Human Neutrophil Elastase Alters Human A-Thrombin Function

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Human Neutrophil Elastase Alters Human a-Thrombin Function: Limited Proteolysis Near the ‘y-Cleavage Site Results in Decreased Fibrinogen Clotting and Platelet-Stimulatory Activity

By Mark S. Brower, Daniel A. Walz, Kimberly E. Garry, and John W. Fenton II

During blood coagulation. polymorphonuclear leukocytes thrombin (T.) retained full amidolytic activity; however. the release elastase in amounts that can exceed 100 nmol/L. concentration of T#{149}causing 50% maximal platelet aggre- We therefore studied the interaction between human gation and adenosine triphosphate (ATP) release was 7.9 leukocyte elastase and human a-thrombin. Elastase nmol/L (1 .1 nmol/L for a-thrombin and 220 nmol/L for cleaved the thrombin B chain (Ala 1 50-Asn 1 51 ) near the “y-thrombin). Fibrinogen clotting activity of T#{149}and ‘y-throm- “y-cleavage site, resulting in two fragments held together bin was 32% and 1 % that of a-thrombin, respectively. by noncovalent interactions. The NH2-terminal fragment Elastase released during the coagulation process may (Fl). mol wt -18,000. was disulfide-linked to the thrombin modulate thrombin activity. In addition, elastase-modified A chain. The COOH-terminal fragment (FlI). mol wt thrombin may be a useful probe of the structure and 1 3.000. contained the active-site serine and formed a function of the “y-cleavage region.

covalent bond with antithrombin Ill. Heparin accelerated S 1987 by Grune & Stratton, Inc. Downloaded from http://ashpublications.org/blood/article-pdf/69/3/813/594640/813.pdf by guest on 26 September 2021 proteolysis of a-thrombin by elastase. Proteolyzed a-

