the chemi~try vol. 255, no. 21, issue of november 10, pp. … · 2001-09-07 · the journal of...

THE JOURNAL OF BIOLOGICAL C H E M I ~ T R Y Vol. 255, No. 21, Issue of November 10, pp. 1 0 0 8 1 - 1 ~ , 1980 Printed in U.S.A.

The Kinetics of Hemostatic Presence of Low Molecular

Enzyme-Antithrombin Interactions in the Weight Heparin*

(Received for publication, December 26, 1979, and in revised form, June 12, 1980)

Robert E. Jordan, Gary M. Oosta, William T. Gardner, and Robert D. RosenbergS From The Sidney Farber Cancer Institute, Beth Israel Hospital, and the Hartlard Medical School, Boston, Massachusetts 021 15

The kinetics of inhibition of four hemostatic system enzymes by antithrombin were examined as a function of heparin concentration. Plots of the initial velocity of factor Xa-antithrombin or plasmin-antithrombin interaction versus the level of added mucopolysaccharide exhibit an ascending limb and subsequent plateau regions. In each case, the kinetic profile is closely correlated with the concentration of the heparin. antithrombin complex formed within the reaction mixture. A decrease in the velocity of inhibition is not observed at high levels of added mucopolysaccharide despite the generation of significant quantities of heparin-enzyme interaction products. The second-order rate constants for the neutralization of factor Xa or plasmin by the mucopolysaccharide *inhibitor complex are 2.4 X lo8 M" min" and 4.0 X lo6 M" min", respectively. These parameters must be contrasted with the similarly designated constants obtained in the absence of heparin which are 1.88 X lo5 1"' min" and 4.0 X lo4 rnin", respectively.

Plots of the initial velocity of the factor IXa-antithrombin or the thrombin-antithrombin interaction versus the level of added mucopolysaccharide exhibit an ascending limb, pseudoplateau, descending limb, and final plateau regions. In each case, the ascending limb and pseudoplateau are closely correlated with the concentration of heparin- antithrombin complex formed within the reaction mixture. Furthermore, the descending limb and final plateau of these two processes coincide with the generation of increasing amounts of the respective mucopolysaccharide-enzyme interaction products. The second-order rate constants for the neutralization of factor IXa or thrombin by the heparinoantithrombin complex are 3.0 x 10' M" min" and 1.7 X IO9 M" min", respectively. The second-order rate constants for the inhibition of mucopolysaccharide-factor 1Xa or mucopolysaccharide-thrombin interaction products by the heparin * antithrombin complex are 2.0 X lo7 M" min" and 3.0 X 10' M" rnin", respectively. These kinetic parameters must be contrasted with similarly designated constants obtained in the absence of mucopolysaccharide which are 2.94 X lo4 M" min" and 4.25 X lo5 M-' min", respectively.

Thus, our data demonstrate that binding of heparin to antithrombin is required for the mucopolysaccharide-dependent enhancement in the rates of neutrali-

* This work was supported by Grant HLB-19131 from the National Institutes of Health. 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 U.S.C. Section 1734 solely to indicate this fact.

$ Recipient of the Established Investigator Award of the American Heart Association. To whom reprint requests should be addressed.

zation of thrombin, factor IXa, factor Xa, or plasmin by the protease inhibitor. Furthermore, a careful comparison of the various kinetic constants suggests that the direct interaction between heparin and antithrombin may be largely responsible for the kinetic effect of this mucopolysaccharide.

Heparin functions as an anticoagulant by dramatically ac- celerating the velocity at which antithrombin neutralizes serine proteases of the hemostatic mechanism (1). We have provided evidence that the mucopolysaccharide acts by binding to antithrombin and thereby increasing the rate of formation of enzyme. inhibitor complexes (2, 3). However, other investigators have claimed that heparin-enzyme interactions are of critical significance. On the one hand, Machovich (4), Smith and Craft (5), Griffith (6), and Griffith et a2. ( 7 ) have proposed that the mucopolysaccharide binds preferentially to hemostatic system serine proteases and that the resultant complexes are rapidly inactivated by free antithrombin. On the other hand, Gitel(8) and Holmer et al. (9) have postulated that heparin is initially bound to antithrombin but is subsequently able to interact with hemostatic system enzymes.

In the present communication, we examine the kinetics of inactivation of four hemostatic system serine proteases by antithrombin as a function of heparin concentration. These data are compared to the levels ofvariousmucopolysaccharide - protein complexes within the respective reaction mixtures. The resultant analyses allow us to estimate the relative importance of these three potential mechanisms of heparin action.

MATERIALS AND METHODS'

I h c , n l ~ d l ~ Ihr I"( I - . i ~ i f z i l n h v d r l d r ~ ~ t l l a \ ~ C L Z ~ I L . d ~ t l i l f y ui ( I X m , I !mq and

\ r k I l l g l i nJ \ o ~ ~ P * I T . lBo \ r>n , h t G ~ s Ihr I'lil-R:iriOlt h ~ f l i * p c ~ l i l i P C C I Y I T I ~ T l ~ l t ) thoroh!dr>dc i h r t > ,I < p e c l ! l ~ i i c t l r ~ t i o f ~ 5 C>immol*cre [ , u r t h r \ c i l r r o ~ ~

u h t i l l l c d t i o m Hloiheml<ol and h u i l e s ~ Corp , l iu rhani . (sllt. l'illvbrene ~ u l > p I l e d h \ the A I d r l i h l h r m l c a l Co. , > l ~ l u a ! l i c c . WI,. lllr ~l-m~I l lv i - l l -manl lO, l r la

~ u i ~ l r - c ~ l b r r ~ r c a ~ r n f cr.'d< or hefler. -15 ~ > r O v l r l v d / I \ ' file \>>:ma (hcrn l ta l L o . , C I I U U I . , '10 i l l u t h c r c I ! c m ~ c ~ I %

L l ~ l d % Hdhhlt h r a l n ihr0rnbopla i t ln * a b i .b f i lned fron O r t h o L,ls*nO\f~~r.Hiil-ltin.

. , f :IO IPI ~:d~ ,and m I ' I I J - I O ~ . ~ ~ ~ O ~ W W ~ I , .pccift i d l t ~ > ) t t o i I Q ; mrlmi .erc

h I l"".lthl" i a 5 I""ct,ircd trom *\>ocI.fed iOnCe"f rore \ , h"ad\ldr. L . I .

