THROMBOSIS RESEARCH 14; 387-397 @Pergamon Press Ltd.1979. Printed in Great Britain

OO&-3848/79/0301-0387 $02:00/o

HEPARIN AND THE INACTIVATION OF THROHBIN BY ANTITHRDHBIN III

Steven Rowalski and Thomas H. Finlay

Departmants of Medicine and Biochemistry New York University Medical Center

New York, N.Y. 10016

(Received 6.6.1978; in revised form 9.11.1978. Accepted by Editor L. Vroman)

We confirm a catalytic role for heparin in the inhibition of thrombin by antithrombin III,* i.e., formation of the inactive thrombin-antithrombin complex is accelerated by a less-than- stoichiometric amount of heparin. Assuming an ordered bi reactant process in which heparin forms a complex with antithrombin that then binds thrombin, we observed the expected direct dependence of the rate of thrombin inactivation on thrombin concentration. The dependence of this rate on antithrombin concentration was anomolous, however, and we attribute this behavior to a kind of “substrate inhibition”.

!NTRODUCTION --

One explanation for the accelerative effect of heparin on the

inhibition of thrombin and other serine proteases by antithrombin

III (AT), is that on binding heparin, AT undergoes a conformational

change which dramatically Increases its rate of reaction with

ser i ne proteases (I -3). In addition, heparln has been reported

to act catalytically, one heparin molecule accelerating the

387

388 HEPARIN & THROMBIN INACTIVATION Vol.lb,No.2/:

formation of many inactive thranbin-AT complexes (4). These suggestions led

US to consider a model for the interaction of thrombln, AT and heparln in

which heparln acts catalytically in an ordered bireactant system where

AT is the obligatory first reactant and thrombin the second, as shown below.

(1) AT + H @AT* H

(2) AT*H + TT&AT*H*T-+AT-T + H

(1+2) AT + T---,AT-T

(H-heparln, Twthrombin)

We hoped to exploit this model to obtain a binding constant of heparin

for AT in the usual way one obtains a Km of an enzyme for its substrate.

This work is part of a larger program whose goal is to determine the binding

constants of various heparin preparations for proteins in the hemostatic

mechanism. We wish to communicate here 1) that heparin does act catalytically,

and 2) that, in its presence, the rate of inactivation of thranbin shown an

unexplained dependence on AT concentration.

MATERIALS AND METHODS

Heparin from porcine gastric mucosa, 158 U/mg (Research plus, Denville,

N.J.) was further purified by affinlty chromatography on AT-agarose by a

modification of the procedure of Hook et al. (5). Fractions eluted with

0.15 M, 0.55 H and 2.0 H NaCl all in 0.05 H trls, pH 7.5, were pooled

separately and the heparln was precipitated with ethanol. The 2.0 M NaCl

pools from several runs were combined to give a preparation with a USP

anticoagulant activity of 330 U/mg. This preparation, which we have called

high affinity heparin (Ka for AT of lo8 M, ref. 3), was used in the present

study. This material did not give rise to the decreased extent of thrunbin

inhibition observed.by Owen with unfractionated preparations of heaprln (6).

The Ka for thrombin, 106 II, was not appreciably different from that of

the starting material (3).

vo1.14,~02/3 HEPARIN & THROMEKCN INACTIVATION 389

AT was purified from cryoprecipitated and aluminum hydroxide-adsorbed

human plasma by affinity chromatography on heparin-agarose essentially as

described by Miller-Andersson et al. (7). An additional ansnonium sulfate

precipitation step followed by gel chromatography on Sephadex G-150 yielded

a preparation homogeneous by SDS gel electrophoresis with an activity of

1400 U/mg, where 1 unit of AT inhibits of 1 unit of thrombin activity.

Human thrombin, 100% A-thrombin, 2300 Wmg, 88.4% activity by titration

with e-nitrophenyl e’-guanidinobenzoate, was generously supplied by

Dr. John W. Fenton II.

Cbz-Gly-Pro-Arg-p-nitroanilide (Chromozym TH) was purchased from

Boehringer-Hannheim. A 1.0 mH stock solution was kept frozen in the dark.

All other chemicals were of the highest grade canaercially available.

Thrombin activity was assessed from the rate of increase in A405 due to

amidolysis of Chromozym TH. Absorbance changes were followed using a Gilford

Model 240 spectrophotometer equipped with a Model 6051 recorder and a

thermostatted cuvette compartment maintained at 37' (experimental details

appear in the legend to Figure 1). The amount of thrombin per assay, kept

constant within a series of experiments by varying the aliquot size

(IO-50 ul), was chosen to give a A ~~~~ of approximately 0.1 A/min in the

uninhibited control samples. Inactivation of thrombin by the heparin-AT

complex was effectively arrested by dilution when added to the cuvette and

by the large molar excess of chromogenic substrate over AT (s IO4 fold).

