Human Blood Coagulation Factor IX PURIFICATION, PROPERTIES, AND MECHANISM OF ACTIVATION BY ACTIVATED FACTOR XI*

(Received for publication, January 19, 1978)

Bjarne Bsterud, Bonno N. Bourna,+ and John H. Griffins

From the ZIemzrtment of Medicine. Ilniversitv of Californsn, and the Department of Zmmunopathology, SWipps Clinic and Research Fo&dation, ~CI Jolla, California i20.37 ’

Human blood coagulation Factor IX was purified from plasma in 27% yield using barium sulfate absorp- tion, DEAE-cellulose chromatography, preparative polyacrylamide gel electrophoresis and heparin-aga- rose affinity chromatography. The specific clotting ac- tivity of purified Factor IX was 207 units/mg, which implies that its concentration in plasma is 5 pg/ml. The purified Factor IX was a single polypeptide with an apparent molecular weight of 55,000 in the presence of sodium dodecyl sulfate on polyacrylamide gels.

Purified human activated Factor XI activated Factor IX in a Ca’+-dependent reaction by limited proteolysis. Kinetic studies of this activation of Factor IX using sodium dodecyl sulfate-polyacrylamide gel electropho- retie analysis in the presence and absence of reducing agents demonstrated a first relatively slow cleavage followed by a second rapid cleavage of the molecule. The appearance of activated Factor IX activity was directly correlated with the extent of cleavage of Factor IX. Activated Factor IX contained a heavy chain of M,. = 27,000 and a light chain of M, = 17,000 which are held together by disultide bonds. Polypeptide fragments of ““I-Factor IX of the same molecular weights were ob- served upon kaolin activation and recalcification of human plasma containing ‘“‘I-Factor IX.

The amino acid composition of human Factor IX and of the heavy and light chains of activated human Factor IX were found to be very similar to the amino acid compositions of the bovine proteins. Eight to ten y- carboxyglutamic acid residues were found in native human Factor IX. These observations emphasize the similar nature of human and bovine Factor IX.

Blood coagulation Factor IX plays an important role in the intrinsic pathway of coagulation and is essential for normal hemostasis (2, 3). Patients with a deficiency of Factor IX (Haemophilia B) have severe bleeding problems in contrast to the mild bleeding tendency of patients with a Factor XI deficiency or to the absence of bleeding in the Factor XII-

* This work was supported in part by Research Grants NHLB- 21544, NIAID-07007, NHLB-16411, and NHLB-18576 from the Na- tional Institutes of Health, a grant from the Council for Tobacco Research, and Contract NR 207-027 from the Office of Naval Re- search. A preliminary report of this work has been presented (1). This is Publication 1399 from the Department of Immunopathology, Scripps Clinic and Research Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact.

$ Supported by the Netherlands Organization for the Advancement of Pure Research (Z.W.O.). Permanent address, Department of Hae- matology, University Hospital, Utrecht, The Netherlands.

8 Recipient of National Institutes of Health Research Career De- velopment Award NHLB-00192.

deficient patients (3, 4). In the in vitro activation of the intrinsic blood coagulation pathway, exposure of plasma to an activating surface, such as kaolin or glass, gives optimal pro- teolytic activation of Factor XII (Hageman factor) in the presence of high molecular weight kininogen and prekallikrein (5-8). Activated Factor XII, with high molecular weight kin- inogen as cofactor, activates Factor XI (5-8). The activated Factor XI (Factor XI,)’ is the intrinsic activator of Factor IX in a Ca’+-dependent reaction (9,lO). The active form of Factor IX, together with activated Factor VIII, phospholipid, and calcium ions, functions as the intrinsic activator of Factor X (11-15).

The activation of purified bovine Factor IX by bovine Factor XI, (10) converts the single chain Factor IX into a two- chain molecule with a heavy chain of M, = 38,500 and a light chain of J4, = 17,700. In the second step which is slower than the first step, an J4, = 11,000 polypeptide is split off from the heavy chain, giving rise to Factor IX, which contains two polypeptide chains of M, = 27,500 and 17,700 held together by disulfide bonds. The study reported herein was undertaken to study the properties of human Factor IX and to examine the mechanism of activation of purified human Factor IX by purified human Factor XI,. Evidence is presented here that the activation of human Factor IX by human Factor XI, is very similar, although not identical, to the bovine system. In addition, observations of the cleavage of ““I-Factor IX in clotting plasma suggest that human Factor IX, in plasma has the same polypeptide structure as that observed for the puri- fied molecule.

