The acceleration by polylysine of the activation of factor X by factor IXa

T;~RG?~BCSISRESE..LSCH25; 319-329, 1982 ?rinted !l0J+9-33~8!82~040319-1iS03.00/0 CGDyvici: (c) 1937 Perganon Press ?_rd. &,--=a

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the CSX. rights reserved.

THE ACCELERATIONBY POLYLYSINE OF TRE ACTIVATION OF FACTOR X BY FACTOR IXa

Roger

Dental

L.

Lundblad

and Harold

R. Roberts

Research Center, Center for Thrombosis & Hemostasis Departments of Pathology, Medicine and Biochemistry The University of North Carolina at Chapel Hill Chapel Hill, North Carolina 27514 (Received

20.7.1981; Accepted

in revised by Editor L.

and the

form 22.12.1981. Hoyer)

ABSTRACT The present study reports that polylysine can function as a cofactor in the conversion of factor X to factor Xa by factor IXa. In the presence of polylysine, factor X is converted to factor Xa by factor IXa as demonstrated by both clotting and amidolytic assays. The activation of factor X by factor IXa requires the prior activation of factor IX to IXa by factor XIa. Conversion of factor X to factor Xa by factor IXa is not observed in the absence of polylysine. The activation reaction proceeds optimally at pH 8.0 with an equal weight ratio of polylysine to factor X. The effect of polylysine is readily reversed by low concentrations of NaCl or elevated temperature suggesting that electrostatic interactions are of primary importance in the polylysine facilitation of the activation of factor X by factor IXa.

INTRODUCTION factor IXa converts factor X to During intrinsic blood coagulation, factor Xa in a reaction which requires the presence of calcium ions, phospholipid and factor VIII (l-6). Early results had suggested that factor VIII was converted by factor IXa to an enzyme (activated factor VIII) which Further investigation, subsequently converted factor X to factor Xa (1,2). revealed that factor VIII served as cofactor in the activation of however, As a result of these and other studies, it is factor X by factor IXa (3). now generally accepted that, during intrinsic blood coagulation, factor X is converted to factor Xa by factor IXa in a reaction requiring the presence of calcium ions, phospholipid and factor VIII as cofactors (7,8). Little information is available regarding the molecular mechanism of this reaction as factor VIII is a complex macromolecule which has only recently been There is still question regarding the relationship of this purified (9). Polylysine, KEY liORDS : Amidolytic Clorting assay,

Factor IXa, assay 319

Factor

X acti\-ation,

Factor

Xa,

preparation to the large mole:ular weight material reported in previous studies ( LO). Regardless of the exact molecular nature of the coagulant portion of the factor VIII complex, the lack of readily available preparations of factor VIII of known properties has frustrated attempts study the interaction of factor IXa with factor X.

to

The activation of factor X by factor IXa is similar to the activation of prothrombin by factor Xa in that calcium ions, phospholipid and protein cofactor (factor V) are required for effective catalysis by factor Xa. We have previously demonstrated that polylysine could replace the cofactors (calcium ions, phospholipid and factor V) required for the activation of prothrombin by factor Xa (11). Considering the similarity between the activation of factor X and prothrombin during intrinsic blood coagulation, it appeared reasonable to examine the effect of polylysine in the activation of factor X by factor IXa. Results presented herein demonstrate that polylysine can participate as a cofactor in the activation of factor X by factor IXa and provide a basis for the development of a further understanding of the nature of the interaction between factor IXa and factor X.

