THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 250, No. 12, Issue of June 25, pp. 4497-4504, 1975

Printed in U.S.A.

The Activation of Coagulation Factor X

IDEXTITY OF CLEAVAGE SITES IS THE ALTERNATIVE ACTIVATIOS PATHWAYS AKD CHARACTERIZATIOS OF THE COOH-TERMIXAL PEI’TIDE*

(Received for publication, December 11, 1974)

JOLYON JESTY, ALLEN I<. SPE?;CER, YASUTSUGU KAKASHIJIA, YALE ~EMERSON, AIYD WILLIAM KONIGSBERG

From the Departments of I&emu1 Medicine and Molecular Biophysics and Biochemistry, Yale University School of Medicine, Sew Haven, Comecticut 06510

SUMMARY

Bovine Factor X can be activated by two alternative path- ways. The first, favored at high concentrations of the complex of tissue factor and Factor VII, is initiated by the action of Factor VII on Factor X to cleave an activation peptide from the NH, terminus of the heavy chain, to produce a-X,. This is then converted autocatalytically to another form of Factor X,, P-X,, by the loss of a 17-residue glycopeptide from the COOH terminus of the heavy chain, in a lipid-dependent re- action. The alternative pathway, favored at lower activator concentrations, is initiated by the action of Factor X, on Factor X, in the presence of lipid, to release the same COOH- terminal peptide as is produced in the conversion of CPX, to /3-X,. The intermediate produced by the loss of this peptide from Factor X, Ir, can be activated directly to P-X, by the tissue factor-Factor VII complex, with the loss of the same NHz-terminal peptide as is produced in the conversion of Factor X to a-X,. The autocatalytic activation of Factor X by Factor X, described previously occurs to a marked extent only at very low activator concentrations, and has been shown to proceed largely by the loss of the normal NH2-terminal peptide from the heavy chain of Ii. Initial experiments show that neither peptide affects the rate of coagulation by either the extrinsic or intrinsic pathways.

The amino acid sequences have been determined on both sides of the peptide cleavages, and it has been shown that the cleavage sites are the same, regardless of the pathway of activation. The amino acid sequence and carbohydrate composition of the COOH-terminal peptide have been deter- mined. The carbohydrate moiety is attached via an O- glycosidic linkage at a threonine residue, and contains galac- tosamine but no glucosamine.

In an earlier paper, we showed that the activation of Factor X by the complex of tissue factor aud Factor VII (TF-VII)’

* This work was supported in part by Grant IIL 16126 from the National Institutes of Uealth.

1 The abbreviations rised are: TF-VII, the tissue factor-Factor VII complex; STI, soybean trypsin inhibitor; BNPS-skatole, 2.(2.nitrophenylsulfenyl)-3-mcthylirldolenine; RVV, Russell’s viper venom.

involves more than one peptide cleavage (1). ;\Iore recently, we have shown that thcrc arc two alteruative path~vays of activa- tion of Factor X, oiie initiated by TF-VII, aud the ot.lier by the product’, Factor X, (2).

The major pathway in fast activations involves first the loss of au KHz-termiual pcptide from the heavy chain of Factor X to give 01-x,, which is apparciitly the same product as that formed by activation with the coagulant protein of Russell’s viper venom (RVV) in the absence of lipid (2, 3). In activations by ‘I’F-VII, however, (Y-X,, acting on itself, produces P-X, with no chauge iu coagulant activity. The convcrsiou of cr-X, to P-X, is almost coml~letely lil)id-depelideiit, and cannot be cata- lyzed by either ‘IF-VII or RVV (2).

Whereas the appearauce of Factor X, activity corresponds with the loss of an XI-I?-tcrmiual pcptide from the heavy chain of Factor X, the conversion of Q-X, to P-X, involves no change in the NIIz-tcrmiual sequence; we, therefore, proposed that the action of either the 01 or @ form of Factor X, led to the loss of a (ZOOH-terminal pcptide from the heavy chaiu of a-X, (2).

The alteruativc pathlvay of Factor X activation, favored at lower rates, is iuitiatcd by the action of Factor X, (either form) on Factor X. This couvcrsion, which is also lipid tlepcndeut, involves no chauge iii the NH-terminal sequcuce of the heavy chain of Factor X, so it seemed that the reactiou involved the loss of a (:OOII-terminal pcptidc, am1 we proposed that the same site of cleavage was involved in this conversion as in the couvcrsiou of G-X, to P-X, (2). The intermediate produced by the loss of the COOKterminal peptide from Factor X, called 11, does not possess prothrombiwconvcrting activity, but can be activated to Factor X, at the same rate as Factor X. On the additiou of ‘IF-VII or RVV (eve11 in the absence of lipid), this intermediate is converted directly to P-X, by the cleavage of the same peptide bond that produces oc-X, from Factor X.

From these observatious, we proposed that the alternative pathways of activation of Factor X reflect the different order of the IiH2- am1 (,“OOII-terminal cleavages of the heavy chain of Factor X, am1 that the same KI12- aud COOII-termiual pep- tides arc produced, regardless of the activation pathway. In the present work, KC have confirmed this scheme (Fig. 1). We report the ammo acid scque~iccs in the immcdiatc region of the two cleavages, aud show that these arc the only reactions that occur under uormal couditious of activation by TF-VII or RVV + lipid. IIowever, under cstremely slow activation conditious, a

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P-X, c----I----- - Intermediate 2

FIG. 1. Pathways of activation of Factor X. Numbers refer to the reactions described in the text (modified from Ref. 2).

third cleavage occurs in the NHz-terminal region of the heavy

chain of I1 to produce a species we call IZ (2). This is a result of the action of Factor X, in the presence of lipid, but in the present

work, we show that the autocatalytic activation of Factor X by

Factor X, (2) proceeds largely by the release of the normal NH2- terminal activation peptide from the heavy chain of Il.

