Phosphate promotes glycation of antithrombin III which interferes with heparin binding

Biochimica et Bwph~sica Act~

993 (1989)217-223

217

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with heparin binding i
(Received20 July 1989)

Key words: Diabetesm=nitus;Activity;Fluorescence;Nt. enzymaticglycosylation None~ymatic glycation of antithrombin lIl has been reported to cause the reduction of heparin-catalyzed thrombin-inhihiting activity in diabetes. The ~.ffect uf in ~iL'o nonenzymatic giycation of pure antithrnmbin Ill on heparin binding and hep~rin.potentiated activity under a variety of buffers and pH values was studied to further darify the physiological significance of this reaction. The extent of glycation, measured by the |ruetosamine assay and [t4C]glueose binding, was enhan~.ed by the presence of phosphate ion (pH 7.45, 8.5 and 9.5) and increased linearly with increasing phosphate ion eoncen~ation from 0.01 to 0~2 M phosphate. Conversely, the heparin.eatalyzed antithrombin activity decreased from 93.1% of controls for 0.01 M phosphate to 73.5% for 0.2 M phosphate as the extent of glyeatinn increased. The increase in int~'insie ~uorescence 'nduced by bindir~g of heparin to anfithrombin 111 was also moderated by glycatinn of antithrumbin m in a dose-dependent mariner w~l'h a negative correlation coefficient of -0.94. Direct measurement of the heparin binding by affinity chromatography showed a decrease in the heparin°binding fraction which correlated with th~ degree of glyca~on and the decrease in hepurin-catalyzed activity. These studies suggest that nonenzymatic glycation u~ay be responsible for the reduction in antithrombin |II activity observed in some diabetics.

Introduction Antithrombin llI (ATII() is the major plasma inhibitor of blood coagulation, inlfibiting the activated serine pr,ateinases of the coagulation system, including thrombin, Factor X!la, Factor Xla, Factor IXa, and kactor Xa. This inhibitory activity is markedly potentiated by binding of heparin to ATIII [1]. Several studies have found that the heparin-catalyzed A Fill activity is reduced in persons with diabetes mellitus, while the total antigenic concentration of ATIII1 is either nomml or mildly elevated [2-5]. In three of these studies [3-5] an inverse correlation with hyperglycemia and glycated serum proteins suggested that glycation (nonenzymatic glycation) may be involved in the reduced antithrombin acti~Sty. Brownlee et al. [~] demonstrated a significant loss of heparin-catalyzed ATIII activity following a 3 day 37°C in vitro glycatiou Abbreviations:ATIII, antithrombin Ill; Mops, morpholinopropanesulfnnicacid. Co~espondence: R.C. Rubens. Mar'~hfieldMedicai Research Foundalion, 510 North St. Joseph Ave!~ue.Marshfield,WI 54449, U.S.A.

(100-400 mM glucose) of pure ATIII in 0.01 M EDTA (pH 9.5), while Ceriello et al. [7] found a 25% reduction of plasma ATIll activity after a 3 day incubation with 250 mM glucose. This reduction in HC activity could be moderated wilh excess hellarin both in vitro and in vivo [8], indicating that glyca~ion was affecting the heparin. binding site of ATIlt. Tb!re is evidence that at least two lysine residues are involved in the binding or" heparin to ATIII [9,10]. In a recent report by Sakurai et al. [11] however, glycation of pure ATIII (100-500 mM glucose) caused only a modest dew,reuse in hepariu binding at 4°C. In this study glytation did not affect the ATIII thronlbin iuhibho.,2¢ an'iv ~y in the absence of heparin. In addition, Busby and ~,~gham [12] found no loss of heparirt cofactor activity when ATlll was incubated at 37°C, 2 days m the presence of 2 M glucose. More recently, however, Ville.nueva and Allen [13] demonstrated that ATIII lost 357o ef its hepariu cofaetc,r during a 7 day incubation in 15 mM ghicose/0.02 M phosphate/0.15 M NaCl (pH 7.5). Because of the uncertainty indicated in these stedies regarding the effect of glycation upon the acdvity and hepariu binding capacity of AT[II, v¢e further studied the in vitro glycation of pure ATIII to determine the

