wash, the precipitate was dissolved in 0.2 M EDTA (pH = 7.01, and the dissolved barium and some contaminating protein were precipitated by the addition of an equal volume of saturated ammonium sulfate. The precipitate was removed by centrifugation, and prothrombin was precipitated from the supernatant by addition of the same volume of saturated ammonium sulfate. The precipitated prothrombin was dissolved in 0.05 M imidazole-HCl (pH = 7.81, 0.02 M sodium citrate, 0.001 M benzamidine-HCl, and 0.2 M ammonium chloride and dialyzed against the same buffer for 3-4 h. The crude prothrombin was chromatographed on QAE-Sephadex (0.9 x 28 cm) using a loo-ml linear (0.2 to 0.45 M) ammonium chloride gradient. At this point, a major contaminant elutes from the column on the trailing edge of the prothrombin peak, and pure prothrombin is found on the ascending edge of the peak. The yield of pure prothrombin can be increased by pooling the appropriate fractions and rechromatographing on an identical QAESephadex column. Following chromatography, the prothrombin fractions were stored at 4°C in the presence of 0.001 M phenylmethylsulfo-
3 Kindly supplied by Dr. C. M. ington University, St. Louis, MO.
4 Abbreviation propyl)aminoethyl.
Jackson,
Wash-
used:
QAE-,
Diethyl-(2-hydroxy-
652
GRANT
AND
nyl fluoride. The elution profile of the second QAESephadex column is shown in Fig. 1. The product has a specific activity of 2200 NIH thrombin units/ mg of protein and appeared homogeneous by both alkaline and sodium dodecyl sulfate gel electrophoresis (Fig. 1). This specific activity is somewhat higher than that usually cited for bovine prothrombin but was highly reproducible under the assay conditions employed and consistent over a large number of prothrombin preparations. In addition, bovine prothrombin assayed in an identical manner gives a similar specific activity (56). The specific activity of the thrombin generated from rat prothrombin was approximately 4500 NIH unitsimg of protein. Sedimentation equilibrium studies of the purified rat prothrombin showed that a plot of the logarithm of the concentration versus the square of the radial distance was linear. RESULTS
ACTIVATION
OF RAT PROTHROMBIN
The time course of activation of rat prothrombin by Factor X, and calcium ions, as visualized by sodium dodecyl sulfate-gel electrophoresis (Fig. 2) demonstrates the formation of four components in addition to prothrombin. By analogy to the reports on bovine prothrombin and on the basis of further characterization (see below), they are identified as follows.” From the top of the gels (largest apparent molecular weight component), prothrombin is followed by a transient band which is prea band containing both thrombin-1, thrombin and prethrombin-2, which coelectrophorese under these conditions, and a band of fragment-l. At the bottom of the gels is a band of fragment-2 which is not really detected in the photographs. Analysis of the reaction mixture by QAE-Sephadex chromatography (Fig. 3) showed four peaks of protein. The first two peaks con: The prothrombin activation component nomenclature used is: prethrombin-1 (P-1) and prethrombin-2 (P-2) to describe the thrombin-containing portion of the molecule corresponding to intermediate-I and intermediate-2 of Jackson and co-workers (7) and Mann and co-workers (54); and fragment-l (F-l) and fragment-2 (F-2) to describe peptides from the nonthrombin-containing portion of the molecule (7). These peptides correspond to intermediate-3 and intermediate-4 of Mann and co-workers (54). Fragment-l ‘2 (F-l .2) is the peptide corresponding to covalently linked F-l and F-2. A comparison of this nomenclature system to those used by other workers can be found in a recent paper by Downing et al. (4).
SU’lTIE
tained thrombin and prethrombin-2 which cochromatograph in this system. Prethrombin-2 was identified by an increase in thrombin activity after further activation with Factor X, or E. carinatus venom and by sodium dodecyl sulfate electrophoresis following reduction with P-mercaptoethanol. After reduction, the thrombin molecule moved with a slightly higher mobility than prethrombin-2. The double-peak elution behavior of thrombin and prethrombin-2 on QAE-Sephadex is similar to that seen with material derived from bovine prothrombin and is the result of loading the column at an ionic strength where thrombin binding to the gel is weak (7). The third peak was identified as fragment2 and the last peak as fragment-l. Although fragment-2 is not readily visible on stained gels (Fig. 2) its existence was confirmed by ion-exchange chromatography. It is of interest that the fragments-l and -2 generated from rat prothrombin elute from ion-exchange chromatograms in the opposite order than do the corresponding fragments from bovine prothrombin (7). In the case of human prothrombin they tend to coelute and must be separated by different means (I). The significance of this species heterology, while apparently charge related, has not been determined. Rat prothrombin was also activated with Factor X, in the presence of Factor V and phospholipid, and the activation fragments, as visualized on sodium dodecyl sulfate-gel electrophoresis, are shown in Fig. 4. The reaction was essentially complete in 20-30 min, as opposed to several hours needed for the reaction with Factor X, and calcium alone, and a new transient band was seen just above the thrombindoublet in the third prethrombin-2 through sixth gels. This component was not characterized but is probably fragment-l .2. When this activation mixture was reduced in this way prior to electrophoresis, it was possible to observe the separation of thrombin and prethrombin-2 and the transient nature of prethrombin-2. When rat prothrombin was digested with thrombin, only two bands appeared following sodium dodecyl sulfate-gel electrophoresis: prethrombin-1 and fragment-l (Fig. 5). Ion-exchange analysis (Fig. 6) of
RAT
653
PROTHROMBIN
06 05
- 1000 - 900
04
-800 - 700
Fraction
FIG. column. alkaline
1. Ion-exchange The gels shown gel electrophoresis
Number
chromatography of rat prothrombin on the second QAE-Sephadex are: left, sodium dodecyl sulfate-disc gel electrophoresis; and right, (pH = 9.5) of 10 pg of the final product (pooled fractions 40-47).
-
Pro
-
Pre-I
c-
Pre-2 ThFornbin
-
FIG. 2. Sodium dodecyl sulfate-gel electrophoresis of prothrombin (100 pg) with Factor X, (10 pg) and calcium left to right, 0,30,60,240, and 1200 min. The reaction was 10 ~1 of sodium dodecyl sulfate to an aliquot of the reaction bath for 5-10 min.
F-i
the time course of activation of rat (10 mM). Incubation times are, from stopped at each time point by adding mixture and heating in a 70°C water
654
GRANT
AND
SU’lTIE
BOO 0 600 2 h 400~ 3 200
FIG. 3. QAE-Sephadex chromatography of the products of the Factor X, activation of rat prothrombin. Rat prothrombin (2 mgl was incubated with Factor X, (24 pg) and calcium (10 mM) for 20 h and then chromatographed on a 0.9 x 2%cm column of QAE-Sephadex. The chromatography was performed in 0.02 M Tris.HCl buffer (pH = 7.51, 0.1 M NaCl, and the column was developed with a loo-ml linear gradient of NaCl from 0.1 to 0.5 M. One centimeter of soybean trypsin inhibitor-agarose was layered over the top of the QAE-Sephadex in order to remove Factor X, from the reaction mixture and prevent further proteolysis during chromatography. The gel inserts are sodium dodecyl sulfate-disc gel electrophoresis of the starting material (far left) and the peak fractions of each protein peak. Both thrombin-prethrombin-2 peaks displayed the same gel pattern. All gels were reduced with P-mercaptoethanol to resolve thrombin and prethrombin-2. Absorbance at 280 nm, (0); thrombin activity, (0).
--
Pro
--
P-l F-k’ Thrombin F-l
-
F-2
FIG. 4. Sodium dodecyl sulfate-gel electrophoresis of the time course of activation of rat prothrombin (200 pg) with Factor X, (10 pg), Factor V (30 pg), phospholipid (300 fig), and calcium (10 mM). Incubation times are, from left to right, 0,40, 70, 100, 140,300,420,600, and 1200 s. Gel samples were treated as described in Fig. 2 except that they were also reduced with /3-mercaptoethanol prior to electrophoresis. The faint protein band between thrombin and fragment-l is factor X,.
RAT
655
PROTHROMBIN
-
Pro
-
P-l
-
F-l
FIG. 5. Sodium dodecyl sulfate-gel electrophoresis of the time course of proteolysis prothrombin (100 pg) with rat thrombin (5 pg). Incubation times are, from left to right, 4, and 60 min. Gel samples were treated as described in Fig. 2.