E LASTASE IS the major neutral protease released by the inhibitor migrated as a single band; >96% of the antithrombin human polymorphonuclear leukocytes (PMNs)’ dun- III formed a higher mol-wt complex with a-thrombin. ing blood coagulation or activation by soluble immune com- Proteolysis ofa-thrombin by elastase. a-Thrombin (- 10 smol/ L) was incubated with elastase (thrombin/elastase molar ratios plexes, C5a, or endotoxin.’3 Elastase is released into the from 1: I to I 00: 1) for various times (37 #{176}C)in 0.02 mol/L of Trizma circulation in acute leukemia, in septicemia,4 and in a variety base (Iris), pH 7.4, or in Iris buffer containing 0. 15 mol/L of NaCI. of other inflammatory conditions.3 Several studies suggest Incubations were performed in the presence or absence of I U/mL of that the decrease in the concentration of coagulation factors sodium heparin (140 U/mg) from porcine intestine (Elkins-Sinn, that occurs in these conditions may be the result of direct Cherry Hill, NJ). The reaction was stopped either by incubating for proteolysis by PMN elastase.56 Elastase also inactivates 10 minutes (37 #{176}C)with 0.7 mmol/L of MeO-Suc-Ala-Ala-Pro- human antithrombin IlI, a2-plasmin inhibitor, and Cl mac- Val-CH2CI (Enzyme Systems Products, Livermore, CA) to inhibit tivator.8 Furthermore, it inhibits thrombin-induced platelet elastase activity#{176}or by boiling in SDS as detailed later. Samples stimulation in association with limited proteolysis of platelet were quantitated for residual elastase activity with MeO-Suc- membrane glycoprotein lb.9 In circulating blood, beukocyte AIa-Ala-Pro-VaI-CH2C1 (Enzyme Systems Products) as de- elastase is inactivated mainly by a,-antitrypsi&#{176}; however, scribed.2’ Only those samples demonstrating >95% inhibition of initial activity were used in the following studies. this inhibitor is readily inactivated by oxidizing agents such SDS-PAGE. Samples for electrophoresis (4 to 8 .tg per lane) as those generated by phagocytosing PMNs.”’2 More were mixed with an equal volume of0.06 mol/L oflris, 2% SDS, 5% recently, studies using cell contact systems have shown that glycerol, 0.001 % bromphenol blue, and 2% dithiothreitol (SDS- a,-antitrypsin does not completely inhibit the proteolytic DTT), solubilized by heating at 100 #{176}Cfor 3 minutes, and stored activity of PMN-denived elastase)3”4 ( - 70 #{176}C).SDS-PAGE was performed by the method of Laemmli,22 In this study, we investigated the effects of PMN ebastase using a 3.8% stacking gel and a 9% to 18% gradient separation gel. on human a-thrombin. PMN elastase cleaved the thrombin Ebectrophoresis was terminated when the buffer front was I cm from B chain at Ala 1 50-Asn I 5 1 , within four amino acids of the the bottom of the gel. Gels were fixed in 25% isopropanol and 10% ‘y-cbeavage site. Cleavage resulted in formation of a larger NH2-terminal and a smaller COOH-terminal fragment held together by noncovabent association. The elastase-modified From the Specialized Center for Research in Thrombosis, Divi- thrombin possessed amidolytic activity similar to that of sion of Hematology-Oncology, Department of Medicine, The New a-thrombin but exhibited reduced fibninogen clotting and York Hospital-Cornell Medical Center; the Department of Physiol- platebet-stimulatony activity. Ebastase degradation of a- ogy, Wayne State University, Detroit; and the Wadsworth Center thrombin was enhanced by hepanin. These findings suggest f or Laboratories and Research, New York State Department of Health, Albany. that elastase may be involved in the physiologic modulation Submitted July /7. 