intrinsic pathway activation of factor x and its activation peptide

THE JOURNAL OF BIOLOGICAL CHEMISTRY 8 1992 by The American Society for Biochemistry and Molecular Bioloa, Inc.

Vol. 267, No. 11, Issue of April 15, pp. 7821-782’7,1992 Printed in U. S. A.

Intrinsic Pathway Activation of Factor X and Its Activation Peptide-deficient Derivative, Factor Xdes-143-191 *

(Received for publication, July 2,1991)

Edward J. Duffy and Pete LollarS From the Division of Hematology-Oncology, Department of Medicine, Emory University, Atlanta, Georgia 30322

The role of the activation peptide in determining the substrate specificity of intrinsic pathway factor X (fX) activation was studied by using a novel derivative of flI in which 49 residues were removed enzymatically from the NH2 terminus of the 52-residue activation peptide by an enzyme from the venom of the snake Agkistrodon rhodostoma. The modified protein, designated fxdes-143-101, is inactive but is activated to a- fXa by either the intrinsic fX activation complex (intrinsic fXase) composed of factor IXa& thrombin-activated factor VI11 (fVIIIaIIa), and phospholipid vesicles or by the fX coagulant protein from Russell’s viper venom (RVV-XCP).

Both the K,,, and kcat for the activation of fX by RVV- XCP were greater than for fXdes-143-191, resulting in less than a 2-fold difference in the catalytic efficiency ( kCat/K,,,) suggestive of nonproductive binding of 143-191 to RVV-XCP. The activation of each substrate by intrinsic fXase revealed that the kc,, was 100-fold greater for fX than fXdes-143-1Dl (16 and 0.16 s-’, respectively), although there was no detectable difference in K,,, (60 and 80 nM, respectively). Activations by fIXa@/phospholipid in the absence of fVIIIaIIa also revealed a difference in kcat but not K,, but the difference in kc,, was smaller (kcat of 0.007 and 0.002 s“ and K , of 220 and 170 nM for fX and fXd”143-191, respectively). Analysis of product versus time curves dem- onstrated that fVIIIaII, promotes formation of the acyl- enzyme intermediate during fx activation. We conclude that the activation peptide plays a critical role during acyl-enzyme formation that is most pronounced in the presence of fVIIIaIIa. The absence of K,,, differ- ences suggests that residues NHz-terminal to P3 do not contribute to the initial formation of the enzyme-substrate complex.

Intrinsic pathway activation of factor X (fX)’ is necessary

*This work was supported by an American Heart Association Established Investigator Award (to P. L.) and by National Institutes of Health Grants HL-40921 and HL-35058. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$.To whom correspondence and reprint requests should be ad- dressed Drawer AJ, Emory University, Atlanta, GA 30322. Tel.: 404- 727-5569; Fax: 404-727-3404.

’ The abbreviations used are: fX, fIX, and NIII, factors X, IX, VIII, respectively; fXa, any species of activated f X ; a-fXa, activated fX that contains residues 143-448 of the heavy chain; fx-des.143-191, fX lacking residues 143-192 from the heavy chain; fIXa& factor IX activated by cleavages at Arg145 and Arg“” using the human factor IX sequence as reference (1); fVIIIaIl,, thrombin (factor 1Ia)-activated factor VIII; PCPs, unilamellar vesicles composed of 75% (w/w) ~ - a - phosphatidylcholine and 25% L-a-phosphatidylserine; intrinsic fXase, intrinsic fX-activating complex composed of fIXap, NIIIa~I.,

during normal hemostasis since deficiencies in two essential proteins in the process (factor VI11 and factor IX) result in a bleeding diathesis. fX is a 59-kDa vitamin K-dependent proenzyme’ consisting of a 17-kDa light chain and a 40-kDa heavy chain (see Ref. 1 for review). The light chain contains 11 post-translationally modified y-carboxyglutamic acid residues that are necessary for calcium-dependent membrane binding and two epidermal growth factor domains. The heavy chain contains the catalytic triad common to all members of the serine protease family and a specificity pocket that confers trypsin-like specificity. fX is activated to a-fXa by a single cleavage at Arg194-Ile195,

which releases a heavily glycosylated 52-residue activation peptide from the NH, terminus of the heavy chain. The activation of fX is catalyzed by either the intrinsic fX acti- vator (intrinsic fXase) consisting of the trypsin-like serine protease fIXap, activated factor VIII, and a phospholipid membrane surface or by the extrinsic fXase consisting of factor VIIa and tissue factor, an integral membrane protein. The conversion of fX to a-fXa also is catalyzed by the fX coagulant protein from the venom of Russell’s viper (RVV- XCP). Phosphatidylserine-phosphatidylcholine (PCPs) vesicles

greatly reduce the K,,, for the activation of fX by fIXaB with little effect on the kcat, whereas activated fVIII greatly increases the k,,, with little effect on the K,,, (2). Similar kinetic effects due to membrane components and protein cofactor components have been observed in prothrombinase and extrinsic fXase (see Ref. 3 for review). Various models have been proposed to explain the membrane-dependent K,,, effect in these vitamin-K dependent protein reactions, which re- mains controversial. In contrast, no explanation has been offered for the kcat effect produced by the protein cofactors in these reactions.

