Inactivation of bovine and human thrombin and factor Xa by antithrombin III studied with amidolytic methods

lXROMBOSIS RESEARCH Printed in Great

l-01. 11, pp. ZOj-Zl6, Pergamon Press,

Britain

INACTIVATION OF BOVIXE AND HCXAN THROMBIS XXD AXTITHROXBIN III STVDIED WITH ANIDOLYTIC

FACTOR Xa WETHODS

1977 Ltd.

BY

0. R. #deg&d. U. Abildgaard, M. Lie and M. MillerAndersson, Medical Department A, Aker Hospital, Oslo j, Norway

(Received 27.4.1977; Accepted by

in revised form 7.6.1.977. Editor H.C. Godal)

ABSTRACT was Antithrombin III (At-III) purified from human plasma incubated with thrombin (T), activated factor X (Xa) or The amount of enzymes inactivated mixtures of T and Xa. by the amount of At-III in one ml of normal plasma was At-III which had inactivated one enzyme, determined. Inactivation of was incapable of inactivating the other. Xa and T by At-III was found to proceed like a second The inactivation rate was highly depenorder reaction. dent on the At-III concentration, the half-time values of the enzyme activities decreasing with increasing At-III concentration. With excess inhibitor, variations in the concentration of enzyme had little influence on the inactivation rate. Bovine Xa was more rapidly inactivated than bovine T. In contrast, human T was more rapidly inactivated than human Xa.

INTRODUCTION Antithrombin III (At-III) inactivates thrombin (T) and acti1,2) by the formation of bimolecular enzyme vated factor X (Xa) inhibitor complexes The active serine sites of the 3,4,5). l clotting enzymes are involved in the reactions (4), and At-III apparently acts as a pseudo-substrate, as was suggested several years ago (6). Yin found that a complete neutralization of At-III with bovine T rendered the inhibitor incapable of subsequent inactivation of bovine Xa and vice versa (2), which suggests that Xa and T bind to the same At-III site. In contrast, (asterud et al (7) reported that At-III which had been preincubated with human its ability to inactivate thrombin, and suggested Xa, retained that T might displace Xa, or that the enzymes were bound to At-III at different sites. Osamo found that anti Xa activity in

205

206

I~ACTIVATTO?i

OF

THRO%fBIx

dc F.Xa

Vol.ll,,'o.2

serum was not reduced by preincubation with T and vice versa (8). As At-III is the main inactivator of both T anti Xa in plasma and serum (4,9,10) these latter results are difficult to reconcile with those of Yin (3j. Yin reported that At-III preferentially reacts with Xa rather origin of the Xa preparathan T, but did not state the species tion used in the experiments (11). As bovine Xa is much more readily inactivated by human plasma than is human Xa, species differences might influence the results (1,l.Z'). The main purpose OF the present experiments was to study the inactivation rates of T and Xa of bovine and human origin by purified human At-III. of At-III to Furthermore, the ability retain inactivating capacity towards one clotting enzyme after reacting with another has been examined. Amidolytic methods have been used in order to measure T and Xa activities separately even when present in mixtures.