0304-a165.'Sq,/$0350 ~ 198° EL~e~..erScience PublishersBV. (Biomedicalt~ivi~;-,)

218 conditions (pH. temperature and glucose concentrationL which result in a Ioss of ATIII activity and heparin binding. The recent report that phosphate ion catalyzes the glycation of ribonuclease and other proteins [14] led us to explore the effect of phosphate concentration on glycation of ATIII, and to examine the resulting changes in heparin binding and heparin-catalyzed activity. Heparin binding was assessed both by the change in intrinsic fluorescence of ATIIL which occurs upon binding heparin, and by the direct measurement of ATIII capable of binding to a heparin-gepharose column. Portions of this research have been previously reported in abstract form (Refs. 24 and 25). Materials and Methods Purified human ATIII with specific HC thrombin iahibitory activity of 48 100 U / m g (see below) was kindly provided by Dr. Robert Jordan of Cutter Laboratories, Berkeley. CA. The sample produced a single homogeneous band when examined by nondenaturing polyacrylanndc gel electrophoresis using the Phast system (Pharmacia, Piscataway, N J). An absorption coefficient, Azsc,-- 6.5, was used to determine ATIII concentration [15]. Human thrombin, specific activity 2062 U / m g , was a gift from Dr. J.W. Fenton of th.~ New York State Department of Health, Albany, NY. Porcine intestinal mucosal heparin (Grade 1. 178 U / m g ; 23 kDa average molecular weight) and the synthetic thrombin substrate Tos-Gly-Pro-Arg-p-nitroanilide acetate were purchased from Sigma, St. Louis. MO.

Measurement of A Till activity ATIII activity was determined by a modification of Abildgaard et at. [16] in which 100 #1 of ATHI (2 /.tg/ml) was added to a polystyrene cuvette containing 1.0 ml of thr~mbin-heparin buffer (0.1 M Tris (pH 8.1))/0.15 M NaC!/thrombin, 0.34 U / m l / s o d i u m heparin, 434 nM (unless otherwise specified). After a 5 rain incubation at 25°C, 100 p.l of thrombin suhstrate (1 9 raM) was added, and the absorbance at 405 nm was mca,~ured at 10 s intervals. Slopes of the resulting linear recordings (r e = 0.999) were determined with thrombin aloae and with on~agla ATIII adaed to yield a 50% decrease in the slope of nonglycated controls. Kinetic analysis of samples wtdch had beet, glycated at different phc'!:ph=.tc concentrations was performed at 25°C (2 min incubation) or 37°C (3 rain incubation) using the kir,efics program of a thermc,~l~tical!y controlled Varian DMS 300 speetrophotometer (Mulgrave, Victoria, Australia). In one experiment, the thrombin-ATIll incubation time was varied from 2 to 15 rain to assess whether extended incubation wkh heparin-thrombin buffer could cahancc the activity of glycated samples. The• nmle~ of thrombin sut~ztrate cleaved were calculated from triplicate assays using a molar absorption

coefficient of 10600 mol - 1- ~- cm- i l'or p-nitroanaline. The coefficient of the variatior, of the ATIII activity was 1.15% (intraday) ~nd 5.3-',% (interday).