Most serine proteases are expressed as zymogens that require proteolytic activation that involves the release of an activation peptide from the NH, terminus of the serine protease domain. By analogy to trypsin and chymotrypsin (see Ref. 4 for review), the activation of fX presumably is associated with the formation of an internal salt bridge between the a-ammonium group of the nascent NH2 terminus of the heavy chain and the carboxylate of Asp194 (in the chymotrypsinogen numbering system). However, the role of residues in the activation peptide that are NHz-terminal to the scissile bond in governing enzyme-substrate interactions is unknown.

An enzyme from the Malayan pit viper (Agkistrodon rhodostoma), designated ARHy, catalyzes cleavages in fIX, fX,

and PCPs; RVV-XCP, Russell’s viper venom fX coagulant protein; ARH, Agkistrodon rhodostoma hydrolase; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; Hepes, 4-(2-hydrox- yethy1)-1-piperazineethanesulfonic acid.

* The molecular masses and sequences of fX and its subunits use human factor X as reference (1).

782 1

7822 Intrinsic Pathway Activation of Factor X and Factor Xdes-143-191

protein C, and prothrombin, resulting in products that remain inactive but can be activated by their respective activators (5). ARHy catalyzes the release of all the activation peptide of fIX, except the 3 residues NHa-terminal to the scissile bond corresponding to residues P1-P3? Thus, the product appears to remain a zymogen because the internal salt bridge cannot form. We now find that ARHy likewise produces a derivative of f X , designated fXdes.143-191, in which all of the activation peptide has been removed, except for residues P1-P3. In this study, we studied the contribution of residues NH2-terminal to P3 in conferring substrate specificity by comparing the steady-state kinetics of the activation of fX and meS-143-191

by intrinsic fXase in the presence and absence of activated factor VIII.

EXPERIMENTAL PROCEDURES

Materials-Crude snake venoms, L-histidine, L-a-phosphatidylcholine, L-a-phosphatidylserine, Sephadex G-25, and bovine factor VII- and X-deficient plasma were purchased from Sigma. Hepes was purchased from BDH Chemicals Ltd. Mono-Q chromatography col- umns were purchased from Pharmacia LKB Biotechnology Inc. Methoxycarbonyl-D-cyclohexylglycylglycylarginine-p-nitroaniline (Spectrozyme Xa) was purchased from American Diagnostica Inc. Unilamellar PCPs (75/25 w/w) vesicles were prepared as described previously (6).

Proteins-Porcine factors IX, IXap, X, prothrombin, and thrombin were prepared from slaughterhouse blood as previously described (7), except dextran sulfate-agarose chromatography in the final step of purification of fX was omitted. Porcine blood was collected into a container that contained 0.1 volume of 3.8% (w/v) trisodium citrate, 3.5% (w/v) e-aminocaproic acid, 0.1 M benzamidine, 5 pg/ml recombinant desulfatohirudin. Recombinant desulfatohirudin was a generous gift from Dr. R. B. Wallis, Ciba-Geigy Pharmaceuticals. Porcine factor VI11 and thrombin (factor 1Ia)-activated factor VI11 (fVIIIalI.) were prepared as described previously (8). fVIIIaII. prepared in this way is indefinitely stable at 4 "C. The hybridoma cell line (W3-3) producing mouse monoclonal anti-factor VI11 used in the isolation of WIII by immunoaffinity chromatography was a generous gift of Dr. D. N. Fass, Mayo Clinic/Foundation. RVV-XCP was isolated as described previously (9). a-FXa was produced by activation of fX with RVV-XCP and was isolated by Mono-Q chromatography (10). ARHy was purified as before (5) with the omission of the preparative gel electrophoresis step that removes ARHP. The venom mixture is, therefore, referred to as ARHyP, and all concentration terms refer to ARHy. ARHp does not catalyze cleavage of fX (data not shown).

in 0.15 M NaC1, 20 mM Hepes, 5 mM CaCI2, pH 7.0, at 37 "c for 30 Fxdae.143.1g1 was prepared by reacting 18 pM fX with 0.18 p M ARHyP

min. After 30 min the mixture was diluted &fold into 20 mM histidine, 10 mM sodium citrate, pH 6.0, and loaded onto a Mono-Q column equilibrated in the same buffer a t a flow rate of 1 ml/min and washed with 10 ml of this buffer. The column was developed with a 20-ml, linear 0-1 M NaCl gradient a t a flow rate of 1 ml/min. ARHy and ARHp did not bind to Mono-Q, as judged by SDS-PAGE. fXdes.143-191 eluted as a single, sharp, symmetrical peak and was >95% pure, as judged by SDS-PAGE. The preparation was free of ARHy activity, as judged by its inability to convert native fX to fXdes-143.191. Mono- Q-purified fXdes.143.191 was pooled and dialyzed against 0.15 M NaC1, 20 mM Hepes, 5 mM CaC12, pH 7.4, and was stored at -80 "c at concentrations ranging from 20-110 pM. In some experiments, fXdes.143-191 was concentrated by ultrafiltration (Centricon 30, Ami- con).

