Studies on the interaction of heparin with thrombin, antithrombin, and other plasma proteins

ARCHIVESOFBIOCHEMISTRY AND BIOPHYSICS Vol. 200, No. 2, April I, pp. 595402, 1980

Studies on the Interaction of Heparin with Thrombin, Antithrombin, and Other Plasma Proteins’ MARIA Department

0. LONGAS, of Biochemistry,

WILLIAM

S. FERGUSON,

New York

University Medical

AND THOMAS Center,

New

York,

H. FINLAY New

York

10016

Received October 2, 1979 The interaction of heparin with plasma proteins was studied by gel chromatography using elution profile analysis to determine binding constants. A commercial heparin preparation was found to bind lOO- to 200-fold more tightly to antithrombin than to either albumin or fibrinogen. With tri- or tetrapeptide nitroanilides as substrates, this heparin preparation proved to be an apparent mixed competitive inhibitor of thrombin and other serine proteases with K,‘s of approximately 10e5 M. At 3 x lo-’ M, it caused 50% loss in the activity of human thrombin on fibrinogen. However, at 1O-5 M it did not inhibit the esterase activity of human thrombin on N-tosyl arginine methyl ester. These observations suggest that in the presence of heparin the size of the substrate able to bind at the thrombin-active site is restricted. When this commercial heparin was chromatographed on immobilized antithrombin, thrombin, albumin, and fibrinogen, measurable binding occurred only to the antithrombin and thrombin affinity materials. The heparin purified by chromatography on immobilized antithrombin had a greater affinity for antithrombin and a greater anticoagulant activity than unfractionated heparin but the same Ki for thrombin (using a peptide nitroanilide substrate). Heparin purified on immobilized thrombin also had the same Ki for thrombin as unfractionated heparin but had a lower affinity for antithrombin and a lower anticoagulant activity. These findings indicate that heparin binds to antithrombin with greater specificity than it does to thrombin.

Although the potent anticoagulant heparin was first isolated more than 60 years ago and was shown to require a plasma protein cofactor for activity more than 40 years ago (l), the relationship between heparin structure and function is still not well understood. This lack of understanding, however, has not precluded the widespread clinical use of heparin, which undoubtedly will increase with the advent of routine “lowdose” heparin therapy (2). A major problem associated with the study of the mechanism of action of heparin is its heterogeneity. Carbohydrate chain length, amino acid content, degree of sulfation, and uranic acid: iduronic acid ratio all vary depending upon source and methods of isolation and purification (3). Furthermore, the physical and chemical properties of heparin do not

seem to correlate well with physiological activity (4). Heparin is postulated to inhibit blood coagulation by binding to and enhancing the activity of its cofactor, antithrombin,3 an irreversible inhibitor of most of the serine proteases of the clotting mechanism (5). In contrast, a second hypothesis holds, at least in the case of thrombin, that heparin first binds to the protease and then serves as a bridge between enzyme and inhibitor (6). Heparin has also been reported to inhibit the esterolytic and proteolytic activities of thrombin in the absence of antithrombin (7,8). In addition to binding to antithrombin, heparin binds to thrombin (‘7), and other proteins of the clotting mechanism (g-11), mobilizes lipoprotein lipase (12), aggregates platelets (13), and inhibits enzymes involved with protein synthesis (14). The question

’ Supported by NIH Grant HL 15596. ’ Recipient of Research Career Development Award HL 00277 from the NIH. Author to whom correspondence should be addressed.

3 Antithrombin is also referred to in the literature as antithrombin III, heparin cofactor, antithrombinheparin cofactor, and factor X, inhibitor (X,1). 595

0003-9361/80/04059508$02.00/0

Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

596

LONGAS,

FERGUSON,

arises as to whether these various activities are all properties of the same chemical entity. In this communication we report on the interaction of commercial heparin with plasma proteins and show that although various heparin fractions bind tightly to thrombin and antithrombin, only binding to antithrombin exhibits a high degree of specificity. MATERIALS