1986; accepted October 3. 1986. of a-thrombin function. Supported by US Public Health Service and by grants No. H1-/8828 (Specialized Center ofResearch in Thrombosis) and ROl MATERIALS AND METHODS HL-32166 (M.S.B.), HL-27073 (D.A. W.), and HL-13160 (J. W.F.). Human polymorphonuclear leukocyte elastase, specific activity Address reprint requests to Dr MS. Brower, Specialized Center 92%, was purified from outdated WBC concentrates or from puru- for Research in Thrombosis, Division of Hematology-Oncology. lent sputum.8”5 Human a-thrombin and ‘y-thrombin were prepared Department ofMedicine, The New York Hospital-Cornell Medical and stored at - 70 #{176}Cin 0.75 mol/L of NaCl at concentrations of 2 Center. 1300 York Aye, New York, NY /002!. to 4 mg/mL.’6”7 As determined by active-site titration (p-nitrophe- The publication costs ofthis article were defrayed in part by page nyl-p’-guanidinobenzoate), the specific activities of the a-thrombin charge payment. This article must therefore be hereby marked and ‘y-thrombin preparations were 95% and 78%, respectively.’8’9 “advertisement” in accordance with 18 U.S.C. §1734 solely to Antithrombin III was obtained as a gift from the American Red indicate this fact. Cross Fractionation Center, Bethesda, MD. As analyzed by sodium © 1987 by Grune & Stratton, Inc. dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 0006-4971/87/6903--0018$3.00/0

Blood, Vol 69, No 3 (March). 1987: pp 813-819 813 814 BROWER ET AL

acetic acid prior to being stained with Coomassie blue (0.1%). AlP with 4 L of firefly luciferin and luciferase (40 mg/mL, Densitometry was performed at 633 nm using an LKB Ultroscan Chrono-Lume No. 395, Chrono-Log) added I to 5 minutes prior to Laser Densitometer and LKB model 22 10 dual-channel recorder. stimulation of the pIateIets.29’#{176} Secretion was measured as the Mol wt markers included phosphorylase b (mol wt 94,000), bovine maximum increase in fluorescence and expressed as the percentage serum albumin (BSA) (mob wt 67,000), ovalbumin (mob wt 43,000), of the increase in the control. carbonic anhydrase (mol wt 30,000), soybean trypsin inhibitor (mol The threshold stimulus determined for individual platelet prepa- wt 20,100), a-lactalbumin (mol wt 14,400), and aprotonin (mol wt rations was defined as the lowest concentration that caused 90% 6,500). increase in light transmission at 5 minutes when added to stirred Interaction of elastase-modified thrombin with antithrombin platelets at 37 0C3O and resulted in fluorescence at least 90% of III. a-Ihrombin (1 1 tmol/L) was incubated either alone or with maximum. Immediately after measurements of aggregation and elastase (1.7 zmol/L) for 60 minutes (37 #{176}C)in 0.02 mol/L of Iris secretion, 40 tmol/L of ATP (Sigma FF-ATP, 1 mg/mL, and 40 pH 7.4 containing I U/mL of porcine intestinal heparin. The mg/mL of MgSO4 . 7 H20) was added for instrument calibration. mixture was incubated for 10 minutes (37 #{176}C)with 0.7 mmol/L of MeO-Suc-Ala-Ala-Pro-Val-CH2CI prior to addition of antithrom- RESULTS bin III (30 zmol/L). Samples were taken at various times for