Extinction Coefficients and Molecular Weights-The published extinction coefficients (E:&') and molecular weights used were porcine fIXap, 1.52, 45,000 (7); ARHy, 1.26, 26,000 (5); and porcine fVIIIaII,, 1.6, 160,000 (8). The extinction coefficient for M was determined by the method of van Iersel et al. (11) and was 1.04; the molecular weight was 57,000 (7). For f)hes.143.191, an extinction coefficient of 1.0 and a molecular weight of 50,000 were assumed.

Steady-state Kinetics of fX Actiuation-Kinetic experiments were performed at room temperature in 0.15 M NaC1, 20 mM Hepes, 5 mM CaCI2, 0.1% polyethylene glycol 8000, pH 7.4. Intrinsic fXase (fIXa@/ WIIIaII./PCPS) reaction solutions were assembled by the addition of

'' The nomenclature of Schechter and Berger (33) is used to designate amino acid residues with respect to the scissile bond.

fVIIIaII. to a solution of fIXap to give a final concentration of 20 PCPs, 5 nM Ixap, and either 25 pM fVIIIaII. for fX or 450 p~ for =des-143-191 reactions. Reactions were initiated by the addition of substrate to yield final concentrations of 15-600 nM substrate. At 15, 30, 45, and 60 s following addition of substrate, 50-pl aliquots were removed from the reaction mixture and added to 5 p1 of 0.5 M EDTA, pH 7.5, to quench the reaction. The amount of a-fXa in each sample was then determined by using a chromogenic substrate assay as described below.

Due to the slower rate of activation of fX and fXdes.143.191 by fIXap (25 IIM) or ffXap/PCPS (2.5 nM/20 pM), samples were taken at 5- min intervals in reactions in which fVIIIaII. was absent. The kinetics of the activation of fX by RVV-XCP were measured as for intrinsic fXase at RVV-XCP concentrations of 50 pM for fX and 1 IlM for %es-143.191. The kinetic constants K,,, and Vmax were determined by unweighted nonlinear least squares fits of initial velocity data to the Michaelis-Menten equation using the Marquardt algorithm (12). Con- stants are expressed as mean f 1 S.D. The reported kinetic constants are representative of experiments involving two separate preparations of both fX and fXdee.143-191.

Chromogenic Assay for fXa-fXa was assayed by mixing 20 p1 of sample with 80 p1 of 0.4 mM Spectrozyme Xa, 0.15 M NaCI, 20 mM Hepes, 50 mM EDTA, 0.1% polyethylene glycol 8000, pH 7.4, in a microtiter plate and measuring the rate of Spectrozyme Xa hydrolysis a t 405 nm in a kinetic microtiter plate reader ( V,.,, Molecular Devices Corp.). Initial rates of p-nitroaniline release were determined under conditions in which less than 10% substrate hydrolysis occurred. fXa concentrations were determined by interpolation on a standard curve that was prepared by using a preparation of purified a-fXa whose concentration was determined by active site titration (13, 14). fXa standard curves were linear between 1 and 100 nM.

fX Clotting Assay-fX was assayed using factor VII- and X-deficient plasma, crude Russell's viper venom (Burroughs Wellcome), and rabbit brain cephalin (Sigma) as previously described (5).

Analytical Velocity Sedimentation of fX and fX~8.143~191-Sedimen- tation analysis was performed using a Beckman model E analytical ultracentrifuge equipped with a photoelectric scanner as previously described (5). The sedimentation coefficients of fX and fXdes.143-191, 0.3 mg/ml, were determined by the methods of second moments and midpoint calculation (15) in 0.15 M NaCI, 20 mM Hepes, pH 7.4.

Electrophoresis-SDS-PAGE was performed using the buffer system of Laemmli (16). Samples containing 1% (w/v) SDS with or without 1-2% (v/v) P-mercaptoethanol to reduce disulfide bonds were heated for 2-5 min in a heating block maintained at 100 "C. Phos- phorylase b (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), and carbonic anhydrase (29 kDa) (Sigma) were used as stand- ards. Proteins were visualized by silver staining (17).