MATERIALS AND METHODS

All chemicals used were of the best grade available from commer- cial sources. DEAE-cellulose, DE11 Whatman, was purchased from Whatman Ltd., Maidstone, Kent, England; agarose, Bio-Gel A-15m, was obtained from Bio-Rad Laboratories. Heparin used for coupling to agarose was purchased from Sigma.

Protein concentrations were determined by the Lowry method (16) using bovine serum albumin (Sigma) as a reference protein.

Human Factor XI and nrekallikrein were nuritied as described elsewhere (17, 18). All purified proteins were -judged to be greater than 95% homogeneous on SDS-polyacrylamide gel electrophoresis.

Factor IX Clotting Assay-The Factor IX clotting activity was measured by a kaolin-activated one-stage partial thromboplastin time assay. One hundred microliters of Factor IX-deficient plasma, 100 ~1 of BaS04-adsorbed ox plasma, and 100 ~1 of kaolin (10 mg/ml) and rabbit brain cephalin (Sigma) were incubated together for 8 min in a glass tube (19) at 37°C. Then a 50-~1 test sample, which had been diluted appropriately, and 100 ~1 of 50 mu CaC12 were added in rapid succession and the clotting time was measured at 37°C. The test sample was diluted lo-fold or more in Tris-buffered saline (0.15 mM NaCl, 0.01 M T&Cl, pH 7.4). The observed clotting time was con-

’ The abbreviations used are: Factor XI., and Factor IX,, the active procoagulant forms of Factor XI and Factor IX, respectively; SDS, sodium dodecyl sulfate.

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Activation of Blood Coagulation Factor IX by Factor Xl, 5947

verted to clotting units by comparison with the clotting activity of serial dilutions of a standard pool of normal human plasma. A linear relationship on a log-log plot existed between the clotting time and the concentration of Factor IX from 0.1 unit/ml to 0.006 unit/ml. One unit of Factor IX clotting activity is defined as the amount present in 1 ml of a standard pool of normal titrated human plasma.

Activated Clotting Factor Assay-Factor IX, activity was mea- sured by incubating 100 ~1 of Factor IX-deficient plasma, 50 ~1 of cephalin, 10 ~1 of thrombin (0.02 unit/ml) for 3 min at 37°C in a plastic tube (10 X 75 mm; Kimble) to allow Factor VIII to be modified by thrombin. Then, 50 pl of test sample and 100 ~1 of 35 rnM CaC12 were added in rapid succession and the clotting time was measured at 37°C. Factor XI, clotting assays were performed as previously de- scribed (17). Kallikrein activity was estimated both in tripeptide amidolytic assays and in coagulation assays (18). The immunologic quantitation of Factor IX using immunoelectrophoresis has recently been described (20).

Purification of Factor IX-A modification of an earlier published purification method was used. The first three steps in this procedure which were identical to the ones previously described in detail (21) involved: 1) BaSO, adsorption of normal plasma anticoagulated with EDTA, benzamidine, and soy bean trypsin inhibitor; 2) batch chro- matography on DE11 Whatman cellulose of the BaS04 eluate; 3) preparative polyacrylamide gel electrophoresis of the concentrated DEAE-cellulose eluate. The following step was introduced. The frac- tions from the preparative polyacrylamide gel electrophoresis were pooled and applied without dialysis to a heparin-agarose (19) column (2 x 10 cm). Heparin (Sigma) was coupled to agarose that had been activated by cyanogen bromide (22). Prior to the application of the sample, the heparin-agarose column was equilibrated with 25 to 40 ml of 0.05 Tris-HCI, pH 7.5, containing 0.15 M NaCl. Factor IX was bound to the column under these conditions and was eluted using the same buffer containing 0.2 M NaCl. The Factor IX from the heparin- agarose column was concentrated by step elution from a small column of DEAE-cellulose. For example, the pooled fractions (25 ml) from the heparin-agarose column were diluted 1:l with distilled water and applied to a DE11 Whatman column (2 x 0.5 cm). Factor IX was eluted in 1 to 2 ml of 0.05 M Tris-Cl, pH 7.5, containing 0.3 M NaCl at a flow rate of 5 ml/h.