The COOH-terminal peptide has been isolated, and we have

determined its amino acid sequence and carbohydrate composi- tion; both show unusual features when compared with the

COOH-terminal sequence and carbohydrate linkages of pro- thrombin.

EXPERIMEKTAL PROCEDURE

Materials

Most reagents used were described previously (2). Additional materials were as follows. Iodoacetic acid (Eastman) was re- crystallized from ethanol and stored at -20” in the dark. 2.(2. Nitrophenylsulfenyl)-3-methylindolenine (BNPS-skatole) and all chemicals for sequence determination were from Pierce Chemical Co. Subtilisin Carlsberg and phosphatidylcholine were products of Sigma. Phosphatidylserine was from ICN Pharmaceuticals. Carboxypeptidases A and B were obtained from Worthington Bio- chemical Co., and carboxypeptidase C was from Nutritional Bio- chemical Co. Soybean trypsin inhibitor (STI) (Sigma) was coupled to Sepharose 4B by the method of Cuatrecasas (4). The Sepharose was activated with 100 mg of CNBr/ml of gel, and ST1 (5 mg/ml of gel) was coupled at pH 8.0 in 0.1 M NaHCOz. The resulting STI- Sepharose was washed well with 1 M NaCl, and suspended in 25 mM Tris-Cl, pH 7.5, for use.

Methods

The following methods were described previously (2): clotting assays, sodium dodecyl sulfate gel electrophoresis (analytical and preparative), purification of Factors VII and X (1, 2), and the preparation of TF-VII. The “prothrombin time” and “partial thromboplastin time” (indications of the state of the extrinsic and intrinsic pathways of coagulation, respectively) were done by the methods of Quick (5) and Proctor and ltapaport (6).

Amino Acid Analysis

Samples were hydrolyzed at 110” in triplicate for 24, 48, and 72 hours in G N HCl, and analyzed with a Beckman 121M analyzer. Values for serine and threonine were obtained by extrapolation to zero time, and half-cystine was determined as the S-carboxy- methyl derivative. Tryptophan was determined (on the COOH- terminal peptide) by the spectrophotometric method of Edelhoch (7).

NHz-terminal Sequence Analysis of Intermediates

Samples were examined by the procedure of Gray (8), as modi- fied by Weiner et al. (9).

COOH-terminal Sequence Analysis

This was done approximately according to the methods of Am- bler (10). Two nanomoles of material were boiled in 20 ~1 of 1% sodium dodecyl sulfate-O.2 M NaHC03, pH 8.2, for 5 min, &nd th& diluted to 0.1 ml with bicarbonate to reduce the dodecyl sulfate concentration. Samples were then digested with either (a) car- boxypeptidase A, (6) carboxypeptidase B, or (c) carboxypeptidase B followed after 2 to 4 hours with carboxypeptidase A. Samples were prepared for analysis by the addition of 2 ~1 of 10 N HCl, cen- trifuged to remove precipitated protein, and diluted to 0.2 ml with 0.2 M sodium citrate, pH 2.2. In order to release amino acids proxi- mal to arginine in the heavy chain of I, or p-X,, it was necessary to use the following procedure. Five nanomoles of protein were digested with 2 pg of carboxypeptidase B for 4 hours in a total volume of 0.1 ml, as described above; the digest was further diluted to 0.25 ml with bicarbonate buffer, and 2 rg of carboxypeptidase A were added. After incubation at 37” for 2 hours, additional enzyme was added and the incubation repeated. Finally, a third addition of 2pg of carboxypeptidase A was made, and the mixture was left for 16 hours at room temperature. The COOH-terminal peptide was studied by digestion with carboxypeptidase A in the absence of dodecyl sulfate, and then by digestion with carboxy- peptidase C in 0.2 M sodium acetate, pH 5.5.

In each case, appropriate controls were run to quantify the contribution of the digesting enzymes and sample contaminants to the baseline values.

Amino Acid Sequence Analysis of the COOH-terminal Peptide

Automatic Seqzcencing-The COOH-terminal peptide (100 nmol) was submitted to automatic sequencing with a JEOL.se- auence analvzer (JAS 47K). Dimethvlallvlamine buffer. DH 9.0. was used in the phenylisothiocyanate-couiling reaction. ‘fhe pep: tide program used the single cleavage system in the heptafluoro- butyric acid reaction. The thiazolinone derivative obtained after each Edman degradation step was converted to the phenylthio- hydantoin in 1.0 N HCl containing 0.01 mM dithiothreitol (11). The phenylthiohydantoin derivatives were analyzed on a Varian 1800 gas chromatograph with a 10% DC560 column (12). They were also hydrolyzed to the free amino acids with hydroiodic acid (ll), and analyzed on an amino acid analyzer.

Manual Degradation-Manual Edman degradation with dansyla- tion (8) was used to determine the sequence of the smaller pep- tides.

Subtilisin Digestion of the COOH-terminal Peptide-The peptide was dissolved in 0.05 M NaHC03. Subtilisin Carlsberg was added to give a final enzyme to protein ratio of 1:lOO. The digestion was allowed to proceed for 4 hours at 37”, and was stopped by lowering the pH to 4 with acetic acid. The peptides produced were separated by paper electrophoresis at pH 1.9 and detected by fluorescamine staining (13) and by the Ehrlich reaction (14).

BNPS-skatole Cleavage of the COOH-terminal Peptide at the Tryptophan Residue-The COOH-terminal peptide (30 nmol) was dissolved in 50yo acetic acid, and 0.05 mg of BNPS-skatole was added in 50% acetic acid. The reaction was allowed to proceed for 40 hours in the dark at room temperature. The excess reagent was extracted three times with diethyl ether (15) and the peptides produced were separated by paper electrophoresis at pH 1.9.