Incubation of a-thrombin with elastase. cr-Thrombin Downloaded from http://ashpublications.org/blood/article-pdf/69/3/813/594640/813.pdf by guest on 26 September 2021 analysis by SDS-PAGE. was incubated alone or with elastase for various times Amino acid sequence of elastase-modified thrombin. Samples were prepared for sequencing as described23 by extensive dialysis (37 #{176}C).After MeO-Suc-AIa-Ala-Pro-Vab-CH2C1 was ad- against 0.1% trifluoroacetic acid (IFA) and freeze-drying. The final ded to inhibit elastase activity, samples were reduced, elec- thrombin product (2 10 tg) was dissolved in 0. 1% IFA, and one-half trophoresed, and stained with Coomassie blue. The B chain (2.6 nmol) was applied to a Beckman 890M spinning cup sequencer. of thrombin migrated as a single band at mob wt 32,500 after The resultant phenylthiohydantoin (PTH) derivatives were resolved incubation for 0 to 30 minutes with buffer alone (Fig 1 , lanes on a Beckman Ultrasphere 5-zm ODS column using a Waters 501 9 through 10). The small (mob wt) a-thrombin A chain was pump, 440 detector (254 nm) and 740 integrating recorder. Isocrac- not visualized in this system.’6 Incubation of thrombin with tic separation of the common PIH amino acids was achieved at elastase (thrombin/ebastase molar ratio 8: 1) resulted in 55 #{176}Cwith 0.01 mol/L of sodium acetate, pH 4.54 and acetonitrile proteobysis (lanes I through 4). After incubation with elas- (38:62, vol/vol) at a flow rate of 1 mL/min. PTH yields were tase for 1 5 seconds, bower mob wt bands were observed (lane automatically calculated by peak area and expressed as a percentage of the respective 200-pmol standard. Duplicate thrombin samples I ). Those at mob wt -28,000 co-migrated with the elastase were subjected to sequence analysis. isomers3’ seen in the elastase controls (lanes I 1 and 1 2). New Amidolytic activity. Samples of a-thrombin and elastase- bands also appeared at mob wt 21 ,000, I 6,000, and 15,000. treated thrombin (>95% conversion to the cleaved form on SDS- These bands were diminished or absent if elastase was PAGE) were analyzed for amidolytic activity with Tos-Gly-Pro- inactivated with a,-antitrypsin (experiment not shown) or Arg-paranitroanalide (GPA, Vega) by modification of the method with greater concentrations of MeO-Suc-Ala-Ala-Pro-Val- of Kirchof and colleagues.24 GPA was dissolved in methanol (14 CH2CI prior to the addition of SDS to the mixture. This mg/mL). Duplicate dilutions of thrombin (0.3 to 270 nmol/L) in suggests that these peptides may result from either (a) Iris 0.2 mob/L, pH 7.4 containing 1 mg/mL of BSA (Sigma) were incubated at 23 #{176}C(final volume 0.2 mL per well) with GPA (0.5 mg/mL) in 96-well microtitration plates (Nunc). Color development at 405 nm was followed on a Titertek Multiscan photometer -Heporin +Heparin r-’---- (Flow Laboratories, McLean, VA). The change in absorbance per minute was subtracted from the values obtained in wells that did not Mr s i0 contain thrombin. Plots of color development with time demon- strated a linear relationship. Fibrinogen clotting activity. Human fibrinogen (Kabi Grade L) was dissolved in 0.3 mol/L of NaCI, 0.1 mol/L of sodium citrate pH

6.4, containing 0.01 mol/L of -aminocaproic acid (Aldrich, Mil- 32.5-

waukee), and stored ( - 70 #{176}C).For thrombin assays, fibrinogen was 25- diluted (6.4 mg/mL) in 10 mmol/L of imidizole, 10 mmol/L of CaC12, 0.15 mol/L of NaCI pH 7.4. Aliquots (0.09 mL) were prewarmed to 37 #{176}Cprior to the addition ofvarious concentrations of I F U) thrombin prepared in 0.2 mL of the same buffer (37 #{176}C)containing 0.1% BSA. Clotting time was determined by fibrometer (Becton Dickinson No. 60415).