NH2-terminal Sequence Analysis of fx~~~.143.191-fXdes. l43.191 was subjected to SDS-PAGE under reducing conditions. The bands corresponding to the ffies.143.191 heavy and light chains visualized with Coomassie Blue were blotted onto polyvinylidene difluoride mem- branes and sequenced as described (18).

RESULTS

Isolation and Characterization of fXdes.143-191-The reaction of ARHy with native porcine fX results in the formation of a species that co-migrates with a-fXa during SDS-PAGE, pos- sesses no coagulant activity, and is activated by RVV-XCP to a product with coagulant activity that is equivalent to a- fXa (5). ARHy-modified fX was isolated as described under "Experimental Procedures." Its electrophoretic mobility during SDS-PAGE was indistinguishable from that of a-fXa (Fig. 1). When allowed to stand at room temperature for several hours at pH 7-7.5 in the absence of protease inhibitors, preparations of porcine a-fXa undergo proteolytic degradation to form a species that appears similar in electrophoretic mobility to a product previously designated as y-fXa (10, 19) (Fig. 1). However, in contrast to a-fXa, ARHy-modified fX underwent no further proteolytic degradation beyond the parent form.

NH2-terminal sequence analysis of the heavy chain and light chain of ARHy-modified fX was performed and could be uniquely aligned to the human fX sequence (Table I). The NH2 terminus of the light chain of ARHy-modified protein

Intrinsic Pathway Activation of Factor X and Factor Xdes.143-191 7823

A B

97.4-

66- - fX

45- - afXa fX HC

- yXa 29- afXa HC

fX.afX0 LC

1 2 3 1 2 3 ”

FIG. 1. SDS-PAGE analysis of fXdes-143-191. Samples (0.7 pg) were subjected to SDS-PAGE followed by silver staining, as described under “Experimental Procedures.” A , lane 1, native f X ; lane 2, a-fXa and y-fXa mixture; lane 3, fxdes.143.191. B, same as panel A (reduced). The markers correspond to apparent molecular masses in kDa. HC, heavy chain; LC, light chain.

TABLE I f X ~ e s . 1 4 ~ - l g l NH2-terminal sequence data

H, human sequence; . , scissile bond (RVV-XCP, intrinsic/extrinsic fXase).

fXdes.148-191 heavy chain LVR. IVGGRDC Homologous sequence (H) 192-LTR. IVGGQEC fXdes.143-191 light chain ANSFW Homologous sequence (H) 1-ANSFL

align’s with residues 1-5 of human fX. The heavy chain of ARHy-modified fX aligns with human sequence residues 192- 201. The first 3 residues align with the COOH-terminal 3 residues on the human fX activation peptide (residues 192- 194). The remaining residues span the scissile bond, which is cleaved in the formation of a-fXa (Arg’94-Ile’9s), and align with the first 7 residues of the human a-fXa heavy chain. Therefore, ARHy leaves a 3-residue tail on the NHz terminus of the fX heavy chain corresponding to residues P1-P3, which prevents the expression of catalytic activity. We designate ARHy-modified fX as fXdes.143-191.

Preparations of fXdes.143-191 possessed measurable activity toward the chromogenic substrate Spectrozyme Xa and av- eraged 0.08% of a-fXa over several preparations. This activity was not significantly different from that present in the starting preparation of fX, which presumably represents trace contamination with a-fXa. The low levels of fXa activity in these preparations were either negligible or, in circumstances where very high concentrations of fX or fXden.143-191 were used, were subtracted from the total activity. Subsequent reaction of fXdes.143-191 with RVV-XCP resulted in a product with coagulant and chromogenic substrate activity that was indistinguishable from a-fXa, consistent with earlier observations with crude activation mixtures containing fXdes.143-191 (5).

Analytical velocity sedimentation of fX and fXdes.143-191

yielded single sharp boundaries for both proteins. ~ ~ 0 , ~ values were 3.74 and 3.67 for fX and fXdes.143-191, respectively. These data indicate that fXdes-143-191 does not self-associate under the concentrations used for this study.

Kinetics of Activation of fX and fxdes.143-191 by Intrinsic fXase-We compared the steady-state kinetic constants for the activation of fX and fXdes.143-191 by intrinsic fXase consisting of fI Xap/fVIIIaIl,/PCPS. These measurements differ from previous determinations in that a stable preparation of fVIIIaIr, of known molar concentration was used. In the presence of fVIIIaIIa, the contributions to the initial velocity made by fIXa/3 free in solution or fIXaP bound to PCPs but not in complex with fVIIIaIr, are negligible (see Table 11). Concentrations of fVIIIaIl, were used that were limiting with respect to the concentrations of fIXaP and PCPs. Initial velocities increased linearly with the concentration of fVIIIaII, up to the concentrations of WIIIaIl, used for the determina-

tion of the kinetic constants K, and V,,, (not shown). The kinetic constants were determined by varying the fX or fXdes.143-191 concentrations and fitting the initial velocities to the Michaelis-Menten equation using nonlinear least squares analysis.

