THE JOURNAL OF BIOLOGICAL Val. 261, No. 28, of 5, pp ...chemically defined systems, we have evaluated the pathways of prothrombin activation in plasma to determine what cleav- ages

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1986 hy The American Society of Biological Chemists, Inc. Val. 261, No. 28, Issue of October 5, pp. 13210-13215 19%

Printed in (j.S.A.

Prothrombin Fragment 1.203, a Major Product of Prothrombin Activation in Human Plasma*

(Received for publication, January 27, 1986)

Marie Josephe RabietS, Alisan Blashill, Bruce Furie, and Barbara C. Furie From the Center for Hemostasis and Thrombosis Research, Division of Hematology-Oncology, Department of Medicine, New England Medical Center and Tufts University School of Medicine, Boston, Massachusetts 02111

The conversion of the blood coagulation zymogen prothrombin to thrombin is associated with the production of several cleavage intermediates and products. In contrast to earlier studies of prothrombin cleavage in chemically defined systems, the current investigation examines the fragmentation of human prothrombin in normal plasma. Radiolabeled prothrombin was added to platelet-poor relipidated normal human plasma, and clotting was initiated with the addition of Ca(I1) and kaolin. Analysis of the radiolabeled prothrombin cleavage products by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate and 8-mercaptoethanol identified a heretofore unobserved product of prothrombin activation with an apparent molecular weight of 45,000. This product was identified as fragment 102.3, the NHz-terminal 286 amino acids of prothrombin. The product was isolated from a prothrombin digest by immunoaffinity chromatography using anti-prothrombin:Ca(II) antibodies and by preparative gel electrophoresis. Its amino-terminal sequence is identical to that of prothrombin. Digestion of this product with either Factor Xa or thrombin yields, at a minimum, fragment 1-22 and fragment 1. Amino-terminal sequence analysis of the products obtained by digestion with Factor Xa of the unknown activation product indicated 3 amino acid residues at each cycle consistent with the presence of fragment 1, fragment 2, and fragment 3. To unambig- uously identify the COOH-terminal amino acid sequence of the product, its factor Xa digestion products were separated by reverse-phase high performance liquid chromatography. Edman degradation of one peptide revealed the complete sequence of fragment 3. On this basis, we identify the M, 45,000 polypeptide as fragment 1-2-3 and indicate that it is a prominent product of prothrombin conversion to thrombin when activation occurs in plasma.

Prothrombin, a plasma glycoprotein, is the zymogen of the serine protease thrombin that catalyzes the conversion of fibrinogen to fibrin as well as several other reactions which may be important to blood coagulation (1, 2). Prothrombin has an apparent molecular weight of about 72,000 and contains a single polypeptide chain. Thrombin has an apparent molecular weight of about 36,000 and is made up of two disulfide-linked polypeptide chains. The proteolytic events

* This work was supported by Grants HL18834 and HL21543 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “adoertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

4 Chargee de Recherche of the Institut National de la Santi et de la Recherche M6dicale.

leading to in uitro activation of both bovine and human prothrombin have been well characterized (3-7). Prothrombin is converted to thrombin by the action of the serine protease Factor Xa. This reaction requires calcium ions. The rate of the reaction is greatly enhanced in uitro by two co-factors, Factor Va, the active form of Factor V, and phospholipids (1, 2). Phospholipids are generally considered to fill in uitro the in uiuo role of the platelet and/or endothelial cell surface.

Bovine Factor Xa cleaves bovine prothrombin at two sites. Hydrolysis of the ArF4-ThP5 peptide bond yields fragment 1.2 and prethrombin 2. Subsequent cleavage of the Ile324 bond in prethrombin 2 produces a-thrombin. Thrombin can itself cleave prothrombin at Arg’s6-Ser’s7 to produce fragment 1 and prethrombin 1. The latter is a substrate for Factor Xa (3-5).

In chemically defined systems, activation of human prothrombin by human Factor Xa in the presence of Factor V, phospholipid, and Ca(I1) produces all of the products described for activation of bovine prothrombin (Fig. l). In human prothrombin, however, the Arg’55-Ser156 bond (cleavage a) may be cleaved by either thrombin or Factor Xa (6-8). Finally, there is an additional peptide bond, Arg286-ThrZS7 (cleavage c), in human prothrombin which appears to be susceptible to hydrolysis by either Factor Xa or thrombin (7, 8). Human prothrombin activation has been demonstrated to result in two additional products: a prethrombin 2 form, prethrombin 2(287-~81), 13 amino acid residues shorter than its bovine counterpart, and a biologically active form of thrombin, thrombin(287-5s1), with 13 residues cleaved from the NHz- terminal end of the A chain (6,9,10). Cleavage of the Arg323- Ile3z4 bond in bovine prothrombin or of the Ar$z2-11e323 bond in human prothrombin (cleavage d) yields species with ester- ase activity known as meizothrombins. The venom of Echis carinatus has been demonstrated to produce such intermediates (11). Recently, Nesheim and colleagues (12) have demonstrated the transient production of meizothrombin from bovine prothrombin in the presence of DAPA,’ Factor V, phospholipid, and Ca(I1). In addition, a mutant form of human prothrombin, prothrombin Barcelona, yields this product (13). It would appear that activation of human prothrombin to thrombin could proceed by multiple pathways, depending upon the sequence order of the different cleavages. By analogy with the bovine system, the pathway commonly assumed for human prothrombin activation in a classically defined in uitro system is defined by cleavage order b, d, c.