Assignment of the Glutamic Acid-Polyamide thin layer chroma- tography of the appropriate phenylthiohydantoin derivative was performed to establish the absence of glutamine according to the method of Summers et al. (16). The mob&ties of the subtilisin- and BNPS-skatole-produced peptides were checked by paper electrophoresis at pH 6.5 (17) to confirm the assignment of glu- tamic acid at position 3 from the NH2 terminus.

p Elimination Method-To identify the amino acid residue in the COOH-terminal peptide to which the carbohydrate is attached, p elimination was performed by treatment of the BNPS-skatole- produced peptides with 0.5 N NaOH for 40 hours at room tempera- ture (18). The solution was neutralized with acetic acid, lyophil- ized, and the product was then hydrolyzed with 6 N HCl for 22 hours at 110”. The hydrolysate was analyzed in the usual way to determine whether any reduction in serine or threonine content had occurred.

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Carbohydrate Analyses

Known amounts of mannitol and norleucine were added to solu- tions of the sample (a-X, heavy chain in 0.2 M NHhHC08; COOH- terminal peptide in water), and the samples were Ivophilized. To determine the protein contents, aliquots were hydroiyzed in 6 N

HCl for 24 hours at 110”. For the estimation of amino-sugar con- tent, aliquots were hydrolyzed in 1.5 N HCl for 24 hours at llO”, and analyzed on the amino acid analyzer, the content being re- ferred to the norleucine standard.

For the estimation of neutral sugars, aliquots were subjected to methanolysis in 0.5 M HCl in anhydrous methanol (Supelco) for 4 hours at 65”. After drying, they were acetylated, and then again subjected to methanolysis as described by Reinhold (19). The samples were then silylated with Tri-Sil (Pierce) before chroma- tography on a B-foot column of SE30 (Pierce) in a Varian gas chromatograph. A temperature program was used from 130~220”, at a rate of 4”/min; development was continued at 220” to complete elution.

For the determination of sialic acids, aliquots were subjected to methanolysis (0.5 N HCl in methanol, 1 hour, 65”), dried, and sub- jected immediately to silylation. The silylated derivatives were analyzed on the gas chromatograph in the same way. Carbohy- drates determined by gas chromatography were related to the internal standard, mannitol, and hence to norleucine and the pro- tein content.

Preparation and Isolation of Intermediates and Products of Activation

Activations of Factor X and intermediate reactions were done, and the products were separated, in the following ways.

Reaction I-Factor X (30mg in 60 ml of 0.2 M ammonium acetate, pH 7.5) was incubated with 0.3 mg of coagulant protein of RVV in the presence of 5 mM C&l% at 37” for 5 min. The reaction was stopped by the addition of acetic acid to a concentration of 1 M.

The mixture was frozen and lyophilized, and taken up in 3 ml of 6 M

guanidine HCl-0.5 M Tris-Cl, pH 8.2, -2 mM EDTA. The pH was adjusted to pH 8.2 again with 3 M Tris, and 2-mercaptoethanol was added to a concentration of 0.1 M. After reduction for 1 hour at 37”, iodoacetic acid was added to a concentration of 0.11 M, and carboxymethylation allowed to proceed for 20 min in the dark at room temperature (20). Excess 2-mercaptoethanol was added to

r ? 1.6

1.4

1.2

I 1.0

s N 0.0 a I

0.6 t

stop the reaction, and the sample was applied directly to a column of Sephadex G-100 equilibrated in 0.2 M NHIHCO~. This column separates the heavy and light chains of a-X, and the NH*-terminal (activation) peptide (Fig. 2). The fractions were pooled as shown and lyophilized.

Reaction S-To determine the nature of the material lost from the heavy chain in the conversion of a-X, to p-X,, we found it easier to activate Factor X with RVV, as described for Reaction 1, and then add lipid to the mixture to convert the a-X, to O-X, (2). The reaction was also done with TF-VII: to ensure that this activation proceeded through Reactions 1 and’2 (rather than 3 and 4), a large amount of TF-VII was used. Under these fast activation conditions, the bulk of the Factor X is activated by this pathway (2).

(a) Factor X (30 mg in 60 ml of 25 mM Tris-Cl, pH 7.5) was ac- tivated with the coagulant nrotein of RVV (0.15 me) in the nres- -, I ence of 5 mM CaClz for 10 mm at 37”. Lipid (an equimolar mixture of phosphatidylcholine and phosphatidylserine) was then added to a final concentration of 0.1 mg/ml, and the reaction was allowed to proceed for an additional 30 min at 37”.

(b) Factor X (12 mg in 25 ml of 25 mM Tris-Cl, pH 7.5) was ac- tivated with TF-VII (Factor VII at a final concentration of 150 units/ml) in the presence of 5 mM CaCls for 30 min at 37”.

Both activations were stopped and processed in the same way. Sodium oxalate was added to a concentration of 5.5 mM; the mix- tures were then centrifuged at 100,000 X g for 1 hour at 4’ to re- move lipid and calcium oxalate. The supernatant was lyophilized, taken up in 6 M guanidine HCl, and reduced and carboxymeth: ylated as described for Reaction 1. The sample was applied di- rectly to a column of Sephadex G-50 to separate the COOH- terminal (proline-rich) peptide from the heavy and light chains of p-X, and the NHz-terminal (activation) peptide (Fig. 3). The two pools were made as shown and lyophilized. The pool of the heavy and light chains and the NHS-terminal (activation) peptide was taken up in 3 M guanidine HCl, and rechromatographed on a col- umn of Sephadex G-100, as described for or-X, (Fig. 2). The separa- tion was very similar to that seen in Fig. 2, and similar pools were made.

Reaction S-Factor X (30 mg in 60 ml of 25 mM Tris-Cl, pH 7.5) was incubated for 110 min at room temperature with 1.5 mg of p-X, in the presence of equimolar phosphatidylcholine and phos- phatidylserine (final concentration, 0.1 mg/ml) and 5 mM CaC12.