In all the purification steps, except the heparin-agarose affinity chromatography, benzamidine (Aldrich) in a final concentration of 10 mM and soy bean trypsin inhibitor (Sigma Chemicals) in a final concentration of 20 pg/ml were present in all buffers to prevent activation of Factor IX during the isolation.

Radiolabeling of Factor IX-Factor IX was labeled with lLiI using the chloramine-T method (23). Radioactivity was measured with a Packard 5320 or Nuclear Chicago y-spectrometer. Radiolabeled Fac- tor IX was separated from free “‘I using a small DEAE-cellulose column as described above. The ‘““I-Factor IX was diluted three times with distilled water prior to application on the column. The column was washed with 150 to 200 ml of 0.05 M Tris-Cl buffer, pH 7.5, containing 0.07 M NaCl. The radiolabeled Factor IX was eluted with 1 to 2 ml 0.05 M Tris-Cl buffer, pH 7.5, containing 0.3 M NaCl. The ““I-Factor IX retained its procoagulant activity after it had been subjected to the radiolabeling procedure and it contained from 1 to 10 pCi/pg.

Polyacrylumide Gel Electrophoresis-Electrophoresis was carried in the presence of SDS on 10%~ gels (6 mm x 8 cm) according to Weber et al. (24) and according to Laemmli (25). The gels were stained for protein with Coomassie blue.

Amino Acid Analysis-Fifteen microgram samples in duplicate were hydrolyzed in uucuo with 6 M HCl at 105’C for 24, 48, and 72h and were analyzed on a Beckman 121M amino acid analyzer (26, 27). Half-cystine was determined as cysteic acid following performic acid oxidation (28). Tryptophan was analyzed in separate alkaline hydrol- ysates (29). y-Carboxyglutamic acid was measured in alkaline hydrol- ysates using a special program for the Beckman 121M amino acid analyzer (30).

Kinetics of Activation und Cleavage of Factor IX-Mixtures of Factor IX and Factor XI, in the presence of Ca2+ ions were incubated for varying times at 37°C. The reaction mixture contained 50 pl of Factor IX (6 pg), 2 d of Factor XI, (0.12 pg) and 5 ~1 of 0.05 M CaCl in a 1.4.ml snap-cap polypropylene centrifuge tube. At various times, a 2-pl aliquot was withdrawn and immediately diluted into 2 ml of Tris-buffered saline containing 1 mg/ml of bovine serum albumin in a plastic tube (12 X 75 mm). Fifty microliters of the diluted reaction mixture was assayed for Factor IX, activity. At the same time 15 ~1 of 10% SDS, 10% P-mercaptoethanol, 0.1 M EDTA was added to the

remaining sample, and the tube containing this mixture was heated in a small boiling water bath for 5 min. Then this sample was subjected to SDS-polyacrylamide gel electrophoresis.

The extent of cleavage of Factor IX was determined by scanning the stained bands on reduced SDS-gels of samples obtained for each time point using a Gilford gel scanner attached to a Gilford 2COO spectrophotometer. In the reported experiments, the observed extent of cleavage reached maximum at 20 min and the stained patterns remained unchanged at least 40 min longer. The extent of cleavage of Factor IX for various incubation times was determined as the per- centage of the maximum observed area of the M, = 27,000 heavy chain. Alternatively, similar results were obtained when the disap- pearance of the M, = 55,000 zymogen stained band was measured.

The extent of activation of Factor IX was quantitated using a Factor IX, standard curve which was constructed in the following manner. Since activation of Factor IX reached a plateau at 20 min and remained constant for at least 40 min thereafter, a reaction mixture which was incubated for 30 min was used as 1004 in the definition of a standard curve by assaying varying dilutions (1:2, 1:4, 1:8,1:16). The standard curve made in this manner was entirely linear on a log-log plot. The clotting times of the samples were expressed in percentages using this standard curve.