Protern. Ilumrn f a L f o r l L 8 , Ihman f d r t o r \ r , Ihuman thromhln . lhman ~ p l a s m ~ r l u id h ; n l m ; l ~ t i r l , r o m h ~ " ~ B T C )p?updrcd I" p h b s l c a l l \ l h o m o ~ e n ~ ~ u ~ form I,? T e ~ h n l y o r \ our-

human l.*Lrc'r ) U Z T L r i n l ~ f v d in a p u r ~ C i e d r t a t c h> - ~ h e n a p ~ w i o u s l r r cpor t~<I Ilncil 111 tile lprucedlei: ~ i n n t i n l i 8 t x o n of f h x l 1>1u11 IIllJ. IIurn~n prothramh~r ,%##d

t r u r T l i l < 1 . 1 h O P a t o ~ 1 ,111 llllmsn factor i l l 1 has ohrslned 111 p ~ f ~ i r l l y Ipurlt iCd t u rn , Ih, ernplui lng Tllc l l r \ t i i t e p r of fhc lp~ocedure of I lmcrmrn, CT a l 11.1 6 n i 1 n e fai.tur \ wai ~prc[lareJ h> IhU method o f l lorts ( 1 3 ) Illman lplafelet I .~ . for 4 L.%\ p c n r r u ~ i \ l i . l p ~ u \ > J c J by IUr Hohcrt Hand,,, Bailor!, hlaar l a c t o r IT*-

' "Materials and Methods" are presented in the miniprint as prepared by the authors. Miniprint is easily read with the aid of a standard magnifying glass. Full-size photocopies are available from the Journal of Biological Chemistry, 9650 Nockville Pike, Bethesda, Maryland 20014. Request Document 79M-2610, cite authors, and include a check or money order for $1.20 per set of photocopies. Full- size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

10081

10082 Inhibition of Thrombin, Factor IXa, Factor Xa, and Plasmin

Inhibition of Thrombin, Factor IXa, Factor Xa, and Plasmin 10083 crlhed hr t w o d~*socldflon ~ ~ n s f a n t s whxch are I ? f o l d FTertcr than t h l f O f

t e . 8 ~ and .mf~fhrornb~n can be m n i l d u r e d a% t w o ~ n d e l w n d e n t equllhrla * f thc the heparln-anf,fhronhln complex. Thur, t h e hindlng of hrp1rm t o l ~ r l l l e lPr0-

r e a c t a n t c o n L r n t r a t ~ o n s employrd ,n the i l n e f ~ c merirurrment~ On th&> ha\,\. iq i ia f lan5 i and LL in the p i e ~ l o u s C U ~ ~ U ~ L C B ~ L U ~ o f fhr, we IllL bere u l i l l -

9 i t i o n producfr The dz9sociatlon C o I ~ ~ t P o t s "if, and k l f { q W e r e ref SI :d t o crlculste fhr conccnfriflon of fhe two f ' CI of hepar>"-thrumhln L n f e r ~

kll x 1 0 " M . ~e,pcct~uel). PuTfhermorP. Lt~uatxon Ill o t h l 4 rem*( x a s e m P l * ~ ~ r . d t o compute the l e v c l i of thc heparin-anfithrombln complrx vlth Kib,, eiiual f O i :4 x lllrd El (II'I

RESULTS AND DISCUSSION

Interactions between Factor IXa, Factor Xu, Thrombin, or Plasmin with Antithrombin-The kinetics of various hemostatic enzyme-antithrombin interactions were examined in the absence of heparin. This was accomplished by incubating equimolar amounts of serine protease with inhibitor and meas- uring the residual levels of enzymatic activity as a function of time. When the inactivation of factor IXa was investigated, the two proteins were employed at the same final concentrations of either 1.4 X 10" M or 7.0 X 10" M. When the neutralization of factor Xa was examined, enzyme and inhibitor were maintained at the same final concentrations of either 2.5 X lo-' M or 1.25 X 10" M, respectively. When the interaction of thrombin with antithrombin was studied, the com- ponents were utilized at the same final concentrations of either 1.0 X M or 2.0 X M, respectively. When the inhibition of plasmin was analyzed, the two reactants were used at the same final concentrations that varied between 5.0 X M and 1.2 X M. The biomolecular rate constants for the four enzyme-inhibitor interactions h: were calculated as described under "Materials and Methods" and are provided in Table I.

Catalytic Nature of the Interaction between Factor IXa, Factor Xu, and Plasmin with Antithrombin in the Presence of Heparin-Heparin functions as a catalyst in the thrombin- antithrombin interaction (3). To determine whether the mucopolysaccharide customarily acts in this manner, we analyzed the kinetics of various enzyme-antithrombin interactions in the presence and absence of trace amounts of heparin. When the neutralization of factor IXa was studied, enzyme and inhibitor were maintained at a final concentration of 1.4 X

M. When the inhibition of factor Xa was investigated, the proteins were employed at a final concentration of 2.5 X M. When the inactivation of plasmin was examined, the reactants were utilized at a final concentration of 3 X 10" M. In the absence of mucopolysaccharide, 10 to 20% of the three enzymes were able to interact with antithrombin if incubation times were limited to 10 min (Fig. 1). Heparin had no direct effect upon the activity of these serine proteases when the inhibitor was omitted (not shown). However, the rates of enzyme neutralization were dramatically augmented when small amounts of mucopolysaccharde were added to reaction mixtures that contained antithrombin. Indeed, the initial velocity of these reactions increased progressively as the molar ratios of heparin to antithrombin were elevated from 0 to 3.5410 (factor IXa), 0 to 20% (factor Xa), and 0 to 20% (plasmin). Furthermore, the final extent of each interaction was independent of mucopolysaccharide concentration since a com- plete loss of enzymatic activity was approached at all levels of added heparin. The latter observations emerged from additional experiments that are not summarized in Fig. 1. Given the 1:l molar binding stoichiometry of this complex polysaccharide (see below), we calculate that heparin must be re- utilized 5 to 100 times during the course of each of these reactions. Thus, our data suggest that the mucopolysaccharide functions as a catalyst in all enzyme-antithrombin interactions.