MATERIALS

AND

METHODS

Bovine thrombin (Tb), Topostasine (Hoche, Basle, Switzerland) batch B 992K9ZA, was dissolved in 0.15 mol/l NaCl to 100 NIH U/ml and stored at -20 "C in polystyrene tubes. The samples were diluted in Verona1 buffer to the concentration indicated, kept on ice and used within one hour. The amidolytic Xa activity was less than 0.1 U per 100 NIH U Tb. Human thrombin (Th), Fibrindex (Ortho Diagnostics, Raritan, New 50 NIH U was Jersey, USA) lot no 5P223. One vial containing dissolved in 1.0 ml 0.15 mol/l NaCl (as recommended by the manufacturer) and diluted in Verona1 buffer to the concentration indicated. Clotting activity and amidolytic activity were found to be identical to that of Topostasine. The amidolytic Xa activity was less than 0.1 U per 100 NIH U Th. Activated bovine factor X (Xab), (Diagnostic Reagents, Thame, Oxon, England) lot no XA6 was obtained as lyophilized powder and dissolved as indicated by the manufacturer. One vial contained 4.25 U compared to the International Xa Standard 75/590 (13). The solution was dialyzed at +4 OC for 60 min in Verona1 buffer. The preparation was found to contain small amounts of T. Compared with Topostasine, the biological thrombin activity determined with fibrinogen as substrate, was 0.07 NIH U/vial, the amidolytic thrombin activity determined with s-2238 (14) was 0.36 NIH U/vial. Activated human factor X (Xa ) batch DqG 32 (Kabi, Stockholm, Sweden). One vial was disso P ved_in 2.2 ml distilled water and dialyzed in Verona1 buffer at 4 oC for 60 min. Assayed by a clotting method (15) it was found to contain 27 U Xa compared to the International Xa Standard. Determined as amidolytic activity one vial contained 22 U. The preparation was found to contain T. When compared with Tb, the biological T activit determined with fibrinogen as substrate. was 4.2 NIH U/vial. 4 he amidolvtic activity determined with s-2238 was equivalent to 6.4 NIk U/vial when compared with Tb.

Antithrombin III (At-III) was prepared as described by MillerAndersson et al (16). This particular batch has also been used for the preparation of the At-III standard (17), and chemical analysis has revealed the absence of heparin. The freeze-dried preparation was dissolved in distilled water to a concentration of 10 U/ml and diluted to the concentration indicated in TrisHCl-EDTA buffer. One unit is defined as the T inhibiting activity of one ml normal titrated plasma (100 per cent At-III activity (18)). Chromogenic substrates. The T susceptible Bz-Phe-Val-Arg-pNA (5-2160) (19) and- H-D-Phe-Pip-Arg-pNA.(S-2238) (14) and the Xa susceptible Bz-Ile-Glu-Gly-Arg-pNA (S-2222) (20) from Xabi, Stockholm, Svedgn, were dissolved in distilled water, stored in the dark at +4 C and diluted with distilled water. Hirudin (Pentapharm, vial containing 2000 fer.

Basle, Switzerland) U vas dissolved and

lot no diluted

1230/70. One in Verona1 buf-

SBTI (Trypsin Inhibitor, Sigma, St. Louis, MO, USA) lot no 94Cm, was dissolved in 0.15 ? ol/l ? NaCl to a concentration of 10 g/l and diluted in Verona1 or Tris buffer. S-2238 with SBTI. Final concentrations: 1.2 ma101/1 s-2238 0.1 mg/ml SBTI. The mixture was used within 4 hours. S-2222 with and 20 U ml X with &udin. VerOMl

hirudin. hirudin.

Final concentrations: The mixture was used

hirudin. Final concentrations: The mixture was kept on ice and buffer,

pH

7.4,

containing

NaCl,

and

1.5 mmol/l s-2222 within 4 hours. 2.6 U/ml Xah and 20 U/ml used within 4 hours.

ionic

strength

0.15

(21).

250 ml 0.2mol/l Tris-HCl-EDTA buffer, pH 8.4, ionic strength 0.2. Tris was mixed with 20 ml 1 mol/l HCl, 30 ml 0.25 mol/l K2EDTA and the final volume adjusted to 1000 ml with 0.26 mol/l NaCl. AMIDOLYTIC