the final reaction mixture also demonstrated that two components were produced. The first peak, which could be activated to thrombin by E. carinatus venom or Factor X,, is prethrombin-1; and the second peak, which cannot be activated to thrombin, is fragment-l. As is the case in all other species studied, thrombin was not generated by thrombin proteolysis. Isolated prethrombin-1 was activated by Factor X, and calcium, and the products produced were identified by electrophoresis and ion-exchange chromatography (data not shown) as prethrombin-2, thrombin, and fragment-2. With the exception of the reversal in elution order of fragment-l and fragment-2 on QAE-Sephadex chromatography, the activation of rat prothrombin by Factor X, and its proteolysis by thrombin (as judged by sodium dodecyl sulfate-gel electrophoresis and ion-exchange chromatography)
of rat 0, 1,2,
appear to proceed in a manner identical to that reported for bovine prothrombin (1420). All of the rat prothrombin activation products appear to correspond to an analogous activation product from bovine prothrombin. Molecular weight determinations (Table I) indicated that, with the possible exception of rat prothrombin itself, all of the protein components display molecular weights similar to those of the bovine system. The molecular weight of rat prothrombin based on sodium dodecyl sulfate-gel electrophoresis has been reported by Olson (27) to be 85,000. In these studies, rat prothrombin had an apparent molecular weight by this method which was only slightly higher than the 70,000-75,000 reported for bovine prothrombin. When rat prothrombin and bovine prothrombin were slightly coelectrophoresed, a single widened band was observed. The molecu-
656
GRANT
Fraction
AND
SUTTIE
Number
FIG. 6. QAE-Sephadex chromatography of the products of thrombin proteolysis of rat prothrombin. Rat prothrombin (1 mg) was incubated with rat thrombin (20 /*g) for 90 min and then chromatographed on a 0.9 x 2%cm column of QAE-Sephadex as described in Fig. 3. The gels shown are sodium dodecyl sulfate gels of the starting material (far left) and the peak fractions of each protein peak. Absorbance at 280 nm, (0); thrombin activity after activation with E. carinatus venom, (0). TABLE MOLECULAR
-
WEIGHTS
Protein Prothrombin equilibrium) Prothrombin Prethrombin-I Prethrombin-2 Thrombin Fragment-l Fragment-2 (I All dod‘ecyl Ir See ” See
(by
OF PROTHROMBIN
I
AND PROTHROMBIN
Rat sedimentation
77,000 (6 = 0.706) 75,000-80,000 58,000 38,000 37,000 25,000 12,000
values exceut those indicated as obtained bv sedimentation sulfate electrophoresis in acelamide gels. ” Ref. (4).and (7). Ref. (31, (4), (19), and (58).
lar. weight of rat prothrombin, as determined by this technique, closely resembles that of bovine prothrombin determined by the same method. The reasons for the discrepancy in the reported molecular weights of rat and bovine prothrombin are not understood, but the results of this study would suggest that the molecular weights are not as different as once thought. The amino acid and carbohydrate compositions of rat prothrombin, calculated on the basis of its sedimentation equilibrium molecular weight, are shown in Table II
ACTIVATION
PRODUCTS"
Bovine”
Human’
75,000 (6 = 0.711) 70,000-75,000 51,000-61,000 38,000-41,000 35,000-41,000 23,000-25,000 ll,OOO-13,000
72,000 (U = 0.711 70,000-75,000 51,000-55,000 37,000-41,000 37,000-41,000 23,000-25,000 13,000-16,000
equilibrium
were
obtained
by sodium
and compared to a previous composition reported for rat prothrombin and several compositions reported for bovine and human prothrombin. The amino acid composition of rat prothrombin appears to be quite similar to that of both bovine and human prothrombin. The amino acid composition of rat prothrombin reported by Li and Olson (27) shows considerably higher values for most residues. This is due in part to the fact that their composition was calculated for a peptide molecular weight of 86,000 with no correction for the amount of carbohydrate in the protein. When these
RAT
PROTHROMBIN
values are recalculated based on the peptide molecular weight determined in this study, somewhat better agreement is obtained (Table II). The amino acid composition of the thrombinand Factor X,-catalyzed digestion products of rat prothrombin were also determined. The data, calculated on the basis of the peptide molecular weights in Table I, are shown in Table III. Within the error of the technique, the sum of the residues found in the products was equal to that found in the respective parent molecules (prothrombin = prethrombin-1 + fragment-l = thrombin + fragment-l + fragment-2). These data support the electrophoretic and ion-exchange analyses of rat prothrombin activation products by confirming the relationship of the various proteins to each other and by accounting TABLE Axnmo --Residue Asp Thr Ser Glu Pro GUY Ala cysi2 Val Met Ile Leu ‘br Phe His LYS Arg Trp
ACID
II
AND CARBOHYDRATE PROTHROMBIN” Rat” Rat’
Bovine”
HLlman’
59.2 36.9 45.8 72.0 37.4 57.8 28.7 15.1 31.0 5.4 22.1 43.6 14.9 21.2 13.4 29.5 35.9 15.4
55-62 25-29 30-41 66-79 32-38 37-48 29-34 16-20 31-33 5-8 17-19 43-45 17-18 16-20 8-10 28-33 38-43 11-19
56-59 32-38 36-38 69-77 31-32 43-46 32-34 21-23 29-32 7-8 21 38-41 18-21 18-20 9-11 26-32 31-36 11-20
90 63 52 94 40 55 34 13 47
(68) (48) (40) (71) (30) (42) (26) (10) (36)
12 (9) 27 53 23 38 15 42 35 15
(21) (40) (17) (29) (11) (32) (27) (11)
COMPOSITIONS
OF
3.9-5.9% 4.1% Hexose 7.5% GlcN 4.5% 2.5-4.5% 2.5% AcNeu 2.8% 3.2-4.4% 3.5% 0 All values for amino acids are expressed as residues per mole. b Experimental values, this study. v From Ref. (27). Based on 86,000 molecular weight. Numbers in parentheses are recalculated using the peptide molecular weight determined in this study. ” See Ref. (7), (541, (55), and (56). ” See Ref. (2) and (4).
657
for all of the prothrombin residues in the various products. The possibility that very small peptides or even single amino acids could be liberated during activation still exists, however, since they probably would not be detected by these methods. Using norleucine as an internal standard, an extinction coefficient expressed as E :Tm was calculated from the amino acid analysis. This procedure gave a value of 15.9 for rat prothrombin which agreed with a value of 16.5 estimated by comparing the protein absorbance at 280 nm to the protein content as determined by the Lowry procedure (47). These values are similar to those reported for bovine prothrombin which range from 13.6 to 16.5 (6,
9, 55, 59). The calcium-binding properties of rat prothrombin are presented in Fig. 7. The Scatchard plot of these data indicates,two distinct classes of binding sites which is in agreement with the bovine prothrombinbinding studies of Benson et al. (48) but is in contrast to several other studies (31, 49, 50) which indicate positive cooperativity at low calcium ion concentrations rather than two noninteracting classes of sites. Similar results were obtained on replicate analyses, but no extensive survey of the calcium-binding properties of different preparations was attempted. The total of 17 Ca2+ binding sites indicated for rat prothrombin is also somewhat higher than the 10 to 12 sites indicated in all bovine studies. The binding constants of 1.55 x lop3 and 2.2 x 10e4 M determined for the two classes of sites from the slopes of the Scatchard plot are similar to those reported for bovine prothrombin. The ability of rat prothrombin to bind to phospholipid is illustrated in Fig. 8. These studies (top and middle profiles) support a calcium-dependent binding of phospholipid to prothrombin, while the bottom profile indicates that this apparent binding was not due to a calcium-dependent aggregation of prothrombin. The effect of other components of the prothrombinase activation complex on the rate of Factor X,-catalyzed activation of rat prothrombin is shown in Fig. 9. Both phospholipid and Factor V were found to accelerate the rate of activation, and, as
658
GRANT
AND
SUlTIE
TABLE Residue ASP Thr Ser Glu Pro GUY Ala cysi2 Val Met Ile Leu ‘br Phe His LYS Arg Trp ” Values
III
ACID
COMPOSITIONS
OF THE
Prothrombin
Thrombin
F-l
F-2
Thrombin + F-l + F-2
16.7 12.3 13.8 22.1 11.2 17.0 7.9 5.2 8.7 1.3 4.1 9.2 4.5 5.1 3.4 5.4 10.5 3.7
12.6 4.8 9.8 16.5 8.0 15.6 7.1 5.0 5.2 0 1.8 10.9 2.8 4.0 1.5 4.2 5.3 2.4
61.2 37.8 52.4 69.0 40.1 58.0 30.9 18.6 29.9 5.4 19.2 42.4 14.2 19.3 11.4 27.4 32.8 14.5
AMINO
59.2 36.9 45.8 72.0 37.4 57.8 28.7 15.1 31.0 5.4 22.1 43.6 14.9 21.2 13.4 29.5 35.9 15.4 expressed
31.9 20.7 28.8 30.4 20.9 25.4 15.9 8.4 16.0 4.1 13.3 22.3 6.9 10.2 6.5 17.8 17.0 8.4 as residues
ACTIVATION
PRODUCTS
OF RAT
PROTHROMBIN”
Prethrombin 46.4 24.8 36.3 53.0 26.9 43.2 25.6 10.7 23.3 4.2 16.2 35.1 8.1 15.4 8.3 23.6 26.3 11.0
1
P-l + F-l 63.1 37.1 50.1 75.1 38.1 60.2 33.5 15.9 32.0 5.5 20.3 44.3 12.6 20.5 11.7 29.3 36.8 14.7
per mole.