MATERIALS AND METHODS Bovine coagulation factors IX and X were purified by existing procedures (12-14). Both of these protein preparations were homogeneous by polyacrylamide gel electrophoresis. Bovine factor XIa was purified as previously described (15). Crude phospholipid (Centrolex P> was obtained from Central Soya. Polylysine hydrobromide (weight average molecular weight : 6900) was a product of Miles-Yeda. RzIleGluGlyArgpNa was obtained from AB Kabi. All other chemicals were of at least reagent grade and used without further purification. Factor IXa was prepared by activation with factor XIa (15). In a typical preparation factor IX (2.0 ml, 1.5 mg/ml in 0.05 M Tris, pH 8.0) was incubated at ambient temperature (24”) with 0.25 ml factor XIa (.4 mg/ml in 0.05 M Tris, pH 8.0) and 0.25 ml 0.05 M CaCl2. Portions were removed at five minute intervals and assayed with human plasma as the substrate. At maximal activation, the reaction mixture was placed at 4’ and used as described below. This material could be stored at -20” for at least one month without apparent loss of activity. In the experiments designed to evaluate the activation of factor X by factor IX the factor X preparations were dialyzed vs. 0.02 M Tris HCl, pH 8.0 prior to use. The polylysine preparations were dissolved in 0.02 M Tris, pH 8.0, prior to use. With the exception of the experiments where the pH dependence of the reaction was evaluated, the reactions were performed in 0.02 M Tris, pH 8.0. A typical reaction mixture contained .05-.lO mg factor X, an equivilant weight amount of polylysine and 0.01-0.025 mg factor IXa in a final volume of 0.5 ml. The reactions were initiated by the addition of factor IXa and portions were removed and assayed for factor Xa activity as described below. The activation of factor X was monitored either by using normal human plasma as the substrate (2,16,17) or by the hydrolysis of BzIleGluGlyArgpNa Protein concentration was determined by either absorbance at 280 nm or (18). by the ninhydrin reaction after alkaline hydrolysis (19) using crystalline bovine serum albumin as a standard.

I‘ol.

25 , x0.4

RESULTS The effect of polylysine on the activation of factor X by factor IXa is It is observed that there was a rapid activation of shown in Figure 1. factor X by factor IXa in the presence of polylysine. Conversion of factor X to factor Xa was not observed with factor IXa alone either in the presence Activation of factor X was not or absence of exogenous calcium ions. observed without the prior activation of factor IX by factor XIa. The development of amidase activity toward BzIleGluGlyArgpNa (18) proceeded at a rate identical to that of the development of clotting activity. The activation of factor X by factor IXa in the presence of polylysine was associated with changes in the covalent structure of Factor X as reported by The electrophoretic analytical system utilized other investigators (21-23). in the present study would not differentiate between factor Xao and factor XaS (23). assays for catalytic activity would also not .Furthermore, differentiate between these two forms of factor Xa since the autocatalytic removal of the small carboxyl terminal peptide associated with the conversion of factor Xacr to factor Xag is not associated with a change in enzymatic activity (23). Complete conversion of factor X to factor Xa was This was based both observed with factor IXa in the presence of polylysine. on electrophoretic analysis and comparison of the activity of the final the level of product with that of homogeneous factor Xa. In addition, activity obtained on the activation of factor X with factor IXa in the presence of polylysine is equivalent with that obtained for the activation The activation of factor X by with Russell’s viper venom (c.f. ref. 9). factor IXa in the presence of polylysine did not require exogenous calcium. In fact, the addition of 5 mm CaC12 resulted in a 80% decrease in the rate This concentration of CaC12 had previously been of factor X activation. shown to be optimal in the activation of factor X in the intrinsic system at low levels of exogenous calcium There is stimulation, however, (2). chloride c.01 mM) and the presence of EDT4 c.2 mY) results in no activation. for calcium ions in this system which can be Thus, there is a requirement fulfilled by the calcium ion present in the factor IXa preparations. Previous studies on prothrombin activation in the presence of polylysine had As indicated above, these also demonstrated inhibition by CaC12 (11). reactions were performed at 24”. Raising the reaction temperature to 37” The activation of resulted in 80% inhibition of the rate of reaction. factor X by factor IXa in the presence of polylysine proceeded with a pH optimum at 8 (Figure 2) similar to that described for the activation of factor X by factor IXa in the presence of factor VIII (2). The reaction mixture containing factor X and polylysine was turbid, but, differing from prothrombin activation, (11) did not clarify during the activation.

80

1

10

I

I

I

I

20

30

40

50

Time (minutes) FIG. 1 The The Activation of Factor X by Factor IXa in the presence of Polylysine. reactions were performed under ambient condition and contained 0.06 mg factor X, 0.015 mg factor IXa, 0.06 mg polylysine in 0.02 M Tris, pH 8.0 in Portions were taken at the indicated time and a final volume of 0.5 ml. assayed either with BzIleGlyGlyArgpNa (o-o) or diluted in 0.06 ?i Tris - 0.09 M NaCl, pH 7.5 and assayed for coagulant activity (e-e).