EXPERIMENTS

were performed at 37 'C. The amidolysis was stopped by the addition of 300 ~1 acetic acid and the absorbance read at 405 run. The experiments were performed at least three times. Unless specified, results of a typical experiment are shown. In preliminary experiments, the efficiency of hirudin and SBTI in blocking T and Xa activities, respectively, were assessed: la. The effect of hirudin oh the T-S-2238 reaction. 100 ill veronal buffer was mixed with 100 ~1 T (4 NIH U/ml) and 100 11 hirudin in Tris-HCl-EDTA buffer (O-30 U/ml) and incubated with 100 ~1 S-2238 (1.2 mmol/l) for 60 set (Table I). lb. The effect of SBTI and hirudin on the ~-s-2238 reaction. 100 ~1 T (4 NIH U/ml) was mixed with 100 ~1 SBTI (0.1 mg/ml) in Tris-HCl-EDTA buffer and 100 @l hirudin (20 U/ml) and incubated with 100 pl s-2238 (1.2 mmol/l) for 60 set (Table I). The effect of hirudin on the Xa-S-2222 reaction. lc. 10.0 tll Verona1 buffer was mixed with 100 ~1 Xa (2.6 U/ml) and 100 ~1 hirudin (O-40 U/ml) in Tris-HCl-EM'A buffer and incubated with 100 11 S-2222 (1.5 mmol/l) for 180 set (Table I). Id. The effect of hirudin on the inactivation of Xa by At-III. 100 pl Xa (2.6 U/ml) with hirudin (O-20 U/ml) was mixed with 200 ~1 At-III (O-1.2 U/ml) and incubated at 37 OC. After 5 lain, 200 ~1 S-2222 (1.5 mmol/l) was added. The amidolysis vas stopped

208

I~ACTIV~TIOS

OF THROMBIS

Zc F.Xa

Vol.ll,No.2

after

240 sec. The effect of SBTI on the Xa-S-2222 reaction. 100 11 Xa ;:I5 U/ml) was mixed with 100 ~1 SBTI (O-O.1 mg/ml) in Tris-HClEUTA buffer and 100 ~1 Verona1 buffer and incubated with 100 ~1 S-2222 (1.5 mmol/l) for 180 set (Table I). The effect of SBTI on the ~-s-2238 reaction. If. 100 j.tlT (2 NIH U/ml) was mixed with 100 ~1 SBTI (O-O.2 mg/ml) in Tris-HCl-EM'A buffer and 100 ~1 Verona1 buffer and incubated with 100 ~1 s-2238 (1.2 mmol/l) f or 60 set (Table I). The effect of hirudin and SBTI on the Xa-S-2222 reaction. lg. 100 ~1 Xa (8.5 U/ml) was mixed with 100 /.~lSBTI (1 g/l) in TrisHCl-EDTA buffer, 100 ~1 hirudin (20 U/ml) and 100 ~1 S-2222 (1.5 mmol/l) for 180 set (Table I). 2. Titration of T with At-III. 200 ~1 T (O-100 NIH U/ml) was incubated with 200 ~1 At-III (0.2-0.4 U/ml) or buffer. After 120 min, 1.6 ml Tris-HCl-EM'A buffer was added. 100 ~1 of this mixture was added to 200 ~1 prewarmed Tris-HCl-EBTA buffer, and the amidolysis started by adding 200 ~1 S-2160 (0.6 mmol/l). Amidolysis was stopped after 60-180 set (depending on initial T concentration), and remaining T calculated using standard curves (Table II). Titration of Xa with At-III. 200 ~1 Xah (O-8.5 U/ml) with 3. hirudin (O-20 U/ml) or X (O-8.5 U/ml) was incubated with 200 ~1 At-III (0.04 and 0.01 U/ma? , respectively) or buffer. After 120 min, 600 /.~lTris-HCl-EDNA buffer was added. The amidolysis was started by adding 200 11 of this incubation mixture to 200 ~1 S-2222 (0.7 mmol/l) and stopped after 300 sec. Remaining Xa was calculated using standard curves (Fig.1). Two step inactivation was performed to see if prior incubation of At-III with Xa reduced its T inactivating capacity and vice versa. 4a. Preincubation of At-III with excess X% 150 ~1 Xab (8.5 U/ml) was incubated with 50 ~1 At-III (0.04 U/ml). After 120 min, After varying incubation 100 ~1 T (2.5 NIH U/ml) was added. time (O-920 min) residual T or X ab activity was measured by ador 100 ul S-2222 (1.5 mmol/l), ding 100 ~1 s-2238 (1.2 mmo?/l) Amidolysis for 60 or 120 set, respectively. (Fig.2a) respectively. 100 ~1 T (18 NIH Preincubation of At-III with excess T . 4b. U/ml) was incubated with 100 ~1 At-III (0.b U/ml). Af%er 120 min, 100 ~1 Xab (4.25 U/ml) was added. After varying incubation time (O-120 min) residual Xab and Tb activities were measured as t; experiment 4a (Fig.Zb). Preincubation of enzyme with excess of At-III. (0104 U/ml) was incubated with 100 ~1 T (4 NM U/ml)'~~ Th2 second incubation U/ml) or veronal buffer for 60 min. min) was initiated by the addition of 100 11 T (4 NM U/ml) or Remaining enzyme ac 5 ivity (T and Xa xab (6.5 U/ml) or buffer. was determined with s-2160 (1.0 mmol/l) and S-2222 (1.5 mmol/l (Table III). 100 ~1 T (2-8 NIH Ulml) was mixed Inactivation rate of T. 5. with 100 ~1 Verona1 buffer and incubated with 100 ~1 At-III (O-03Residual T activity was determined by 3.0 U/ml) for O-240 min. incubation with 100 ~1 s-2238 with SBTI for 60 sec. Inactivation rate of Xa. 6. U/ml with hirudin ) was mixed