/a -a I-

0 20 40 60 80 100 120

FRACTION NUMBER

FIG. 2. Separation of heavy and light chains of or-X,, and NHz- brated in 0.2 M NHIHC03, and eluted at the same flow rate at 4”. terminal (activation) peptide. Factor X was activated, the prod- Pools indicated (u) were, in order of elution, heavy chain, light ucts lyophilized, reduced, and carboxymethylated as described chain, and activation peptide. Final peak consisted of low molecu- under “Methods” (Reaction 1). The mixture (3 ml) was applied lar weight solutes. at 10 ml/hour to a column of Sephadex G-100 (2 X 110 cm) equili-

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TABLE I

Amino acid and carbohydrate analyses of Factor X, and the inter- mediates and products of activation

The heavy chains (HC) of 11, a-X,, and P-X, were prepared as described under “Methods” (Reactions 3, 1, and 2). Factor X heavy chain was isolated after reduction and carboxymethylation by gel filtration, as described for cu-X, under Reaction 1. The NH2- and COOH-terminal peptides were prepared as described under “Methods.” Reactions 1 and 2.

I-X, T

HC B -x, N

HC P

14.9 5.8

14.6 20.6 18.0 13.3 25.1

8.4 22.3 17.1

8.6 16.9

4.9 8.2

14.4 6.3

11.4

11 HC

15.8 15.3 7.8 6.9

17.1 15.5 25.6 19.2 22.6 20.2 19.4 13.3 32.5 26.3 10.9 12.6 26.8 23.7 22.8 19.5

7.7 9.3 19.7 18.4

5.3 4.9 9.2 8.0

20.9 16.3 6.4 5.9

13.1 12.3

hino a X acid HC

0.2 0 15.1 1.9 1.2 8.9 2.0 0 16.6 7.2 0.1 27.3 2.9 1.9 22.8 6.8 1.0 21.1 7.2 1.1 33.4 3.0 5.0 16.4 3.6 1.0 27.5 5.0 2.0 24.1

0 0 8.6 1.9 1.0 19.8

0 0 4.9 0.9 0 9.1 6.8 1.7 22.3 1.0 0 7.3 0.2 0 11.6

-term. C -term. eptide P eptide

LYS His Arg ASP Thr SeK Glu PKO GUY Alab CYS va1 Met 11e Leu T Y ~ Phe

15.7 8.8

17.8 25.7 24.1 18.9 33.4 15.4 27.3 24.6

9.4 20.0

4.7 9.0

23.1 6.9

12.4 I 1

"arbohydratea

1 t 0 L-2 v I- t- -

80 0 20 40 60 FRACTION NUMBER

FIG. 3. Separation of the COOH-terminal (proline-rich) peptide from heavy and light chains of p-X, and activation peptide. Factor X was activated in the presence of lipid, and the resulting mixture was further processed as described under “Methods” (Reaction 2a). The sample (2 ml) was applied at 5 ml/hour to a column of Sephadex G-50 (1.5 x 70 cm) previously equilibrated in 0.2 M NHIHCOZ, and elllted at the same flow rate at 4’. The two pools indicated (t) were, in order of elution: (a) heavy and light chains of P-X, and NH*-terminal peptide, and (h) COOH-terminal peptide. Final peak, of which the leading edge is shown, consisted of low molecular weight solutes.

The reaction was stopped by the addition of 5.5 mM sodium oxalate, and the lipid and calcium oxalate were removed by centrifugation at 100,000 X 9 for 1 hour. STI-Sepharose, 1.2 ml, was added to the supernatant, stirred for 20 min at room tempera- ture to remove most of the Factor X,, and removed by filtration. The filtrate was lyophilized, the product reduced, and carboxy- methylated as described for Reaction 1, and chromatographed on Sephadex G-50. The chromatogram resembled closely that shown in Fig. 3, except that in this case, the NHz-terminal (activation) peptide was not present. The pool of the heavy and light chains of 11 was lyophilized and rechromatographed on Sephadex G-100.

Reaction Q-Factor X was converted to 11, and the Factor X, was removed from the mixture, as described for Reaction 3. The resulting solution of 11 (+ COOH-terminal peptide) was activated with TF-VII as follows. 11 (15 mg in 30 ml of 25 mM Tris-Cl, pH 7.5) was incubated at 37” for 30 min with Factor VII (as the active TF-VII complex) at a concentration of 100 units/ml in the pres- ence of 5 mM CaCL The reaction was stopped and the mixture processed as described for Reaction 2(b), the reduced and car- boxymethylated material being chromatographed on Sephadex G-50 and G-100 to separate the heavy and light chains and the two peptides.

Reaction 5-The heavy chain of 12 was isolated from 11 prepara- tions by preparative sodium dodecyl sulfate gel electrophoresis, as described previously (2).

The isolated carboxymethylated heavy and light chains of cu-X, and p-X, were shown to be single bands on sodium dodecyl sulfate gel electrophoresis. The heavy chain of 11 was about 57, contaminated with the heavy chain of 1~. Neither peptide was de- tectable with this technique.

Mannose 0 0 Galactose 3.2 1.6 Glucosamine 0 0.1 Galactosamine 0.7 0.8 Sialic acid 1.4 1.3

aBoth amino acid and carbohydrate contents are ex- pressed as moles per mole of chain or peptide (Table II).

b Half-cystine was determined as S-carboxymethylcysteine.

'This column is the sum of the three previous columns.

obtain cu-X, not contaminated with P-X, requires the venom

fraction at only 1 yc of the concentration (by weight), so that contamination of t,he reaction products by RVV is negligible

(the molecular weight of the RVV fraction is high enough to contaminate only the heavy chain). However, even though lipid is absent, it is necessary to stop the reaction completely with acetic acid to prevent the subsequent formation of P-X,.