Cleavage of ‘““Z-Factor IX in Clotting P&mu--The cleavage of ‘““I-Factor IX in plasma was studied in order to assess whether the same fragments of Factor IX as seen in purified systems are produced in whole clotting plasma. Trace amounts of ““I-Factor IX (5 ~1) were added to 10 ~1 of normal human plasma. Then 5 pl of kaolin (20 mg/ml) was added followed by 5 ~1 of 50 mM CaC12. Clotting was allowed to occur at 37’C and, after 20 min at 37”C, 20 pl of 0.3 M

EDTA was added followed by 50 ~1 of 3% SDS, 5% mercaptoethanol. The reaction mixture was placed in a boiling water bath for 5 min and then subjected to polyacrylamide gel electrophoresis.

In studies using ““I-Factor IX both in purified systems and in plasma, it was observed that the light chain of Factor IX, of M, = 17,060 formed large aggregates in the presence of CaCIL and these aggregates did not enter, or remained at the top of the SDS-polyacryl- amide gels. Hence, high concentrations of EDTA (0.1 M) were in- cluded in the SDS solution added to the samples prior to polyacryl- amide gel electrophoresis to overcome this calcium-dependent aggre- gation

RESULTS

Preparation of Human Factor XI-The first two steps in the purification of Factor IX, BaS04 adsorption and elution and DEAE-cellulose batch chromatography, are used to re- move the bulk of plasma proteins (Table I). Then the prepar- ative polyacrylamide gel electrophoresis separates Factor IX completely from prothrombin, Factor VII, and several other plasma proteins. At this stage Factor IX is still contaminated with albumin and Factor X. The heparin-agarose affinity column separated Factor IX from these contaminants. Both Factor X and albumin do not bind to the heparin-agarose under the conditions used and are eluted with the 0.05 M Tris- HCl buffer, pH 7.5, containing 0.15 M NaCl before Factor IX is eluted with the same Tris buffer containing 0.2 M NaCl. The final preparation of Factor IX contained no measurable acti- vated Factor IX (less than 0.01 units/ml) as measured in a Factor IX, clotting assay. Based on the observed spec3ic activity of 207 clotting units/mg, the concentration of Factor IX in plasma is 5 pg/ml. Immunization of rabbits with purified Factor IX gave a rnonospecific antiserum.’

Polyacrylamide Gel Electrophoresis of Factor IX-A single protein band was observed when preparations of Factor IX were analyzed on SDS-polyacrylamide gels in the presence or absence of reducing agents using two different buffer systems (Fig. 1). Determination of the apparent molecular weight of Factor IX from standard reference curves for each buffer system gave 55,000 in the phosphate buffer system (24) and 70,000 in the Tris buffer system (25). To illustrate this anom- aly, the apparent relative mobility of Factor IX in the two buffer systems is compared in Fig. 1 with the mobjlity of bovine serum albumin and of the heavy and light chains of

’ B. gsterud, unpublished observation.

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5948 Activation of Blood Coagulation Factor IX by Factor XL

TABLE I

Purification of human Factor IX

Total Total Specific

Volume units

protein clotting clotting Recovery

activity activity

Plasma BaS04 DEAE-cellulose Polyacrylamide

gel electropho- resis

Heparin-agarose

umts/mg protem 70

600 43.2 615 0.0142

220 0.19 468 2.46 76 410 0.036 393 10.1 59

30 0.0034 221 65.0 36

24 0.0008 166 207 27

FIG. 1. Human coagulation Factor IX and y-globulin on 10% SDS- polyacrylamide gels in both a Tris buffer system (Gels A and B) and a phosphate buffer system (Gels C and D). The gels were run in presence of reducing agent. The gels with Factor IX (Gels A and C) contain 4 pg of protein each, and the gels with y-globulin (Gels B and D) contain 5 pg of protein each.

IgG (Fig. 1). A molecular weight value of 57,000 for human Factor IX has recently been obtained using sedimentation equilibrium techniques (31), suggesting that the apparent molecular weight of 70,000 is an artifact of the Tris-buffer system.