In contrast to prior studies of prothrombin activation in chemically defined systems, we have evaluated the pathways of prothrombin activation in plasma to determine what cleavages occur in this milieu. Potentially, the presence of protease

’ The abbreviations used are: DAPA, dansylarginine-N-(3-ethyl- 1,5-pentanediyl)amide; dansyl, 5-dimethylaminonaphthalene-1-sul- fonyl; SDS, sodium dodecyl sulfate; HPLC, high performance liquid chromatography.

13210

Prothrombin Fragment 1.2.3 13211

C v I PROTHROMBIN I

A A A a b d

FIG. 1. Intermediates and products of prothrombin activation. All the known intermediates and products of human prothrombin activation are identified. The two potential forms of prethrombin 2 and of thrombin are dis- tinguished by a subscript indicating the amino acid residues contained. Cleavage a, Arg'55-Ser'56; cleavage b, Arg273-Th?'4; cleavage c , ArP-Th?; cleavage d, Arg122-Ile323.

I I ,

FRAGMENT 1 I 1

PRETHROMBIN 1 I

i I 1 1 I 1

FRAGMENT 1.2 PRETHROMBIN 2 (274-581)

A CHAIN,,/! B CHAIN I SP (287-581)

I

S II/ I I 1 I

MEIZOTHROMBIN S P

inhibitors and other regulatory plasma proteins might influence the pathway of prothrombin activation. Indeed, activation of '251-labeled human prothrombin in plasma yields a product (peak B, see below) which has not been observed previously. In this paper, we describe the isolation of this product, and identify it as fragment 1.2 .3 .

MATERIALS AND METHODS

Human prothrombin (7,14), bovine prothrombin, and bovine Fac- tor X (15-17), the coagulant protein of V i p r a russelli (18), and bovine Factor Xa (18-20) were prepared as previously described. Bovine thrombin was purified from Topical Thrombin (Parke-Davis) (21). Prethrombin 1 and fragment 1 were obtained by digestion of human prothrombin by bovine thrombin and separated according to the method of Mann (15) except that the reaction was terminated by addition of diisopropylphosphofluoridate to a final concentration of 5 mM. Prethrombin 2 was prepared by digestion of human prethrombin 1 with bovine Factor Xa (15). Fragment 1 .2 was obtained from

that DAPA was used to inhibit thrombin (22). The isolated prothrom- activation of human prothrombin as described by Mann (15) except

bin activation products were concentrated by ultrafiltration using an Amicon PM-30 membrane and stored at -40 "C in the presence of 1 mM benzamidine. DAPA, a competitive inhibitor of thrombin, was synthesized using the procedure of Nesheim et al. (22). Synthetic fragment 3 was a generous gift from Dr. George Wilner, Washington University, St Louis, MO.

Activation of Radiolabeled Prothrombin in Plnsma-The activation of prothrombin in citrated normal human platelet-poor plasma was carried out at 37 "C. Radiolabeled prothrombin (50 pg in 0.04 ml of 0.1 M Tris-HC1, pH 7.6) was added to 0.4 ml of normal human platelet-poor plasma in a polystyrene test tube. Reactions leading to clot formation were initiated by the simultaneous addition of 0.04 ml of a suspension of kaolin (0.2 g/ml) in rabbit brain cephalin (Sigma, stock solution diluted 1:1, v/v) and 0.4 ml of 0.025 M CaC12. The reaction mixture was thoroughly mixed. At the specified time the reaction was quenched by addition of sodium dodecyl sulfate polyacrylamide gel incubation buffer (3.5% SDS, 10% fl-mercaptoethanol, 0.063 M Tris-HCl,20 mM EDTA, pH 6.8) maintained at 80 "C. These conditions were sufficient to terminate instantaneously the proteolytic cleavage of prothrombin.