In vitro glycation of A TIll Samples of human ATIH (1 mg/ml) were prepared in one of the following buffers containing 0-400 mM glucose/0.02% NaN 3, sterilized by passage through a 0.20 ~M Minipore filter into sterile capped plastic tubes, and incubated at 37°C for 3-4 days: 0.2 M NazHPO4/0.01 M EDTA (pH 7.45, 8.5, and 9.5) or 0.0l M Tris (pH 8.5) or 0.l M MOPS (pH 7.45)/0 0.1 M Na2HPO 4. Because the pH of Mops is highly temperature-dependent, the pH of all buffers was adjusted at 37°C. The pH was checked at the end of each incubation and was found to vary by less than 0.1 unit. The percent glycated ATIII was determined by phenylboronate affinity chromatography (Glycol Gel-B, Pierce, Rockford, IL) following extensive dialysis against the respective buffers containing no glucose [17]. The number of moles of glucose bound to ATIII was determined by a modification of the fructosamine assay to accommodate small protein samples [18]. Aliquots of ATlll (100 /H) which had been dialyzed extensively against 0.1 M Tns (pH 8.0) and 100 td of 1-deoxy-1morpholinofruetose (Sigma) standards (0.04-0.2 raM) were incubated at 37 ° C with 1.0 ml of 0.25 M nitroblue tetrazolium/0.1 M Na2CO 3 (pH 10.8). After 1 h the tubes were placed in an ice bath. The moles of fruetosamine, a ketoantine derived from bound glucose, were calculated from the absorbance at 530 nm. Bound glucose was also determined utilizing o-[U-14C]glucose (DuPont, Wilmington, DE) spec. act. 265 mCi/mmol, wtfich had been purified to remove radioactive impuritier which are known to bind eovalently to protein [19]. The [~4C]glucose was purified essentially as described by Villanueva and Allen [13] by pretreatment with bovine serum albumin. Samples of purified ATIII (0.75 mg/ml in various buffer containing 0.02% NaN 3 as a preservative) were incubated 4 days at 37°C with 200 mM or 10 aiM glucose containing 50 ~1 of purified D-[U-14Clglucose. The glycated ATIII was separated from the nonbound glucose by chromatography on a 1.5 × 11 cm Bio-Gel P-10 column and dialyzed against 0.1 M Tris/0.02% NaN 3 (pH 8.0). The moles of glucose bound to ATHI were calculated from the net radioactivity measured with a 2000 CA Tri-Catb scintillation couate (Packard, Downers Grove, IL). Fluorescence increase upon binding hepurin Fluor~.scence studies were performed with an Aminco-Bowman J4 spectrofluorometer using the conditions of Jordan etal. [15] to measure the fluorescence increase, A F upon binding of heparin to ATIII. Glycated ATIII samples and c,'mtrols were diluted to 200 nM ATIII with 0.2 M NaCt/0.01 M Na2HPO 4 (pH

219 7.45). After establishing a baseline relative fluorescence following excitation at 280 nm and emission at 330 nm, the /tF was determined in triplicate 0.24% coefficient of variation) by adding 100 ~1 of 2.5 m~,',lheparin to 2.4 ml of ATlll solution.

Direct measurement of hepurin binding The fraction of ,*,Till incapable of binding heparin was determined by affinity chromatograFhy. To 1.0 ml of heparin agarose (Sigma Type 1) equilibrated with 0.05 M Tris (pH 8.0) was added 10 ttl of 1.0 mg/ml ATII1 (glycated samples and controls in 0.1 M Mops or sodium phosphate buffers). Following the elution of the nonbound ATIll with 3.0 rnl of 6.05 M Tds (pH 8.0), the bound fraction was eluted with the same buffer containing 2 M NaCh Both the bound and nonbound fractions were desalted, concentrated (Centricon Microconcentrator, Amicon, Danvers, MA) and assayed for ATIII by rocket immunoe!ectrophoresis [20] using IgG fraction antisera to ATIII (Atlantic Antibodies, Scarborough, ME) and ATIIt standards (Nor-Partigen Standard iV, Behring Diagnostics, La Jolla, CA). The percent ATIII nonbound to hepadn was calculated from duplicate rocket measurements of tl,e total ATIII and nonbound fractions. To assess the effect of temperature on heparin binding, column elution was performed at both 25 and 4°C. Two sets of five replicate samples chromatographed and then assayed t~y rocket immunoeleetrophoresis yielded a coefficient of variation of 3.34%. Results