Heparin could act in this manner if it were released from antithrombin upon formation of the enzyme.inhibitor complex. In this case, the discharged mucopolysaccharide could bind anew to free antithrombin and thus promote multiple

10084 Inhibition of Thrombin, Factor IXu, Factor Xu, and Plasmin

TABLE I Summary of kinetic and bindingparameters

See text and "Materials and Methods" for additional details. Kinetic or binding parameters Thrombin Factor IXa Factor Xa Plasmin

kl* (rate constant in the absence of 4.25 X 10:' M" min" 2.94 X 104 M" min" 1.88 X 10" M " min" 4.0 X lo4 M" min I

hl (rate constant with the inhibitor 1.7 X 10q M " min"' 3.0 X 1 0 ~ M" min" 2.4 X 1 0 ~ M" min" 4.0 X IO" M min" heparin)

saturated with heparin) k'l =,

kI 4,000 10,200 1,280 100 kl

kz (rate constant with the enzyme 3.0 X IO" M-' min- I 2.0 X 10' M" min" 2.4 x IO" min" 4.0 X 10" M" min I

and inhibitor saturated with heparin)

k2 k1

k t p =7

KI,~ss" 8.0 X 10" ~ / 8 . 0 X 10" M 2.58 X 10" M 8.73 X lo-'' M -1.0 x M

700 680 1,280 100

The K ~ , I S S values were determined as described in the preceding paper (10).

0 2 4 6 8 1 0 1 0

c

2 4 6 8 1 0

FIG. 1. The acceleration ofhemostatic enzyme-antithrombin interactions with limiting concentrations of heparin. A, factor IXa-antithrombin interactions (A). The inhibition of enzyme by inhibitor in the absence of heparin with the concentrations of the two proteins set at 1.4 X 10 ' M (-j; the effects of heparin when present at concentrations of 1.4 X lo-' M ( 1 1 1 1 1 1 ) and 5.0 X 1 0 ~ '' M (-//-) are also shown. The uppermost curue ( \ \ \ \ ) represents a reaction mixture identical with that of -//- except that purified factor IXa. antithrombin complex was added at a level of 2.8 X IO-" M. E , factor

rounds of enzyme neutralization. To examine this hypothesis, we employed heparin that had been radiolabeled with [14C]acetic anhydride or conjugated with l"I-containing acy- lating agent. The resultant mucopolysaccharide possessed from 0.01 to 0.30 radiolabeled groups/molecule of mucopolysaccharide. These modifications of the heparin molecule do not significantly affect the specific anticoagulant activities of the mucopolysaccharide (not shown).

These preparations of mucopolysaccharide were utilized to examine the stoichiometry of the heparin. antithrombin interaction. This was accomplished by mixing radiolabeled heparin with inhibitor at concentrations of 1.23 X M and 6 X M, respectively, and chromatographing the resultant solution at flow rates of 1.5 ml/h on a column of Sephadex G-100 (0.9 X 200 cm) equilibrated with 0.2 M NaCl in 0.01 M Tris-HC1, pH 7.5. The column effluents were monitored for protein by absorbance measurements a t 280 nm and for mucopolysaccharide by p or y counting. These measurements revealed two discrete peaks. The first peak contained protein as well as mucopolysaccharide and represented the heparin-antithrombin complex. The second peak consisted only of labeled heparin and signified the presence of free mucopolysaccharide. The molar ratios of heparin/antithrombin within the first peak were 0.95 k 0.02. This value is based upon the known molecular weights of the two reactants, their extinction coef-

Xa-antithrombin intereactions (0). The inhibition of enzyme by antithrombin in the absence of heparin with protein concentrations set at 2.5 X lo-" M (-). The effect of heparin when present at concentrations of 2.5 X IO"" M ( 1 I I 1 1 I ), 5.0 x 10"" M (- - -), 2.5 X IO M (. . . .), and 5.0 x M (-//-j. C, plasmin-antithrombin interactions (0). The inhibition of enzyme by antithrombin-in the absence of heparin with protein concentrations set at 5.0 X 10 ' M (-1. The effect of heparin when present at 1.0 X lo" M (-//-) (see text for additional details).

ficients, and the specific radioactivity of the mucopolysaccharide.

The radiolabeled heparin- antithrombin complex isolated during the previous set of experiments was rechromato- graphed on Sephadex G-100 under conditions identical with those utilized above. As shown in Fig. 2, only -6% of the bound mucopolysaccharide dissociated from the protease inhibitor. This feature of the interaction permitted us to determine whether tightly bound heparin is discharged from antithrombin foilowing inactivation of enzyme by the mucopolysaccharide. protease inhibitor complex.

To this end, multiple reaction mixtures were prepared in which the molar ratios of factor IXa, factor Xa, or plasmin added to radiolabeled heparin. antithrombin complex were progressively increased from 0 to 1.0. The resultant solutions were examined by appropriate enzymatic assays, sodium do- decyl sulfate gel electrophoresis, and gel filtration on Sepha- dex G-100. As anticipated, no free enzyme could be detected in any of the samples (not shown). In addition, an electropho- retic examination of these solutions showed the expected increase in the intensity of the various enzyme-inhibitor bands uis-6-uis the antithrombin band as the amount of added enzyme was elevated (not shown). Furthermore, Sephadex G- 100 g t l filtration patterns revealed that an increasing fraction of the isotopically labeled heparin was discharged from anti-

Inhibition of Thrombin, Factor IXa, Factor Xu, and Plasmin 10085

thrombin as the level of added enzyme was raised and that this ligand appeared in a region of the chromatogram typical of free mucopolysaccharide (Fig. 2, A, B, and C). Fig. 2 0 depicts the relationship between the presence of heparin released from antithrombin and the molar ratios of factor IXa, factor X, or plasmin individually added to the mucopolysaccharide. protease inhibitor complex. These data were obtained from chromatograms similar to those previously described. It is evident that these two parameters are linearly related with an associated r value of 0.98. The slope of the line by least square fit is -0.93, which suggests that one molecule of heparin is released from antithrombin by each molecule of factor IXa, factor Xa, or plasmin added. This interpretation is supported by the x intercept of the plot (extrapolated) which indicates that full release of heparin occurs at an average enzyme-antithrombin molar ratio of 1.03.

The radiolabeled heparin released from antithrombin during each of the previous sets of experiments was collected, concentrated, and individually dialyzed against 0.2 M NaCl in 0.01 M Tris-HC1, pH 7.5. A slight molar excess of antithrombin (10%) was added to the mucopolysaccharide and the resultant mixture was subsequently examined by Sephadex G-100 chromatography. We observed that the labeled heparin eluted with the protease inhibitor (not shown). These results demonstrate that mucopolysaccharide discharged from the various enzyme. antithrombin complexes retains its ability to bind anew to free antithrombin.