Preparation of Peak B (Fragment 1'2'3)"The polypeptide corresponding to peak B was prepared by digestion of human prothrombin by bovine Factor Xa. Prothrombin (1-5 mg), a t a concentration of

0.6 mg/ml in 0.05 M Tris-HC1, pH 7.2, 0.15 M NaC1, was incubated with bovine Factor Xa (6.5 units/mg of prothrombin) in the presence of phospholipid vesicles (phosphatidylcholine:phosphatidylserine, 1:l; 0.45 p M final concentration), 2.5 mM CaC12, and 1.2 X io" M DAPA. After incubating the reaction mixture for 45 min at 37 "C, the pH of the mixture was adjusted to 6.0 and p-amidinophenylmethanesulfonyl fluoride (Chemicon) was added to inactivate Factor Xa ( M , w/w, ratio of p-amidinophenylmethanesulfonyl fluoride:Factor Xa). Antithrombin I11 (65 pg) and heparin (65 pg) were added to the reaction mixture to inactivate the thrombin produced. The mixture was then applied to an anti-human prothrombin:Ca(II)-agarose column (2 ml) previously equilibrated in 0.05 M Tris-HC1, pH 7.5, 0.15 M NaCl, 8 mM CaCl,, 1.2 X M DAPA (23). After extensive washing with the same buffer, the desired material was eluted with 0.05 M Tris-HC1, pH 7.5, 0.15 M NaC1, 16 mM EDTA, 1.2 X 10" M DAPA. The EDTA eluate was concentrated to 1 ml, dialyzed against 0.07 M Tris-HC1, pH 6.8, 1 mM EDTA and applied to a preparative gel electrophoresis column, containing a stacking gel (1.5 X 1.6 cm) and a resolving gel (1.5 X 6 cm) with an acrylamide concentration of 8.8% and acry1amide:bisacrylamide ratio of 401. The gel was run in 0.05 M Tris glycine, pH 8.2, 1 mM EDTA at a flow rate of 12 ml/h. Protein elution was monitored by optical density at 280 nm. The product corresponding to peak B was identified by its migration in polyacrylamide gels containing dodecyl sulfate (see below). The polypeptide was stored in 5 mM benzamidine at -40 "C.

Proteolytic Digestion of Peak B (Fragment 1.2.3)"Peak B was subjected to proteolysis by either bovine Factor Xa or bovine thrombin, and the products were analyzed by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate. Thrombin, 5 pl (100 NIH units/ml), or factor Xa, 5 p1 (0.7 mg/ml), was added to 40 pg of '9- labeled peak B in 100 p1 of 0.02 M Tris-HC1, pH 7.4, 0.15 NaCl, and the reaction mixture incubated at 37 'C for varying time periods. The reactions were quenched by adding aliquots of the reaction mixtures to dodecyl sulfate sample buffer containing 10% 8-mercaptoethanol and 3 mM EDTA. The samples, in sample buffer, were heated at 80 "C for 1 min.

Peptide fragments of peak B were prepared for automated sequence analysis by digestion of the polypeptide by human Factor Xa. Peak B and Factor Xa (1000:1, w/w) were incubated in 0.05 M NH4HC03 at 37 "C for 36 h. The reaction mixture was lyophilized, resuspended in acetic acid, and subjected to amino acid sequence analysis. The peak B digest was also subjected to high performance liquid chromatography (HPLC) under reverse phase conditions on a C18 column (Beckman) using a 0.1% trifluoroacetic acid/water/acetonitrile gra- dient from 0 to 40% acetonitrile. The peak corresponding in migration

13212 Prothrombin Fragment 1 .2.3

to that of synthetic fragment 3 was collected and subjected to automated amino acid sequence analysis.

Protein Concentration-Protein concentrations were determined spectrophotometrically using extinction coefficients, E$& of 14.4, 16.4, 19.5, 10.1, and 12.9 for prothrombin, prethrombin 1, prethrombin 2, fragment 1, and fragment 1.2, respectively (8, 15). The extinction coefficient of fragment 1.2.3 (peak B) was estimated as that of fragment 1.2. The concentration of thrombin solutions was calculated from its activity (2400 NIH units = 1 mg). A unit of bovine Factor Xa was defined as that quantity of enzyme which hydrolyzed 1 X W 5

mol of 5,5'-dithiobis-(2-nitrobenzoic acid)/min. lodination of Proteins-Proteins were labeled with either Na'"I

(17 Ci/mg) or Na13'I (25 Ci/mg) using chloramine T (24) or lactoper- oxidase (25).

Preparation of Phospholipid Vesicles-Phospholipid vesicles were prepared by the method of Barenholz et al. (26). Phosphatidylserine (10 mg from bovine brain, Sigma) and phosphatidylcholine (10 mg from egg yolk, Sigma) were combined, and the mixture was freed from solvent under a stream of nitrogen, redissolved in benzene, and lyophilized. The lyopholized powder was dispersed in 5.2 ml of 0.1 M NaCl, placed on ice under a stream of nitrogen, and subjected to direct probe sonication for 1.5 min followed by a 1-min cooling period. The sonication cycle was repeated 20 times. The vesicle dispersion was separated by centrifugation at 100,000 X g for 30 min, and the supernatant again subjected to centrifugation at 159,000 X g for 3 h. The clear supernatant contains single bilayer micelles of homogene- ous size. The phospholipid concentration of the vesicles was quantitated by the method of Chen et al. (27).