.4 T i l l thromb~n-inhibitory activi O, The HC thrombin-inhibitory activity of ATIlI samples incubated 3 days at 37°C in the presence of glucose was found to depend on the pH of incubation, the buffer salt, and the glucose concentration. As shown in Fig. 1, the heparin-catalyzed activity expressed as the percentage of control samples (without glucose) decreases as the pH is increased. At pH 7.45, 0.01 M EDTA, both 200 mM and 400 mM glucose-incubated samples retained the full activity of the controls. However, as seen in Fig. 1, when the pH is increased to 8.5 and then 9.5 in either Tris or EDTA buffers, a marked decrease in antithrombin activity occurs and becomes more pronounced for solutions incubated in 400 mM glucose. The most significant activity reduction ',-,as obtained wihh 0.2 M phosphate buffer. In 200 mM glucose the activities were reduced to 70.5 +_ 3.8% (+1 S.D.) (pH 7.45), 33.6 _+ 1.8% (pH 8.3), and 32.1 + 2.0% (pH 9.5) of controls. The effect was enhanced with 400 mM glucose, which lowere~l activities to 40.4 _+ 1.7% of controls at pH 7.45 and further to only 5.7 ± .3.2% at pPl 9.5. The influence of phosphate in the incubation buffer is

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Fig. l. Activityof ATilt as a funClionof pH duringglucoseincubation withdifferentanions.ATlll samples(2 mg/ml)were incubated 3 days at 37°C xith the indicatedconcentrationof glucoseqhe buffer sysle,l"tiJ~"luded0.01 M EDTA, a; 90l M Tns. O: or 0.l M sodium ?hospha~e, e, The ATilt assay was run in the presence of 434, nM heparin. Err2r bars indicate:Ll S.D. Tile activityis expressed as the percentage of a control sample incubated under identicalconditions ill Ihe abserce of glucose. further illustrated in Fig. 2, which depicts an inverse relationship between ATIII activity and phosphate molarity for samples incubated 3 days in 200 mM glucose at pH 7.45. Although the activity in 0.1 M Mops was 94.28 ± 1.05% of the nonglycated control, , buffer containing both 0.1 M phosphate and 0.1 M Mops reduced the activity to 80.66 ± 3.57% and further to 73.54_+ 1.18% with 0.2 M phosphate. As shown in Table I, even 10 mM glucose, a blood glucose level often experienced by d',betics (180 mg/dl), resulted in an appreciable loss of HC-ATIII activity compared to nonglycated controls. Activity measurements performed at 37°C in the absence .of heparin resulted in no difference between the nonglycatec controls and the samples incubated 3 days with 200 mM glucose in 0.05-0.2 M phosphate buffer containing 0.1 M Mops (pH 7.45). In contrast, a marked decrease in the heparin-potentiation effect on the assay

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220 TABLE lI

FABLE 1 A Till heparin-catalgzed actici(v at pit Z45 Glucose (raM) 10

Buffer

Incubation (37°C)

Effect of pH and buffer on AT III glvcation and effect of heparin on glucose binding HeDarin Activity (nM) (% conirol)

4d

43

82.7+ 5.5

10

5 mM phosphate 50 mM Mops 10 mM phosphate

3d

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200 mM phosphate 100 mM phosphate

20 h 3d

43 434 434 43

76.4 +4.3 89.5+0.1 85.95-5.5 65.1 _+0.1

434 4340

85.5±3.5 97.4+1.9

c o u l d be observed in the glycated s a m p l e (65% less t h a n control) at 43 n M heparin (Table 1). However, this effect was o v e l c o m e for the most part w h e n the h e p a r i n concentration was increased 100-fold (4300 nM), where the activity of the glycated A T I l t w a s 97.4% of the control nonglycated sample. T o assess whether phosp h a t e alone affected heparin-catalyzed activity, A T I I I samples were i n c u b a t e d in 0.1 M or 0.2 M p h o s p h a t e without glucose for 4 days. These retained 96.8 a n d 95.7% of the activity of a control i n c u b a t e d with 0.2 M NaC.I/0.05 M M o p s ( p H 7.45). Glucose b i nding T h e results of the [ructosamine assay for the m o l a r b i n d i n g ratio of g l u c o s e - A T I l I are s h o w n in Fig. 2. A n inverse correlation between ATII1 activity a n d the mol es of gIuco,e b o u n d is evident ( r 2 - - 0 . 9 9 2 ) . Also illustrated is the positive relationship between the m o l a r ity of p h o s p h a t e buffer a n d the g l u c o s e - b i n d i n g ratio. A similar trend was observed between acti'dty a n d the percent glycated A T I I I d e t e r m i n e d by p h e n y l b o r o n a t e