If formation of the enzyme.inhibitor complex induces release of mucopolysaccharide, then heparin must possess a low avidity for these interaction products as compared to free

antithrombin. To examine this hypothesis, we determined whether the factor IXa-antithrombin interaction product could compete with free inhibitor for available heparin. This analysis was conducted by adding factor IXa at final concentrations of 1.4 X 10" M to two separate reaction mixtures. The first reaction mixture contained factor IXa. antithrombin complex, antithrombin, and heparin a t final concentrations of 2.8 X M , 1.51 X lo-' M, and 5.0 X M , respectively. The second reaction mixture was of identical composition except that the factor IXa-antithrombin interaction product was omitted. Both reaction mixtures exhibited identical rates of enzyme neutralization (Fig. 1A). We have calculated that if the factor IXa. inhibitor complex were capable of binding to heparin with a dissociation constant 170-fold weaker than that of antithrombin, then the concentration of free mucopolysaccharide within the first reaction mixture would be decreased to 4.2 X M. Additional experiments (not shown) indicate that a reduction in heparin concentration of this magnitude would result in a statistically significant decrease in the velocity of factor IXa inactivation. This suggests that the interaction between heparin and factor IXa + antithrombin complex must be characterized by a dissociation constant which is 1.5 X lo-> M or higher.

Attempts were made to analyze the binding of mucopolysaccharide to the other enzyme. inhibitor complexes in a manner similar to that described above. Meaningful data could not be obtained due either to the instability of enzyme-inhibitor interaction products or to the relative insensitivity of the specific assay system to catalytic phenomena.

The Kinetics of Interaction of Heparin and Antithrombin

EFFLUEM WLUME(rnl)

FIG. 2. Sephadex G-100 chromatography of the heparin-an- tithromhin complex and the displacement of bound mucopolysaccharide from the protease inhibitor caused by the admix- ture of specific serine proteases. A, results obtained with the addition of factor IXa (M). Isotopically labeled heparin. antithrombin complex alone (- - -). Isotopically labeled heparin. antithrombin complex to which 0.45 (-), 0.63 (- - -), or 0.80 (-. . .-) molar equivalents of factor IXa were added. E , results obtained with the addition of factor Xa (m). Isotopically labeled heparin-antithrombin alone (- - -). Isotopically labeled heparin.antithrombin complex to which 0.20 (-), or 0.35 (- - -), or 0.60 (-. . .-) molar equivalents of

MOLES ENZYME ADDED MOLES HEPARIN+iNTITHROMBIN COMPLEX

factor Xa are added. C, results obtained with the addition of plasmin (0). Isotopically labeled heparin. antithrombin complex alone (- - -). Isotopically labeled heparin.antithrombin complex to which 0.33 (- --),or 0.90 (-. . .-) molar equivalents of plasmin are added. D, plot of the percentage of isotopically labeled heparin that remains bound to antithrombin uersus the molar equivalents of factor IXa (A), factor Xa (m), or plasmin (e) which are added to the mucopolysaccharide .protease inhibitor complex. Each point represents data derived from individual reaction mixtures that have been analyzed by Sephadex G-100 chromatography (see text for additional details).

10086 Inhibition of Thrombin, Factor IXa, Factor Xu, and Plasmin

with Factor ZXa, Factor Xa, Plasmin, and Thrombin-In the sections below, we provide a detailed kinetic examination of the various heparin-antithrombin-enzyme interactions. In each case, the initial velocity of a given reaction was experimentally determined as a function of mucopolysaccharide concentration. Subsequently, we at,tempted to correlate these kinetic profiles with the levels of heparin. antithrombin complex or mucopolysaccharide-enzyme interaction product within the respective incubation mixtures. Concentrations of the above species were calculated from experimental data provided in the preceding communication (10).

We hoped that this analytic approach would allow us to ascertain whether the interactions of heparin with enzymes or with antithrombin, or with both were critical to the mucopolysaccharide-dependent acceleration of these processes. In cer- tain instances, these issues could be settled by qualitatively comparing the various kinetic profiles with the levels of the specific mucopolysaccharide-protein interaction products. On other occasions, quantitative correlations of these two types of data were required. In these instances, Equation IV (see below) was utilized to predict the initial velocities of enzyme- inhibitor interactions from the known concentrations of reactants. These theoretical values could then be directly contrasted with those obtained from actual experimental observations.

The following equation provides the relationship between the initial velocity of enzyme neutralization, the concentration of mucopolysaccharide added, and the level of enzyme employed. This algebraic expression is valid for reactant concentrations and experimental conditions utilized in the studies cited below’:

Initial velocity = h ( l - C ) / ? ~ [ E , , ~ ’ + hc kL[E , i ]L (IV)

where [E,,] is the initial concentration of enzyme, h , is the bimolecular rate constant for the interaction of a given enzyme with the heparin. inhibitor complex, hr is the bimolecular rate constant for the interaction of a given heparin.enzyme complex with the heparin.inhibitor complex, 6 is the fractional saturation of antithrombin with mucopolysaccharide, and c is the fractional saturation of enzyme with mucopolysaccharide. The latter two parameters are functions of heparin and protein concentrations and can be directly calculated from the dissociation constants KZks and KEFss, respectively.

It should be noted that KEks is significantly smaller than K::;,kh (10) and h1 is often larger than k , (see below). On this basis, Equation IV predicts that plots of initial velocity uersus heparin concentration should exhibit four phases. The first phase would constitute the ascending limb of the kinetic profile that is produced by increasing levels of heparin. an- ’ We have assumed that hemostatic enzyme-antithrombin inter-

actions can be quantitatively described by the following equilibria: k

E + A - I . E - A k ,

This mechanism is in excellent accord with the kinetic analyses of other investigators (17, 18). If this model were appropriate, the rate constants measured throughout our study would be equivalent to h .

A plausible alternative to the simple situation outlined above is provided by the following equilibria:

1, h

E + A i E * A L E - A ’, , 1, I,

Where E*A represents a “loose” complex and /ZI >> k I >> k11. This mechanism is similar to those proposed for other protease inhibitors. If this model were appropriate, the rate constants quantitated throughout our investigation would be equivalent. to hu X ( k I / ~+ I ) . However, this should not alter our conclusions to any significant extent.

tithrombin complex (0 < 6 < 1). The second phase would represent a plateau or pseudoplateau of constant initial velocity which reflects the saturation of antithrombin by mucopolysaccharide (6 = 1). The third phase would encompass the descending limb of the kinetic profile caused by the formation of increasing amounts of heparin-enzyme interaction product (6 p 1; 0 < c < 1). The fourth phase would consist of a plateau of constant initial velocity generated by the saturation of serine protease with mucopolysaccharide ( h E 1, c = I).

The experimental data provided below will show that the first two phases of the initial velocity plot were observed for all enzyme-inhibitor interactions. It will also reveal that the third and fourth phases of this profile were absent when the kinetics of inhibition of factor Xa and plasmin were examined. The latter result would occur if mucopolysaccharide. enzyme complexes were inhibited by heparin-antithrombin interaction product a t reaction rates similar to that of free enzyme ( k , = k z ) . Alternately, the above phenomena might happen if neg- ligible amounts of mucopolysaccharide. enzyme complex were generated under the conditions of the assay (c = 0).