Polyac ylumide Gel Electrophoresis in the Presence of SDS-Poly- acrylamide gel electrophoresis was carried out according to the procedure of Laemmli (28). Stacking gels contained 7.5% acrylamide at an acry1amide:bisacrylamide ratio of 301. Resolving gels contained 12% acrylamide with an acry1amide:bisacrylamide ratio of 301. Res- olution of bands was improved by increasing the electrophoresis time to 1.5 times the length of time required for tracking dye (bromphenol blue) to migrate through the resolving gel. Gels were either stained for protein with Coomassie Blue or assayed for radioactivity. In the latter case, gels were sliced into 1-mm segments using a Bio-Rad Gel Divider, Model 190. The segments were assayed for 1251 or, when radiolabeled protein markers were included, for lZ5I and 13'1 simulta- neously in a Beckman Gamma 8000 scintillation spectrometer. The radioactivity profiles from the gels were calculated, normalized, and visualized with a program developed incorporating MLAB (29). The gel profiles were visualized on a Tektronix 4006 display terminal and drawn using a Tektronix 4662 digital plotter.

Amino Acid Sequence Anulys&"utomated Edman degradation was carried out using either a Beckman 890C Sequencer with a 0.1 M Quadrol program or an Applied Biosystems gas-phase Sequencer. Phenylhydantoins of the hydrolyzed and isolated amino acids were identified by HPLC.

RESULTS

Prothrombin Cleavage Products in Plusma-The products of prothrombin conversion to thrombin in clotting plasma were identified by gel electrophoresis of aliquots of this plasma. Platelet-poor relipidated plasma was supplemented with l3II-labeled prothrombin. Clotting was initiated by the addition of kaolin and calcium to the plasma at concentrations which simulate the activated partial thromboplastin time. Three minutes after clot formation, the proteolytic processes affecting prothrombin, its activation intermediates, and products were terminated by the addition of the dodecyl sulfate sample buffer at 80 "C. The products of prothrombin conversion were then analyzed by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate and P-mercaptoethanol. Gels were sliced, and the migration of each of the radiolabeled degradation products was determined. The migration of the prothrombin cleavage products generated in plasma were directly compared to lZ5I-labeled prothrombin cleavage product standards. The results of a typical experiment are shown in Fig. 2. We have employed phospholipid vesicles as substi- tutes for activated platelets, but we should emphasize that

- .2 0 40 80 GEL SLICE

FIG. 2. Dodecyl sulfate polyacrylamide gel electrophoresis in the presence of 0-mercaptoethanol. Clotted plasma containing '311-labeled human prothrombin at 3 min after clot formation (upper panel) and composite showing migration of 1251-labeled prothrombin fragments run individually on gels (lower panel) are as follows. Prethrombin 1, -----; fragments 1-2, - - -; prethrombin 2, -; B chain of thrombin, -.-.-; fragment 1, - -. The profiles are plotted as normalized counts.

experiments in platelet-rich plasma yielded the same results.' The radioelectrophoretogram of '311-labeled prothrombin activated in plasma contains six peaks, labeled A through F. Except for peak A, the peaks were identified by their position in the gel relative to that of the '251-labeled standards. By this analysis, peak C is identified as fragment 1.2 , peak D as prethrombin 2, peak E as the B chain of thrombin and peak F as fragment 1. Peak A is prothrombin, the only peak in the electrophoretogram of a zero time gel. Under the conditions employed, fragment 2 and the A chain of thrombin migrate off the gel and are not observed. No peak in the plasma clot corresponds to prethrombin 1. Furthermore, peak B does not co-migrate with any of the prothrombin fragment standards.

In a further attempt to identify peak B, human prethrombin 2(287-581), human prethrombin 2(274-581), and bovine prethrombin 2 were examined. However, these prethrombin 2 species had anodal mobilities significantly greater than peak B.

Isolation of Peak B-Since peak B did not migrate with the previously identified products of prothrombin cleavage in chemically defined systems, we considered that this product might contain a labile peptide bond or it might be a transient intermediate that was rapidly degraded in the absence of plasma protease inhibitors. As a first step in the purification of peak B, we identified conditions under which the yield of peak B from prothrombin was optimal in an activation system including purified prothrombin, activated Factor X, phospholipid, and calcium. Human prothrombin was incubated with activated bovine Factor X in the presence of phospholipid and calcium. In order to inhibit any thrombin formed in the course of the reaction, DAPA was included in the reaction mixture. The desired product, peak B, was obtained by affinity chromatography using anti-prothrombin:Ca(II) antibodies bound to agarose. These antibodies are specific for the calcium-stabilized conformer of prothrombin and recognize antigenic sites which are located within the first 42 amino acids in the protein (30). Among the products of activation of prothrombin in the reaction mixture, only those which con-