Buffer

mol glucose bound/mol ATIII ATIII with glucose ~ ATIII with fructos14Cheparin amine assay glucose and [1~Clglucose b assay

0.l M Mops (pH 7.45) 0.1 phosphate (pH 7.45) 0.1 M Tris (pH 8.5) 0.1 M ED'I'A (pH 9.5) 0.01 EDTA (pH 95) 0.1 M Mops+heparin (pH 7.45) 0.1 phosphate+ hepann (pH 7.45)

0.84 1.54 1.82 4.77 2.94

ATilt incubated calculated as net of glucose. s ATIlt incubated incubation with tracer.

0.79

1.10

A

3 days at 37°C with 200 mM glucose. Ratio increase over control ATIII incubated in absence 5 rnin with 434 nM heparin prior to 3 day 20fl mM glucose containing [~4C]glucose as a

affinity c h r o m a t o g r a p h y (data n o t shown). A s s h o w n in T a b l e 11, p H h a s a m a j o r effect o n ' h e extent o f glycation. A t p H 7.45, b o t h the fructosamine a n d [t4C]glucose assays d e m o n s t r a t e d that approx. 0.8 m o l of ~ u c o s e are i n c o r p o r a t e d p e r tool A T I I I d u r i n g a 3 day, 37 ° C i n c u b a t i o n in 0.1 M M o p s / 2 0 0 m M glucose buffer. T h e g l u c o s e - b i n d i n g ratio (by the fructosamine assay) increases with the p H o f the buffer to 4.77 at p H 9.5. T h e result of i n c u b a t i n g A T I I I 5 rain with h e p a r i n prior to glucose i n c u b a t i o n is also given in T a b l e If. A h h o u g L h e p a r i n i n c u b a t i o n resulted in a 40% decrease

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Fig. 3. (a) Heparin-induced fluozescence increase ezpressed as percent control for ATIII samples (2.0 nlg/ml) incubated 3 days at 37°C in 0.2 M Na ~HPO4/3 mM NaN~ at the indicated pH in 200 mM (striped bars) or 400 mM (dark bars) glucose. The error bars indicate t t S.EI. of the mean of triplicate values. The relative fluores~nce increaze was measure.d at 280 nm txcitat~en, 330 nm emission following the addition of 0.10 ml of 0.(]025 M hepeml to 2.4 ml of 2.0-I0 -7 M ATIII diluted from the ~l~k solution with 0.2 M NaCI/0.01 M NaHzPOa (pH 7.45). (b) Heparin-induced fluorescence increase ,gf ATIII incubated 3 days at 37°C with 200 mM glucose, 0.1 M Mops, pH 7.45 at the given phosphate cr ,ce~t~ ati_-n~

221 in the glucose-binding ratio, no difference was evident between ATIII incubated in Mops or phosphate p H 7.45 buffers, suggesting heparin overcame the phosphate enhancement of glycation.

Mops was 80.46 _+ 2.03% of the control, ever, the prese l c e of a low (0.01 M) phoshhate coric'mtration reduced tlte £ F further to 71.01 5- 0.88%. U n d e r these conditions the sample glycated in 0.2 M phosphate yielded a A F of only 47.53 + 1.36% of the control.

Fluorescence studies Direct measurement of heparin binding

The addition of heparin to A T I I I causes an increase in fluorescence, AF, due to the exposure of t~3,ptophan residues within the proteinase inhibitor [151. As shown in Fig. 3a, glycation of A T I I I produces a smaller fluorescence increase than displayed by the control upon exposure to heparin. Samples glycated in 0.2 M phosphate (pH 7 . 4 5 ) / 2 0 0 m M or 400 m M glucose showed only 6 8 . 0 + 4 . 6 % and 5 6 . 2 + 0 . 7 % z~F of the control, rcspeeti-¢ely. In contrast, samples incubated with glucose in 0.01 M E D T A (pH 7.45) retained 100% ,~F of the control. At higher pH with 0.2 M phosphate and 200 m M glucose, the z~F is reduced further to 43.1 :i: 1.4% of control at p H 8.5 and to only 11.7 5- 1.3% at p H 9.5. Since no further decrease occurred for samples incubated in 400 m M glucose at pt-I 8.5 or 9.5, the glycation effect on Z~F appears to be saturated with 200 m M glucose at these p H values. The effect of phosphate concentration upon the heparin-induced fluorescence increase, AF, is depicted in Fig. 3b for A T I I I incubated 3 days at p H 7.45 with 200 m M glucose. The percentage AB increase is relative to that obtained with 0.1 M M~ps con]rc, l. Although the fluorescence increase of the sample glycated in 0.1 M