Thrombin activity (AA 405/min) was taken from the initial slopes of the

recorder tracings.

The heparin level used in these experiments was established by

incubating decreasing amounts of high-affinity heparln with an amount of AT

Sufficient to completely neutralize a given quantity of thrunbin in the

presence of excess heparin; thrombin was then added, and after 30 set the

390 HEPARIN & THROMBIN INACTIVATION Vo1.14,No.2/3

residual amidolytic activity was determined. A range of heparin concentra-

tions was found in which appreciable thrombin activity remained after

sufficient incubation to permit convenient experimental manipulation

( 210 set). The heparin concentration under these conditions was at least

IO-fold lower than that of AT or thrombin.

The mol wt of our heparln preparation, determined by viscometry (8)

before affinity chromatography, was 10,000. We have assuned a mol wt of

36,000 for thrombln (91, and 56,000 for AT (IO). We have also assumed a

theoret’i ca 1

thrombin in

3000 U/mg for pure thrombin and a 1:l stoichianetry of AT to

the inhibited complex (1).

RESULTS

Figure 1 shows that a less-than-stoichiometric quantity of heparin

accelerates the AT-mediated inactivation of thrombin and that increasing

the hepar i n concentration increases this acceleration. Control samples

without AT showed little effect of heparin on thrombin activity. In

controls without heparin, more than 75% of the initial thrombin activity

remained after 120 set of Incubation with AT alone. For various reasons

we have not corrected the values of thrombin activity determined in the

presence of hsparin for those observed in its absence (the so-called

“progressive” antithrombin activity). First, because of the short incubation

times employed, only a small fraction of the observed inhibition was due

to “progressive” antithrcmbin activity. Second, AT may exist as an

equilibrium mixture of active and inactive conformers; if heparin were

to bind more tightly to the active conformer, there would be no “progressive”

antithrombin activity in the presence of heparin.

vol.lb,No.2/3 IiEPARIN & THROMBIN'INACTIVAT~ON 391

0.10 z - 2 0.08

6 ‘E a ‘, 0.06

=$ g 0.04

is-

z 0.02

20 40 60 80 100 120

INCUBATION TIME (Sec.)

FIGURE 1

Decrease in thrombin activity on incubation with AT and heparin. Appropriate

dilutions of AT, heparin and thrcmbin were made in 0.15 H NaCl, 0.05 M tris,

PH 7.5. AT and heparin were preincubated for 60 set In plastic coagulation

cups in a 37’ -thermostatted block. Thrombin was added and, at the indicated

intervals, aliquots were introduced into semi-microcuvettes containing 0.1 mM

substrate, 0.15 H NaCl, 0.05 H tris, pH 8.3, in final volume of 750 ul.

Absorbance at 405 nm was followed as a function of time, and thrombin

activity ( AA405 /min) was taken from initial slopes. AT - 2.8 X 10m7 H;

O-- 4 X 10” M heparin; A-- 8 X lo-’ M heparin; O-- 12 X lo-’ M heparin.

Figure 2 shows the effect of heparin concentration on the rate of AT-

inactivation of thrombin. The data were taken from the initial slopes of

the curves in Figure 1 and other experiments not shown. Noe that for a

given heparin concentration, increasing the AT concentration decreases the

rate of thrombin inactivation, a point more fully illustrated in Figure 3.

392 HEPAFUN & THROMBIN INACTIVATION Vol.lk,No.2/3

4 8 12 HEPARIN (X IO-‘Ml

FIGURE 2

Effect of heparin concentration on the rate of.inactivation of thrombin by

AT. Thrombin = 1.2 X 10 -7 H; AT = 2.8 x 10 -7 M (a), or 5.6 X 10m7 H ( 0).

Figure 3 shows the effect of varying AT concentration on the rate of

inactivation of thrombln at constant heparin and thrombin concentrations.

In the region below 1.4 X 10m7 H AT, it was technically difficult to obtain

reliable experimental data, hence the dotted portion of the curve is inferred.