AND METHODS

Materials. Peptide substrates for protease assays, Bz-Phe-Val-Arg-p-nitroanilide (S-2160) and BzIle-Glu-Gly-Arg-p-nitroanilide (S-2222), were obtained from Bofors. Cbz-Gly-Pro-Arg-p-nitroanilide (Chromzym TH) was from Boehringer-Mannheim. Heparin from porcine gastric mucosa (Research Plus) was chromatographed on Sephadex G-75 in 0.2 M NaCl; fractions from the central portion of the heparincontaining peak (assayed with azure A (15)) were combined and the polysaccharide was precipitated with ethanol and dried. The molecular weight of this preparation was determined to be 8900 2 1000 by viscometry (16). (The range was estimated from the width of the heparin-containing peak from the Sephadex G-75 column.) This heparin, or some fractions thereof, is the only heparin used in this study. Molar concentrations were based on this molecular weight. Antithrombin was purified from human plasma essentially as described by Miller-Andersson et al. (17) except that the immobilized heparin was prepared with triazine-activated agarose.4 The purified antithrombin inactivated 1200 to 1450units of thrombin per milligram and demonstrated only a single proteinstaining band following reduction and electrophoresis on 7.5% polyacrylamide gels in the presence of sodium dodecyl sulfate (18). Human thrombin (2440 NIH units/ mg, 85% active enzyme, 99% a-thrombin) was a gift from Dr. J. W. Fenton II. Bovine trypsin, 2X crystallized, was obtained from Worthington and used without further purification. Human plasmin was the CTA standard obtained from the American National Red Cross. Bovine factor X,, generously supplied by Dr. Craig Jackson, was the research reference preparation of the International Committee on Thrombosis and Haemostasis. Bovine thrombin was purified from Bovine Topical Thrombin (Park Davis) by chromatography on sulfopropyl Sephadex (19) and had a specific activity of 1600 units/mg. Human fibrinogen was prepared by differential polyethylene glycol precipitation of aluminum hydroxide gel-adsorbed cryoprecipitated human plasma.5 The fibrinogen was over 95% clottable, had only N-terminal alanine and * T. H. Finlay, V. Troll, and L. T. Hodgins, manuscript in preparation. 5 M. 0. Longas and T. H. Finlay, manuscript in preparation.

AND FINLAY

tyrosine residues, and showed only three proteinstaining bands (corresponding to the Aa, B/3, and y subunits) following reduction and SDS-gel electrophoresis (18).6 All other chemicals were of the highest purity commercially available. Assay of antithrombin. Rapid neutralization of thrombin by antithrombin in the presence of heparin was determined at 37°C by a modification of the twostage assay of Odegard et al. (20). Heparin (0.6 unit), human thrombin (0.3 to 0.5 unit), and antithrombin were incubated for 30 s in 0.45 ml of 0.067 M Tris-Cl, 0.2 M NaCl, pH 8.3. Then 0.15 ml of a solution containing 0.5 ITIM substrate (S-2160) and 0.33 mgiml polybrene was added and residual thrombin activity was determined from the change in absorbance at 405 nm as a function of time. The amount of thrombin neutralized was determined from the tangent to the curve of thrombin inhibition at several antithrombin dilutions. One unit of antithrombin neutralizes one unit of thrombin under the conditions of the assay. In some experiments thrombin activity was determined using fibrinogen as a substrate. These assays were conducted by the method of Lunblad et al. (21) except for the use of the highly purified fibrinogen described above (1.8 mgiml) and the determination of clotting times with a fibrometer (Bio Quest). A thrombin concentration of 0.4 unit/ml gave a clotting time of 18.8 s. Plots of thrombin concentration against the reciprocal of the clotting time were linear for thrombin concentrations between 0.05 and 1 unit/ml. Protease assays. Esterase activity of the serine proteases used in this study was determined with N-atosyl-lrarginine methyl ester as a substrate either by the radioactive substrate assay of Roffman et al. (22) or by pH stat titration (23). Proteases were also assayed with polypeptide nitroanilides as substrates essentially as described for the assay of antithrombin. Preparation of immobilized plasma proteins. Human thrombin, antithrombin, and fibrinogen, purified as described, and bovine serum albumin were coupled to triazine-activated Sepharose CL-6B by the method of Finlay et al. (24). The amount of protein coupled to the support was determined with ninhydrin following alkaline hydrolysis of a dried sample (24). The thrombin gel contained 1.1 mg and 510 NIH thrombin units (determined by pH stat titration) per milliliter of packed resin. The antithrombin gel contained 2.1 mg of protein and inactivated 225 NIH thrombin units per milliliter of gel. The fibrinogen gel contained 2.4 mg of protein per milliliter and after conversion of the immobilized fibrinogen to fibrin with thrombin (24), adsorbed 1.6 mg of free fibrinogen per milliliter. The albumin gel contained 3.7 mg of protein per milliliter. Fluorescence measurements. Uncorrected fluorescence spectra were taken at room temperature (20 6 Abbreviations used: SDS, sodium dodecyl sulfate; AT, antithrombin.

