Alpha-1-antitrypsin-Pittsburgh. A potent inhibitor of human plasma factor XIa, , and factor XIIf.

C F Scott, … , J H Lewis, R W Colman

J Clin Invest. 1986;77(2):631-634. https://doi.org/10.1172/JCI112346.

Research Article

Alpha-1-antitrypsin-Pittsburgh is a human variant that resulted from a in the plasma inhibitor, alpha 1-antitrypsin (358 Met----Arg). This defect in the alpha 1-antitrypsin molecule causes it to have greatly diminished anti-elastase activity but markedly increased activity. In this report, we demonstrate that this variant also has greatly increased inhibitory activity towards the -specific enzymes of the contact system of plasma (Factor XIa, kallikrein, and Factor XIIf), in contrast to normal alpha 1-antitrypsin, which has modest to no inhibitory activity towards these enzymes. We determined the second-order-inactivation rate constant (k'') of purified, human Factor XIa by purified alpha 1-antitrypsin-Pittsburgh and found it to be 5.1 X 10(5) M-1 s-1 (23 degrees C), which is a 7,700-fold increase over the k'' for Factor XIa by its major inhibitor, normal purified alpha 1-antitrypsin (i.e., 6.6 X 10(1) M-1 s-1). Human plasma kallikrein, which is poorly inhibited by alpha 1-antitrypsin (k'' = 4.2 M-1 s-1), exhibited a k'' for alpha 1-antitrypsin-Pittsburgh of 8.9 X 10(4) M-1 s-1 (a 21,000-fold increase), making it a more efficient inhibitor than either of the naturally occurring major inhibitors of kallikrein (C-1-inhibitor and alpha 2-). Factor XIIf, which is not inhibited by normal alpha 1-antitrypsin, displayed a k'' for alpha 1-antitrypsin-Pittsburgh of 2.5 X 10(4) M-1 s-1. This enhanced inhibitory activity is similar […]

Find the latest version: https://jci.me/112346/pdf Alpha-1 -antitrypsin-Pittsburgh A Potent Inhibitor of Human Plasma Factor XIa, Kallikrein, and Factor Xllf Cheryl F. Scott, Robin W. Carrell, Charles B. Glaser, Friedrich Kueppers, Jessica H. Lewis, and Robert W. Colman Research Center, and the Hematology/Oncology and Pulmonary Sections ofthe Department ofMedicine, Temple University Health Sciences Center, Philadelphia, Pennsylvania 19140; Medical Research Institute ofSan Francisco, at Pacific Medical Center, San Francisco, California 94115; Clinical Biochemistry, Department ofPathology, Christchurch Hospital, Christchurch, New Zealand; and Department ofMedicine, University ofPittsburgh, and Central Blood Bank Pittsburgh, Pennsylvania 15261