FIG. 7. Calcium binding of rat prothrombin. The ability of rat prothrombin (1 mgiml) to bind calcium ions was determined by equilibrium dialysis in the presence of 0.05 M Tris HCl, 0.1 M NaCl (pH = 8.01. Calcium and prothrombin were placed on opposite sides of the membrane in 0.1 ml of cells and dialysis was allowed to proceed for 16 h at room temperature. The upper plot indicates the moles of calcium bound 3s a function of the calcium concentration. The lower plot is a Scatchard plot of the same data.
expected from similar studies on bovine prothrombin, the presence of both Factor V and phospholipid indicates an additive effect which is consistent with the binding of each component at a different site on the prothrombin molecule. The plateau effect seen when phospholipid or Factor V was added separately is most likely due to the conversion of all of the prothrombin present in the incubation to thrombin precursors (prethrombin-1 and -2) which no longer bind the added component and are thus no longer accelerated by it. The activation of prethrombin-1 by Factor X, (Fig. 9) in the presence and absence of phospholipid indicates that, as in other species studied, phospholipid binding is associated with the fragment-l portion of prothrombin. Reaction of rat prothrombin (100-200 pg) with dansyl chloride followed by hydrolysis and chromatography of the hydrolysate on thin-layer polyamide sheets revealed alanine as the amino-terminal residue. This is the same amino terminus as found on bovine (11, 51, 52) and human prothrombin (4, 12). The amino acid sequence of the amino terminal region of rat prothrombin was also determined (Table IV) for the purpose of comparison to other species. The se-
RAT
quence of the first nine residues in rat prothrombin shows a high degree of homology with the same region in bovine prothrombin, human prothrombin, Factor IX, and the light chain of Factor X. These data confirm the presence of alanine as the amino-terminal residue of rat prothrombin and suggest that the first two y-carboxyglutamic acid residues are in the same positions (7 and 8) as in the bovine clotting factors. The methods utilized would not detect y-carboxyglutamic acid, but the data do indicate that positions 7 and 8 are occupied by glutamic acid. Since these are the same two positions occupied by the first two y-carboxyglutamic acid residues in bovine prothrombin, it is reasonable to expect that further chemical studies would confirm this homology. These data have provided a description of the chemical nature of rat prothrombin PL,
Co”
-20 - I5 - IO -5
PRO,
PL , EDTA
PRO.
Fraction
6
Ca”
number
FIG. 8. Phospholipid binding to rat prothrombin. Rat prothrombin (20 fig) was incubated with phospholipid (250 yg) for 30 min and then chromatographed on a 0.9 x 28-cm column of Bio-Gel A-0.5m in 40 mM Tris’HCl, 70 mM NaCl. When included in the incubation and column, calcium was 10 mM and EDTA was 1.5 mM. The apparent increased size of rat prothrombin in the presence of phospholipid and EDTA over that of prothrombin in the presence of Ca2+ alone is due to an incomplete inhibition of phospholipid binding by this level of EDTA. The abbreviations used are PRO, prothrombin; PL, phospholipid; and EDTA, ethylenediaminetetraacetic acid.
0
4 0
2 6 L
DISCUSSION
PRO.
659
PROTHROMBIN
5
10 15 INCULIATION
20 25 TlME h”I
30
FIG. 9. The effect of phospholipid and Factor V on the Factor X,-catalyzed activation of rat prothrombin and rat prethrombin-1. Rat prothrombin (6 Kg) or rat prethrombin-1 (20 pg) was incubated with Factor X, (1 pgl in the presence of 10 mM calcium and either phospholipid (300 pg), Factor V (10 wg), or both in a volume of 1 ml. Aliquots were removed at time intervals and assayed for thrombin. Top: prothrombin and Factor X, alone (01; plus phospholipid, (ml; plus Factor V, (0); plus phospholipid and Factor V, (0). Bottom: prethrombin-1 and Factor X, alone, (0); plus phospholipid,
and serve to demonstrate its similarity to bovine and human prothrombin which have been extensively studied by several groups. The amino acid and carbohydrate compositions of rat, bovine, and human prothrombin are very similar, as are the compositions of the activation products. The most striking difference between rat and bovine prothrombin is seen in the type of calcium binding observed and the total number of sites participating as well as the ionic properties of fragment-l and fragment-2. The apparent increased acidity of rat prothrombin fragment-l may be related to the apparent increased number of calcium-binding sites. Rat prothrombin also appears to be activated in vitro in a manner identical to that of bovine prothrombin. Both proteins produce similar activation components in re-
660
GRANT
AND
SUTI’IE
TABLE HOMOLOGY
Protein
Rat prothrombin” Bovine prothrombin’ Human prothrombin” Bovine Factor IX Bovine Factor X’
H,N-1 Ala Ala Ala ‘br Ala
OF VITAMIN
2 ASX
Asn Asn Asn Asn
3 Ser LYS Thr Ser Ser
IV K-DEPENDENT
4
PROTEINS
5
GUY GUY - ‘1
Phe Phe Phe
GUY - (1
LYS Phe
” This study. ’ The abbreviation Glx indicates that glutamic acid has been indicates a positive identification of y-carboxyglutamic acid at this c Magnusson et al. (23); Stenflo et al. (21). ” Downing et al. (4). ’ Fujikawa et al. (57). I Light chain, Enfield et al. (53); Howard and Nelsestuen (60). ‘I Gaps are inserted at these positions to optimize homology
sponse to Factor X, and thrombin, and both appear to participate in the same type of protein-protein and lipid-protein interactions during activation. There was no indication in the studies that rat prothrombin activation proceeds via a prethrombin-2 and prethrombin-2’ conversion as has been suggested (4) for human prothrombin. It is unlikely, however, that the 13-amino-acid peptides which account for the difference in these two forms would have been detected in our studies. While prothrombins from all species have usually been considered to perform identical functions in the clotting process, some question has been raised in the past concerning the degree to which rat prothrombin might differ structurally from t?le prothrombins of other mammalian species. This concern has stemmed largely from the observations of an apparently higher molecular weight for rat prothrombin as determined from sodium dodecyl sulfate-gel electrophoresis. The molecular weight data presented here, however, indicate that this difference is probably not as great as once thought. A similarity in molecular weight is also supported by the apparent identical molecular weights of the prothrombin activation products from the various species. Olson (28) has reported observing both an 85,000- and a 73,000-molecular weight rat prothrombin on sodium dodecyl sulfate gels and has suggested that the smaller molecule is an autodigestion product of the
6
7
8
9
Leu Leu Leu Leu Leu
Glx” GLA Glx Glx Glx
Glx GLA Glx Glx Glx
Leu Val Val Phe Val
reported position.
at this
position,
while
( -LA
larger. Direct evidence of this conversion has, however, not been presented. During the course of this study, prothrombin was purified numerous times from separate lots of plasma. These preparations have consistently yielded only a single form of prothrombin. Neither a lower apparent molecular weight prothrombin (73,000) nor the peptide that would be produced as a result of its formation has ever been observed. When isolated prothrombin preparations have been observed to be unstable (a common problem in prothrombin isolation from all species), it has always been due to trace amounts of thrombin in the preparation and the breakdown products are always observed to be primarily prethrombin-1 and fragment-l. Furthermore, the rat prothrombin purified for use in these studies possesses an amino-terminal sequence which is homologous to that of human and bovine prothrombin. While the existence of two molecular weight forms of rat plasma prothrombin cannot be completely ruled out, there was no evidence from this study to confirm the earlier observations, and it is more likely that only a single form with a molecular weight similar to bovine and human prothrombin is present. ACKNOWLEDGMENTS The authors thank Dr. H. F. Deutsch for his aid in obtaining amino acid analyses of rat prothrombin and its degradation products and for determining the sequence of the amino-terminal residues of pro-
RAT
thrombin. They also acknowledge the technical assistance of Judith Hageman and the aid and advice of Dr. Charles Esmon during the studies. REFERENCES
MAGNIJSSON,
S.,
SOTTRUP-JENSEN,
L.,
PETER-
T. E., AND CLAEYS, H. (1975) in Prothrombin and Related Coagulation Factors Boerhave Series (Hemker, H. D., and Veltkamp, J. J., eds.), Vol. 10, pp. 25-46, Leiden University Press, Leiden. PIRKLE, H., MCINTOSH, M., THEODOR, I., AND VERNON, S. (1973) Thromb. Res. 2,461-466. STENN, K. S., AND BLOUT, E. R. (1972) BiochemiStFy 11, 4502-4515. HELDEBRANT, C. M., AND MANN, K. G. (1973) J. Biol. Chem. 248, 3642-3652. ESMON, C. T., OWEN, W. G., AND JACKSON, C. M. (1974) J. Biol. Chem. 249, 7798-7807. ESMON, C. T., OWEN, W. G., AND JACKSON, C. M. (1974) J. Biol. Chem. 249, 8045-8047. KISIEL, W., AND HANAHAN, D. J. (1974) Biothem. Biophys. Res. Commun. 59, 570-577. SEN,
13. 14. 15. 16. 17. 18. 19.
ROSENBERG, J. S., BEELER, D. L., AND ROSENBERG, R. D. (1975) J. Biol. Chem. 250, 1607-
1617. 20. SEEDERS, W. H., E., SEDENSKY, (1972) Thromb.
SAKURAGAWA,
21.
STENFLO, J., ROEPSTORFF,
22.
NELSESTUEN,
G. L.,
J.
B. (1974)
USA ARD,
1. ARONSON, D. L., AND MENACHE, D. (1966) Biochemistry 5, 2635-2640. 2. LANCHANTIN, G. F., HART, D. W., FRIEDMANN, J. A., SAAVEDRA, N. V., AND MEHL, J. W. (1968) J. Biol. Chem. 243, 5479-5485. 3. KISIEL, W., AND HANAHAN, D. J. (1973) Biochim. Biophys. Actu 329, 221-232. 4. DOWNING, M. R., BUTKOWSKI, R. J., CLARK, M. M., AND MANN, K. G. (1975) J. Biol. Chem. 250, 8897-8906. 5. MILLER, K. D., AND SEEGERS, W. H. (1956) Arch. Biochem. Biophys. 60, 398-401. 6. INGWALL, J. S., AND SCHERAGA, H. A. (1969) Biochemistry 8, 1860-1869. 7. OWEN, W. G., ESMON, C. T., AND JACKSON, C. M. (1974) J. Biol. Chem. 249, 594-605. 8. MANN, K. G., HELDEBRANT, C. M., AND FASS, D. N. (1971) J. Biol. Chem. 246, 6106-6114. 9. Cox, A. C., AND HANAHAN, D. J. (1970) Biochim. Biophys. Acta 207, 49-64. 10. MAGNUSSON, S. (1970) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 19, pp. 157-184, Academic Press, New York. 11. MAGNUSSON, S., PETERSEN, T. E., SOTTRUP-JENSEN, L., AND CLAEYS, H. (1975) in Proteases and Biological Control (Reich, E., Rifkin, D. B., and Shaw, E., eds.), pp. 123-149, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 12.