HEPARIN-PLASMA

PROTEIN

t 1°C) with a Perkin-Elmer MPF-3 fluorescence spectrophotometer. Square cells with l.O-cm pathlengths were used. The slits of the excitation and emission monochromators were set at 4 mm. In preliminary experiments it was determined that the excitation maximum was 285 nm and emission spectra were taken at this excitation wavelength. Fluorescence intensities of antithrombin and mixtures of heparin and antithrombin were measured at the wavelengths of maximum excitation and emission (285 and 335 nm, respectively) at room temperature with an Aminco-Bowman spectrofluorometer. Square cells with a l-cm pathlength were used. Monochromator slits were 5 mm. RESULTS

Binding of heparin to plasma proteins studied by gel chromatography. The inter05

ANTITHROMEIN + HEPARIN

m

%

62 5z

2010

3. 22

go05 ?

f

’I

i?

FRACTION No FIBRINOGEN

t HEPARIN

14 1 D '2 2 10 f 8 03

% 05 04

E 03

61 5 P & a

TABLE

I

ASSOCIATION CONSTANTS FOR HEPARIN-PLASMA PROTEIN COMPLEXES ASDETERMINEDBY GEL CHROMATOGRAPHYON SEPHADEX G-150 Protein

K, CM-‘)

Antithrombin III (human) Serum albumin (bovine) Fibrinogen (human)”

2.2 x 106 2.4 x lo4 5.3 x 104

a Determined by the method of Gilbert and Kellett (25). Elution volumes of the pure species at infinite dilution were determined in a separate experiment from the concentration dependence of the finite difference boundary. * Determined by the method of Nichol et al. (27) from a Scatchard plot (26) of the data from six binding experiments in which the constituent heparin:fibrinogen ratio varied from 1.2 to 15. The number of heparin binding sites per fibrinogen molecule (340,000 M,) was found to be approximately 6.

4:

F

E

597

INTERACTIONS

02

4;

2;

01 10

20 30 FRACTION No

40

FIG. 1. Elution profiles for the chromatography of heparin and AT (upper panel) and heparin and fibrinogen (lower panel) on a 0.9 x 59-cm column of Sephadex G-150. Chromatography was conducted at 22°C. V, for the column was 14.72 ml and V, was 36.42 ml. In the AT + heparin experiment, 3.3 mg of AT and 1.25 mgofheparin in30.0 ml ofO.10 M NaCl, 0.05 MTris-Cl buffer (pH 7.4) were loaded onto the column followed by this same buffer without either protein or heparin. Fractions of 1.5 ml were collected at a flow rate of about 10 ml/h. A constant pressure of 20 cm of water was maintained with a Marriott bottle. Protein was determined by the Lowry method (36) and heparin with azure A (15). In the fibrinogen + heparin experiment, 12.0 mg of fibrinogen and 3.0 mg of heparin in 30 ml of buffer were loaded onto the column. Other conditions were the same as in the AT + heparin experiment.

action of two macromolecules in solution can be studied by gel chromatography providing the concentrations of both molecules are known independently through the system (25). As heparin and protein concentrations can easily be determined independently, we have used this technique to determine the binding constants for the complexes of heparin with several plasma proteins. Figure 1 shows typical elution profiles for the chromatography of mixtures of either heparin and antithrombin or heparin and fibrinogen on columns of Sephadex G-150. In Table I are the association constants for the complexes of heparin with antithrombin, albumin, and fibrinogen as determined by gel chromatography experiments. For albumin and antithrombin, K, was calculated from the trailing edge of the elution profile (25). For fibrinogen, K, was calculated from a Scatchard plot (26) of the data from six different binding experiments using the method outlined by Nichol et al. (27). This approach is valid as the elution volumes of the heparin-fibrinogen complex and fibrinogen alone are the same under these conditions. Inhibition

of serine proteases by heparin.