100

80

20

1

1

I

I

6

7

8

9

PH FIG. 2 The Effect of pH on the Activation of Factor X by Factor IXa in the Presence The reactions were performed as described under Figure 1 of Polylysine. except that the reaction contain 0.100 mg factor X, 0.100 mg polylysine and 0.025 mg factor IXa in a final volume of 0.5 ml of 0.02 m Tris-acetate buffer at the indicated pY. Initial rate measurements were obtained using the clotting assay.

The

activation proceeded corresponds

effect by

of polylysine factor

optimally to

a

IXa

concentration was

examine,d

on as

the

siok-;?

at

a equal

weight

ratio

L?-fold

molar

excess

of

of

in

rate Figiire

polylysine

polylysine

to

of

factor The

3.

to factor

factor

X react

ion

9.

This

X.

100

80

-04

.08

.I2

Polylysine I

.32

.20

(mglml) 1

I

.65

.16

.97

1

1

1.29

1.6

Polylysine/F.X(w/w) FIG.

3

of Factor X by The Effect of Polylysine Concentration on the Activation The reactions were performed as described under Figure 1 in Factor IXa. pH 8.0 and contained 0.100 mg/ml factor X, 0.025 mg/ml factor O.02 H Tris, Initial rate measurement IXa and polylysine at the indicated concentration. were obtained using the clotting assay.

Figure 4 shows the activation of factor X by factor concentrations of factor IXa. As would be expected, is dependent upon the factor IXa concentration.

IXa at various the rate of activation

8(

2(

(

10

20

30

40

50

60

Time (minutes) FIG.

4

The Effect of Factor IXa Concentration on the Activation of Factor X in the The reactions were performed as described under Presence of Polylysine. Figure 1 and contained 0.040 mg (o-o), 0.02 mg (e-e) or 0.01 mg (0-n) of factor IXa. Factor Xa activity was assayed using the clotting assay.

The reaction mixture was turbid with polylysine, factor X and factor IXa at the respective protein concentrations employed in the present experiments. This turbidity could be obviated by the addition of 0.1 M NaCl. Figure 5 shows that this effect of NaCl results in inhibition of the activation of factor X by factor IXa in the presence of polylysine. Likewise, turbidity did not develop under conditions which did not result in factor X activation, such as in the presence of 0.1 ?l sodium phosphate, pH

8.0.

100

80

20

-1

.030

.015

[NaCI] FIG.

.045 M

5

The Effect of NaCl on the Rate of Factor X Activation by Factor IXa in the Presence of Polylysine. The reactions were performed as described under Figure 1 with the indicated final concentration of NaCl. Factor Xa activity was assayed using the clotting assay.

To1.25,

X0.4

327

DISCUSSION The similarity between the present study and the results obtained in our laboratory for prothrombin activation (11) would suggest that polylysine substitutes for calcium ions, phospholipid and factor VIII in the activation of factor X by factor IXa. In support of this concept, inhibition of the activation of factor X by calcium ions in the presence of polysine was observed suggesting that calcium ions and polylysine compete for the same site on the factor X molecule. The stimulation seen at low levels of exogenous calcium and the inhibition observed with FDTA does suggest that the presence of calcium ions is essential for this reaction. It is likely that this represents the necessity of calcium ions for the preservation of factor IXa activity as previously reported from this laboratory (15). In our previous studies on the effect of polylysine on prothombin activation, it was postulated that polylysine bound to site (or sites) in the amino terminal portion of the prothrombin molecule increasing the susceptibity of the peptide bonds cleaved by factor Xa in the formation of tb 1 bin. Considering the present results, we would suggest that polylysine bit c) to the light chain of factor X increasing the susceptibity of the peptide bonds cleaved during the formation of factor Xa. We would further suggest that electrostatic forces are primarily responsible for this interaction. This concept is supported by the observed effect of NaCl in reversing the physical association of polylysine and factor X with concomitant inhibition of the activation reaction. A similar effect of NaCl has been reported by Anderson and coworkers in the dissociation of polylysine-pepsin complexes (24). These investigators also reported that the formation of the polylysine-pepsin complex which results in the inhibition of pepsin, was reversed at an excess concentration of polylysine. A similar effect in the polylysine-factor X interaction could explain the concentration dependence observed in the present experiments. The inhibition of the rate of reaction observed at 37” compared to that at 24” is also consistant with this hypothesis. Although we consider the direct interaction of polylysine with factor X to be the most likely mechanism for polylysine in the factor IX-catalyzed activation of factor X, the present results do not exclude the possibility that polylysine could provide a “surface” in solution which would bind both factor IXa and factor X thus promoting the activation of factor X. The above observations also provide the basis for the development of a specific assay for factor IXa which is not dependent upon the availability of a factor VIII preparation. This will, for example, allow direct studies of the catalytic activity of factor IXa using factor X as a substrate. Such studies might involve the factor IXa preparations obtained from the activation of abnormal forms of factor IX. This study provides a strong basis for further studies of the relationship between structure and function in factor IXa.