M. J. Rabiet, A. Blashill, B. Furie, and B. C. Furie, unpublished observations.


tain the amino-terminal portion of the molecule bind to the column. As indicated by SDS gel electrophoresis (Fig. 3, lane 2), prothrombin, peak B, fragment 1.2, and fragment 1 were found in the EDTA eluate. These components were separated by preparative gel electrophoresis and the constituents of the fractions containing material absorbing a t 280 nm analyzed by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate and p-mercaptoethanol. The polypeptide corresponding to peak B was eluted shortly after fragment 1 (Fig. 3). Prothrombin and fragment 1.2 were both found in the late fractions eluted from the preparative gel electrophoresis column (Fig. 3). The fact that peak B binds to the anti- prothrombin:Ca(II)-agarose column indicates that it includes the NH,-terminal y-carboxyglutamic acid-rich region of prothrombin, fragment 1. In initial studies, 12sII-labeled prothrombin was employed in digestion mixtures. In later preparations, unlabeled prothrombin was employed and the desired product subsequently radiolabeled. The peak B products from labeled and unlabeled prothrombin co-migrated when subjected to gel electrophoresis and appeared identical.

T o assure that the desired polypeptide had been isolated from the prothrombin digestion mixture, purified peak B was labeled with '251 and was then mixed with a plasma clot containing the cleavage products of '"I-labeled prothrombin. After treatment with SDS, this mixture was examined by gel electrophoresis in dodecyl sulfate and P-mercaptoethanol. As shown in Fig. 4, purified peak B contains only one polypeptide chain. This polypeptide co-migrates with peak B in the plasma clot. The apparent molecular weight of peak B is 45,000.

Peak B as a Substrate for Factor Xa and Thrombin- Cleavage of '2sI-labeled peak B by Factor Xa or thrombin yielded cleavage products that co-migrated upon gel electrophoresis in dodecyl sulfate with known fragments of prothrombin activation. As seen in Fig. 5, there is a time- dependent appearance in the digestion mixture of fragments that co-migrate with fragment 1.2 and fragment 1. Under conditions of electrophoresis which resolve peak B from fragment 1.2, fragment 2 is eluted from the gel and not observed. These results suggest that fragment 1 e 2, fragment 1, and, by inference, fragment 2 or a related form, are regions of peak B.

'2sI-Labeled peak B was also digested with thrombin. As with Factor Xa, thrombin-treated peak B digests also contained fragment 1.2 and fragment 1. Although the kinetics of peptide bond cleavage by Factor Xa or thrombin give different

- II - F1-2.3 - F1-2 - F1

FIG. 3. Fragment 1.203 preparation. Dodecyl sulfate polyacrylamide gel analysis of the activation mixture (lane I ) , EDTA eluate from the anti-prothrombin: Ca(I1) antibody column (lanes 2 and 3) . fractions eluted from the preparative gel electrophoresis column (lanes 4-7) are shown. The order of elution is fragment 1 (lane 4), fragment 1 . 2 . 3 (lane 6), prothrombin and fragment 1 . 2 (lane 7).

1

, {', 5 ,", I I ; ; 1i I I

,.

0 /""" . ..&"'! \.<.."-<>A-

0 40 80 GEL SLICE

l l ""_ FIG. 4. Dodecyl sulfate polyacrylamide gel electrophoresis

in the presence of 8-mercaptoethanol. Clotted plasma containing lR'I-labeled prothrombin at 3 min after clot formation (-----); and fragment 1 .2 .3 isolated from activation mixtures of prothrombin and subsequently labeled with '*'I (-) are shown. The profiles are plotted as normalized counts.

product concentrations under the conditions employed, it appears that both enzymes cleave the same peptide bonds. Peak B is a single-chain polypeptide that is somewhat larger than fragment 1.2. It binds to anti-prothrombin:Ca(II) antibodies and therefore must bind calcium and undergo the conformational change resulting in exposure of the antigenic determinants recognized by the conformation-specific antibody population. The data in sum suggested that peak B was fragment 1.2.3.

Amino Acid Sequence of Peak B-The NH2-terminal sequence of peak B was determined by automated Edman degradation (Table I). The sequence is identical to that established for prothrombin and prothrombin fragment 1, confirm- ing that peak B is formed from the NH2-terminal half of prothrombin. The NH2-terminal amino acid sequence analysis was also performed on the Factor Xa digest of peak B. The anticipated results, the appearance of 3 residues during each cycle, were obtained (Table I). The residues correspond to the NH2 termini of fragment 1, fragment 2, and fragment 3.