Gel filtration of the reduced and carboxymethylated mixture on Sephadex G-50 showed that no material smaller than the NHz-terminal (activation) peptide had been formed during the reaction. Therefore a single gel filtration step on Sephadex G-100 was used to separate the heavy and light chains of a-X, and the activation peptide (Fig. 2).

Fujikawa et al. (3) reported the molecular weight of the pep- tide to be 10,800, 757, of which was protein (Al, = 8,100). However, the assumption of this molecular weight for the peptide protein did not agree with our calculation based on the adjust- ment of the analysis of the peptide to nearest integer values for the amino acids. This method can generally be used satisfac- torily for smaller peptides. When the molecular weight of the protein portion of the NHz-terminal (activation) peptide is calculated in this way, we find that the sum of the amino acid contents of the heavy chain of a-X, and the peptide agrees more closely with the amino acid composition of the heavy chain of Factor X (Table I). Therefore, we consider that the molecular weight of the NHz-terminal (activation) peptide is probably 5700. This value also agrees closely with the amount of arginine re- leased by carboxypeptidase B from this peptide (assuming M, = 5700, we find 1 mol of arginine released/2.05 mol of total arginine).

The NHz-terminal sequence of each component of the mixture

RESULTS

RVV Reaction 1: Factor X ------+ CU.X, + NH2-terminal Pep-

tzde-The activation of Factor X by RVV at rates sufficient to

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TABLE II

NHZ- and COOH-terminal sequences and molecular weights of Factor X and the intermediates and products of activation

Heavy chain or peptide

Factor X I1 CX-X, 8-X, NHS-terminal

peptide COOH-terminal

peptide

- NH*-terminal sequence

(-)-Ala-Ile-Gly- (-)-Ala-Ile-b Ile-Val-Gly-Gly- Ile-Val-Gly-Gly- (-)-Ala-Ile-

Gly-His-Glx-Ser

COOH-terminal sequence

MOkCUl?U weighta

Leu 33,200 Argc 31,300 Leu 27,500

Ax 25,600 Val-Arg 5,700

Pro-Leu 1,900

a All molecular weights refer to the protein portion, i.e. exclude carbohydrate. The molecular weight of Factor X is from Fujikawa et al. (3)) and those of the peptides were determined by quantifica- tion of release of COOH-terminal residues by carboxypeptidase A

(leucine) and carboxypeptidase B (arginine). The molecular weights of 11, OL- X,, and p-X, were determined by subtraction of the appropriate peptide weights (see under “Results”).

b Tryptophan cannot be detected after acid hydrolysis of dan- sylated protein; (-) corresponds with tryptophan reported by Fujikawa et al. (3) for Factor X heavy chain and the activation peptide.

c See Table III.

is shown in Table II and agrees with the results of Fujikawa et al. (3). The NHB-terminal sequence of the heavy chain, Trp- Ala-Ile-, is also found in the activation peptide, and LY-X, heavy chain has the new NHz-terminal sequence, Ile-Val-Gly-Gly-, Leucine, which is the COOH-terminal amino acid of the heavy chain of Factor X, was also released from the heavy chain of a-X,. Thus, it is very likely that the COOH terminus of the heavy chain of Factor X remains intact during activation with RVV in the absence of lipid.

Reaction 2: cr-X, xa + Lipid f /3-X, + COOH-terminal Peptide-Most of the P-X, and COOH-terminal (proline-rich) peptide was prepared by activation of Factor X by RVV with the addition of lipid. To minimize contamination of the products with extraneous peptides, which tend to occur in preparations of mixed brain lipids, we used an equimolar dispersion of phos- phatidylcholine and phosphatidylserine. The reaction was allowed to proceed for 30 min after the formation of (Y-X,, to ensure complete conversion to P-X, (2). The peptide released in the conversion of cu-X, to P-X, is of considerably lower molecular weight than the NHz-terminal (activation) peptide, necessitating two gel filtration steps to separate all of the components of the mixture. The first step, on Sephadex G-50, separates a peptide that is not present in mixtures where only cr-X, is formed (Fig. 3) and appears to be the only peptide produced in the conversion of cr-X, to P-X,; no other material can be detected in the chroma- togram before 2-mercaptoethanol (by spectrophotometry at 220 nm) other than the mixture of heavy and light chains and the activation peptide.

The NHz-terminal peptide obtained had the same amino acid composition, and NH2- and COOH-terminal sequences, as the peptide produced by RVV in the absence of lipid. Moreover, the NHz-terminal sequence of P-X, was the same as that of cu-X, (Table II). The other peptide produced, of lower molecular weight, contained the NH*-terminal sequence Gly-His-Glx-Ser-, which is not found in any of the precursors, and also contained carbohydrate. This is in agreement with the reported differences

TABLE III

Carboxypeptidase digestion of the heavy chains of Ix and P-X,

11 and P-X, heavy chains were digested with carboxypeptidases A and B as described under “Methods.” The release of residues, expressed as mol/mol of heavy chain, was determined by amino acid analysis, and was corrected for appropriate controls.

Amino acid released I 11 I B-X,

Arg 0.79 0.89 Ala 0.45 0.47 Ser or Asn 0.49 0.45

GUY 0.24 0.25 Leu 0.21 0.18 Val 0.19 0.12

in the carbohydrate contents of ar-X, (3) and P-X, (al), the latter not containing any carbohydrate. Confirmation that this peptide is derived from the COON terminus of the heavy chain of a-X, was obtained from its COOK-terminal amino acid, leucine, which is the same as that of Factor X and a-X, heavy chains (Table II). Finally, we showed that P-X, heavy chain has a new COOH terminus, arginine. Other amino acids released from P-X, heavy chain upon prolonged digestion with carboxy- peptidase A and carboxypeptidase B are shown in Table III, but an unambiguous sequence cannot be deduced from these results.