ACKNOWLEDGEMENTS This National

work was supported in Heart, Lung and Blood

part by research Institute.

grant

HL-06350

from the

32s

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2.

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3.

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4.

HULTIN, M.B. and NEMERSON, Y. "Activation of Factor X by Factors IXa and VIII; A Specific Assay for Factor IXa in the Presence of Thrombin-Activated Factor VIII." Blood 52, 928-940, 1978.

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NEAL, G.G. and CHAVIN, S.L. "The Qole of Factors VIII and IX in the Activation of Bovine Blood Coagulation Factor X." Thromb. Res. 16, 473-484, 1979.

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Y.

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"Simultaneous Purification of Bovine J. Biochem. Chem. 248, 7729-7741, 1973.

13. FUJIKAWA, I<., THOMPSON, A.Q., LEGAZ, M.E., MEYER, R.G. and DAVIE, E.Y. "Isolation and Characterization of Sovine Factor IX (Christmas Factor)." Biochemistry 12, 4938-4945, 1973. 14. FUJIKAWA, K. and DAVIE, F.W. "Bovine Factor IX (Christmas Factor)." Methods in Enzymology 45, 74-83, 1976.

15. LLTNDBLAD, R.L. and KINGDON, H.S. "Biochemistry of the Interactions Bovine Factors IXa and IX." Thromb. Haemorrh. Diath. Suppl. 57, 315-332, 1974.

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of Human Factor 1978.

18. AURELL, L., FRIBERGER, P., KARLSSON, ?. and CLAESON, G. "A New Sensitive and Highly Specific Chromogenic Peptide Substrate for Factor Xa." Thromb. Res. 11, 595-609, 1977. 19. MOORE, S. "Amino Acid Analysis Aqueous Dimethyl Sulfoxide as Solvent for the Ninhydrin Reaction." J. Biol. Chem. 243, 6281-6283, 1968. 20. FUJIKAWA, K. and DAVIE, E.W. “Bovine Factor X (Stuart Factor)." Methods in Enzymology 45, 89-95, 1976. 21. RADCLIFFE, R.D. and BARTON, P.G. "Comparisons of the Molecular Forms of Activated Bovine Factor X. Products of Activation with Russell's Viper Venom, Insoluble Trypsin, Sodium Citrate, Tissue Factor, and the Intrinsic System." J. Biol. Chem. 248, 6788-6795, 1973. 22. FUJIKAWA, K., TITANI, Y. and DAVIE, E.W. "Activation of Bovine Factor X (Stuart Factor): Conversion of Factor Xa to Factor Xa .ll Proc. Nat. Acad. Sci. USA 2, 3359-3363, 1975. 23. SILVERBERG, S.A., NEMERSON, Y. and ZUR, M. "Kinetics of the Activation of Bovine Coagulation Factor X. Kinetic Behavior of Two-Chain Factor VII in the Presence and Absence of Tissue Factor." J. Biol. Chem. 252, 8481-8488, 1972. 24. ANDERSON, W., HARTHILL, J.E. and RABMATALL, R. "The Influence of Molecular Size and pH on the Macrocationic Inhibition of Pepsin by Polylysine." 3. Pharm. Pharmacol. 32, 248-255, 1979.