Fig. 2 shows velocity versus substrate and Eadie-Scatchard plots along with least squares fits for the activation of fX (panel A) and fXdes.143-191 (panel B ) by intrinsic fXase. Be- cause the activation of fXdes.143-191 was slower, the concentration of fVIIIalIa used to activate fXdes.143-191 was 17-fold higher than for fX. Preliminary experiments using fIXaP modified with a fluorescent dye to monitor the assembly of intrinsic fXase have indicated that under the conditions used for the activations of both fX and fXdes.143-191 by intrinsic fXase, approximately 50% of the total fVIIIalI. is bound in the complex and that the stoichiometry of the association of fVIIIaIl, with fIXa@ is L 4 Therefore, values of kc,, were calculated by dividing V,,, values by the concentration of fVIIIalr, and then multiplying by 2. Table I1 shows there is no significant difference in the K,,, between the two substrates; however, the kc,, for fX is approximately 100-fold higher than for fXdes.143-191. This suggests that residues in the fX activation peptide that are NHz-terminal to the P3 residue participate in the rate-limiting catalytic step in fX activation. However, as discussed below, because K, does not appear to be a function of the catalytic steps in the reaction (acylation and deacylation), this region of the activation peptide does not appear to be involved in Michaelis complex formation.

Activation of fX and fXdes.143-191 in the Absence off VIIICZ~~~- We also measured the kinetic constants for the activation of fX (Fig. 3A) and fXdes.143-191 (Fig. 3B) by fIXaP and PCPs in the absence of fVIIIall,. The fitted constants for the activation of fX and fXdes.143-191 by fIXap/PCPS are listed in Table 11. The calculation of kcat from V,,, assumes that all fIXaP is bound to PCPs under these conditions. A similar kc,, was determined using a 10-fold higher concentration of fIXaP and a 2-fold higher concentration of P C P s (Fig. 4), which argues against significant amounts of free fIXaP under the conditions used in Fig. 3. As for the completely assembled fXase complex, the K, values of the two substrates are essentially the same. The kat for fX is significantly greater than for fXdes.143-191, but the difference was only %fold. The implications of this are considered under “Discussion.”

The activation of fX and fXdes.143-191 by fIXaP in the absence of P C P s was also studied (Table 11). The K, for fXdes-143-191

was not determined due to lack of a preparation of fX~es.143..191 sufficiently concentrated (>lo0 PM) to make the determination. However, the catalytic efficiency ( k,.,/K,) was determined by linear regression of the plot of initial velocity uersus fXdes.143-191 concentration and was &fold less for fX (Table 11). A similar decrease in catalytic efficiency was observed in the presence of PCPs, implying a similar defect exists in the activation of fXdes-143-191 in the presence and absence of PCPs.

The kinetic constants for the activation of native fX by fIXap/fVIIIaII,/PCPS show -500-fold reduction in K, and -6000-fold increase in kcat, as compared with fI Xap, resulting in an overall increase of =3 X lo6. The results with fIXa@/ fVIIIalI,/PCPS are in good agreement with values previously determined in the bovine system using partially purified WIII and PCPs in which a K, of 63 nM and a kc,, of 8 s-* were reported5 (2). The overall increase in catalytic efficiency due

E. J. Duffy and P. Lollar, unpublished observations. kcat was calculated from the published value of 8 mol of fXa/mol

of ffXap/s under conditions in which the concentration of ffXaP was limiting.

7824 Intrinsic Pathway Activation of Factor X and Factor Xdes.143-191 TABLE I1

Kinetic constants for the activation of native fX and fXdes.143.191 Values are means & S.D.; ND, not determined.

Enzyme Substrate K , kat k d L M s"

IXaB/VIIIa/PCPS Native fX 6.0 f 1.1 X lo-' 16.6 f 0.4

IXaB/PCPS 8.0 f 0.8 X lo-@ 16.2 f 0.4 X lo-'

IXaP

fXdes-113-191 Native fX

Native X 3.3 f 0.8 X 10-5 3.0 f 0.1 X f%ea-143-191 ND ND Native X 2.4 f 0.5 X 2.3 f 0.4

2.2 f 0.32 X 10-7 7.0 f 0.3 X fXdes-143-191 1.7 f 0.20 X 10-7 2.0 f 0.1 X 10-~

RVV-XCP

"1 s-l

2.8 X 10' 2.0 x lo6 3.2 X 104 1.2 X lo4

91 16

9.6 X lo6 fXdes-143-1531 3.8 rt 0.5 X lo-' 0.18 f 0.003 4.7 x lo6

v ("u/mlJ

0 0 100 200 300 400 500 606

Factor X (nM)