Evidence that the COOH-terminal (proline-rich) peptide is the only material released in the conversion of cr-X, to P-X, was provided by summing the amino acid compositions of this peptide, the NHz-terminal peptide, and the heavy chain of P-X,, and comparison with the composition of Factor X heavy chain (Table I) The number of amino acid residues in the COOH- terminal peptide was estimated by adjustment of the amino acid composition to nearest integer values for each amino acid. The molecular weight calculated in this way (M, = 1900) does not correspond with the change in apparent molecular weight obtained by sodium dodecyl sulfate gel electrophoresis of cu-X, and P-X, heavy chains (Mr 4500) (2). However, we know that the peptide contains carbohydrate, which may account for this difference.

Reaction 3: Factor X X, + Lipid

) II f COOH-terminal Peptide-This is the first reaction of t,he alternative pathway of activation of Factor X, and leads to the release of the proline- rich peptide identical with that produced in the conversion of a-X, to P-X, (Reaction 2). Identity of the peptides was shown by amino acid analysis, and by NH2- and COOH-terminal analysis. We also confirmed that I1 heavy chain has the same NI&terminal sequence as Factor X, but a new COOH-terminal amino acid, arginine. Moreover, on prolonged digestion with carboxypcptidases A and R, the same group of amino acids is released from the COOH terminus as with P-X, heavy chain (Table III).

Reaction 4: II TF-VII or RVV ) P-X, + NH2-terminal

Peptide-The activation of I1 with TF-VII or RVV produces P-X, and the normal NHz-terminal (activation) pcptidc. This has been confirmed by amino acid analysis, and NH2- and COOH- terminal analysis of both the heavy chain of P-X, and the pep- tide; these are identical with those produced by Reactions 1 and 2, as shown in Fig. 1. It is, therefore, clear that Reactions 3 and 4 involve the same cleavages of the heavy chain as Reactions 1 and 2.

P-X, produced by fast activation of Factor X with TF-VII

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4502

FIG. 4. Proposed sites of cleavage of Factor X during activation. Cleav- age at A releases the activation pep- tide and results in the appearance of Factor X, activity. Cleavage at B re- leases the COOH-terminal nentide. Cleavage at C, in addition to i, occurs slowly in the presence of Factor X, and results in the formation of 1~.

14 u H

ax0 u 2(

Activation Peotide 6x0 Carbo;;-tt;mmlnol

X,) I

TF-m RVV

(X0)

I

X0

I

is known to derive mainly by Reactions 1 and 2 (2). The products of this activation (P-X, heavy chain and the NHz- and COOH- terminal peptides) are identical with those produced in activa- tions by RVV + lipid, as shown by NH*- and COOK-terminal analysis. P-X, was also produced by conversion of Ii by TF-VII, and again, the products were identical with those produced by RVV. Thus, we confirmed that TF-VII cleaves the same bond as RVV, and that, as expected, the site of cleavage of the proline- rich (COOH-terminal) peptide is the same as that in Reaction 2 (Fig. 4).

Finally, we have shown by NH2- and COOH-terminal analysis of the light chains of Factor X and P-X,, produced by either RVV + lipid or TF-VII, that the light chain is not cleaved during activation.

Reaction 5: Ii xa ’ Lipid > ZzpWe reported in an earlier paper (2) that Factor X, can activate Factor X (via Ii)-ap- parently to P-X,-and that during this slow activation, a new species appears that we called I*. This seemed to be a result of the cleavage of the heavy chain of I1 in the NHz-terminal region. We have studied the peptides released during the autocatalytic activation and we find that the major portion of P-X, formed can be accounted for by the cleavage of the normal NHz-terminal (activation) peptide from the heavy chain. Therefore, it is clear that I$ is not an obligatory intermediate in the activation of I1 by Factor X, + lipid, and it is doubtful whether 12 can be con- verted to P-X,, even though it eventually disappears in these mixtures (2).

Effect of the Peptides on the Intrinsic and Extrinsic Pathways- If either the NH2- or COOH-terminal peptide had a control function in coagulation, we would expect the addition of the peptides to plasma at a high concentration (relative to Factor X) to affect the rate of coagulation by either the intrinsic or extrinsic pathways. To test this, the peptides were added, separately and together, to titrated bovine plasma at approxi- mately 10 times the molar concentration of plasma Factor X. Neither peptide separately, nor both together, affected either the partial thromboplastin time (6) or the prothrombin time (5).

Carbohydrate Analysis of (Y-X, Neavy Chain and the COOH- terminal Peptide-The results of Fukikawa et al. (3) and Rad- cliffe and Barton (21) suggested that the small amount of car- bohydrate remaining on cu-X, heavy chain is released in the conversion of a-X, to P-X,. We, therefore, determined the car- bohydrate compositions of cr-X, heavy chain and the COOH- terminal peptide (Table I), and these results suggest that all of the carbohydrate of a-X, heavy chain is lost in the COOH-

H,N-TRP-ALA-ILE -‘THR - VAL-ARG:ILE-VAL-GLY-GLY

T ARG:GLY-HIS - PRO-LEU-COOH

0 @

S Ill HOOC-ARG 2 SER-ASX-ALA-NH2

Sb ENPS I

Gly-His-Ser-Glu-Alo-Pro-Alo-Thr!Trp-T~~-Vol-P~o-P~o-P~o-L~~-P~o-Le~

CHO

==F==P==%-==T==v-===7====?-- LL

FIG. 5. Amino acid sequence of the COOH-terminal peptide. Sequences shown were obtained by automatic sequencing (T), manual Edman degradation (-), and by carboxypeptidase di- gestion (-). Sb and BNPS indicate the sites of cleavage by sub- tilisin and BNPS-skatole. CHO indicates the carbohydrate moiety.

terminal peptide. We also confirmed that P-X, heavy chain and the light chain of Factor X do not contain carbohydrate, in agreement with the results of Radcliffe and Barton (21) and Jackson (22).