However, the curve intersects the vertIca1 axis at the rate due to heparin

alone which at low heparin is essentially zero. At the other extreme,

as the AT: heparin ratio increases, the inactivation rate should

assymptotically approach that due to AT alone.

vo1.14,No.2/3 HEPARIN & TI-IROMBIN INACTIVATION

AT (XiO%l)

FIGURE 3

Effect of AT concentration on the rate of heparin-accelerated

of thrombin. Thrombin - 1.2 X 10 -7 II; heperin = 8 X 10” H.

1 2 3 4 5 TH ROMBIN ( X 10’‘Id

FIGURE 4

393

6

inectivation

Effect of thrombin concentration on the heparfn-accelerated rate of thrombin

inactivation by AT. AT - 2.8 X 10 -7 M; hepartn - 8 X 10” H.

394 HEPARIN & THROMBIN INACTIVATION VO~.~'+,NO.~/J

As shown in Figure 4, ‘at constant levels of AT and heparin, the rate of

thrombin inactivation is directly dependent on thrombin concentration.

DISCUSSION

. In our model for the heparin-accelerated AT, inactivation of thrombin,

we postulate an ordered bireactant scheme in which an initial heparin-AT

complex combines with thrombih to form an inactive AT-thrombin conjugate

with concomitant release of heparin. This model is plausible on experlmentai

grounds (11) and on the circumstantial evidence that heparin accelerates the

slow inhibition by AT of various serine proteases suggesting an effect of

heparin on AT rather than on protease. We felt that the use of high

affinity heparin with its stronger binding to AT than to thrombin (Ka of

IO8 M ~9 lo6 M for thrombin) would minimize possible complicating effects

of heparin binding to thrombin on the interpretation of experimental results.

Our observations, confirming a catalytic

explained by a weaker affinity of heparin for

the initial reactants (12), the net effect of

the heparin.

role for heparin, may be

the inhibited product than for

which would be a recycling of

The decrease in the thrombin inactivation rate on increasing AT

concentration in the presence of heparin would appear to be inconsistent with

our postulated scheme in which heparin and AT form a complex that then reacts

with thrombin in an overall stoichiometry of l:i:l.

However, we retain the model and will consider several explanations for

the apparent inconsistency.

WI thin the framework of the postulated ordered scheme a higher-than-first

order dependence on heparin concentration of the rate of AT-heparin complex

formation, i .e., involvement of more than one molecule of heparin in the

reactive complex with AT, could explain the observed decrease in the rate of

vo1.14,No.2/3 HJZPARIN & THROMBIN INACTIVATION

thrombin inactivation on increasing AT concentration. However, only one

heparin binding site per AT molecule has been detected with highly

fractionated or high affinity preparations of heparin such as used in this

study (2) and consequently we reject this explanation.

Our inclination is to ascribe the decreased rate of thrombin

inactivation on increased AT concentration to unproductive binding of AT

to heparin which in pursuing the analogy to enzymatic reactions we term

“substrate inhibition”. Heparin is generally considered to be a linear

mucopolysaccharide with a more-or-less regular repeating structure. It

is conceivable then, that an AT molecule could bind at several places along

the heparin chain wlth a Ka for a given site dependent on the local

microenvironment. Binding of an AT molecule at a particular lpcus might

stericaliy hinder the binding of another AT molecule at an adjacent, more

reactive site on the heparin molecule. Such interference wi th reactive

intermediate formation would be expected to increase with increasing AT

concentration.

The observed effect could also be explained by another type of

“substrate inhibition”. AT could bind reversibly to thrombin to form a

non-covalent AT-thrombin complex (AT T) as shawn belar. This of course

happens during the so-called “progressive” antithrombin reaction. If the

rate constants for this reaction were of the right

AT+ T= AT*T P AT-T

magnitude, then in the presence of excess AT, the effective thrombin

concentration would be reduced. As we have shown a direct dependence of

the rate of thrombin inactivation on thrombin concentration (Fig. 4),

increasing the AT concentration in the presence of catalytic amounts of

heparin could also result in a decreased rate of thrombin inactivation.

395

396 HEPARIN & THROMBIN INACTIVATION Vo1.14,No.2/3

WC have not as yet devised a strategy to distinguish

two types of “substrate inhibition” to which we attribute

effect.

between these

the observed

Although frustrated in this inltial attempt at obtaining a Ka of

heparin for AT by a kinetic method, we consider it useful to communicate

the unanticipated dependence on AT concentration of the heparin-accelerated

inactivation of thrombin.

ACKNOWLEDGMENTS

This work was supported in part by Program Project Grant

the National Heart, Lung and Blood Institute and NIH Research

Deveiopmant Award HL 00277 (to T.H.F.).

1.

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3.

4.

5.

6.

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8.

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