.i C v ("u/na"I -

0.0 0 100 200 300 400 500 600

Factor '(do8 143-191) (nM)

FIG. 2. Activation of fX and fXdes.143-1Bl by intrinsic fXase. The initial velocities of the activation of fX ( A ) and fXdes.143-191 ( B ) were determined and analyzed by using conditions described under "Experimental Procedures." The concentrations of fIXa6 and PCPs were 5 nM and 20 pM, respectively. The fVIIIarr. concentration was 25 pM in A and 450 pM in B. The curves are drawn by using the fitted kinetic constants. Inset, Eadie-Scatchard transformation of the data.

to fVIIIa~I. and PCPs reported here is less than that found in the bovine system, since in the absence of fVIIIa,l, and PCPs, their K,,, was higher (299 PM) and kc, lower (4 X s-').

Formation of the Acyl-Enzyme Is Rate-limiting in the Acti- vation of fX and fXdes.143-191 by fZXap/PCPS-The low kcat values for the activation of fX and fXdes.143-191 by fIXaP/PCPS in the absence of fVIIIaII, and the generally poor reactivity of fI Xa@ toward small substrates and serine protease inhibitors (20, 21) implies a catalytic defect, the nature of which is poorly understood. Peptide bond hydrolysis catalyzed by serine proteases proceeds by an acyl-enzyme mechanism given by Scheme I, which follows

kl kz E + S + ES E-acyl 3 E + P2

k 3

Kr; X p 1

where E and S represent enzyme and substrate, and P1 and P2 represent products derived from the COOH-terminal and NH2-terminal sides of the scissile bonds, respectively. When fX or fXdes.143-191 is substrate, P1 corresponds to a-fXa; the activation peptide is attached in the acyl-enzyme complex and is released to form P2 during deacylation. Acylation is typically but not always (22) rate-limiting during the hydrol-

1000 p, ,

Factor X (nM)

L

0 200 400 600 800 1000 1200

Factor '(des 143-191) (nM)

FIG. 3. Activation of fX and fXdes-143-lsl by fIX@/PCPS. The initial velocities of the activation of fX ( A ) and fXdes.113.191 ( B ) were determined by using conditions described in "Experimental Proce- dures.'' The concentrations of fIXaP and PCPs were 2.5 nM and 20 p ~ , respectively.

ysis of peptide or amide bonds, whereas deacylation is generally rate-limiting during the hydrolysis of small ester substrates. When substrate depletion is negligible and formation of the Michaelis ( E S ) complex is in rapid equilibrium relative to the subsequent catalytic steps (which seems reasonable, given the low kcat values in the absence of fVIIIaIIa), the measured kinetic constants kat and K,,, are related to the constants in Scheme I by the following.

kcat = k z k 3 / ( k 2 + k 3 ) (1)

K , = K s k 3 / ( k 2 + k 3 ) (2)

where K, = k-Jkl is the dissociation constant for the formation of the Michaelis complex (23). When acylation is rate- limiting (kz << k3), K,,, is approximately equal to K, and is independent of the catalytic steps represented by kz and k3.

However, when acylation is not rate-limiting, then this sim- plifying assumption cannot be made. Thus, to interpret the difference in kinetic constants between fX and fXdes-143-191

and the effect of fVIIIaII,, it is desirable to determine whether acylation is rate-limiting in the absence of fVIIIaI~,. To do this, we measured fX and fXdes.143-191 activation as a function of time under conditions where an exponential burst of prod-

Intrinsic Pathway Activation of Factor X and Factor Xdes-143-191 7825

Time (s)

25 - 20 f

0

9 5 ””

””

0 50 100 150 200 250 SO0 Time (s)

FIG. 4. Rate-limiting step during the activation of fX and fX.+e..ll~-lsl by fIXa@/PCPS. A , the activation of fX (1 pM) by fIXaa (25 nM), PCPs (40 phf) was measured as described under “Experimental Procedures.” In a separate experiment, kc,, and K,,, were determined to be 0.005 s-l and 450 nM, respectively, at these concentrations of fIXaa and PCPs. B, the activation of f X d e a . 1 4 3 - 1 ~ ~ (1 p ~ ) by fIXa/3 (50 nM), PCPs (40 p ~ ) was measured as described under “Experimental Procedures.” In a separate experiment, kcat and K,,, were determined to be 0.0014 s-l and 450 nM, respectively, at these concentrations of fIXaP and PCPs. Curves were constructed by using these values of kat and K,,, and Equation 3, assuming kcat = k, << k3 (solid line), kcat = k3 << kz (upper dashes), and kz = ka = k,.J 2 (lower dashes).

uct formation would be expected if deacylation were rate- limiting by using the following relationship derived from Scheme I (24).