Heparin has been reported to inhibit the amidolytic activity of thrombin on peptide nitroanilides (28). We have found heparin

598

LONGAS, FERGUSON.

[HEPAWN]

tions (Table II). When heparin was tested as an inhibitor of the e&erase activity of several proteases, using either the radioactive substrate or pH stat titration assays, it proved to be a poor inhibitor of human plasmin and bovine factor X, (Ki > lo4 M) and failed to inhibit bovine trypsin or human thrombin at 10e5 M. When the heparin fractionated by chromatography on immobilized thrombin or antithrombin was tested as a thrombin inhibitor in a clotting assay using fibrinogen as a substrate, each of the four heparin preparations shown in Table III as well as unfractionated heparin caused a 50 + 10% loss in thrombin activity at a heparin concentration of 3 X IO-’ M. Chromatography of heparin on immobilized plasma proteins. In order to fractionate heparin into classes with affinities for different plasma proteins, the partially purified heparin used in the gel chromatography and inhibition studies was chromatographed on columns of immobilized antithrombin, thrombin, fibrinogen, and albumin. In these experiments, 1.0 mg of heparin was loaded onto a 5-ml(0.9 x ES-cm)column containing the immobilized protein which had been equilibrated with 0.10 M NaCl, 0.05 M Tris-Cl, pH 7.4. Columns were first eluted with 15 ml of the equilibrating buffer, then with 15 ml of the buffer containing 0.55 M NaCl, and finally with 15 ml of the buffer containing 2.0 M NaCl. Fractions were assayed for the presence of heparin by either the azure A assay (15) or the carbazole assay for uranic acid (29) with similar results.

x 10-5~

[SUBSTRATEI ~10-5~

FIG. 2. Hanes-Woolf plot for the inhibition of the thrombin-catalyzed hydrolysis of Bz-Phe-Val-Argp-nitroanilide by heparin. Reactions were conducted at 37°C in 0.10 M NaCl, 0.05 M Tris-Cl, pH 7.4: 0, no heparin; 0, 4.65 x 1O-6 M heparin; A, 4.65 X lo+ M heparin; q , 9.3 x 10” M heparin. Inhibition constants were calculated from a secondary plot of s/v versus s, as shown in the upper panel.

to be a mixed-competitive inhibitor of human thrombin with a Ki of 1 to 5 x 10m5 M when peptide nitroanilides were used as substrates (Fig. 2, Table II). We also found heparin to be a mixed-competitive inhibitor of a number of other serine proteases when these enzymes were assayed under similar condiTABLE INHIBITION

Enzyme Thrombin (human) Thrombin (bovine) Trypsin (bovine) Plasmin (human) Factor X, (bovine)

OF SERINE

AND FINLAY

II

PROTEASES

Substrate Bz-Phe-Val-Arg-pNA Bz-Phe-Val-Arg-pNA Bz-Phe-Val-Arg-pNA Bz-Phe-Val-Arg-pNA Bz-Ile-Glu-Gly-Arg-pNA

BY HEPARIN~

Km (M) 1.5 x 10-S 4.8 7.6 1.8 8.2

x x x x

10” 10” 104 10-4

Kib (M) 4.5 1.5 1.4 6.5 3.5

x x x x x

10-b 10-S 10-S 1O-5 10”

a Reactions were conducted in 0.05 M Tris-Cl, 0.15 M NaCl, pH 8.3 at 37°C. The heparin was partially purified by chromatography on Sephadex G-75 as described under Methods. b In each case inhibition was judged to be mixed. Inhibition constants were determined from HanesWoolf plots (Fig. 2) assuming a molecular weight of 8900 for the heparin.