Interaction of Heparin with Factor Xu and Antithrombin As Well As Plasmin and Antithrombin-Fig. 3, A and B , shows plots of the initial velocities of the factor Xa-antithrombin interactions as well as the plasmin-antithrombin interactions as functions of mucopolysaccharide concentration. We note that increasing rates of enzyme neutralization are apparent as the levels of heparin are raised from -1.0 X lo-“ M to -5 X 10”’ M. Thereafter, this parameter is relatively constant with further increments in mucopolysaccharide concentration. The levels of heparin. antithrombin complexes, heparin-enzyme interaction products, and free enzymes are also provided as functions of mucopolysaccharide concentration. The levels of these molecular species have been calculated as described under “Materials and Methods.”:’

It is obvious that a remarkable correlation exists between the ascending limbs of the initial velocity profies of factor Xa and plasmin neutralization and the levels of heparin-antithrombin complex. I t should also be noted that the regions of constant initial velocity coincide with the saturation of inhibitor by mucopolysaccharide. No such correspondence is seen between the initial velocity plots and the concentrations of heparin-enzyme interaction products. It is also apparent that a progressive reduction in the levels of free enzymes does not result in a proportional decrease in the initial velocities of factor Xa or plasmin inactivation. These results st,rongly suggest that the heparin. antithrombin complex, rather than heparin-enzyme interaction products must be responsible for the mucopolysaccharide-dependent acceleration of these reactions. In addition, the heparin. antithrombin complex appears able to neutralize heparin-factor Xa or heparin-plasmin interaction products at approximately the same rate as free enzyme. This conclusion is tentative in the case of the heparin. plasmin complex since there is some uncertainty as to the absolute levels of this mucopolysaccharide-enzyme interaction product (see Footnote 3) . Furthermore, by utilizing data within the plateau regions of the initial velocity plots, it is possible to calculate valid bimolecular rate constants for the interactions of factor Xa or plasmin with the heparinsanti- thrombin complex. This is permissible since the inhibitor is saturated with mucopolysaccharide and recycling of heparin

I As demonstrated in the previous communication (IO), the avidity of plasmin for heparin is minimal. Indeed, it has been difficult to accurately define KEY;:, with our current methodology. However, we calculate that this parameter is -1 X 10.‘ M (IO). Based upon this estimate, we present profiles of the maximum possible levels of heparin. plasmin complex and free enzyme as functions of mucopolysaccharide concentration.

Inhibition of Thrombin, Factor IXa, Factor Xa, and Plasmin 10087

has been eliminated. The values of these parameters are given in Table I and are compared to the bimolecular rate constants of these interactions obtained in the absence of mucopolysaccharide.

Interaction of Heparin with Factor IXa and Antithrombin A s Well As Thrombin and Antithrombin-Fig. 4A provides a plot of the initial velocity of factor IXa-antithrombin interaction versus the concentration of mucopolysaccharide utilized. It is evident that this profiie exhibits the four phases predicted by Equation IV. In the same figure, the levels of heparin antithrombin complex, heparin. factor IXa complex,

HEPARIN CONCENTRATION(M)

HEPARIN CONCENTRATION (Mi

FIG. 3. Correlation between the binding of heparin to factor Xa or plasmin as well as antithrombin, and the ability of the mucopolysaccharide to accelerate enzyme-inhibitor interactions. A, correlation between the binding of heparin to factor Xa as well as antithrombin and the ability of the mucopolysaccharide to accelerate factor Xa-antithrombin interactions. 0- - -0, initial velocity; A- . .-A, concentration of heparin. antithrombin complex; 0- . -0, concentration of free factor Xa (factor Xa-[heparin . factor Xa complex]); A. - . .A, concentration of heparin. factor Xa complex. B, correlation between the binding of heparin to plasmin as well as antithrombin and the ability of the mucopolysaccharide to accelerate plasmin-antithrombin interactions. O " - O , initial velocity; A- .A, concentration of heparin. antithrombin complex; 0- . -0, concentration of free plasmin (plasmin-[heparin- plasmin complex]); A- . - . .A, concentration of heparin. plasmin complex. The concentrations of the various heparin. protein complexes at given levels of mucopolysaccharide were calculated from the binding equations and dissociation constants provided in the preceding communication (10). These procedures are summarized under "Materials and Methods." The kinetics of enzyme-antithrombin interactions were also examined in the presence of varying concentrations of heparin. The initial velocities of these processes are plotted as kE? with k evaluated according to Equation I and Eo equivalent to the nominal starting concentration of enzyme. The solvent employed in the kinetic analyses was identical with that used for obtaining binding curves cited above. Each point in the kinetic profiles represents the average of at least two separate enzyme inactivation studies (see text for additional details).

HEPARIN CONCENTRATION (MI

588~10.~ t 111 I LIIIIII4 I 1 1 1 1 1 1 1 1 I l l l l l U L 1 1 1 1 1 1 1 1 1

10-9 10-8 10-7 10-6 10-5 10-4

HEPARIN CONCENTRATION (M)

FIG. 4. Correlation between the binding of heparin to factor IXa as well as antithrombin and the ability of the mucopolysaccharide to accelerate factor IXa-antithrombin interactions. A, comparison of the levels of heparin-protein complexes and free factor IXa with the kinetics of the factor IXa-antithrombin interaction. o " - o , initial velocity; A-. . .-A, concentration of heparin- antithrombin complex; A - - . + - . A, concentration of heparin- factor IXa complex; 0 - e - 0 , concentration of free Factor IXa-(factor IXa- [heparin. factor IXa complex]). B, comparison of the observed ( O " 3 ) and theoretically predicted (*- -e) kinetics of the factor IXa-antithrombin interaction. The theoretical kinetic curve was obtained by utilizing Equation IV in conjunction with estimates of the various heparin-protein complexes and values of kl and k2 listed in Table I (see Fig. 3 and text for additional details).

and free factor IXa are also presented as functions of mucopolysaccharide concentration. Both types of heparin-protein interaction products exhibit stoichiometries of 1:l (10). Peru- sal of these data indicates that the ascending limb of the initial velocity plot possesses a higher degree of correspondence to the level of heparin. antithrombin complex than to heparin-factor IXa interaction product. More detailed inspection reveals that the descending limb of the initial velocity plot roughly coincides with the level of free factor IXa. These results suggest that generation of the heparin-antithrombin complex must be responsible for the mucopolysaccharide- dependent acceleration of this reaction. The data further imply that formation of the heparin-factor IXa complex is causally related to the progressive decline in the velocity of enzyme inhibition. This phenomenon must be due to the significantly slower neutralization rate of the latter interaction product as compared to free factor IXa.