P1 = A . t + B. (1 - e-bt) (3)

where A, B, and b are given by

A = kcat.Eo.So/(So + K m ) (4)

b = k3 + kz/[l + (Ks/So)l (6)

and where Eo and So are the nominal concentrations of enzyme and substrate. When kz >> k3, the curve resulting from Equation 3 is initially nonlinear due to the exponential term but becomes linear as t approaches infinity. When kz << k3, the amplitude of the exponential, B, becomes negligible and linear initial velocities are observed. Additionally, to observe the exponential phase readily, Eo must be sufficiently large and So > K,,,.

Fig. 4 shows plots of the activation of fX and fXdes.143-191 by fIXap/PCPS versus time under conditions in which an exponential phase would be observed if deacylation were rate- limiting. The fIXaB concentration was increased 10-fold relative to previous conditions to increase the value of B. The kinetic constants under these conditions were determined by measuring the velocity of fX activation as a function of fX concentration and were similar to those reported in Table I1 (see legend). These constants were used to calculate curves using Equations 1 and 3-6 under three conditions: kz << k3 (kcat = k2), kz >> k3 (k,,, = k3) , and kZ = k3 = kJ2. To look

for the exponential phase, samples were taken at shorter times than for the determination of kinetic constants. Fig. 4 shows that for both fX and fX&.143-191, there is no evidence for an exponential burst (top curue) and that the data lie within experimental error of the curve calculated assuming 122 << k3. Additionally, the data do not fit the curve (bottom) constructed assuming kz = k,. Thus, we conclude that acylation is rate-limiting during the activations of fX and =des-143-191 by fIXap/PCPS.

Activation of fX and fX&s.143-191 by RVV-XCP-The kinetic constants for the activation of fX and fXdes.143-191 by RVV- XCP were also determined (Fig. 5 and Table 11). The values for the activation of native fX agree well with a K,,, of 240 nM and a kcat of 1.9 s” published for bovine fX (25), but not the K,,, of 10 nM published for human fX (26). In contrast to experiments with fIXap, the K,,, for the activation of fxdes.143- 191 is 6-fold lower than for native fX. This is offset by a 12- fold lower kcat for f&es.143-191. The overall catalytic efficiency is only slightly greater for native fX. This result is suggestive of nonproductive binding of f&es.143-191 to RVV-XCP. Overall, the difference between intrinsic fXase and RVV-XCP indicates a different catalytic mechanism for the activation of fX.

DISCUSSION

In this study, the kinetics of activation of native fX and a novel derivative of fX that was prepared enzymatically via the action of ARHy from the Malayan pit viper were compared. NH2-terminal sequence analysis revealed that ARHy modified fX retained residues Pl-P3 of the activation peptide (Table I), as previously found for ARHy-modified fIX (5). ARHy-modified fX, designated f)14Les-1&191, contains no detectable enzymatic activity but can be activated by intrinsic fXase and RVV-XCP. Thus, fXdes.143-191 is an interesting substrate with which to evaluate the contribution of residues

6 1 . I

Factor X (nM)

2.0 .- c 1” ’ 1

0 100 200 300 400 500 600

Factor ‘(des 143-191) (nM)

FIG. 5. Activation of fX and tXdea.ll+-lsl by RVV-XCP. The initial velocities of activation of fX (A) and fXdes.143.191 ( B ) were determined by using conditions described under “Experimental Pro- cedures.” The concentrations of RVV-XCP were 50 p~ and 1 nM for fX and f)(dea.143-191, respectively.

7826 Intrinsic Pathway Activation of Factor X and Factor Xdes.143-191

P4-P52 of the fX activation peptide toward substrate specificity of fX.

Intrinsic fXase kinetics were done by using highly purified porcine fIXap, fVIIIaII,, fX, and fXdes.143-191. A preparation of stable porcine fVIIIaII, has recently become available (8), and some of the properties of pure fVIIIaIIa in promoting fX activation are reported here for the first time. As has been observed previously using bovine proteins and partially purified fVIII, we found that fX activation by fIXap obeys Mi- chaelis-Menten kinetics and that the catalytic efficiency (kcat/ K",) is enhanced by phospholipid mainly through a large decrease in K, and is enhanced by fVIIIaII, mainly through a large increase in kcat (2). This pattern has also been observed in the homologous prothrombinase reaction (27). However, for reasons that are not clear, these results are not consistent with another study of bovine intrinsic fXase that found that phospholipid increased the kcat but did not affect the K, for the activation of fX by fIXaP (28).

The kcat of the activation of fXdes.143-191 by fIXap/fVIIIaII./ PCPs was 100-fold less than for fX, but the K,,, was not significantly different. In the absence of fVIIIaII,, the activation of fXdes-143-191 also was associated with a decrease in kcat and no change in K,, although the difference in kc,, was much lower than observed in the presence of fVIIIaII, (Table 11).