Amino Acid Sequence of the COON-terminal Peptide-The peptide was prepared by treatment of Factor X with P-X, in the presence of lipid and Ca 2+ (Reaction 3). After removal of the lipid by centrifugation, and the Factor X, with STI-Sepha- rose, the mixture was lyophilized and chromatographed, without prior carboxymethylation, on Sephadex G-50. The COOH- terminal sequence of the peptide was shown to be Pro-Leu, which is consistent with the heavy chains of Factor X and Cl-X,.

The COOH-terminal peptide was first submitted to automatic sequence analysis, the results of which established the sequence of the first 10 residues from the NHz terminus (Fig. 5). The peptide was then digested with subtilisin at pH 8.5. Two pep- tides were obtained whose amino acid compositions are shown in Table IV. The composition and NHz-terminal sequence are in good agreement with the results obtained with the whole peptide. Edman degradation with dansylation on the second subtilisin- produced peptide gave the amino acid sequence from residues 11 to 17. This peptide, like the intact COOH-terminal peptide, contained galactosamine, but was not analyzed for neutral sugars or sialic acid. In order to identify the amino acid residue to which the carbohydrate is attached, fi elimination was performed on this second subtilisin-produced peptide. The threonine content was markedly reduced by this t.reatment, showing that the carbohydrate is bound to the threonine residue of this peptide.

To confirm the amino acid sequence, the COOH-terminal peptide was cleaved at the tryptophan residue with BNPS- skatole. Two peptides were obtained; the amino acid composition and sequences and the amino-sugar contents were fully consistent

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TABLE IV Amino acid composition of the peptides obtained from the COOH-

terminal peptide by cleavage with subtilisin and BNPS-skatole

The cleavages of the COOH-terminal nentide and the methods of analysis are described under “Methods.”

Amino acid Sb 1 Sb 2 BNPS 1 BNPS 2

1.13 Thr 0.85 1.17 Ser 0.93 0.94 GlU 1.10 1.12 Pro 1.08 1.04

GUY 0.94 1.01 Ala 2.13 1.90 Val Leu His 0.97 0.82

‘h Amino-sugar - -

No. residues 8 8 Ehrlich reaction” - Mobilityc N N Yield (%) 25 40

1.05

3.79 3.96

0.91 2.17

0.84 2.15

0.13 9 + N

42

0.95 8

N 60

- ntact COOH-

terminal peptide

1.9 1.0 1.1 5.0 1.0 2.0 1.0 1.7 1.2 1.28 0.8

17 + N

a Tryptophan content was determined only on the intact pep- tide.

* A positive Ehrlich reaction indicates tryptophan. c Mobility refers to the electrophoretic mobility at pH 6.5; (N)

indicates that the peptides remained at the neutral area.

with the results from automatic sequencing of the intact peptide and with the sequences of the subtilisin-produced peptides (Table IV, Fig. 5).

DISCUSSION

In an earlier paper, we showed that the activation of Factor X by either TF-VII or RVV + lipid can proceed by two alternative pathways (Fig. 1). In the present work, we have shown that two sites of cleavage of the heavy chain are involved, which are independent of the activation pathway. The cleavage of the activation peptide from the NH2 terminus of the heavy chain of Factor X is catalyzed by either TF-VII or RVV, resulting in the appearance of a new NHz-terminal sequence, Ile-Val-Gly- Gly-, in agreement with the results of Fujikawa et al. (3), who used RVV in the absence of lipid. It is this cleavage that results in the appearance of Factor X, (prothrombin-converting) ac- tivity, producing the first species seen in rapid activations, Q-X,. Titani et al. (23) have shown that, cu-X, has extensive regions of homology with the amino acid sequences of trypsin, chymotrypsin, elastase, and the B chain of thrombin.

In activations in the absence of lipid, a-X, is initially the only product of activation; but in the presence of lipid (either RVV + lipid, or TF-VII). we find that the cr-X, formed cleaves a COOH-terminal peptide from its own heavy chain, producing P-X,, which has equal coagulant activity to (Y-X, (2).

Another activation pathway, seen at lower activator concen- trations, involves the same cleavages of the heavy chain of Factor X as we find in fast activations, but in the reverse order. This pathway is initiated by the action of Factor X, on Factor X to release the same COOII-terminal peptide. The intermediate produced in this reaction, 11, can be converted directly to P-X, by either activator, even in the absence of lipid, with the release of the normal NHz-terminal activation peptide.

We have isolated the heavy and light chains of Factor X,

4503

Ii, (Y-X,, and P-X,, and the two peptides. We have shown that these are the only peptides released from Factor X during activa- tion. This conclusion is based on: (a) the sum of the amino acid compositions of P-X, heavy chain and the two peptides is very close to the composition of Factor X heavy chain; and (b) the NH2 and COOH termini of Factor X heavy chain match the termini of the appropriate intermediate heavy chains and the peptides. Moreover, the sites of cleavage are independent of both the activation pathway and the activator used. In addition, we have shown that the autocatalytic activation of Factor X by Factor X, also proceeds by the loss of these peptides.

We have confirmed that the bond broken by RVV, reported by Fujikawa et al. (3), is the same as that broken by TF-VII, in agreement with the results of Radcliffe and Barton (21). As shown previously, the removal of the COOH-terminal peptide by the action of Factor X, has no effect on the coagulant activity of Factor X or (Y-X, (2). Moreover, we have shown that neither peptide affects the rate of activation of Factor X by TF-VII. Thus, as far as we can tell, the only means of control of Factor X activation is the inactivation of TF-VII, already described (a), which has been studied extensively by Radcliffe and Nemer- son (24). This inactivation causes the yield of Factor X, to be a function of the concentration of TF-VII. The possible significance of this type of control of Factor X activation is discussed else- where (25).