loo ~1 x9 with 100 p

(8.5 u/ml) or Xah (2.6 Verona1 buffer and'incu-

Vol.ll,No.2

bated dual

for

I?;ACT~XTTOS

with 100 ~1 At-III (0.03-3.6 activity was determined 120 sec. Xa

& F.Sa

OF THRO>iBIN

U/ml) with 100

for O-1200 ~1 S-2222

min. Resiwith hirudin

Inactivation rate in a mixture of Xa 100 &Al X% 7. (8.5 U/ml) was mixed with 100 ul Tb (4 N!?mTAand incubated with 100 ul At-III (0.03-3.0 U/ml) for O-240 min. Residual Xa and T activities were determined with S-2222 and s-2238 as described above. Inactivation rate in a mixture of Xal and T,.. 100 pl x U/ml) containing 0.5 NIH U/ml T was mixed with 100 ~1 aP 3.5 NIH U/ml) and incubated with 10 8 ul At-III (0.03-3.0 U/m?) for O-1200 min. were determined Residual Xah and Th activities as described above. with S-2222 and s-2238 8.

2.6

RESULTS Hirudin and SBTI were studied to examine if these reagents allowed separate amidolytic analysis of T and Xa activities when present together. Hirudin inhibited about 90 per cent of the T activity without significantly influencing the Xa-At-III (Cf. Methods, exp.no. Id) or the Xa-S-2222 (Cf. Methods, exp.no. lc) reactions. SBTI inhibited about 90 per cent Xa activity without Higher concentrainfluencing the T-S-2238 reaction (Table I). tions of hirudin or SBTI (Cf. Methods, exp.no. la-f) did not The residual result in lower residual amidolytic activities. activities were not blocked by a combination of SHTI and hiNdin These results were (Cf. Methods, exp.no. lb and lg) (Table I). found satisfactory for the use of hirudin and SBTI for the purpose mentioned.

Inhibition

TABLE I of amidolytic activity by (Cf. Methods, exp.no.

hirudin and SBTI la-c and e-g)

Factor Final

cont.