661
PROT ‘HROMBIN
N.,
MCCOY,
J. A., AND DOMBROSE, Res. 1, 293-310.
F.
L. A.
FERNLUND, P. (1974)
P., EGA&, W., PFOC. Nat. Acad.
AND Sci.
71, 2730-2733. ZYTKOVICZ,
J. Biol.
T. H., AND HowChem. 249, 6347-
6350. 23. MAGNUSSON, S., SOTTRUP-JENSEN, L., PETERSEN, T. E., MORRIS, H. R., AND DELL, A. (1974) FEBS Lett. 44, 189-193. 24. ESMON, C. T., JACKSON, C. M., AND SUTTIE, J. W. (1975) J. Biol. Chem. 250, 4095-4099. 25. ESMON, C. T., SADOWSKI, J. A., AMD SUTTIE, J. W. (1975) J. Biol. Chem. 250, 4744-4748. 26. ESMON, C. T., GRANT, G. A., AND SUTTIE, J. W. (1975) Biochemistry 14, 1595-1600. 27. LI, L. F., AND OLSON, R. E. (1967) J. Biol. Chem. 242, 5611-5616. 28. OLSON, k. E. (1974) Vitam. Horm. 32,483-511. 29. SHAPIRO, S. S., AND WAUGH, D. F. (1966) Thromb. Diath. Haemorrh. 16, 469-490. 30. SHAH, D. V., SUTTIE, J. W., AND GRANT, G. A. (1973) Arch. Biochem. Biophys. 159, 483-491. 31. HENRIKSEN, R. A., AND JACKSON, C. M. (1975) Arch. Biochem. Biophys. 170, 149-159. 32. GITEL, S. N., OWEN, W. G., ESMON, C. T., AND JACKSON, C. M. (1973) Proc. Nat. Acad. Sci. USA 70, 1344-1348. 33. DAVIS, B. J. (1964) Ann. N.Y. Acad. Sci. 121, 404-427. 34. LAEMMLI, U. K. (1970) Nature (London) 227, 680-685. 35. YPHANTIS, D. A. (1964) Biochemistry 3, 297-317. 36. MCMEEKIN, T. L., GROVES, M. L., AND HIPP, N. J. (1949)J. Amer. Chem. Sot. 71,3298-3300. 37. BEZKOROVAINY, A., AND DOHERTY, D. G. (1962) Arch. Biochem. Biophys. 96, 491-499. 38. BENCZE, W. L., AND SCHMID, K. (1957) Anal. Chem. 29, 1193-1196. 39. HIRS, C. H. W. (1967) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 11, pp. 59-62, Academic Press, New York. 40. GRAY, W. R. (1972) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 25, pp. 121-138, Academic Press, New York. 41. HARTLEY, B. S. (1970) Biochem. J. 119,805-822. 42. LIN, K. D., AND DEUTSCH, H. F. (1973) Anal. Biochem. 56, 155-164. 43. DUBOIS, M., GILLES, K. A., HAMILTON, J. K., REBERS, P. A., AND SMITH, F. (1956) Anal. Chem. 28, 350-356. 44. WARREN, L. (1959) J. Biol. Chem. 234, 19711975. 45. STENFLO, J., AND GANROT, P. 0. (1972) J. Biol. Chem. 247, 8160-8166. 46. CUATRECASUS, P. (1970) J. Biol. Chem. 245, 3059-3065. 47. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L.,
662
48. 49. 50. 51. 52.
53.
54.
GRANT
AND
AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. BENSON, B., KISIEL, W., AND HANAHAN, J. (1973) Biochim. Biophys. Acta 329, 81-87. STENFLO, J., AND GANROT, P. 0. (1973) Biochem. Biophys. Res. Commun. 50, 98-104. NELSESTUEN, G. L., AND SUTTIE, J. W. (1972) Biochemistry 11, 4961-4964. ESMON, C. T., OWEN, W. G., AND JACKSON, C. M. (1974) J. Biol. Chem. 249, 606-611. HELDEBRANT, C. M., Noyes, C., KINGDON, H. S., AND MANN, K. G. (1973) Biochem. Biophys. Res. Commun. 54, 155-160. ENFIELD, D. L., ERICSSON, L. H., WALSH, K. A., NEURATH, H., AND TITANI, K. (1975) hoc. Nat. Acad. Sci. USA 72, 16-19. HELDEBRANT, C. M., BUTKOWSKI, R. S., BAJAJ,
SU’ITIE S. P., AND MANN, 248, 7149-7163. 55. STENFLO, 8175.
J.
(1972)
K. G. (1973) J.
Biol.
J. Biol.
Chem.
56. NELSESTUEN, G. L., AND SUTTIE, Biol. Chem. 247, 8176-8182.
247,
Chem. 8167-
J. W. (1972)
J.
57. FUJIKAWA, K., COAN, M. H., ENFIELD, D. L., TITANI, K., ERICSSON, L. H., AND DAVIE, E. W. (1974) Proc. Nat. Acad. Sci. USA 71, 427430. 58. KISIEL, W., AND Biochim. Biophys.
HANAHAN, D. J. Acta 304, 103-113.
59. BENSON, B. J., AND HANAHAN, chemistry 14, 3265-3277.
D. J. (1975)
(1973) Bio-
60. HOWARD, J. B., AND NELSESTUEN, G. L. (1975) Proc. Nat. Acad. Sci. USA 72, 1281-1285.
OF
BIOCHEMISTRY
Rat Prothrombin:
AND
BIOPHYSICS
176,
Purification, G. A. GRANT’
Department
of Biochemistry,
650-662
College
of Agricultural Madison, Received
(1976)
Characterization, AND
J. W. SUTTIE
and Life Sciences, Wisconsin 53706 March
and Activation’
University
of Wisconsin-Madison,
15, 1976
Prothrombin has been purified from rat plasma and its properties compared to prothrombin isolated from other species. The molecular weight, amino acid composition, and amino-terminal sequence of rat prothrombin are similar to human and bovine prothrombin. Rat prothrombin binds to phospholipid in the presence of calcium ions, and calcium-binding measurements indicate that it may bind somewhat more calcium than does bovine prothrombin. The proteolytic cleavage of purified rat prothrombin by Factor X, or thrombin yields the same peptides that are formed from similar proteolysis of bovine prothrombin. Factor V and phospholipid were shown to enhance the rate of Factor X, and calcium ion generation of thrombin from rat prothrombin.
Prothrombin, the plasma zymogen of thrombin, has been purified from human (l-4) and bovine (5-10) plasma and extensively characterized. Prothrombin from these two species is a glycoprotein composed of a single polypeptide chain with an apparent molecular weight of 68,00074,000. The complete amino acid sequence of bovine prothrombin (11, 12) and the sequence of portions of human prothrombin (4, 13) have recently been reported. Thrombin is generated from prothrombin by the action of Factor X, in the presence of Factor V, Ca*+, and phospholipid; and the pathway of this activation has been the subject of intensive investigation in recent years (3, 4, 14-20). Prothrombin and Factor X, contain a number of y-carboxyglutamic acid residues (21-23) in their amino-terminal region, and, during the activation of prothrombin, these proteins are bound to the negatively charged surface by Ca*+ interactions to the y-carboxy-
glutamyl residues (241. These residues arise from a post-translational vitamin Kdependent carboxylation of glutamyl residues in liver microsomal precursors of these proteins (25). We have recently reported (26) the purification and partial characterization of one form of the prothrombin precursor from the rat liver and have additional evidence for the existence of multiple forms of this protein. An understanding of the relationship of these liver precursor proteins to the final carboxylated product depends on a knowledge of the structural and functional properties of rat plasma prothrombin. Since it has been claimed (27, 28) that rat prothrombin differs significantly in some of its structural properties from human and bovine prothrombin, we are therefore reporting a purification and characterization of this protein. In addition, the activation of rat prothrombin by Factor X, and its proteolysis by thrombin have been studied; and the peptides released from these digestions have been compared to similar products obtained from bovine prothrombin digestion.
’ Research supported by the College of Agricultural and Life Sciences, University of WisconsinMadison, and in part by Grant No. AM-14881 from the National Institutes of Health, USPHS, and in part by National Institutes of Health Training Grant No. GM-00236 BCH. ’ Present address: Department of Biological Chemistry, Division of Biology and Biomedical Sciences, Washington University, St. Louis, MO. 63110.