Platelet stimulation studies. Washed platelet suspensions were Lone 234 5678 9 0 11 12 prepared by modification of the technique of Mustard and co- Time (mm) 1/4 3 0 30 1/4 3 10 30 0 30 0 30 workers as described.6227 Platelet stimulation studies were performed at 37 #{176}Cwith 0.45-mL aliquots of washed platelets (2 x Fig 1 . Effect of heparin on thrombin proteolysis. a-Thrombin 108/mL) stirred at 1,000 rpm in a Chrono-Log model 460 Lumi- (10 .tmol/L) was incubated (37 ‘C) with elastase (1 smol/L) in 0.02 mol/L of Tris pH 7.4 with (lanes 1 through 4) or without (lanes 5 Dual double-channel aggregometer. Aggregation and luminescence through 8) of heparin (1 U/mI). After various times, MeO-Suc- in each channel was recorded with a Chrono-Log model 703 strip Ala-Ala-Pro-Val-CH2CI (0.7 mmol/L) was added to inhibit elastase chart dual-pen recorder. Aggregation was quantitated by planime- activity. Samples were electrophoresed (5 ig thrombin per lane) try. Results were expressed as percentage of the area under the and stained with Coomassie blue. The a-thrombin controls at 0 control curves. and 30 minutes are shown in lanes 9 and 1 0, respectively; those for Secretion was measured by the luminescence reaction of secreted elastase are shown in lanes 1 1 and 12. PROTEOLYSIS OF a-THROMBIN BY ELASTASE 815 elastase-proteolysis of SDS-denatured thrombin or (b) slim- ubation of ebastase activity by SDS.3234 With longer incubations (lanes 2 through 4), the concentration of thrombin B chain at mob wt 32,500 was diminished; by 30 minutes proteolysis of 50% of the thrombin B chain occurred. The three bands at mol wt 21 ,000, I 6,000, and 15,000 were no longer apparent after 30 minutes, and two additional bands representing core fragments appeared at mob wt I 8,500 and I 3,500, designated fragment I (Fl) and fragment lb (FII), respectively. In four separate expeni- ments, the mob wt of Fl ranged from 1 7,000 to I 8,500, and that of FII ranged from 12,000 to 13,500. In experiments not shown, Fl and FbI could not be separated under nondenatuning conditions (0% to 1 mol/L of Downloaded from http://ashpublications.org/blood/article-pdf/69/3/813/594640/813.pdf by guest on 26 September 2021 NaC1; 0% to 3% Triton X-lOO, Sigma, St Louis). This suggests that Fl and Fil are held together by strong noncovabent interactions, as reported for the ‘y-thrombin B chain fragmentation peptides.3538 Hepanin binds to both thrombin and elastase.393 There- fore, the effect of hepanin on elastase-mediated proteolysis of Fig 2. Interaction of elastase-modified thrombin with antithrombin Ill. a-Thrombin (1 1 pmol/L) was incubated either alone a-thrombin was examined. a-Thrombin was incubated with or with 1 .7 mol/L of elastase at 37 ‘C prior to incubation at 37 ‘C elastase in Iris buffer with I U/mL of hepanin (lanes 5 (10 minutes) with MeO-Suc-Ala-AIa-Pro-Val-CH2CI (0.7 mmol/L). through 8). Within 15 seconds after the addition of elastase Antithrombin Ill (30 smol/L) was added to the mixtures, and to a-thrombin, Fl and FII were observed (lane 5). The samples were taken for analysis at various times. Lane 1 . elastase- modified a-thrombin; lane 2, a-thrombin control; lane 3. anti- breakdown products at mol wt 2 1 ,000, I 6,000, and 14,000 thrombin Ill control; lanes 4 through 6. incubation of antithrombin seen in lanes I and 2 were not observed in the presence of Ill with elastase-modifled thrombin shown in lane for 1 5 seconds. hepanin. Within 3 minutes (bane 6), >50% ofa-thrombin was 5 minutes, and 60 minutes. respectively; lanes 7 through 9, proteolyzed to Ft and FII; by 30 minutes (lane 8), the incubation of antithrombin Ill with a-thrombin shown in lane 2 for conversion ofa-thrombin to Fl and FIb was complete. 