The Factor Xa digest of peak B was separated by high performance liquid chromatography on a reverse-phase C18 column. The peak corresponding in migration to that of synthetic fragment 3 was subjected to amino acid sequence analysis. Its sequence corresponds to that of fragment 3 (Table I). On this basis, we have established that peak B is fragment 1.2.3, an elongated form of fragment 1-2 that has the additional 13-residue peptide (fragment 3) on its COOH terminus.

DISCUSSION

The conversion of prothrombin to thrombin has been ex- tensively studied in chemically defined systems (3-7). These systems include, in varying ratios, prothrombin as substrate and Factor Xa as enzyme. Phospholipid vesicles or activated platelets, calcium, and Factor V significantly enhance the rate of thrombin generation. The concentration of these cofactors relative to the substrate and enzyme may alter the rate of a particular bond cleavage, and thus the concentration of prothrombin intermediates and products. For example, high con-

13214 Prothrombin Fragment 1 2.3

Fragment 1.2

10 20 30 40 50 60 70

Gel Slice FIG. 5. Products of fragment 1.2-3 digestion with activated

bovine Factor X. Aliquots from a reaction mixture containing '9- labeled fragment 1 .2 .3 and activated bovine Factor X were removed at various time points, and the product was analyzed by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate and 8- mercaptoethanol. Electrophoretograms of '*sI-labeled fragment 1 . 2 and 1261-labeled fragment 1 are included for reference.

TABLE I Amino-terminal seauences

Determined sequences Residue

Peak B Peak B HPLC pep- digest tide

1 Ala 2 Asn 3 Thr 4 Phe 5 Leu 6 -a

7 -a

8 Val 9 Arg

10 Lys 11 12 13

P m l 595 340 309 35 1 385

235

233 c -

P m l Ala, Ser, Thr Thr 316 Asn, Glu, Ala Ala 555 Thr, Gly Thr 240

Ser 92 Glu 276 Tyr 166 Gln 233 Thr 120 Phe 309 Phe 260 Asn 188 Pro 147 Arg -'

Known sequences

F1 F2 F3

Ala Ser Thr Asn Glu Ala Thr Gly Thr Phe Ser Ser Leu Ser Glu Gla* Val Tyr Gla* Asn Gln Val Leu Thr Arg Ser Phe Lys Pro Phe Gly Pro Asn Asn Leu Pro Leu Glu Arp

The phenylthiohydantoin derivative of y-carboxyglutamic acid is not recovered from the filter of the gas phase Sequencer.

* Gla, y-carboxyglutamic acid. e Identified but not quantitated.

centrations of calcium have been shown to inhibit thrombin- mediated cleavage of bovine prothrombin between residue 156 and residue 157 by altering the K,,, of the reaction from 0.25 to 1.8 pM (31). Calcium can thus influence the amount of fragment 1 that appears during prothrombin activation. Sil- verberg (31) has emphasized that the K, values of many enzymatic reactions in blood coagulation are of the same magnitude as the concentration of the zymogen substrate. Thus, the relative concentrations of the components of the prothrombin activation system may determine the concentrations of the products of prothrombin activation.

As an approach to elucidating the pathways of prothrombin activation under conditions which mimic more closely the physiologic concentration of reactants and cofactors, we have examined the conversion of prothrombin to thrombin in plasma. Calcium, Factor V, and prothrombin concentrations in this system approximate those in whole clotting blood. The concentration of Factor Xa generated at the site of tissue injury is unknown. We have arbitrarily chosen to use kaolin to activate the intrinsic pathway. The amount of Factor Xa developed in this assay is typical of that generated in a partial thromboplastin time. Most importantly, we have chosen to examine prothrombin activation in the presence of all of the plasma proteins including those proteins which regulate hemostasis (e.g. protein protease inhibitors). The influence of one physiologically important regulatory process, that mediated by activated protein C and protein S, may not be partic- ipating in the molecular events studied here, as the endothelial cell cofactor required for protein C activation is not present (32).

We have shown that clotting plasma contains a heretofore unobserved prothrombin cleavage product. Although this product is easily detected in plasma, it had not been observed in the extensive studies of prothrombin cleavage in chemically defined systems. This product, fragment 1.2 .3 , is very labile and, in the absence of protease inhibitors, is rapidly hydrolyzed by Factor Xa and thrombin to other known prothrombin cleavage products. Plasma contains protease inhibitors which apparently protect this product. When efforts are made to control proteolysis in chemically defined systems using DAPA or diisopropylphosphofluoridate, fragment 1 . 2 - 3 can be detected and isolated in reasonable yield. Fragment l + 2 - 3 prob- ably appears transiently as an intermediate in prothrombin activation in purified systems. Failure to detect this fragment in such systems is most likely due to its lability and to the difficulty in resolving it from fragment 1 - 2 by gel electrophoresis.