The discrepancy between the ascending limb and the subsequent pseudoplateau region of the initial velocity plot vis-ci- vis the concentration profie of heparin antithrombin complex is a result of the fact that Kg& is only 4-fold smaller than

~ 1 s ~ . For this reason, significant amounts of heparin - factor IXa complex are formed prior to saturation of antithrombin K H I X ~

10088 Inhibition of Thrombin, Factor IXa, Factor Xu, and Plasmin

I / UI , 1-Y . , I . I L L . , . .,,..,

10.9 10-8 10.7 10-6 10-5 HEPARIN C0h:;ENTRATION (MI

FIG. 5. Correlation between the binding of heparin to thrombin as well as antithrombin and the ability of the mucopolysaccharide to accelerate thrombin-antithrombin interactions. A, comparisons of the levels of heparin-protein complexes with the kinetics of the thrombin-antithrombin interaction. o " 0 , initial velocity; A-. . .-A, concentration of heparin-antithrombin complex; & -4, concentration of heparin. thrombin complexes (THI + TH2); 0-. -0, concentration of tertiary complex of heparin and thrombin (TH,); A.. . . . .A, concentration of binary complex between heparin and thrombin (THI). B , comparison of various combinations of free thrombin and heparin. thrombin complexes with the kinetics of the thrombin-antithrombin interactions. M, initial velocity; & - 4, concentration of free thrombin (T-THI-THd, O-.-, concentration of free thrombin THI (T-THr); A.. . . . .A, concentration of free thrombin and TH2 (T-THI). C, comparison of the observed (M) and theoretically predicted (+- - 4 ) kinetics of the thrombin-antithrombin interaction (see text and legends to Fig. 3 and 4 for additional details).

with mucopolysaccharide. The resultant decrease in the rate of enzyme neutralization at heparin concentrations above 1.0 X lo-' M leads to a disparity between these two profiles.

The lack of concordance between the descending limb and subsequent plateau region of the initial velocity plot vis-&vis the level of free factor IXa is caused by the interplay of two sets of events. On the one hand, increasing concentrations of both heparin. antithrombin complex as well as heparin-enzyme interaction product are generated as the level of mucopolysaccharide ranges from 1 X lo-' M to 1 X M (see below). These two species have opposite kinetic effects upon

reaction velocity. Therefore, the rate of enzyme neutralization within this region of the profiie is dependent upon the concentration of both interaction products.

On the other hand, the velocity of factor IXa inhibition a t heparin concentrations of 1 X M or greater is predomi- nantly a function of the level of mucopolysaccharide - enzyme complex. For example, 99.5% of the observed reaction velocity a t a heparin concentration of 1 X M is due to the neutralization of heparin. factor TXa complex by heparin-antithrombin interaction product. Given that kz has a non-zero value different from k I , the rate of enzyme inhibition becomes virtually independent of the concentration of free factor IXa.

Due to these kinetic complexities, Equation IV was employed to construct a theoretical plot of the initial velocity of this interaction which takes into account the binding of mucopolysaccharide to enzyme as well as inhibitor. The experimentally determined values of Kg& and KE;:; as well as numerous estimates of k , and k2 were utilized. As shown in Fig. 5 B , this approach generates a kinetic plot which is vir- t u d y indistinguishable from the experimental profile when the bimolecular rate constants provided in Table I are employed. We observe that k l is approximately 2.2-fold higher than that maximal reaction velocity experimentally attained within the second phase of this profile. Furthermore, we note that k2 is virtually identical with the minimal rate of enzyme neutralization achieved within the fourth phase of this plot. Table I also compares these two bimolecular rate constants to the similarly designated parameter obtained in the absence of mucopolysaccharide.

Fig. 5 A shows a profiie of the initial velocity of the thrombin-antithrombin interaction as a function of heparin concentration. This plot exhibits the four phases previously noted in the factor IXa-antithrombin interaction. In the same figure, we also depict the levels of heparin.antithrombin complex and various types of heparin-thrombin interaction products uersus the concentration of mucopolysaccharide. Profiles of the latter species include those for binary complexes of heparin and thrombin [THI], tertiary complexes of two molecules of heparin and one molecule of thrombin [TH,], and a sum- mation of both interaction products, [THI + TH2]. Examina- tion of these data reveals that the ascending limb of the initial velocity plot exhibits a higher degree of correspondence to the level of heparin. antithrombin complex than to the concentration of any heparin-thrombin interaction product. Further- more, the pseudoplateau of relatively constant initial velocity roughly coincides with the saturation of antithrombin by mucopolysaccharide. These results suggest that formation of the heparin. antithrombin complex must be responsible for the mucopolysaccharide-dependent acceleration of this reaction. In Fig. 5 B , we compare the initial velocity profile with the levels of [T,,]-[THI], [To]-[TH2], and [TO]-[THII - [TH2], respectively. Inspection of this figure indicates that the descending limb of the kinetic plot corresponds best to the profile of [To-TH2]. Thus, our data suggest that the THz complex rather than the THI interaction product is neutral- ized at a significantly slower rate than free enzyme."

It should be noted that mucopolysaccharide located within the T H I complex may be bound a t one of two spectrally indistinguishable sites. The kinetic consequences might be minimal when the first site is occupied whereas binding to the second site could result in a greatly reduced rate of enzyme-inhibitor interaction. In this case, we should contrast the descending limb of the initial velocity plot with a Profile of the concentration of [To]-+[THI]-[TH2]. The parameter + would represent the fraction of THI molecules with heparin bound to the kinetically relevant site. It is obvious that the two profiles would be in reasonable accord for specified values of 6. Unfortunately, we cannot estimate individual site occupancy with our present experimental techniques. Therefore. we shall assume that only the TH,

Inhibition of Thrombin, Factor IXa, Factor Xa, and Plasmin 10089

Detailed analysis of Fig. 5A reveals a small discrepancy between the ascending limb and subsequent pseudoplateau region of the kinetic plot vis-a-vis the concentration profiie of the heparin. antithrombin complex. Careful examination of Fig. 5B indicates a lack of concordance between the descending limb and subsequent plateau region of the initial velocity plot vis-u-vis the level of To-TH2. These aberrant findings are caused by the binding of mucopolysaccharide to enzyme as well as inhibitor as outlined for the factor IXa-antithrombin interaction. To take these kinetic complexities into account, we also provide a theoretical profiie of the initial velocity of thrombin neutralization constructed by utilizing Equation IV. This plot was generated by employing experimentally determined values of K ~ ~ s s , KE&, and as well as numerous estimates of k l and k2. To conduct this analysis, we have assumed that free thrombin and THI interact with the heparin. antithrombin complex at a similar rate whereas TH, is inhibited by this species at a greatly reduced velocity. As shown in Fig. 5C, the theoretical values generated by this model are in excellent agreement with our experimental data when the bimolecular rate constants provided in Table I are utilized.