Amino Acid Composition of Factor IX and Factor IX,-The amino acid compositions of Factor IX and of the light and heavy chains of Factor IX, are shown in Table II together with the amino acid compositions of bovine Factor IX and bovine Factor IX, as reported by Fujikawa et al. (10). The marked similarity in the amino compositions of human and bovine Factor IX and Factor IX, are apparent (Table II).

y-Carboxyglutamic Acid Content of Human Factor IX-y- Carboxyglutamic residues were determined in alkaline hy- drolyzates of Factor IX. Eight to 10 y-carboxyglutamic acid

TABLE II

Comparison of amino acid compositions of human and bovine Factor IX and of heavy and light chains of Factor IX,

Data for bovine Factor IX and Factor IX, are taken from Fujikawa et al. (10). Amino acid composition data were rounded off to the nearest integer and, for the sake of comparison only, it was assumed that human Factor IX has approximately 370 residues and that the heavy and light chains of human Factor IX, contain approximately 215 and 120 residues, respectively. These values may vary, depending on a more precise evaluation of molecular weight and carbohydrate content (31). The values for human Factor IX, were obtained from amino acid analyses of heavy and light chains which had been separated from each other on SDS-polyacrylamide gels. Stained bands of each protein in polyacrylamide gel sections were subjected to hydrolysis as described elsewhere (32).

Factor IX

Human Bovine

23 28 8 8

13 17

42 36 24 20 28 29 51 47

10 13 37 30 19 19

13 17

27 25 2.4 2.6

17 19 18 19

10 17 16 15 12 11

Factor IX,

Amino acid Heavy chain Light chain (M, = 27,ooO) (MT = 17,lm)

Human Bovine Human Bovine

LYS 14 18 HIS 9 8 ARG 7 9

ASX 22 16 THR 15 13 SER 11 15 GLX 23 22

PRO 7 8 6 2

GLY 27 23 15 10 ALA 14 14 5 5

‘4CYS VAL MET

20 17 2 2

7 7 1 1

ILE 13 16 2 3 LEU 13 14 6 6

TYR PHE TRP

7 12 5 2

8 7 6 6

8 13 0.3 1

7 7

15 17 7 7

11 9 22 23

residues/molecule of Factor IX were found. Mechanism of Activation of Factor IX by Activated Factor

XI-The rate of activation and of limited proteolytic cleavage of Factor IX by purified Factor XI, was studied. In the presence of calcium ions Factor IX was incubated with Factor XI, for varying times at 37°C. At varying incubation times, aliquots of the mixture were analyzed for the extent of cleav- age of Factor IX and for Factor IX, clotting activity. During the incubation, Factor IX was cleaved at two sites as shown on SDS-polyacrylamide gels. The M, = 55,000 polypeptide chain of Factor IX disappears with concordant appearance of a M, = 44,000 polypeptide on SDS-gels in the absence of reducing agents (Fig. 2). In the presence of reducing agents, two new protein bands appear, corresponding to apparent molecular weights of 27,000 and 17,060. Thus, Factor IX, at M, = 44,060 consists of two chains held together by disulfide bonds. As seen in Fig. 3, the extent of cleavage of Factor IX was directly correlated with the appearance of procoagulant Factor IX, activity. The maximum observed extent of cleavage of Factor IX varied from 60 to 95% in different experiments using six different preparations of Factor IX and three differ- ent preparations of Factor XI,.

Cleavage of ‘251-Factor IX in Plasma--The molecular weight pattern of ‘251-Factor IX in plasma subjected to contact activation was studied in order to see whether contact acti- vation which generates Factor XI, and which produces Factor IX, activity involves limited proteolytic cleavage of Factor IX as described above. Normal human plasma containing ‘251-

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Activation of Blood Coagulation Factor IX by Factor XL 5949

Factor IX was incubated with kaolin in the presence of Ca2+ and allowed to clot. Then the reaction mixture was analyzed on SDS-gels in the presence of reducing agent. The data seen in Fig. 4 show that the contact activation of plasma results in the cleavage of ‘251-Factor IX in plasma to yield similar M; = 27,000 and 17,000 polypeptide chains as seen in Fig. 2 for the activation of Factor IX by purified Factor XI,. In Fig. 4C, approximately 60% of the ‘251-Factor IX molecules were cleaved at 20 min after clot formation. At the time of clot formation under these conditions, less than 10% of the 1251- Factor IX was cleaved. Cleavage of ‘251-Factor IX in serum continued throughout the 60 min following clot formation and finally resulted in cleavage of greater than 90% of the Factor IX molecules into fragments similar to those seen in Fig. 4C.