In the course of sequencing human prethrombin 2 and human thrombin, it became apparent that these molecules were shorter by 13 amino-terminal residues than their bovine counterparts (9, 10). As a complete pro-piece, fragment 1 .2 . 3, was never identified, it has been assumed that the pro- piece is released from the zymogen by cleavage of the peptide bond between the fragment 2 domain and the fragment 3 domain, yielding fragment 1 . 2 and the extended prethrombin 2(274-581). Prethrombin 2(287-581), lacking the 13-residue NH2- terminal extension, has often been identified in purified prothrombin activation mixtures, supporting speculation that prethrombin 2(274-581) is degraded to prethrombin 2(287-581). Our experiments indicate that fragment 1 . 2 . 3 may be the predom- inant product of human prothrombin activation. However, there appears to be only one prethrombin 2 species in our reaction mixtures. Either the two forms of prethrombin 2 are not separated in our gel electrophoresis system or only the shorter form is produced under our reaction conditions. We cannot conclude from the current data whether cleavages b and c (Fig. 1) both occur during prothrombin activation in our system or whether fragment 1 - 2 appears as a result of


degradation of fragment 1-2.3. Furthermore, prethrombin 1 does not appear in our plasma reaction mixture. The fragment 1 observed must arise from cleavage of fragment 1 - 2 or fragment 1 12.3.

Walz and Brown (33) developed a radioimmunoassay for fragment 3 using antibodies developed against the free fragment. With this assay they were able to detect thrombin- bound fragment 3 after whole blood clotting. Free fragment 3 was not detected. However, at most 6% of the theoretical concentration of fragment 3 in the serum, all thrombin-bound, can be accounted for with this assay. It is possible that fragment 1 . 2 . 3 is a major product of prothrombin activation in this whole-blood clotting system but that the fragment 3 domain of fragment 1 .2 .3 is not recognized by the antibody. This explanation is consistent with the apparent poor recov- ery of antigen and with the failure to identify fragment 1 - 2' 3 in serum. Aronson et al. (34) have isolated the pro-piece cleaved from prothrombin upon activation of human blood. They established the identity of the product as fragment 1 . 2 by NH2-terminal analysis, amino acid analysis, migration in polyacrylamide gels with the Weber-Osborn formulation, and by proteolytic degradation. Based upon our own experience, these analyses may not distinguish between fragment 1 .2 and fragment 1.2.3. Alternatively, due to its extreme lability, fragment 1 .2 .3 produced during the clotting of plasma may have been degraded during the isolation procedure employed. Thus, part or all of the product isolated from their reaction mixtures may have been fragment 1.2.3.

Our results indicate that, in plasma, prothrombin is activated to thrombin by the cleavage of two peptide bonds, c and d (Fig. 1). Fragment 1 .2 .3 is a major product of the reaction. Although several laboratories have previously searched for a functional role for fragments of the prothrombin propiece without success, fragment 1 . 2 . 3 has never been observed or isolated before. It will be of interest to determine whether it plays any role in hemostasis or inflammation, for example. Our observations confirm that cleavage a (Fig. 1) does not occur during prothrombin activation in plasma and lead us to propose cleavage c as the preferred cleavage site for removal of the pro-piece of human prothrombin in plasma. The results also lead us to question the strict specificity of Factor Xa for cleavage b and of thrombin for cleavage c. Fragment 1.2.3 can be obtained in the presence of the specific thrombin inhibitor DAPA, suggesting that cleavage c under these conditions is a Factor Xa-catalyzed cleavage. On the other hand, proteolytic digestion of fragment 1 . 2 . 3 by thrombin leads to the appearence of fragment 1.2, suggesting that thrombin can cleave bond b. It therefore appears that, under the conditions employed in our experiments, the strict specificity for cleavages b and c is not adhered to by Factor Xa and thrombin.

Whether fragment 1 - 2 . 3 is a product of prothrombin activation in vivo during normal hemostasis is unknown. Al- though clotting plasma or clotting whole blood may offer first approximations of the events which occur in uiuo, neither truly simulate the environment around the site of intravas- cular injury. A major role has recently been established for the endothelial cell as a cofactor in the activation of protein c, a protein with important regulatory function (32). These cells may also play a role as a site of activation of prothrombin (35). Substrates, cofactors, enzymes, and enzyme inhibitors may all have significantly altered concentrations at the inter- face of the platelet plug and flowing blood from those in isolated blood or plasma. These alterations will bear directly on the chemical structure and quantity of the products of zymogen activation occurring at such a site. Demonstration of a heretofore unrecognized product of prothrombin activation suggests that the pathways and kinetics observed for such

processes as measured in reaction mixtures reconstituted from isolated reaction components, while vital in dissecting complex biological processes, must be extrapolated to physiologic conditions with caution. Even our current system employing plasma, an artificial stimulus (kaolin), and phospholipid vesicles in the absence of the vessel wall is an imperfect model. Although these studies provide the ground work for under- standing a complex biological phenomenon, it will be neces- sary to extend these studies into more physiologic systems.