HEPARIN-PLASMA

PROTEIN TABLE

BINDING

OF HEPARIN

K, (X lo6

Heparin “Unfractionated” Antithrombin-Sepharose, Antithrombin-Sepharose, Thrombin-Sepharose

FRACTIONS

peak I peak II

M-l)"

599

INTERACTIONS

III

TO ANTITHROMBIN

AND THROMBIN

KT (X10-' M)b

V,,,axb

1.15 1.11 1.11 1.45

3.93 7.12 9.05 2.43

0.42 1.3 5.2 0.30

Ki(XW5

M)C

1.6 1.2 0.8 1.1

a Binding of heparin to antithrombin as determined by tryptophan fluorescence enhancement. K, was assumed to be equal to the heparin concentration at half-saturation (log F - F,/F,,, = 0), as shown in Fig. 4. b Apparent binding constants and maximum velocities for the inactivation of thrombin by antithrombin as determined from the intercepts of the double-reciprocal plots shown in Fig. 5. The maximum velocities (arbitrary units) have been normalized by division by the heparin concentration. c Inhibition of thrombin amidolytic activity by heparin in the absence of antithrombin as shown in Fig. 2, except that the substrate was Cbz-Gly-Pro-Arg-p-nitroanilide. In each case inhibition was mixed.

All of the heparin applied to either the fibrinogen or albumin affinity columns eluted in the breakthrough volume, indicating little or no binding under the conditions of the experiment. These results were not unexpected even though the dissociation constants for the heparin complexes of these proteins suggest that there should be some interaction. The failure to detect heparin binding is probably due to the fact that the lysine residues at least partially responsible are buried or attached to the gel matrix by the linking agent. Figure 3 shows the elution patterns for the chromatography of heparin on immobilized thrombin and antithrombin. Analysis of the elution pattern from the thrombin affinity column indicates that approximately two-thirds of the heparin applied to the column eluted in the breakthrough peak while one-third (38 nmol) was bound to the gel and required buffer containing 0.55 M NaCl for desorption. This value lies between the amount of thrombin bound to the support calculated from activity measurements (23 nmol) and from covalently linked protein (150 nmol). No further heparin was eluted with 2.0 M NaCl. Chromatography of heparin on immobilized antithrombin resulted in the binding of approximately 30 nmol to the gel. Of the heparin bound, 20 nmol eluted with buffer containing 0.55 M NaCl while 10 nmol required buffer containing 2.0 M NaCl for desorption. This column contained 153 nmol of antithrombin as calculated from the

amount of protein bound to the support or 13 nmol as calculated from its thrombin neutralizing activity. Binding of heparin to antithrombin. In order to differentiate between the heparin fractions purified by affinity chromatography on immobilized thrombin and antithrombin, binding constants for the interaction of these fractions with antithrombin were determined from their enhancement of the intrinsic antithrombin tryptophan fluorescence and from their effect on the apparent second-order rate constant for the reaction between antithrombin and thrombin. Binding constants were not determined by gel chromatography as only limited amounts of the purified heparins were available.

g 602 ?F 40ZO2.J 0

I

I

I 1

I

5 10 15 XJ 25 30 0 5 10 15 20 25 Xl FRACTION NUMBER (1 4 ml 1

FIG. 3. Chromatography of heparin on 0.9 x 8-cm columns of immobilized antithrombin and thrombin. Experimental conditions are described in the text. Heparin was determined by the azure A assay; protein was determined by the Lowry method.

600

LONGAS,

FERGUSON,

Binding constants for the interaction of heparin with antithrombin were determined by fluorescence spectroscopy from the intercept on the ordinate of plots of log (8’ - Fd F max- F ) against log (free heparin concentration) where F is the fluorescence intensity of the antithrombin at a particular heparin concentration, F,, is the fluorescence intensity in the absence of heparin, and F maxis the fluorescence intensity in the presence of saturating amounts of heparin (30). Typical plots for the interaction of unfractionated heparin and heparin eluted from antithrombin-Senharose with 2 M NaCl anpear in Fig. 4. fiinding constants appear in Table III. Initial velocities for the reaction of antithrombin with thrombin in the presence and absence of heparin as a function of antithrombin concentration were determined from plots of residual thrombin activity against time. Apparent binding constants and maximum velocities for the interaction of thrombin, antithrombin, and heparin (Table III) were calculated from the intercepts of secondary plots of the reciprocal of the initial velocities against the reciprocal of the antithrombin concentration (Fig. 5).