1 5 seconds, 5 minutes. and 60 minutes. respectively; lanes 10 and 1 1 , a-thrombin was incubated with antithrombin for 5 minutes as These results indicate that hepanin accelerates elastase- in lane 8 prior to the addition of elastase (1 .7 Mmol/L). Samples mediated proteolysis of a-thrombin. They also suggest that were taken at 1 5 seconds (lane 10) and 5 minutes (lane 1 1). hepanin protects a-thrombin from conversion into the mob wt Positions of the mol wt markers are indicated on the right. 2 1 ,000, I 6,000, and 14,000 fragments. Elastase cleavage of factor IX is bess extensive in the wt 72,000, the mol wt compatible with that of a complex absence of calcium.6 Therefore, the above experiments were between antithrombin III (mol wt 58,000) and FII (mob wt performed in the presence of CaCI2 (2 to 6 mmob/L). 13,500). In addition, after 15-second incubation (lane 4) the Calcium did not alter or inhibit proteolysis of thrombin by intensity of free FII was diminished relative to Fl; within 5 elastase. minutes of incubation (lane 5), FII was no longer apparent. When analyzed on SDS-PAGE without disulfide bond These findings suggest that the active-site senine is contained reduction, thrombin fragments Fl and FlI were identified at within FII. mob wt 23,000 and I 3,500 (gel not shown). The increase in For comparison, antithrombin III was incubated with the mob wt of Fl prior to reduction suggested that it was a-thrombin for I 5 seconds, 5 minutes, and 60 minutes, covabently linked to the thrombin A chain. respectively (lanes 7 through 9). The a-thrombin-antithrom- Interaction of elastase-modified thrombin with anti- bin III complex was visible at mob wt 88,500. A faint band thrombin III.- Identification of the active-site--containing was apparent at mob wt -72,000, with ebectrophoretic mobil- fragment. To determine if F! or FIl contained the active- ity similar to the FII-antithrombin Ill complex. This mob wt site senine, antithrombin III was incubated either with -72,000 complex has been shown to be the result of degrada- a-thrombin or with the elastase-modified protease in the tion of the a-thrombin-antithrombin complex by free throm- presence of I U/mL of hepanin (inhibitor/enzyme molar bin.”45 The relationship of this complex to the FIb- ratio -3:1). After various times, samples were analyzed by antithrombin complex remains undetermined. SDS-PAGE (Fig 2). ct-Thrombin incubated in the presence These results demonstrate that antithrombin III inhibits of elastase (lane I ) was converted into the elastase-modified elastase-modified thrombin by combining with the active- form, as indicated by the presence of Fl and FII. A faint site senine within FII. These findings suggest that elastase band representing ebastase was seen at mob wt 28,000. Lane 2 cleaves the a-thrombin B chain in the vicinity of the -y- contained a-thrombin incubated in the absence of ebastase cleavage site.46 This results in the formation of an NI-I2- for comparison, and bane 3 contained antithrombin Ill (mob terminal fragment (Fl), mob wt 18,000, that is disulfide wt 58,000). Incubation ofantithrombin III with the modified linked to the thrombin A chain and a COOl-I-terminal thrombin for I 5 seconds, 5 minutes, and 60 minutes (bane 4, fragment (FbI), mol wt 13,000, containing the active-site 5, and 6, respectively) resulted in formation of a band at mol senine. 816 BROWER ET AL