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Because the fluorescence results suggested a reduced heparin interaction with glycated A T I l l , we used heparin-agarose affinity chromatography to directly measure the percentage A T l l l capable of binding heparin. The results of this study are shown in the rocket immunoelectrophoresis patterns of Fig. 4a for samples glycated in 0.2 M phosphate at three p H values with 200 m M and 400 m M glucose. The percentage A T ! I t not bound to heparin was increased with both the p H and the molarity of glucose during incubation. The bar graph of Fig. 4b illustrates the effect of phosphate concentration on heparin binding for samples incubated at p H 7.45 in 200 m M glucose. For elution at 2 5 ° C , the percentage nonbound increases from 2.71% for the 0.1 M Mops control to 30.3% for the A T I I I sample glyeated in 0.2 M phosphate. At the lower elution temperature ( 4 ° C ) the same trend continues, although the percentage nonbound is considerably lower. The 0.1 M Mops control had 2.26% nonbound, while that of the 0.2 M phosphate glycated sample was 17.2%. The presence of phosphate alone was also found to affect the heparin binding. The 0.2 M phosphate control

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bis. 4. (a) Rocket immunoelectrophvr=sls of ,iumieparm-bindmg Attll ,i'mctionzclutexl from a hepatin-agarose columJ~ ~ith pt,~ S I 0.c~5M Tfis. Samples wcrc incubated 3 days at 37 ~ C in 0.2 M Na2HPO4/0.02 NaN 3 with the indicated molari~y of glucose. Wells 1, 4 and 7: con£rois at pH 7.45, 8.50, and 9.50; wells 2, 5 and 8:200 mM glucose at pH 7.45, 8.50 and 950; wells 3, 6 and 9:400 mM glucose at pH 7.45, 8.50 and 9.50; v,elis 10-]3 contain ATI|] standards of 11.4, 5.7, 2.9, and 1.4 mg/dl. (b) The effect of glycation and elutinn temperature on the ability of ATI[I to bind heparin. A'I Ill (1 mg/ml) was incubated 3 days at 37 °C in 3 mM NAN3/200 mM glucose (pH 7.45) with h, 0.l M Mops; c, 0.] M Mops/f,.0~ M phosphate; d, 0.1 M Mops/0.0S M phosphate; e, 0.] M Mops/0.10 M phosphate: arid f, 0.2 M phosphate. 1he controls are: a. 0.1 M Mops and g, 0.2 M phosphate in the absence of ~Juco~e. The nonbound fractlv,~was eluted from a 2.5×0.8 em heparin agarose column with 0.05 Tris tpH 8.0) at 25°C or 4°C.

222 produced 12.6% nonbinding at 25°C and 9.13% at 4 ° C. However, the phosphate effect accounts for a fraction of the effect seen in the glycated samples. Discussion