Effect of Kdikrein on Factor IX-Kallikrein has been reported to activate Factor IX in the absence of calcium ions (33). Therefore, a quantitative comparison of the activation of Factor IX by kallikrein and Factor XI, was made. The effect of plasma kallikrein on Factor IX was studied in one series of experiments by incubating 10 ~1 of purified kallikrein (135 pg/ml) with 50 ~1 of Factor IX (35 pg/ml) in a plastic tube at 37’C for 90 min in the presence or absence of 5 mM CaCl2. At various times, 2-~1 aliquots were withdrawn from the reaction mixture, diluted 1:lOO with Tris-buffered saline containing 1 mg/ml of bovine serum albumin, and assayed in a Factor IX, clotting test. During the 90 min a linear activation of Factor IX occurred in the absence or presence of CaC12, reaching a maximum of 5.4% of the available Factor IX. For comparison, 1 ~1 of purified Factor XI, (32 pi/ml) was incubated with 50 ~1 of Factor IX (35 pg/ml) with 5 mu CaC12. Aliquots (2 ~1) of this reaction mixture were withdrawn at various times and diluted 1 to 1000 with Tris-buffered saline containing 1 mg/ml of bovine serum albumin before being assayed for Factor IX,.

HUMAN FACTOR IX HUMAN FACTOR IX,

(REDUCED] (NON-REDUCED] (REDUCED]

55,000

, * .A 17,000 I

I 1 i .1 I ! !

1 I I

i 1 -A L i ! i

FIG. 2. Human Factor IX and Factor IX, on 10% SDS-polyacryl- amide gels.

TIME jmbnj

FIG. 3. Correlation of the kinetics of activation of Factor IX with its cleavage by activated Factor XI,. The appearance of Factor IX, activity is correlated with the cleavage of Factor IX. For details, see “Materials and Methods.”

55.clclrl MW

x

10 20 30 40 50 60 70 80

SDS GEL SLICE NUMBER

FIG. 4. Activation of ““I-Factor IX. SDS-polyacrylamide gel elec- trophoresis of (A) ““I-Factor IX, (B) ‘““I-Factor IX activated with partial purified Factor XI,, and (C) ‘251-Factor IX in normal plasma activated with kaolin and calcium. For details, see “Materials and Methods.”

In this reaction mixture, activation of Factor IX occurred rapidly. For example, the initial rate of activation was 20% of the available Factor IX/min. Thus, based on a comparison of the initial rates of activation of Factor IX, it was calculated that Factor XI, is approximately 20,000 times more active than kallikrein in activating Factor IX under these conditions. Noteworthy is the fact that the action of kallikrein was independent of calcium, whereas the activation of Factor IX by purified Factor XI, was calcium-dependent,

DISCUSSION

Human blood coagulation Factor IX plays an important role in the intrinsic pathway of blood coagulation. The intrin- sic pathway is initiated in vitro after exposure of plasma to an activating surface, such as kaolin or glass, which results in the activation of Factor XII (Hageman Factor) in the presence of high molecular weight kininogen and prekallikrein (5-8). Sur- face-bound activated Factor XII can then activate Factor XI that is complexed with high molecular weight kininogen on the surface (6). This activation of Factor XI involves limited proteolysis (17). Factor IX is activated in vitro by the acti- vated form of Factor XI in the presence of calcium ions. Alternatively, Factor IX can be activated by tissue Factor and Factor VII (34). Factor IX, binds to phospholipid surfaces in the presence of calcium ions and together with Factor VIII it

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5950 Activation of Blood Coagulation Factor IX by Factor XIa

activates Factor X (11-15). From there a sequence of reactions involving Factor X,, Factor V, and prothrombin finally leads to fibrin formation (35). This report is concerned with the properties of human Factor IX and with the mechanism of activation of purified human Factor IX by purified human Factor XI,.