To determine if elastase can degrade preformed a-throm- 0.6 ---T bin-antithrombin complexes, thrombin was incubated with 0.5 antithrombin for 5 minutes as in lane 8 prior to incubation 0 with elastase (thrombin/elastase molar ratio 9:1). Fifteen 0.4 E seconds after addition of ebastase (bane 10), a complex was E 0.3 observed at mob wt 72,000; by 5 minutes (lane I 1), the U) 0.2 a-thrombin-antithrombin complex at mob wt 88,500 was 0 .6 markedly reduced in intensity, and protein at mol wt 72,000 63 0.1 was increased. A faint band migrated at mol wt 16,000. Neither fragment Fl nor fragment FIl was observed in lanes 100 10 or 1 1, and there was an increase in stained material [Thrombin] moving just behind the buffer front. These findings suggest Fig 3. Amidolytic activity of elastase-modified thrombin. a- that elastase cleavage of the a-thrombin-antithrombin com- Thrombin (T) or elastase-modified thrombin (T,) was diluted (0.3 plex may differ from cleavage of free a-thrombin.

to 270 nmol/L) in buffer and incubated at 23 ‘C with Tos- Downloaded from http://ashpublications.org/blood/article-pdf/69/3/813/594640/813.pdf by guest on 26 September 2021 Amino acid sequence of elastase-modified throm- Gly-Pro-Arg-paranitroanalide (GPA) (0.5 mg/mI). Color develop- bin. Because the thrombin samples were analyzed directly ment was analyzed as detailed in the Materials and Methods section. Each point is the average of duplicate determinations. after proteolysis, at beast one new amino terminal sequence Curves were determined by linear regression analysis. For T. y = was anticipated, in addition to the usual A chain and B chain 39.2x - 38.2 (r = 0.988); for T#{149},y = 39.7x - 41 .4 (r = 0.968). sequences. Amino acid analysis confirms the presence of a new NH2-terminal sequence corresponding to residues I 51 of a-thrombin but less than that of y-thrombin. As deter- through 156 of the human B chain of thrombin (Table I). mined by comparing the slopes of the three lines obtained by Residues 1 (Asn), 4 (Lys), and 6 (GIn) are unequivocal; the linear regression analysis (Fig 4 legend), elastase-modified yields at residues 2 (Val), 3 (Gly), and 5 (Gly) are in excess thrombin was 32% as potent as a-thrombin in clotting of theoretical yields, assuming that only one or two peptides fibrinogen; ‘y-thrombin was I % as potent as a-thrombin. The exist. Further efforts are under way to resolve these compo- relative slopes of the three curves were unaltered by the nents so that peptide yields and the extent, if any, of protein presence or absence of hepanin or by the addition of elastase fragmentation at the carboxy terminal regions of the three to a-thrombin immediately prior to mixing with fibninogen constituent polypeptides detected there can be quantitatively (experiments not shown). determined. Platelet stimulatory activity ofelastase-modified throm- Amidolytic activity of elastase-modified thrombin. In- bin. To compare the platebet-stimulatory activity of elas- creasing concentrations of elastase-modified thrombin were tase-modified thrombin with that of a-thrombin and ‘y- added to microtitration plate wells containing GPA (Fig 3). Color generation was proportional to the concentration of enzyme in the range of 13.6 to 272 nmol/L. The amidolytic 0 Te 0 (2 activity of the modified enzyme appeared to be nearly a, U T identical to that of a-thrombin. U) Fibrinogen clotting activity of elastase-modified throm- a, E bin. The fibrinogen clotting activity of elastase-modified thrombin (>98% conversion to the modified form in the 0’ C presence of heparin 1 U/mL) was compared with that of a- 0 and ‘y-thrombin (Fig 4). At each thrombin concentration, the C.) clotting time of the modified thrombin was greater than that C a, a, 0 Table 1 . Sequence of E lastase-Treated Hum an a-Thrombin C .0 Amino Acid Detected LI- Cycle A-chain B-chain Additional Peptide 0 0.5 1.0 1.5 1 Thr(0.6) lle(1.2) Asn(O.8) 1 I 2 Phe(1.0) Val(1.9) Val(1.9) [Thrombin] (nM1) 3 Gly(1.6) GIu(O.5) Gly(1.6) Fig 4. Fibrinogen clotting activity of elastase-modified throm- 4 Ser(NQ) GIy(O.7) Lys(O.8) bin. a-Thrombin (T). elastase-modified thrombin (Ta). or ‘y-throm- 5 Gly(1.4) Ser(NQ) Gly(1.4) bin (T.) was diluted in buffer and prewarmed to 37 ‘C. After 6 Glu(0.6) Asp(0.4) GIn (0.3) fibrinogen was added (2.0 mg/mL). fibrin clotting time was deter- Human a-thrombin (2.6 nmol) was subjected to sequence analysis. mined as detailed in the Materials and Methods section. Each point represents an individual clotting time measurement. Curves were Yields (in parentheses) are corrected to final yield, expressed in nano- determined by linear regression analysis. For T. y = 77.7x + 17.02 moles. The sequence, Additional Peptide, corresponds to residues 151 (r 0.968); for T,, y - 245.6x + 8.66 (r = 0.992); and for T.,, y through 156 of the human thrombin B-chain. 6629x + 12.44 (r = 0.941 ). Study shown is representative of Thr, threonine; lIe, isoleucine; Asn, aspargine; Phe, phenylalanine; Val, studies on four separate T#{149}preparations. Equivalent concentra- valine; Gly. glycine; Glu, glutamic acid; Sac, serine; Lys, lysine; GIn, tions of T. T,, and T. had similar amidolytic activities against glutamine, NQ, not quantitated. Tos-Gly-Pro-Arg-paranitroanilide (GPA). PROTEOLYSIS OF a-THROMBIN BY ELASTASE 817