Human

[

4.6

U/ml

SBTI

0.03

mg/ml

95%

97%

Hirudin + SBTI

6.6 0.03

U/ml m&/ml

95%

97%

<

cent

Thrombin

Xa Bovine

Hirudin

( 1%

in per

Human

1%

Bovine 92%

91% < 1%

:

91%

<

1% 91%

Titration experiments were perrormed to study stoichiometric relations and to find the appropriate enzyme concentration to be used in the inactivation rate studies (Fig.1). With the At-III concentration used, about 120 min incubation was required to obtain completion of the reaction. The amount of enzyme inactivated by one At-III unit was expressed in enzyme units (Table II), as the exact quantity of pure enzymes present was not known

I?r'ACTIVATIOK OF TKRO!+[BI~ Zs F.Xa

(Cf. vine

Discussion). preparations

Distinct differences between were observed, notably for Xa

Vol.ll,No.2

the human and (Table II).

TABLE II Amounts of enzyme expressed in units (Cf. Methods, exp.no.2 inactivated by one unit At-III. Incubation time 120 min. Results of two titration experiments. Factor

Xa

Bovine 24%

and

3)

Thrombin Human

251

bo-

102,

Bovine

93

147,

‘50

Human 227,

228

Preincubation experiments. To ensure complete saturation of At-III vith enzyme, it was incubated with three times as much enzyme as had been found equivalent to the amount of At-III used in the titration experiments. 120 min incubation with one enzyme rendered At-XII completely incapable of inactivating the other enzyme (Fig.2).

Xa*+ buffer /

??

+

At-III

,

5; 0

2

4

6

Original

Xa

activity,

9 U/ml

of incubation Fig.1. Effect of Xa with At-III. Varying and Xah vere amounts of X amount incubated wit"B constant of At-III or buffer for 120 min (Cf.Methods, exp.no.3). The length of the lines a and b reflects the amount of Xa inactivated.

OF THROYBIS

& F.Sa

Buffer +Xa (1ZOmin)+T At-Ill +Xa

211

Buffer *T WOrnin)

+ Xa

(120min)+T

At-III +buffer (120min) +T At-Ill+ 0

’

t 30

60

90

30

lti

buffet(120min)rXa

of preincubation Fig.2a. Effect excess Xa of At-III with sequent inactivation of p,"i,',""Methods, exp.no. ka).

&I

$0

Incubation,

Incubation.min

120

min

Effect of preincubaFig.2b. tion of At-III with excess T on subsequent inactivation o pi Xab (Cf. Methods, exp.no. 4b).

Even when the amount of enzyme was reduced and the incubation decreased the preincubation of At-III with T time shortened, that was subsequently inactivate b( Table 111,exp.e) amount of Xa and vice verka (Table III, exp. f). Xa was more rapidly inactivated than Tb (Table III, exp. a,c, the inactivation rates were higher in the As expected, b,d). initial 60 min than in the subsequent 60 min (Table III, exp. a, b,c,d). These results indicated that reaction with one enzyme interfered with the ability of At-III to inactivate the other enzyme. it was found necessary to study For a quantitative evaluation, the inactivation rates of the enzymes in more detail.

TABLE III Influence of preincubation of At-III with Tb and Xa Qc;n lity of further inactivation (Cf. Methods, exp.no. Exp.

Incubated initial

for

60 min

a. _At-III

+ buffer

b.

At-III

+ Tb

C.

At-III

+

d.

A t-111

+ Xa,,

e.

At-III

+ Tb

f.