MATERIALS
650 Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
AND
METHODS
Assays and measurements. Prothrombin was measured by the two-stage assay of Ware and Seegers as modified by Shapiro and Waugh (29) and by
RAT
651
PROTHROMBIN
activation with&his carinatus venom as previously described (30). Clotting times were converted to thrombin activity (NH-I units) by comparison with a standard curve prepared from NIH standard thrombin (Lot 3B). Cephalin was the source of lipid for all activations done in the presence of phospholipid. Calcium binding was performed by equilibrium dialysis (31). Phospholipid vesicles (dioleoylphosphatidylglycerol:dioleoylphosphatidylcholine, 1:l molar ratio) used for binding studies were prepared as described by Gitel et al. (32).3 Rat prothrombin was incubated with labeled phospholipid vesicles in the presence of either calcium ions or EDTA for 30 min and then applied to a 0.9-cm column of Bio-Gel A0.5m. Polyacrylamide disc gel electrophoresis was performed by the method of Davis (33). Polyacrylamide disc gel electrophoresis in the presence of sodium dodecyl sulfate was performed according to Laemmli (34), and peptide molecular weight was estimated from these gels by comparison to a curve prepared with standard proteins after disulfide bond reduction with P-mercaptoethanol prior to electrophoresis. Molecular weight estimation by sedimentation equilibrium was performed by the meniscus depletion technique on a Spinco Model E ultracentrifuge employing Raleigh interference optics (35). Partial specific volumes were calculated from amino acid and carbohydrate compositions using residue specific volumes (36, 37). Amino acid analyses were performed on a Durrum Model D-500 analyzer using single-column technology. Tryptophan was determined spectrophotometrically by the procedure of Bencze and Schmid (38). Cysteine and methionine were determined as cysteic acid and methionine sulfone alter oxidation with performic acid (39). Serine and threonine were determined by extrapolation to zero time assuming a first-order rate of destruction. Hydrolysis in 6 N HCl was allowed to proceed for 24, 48, and 96 h at 110°C in sealed evacuated tubes. Aminoterminal amino acid analysis was performed using the dansyl method as described by Gray for proteins (40). The dansyl amino acids were identified by thinlayer chromatography on polyamide sheets as described by Hartley (41). Amino-terminal amino acid sequencing was performed (42) on a Beckman Model 890-C sequencer using a protein program employing Quadrol buffer. The phenylthiazolinone derivatives were converted to the free amino acids by acid hydrolysis and identified on a Durrum D-500 analyzer. Hexose analyses were performed by the procedure of DuBoiset al. (43). Sialic acid was determined by the procedure of Warren (44) after hydrolysis of the glycoprotein for 1 h at 80°C in 0.1 N H,SO,, and hexosamines were determined by amino acid analy-
sis after hydrolysis for 4 h at 110°C in 1 N HCl. All hydrolyses were done in sealed evacuated tubes. Reagents. Phenylmethylsulfonyl fluoride, soybean trypsin inhibitor (type II-S), and sodium heparin were purchased from Sigma Chemical Company, St. Louis, MO. Soybean trypsin inhibitor was covalently linked to agarose by the method of Cuatrecasus (46) as previously described (26). BenzamidineHCl was purchased from Aldrich Chemical Company, Milwaukee, Wis. QAE-Sephadex’ was a product of Pharmacia (Uppsala). Bovine Factor X, Factor X-activating enzyme, bovine Factor V, and cephalin were obtained and prepared as previously described (26).3 Bovine prothrombin was prepared as described by Stenflo (45). Ratprothrombin isolation. For the isolation of rat prothrombin, blood was drawn by cardiac puncture from large male donor rats under ether anesthesia into one-tenth volume of 2.85% sodium citrate and centrifuged at 3600g for 16-15 min, and the plasma was immediately frozen. After thawing, the plasma was centrifuged at 8OOOg for 5 min to remove insoluble material and treated with sodium heparin (10 units/ml) and soybean trypsin inhibitor (20 pg/ml). All subsequent steps were performed at 4°C except for the ion-exchange chromatography which was performed at room temperature. Ten milliliters of 1 M BaC12.H,0 was added slowly to each 125 ml of gently stirring plasma, and stirring was continued for 15-30 min. The barium citrate precipitate, with adsorbed prothrombin, was collected by centrifugation and washed three times with 0.9% NaCl, 0.005 M sodium citrate. After the final wash, the precipitate was dissolved in 0.2 M EDTA (pH = 7.01, and the dissolved barium and some contaminating protein were precipitated by the addition of an equal volume of saturated ammonium sulfate. The precipitate was removed by centrifugation, and prothrombin was precipitated from the supernatant by addition of the same volume of saturated ammonium sulfate. The precipitated prothrombin was dissolved in 0.05 M imidazole-HCl (pH = 7.81, 0.02 M sodium citrate, 0.001 M benzamidine-HCl, and 0.2 M ammonium chloride and dialyzed against the same buffer for 3-4 h. The crude prothrombin was chromatographed on QAE-Sephadex (0.9 x 28 cm) using a loo-ml linear (0.2 to 0.45 M) ammonium chloride gradient. At this point, a major contaminant elutes from the column on the trailing edge of the prothrombin peak, and pure prothrombin is found on the ascending edge of the peak. The yield of pure prothrombin can be increased by pooling the appropriate fractions and rechromatographing on an identical QAESephadex column. Following chromatography, the prothrombin fractions were stored at 4°C in the presence of 0.001 M phenylmethylsulfo-
3 Kindly supplied by Dr. C. M. ington University, St. Louis, MO.
4 Abbreviation propyl)aminoethyl.
Jackson,
Wash-
used:
QAE-,
Diethyl-(2-hydroxy-
652
GRANT
AND
nyl fluoride. The elution profile of the second QAESephadex column is shown in Fig. 1. The product has a specific activity of 2200 NIH thrombin units/ mg of protein and appeared homogeneous by both alkaline and sodium dodecyl sulfate gel electrophoresis (Fig. 1). This specific activity is somewhat higher than that usually cited for bovine prothrombin but was highly reproducible under the assay conditions employed and consistent over a large number of prothrombin preparations. In addition, bovine prothrombin assayed in an identical manner gives a similar specific activity (56). The specific activity of the thrombin generated from rat prothrombin was approximately 4500 NIH unitsimg of protein. Sedimentation equilibrium studies of the purified rat prothrombin showed that a plot of the logarithm of the concentration versus the square of the radial distance was linear. RESULTS
ACTIVATION
OF RAT PROTHROMBIN
The time course of activation of rat prothrombin by Factor X, and calcium ions, as visualized by sodium dodecyl sulfate-gel electrophoresis (Fig. 2) demonstrates the formation of four components in addition to prothrombin. By analogy to the reports on bovine prothrombin and on the basis of further characterization (see below), they are identified as follows.” From the top of the gels (largest apparent molecular weight component), prothrombin is followed by a transient band which is prea band containing both thrombin-1, thrombin and prethrombin-2, which coelectrophorese under these conditions, and a band of fragment-l. At the bottom of the gels is a band of fragment-2 which is not really detected in the photographs. Analysis of the reaction mixture by QAE-Sephadex chromatography (Fig. 3) showed four peaks of protein. The first two peaks con: The prothrombin activation component nomenclature used is: prethrombin-1 (P-1) and prethrombin-2 (P-2) to describe the thrombin-containing portion of the molecule corresponding to intermediate-I and intermediate-2 of Jackson and co-workers (7) and Mann and co-workers (54); and fragment-l (F-l) and fragment-2 (F-2) to describe peptides from the nonthrombin-containing portion of the molecule (7). These peptides correspond to intermediate-3 and intermediate-4 of Mann and co-workers (54). Fragment-l ‘2 (F-l .2) is the peptide corresponding to covalently linked F-l and F-2. A comparison of this nomenclature system to those used by other workers can be found in a recent paper by Downing et al. (4).
SU’lTIE
tained thrombin and prethrombin-2 which cochromatograph in this system. Prethrombin-2 was identified by an increase in thrombin activity after further activation with Factor X, or E. carinatus venom and by sodium dodecyl sulfate electrophoresis following reduction with P-mercaptoethanol. After reduction, the thrombin molecule moved with a slightly higher mobility than prethrombin-2. The double-peak elution behavior of thrombin and prethrombin-2 on QAE-Sephadex is similar to that seen with material derived from bovine prothrombin and is the result of loading the column at an ionic strength where thrombin binding to the gel is weak (7). The third peak was identified as fragment2 and the last peak as fragment-l. Although fragment-2 is not readily visible on stained gels (Fig. 2) its existence was confirmed by ion-exchange chromatography. It is of interest that the fragments-l and -2 generated from rat prothrombin elute from ion-exchange chromatograms in the opposite order than do the corresponding fragments from bovine prothrombin (7). In the case of human prothrombin they tend to coelute and must be separated by different means (I). The significance of this species heterology, while apparently charge related, has not been determined. Rat prothrombin was also activated with Factor X, in the presence of Factor V and phospholipid, and the activation fragments, as visualized on sodium dodecyl sulfate-gel electrophoresis, are shown in Fig. 4. The reaction was essentially complete in 20-30 min, as opposed to several hours needed for the reaction with Factor X, and calcium alone, and a new transient band was seen just above the thrombindoublet in the third prethrombin-2 through sixth gels. This component was not characterized but is probably fragment-l .2. When this activation mixture was reduced in this way prior to electrophoresis, it was possible to observe the separation of thrombin and prethrombin-2 and the transient nature of prethrombin-2. When rat prothrombin was digested with thrombin, only two bands appeared following sodium dodecyl sulfate-gel electrophoresis: prethrombin-1 and fragment-l (Fig. 5). Ion-exchange analysis (Fig. 6) of
RAT
653
PROTHROMBIN
06 05
- 1000 - 900
04
-800 - 700
Fraction
FIG. column. alkaline
1. Ion-exchange The gels shown gel electrophoresis
Number
chromatography of rat prothrombin on the second QAE-Sephadex are: left, sodium dodecyl sulfate-disc gel electrophoresis; and right, (pH = 9.5) of 10 pg of the final product (pooled fractions 40-47).