These results can be interpreted in terms of the mechanism for reactions catalyzed by serine proteases (Scheme I). The experiment described by Fig. 4 strongly indicates that formation of the acyl-enzyme during fX and fXdes.143-191 activation is rate-limiting in the absence of fVIIIaIIa.

These conclusions are also consistent with results obtained when fVIIIaII, is added. If deacylation were rate-limiting in the absence of fVIIIaII,, then according to Equation 2, the effect of fVIIIaIIa would be to accelerate k3, which would result in a corresponding increase in the K,, which is not observed. Therefore, it is evident that fVIIIaII, increases the rate of activation of fX and fXdes.143-191, at least in part by increasing the acylation rate, although the results do exclude contributions by fVIIIaII, at other steps of the catalytic mechanism.

Scheme I does not include the multiple equilibria that are involved due to the presence of PCPs and fVIIIarI.. These equilibria define the process that leads to formation of the fIXa(3-fVIIIa~I,-fX-PCPS complex, which is the only complex that contributes significantly to the overall formation of product. In the absence of fVIIIaII,, because kcat is small, it seems reasonable to assume that the catalytic step is slow, relative to the dissociation of fX from the Michaelis complex. Under these conditions, K, reflects the equilibria leading to the formation of the Michaelis complex but is not influenced by the catalytic steps of acylation and deacylation. Furthermore, fVIIIaII, increases the kcat for the activation of fX over 1000- fold but only reduces the K, 3.7-fold. The relative insensitiv- ity of the K, implies that the rate constants involving the catalytic steps do not influence the K,. In summary, without proposing a model for assembly of intrinsic fXase, it appears appropriate to compare the activation of fX and =des-143-191

by assuming that K, values reflect dissociation constants for the formation of the quaternary fIXap/fVIIIaII,/PCPS/fX Michaelis complex, whereas kcat values reflect the chemical steps of acylation and deacylation. Thus, since the K, for the activation of fXdes.143-191 and fX by either fIXa@/PCPS/ fVIIIaII, or fIXaP/PCPS are not different, it follows from making this rapid equilibrium assumption that activation peptide residues P4-PS2 are not involved in binding events that govern the concentration of Michaelis complexes.

However, it is clear that residues within P4-P52 are critical in the acylation step and thus are required to stabilize the

tetrahedral intermediate in the transition state during the production of the acyl-enzyme from the Michaelis complex. The 100-fold increase in kcat for native fX, as compared with =des-143-191 using fIXap/PCPS/fVIIIaII, corresponds to a decrease in free energy of 2.9 kcal/mol to reach the transition state. Since the catalytic step involves cleavage of the scissile bond between residues P1-PI,, it is puzzling at first glance that residues as remote as P4 are involved in the catalytic step. However, our results are consistent with those obtained with elastase in which the addition of P4 residues to several peptide substrates resulted in a 100-fold increase in kcat with no change in K, (29, 30). Furthermore, by using transition state analogs at the P1 site in these studies, it was inferred that the P4 residue interacted with the enzyme but that this binding energy was not reflected in the dissociation constant for the formation of the Michaelis complex but rather in destabilizing the substrate toward the transition state. The hypothesis that substrate residues remote from the scissile bond can alter the structure of the active site of the enzyme has subsequently been supported in the case of elastase by crystallographic data (31). This effect, termed induced desta- bilization by Jencks (32), is one way available binding energy in an enzyme-substrate interaction can be used to drive or pay for the catalytic step and appears to explain our results.

The addition of fVIIIaII, enhances the kcat of native fX activation 1200-fold but only 40-fold of fXdes.143-191. Thus, fVIIIaII, appears to influence the interaction of the fX activation peptide with fIXap. However, it is not possible to distinguish whether this is due to a fVIIIaIIa-fX interaction that enhances the binding of the activation peptide to fIXaP or due to a fVIIIaII,-fIXap interaction that results in a struc- tural change in fIXaP that promotes its interaction with the activation peptide.

Although the results of this study clearly implicate the role of fX activation peptide residues NH2-terminal to P3 in the catalytic step, it is not possible to determine how much of the activation peptide can be eliminated before a decrease in kcat is observed. In fact, it is possible that only the P4 residue is required to eliminate an unfavorable interaction between the positively charged a-ammonium group on P3 and the enzyme. Further evaluation of the structure of the activation peptide of fX in governing the kinetics of fXa production probably will require the use of site-directed mutants of fx. However, the results of the present study would seem to provide an important starting point for the evaluation of mutants, since the mutant corresponding to fXdes.143-191 should give the results obtained in this study barring folding artifacts or functional abnormalities due to alterations in post-translational modi- fication resulting from the use of a heterologous expression system.

Acknowledgment-We thank Dr. Sriram Krishnaswamy for stim- ulating discussions and reviewing the manuscript.

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