Log

Heporin

Concentration

FIG. 4. Hill plots for the interaction of heparin with antithrombin. Aliquots (5-20~1) ofheparin were added to 2 ml of antithrombin (2.9 x 10m6M) in 0.05 M Tris, 0.10 M NaCl, pH 7.4 and the fluorescence emission intensity was measured with an Aminco-Bowman spectrofluorometer as outlined under Methods. a, “unfractionated” heparin; 0, peak I heparin from immobilized antithrombin; A, peak II heparin from immobilized antithrombin; 0, heparin from immobilized thrombin.

AND FINLAY

0

5 ‘/

10

[Antithrombin]

15 lX106

20 ,.-‘I

FIG. 5. Double-reciprocal plots for the inactivation of thrombin by antithrombin in the presence of heparm. Antithrombin was incubated with heparin in 0.05 M Tris, 0.10 M NaCl, pH 7.4 in a total volume of 0.95 ml at 37°C for 2 min. At zero time, thrombin in 50 ~1 (to make the final thrombin concentration 1.0 x lo-’ M) was added. At various intervals 100~~1 aliquots from the reaction mixture were withdrawn and transferred to 600 ~1 of 0.166 rnw Cbz-Phe-Val-Argp-nitroanilide in the same buffer and the change in A,,, was recorded as a function of time. Thrombin activity was determined from the initial slopes of the recorder tracings. 0, peak I heparin from immobilized antithrombin, 9.97 x 10-l” M; A, peak II heparin from immobilized antithrombin, 6.96 x lO-Lo M; H, “unfractionated” heparin, 10.17 x lo-‘O M; 0, heparin from immobilized thrombin, 10.66 x 10-l” M. DISCUSSION

As would be expected for a highly negatively charged macromolecule, heparin binds readily to many proteins under physiologic conditions (‘7, 9-11). Using analytical gel filtration, we have examined the binding of commercial heparin to several plasma proteins (Fig. 1, Table I). Commercial heparin, purified only by chromatography on Sephadex G-75, was found to bind more tightly to antithrombin than to albumin or fibrinogen, Our data indicate that a commercial heparin concentration of approximately 3 pg/ml will half-saturate the antithrombin in normal human plasma. Although the K, for the interaction of commercial heparin with albumin is 150-fold smaller than for its interaction with antithrombin, the 300fold greater concentration of albumin in plasma would suggest that in plasma containing 3 pug/ml of heparin, a significant fraction of the heparin would exist as a complex with albumin. The fact that a heparin concentration of 3 pg/ml (approximately 0.5 unit/ml) makes normal human plasma essentially uncoagulable is consistent with the

HEPARIN-PLASMA

PROTEIN

observation that only a fraction of the heparin specifically binds to and activates antithrombin (Fig. 3; and also Refs. (31) and (32)) and that heparin acts catalytically, one heparin molecule activating many antithrombin molecules (33). It is also likely that the binding of heparin to albumin does not exhibit a high degree of specificity. Table III lists the binding constants for the interaction of antithrombin with the heparin fractions prepared by affinity chromatography. These values should, of course, be regarded with some caution as even these affinity-purified heparins are certainly heterogeneous. However, the binding constants should be fairly self-consistent as a single heparin fraction with a narrow molecular weight range was used as a starting material for all experiments. To calculate the values for KT and V,,, (Table III, columns 3 and 4), we have used a model for the heparin-mediated inactivation of thrombin by antithrombin in which heparin acts catalytically (33) as shown below: At-H A.H

KA e A.H

KT + T G A.H *T + A-T

+ H

where A is antithrombin, T is thrombin, and H is heparin. It is interesting that the value determined for the apparent K, is essentially the same for each of the heparin preparations while the values for apparent V,,,/heparin concentration (which is a function of KA) vary. The simplest explanation for this observation is that each heparin preparation contains different amounts of a particular heparin fraction with the ability to bind to and activate antithrombin. If, however, the heparin preparations contained different antithrombin activators which bound to antithrombin with differing affinities, this would imply that heparin and thrombin interact with antithrombin at separate sites. Our results for heparin chromatographed on immobilized antithrombin are similar to those reported by Hook et al. (31). That is, the more strongly a heparin fraction binds to the immobilized antithrombin, the higher its anticoagulant activity. Heparin in peak II from our antithrombin affinity column