-
Pro
-
Pre-I
c-
Pre-2 ThFornbin
-
FIG. 2. Sodium dodecyl sulfate-gel electrophoresis of prothrombin (100 pg) with Factor X, (10 pg) and calcium left to right, 0,30,60,240, and 1200 min. The reaction was 10 ~1 of sodium dodecyl sulfate to an aliquot of the reaction bath for 5-10 min.
F-i
the time course of activation of rat (10 mM). Incubation times are, from stopped at each time point by adding mixture and heating in a 70°C water
654
GRANT
AND
SU’lTIE
BOO 0 600 2 h 400~ 3 200
FIG. 3. QAE-Sephadex chromatography of the products of the Factor X, activation of rat prothrombin. Rat prothrombin (2 mgl was incubated with Factor X, (24 pg) and calcium (10 mM) for 20 h and then chromatographed on a 0.9 x 2%cm column of QAE-Sephadex. The chromatography was performed in 0.02 M Tris.HCl buffer (pH = 7.51, 0.1 M NaCl, and the column was developed with a loo-ml linear gradient of NaCl from 0.1 to 0.5 M. One centimeter of soybean trypsin inhibitor-agarose was layered over the top of the QAE-Sephadex in order to remove Factor X, from the reaction mixture and prevent further proteolysis during chromatography. The gel inserts are sodium dodecyl sulfate-disc gel electrophoresis of the starting material (far left) and the peak fractions of each protein peak. Both thrombin-prethrombin-2 peaks displayed the same gel pattern. All gels were reduced with P-mercaptoethanol to resolve thrombin and prethrombin-2. Absorbance at 280 nm, (0); thrombin activity, (0).
--
Pro
--
P-l F-k’ Thrombin F-l
-
F-2
FIG. 4. Sodium dodecyl sulfate-gel electrophoresis of the time course of activation of rat prothrombin (200 pg) with Factor X, (10 pg), Factor V (30 pg), phospholipid (300 fig), and calcium (10 mM). Incubation times are, from left to right, 0,40, 70, 100, 140,300,420,600, and 1200 s. Gel samples were treated as described in Fig. 2 except that they were also reduced with /3-mercaptoethanol prior to electrophoresis. The faint protein band between thrombin and fragment-l is factor X,.
RAT
655
PROTHROMBIN
-
Pro
-
P-l
-
F-l
FIG. 5. Sodium dodecyl sulfate-gel electrophoresis of the time course of proteolysis prothrombin (100 pg) with rat thrombin (5 pg). Incubation times are, from left to right, 4, and 60 min. Gel samples were treated as described in Fig. 2.
the final reaction mixture also demonstrated that two components were produced. The first peak, which could be activated to thrombin by E. carinatus venom or Factor X,, is prethrombin-1; and the second peak, which cannot be activated to thrombin, is fragment-l. As is the case in all other species studied, thrombin was not generated by thrombin proteolysis. Isolated prethrombin-1 was activated by Factor X, and calcium, and the products produced were identified by electrophoresis and ion-exchange chromatography (data not shown) as prethrombin-2, thrombin, and fragment-2. With the exception of the reversal in elution order of fragment-l and fragment-2 on QAE-Sephadex chromatography, the activation of rat prothrombin by Factor X, and its proteolysis by thrombin (as judged by sodium dodecyl sulfate-gel electrophoresis and ion-exchange chromatography)
of rat 0, 1,2,
appear to proceed in a manner identical to that reported for bovine prothrombin (1420). All of the rat prothrombin activation products appear to correspond to an analogous activation product from bovine prothrombin. Molecular weight determinations (Table I) indicated that, with the possible exception of rat prothrombin itself, all of the protein components display molecular weights similar to those of the bovine system. The molecular weight of rat prothrombin based on sodium dodecyl sulfate-gel electrophoresis has been reported by Olson (27) to be 85,000. In these studies, rat prothrombin had an apparent molecular weight by this method which was only slightly higher than the 70,000-75,000 reported for bovine prothrombin. When rat prothrombin and bovine prothrombin were slightly coelectrophoresed, a single widened band was observed. The molecu-
656
GRANT
Fraction
AND
SUTTIE
Number
FIG. 6. QAE-Sephadex chromatography of the products of thrombin proteolysis of rat prothrombin. Rat prothrombin (1 mg) was incubated with rat thrombin (20 /*g) for 90 min and then chromatographed on a 0.9 x 2%cm column of QAE-Sephadex as described in Fig. 3. The gels shown are sodium dodecyl sulfate gels of the starting material (far left) and the peak fractions of each protein peak. Absorbance at 280 nm, (0); thrombin activity after activation with E. carinatus venom, (0). TABLE MOLECULAR
-
WEIGHTS
Protein Prothrombin equilibrium) Prothrombin Prethrombin-I Prethrombin-2 Thrombin Fragment-l Fragment-2 (I All dod‘ecyl Ir See ” See
(by
OF PROTHROMBIN
I
AND PROTHROMBIN
Rat sedimentation
77,000 (6 = 0.706) 75,000-80,000 58,000 38,000 37,000 25,000 12,000
values exceut those indicated as obtained bv sedimentation sulfate electrophoresis in acelamide gels. ” Ref. (4).and (7). Ref. (31, (4), (19), and (58).
lar. weight of rat prothrombin, as determined by this technique, closely resembles that of bovine prothrombin determined by the same method. The reasons for the discrepancy in the reported molecular weights of rat and bovine prothrombin are not understood, but the results of this study would suggest that the molecular weights are not as different as once thought. The amino acid and carbohydrate compositions of rat prothrombin, calculated on the basis of its sedimentation equilibrium molecular weight, are shown in Table II
ACTIVATION
PRODUCTS"
Bovine”
Human’
75,000 (6 = 0.711) 70,000-75,000 51,000-61,000 38,000-41,000 35,000-41,000 23,000-25,000 ll,OOO-13,000
72,000 (U = 0.711 70,000-75,000 51,000-55,000 37,000-41,000 37,000-41,000 23,000-25,000 13,000-16,000
equilibrium
were
obtained
by sodium
and compared to a previous composition reported for rat prothrombin and several compositions reported for bovine and human prothrombin. The amino acid composition of rat prothrombin appears to be quite similar to that of both bovine and human prothrombin. The amino acid composition of rat prothrombin reported by Li and Olson (27) shows considerably higher values for most residues. This is due in part to the fact that their composition was calculated for a peptide molecular weight of 86,000 with no correction for the amount of carbohydrate in the protein. When these
RAT
PROTHROMBIN
values are recalculated based on the peptide molecular weight determined in this study, somewhat better agreement is obtained (Table II). The amino acid composition of the thrombinand Factor X,-catalyzed digestion products of rat prothrombin were also determined. The data, calculated on the basis of the peptide molecular weights in Table I, are shown in Table III. Within the error of the technique, the sum of the residues found in the products was equal to that found in the respective parent molecules (prothrombin = prethrombin-1 + fragment-l = thrombin + fragment-l + fragment-2). These data support the electrophoretic and ion-exchange analyses of rat prothrombin activation products by confirming the relationship of the various proteins to each other and by accounting TABLE Axnmo --Residue Asp Thr Ser Glu Pro GUY Ala cysi2 Val Met Ile Leu ‘br Phe His LYS Arg Trp
ACID
II
AND CARBOHYDRATE PROTHROMBIN” Rat” Rat’
Bovine”
HLlman’
59.2 36.9 45.8 72.0 37.4 57.8 28.7 15.1 31.0 5.4 22.1 43.6 14.9 21.2 13.4 29.5 35.9 15.4
55-62 25-29 30-41 66-79 32-38 37-48 29-34 16-20 31-33 5-8 17-19 43-45 17-18 16-20 8-10 28-33 38-43 11-19
56-59 32-38 36-38 69-77 31-32 43-46 32-34 21-23 29-32 7-8 21 38-41 18-21 18-20 9-11 26-32 31-36 11-20
90 63 52 94 40 55 34 13 47
(68) (48) (40) (71) (30) (42) (26) (10) (36)
12 (9) 27 53 23 38 15 42 35 15
(21) (40) (17) (29) (11) (32) (27) (11)
COMPOSITIONS
OF
3.9-5.9% 4.1% Hexose 7.5% GlcN 4.5% 2.5-4.5% 2.5% AcNeu 2.8% 3.2-4.4% 3.5% 0 All values for amino acids are expressed as residues per mole. b Experimental values, this study. v From Ref. (27). Based on 86,000 molecular weight. Numbers in parentheses are recalculated using the peptide molecular weight determined in this study. ” See Ref. (7), (541, (55), and (56). ” See Ref. (2) and (4).