At-III

+ Xab

buffer

Added prior to subsequent 60 min incubation Tb buffer

Per cent activity Tb

the

of initial inactivated xab

50 85 68

xab buffer

88

xaa

82

28

*b

30

83

abienzyme

212

IWACTIVATIOX

OF THRO?GIN

Vol.ll,No.2

B F.Xa

This was studied in two-stage Inactivation rate experiments. Ideally, systems and substrate added at the start of stage II. the substrate should block the enzyme-At-III reaction, so that inactivation of enzyme terminated at the start of stage II. Whereas s-2238 nearly abolishes the T-At-III reaction, S-2222 has little influence on the Xa-At-III reaction, making the half-time values for Xa at 0.01 U of At-III somevhat inaccurate (Table IV, without affecting the other results of Table IV left column), (BdegArd et al, to be published). changes in the enzyme concentration had With excess At-III, little influence on the inactivation rate (Fig.3). Approximately equimolar concentrations of enzyme (as determined by titration studies and assuming bimolecular Complex formation) were used in a comparison of the inactivation rate of the Four different At-III concentrations were used; various enzymes. at the lowest At-III concentration the molar concentration of each enzyme (Cf. Discussion) was about 0.7 of the molar At-III concentration. Half-time values were calculated from graphs (Fig.3 and 5) obtained with one enzyme and At-III (Table IV). As expected, the half-time values for the enzyme decreased Bovine Xa with increasing inhibitor concentration (Table IV). was more rapidly inactivated than bovine T. In contrast, human T was more rapidly inactivated than human Xa. When two enzymes were incubated with At-III, the inactivation of each enzyme proceeded more slowly (Fig.S), and the results confirmed that of the bovine enzymes Xa was more rapidly inactivated, and that human T was more rapidly inactivated than human Xa. The results of a plot of the reciproke values of remaining enzyme activity at the time t, versus t; suggest that the inactivation of Xa with the concentrations used proceeds as a second order reaction (Fig.4).

80 60

40

i)

20

C

C1

$0

,,,I

10

Incubation,

20

30

min

thrombin activity Fig-J. Residual as a function of time at various actiInitial T Th activities. vlty indicated, initial A 9 -111 activity 0.1 U/ml.

0

60

120

Incubation.

180

, 240

min

Inactivation of Xa Fig.4. Initigl and Xa 0.84, activi$iZ tG/Z;I Xa 2.8, At-III 0.01 ?Cf. Xa 6). Second Methods , exp.no. order rate plot.

Vol.ll.zio.2

IKSCTIVATIO1

OF THROYBIX

& F.Sa

213

Inactivation rates with At-III 0,Ol u/ml Bovine T and Xa

Human T and Xa

0’ 0

OOsb

60

Incubation, min.

7 I*

120 180 Incubation, min

240 1200

Inactivation of T and Xa alone or in combination by excess Fig.5. A$-111 with bovine (left) and human (right) enzymes. Initial activities (U/ml) X 0.87, Xab 2.8, Th l-3 and Tb 1.3 (Cf. Methods, exp.no.

Influence values. indicated

TABLE IV of At-III concentration on the T and Xa half-time Initial At-III concentration in the reaction mixtures (Cf. Methods, exp.no. 5 and 6). At-III

Initial Th

activity

concentration,

0.01

0.1

1.3

NIH

U/ml

3080

set

300

NIH

U/ml

set

Tb

1.3

3220

set

240 set

Xah

0.86

U/ml

7200

set

690

X%

2.8

U/ml

2700 set

1.0

0.5 78

U/ml

SAC

45 set

37 set 40

set

set

150

set

90

set

150 Eec

20

set

21 set

DISCUSSION Vascular patency depends inter alia on the neutralization of activated clotting factors. At-111 is the main plasma inactivator of T (9) as well as Xa (22). As conventional clotting methods do not allow accurate monitoring of Xa inactivation in presence of thrombin, chromogenic substrates were used in the present study. The specificity of these substrates can be sum-