We note that k , is approximately 1.3-fold higher than the maximal rate of enzyme neutralization experimentally attained within the second phase of the kinetic plot. Further- more, we observe that k2 is virtually identical with the reaction velocity achieved within the fourth phase of this profiie. Table I also compares these two bimolecular rate constants to the similarly designated parameter obtained in the absence of mucopolysaccharide.

The experimental data summarized in this communication permit us to draw three important conclusions.

Firstly, heparin functions as a catalyst in each enzyme- inhibitor interaction. Our present studies indicate that mucopolysaccharide is initially complexed with antithrombin. This species is subsequently released from the protease inhibitor on a 1:l molar basis when factor IXa, factor Xa, or plasmin are inactivated by the heparin.antithrombin complex. The discharged mucopolysaccharide retains its ability to bind to antithrombin and thus is able to catalyze multiple rounds of factor IXa, factor Xa, or plasmin neutralization by protease inhibitor. A previous report from our laboratory reached similar conclusions concerning the thrombin-antithrombin interaction ( 3 ) . In the latter communication, we showed that the physical basis for this remarkable property of heparin is the greatly reduced avidity of mucopolysaccharide for the thrombin -antithrombin complex. Experimental data obtained in the present study suggest that this is also the case for other hemostatic-enzyme-inhibitor interactions.

Secondly, we have demonstrated that the ability of heparin to accelerate serine protease neutralization by antithrombin is dependent upon the formation of mucopolysaccharide. inhibitor complex. To this end, the initial velocities of the four interactions were determined as functions of heparin concentration. These kinetic profiles were subsequently compared to the levels of the various mucopolysaccharide. protein complexes present within the reaction mixtures. The concentrations of interaction products were computed from the binding isotherms provided in the preceding communication (10). It was apparent that an excellent correlation existed between the ascending limbs and pseudoplateau regions of the initial velocity plots and the levels of heparin. antithrombin complex. NO such corespondence was noted between these same regions

complex is inhibited at a reduced rate by the heparin-antithrombin interaction product. This simplification will not alter our conclusions to any significant extent since the To-TH. profde represents the limiting case of the situation described above.

of the kinetic profiies and the concentrations of various mucopolysaccharide-serine protease interaction products. Based upon these data, we can dismiss the claims of other investigators that formation of heparin - enzyme complex is responsible for the anticoagulant action of this mucopolysaccharide (4-7).

Thirdly, we have undertaken a detailed examination of the molecular mechanism by which heparin exerts its biologic effect. Our studies suggest that the mucopolysaccharide is able to accelerate enzyme neutralization via its direct binding to antithrombin. Potential interactions between free serine protease and heparin attached to inhibitor appear to be of lesser kinetic importance. These conclusions are based upon careful comparisons of K~??s , k’ , , and k’2 for the various hemostatic enzyme-antithrombin interactions.

The K&s describes the binding of a serine protease to mucopolysaccharide in a quantitative manner. However, this parameter does not indicate whether such events will ulti- mately lead to a significant reduction in the rate of enzyme neutralization by the heparin-antithrombin complex or be kinetically silent. In the former instance, binding of mucopolysaccharide to serine protease might saturate sites on this protein essential for the interaction between heparin attached to inhibitor and free enzyme (“approximation” site). Alter- nately, mucopolysaccharide could bind to serine protease and thereby interfere in a steric or electrostatic manner with the neutralization of enzyme by the heparineantithrombin complex (“interference” site). In the latter case a kinetic effect is absent when mucopolysaccharide is bound to serine protease. This would suggest that neither approximation nor interference sites were present on the enzyme.

The kinetic constants k‘l and R2 quantitate the action of heparin upon hemostatic enzyme-inhibitor interactions (Ta- ble I). These parameters have been normalized to eliminate pre-existing differences among serine proteases with respect to their rates of neutralization by antithrombin in the absence of mucopolysaccharide. Therefore, the above constants can be utilized to compare the effect of heparin upon different inhibition reactions.

The parameter kf l provides an estimate of all kinetic actions of the mucopolysaccharide on a given neutralization process. This would include contributions from the direct binding of heparin to antithrombin as well as from potential interactions between free enzyme and mucopolysaccharide attached to inhibitor. The parameter k‘a represents the normalized rate at which mucopolysaccharide -enzyme complex is inhibited by heparin-antithrombin interaction product. This constant is subject to several possible molecular interpretations. If k’z = k’,, mucopolysaccharide bound to serine protease is kinetically silent and the absence of approximation or interference sites on the enzyme is likely. If K’z < k’ , , the attachment of heparin to serine protease has significantly reduced the rate of enzyme neutralization and the presence of approximation or interference sites on this protein should be suspected.

The occurrence of an approximation site is suggested when either k ’ l / k ’ ~ or k’] - k‘a and Ks:qs are proportional for two or more enzyme-inhibitor interactions (see below). The extent of correlation will be dependent upon the manner in which

DISS reflects the rates of association or dissociation between free enzyme and mucopolysaccharide bound to antithrombin. The above proportionality would not be expected if heparin were to interact with interference sites on the enzyme. Given the presence of approximation sites on the serine protease, kIP would represent a minimal estimate of the kinetic effect due to the direct binding of heparin to antithrombin. This interpretation is justified since approximation sites on the enzyme would be saturated by mucopolysaccharide and unable to

KHE

10090 Inhibition of Thrombin, Factor IXa, Factor Xa, and Plasmin

function in their usual manner. The above conclusion would not be valid if interference sites on the serine protease where occupied by heparin.

Based upon the binding and kinetic parameters described above, we can divide the four enzyme-inhibitor interactions examined in this communication into two major groups.