Thus, in the case of Factor X activation, although there are alternative pathways, one of which is initiated by the product, they have no significance per se in regulating the final yield of Factor X,. This is in contrast with the activation of prothrombin, where there is also an alternative activation pathway initiated by the product. In that case, the site of the feedback cleavage is in the NHz-terminal region of the molecule, and the concomitant loss of the calcium- and lipid-binding sites renders it partially refractory to activation by the activating complex of X,, V, lipid, and Ca2+ (26). It is interesting to note that the peptide lost from prothrombin in this reaction, Fi, is very similar to the light chain of Factor X (27). As suggested by Mattock and Esnouf (28), it seems likely that Factor X is formed from a single chain precursor homologous with prothrombin. The major difference is that Fi is not linked by disulfide bonds to the region of prothrombin containing the thrombin active site, whereas the light chain of Factor X is linked to the region that becomes the heavy chain of P-X,. Finally, we can relate this with the observation that, whereas thrombin is released from the pro- thrombin-converting complex (29), the product of Factor X activation is still bound to phospholipid, forming, with Factor V, the prothrombin-converting complex. This is presumably due to the location of the lipid-binding sites, which, by comparison with prothrombin, would be located in the light chain of Factor X.

In an earlier paper (a), we noted that the product-initiated pathway of activation occurs to a greater extent in slower ac- tivations, as would be expected on theoretical grounds, and it occurred to us that such alternative pathways are suitable sites for feedback control. For instance, a peptide released from a zymogen by a product might be an inhibitor of the activator, or of a preceding stage in a system. We, therefore, examined both the intermediates and the peptides produced during the activa- tion of Factor X in terms of their activity in the clotting system. We previously showed that I1 can be activated at the same rate as Factor X by TF-VII, and that cr-X, and P-X, have identical activities (2). In this work, we have shown that neither peptide has any effect on either the prothrombin time or the partial

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4504

thromboplastin time of normal bovine plasma. These are indi- cators of the state of the extrinsic and intrinsic pathways of coagulation.

Jackson (22) showed that all of the carbohydrate of Factor X is bound to the heavy chain, and Fujikawa et al. (3) showed that about 70% of this is lost in the NHz-terminal activation

peptide on the formation of o-X,. In contrast, Radcliffe and

Barton (21) examined Factor X, produced by RVV and by the intrinsic and extrinsic pathways and found all the forms of Factor X, to contain little or no carbohydrate. However, as we discussed previously (2), the inclusion of a purification step after activation would probably have resulted in the conversion of any cr-X, to P-X,. Therefore, it seemed that the carbohydrate of (Y-X, is lost in the conversion to P-X,, i.e. in the COOH- terminal peptide.

Carbohydrate analyses of a-X, heavy chain and the COOH- terminal peptide confirmed that they have approximately the same carbohydrate composition (Table I). Notably, both contain galactosamine but no glucosamine, and neither contains mannose. It should be pointed out that under the hydrolysis conditions used for amino-sugar analysis (1.5 N HCl, 110” for 22 hours), any N-acetylgalactosamine would be deacylated.

It was shown by p elimination, performed on the subtilisin- produced peptide that contained galactosamine (Residues 9 to 17), that the threonine residue (position 10) bears the carbo- hydrate. It has been found by other workers that in most plasma glycoproteins studied, the carbohydrate moieties are bound to the protein chain through an N-glycosidic linkage between asparagine and N-acetylglucosamine (30). This linkage also ap- pears to be the way the carbohydrate groups of prothrombin are attached, since no 0-glycosidic linkages to threonine or serine could be shown by /3 elimination in a study of protease digests of prothrombin (31). In contrast, an 0-glycosidic bond to a threonine residue is evidently the mode of linkage of the carbo- hydrate moiety in the COOH-terminal peptide. Although rare in plasma glycoproteins, this type of linkage is common in mucins and cell membrane glycoproteins; the sugar component, however, is almost invariably N-acetylgalactosamine (30). It is, therefore, of interest that the COOH-terminal peptide contains galactosaminc, but no glucosamine. Thus, it seems likely that, unlike the protein-carbohydrate linkages known in prothrombin, fibrinogen, and the majority of other plasma glycoproteins studied, the sugar-protein bond in the COOH-terminal peptidc is N-acetylgalactosaminyl-threonine.

Another interesting feature of the COOH-terminal peptide is the high proline content of the last 6 residues (Fig. 5), and the fact that the amino acid sequence of the intact COOH-terminal peptide shows no homology with the COOH-terminal sequences of chymotrypsinogen, trypsinogen, proelastase, and prothrombin. In these four cases, the COOH terminus remains intact during activation, and all four enzymes show considerable sequence homology in the immediate region of their COOH termini (32). Moreover, it is quite clear from the NHp-terminal amino acid sequence of the heavy chain, and the sequence in the region of the active site, that Factor X, is closely related to the other serine esterases mentioned above (23).

Preliminary experiments indicate that neither the NH2-

terminal nor the COOH-terminal peptide produced during Factor X activation has any control function, inasmuch as they do not affect detectably the rate of coagulation by either the extrinsic or intrinsic pathways. This does not exclude the possibility, however, that either peptide may have a biological function.

Acknowledgments-We are grateful to Mr. Ron Bach for supplying the tissue factor, and to Ms. Audrey Lee for doing the gas chromatography.

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J Jesty, A K Spencer, Y Nakashima, Y Nemerson and W Konigsbergactivation pathways and characterization of the COOH-terminal peptide.

The activation of coagulation factor X. Identity of cleavage sites in the alternative

1975, 250:4497-4504.J. Biol. Chem. 

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