clotting activity and diminished platelet stimulatory activity, S as measured by platelet aggregation and platelet ATP C 0,, release. Loss of a-thrombin function is associated with limited cleavage of the thrombin B chain. WC , .2 The concentration of prothrombin in plasma is sufficient to generate I 50 U/mL of a-thrombin.47 In addition to C.) Ew ,u) being inactivated by circulating inhibitors, a-thrombin selec-

.; 0 tively binds to fibrin, thereby becoming less susceptible to 0 E inactivation by its plasma inhibitors.5#{176} These studies suggest that fibnin acts as a reservoir for enzymatically active thrombin. Human PMNs release ebastase as blood clots2”; in [Thrombin] addition, PMNs penetrate preformed blood clots, accumu- late within thrombin, and ingest fibrin.5’5’ Recently, Weitz Fig 5. Platelet stimulation by elastase-modifled thrombin. and co-workers documented that the fibrinogen peptide Downloaded from http://ashpublications.org/blood/article-pdf/69/3/813/594640/813.pdf by guest on 26 September 2021 Washed platelets (2 x 10’/mL) were stirred in the presence of Aal-21 is a specific in vivo indicator of elastase-mediated luciferin and luciferase for 1 minute at 37 ‘C prior to the addition of proteolysis of fibninogen and higher levels of Aal-2l were elastase-modified thrombin (T.), a-thrombin (T). or ‘y-thrombin found in patients with a,-antiprotease deficiency than in (T.,, inset). Aggregation was recorded as the increase in light transmission and quantitated by planimetry. Results were healthy controls.54 In a similar manner, the limited digestion expressed as percentage of the area under the control curves. of a-thrombin by ebastase could be an important physiologic Secretion was expressed as the maximum increase in fluores- mechanism for regulation of the activity of both free and cence expressed as a percentage of the increase in the control. fibrin-bound thrombin. Equivalent concentrations of T. T and T. had similar amidolytic PMN elastase cleaves the thrombin B chain at Ala activity against Tos-GIy-Pro-Arg-paranitroanilide (GPA). l50-Asn 151. This site is four amino acids to the NH2- terminal side of the autoproteolytic or tryptic ‘y-thrombin thrombin, platelet aggregation and secretion were measured cleavage site; the f. domain is not cleaved. Proteolysis by simultaneously with a lumi-aggregometer. Examples of stan- PMN elastase occurs in areas rich in apolar amino dard curves of a-thrombin-induced platelet aggregation and acids.32’557 In contrast to the positively charged thrombin fi ATP-mediated luminescence are shown in Fig 5. The con- cleavage domain, the ‘y cleavage site (Lys 1 54-Gly I 55) centration of a-thrombin required to liberate 50% of maxi- resides within a domain containing several apolar amino mally releasable ATP from platelets was 1.1 nmob/L. This acids.338 This area should be susceptible to cleavage by was similar to the concentration required to cause 50% of elastase.’2557 These considerations are compatible with out’ maximal aggregation. In contrast, the concentration of modi- findings that the larger NH2-terminab fragment, Fl (mob wt fled thrombin required to liberate 50% of releasable ATP 1 8,000) is disulfide linked to the thrombin A chain; and the and to cause 50% of maximal aggregation was -7.9 nmol/L. smaller COOH-terminal fragment, FII (mob wt -13,000) As a result, both curves representing aggregation and contains the active-site senine, as indicated by the recovery of secretion were shifted to the right. Using the concentration a covalent bond between FII and antithrombin Ill. These required to produce 50% maximal stimulation for compani- results are supported by the recent report that porcine son, the modified thrombin was -14% as potent as a- pancreatic elastase also cleaves a single peptide linkage in thrombin in stimulating both platelet aggregation and plate- prothrombin corresponding to Ala I 50-Asn I 5 1 within the let release. In three additional experiments using three a-thrombin B chain, thereby facilitating localization of the different preparations of the modified thrombin, the modi- staphybocoagulase-binding region to the COOH-terminal fled thrombin was 10% to 19% as potent as a-thrombin. fragment.58 -y-Thrombin was a less potent platelet stimulus than PMN elastase binds to glycosaminoglycans of biologic elastase-modified thrombin (Fig 5 insert). The concentration importance, including heparin, heparin sulfate, dermatan required to cause 50% of maximum platelet aggregation and sulfate, chondroitin sulfate, and hyaburonic acid.43 Although release was -220 nmol/L (0.5% as potent as a-thrombin). glycosaminoglycans inhibit the amidolytic activity of elas- Elastase has been reported to inhibit thrombin-induced tase against a small oligopeptide substrate,4’ heparin stimu- platelet aggregation and secretion.9 The final concentration bates elastase-mediated proteobysis of elastin.59 Thus, our of elastase in the cuvette (3.5 nmol/L), however, was less finding that hepanin accelerates ebastase-mediated proteoly- than that required to inhibit platelet function in this system.9 sis of a-thrombin is not without precedent. It also suggests In addition, incubation of similar concentrations of chboro- that the physiologic interaction of ebastase with thrombin methyl ketone inhibited elastase (95% inhibited by MeO- might be a surface-mediated phenomenon. Finally, because Suc-Ala-Ala-Pro-Val-CH2C1) with a-thrombin (0 to 60 mm- heparin binds to both thrombin and elastase,43#{176} a template utes) did not inhibit thrombin-induced platelet aggregation mechanism similar to that for the thrombin-antithnombin or secretion. III reaction is implied, whereby the interaction of both thrombin and elastase with heparin are involved in the DISCUSSION expression of catalytic activity.62’ These studies demonstrate that human a-thrombin, upon Elastase-modified thrombin might be an important probe proteolysis by PMN elastase, exhibits diminished fibninogen of the thrombin ‘y cleavage domain. In marked contrast to 818 BROWER ET AL autolytic or tryptic digestion of a-thrombin,’6’ elastase- that thrombin-induced fibninogen clotting and platelet stim- mediated proteolysis of the ‘y region does not require proteol- ulation are altered by cleavage near the ‘y cleavage site. In ysis near the $ cleavage site prior to cleavage in the ‘y region. contrast, cleavage near the ‘y cleavage site does not appear to Cleavage at the 3 site reduces fibrinogen clotting activi- inhibit inactivation by antithrombin 111.69 The limited digest ty.”7 Therefore, it is not surprising that the elastase- of a-thrombin by elastase should prove useful in the investi- modified enzyme, with an intact 9 domain, retains some gation of the structure and function of human thrombin. fibrinogen clotting activity. Likewise, we found (Fig 5) that ACKNOWLEDGMENT the platelet stimulatory activity of elastase-modified throm- The advice of Dr Peter Harpel is most gratefully acknowledged. bin (14% that of a-thrombin) is greater than that of ‘y- The technical expertise of Sarah Carson, Aaron Cormier, and Susan thrombin (0.5% that of a-thrombin). These findings, con- Gershen in the preparation of photographs and figures used in this firming the results of others for human ‘y-thrombin, suggest manuscript is gratefully acknowledged.

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