The first group consists of the thrombin-antithrombin and factor IXa-antithrombin interactions. The mucopolysaccharide-induced acceleration of these two processes appears to be dependent upon the direct binding of heparin to antithrombin as well as upon interactions between free enzyme and complex polysaccharide attached to inhibitor.s The presence of approximation effects are suggested by the fact that both neutralization events exhibit < k’l (the occurrence of a descending limb in the initial velocity plot) as well as a proportionality between (k’ l /k’z) or - k’2) and Kg& (Table I). The relative contributions of the “direct” and approximation phenomena may be computed from our kinetic data. A minimal estimate of the direct effect is provided by k’z. The kinetic importance of approximation phenomena can be calculated as either (k ’ l /k ’ z ) or ( K I - k’2), respectively. We favor the use of Pl/k; based upon our belief that the approximation of factor IXa or thrombin via mucopolysaccharide bound to antithrombin and the assembly of enzyme. inhibitor complexes are likely to represent sequential events in the neutralization of these two serine proteases.5 If such is the case, our data would suggest that approximation phenomena are responsible for no more than 1 to 2% of the direct effect of heparin upon antithrombin (Table I). Of course, the alternate method of calculating the contributions of approximation phenomena might be more appropriate if the above events were not sequential and would lead to the conclusion that these latter effects are of considerably greater importance than the direct binding of heparin to antithrombin. We also note that the descending limbs of the two kinetic profdes coincide with the respective concentrations of thrombin or factor IXa minus the appropriate mucopolysaccharide-enzyme interaction product. These observations indicate that binding of heparin to antithrombin does not increase the avidity of the mucopolysaccharide for free serine protease. If this were not the case, a significant displacement of the descending limb of the initial velocity plot toward higher levels of heparin would be observed.

The second group is composed of factor Xa-antithrombin and plasmin-antithrombin interactions. The mucopolysaccharide-induced acceleration of these two processes appears to be solely dependent upon the direct binding of heparin to the protease inhibitor. In the case of the factor Xa-antithrombin interaction, this conclusion is suggested by the lack of a descending limb in the kinetic profile (k’2 = k’ , ) despite the formation of substantial amounts of mucopolysaccharide. enzyme complex as judged by the value of Kg&%. Of course, one might argue that heparin attached to antithrombin exhibits a greatly increased avidity for factor Xa as compared to free mucopolysaccharide. This could lead to the displacement of heparin bound to an approximation site on the serine protease without significant reduction in reaction velocity. However, this hypothesis is absolutely contrary to results

,” It seems most reasonable to suggest that thrombin possesses two different types of mucopolysaccharide binding sites. The first of these sites would be identical with that present on factor IXa (approximation site). The second of these sites would be similar to those present on factor Xa or plasmin (kinetically silent site). However, we cannot exclude the possibility that both of these sites may function to approximate enzyme and inhibitor.

obtained with thrombin and factor IXa (see above). Further- more, the value of k’l for the factor Xa-antithrombin interaction is similar to the estimates of PZ for the thrombin- antithrombin and factor IXa-antithrombin interactions. This strengthens our claim that factor Xa differs from the latter two homologous enzymes due to the absence of an approximation site. In the case of the plasmin-antithrombin interactions, the preceding interpretation is somewhat less secure because of our inability to form large amounts of mucopolysaccharide. enzyme complex within the reaction mixtures. This situation is secondary to the relatively highE[& which characterizes the binding of heparin to plasmin. However, the minimal avidity of mucopolysaccharide for this serine protease itself suggests that approximation effects are not likely to be of kinetic importance.

On the basis of the above evidence, we believe that the direct binding of heparin to antithrombin is probably responsible for the mucopolysaccharide-dependent acceleration of hemostatic enzyme-inhibitor interactions. We suspect that the heparin-induced augmentation in reaction velocity is due to a conformational transition within antithrombin induced by the mucopolysaccharide. However, this suggestion requires direct experimental validation. It is of interest to note that k’, of the plasmin-antithrombin interaction is significantly smaller than k’, of the factor Xa-antithrombin interaction. These differences may reflect the precise molecular nature of the postulated conformational transition.

We should emphasize that all of the above analyses were conducted with heparin fractions which possess a single binding site for antithrombin. The existence of mucopolysaccharide species that bear multiple binding sites for the inhibitor has recently been reported from our laboratory (19). It remains to be determined whether additional kinetic complexities will be observed when the latter species are examined.

2. 1.

3.

4. 5.

6. 7.

8.

9

10.

REFERENCES Rosenberg, R. D. (1977) Semin. Hematol. 14,427-440 Rosenberg, R. D., and Damus, P. S. (1973) J. Biol. Chem. 248,

Jordan, R., Beeler, D., and Rosenberg, R. D. (1979) J. Biol. Chem.

Machovich, R. (1975) Bzochim. Biophys. Acta 412, 13-17 Smith, G. F., and Craft, T. J. (1976) Biochem. Biophys. Res.

Griffith, M. J. (1979) J. Biol. Chem. 254, 3401-3406 Griffith, M. J., Kingdon, H. S., and Lundblad, R. L. (1979) Arch.

Biochem. Biophys. 195,378-384 Gitel, S . N. (1975) in Heparin: Structure, Function and Clinical

Implications (Bradshaw, R. A., and Wessler, S., eds) pp. 243- 247, Plenum Press, New York

Holmer, F., Soderstrom, G., and Andersson, L-0. (1979) Eur. J .

6490-6505

254,2902-2913

Commun. 71, 738-745

Jordan. R. E.. Oosta, G. M., Gardner, W., and Rosenberg, R. D. Biochem. 93, 1-5

(1980) J. Bzol. Chem. 255, 10073-10080

Biol. Chem. 250, 1607-1617

Clin. Inuest. 50, 244-254

11. Rosenberg, J. S., Beeler, D. L., and Rosenberg, R. D. (1975) J.

12. Zimmerman, T. S., Ratnoff, 0. D., and Powell, A. E. (1971) J.

13. Oosta, G. M. (1975) Ph.D. thesis, MIT Press, Cambridge, Mass. 14. Rosenberg, R. D., and Lam, L. H. (1979) Proc. Natl. Acad. Sci.

15. Pepper, D. S., and Dawes, J. (1977) Thromb. Huemostasis 38,

16. Silverberg, S. A., Nemerson, Y., and Zur, M. (1977) J. Biol. Chem.

17. Jesty, J. (1978) Arch. Biochem. Biophys. 185,165-173 18. Jesty, J. (1979) J. Biol. Chem. 254, 10044-10050 19. Rosenberg, R. D., Jordan, R., Favreau, L. V., and Lam, L. 14.

(1979) Biochem. Biophys. Res. Commun. 86, 1319-1324

U. S. A . 76, 1218-1222

Supplement 1, 15

252, 8481-8488

the chemi~try vol. 255, no. 21, issue of november 10, pp. … · 2001-09-07 · the journal of...

Documents