incubation of ATIII in glucose leads to a reduction in its HC antithrombin activity. In the original report that heparin cofactor activity of ATIII was reduced by glycation, the glucose incubation was done at the unphysiological pH of 9.5 [5]. Our data show that as the pH of incubation decreased to pH 7.4, the effect on the heparin-ccfactor act.vity and the extent of g!ycation decreases. The rate of giycation of proteins would be expected to increase as the pH becomes more alkaline, since Schiff base formation requires an unprotonated amino group and the formation of the stable kctoamine (Amadori rearrangement product) is accelerated at higher pH [21]. However, significant reduction in antithromt,hi activ;ty at pH 7.45 was also observed when the anionic buffeting ion, phosphate, was present. Sakurai et al. [11] did not find a decrease in ATIII activity following glycation under very similar conditions (0.1 M phosphate (pH 7.7)/100-500 ruM glucose. 3 days at 37°C). However~ their assay differed from ours in that they did not include heparin. Thus, our assay was measuring HC ATII1 activity while their procedure would not show the influence of heparin bindi~ag co ATIII activity. When we left heparin out of our assay mixtures, we also found no decrease in the ATIII activity following incubation with glucose. Most o, the glycation experiments in this study involved 3-day incubation with glucose concentrations 10-20-tittles the usual hyperglycemic levels seen in diabetics, in order to produce pronounced etfects in the parameters being compared. However. in an experiment designed to approximate physiologically attainable levels of glucose (10 mM) in diabetes at pH 7.45 after a 3-day incebation, a 17% reduction in heparin-eatalyzed antithrombin activity was observed when 5.0 mM phosphate was present. The half-hfe of ATIII in blood has been estimated to be 66.2 h [22]. This reduction in activity is in the same range reported in studies on groups of diabetics. These results support the conclusions drawn :n a recent report by Vil!anueva and Allen [13] that significant heparin-catalyzed activity reduction occurs when ATIII is incubated at glucose conce~Jtrations (15 mM) fouv.d in severe diabetics. The ~eductlon in heparin-catalyzed ATilt activit> paralleled an increase in the number of moles of glucose incorporat ~d into ATIII as evidenced by the fructosamine and t 14C]~ucose assays. In addition, the moles of glucose reacting with ATIII increased progressively with the phosphate concentration ~n the buffer, whether measured by [a4C]glueose binding or fruetosamine assays. This is evidence that the observed reduction in activity

is due to nonenzymatic glycation. Free glucose in the assay mixtures did not contribute to ~ile reduction in activity, since dialysis of the samples to remove the glucose before assay did not change the results. That the reduclion in antithrombin activity following giycation was due to reduced heparin binding is subztantiated by the experiments showing a reduction in the heparin-induced intrinsic flporescence change and the direct measurement of the fraction of A T i l l bound to heparin-Sepharose columns. An inverse linear correlation (r 2 ~ --0.999) between ATII1 activity and the percent nonbinding to heparin exists. In addition, a negative correlation (r 2= -0.941) was found between AF and the percentage nonbinding ATIll. The reduction in the heparin-induced fluorescence increase is evidence that the conformation change caused by hepadr~ binding is not nccuning to the same extent, leading to reduced heparin catalyzed activity. The reduction in the percentage of ATIII molecules binding to the heparin affinity column is direct evidence of reduced heparin binding following the giycation reaction. Our results at 4 ° C compare favorably to those of Sakurai et al. [ll[, who found only a 6% difference between glycated and nonglycated samples following a 2 day incubation with 100 mM glucose. At 25°C, however, we found an 18% difference between glycated and non-glycated ATIll in 0.2 M phosphate. Clearly, heparin binding is temperature-dependent. In agreement with Villanueva and Allen [13], we found that preinenbation of A T l l l with heparin reduces glucose bLading. This evidence supports the theory that ATIII has glucose- and heparin-binding sites in similar regions of the molecule. Since phosphate did not increase the [14C]ghicose incorporation following heparin incubation, we speculate that the heparin competes for the sites most affected by phosphate-enhanced glycation. The facts that phosphate ion enhances the effect of glycation on reducing the heparin-catalyzed activity of ATIII in vitro, and that the initial groups glyeated are those directly involved in this activity reduction, suggest that there may be factors in plasma during period~ of hyperglycemia that promote giycation to the extent necessaD to e:~plaip the rapid reduction ,in ATIII activity reported by Cerielhi and co-workers [23]. Further wc,rk ',,.'ill be needed to determine if glycation is the cause tff the rapid reductions in AT1H activity in byperglycemia, or whether simple Sehiff base formation, withnnt the subsequent Amadori rearrangement, is all that is required to result in the in vivo ATIII activity reduc!ion. Acknowledgements This work was supported it1 part by the Marshfield Medical Research Foundation. The auth~r~ w(sh to express their app,'eciaqon to Caroline Farainb~; for her

223 technical support and to Doreen Luepke for typing this manuscript.

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