Factor IX can be purified to greater than 95% homogeneity. The isolated Factor IX is composed of a single polypeptide chain and has an apparent molecular weight of 55,000 as determined by SDS-polyacrylamide gel electrophoresis. Pre- viously reported molecular weight values based on SDS-gel analyses have ranged from 57,000 to 72,000 (21, 31, 36-39). The minimum molecular weight of human Factor IX deter- mined using sedimentation equilibrium techniques was 57,000 (31). Therefore, the value of M, = 55,000 seen in the phos- phate-buffered SDS-gel system is used in our analyses and it is concluded that the mobility of Factor IX in the Tris- buffered SDS-gel system is very anomalous. The observed specific clotting activity of the purified Factor IX of 207 clotting units/mg of protein suggests that the concentration of Factor IX in normal human plasma is 5 pg/ml, a value similar to that estimated using immunologic techniques (20, 38). The amino acid composition of human Factor IX is in agreement with those reported by others for human and bovine Factor IX (10, 36, 39). In addition, human Factor IX, as reported for the vitamin K-dependent coagulation factors (40), contains 8 to 10 y-carboxyglutamic acid residues (41).

The mechanism of activation of Factor IX by Factor XI, was studied in the presence of Ca’+ ions. Based on SDS- polyacrylamide gel analysis, two proteolytic cleavages were shown to take place. On SDS-gels, in the absence of reducing agents, the M, = 55,000 single peptide chain of Factor IX disappeared with concordant appearance of a M, = 44,000 polypeptide when Factor IX was activated by Factor XI,. In the presence of reducing agents, the M, = 44,000 polypeptide was shown to consist of two polypeptides with molecular weights of 27,000 and 17,000. The extent of cleavage of Factor IX was directly correlated with the appearance of procoagu- lant Factor IX, activity. Thus, as depicted in Fig. 5, the activation of Factor IX by Factor XI;, involves two cleavages. One cleavage changes the single chain into a two-chain mol- ecule held together by disulfide bonds and the second cleavage results in the splitting off of an M, = 11,000 polypeptide. The resultant Factor IX,, consists of a heavy chain of M, = 27,000 and a light chain of M, = 17,000 held together by disulfide bonds. Rosenberg et al. (37) reported similar molecular weight values for the polypeptide structure of human Factor IX,. Under our experimental conditions, no intermediate form of Factor IX was detected during the activation by Factor XI,, suggesting that the first cleavage is relatively slow and that the second cleavage is much faster. This is in contrast to the activation mechanism reported for bovine Factor IX involving similar cleavages where the fist cleavage step appeared to be faster than the second step, thus giving rise to observable quantities of an intermediate (10). The lack of observable quantities of intermediate forms of human Factor IX makes it impossible to distinguish between two intermediates as shown in Fig. 5.

Studies of the cleavage of radiolabeled coagulation factors in clotting plasma are necessary in order to compare the molecular mechanisms which are observed using purified pro- teins with the mechanisms which are operative in the complex milieu of plasma. Previous studies of the cleavage of ‘?- Factor XII, ‘““I-Factor XI, and ‘““I-prekallikrein in clotting plasma have shown that the same forms of each molecule produced by limited proteolysis are observed in both clotting plasma and purified reaction mixtures (6, 7, 17, 42). The

FIG. 5. Schematic model of the polypeptide structure of Factor IX and activated Factor IX. Activation of human Factor IX by activated human Factor XI occurs by limited proteolytic cleavage in a two-step reaction. In the first step Factor IX is slowly cleaved giving rise to an intermediate which may be either of the two intermediate forms shown. In the second step, the intermediate form is cleaved at one of the sites designated by the small arrows.

studies of the cleavage of ‘““I-Factor IX in normal plasma during contact activation which are reported here suggest that the same limited proteolytic cleavages of Factor IX occur in plasma as seen using purified proteins under conditions which generate Factor IX.

Achnowledgments-The support and advice of Drs. Charles G. Cochrane and Samuel I. Rapaport are gratefully acknowledged. The very skillful help of Katherine K. Lavine, Gregory Beretta, and Pete Segrist is most appreciated.

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21. 22.

23.

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B Osterud, B N Bouma and J H Griffinactivation by activated factor XI.

Human blood coagulation factor IX. Purification, properties, and mechanism of

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