657
for all of the prothrombin residues in the various products. The possibility that very small peptides or even single amino acids could be liberated during activation still exists, however, since they probably would not be detected by these methods. Using norleucine as an internal standard, an extinction coefficient expressed as E :Tm was calculated from the amino acid analysis. This procedure gave a value of 15.9 for rat prothrombin which agreed with a value of 16.5 estimated by comparing the protein absorbance at 280 nm to the protein content as determined by the Lowry procedure (47). These values are similar to those reported for bovine prothrombin which range from 13.6 to 16.5 (6,
9, 55, 59). The calcium-binding properties of rat prothrombin are presented in Fig. 7. The Scatchard plot of these data indicates,two distinct classes of binding sites which is in agreement with the bovine prothrombinbinding studies of Benson et al. (48) but is in contrast to several other studies (31, 49, 50) which indicate positive cooperativity at low calcium ion concentrations rather than two noninteracting classes of sites. Similar results were obtained on replicate analyses, but no extensive survey of the calcium-binding properties of different preparations was attempted. The total of 17 Ca2+ binding sites indicated for rat prothrombin is also somewhat higher than the 10 to 12 sites indicated in all bovine studies. The binding constants of 1.55 x lop3 and 2.2 x 10e4 M determined for the two classes of sites from the slopes of the Scatchard plot are similar to those reported for bovine prothrombin. The ability of rat prothrombin to bind to phospholipid is illustrated in Fig. 8. These studies (top and middle profiles) support a calcium-dependent binding of phospholipid to prothrombin, while the bottom profile indicates that this apparent binding was not due to a calcium-dependent aggregation of prothrombin. The effect of other components of the prothrombinase activation complex on the rate of Factor X,-catalyzed activation of rat prothrombin is shown in Fig. 9. Both phospholipid and Factor V were found to accelerate the rate of activation, and, as
658
GRANT
AND
SUlTIE
TABLE Residue ASP Thr Ser Glu Pro GUY Ala cysi2 Val Met Ile Leu ‘br Phe His LYS Arg Trp ” Values
III
ACID
COMPOSITIONS
OF THE
Prothrombin
Thrombin
F-l
F-2
Thrombin + F-l + F-2
16.7 12.3 13.8 22.1 11.2 17.0 7.9 5.2 8.7 1.3 4.1 9.2 4.5 5.1 3.4 5.4 10.5 3.7
12.6 4.8 9.8 16.5 8.0 15.6 7.1 5.0 5.2 0 1.8 10.9 2.8 4.0 1.5 4.2 5.3 2.4
61.2 37.8 52.4 69.0 40.1 58.0 30.9 18.6 29.9 5.4 19.2 42.4 14.2 19.3 11.4 27.4 32.8 14.5
AMINO
59.2 36.9 45.8 72.0 37.4 57.8 28.7 15.1 31.0 5.4 22.1 43.6 14.9 21.2 13.4 29.5 35.9 15.4 expressed
31.9 20.7 28.8 30.4 20.9 25.4 15.9 8.4 16.0 4.1 13.3 22.3 6.9 10.2 6.5 17.8 17.0 8.4 as residues
ACTIVATION
PRODUCTS
OF RAT
PROTHROMBIN”
Prethrombin 46.4 24.8 36.3 53.0 26.9 43.2 25.6 10.7 23.3 4.2 16.2 35.1 8.1 15.4 8.3 23.6 26.3 11.0
1
P-l + F-l 63.1 37.1 50.1 75.1 38.1 60.2 33.5 15.9 32.0 5.5 20.3 44.3 12.6 20.5 11.7 29.3 36.8 14.7
per mole.
FIG. 7. Calcium binding of rat prothrombin. The ability of rat prothrombin (1 mgiml) to bind calcium ions was determined by equilibrium dialysis in the presence of 0.05 M Tris HCl, 0.1 M NaCl (pH = 8.01. Calcium and prothrombin were placed on opposite sides of the membrane in 0.1 ml of cells and dialysis was allowed to proceed for 16 h at room temperature. The upper plot indicates the moles of calcium bound 3s a function of the calcium concentration. The lower plot is a Scatchard plot of the same data.
expected from similar studies on bovine prothrombin, the presence of both Factor V and phospholipid indicates an additive effect which is consistent with the binding of each component at a different site on the prothrombin molecule. The plateau effect seen when phospholipid or Factor V was added separately is most likely due to the conversion of all of the prothrombin present in the incubation to thrombin precursors (prethrombin-1 and -2) which no longer bind the added component and are thus no longer accelerated by it. The activation of prethrombin-1 by Factor X, (Fig. 9) in the presence and absence of phospholipid indicates that, as in other species studied, phospholipid binding is associated with the fragment-l portion of prothrombin. Reaction of rat prothrombin (100-200 pg) with dansyl chloride followed by hydrolysis and chromatography of the hydrolysate on thin-layer polyamide sheets revealed alanine as the amino-terminal residue. This is the same amino terminus as found on bovine (11, 51, 52) and human prothrombin (4, 12). The amino acid sequence of the amino terminal region of rat prothrombin was also determined (Table IV) for the purpose of comparison to other species. The se-
RAT
quence of the first nine residues in rat prothrombin shows a high degree of homology with the same region in bovine prothrombin, human prothrombin, Factor IX, and the light chain of Factor X. These data confirm the presence of alanine as the amino-terminal residue of rat prothrombin and suggest that the first two y-carboxyglutamic acid residues are in the same positions (7 and 8) as in the bovine clotting factors. The methods utilized would not detect y-carboxyglutamic acid, but the data do indicate that positions 7 and 8 are occupied by glutamic acid. Since these are the same two positions occupied by the first two y-carboxyglutamic acid residues in bovine prothrombin, it is reasonable to expect that further chemical studies would confirm this homology. These data have provided a description of the chemical nature of rat prothrombin PL,
Co”
-20 - I5 - IO -5
PRO,
PL , EDTA
PRO.
Fraction
6
Ca”
number
FIG. 8. Phospholipid binding to rat prothrombin. Rat prothrombin (20 fig) was incubated with phospholipid (250 yg) for 30 min and then chromatographed on a 0.9 x 28-cm column of Bio-Gel A-0.5m in 40 mM Tris’HCl, 70 mM NaCl. When included in the incubation and column, calcium was 10 mM and EDTA was 1.5 mM. The apparent increased size of rat prothrombin in the presence of phospholipid and EDTA over that of prothrombin in the presence of Ca2+ alone is due to an incomplete inhibition of phospholipid binding by this level of EDTA. The abbreviations used are PRO, prothrombin; PL, phospholipid; and EDTA, ethylenediaminetetraacetic acid.
0
4 0
2 6 L
DISCUSSION
PRO.
659
PROTHROMBIN
5
10 15 INCULIATION
20 25 TlME h”I
30
FIG. 9. The effect of phospholipid and Factor V on the Factor X,-catalyzed activation of rat prothrombin and rat prethrombin-1. Rat prothrombin (6 Kg) or rat prethrombin-1 (20 pg) was incubated with Factor X, (1 pgl in the presence of 10 mM calcium and either phospholipid (300 pg), Factor V (10 wg), or both in a volume of 1 ml. Aliquots were removed at time intervals and assayed for thrombin. Top: prothrombin and Factor X, alone (01; plus phospholipid, (ml; plus Factor V, (0); plus phospholipid and Factor V, (0). Bottom: prethrombin-1 and Factor X, alone, (0); plus phospholipid,
and serve to demonstrate its similarity to bovine and human prothrombin which have been extensively studied by several groups. The amino acid and carbohydrate compositions of rat, bovine, and human prothrombin are very similar, as are the compositions of the activation products. The most striking difference between rat and bovine prothrombin is seen in the type of calcium binding observed and the total number of sites participating as well as the ionic properties of fragment-l and fragment-2. The apparent increased acidity of rat prothrombin fragment-l may be related to the apparent increased number of calcium-binding sites. Rat prothrombin also appears to be activated in vitro in a manner identical to that of bovine prothrombin. Both proteins produce similar activation components in re-
660
GRANT
AND
SUTI’IE
TABLE HOMOLOGY
Protein
Rat prothrombin” Bovine prothrombin’ Human prothrombin” Bovine Factor IX Bovine Factor X’
H,N-1 Ala Ala Ala ‘br Ala
OF VITAMIN
2 ASX
Asn Asn Asn Asn
3 Ser LYS Thr Ser Ser
IV K-DEPENDENT
4
PROTEINS
5
GUY GUY - ‘1
Phe Phe Phe
GUY - (1
LYS Phe
” This study. ’ The abbreviation Glx indicates that glutamic acid has been indicates a positive identification of y-carboxyglutamic acid at this c Magnusson et al. (23); Stenflo et al. (21). ” Downing et al. (4). ’ Fujikawa et al. (57). I Light chain, Enfield et al. (53); Howard and Nelsestuen (60). ‘I Gaps are inserted at these positions to optimize homology
sponse to Factor X, and thrombin, and both appear to participate in the same type of protein-protein and lipid-protein interactions during activation. There was no indication in the studies that rat prothrombin activation proceeds via a prethrombin-2 and prethrombin-2’ conversion as has been suggested (4) for human prothrombin. It is unlikely, however, that the 13-amino-acid peptides which account for the difference in these two forms would have been detected in our studies. While prothrombins from all species have usually been considered to perform identical functions in the clotting process, some question has been raised in the past concerning the degree to which rat prothrombin might differ structurally from t?le prothrombins of other mammalian species. This concern has stemmed largely from the observations of an apparently higher molecular weight for rat prothrombin as determined from sodium dodecyl sulfate-gel electrophoresis. The molecular weight data presented here, however, indicate that this difference is probably not as great as once thought. A similarity in molecular weight is also supported by the apparent identical molecular weights of the prothrombin activation products from the various species. Olson (28) has reported observing both an 85,000- and a 73,000-molecular weight rat prothrombin on sodium dodecyl sulfate gels and has suggested that the smaller molecule is an autodigestion product of the
6
7
8
9
Leu Leu Leu Leu Leu
Glx” GLA Glx Glx Glx
Glx GLA Glx Glx Glx
Leu Val Val Phe Val
reported position.