is

21h

marized

IXACTIVATIOX

OF THROMBIX

k F.Sa

v01.11,s0.2

as follows: At equimolar enzyme concentrations T splits about hundred times faster than does X%; whereas &I splits S-2222 about fourteen times faster than does T (14). The small errors resulting from nunspecificn amidolysis (e-2222 by T, s-2238 by Xa) were reduced to about l/l0 by the use of SBTI and hirudin. We have found that some enzyme preparations of high purity probably contained substantial fractions of denaturated enzyme resulting in a diminished proteolytic relative to amidolytic For this reason, relatively imactivity (unpublished results). pure, but possibly less denaturated preparations were used, although they had been purified by different methods. Preincubation of At-III with excess Xa showed that the ability to inactivate thrombin was completely blocked, and vice versa. Preincubation with excess At-III, and particularly the inactivation rate studies indicated that incubation of At-III with one enzy-me reduced its ability to inactivate the other. These results are in contradiction to the results of Osterud who were unable to measure a decrease in At-III actiet al (7), vity following incubation of At-III with human Xa, and to our pilot experiments. Further studies revealed that our initial experimental conditions had been inadequate (too short incubation and too low amounts of Xa). A relatively low Xa-At-III ratio and too short incubation might also explain the results of Osterud et The present results support the conclusion of Yin (11) al (7). that complete neutralization of At-III with T render the inhibitor incapable of subsequently inactivating Xa and vice versa. The most simple explanation for these findings is that thrombin and Xa compete for the same binding site on At-III. It appears less probable that At-III undergoes sterical changes when bound to one enzyme, which preclude reaction with the other enzyme. The inactivation rate of the enzymes were highly dependent on the At-III concentration. With excess inhibitor, enzyme concentration had little influence on the half-time values. The second order reaction of'Xa inactivation by At-III (Fig. 4). fits with the hypothesis of bimplecular complex formation, similar to that found for thrombin (3). With human rear ents, thrombin was more rapidly inactivated than Xa, even when the two enzymes were present together (Fig.5, Table IV). The finding contrasts with the conclusion of Yin (11) about the preference of At-III. With bovine enzymes and human At-III, Xa was more rapidly inactivated than T (Table IV), in accordance with the conclusion of Yin (11). It will be of interest to repeat these experiments with bovine At-III. the amount of enzymes inIn the present "titration studies", activated at equilibrium by a certain amount of At-III were expressed in units (Table II). As the molar concentrations of the reac ants are not definitely known, the following calculations are based on somewhat assumptive values for specific activity Assuming a molecular weight of and molecular weight. At-III of about 65 000 (p), and a plasma concentration of 0.20 human plasma contains about 3 nmol Atmg/ml, one ml titrated III, which then is the amount in one At-III unit. Assuming molecular weights of bovine anti human thrombin of about 30 000 (3,23) as specific activity of about 2 200 U mg protein (24), the amounts inactivated by 3 nmol At-III corresponds to 2.3-3.3 nmol of these enzymes. These values are not too far from those

s-2238

INACTn..\TIOS

Vol.ll,No.2

OF THROMBIS

215

& F.Xa

The specific actiexpected according to bimolecular reactions. There is, vity of bovine and human Xa is less well established. good reason to believe that approximately identical however, molar amounts of human and bovine Xa were inactivated by a The "amounts" of bovine and human constant amount of At-III. by a factor of expressed in units, deviated Xa inactivated, The unit strength of the Xa solutions about 2.5 (Table II). is conventionally defined by its ability to convert prothromprothromPresumably then, human Xa converts bin to thrombin. bin to thrombin at a slower rate than equimolar amounts of bovine Xa does. The Xa-At-III reaction probably involves an initial proteoThe relatively slower reaction rate for human Xa lytic step. compared to bovine Xa versus both human prothrombin and At-III may then reflect a species difference in proteolytic activity. In a subsequent publication the inactivation of thrombin and Xa in plasma will be reported.

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2.

Identity of plasma YIN, E.T., WESSLER, S. and STOLL, P.J. activated factor X inhibitor with antithrombin III and heparin cofactor. J.Biol.Chem. 246, 3712, 1972.

3.

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4.

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5.

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Antithrombin III, heparin O.R. and TEIEN, A.N. and antifactor Xa in a clinical material. Res. 2, 173, 1976.

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INACTIVATION

OF THROMBIX

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'3.

DENSON,

K.E.lr'.

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