INTERACTIONS

601

(Fig. 3) with an activity of 365 units/mg corresponds to the “high-affinity” heparin of Hook et al. while the heparin from our peak I, with an activity of 285 units/mg, corresponds to their “medium-affinity” heparin. The heparin eluted from the immobilized thrombin column had an activity of 98 units/mg. We found heparin to be a mixed-competitive inhibitor of thrombin and other serine proteases when tri- and tetrapeptide nitroanilides were used as substrates (Table II). With tosyl arginine methyl ester as a substrate, inhibition by heparin was poor with factor X, and plasmin and not measurable at 1O-5 M heparin with trypsin and thrombin. These results conflict with those of Li et al. (7) who found heparin to be a noncompetitive inhibitor of bovine thrombin with a Ki of 3 X 1OP M. We can offer no explanation for this discrepancy except that the heparin preparations were from different suppliers and the thrombin preparations from different species. Neither the Kt for thrombin inhibition by heparin of peptide nitroanilide hydrolysis nor the inhibition of thrombin in a clotting assay seemed to be affected by prior chromatography of the heparin on either immobilized thrombin or antithrombin (Table III). However, the ability of heparin to activate antithrombin was affected by prior affinity chromatography. These findings suggest that inhibition of thrombin does not require as high a degree of specificity in the heparin molecule as does activation of antithrombin. Similar results obtained from a study of the interaction of high- and low-activity heparin with immobilized thrombin (34) and by a crossed immunoelectrophoretic technique (35) have recently been reported. One possible conclusion that could be drawn from these studies is that if heparin binds to plasma proteins other than antithrombin nonspecifically, then clinical administration of a less highly purified heparin might be advantageous in order to saturate nonspecific binding sites. REFERENCES 1. BRINKHOUS, K. M., SMITH, H. P., WARNER, E. D., AND SEEGERS, W. H. (1939) Amer. J. Physiol. 125, 683487.

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FERGUSON,

2. WESSLER, S. (1977) Fed. Proc. 36, 67-71. 3. JAQUES, L. B. (1975) Adv. Exp. Med. Biol. 52, 139-147. 4. CIFONELLI, J. A. (1974) Carbohyd. Res. 37, 145154. 5. ROSENBERG, R. D. (1977) Fed. Proc. 36, 10-18. 6. MACHOVICH, R., STAUB, M., AND PATHY, L. (1978) Eur. J. Biochem. 83, 473-477. 7. LI, E. H. H., ORTON, C., AND FEINMAN, R. D. (1974) Biochemistry 13, 5012-5017. 8. ABILDGAARD, U. (1968) &and. J. Haematol. 5, 432-439. 9. MARCINIAK, E. (1974)J. Lab. Clin. Med. 84,344356. 10. FUJIKAWA, K., THOMPSON, A. R., LEGAZ, M. E., MEYER, R. G., AND DAVIE, E. W. (1973) Biochemistry 12, 4938-4945. 11. GENTRY, P. W., AND ALEXANDER, B. (1973) Biothem. Biophys. Res. Commun. 50, 500-506. 12. OLIVERCRONA, T., BENGTSSON, G., MARKLUND, S.-E., LINDAHL, U., AND HOOK, M. (1977) Fed. Proc. 36, 60-65. 13. FRATANTONI, J. C., POLLET, R., AND GRALNICK, H. R. (1975) Blood 45, 395-401. 14. HRADEC, J., AND DUSEK, Z. (1978) Biochem. J. 172, l-7. 15. JAQUES, L. B., AND WOLLIN, A. (1967) Canad. J. Physiol. Pharmacol. 45, 787-794. 16. RODEN, L., BAKER, J. R., CIFONELLI, A. J., AND MATHEWS, M. D. (1972) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 28, pp. 73-140, Academic Press, New York. 17. MILLER-ANDERSSON, M., BORG, H., AND ANDERSSON, L. 0. (1974) Thromb. Res. 5,439-452. 18. WEBER, K., AND OSBORN, M. (1969) J. Biol. Chem. 244, 4406-4412. 19. LUNDBLAD, R. L., HARRISON, J. H., AND MANN, K. G. (1973) Biochemistry 12, 409-413. 20. ODEGARD, 0. R., LIE, M., AND ABILDGAARD, U. (1975) Thromb. Res. 6, 287-294.

AND

FINLAY

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