at this
position,
while
( -LA
larger. Direct evidence of this conversion has, however, not been presented. During the course of this study, prothrombin was purified numerous times from separate lots of plasma. These preparations have consistently yielded only a single form of prothrombin. Neither a lower apparent molecular weight prothrombin (73,000) nor the peptide that would be produced as a result of its formation has ever been observed. When isolated prothrombin preparations have been observed to be unstable (a common problem in prothrombin isolation from all species), it has always been due to trace amounts of thrombin in the preparation and the breakdown products are always observed to be primarily prethrombin-1 and fragment-l. Furthermore, the rat prothrombin purified for use in these studies possesses an amino-terminal sequence which is homologous to that of human and bovine prothrombin. While the existence of two molecular weight forms of rat plasma prothrombin cannot be completely ruled out, there was no evidence from this study to confirm the earlier observations, and it is more likely that only a single form with a molecular weight similar to bovine and human prothrombin is present. ACKNOWLEDGMENTS The authors thank Dr. H. F. Deutsch for his aid in obtaining amino acid analyses of rat prothrombin and its degradation products and for determining the sequence of the amino-terminal residues of pro-
RAT
thrombin. They also acknowledge the technical assistance of Judith Hageman and the aid and advice of Dr. Charles Esmon during the studies. REFERENCES
MAGNIJSSON,
S.,
SOTTRUP-JENSEN,
L.,
PETER-
T. E., AND CLAEYS, H. (1975) in Prothrombin and Related Coagulation Factors Boerhave Series (Hemker, H. D., and Veltkamp, J. J., eds.), Vol. 10, pp. 25-46, Leiden University Press, Leiden. PIRKLE, H., MCINTOSH, M., THEODOR, I., AND VERNON, S. (1973) Thromb. Res. 2,461-466. STENN, K. S., AND BLOUT, E. R. (1972) BiochemiStFy 11, 4502-4515. HELDEBRANT, C. M., AND MANN, K. G. (1973) J. Biol. Chem. 248, 3642-3652. ESMON, C. T., OWEN, W. G., AND JACKSON, C. M. (1974) J. Biol. Chem. 249, 7798-7807. ESMON, C. T., OWEN, W. G., AND JACKSON, C. M. (1974) J. Biol. Chem. 249, 8045-8047. KISIEL, W., AND HANAHAN, D. J. (1974) Biothem. Biophys. Res. Commun. 59, 570-577. SEN,
13. 14. 15. 16. 17. 18. 19.
ROSENBERG, J. S., BEELER, D. L., AND ROSENBERG, R. D. (1975) J. Biol. Chem. 250, 1607-
1617. 20. SEEDERS, W. H., E., SEDENSKY, (1972) Thromb.
SAKURAGAWA,
21.
STENFLO, J., ROEPSTORFF,
22.
NELSESTUEN,
G. L.,
J.
B. (1974)
USA ARD,
1. ARONSON, D. L., AND MENACHE, D. (1966) Biochemistry 5, 2635-2640. 2. LANCHANTIN, G. F., HART, D. W., FRIEDMANN, J. A., SAAVEDRA, N. V., AND MEHL, J. W. (1968) J. Biol. Chem. 243, 5479-5485. 3. KISIEL, W., AND HANAHAN, D. J. (1973) Biochim. Biophys. Actu 329, 221-232. 4. DOWNING, M. R., BUTKOWSKI, R. J., CLARK, M. M., AND MANN, K. G. (1975) J. Biol. Chem. 250, 8897-8906. 5. MILLER, K. D., AND SEEGERS, W. H. (1956) Arch. Biochem. Biophys. 60, 398-401. 6. INGWALL, J. S., AND SCHERAGA, H. A. (1969) Biochemistry 8, 1860-1869. 7. OWEN, W. G., ESMON, C. T., AND JACKSON, C. M. (1974) J. Biol. Chem. 249, 594-605. 8. MANN, K. G., HELDEBRANT, C. M., AND FASS, D. N. (1971) J. Biol. Chem. 246, 6106-6114. 9. Cox, A. C., AND HANAHAN, D. J. (1970) Biochim. Biophys. Acta 207, 49-64. 10. MAGNUSSON, S. (1970) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 19, pp. 157-184, Academic Press, New York. 11. MAGNUSSON, S., PETERSEN, T. E., SOTTRUP-JENSEN, L., AND CLAEYS, H. (1975) in Proteases and Biological Control (Reich, E., Rifkin, D. B., and Shaw, E., eds.), pp. 123-149, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 12.
661
PROT ‘HROMBIN
N.,
MCCOY,
J. A., AND DOMBROSE, Res. 1, 293-310.
F.
L. A.
FERNLUND, P. (1974)
P., EGA&, W., PFOC. Nat. Acad.
AND Sci.
71, 2730-2733. ZYTKOVICZ,
J. Biol.
T. H., AND HowChem. 249, 6347-
6350. 23. MAGNUSSON, S., SOTTRUP-JENSEN, L., PETERSEN, T. E., MORRIS, H. R., AND DELL, A. (1974) FEBS Lett. 44, 189-193. 24. ESMON, C. T., JACKSON, C. M., AND SUTTIE, J. W. (1975) J. Biol. Chem. 250, 4095-4099. 25. ESMON, C. T., SADOWSKI, J. A., AMD SUTTIE, J. W. (1975) J. Biol. Chem. 250, 4744-4748. 26. ESMON, C. T., GRANT, G. A., AND SUTTIE, J. W. (1975) Biochemistry 14, 1595-1600. 27. LI, L. F., AND OLSON, R. E. (1967) J. Biol. Chem. 242, 5611-5616. 28. OLSON, k. E. (1974) Vitam. Horm. 32,483-511. 29. SHAPIRO, S. S., AND WAUGH, D. F. (1966) Thromb. Diath. Haemorrh. 16, 469-490. 30. SHAH, D. V., SUTTIE, J. W., AND GRANT, G. A. (1973) Arch. Biochem. Biophys. 159, 483-491. 31. HENRIKSEN, R. A., AND JACKSON, C. M. (1975) Arch. Biochem. Biophys. 170, 149-159. 32. GITEL, S. N., OWEN, W. G., ESMON, C. T., AND JACKSON, C. M. (1973) Proc. Nat. Acad. Sci. USA 70, 1344-1348. 33. DAVIS, B. J. (1964) Ann. N.Y. Acad. Sci. 121, 404-427. 34. LAEMMLI, U. K. (1970) Nature (London) 227, 680-685. 35. YPHANTIS, D. A. (1964) Biochemistry 3, 297-317. 36. MCMEEKIN, T. L., GROVES, M. L., AND HIPP, N. J. (1949)J. Amer. Chem. Sot. 71,3298-3300. 37. BEZKOROVAINY, A., AND DOHERTY, D. G. (1962) Arch. Biochem. Biophys. 96, 491-499. 38. BENCZE, W. L., AND SCHMID, K. (1957) Anal. Chem. 29, 1193-1196. 39. HIRS, C. H. W. (1967) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 11, pp. 59-62, Academic Press, New York. 40. GRAY, W. R. (1972) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 25, pp. 121-138, Academic Press, New York. 41. HARTLEY, B. S. (1970) Biochem. J. 119,805-822. 42. LIN, K. D., AND DEUTSCH, H. F. (1973) Anal. Biochem. 56, 155-164. 43. DUBOIS, M., GILLES, K. A., HAMILTON, J. K., REBERS, P. A., AND SMITH, F. (1956) Anal. Chem. 28, 350-356. 44. WARREN, L. (1959) J. Biol. Chem. 234, 19711975. 45. STENFLO, J., AND GANROT, P. 0. (1972) J. Biol. Chem. 247, 8160-8166. 46. CUATRECASUS, P. (1970) J. Biol. Chem. 245, 3059-3065. 47. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L.,
662
48. 49. 50. 51. 52.
53.
54.
GRANT
AND
AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. BENSON, B., KISIEL, W., AND HANAHAN, J. (1973) Biochim. Biophys. Acta 329, 81-87. STENFLO, J., AND GANROT, P. 0. (1973) Biochem. Biophys. Res. Commun. 50, 98-104. NELSESTUEN, G. L., AND SUTTIE, J. W. (1972) Biochemistry 11, 4961-4964. ESMON, C. T., OWEN, W. G., AND JACKSON, C. M. (1974) J. Biol. Chem. 249, 606-611. HELDEBRANT, C. M., Noyes, C., KINGDON, H. S., AND MANN, K. G. (1973) Biochem. Biophys. Res. Commun. 54, 155-160. ENFIELD, D. L., ERICSSON, L. H., WALSH, K. A., NEURATH, H., AND TITANI, K. (1975) hoc. Nat. Acad. Sci. USA 72, 16-19. HELDEBRANT, C. M., BUTKOWSKI, R. S., BAJAJ,
SU’ITIE S. P., AND MANN, 248, 7149-7163. 55. STENFLO, 8175.
J.
(1972)
K. G. (1973) J.
Biol.
J. Biol.
Chem.
56. NELSESTUEN, G. L., AND SUTTIE, Biol. Chem. 247, 8176-8182.
247,
Chem. 8167-
J. W. (1972)
J.
57. FUJIKAWA, K., COAN, M. H., ENFIELD, D. L., TITANI, K., ERICSSON, L. H., AND DAVIE, E. W. (1974) Proc. Nat. Acad. Sci. USA 71, 427430. 58. KISIEL, W., AND Biochim. Biophys.
HANAHAN, D. J. Acta 304, 103-113.
59. BENSON, B. J., AND HANAHAN, chemistry 14, 3265-3277.
D. J. (1975)
(1973) Bio-
60. HOWARD, J. B., AND NELSESTUEN, G. L. (1975) Proc. Nat. Acad. Sci. USA 72, 1281-1285.