REFERENCES 1. Nemerson, Y., and Furie, B. (1980) CRC Crit. Reu. Biochem. 9,

2. Jackson, C. M., and Nemerson, Y. (1980) Annu. Reu. Biochem.

3. Stenn, K. S., and Blout, E. R. (1972) Biochemistry 11 , 4502-

4. Heldebrant, C. M., Butkowski, R. J., Bajaj, S. P., and Mann, K.

5. Esmon, C. T., Owen, W. G., and Jackson, C. M. (1974) J. Biol.

6. Downing, M. R., Butkowski, R. J., Clark, M. M., and Mann, K.

7. Rosenberg, J. S., Beeler, D. L., and Rosenberg, R. D. (1975) J.

8. Lau, H. K., Rosenberg, J. S., Beeler, D. L., and Rosenberg, R. D.

9. Thompson, A. R. (1975) Bhchim. Biophys. Acta 422 , 200-209 10. Walz, D. A., and Seegers, W. H. (1974) Biochim. Biophys. Res.

11. Moritz, T., Iwanaga, S., and Susuki, T. (1976) J. Biochem. (Tokyo)

12. Nesheim, M. E., Krishnaswamy, S., and Mann, K. G. (1985)

13. Rabiet, M. J., Elion, J., Labie, D., and Josso, F. (1979) Biochim.

14. Miletich, J. P., Jackson, C. M., and Majerus, P. W. (1978) J. Biol.

15. Mann, K. G. (1976) Methods Enzymol. 46, 123-155 16. Bajaj, S. P., Byme, R., Nowak, T., and Castellino, F. J. (1977) J.

17. Fujikawa, K., and Davie, E. W. (1976) Methods Enzyml. 46,74-

18. Furie, B. C., and Furie, B. (1975) J. Biol. Chem. 2 5 0 , 601-608 19. Furie, B., and Furie, B. C. (1976) J. Biol. Chem. 261,6807-6814 20. Esnouf, M. P., Lloyd, P. H., and Jesty, J. (1973) Biochem. J.

21. Lundblad, R. L., Kingdon, H. S., and Mann, K. G. (1976) Methods

22. Nesheim, M. E., Prendergast, F. G., and Mann, K. G. (1979)

23. Borowski, M., Furie, B. C., Goldsmith, G. H., and Furie, B. (1985)

24. Hunter, W. M., and Greenwood, F. C. (1962) Nature 194,495-

25. Morrison, M. (1980) Methods Enzymol. 7 0 , 214-220 26. Barenholz, Y., Gibbs, D., Litman, B. J., Goll, J., Thompson, E.,

and Carlson, F. D. (1977) Biochemistry 16 , 2806-2810 27. Chen, P. S., Jr., Toribara, T. Y., and Warner, H. (1956) Anal.

Chem. 28,1756-1758 28. Laemmli, U. K. (1970) Nature 227,680-685 29. Knott, G. D., and Rhee, D. K. (1972) Proceeding of the Online '72

International Conference, Vol. 1, pp. 497-526, Brunel Univer- sity, England

30. Tai, M. M., Furie, B. C., and Furie, B. (1984) J. Bwl. Chem. 2 6 9 ,

31. Silverberg, S. A. (1979) J. Biol. Chem. 264,88-94 32. Esmon, C. T., and Owen, W. G. (1981) Proc. Natl. Acad. Sci. U.

33. Walz, D. A., and Brown, T. R. (1979) in Vitamin K Metabolism and Vitamin K Dependent Proteins (Suttie, J. W., ed) pp. 115- 119, University Park Press, Baltimore

34. Aronson, D. L., Stevan, L., Ball, A. P., Franza, B. R., and Finlayson, J. S. (1977) J. Clin. Invest. 6 0 , 1410-1418

35. Rodgers, G., and Shuman, M. (1983) Proc. Natl. Acad. Sci. U. S.

45-85

49,765-811

4515

G. (1973) J. Bwl. Chem. 2 4 8 , 7149-7162

Chem. 249,606-611

G. (1975) J. Biol. Chem. 260,8897-8906

Biol. Chem. 260,1607-1617

(1979) J. Bwl. Chem. 264,8751-8761

Commun. 60,717-722

79, 1089-1108

Blood 66,341a

Bwphys. Acta 684,66-75

Chem. 263,6908-6919

Bwl. Chem. 252,4758-4761

83

131,781-789

Enzymol. 46,156-176

Biochemistry 18,996-1003

J. Bwl. Chem. 260,9258-9264

496

4162-4168

S. A. 78,2249-2252

A. 80,7001-7005

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THE JOURNAL OF BIOLOGICAL Val. 261, No. 28, of 5, pp ...chemically defined systems, we have evaluated the pathways of prothrombin activation in plasma to determine what cleav- ages