Abstract 30-40% of normal results in a predisposition to early venous thromboembolic disease. The most abundant protease inhibitor Alpha-l-antitrypsin-Pittsburgh is a human variant that resulted in plasma, however, is a,-antitrypsin (a, protease inhibitor) (a,- from a point mutation in the plasma protease inhibitor, a,-an- AT),' which exerts little influence on the cascade, titrypsin (358 Met - Arg). This defect in the a,-antitrypsin except on Factor XIa ofthe contact system, where it is the major molecule causes it to have greatly diminished anti-elastase ac- inhibitor of that enzyme (5), accounting for at least 50% of its tivity but markedly increased antithrombin activity. In this report, inhibition (6) when the value of 25 ,M is used for the concen- we demonstrate that this variant protein also has greatly in- tration of a,-AT in normal plasma (7). creased inhibitory activity towards the arginine-specific enzymes A patient has been described, whose plasma contained, along of the contact system of plasma proteolysis (Factor XIa, kalli- with normal a,-AT, a variant of a,-AT, designated antithrom- krein, and Factor XIIf), in contrast to normal cal-antitrypsin, bin-Pittsburgh (8), and later a,-antitrypsin-Pittsburgh (AT-P) which has modest to no inhibitory activity towards these enzymes. (9). This variant protein was found to result from a point mu- We determined the second-order-inactivation rate constant (k") tation at position 358 in the reactive center ofthe molecule that of purified, human Factor XIa by purified a,-antitrypsin-Pitts- substituted arginine for methionine (9), allowing the protein to burgh and found it to be 5.1 X 10' M- s'1 (230C), which is a function as a potent inhibitor, while greatly diminish- 7,700-fold increase over the kV for Factor XIa by its major in- ing its anti-elastase activity (10). Since a,-AT is an acute-phase hibitor, normal purified al-antitrypsin (i.e., 6.6 X 101 M-l s-1). reactant that can increase as much as fourfold during periods Human plasma kallikrein, which is poorly inhibited by a,-an- of stress (1 1), it is not surprising that the concentration of the titrypsin (k" = 4.2 M-l s1), exhibited a k" for a,-antitrypsin- mutant protein, AT-P, in the patient's plasma was elevated after Pittsburgh of 8.9 X 104 M- s'1 (a 21,000-fold increase), making minor trauma (9). The elevated level of AT-P in the patient it a more efficient inhibitor than either of the naturally occurring resulted in numerous severe bleeding episodes, ultimately leading major inhibitors of kallikrein (Cl-inhibitor and a2-macroglob- to his death (9). The total antitrypsin content in the patient's ulin). Factor XIIf, which is not inhibited by normal a,-antitrypsin, plasma (normal a,-AT = 25 ,gM) ranged from -9.2 MM, in the displayed a k" for a,-antitrypsin-Pittsburgh of 2.5 X 104 M- quiescent phase of the disorder, to -40 MM, in the acute phase s'1. This enhanced inhibitory activity is similar to the effect of (9). AT-P was 4,000-fold more efficient as an inhibitor ofthrom- a,-antitrypsin-Pittsburgh that has been reported for thrombin. bin than normal a,-AT (9), and 40-fold more efficient than the In addition to its potential as an , this recently major thrombin inhibitor, antithrombin III, in the absence of cloned protein may prove to be clinically valuable in the man- (10), thus, offering a possible explanation for the marked agement of septic shock, hereditary angioedema, or other syn- decrease in coagulability of the patient's blood. dromes involving activation of the surface-mediated plasma pro- Since the enzymes ofthe contact phase ofplasma proteolysis teolytic system. (Factors XIa, activated Factor XII, and kallikrein) cleave pri- marily bonds containing arginine in the PI position (12-14), we Introduction hypothesized that since the variant protein now contained ar- ginine in its PI position, that it might function as an efficient Blood coagulation is a multifaceted process involving zymogen inhibitor of these enzymes. In this paper, we present evidence and co-factor activation as well as enzyme inhibition, with many that AT-P is a more potent inhibitor than any of the naturally steps of this integrated series of reactions occurring on the surface occurring plasma protease inhibitors of all ofthe contact enzymes of . Interference with certain critical steps in the "cas- (Factor XIa, kallikrein, and Factor XIIf), especially Factor XIa, cade" can lead to either the pathologic processes of thrombosis where it displayed a greater affinity than that reported for in co- or hemorrhage. The most influential inhibitor the blood thrombin. Therefore, the presence of this mutant protein would agulation cascade is antithrombin III, which is the major inhib- result in greatly increased total plasma inhibitory capacity to- itor of thrombin (1, 2), the enzyme responsible for for- wards each contact enzyme, and would dramatically inhibit the mation as well as aggregation (3). It has been demon- contact system of plasma proteolysis. strated that a level of antithrombin III equal to at least 80% of normal is necessary to maintain hemostasis (4), and a level of Methods

Receivedfor publication 9 July 1985. Materials.

Alpha-l-antitrypsin-Pittsburgh Inhibits Contact Enzymes 631 and antithrombin III were purchased from Helena Laboratories, Beau- This contrasts with the k" for the inactivation of Factor XIa by mont, TX. Polyethylene glycol (PEG) (PEG 8000) was obtained from normal a, -AT, which was 6.6 X 10' M' s-', in agreement with Chemical Sigma Co., St. Louis, MO. BCA protein determination reagent previous results (6), and normal antithrombin III, which was was obtained from Pierce Chemical IL. All Co., Rockford, other reagents 1.7 X 102 M-' s-'. Therefore, AT-P is 7,700-fold more potent were the best grade available. than normal and more Purified . Factor XI, kallikrein, and Factor XIIf (the catalytic a,-AT, 3,000-fold potent than anti- subunit of activated Factor XII) were prepared from purified zymogens, thrombin III towards inactivation of Factor XIa. as previously described (6, 15, 16). Radial immunodiffusion, to measure Effect of A T-P on plasma kallikrein. Kallikrein (0.24 AtM) aI-antitrypsin antigen, was performed according to the method of Man- was incubated with AT-P under second-order conditions (0.25- cini et al. (17), using monospecific antiserum (7), assigning a value of 0.35 jsM) at 230C. The rapid inactivation yielded a k' = 8.9 25 AM to normal, pooled plasma (1.36 mg/ml al-AT) (7). X 104 M-' s'l, whereas normal a,-AT is a weak inactivator, The protein determinations of purified al-AT and AT-P were per- under the same conditions (k" = 4.2 M' s-') (24), and anti- formed with the BCA protein determination reagent. thrombin III has moderate inhibitory activity towards kallikrein al Protease inhibitor (al-AT) was prepared by the reductive-salting (k" = 3 X 102 M-' s-') (24). Therefore, AT-P is also an efficient method of Glaser et al. (18), with the following modifications: (a) Plasma inhibitor of plasma kallikrein. from normal individuals was used as the starting material (MM-type) ofA T-P on the inactivation rate Factor . The instead of Cohn fraction IV-I. (b) In the DEAE-cellulose step, a gradient Effect of XIIf of 10 mM sodium phosphate, pH 7.6, to 10 mM sodium phosphate, pH k" for the inactivation of Factor XIIf by AT-P, determined with 6.0, containing 0.2 M NaCl was used. (c) A further purification step was 0.4 AM Factor XIIf and AT-P equalling 0.4-1.15 AM (second- carried out on QAE-Sephadex at pH 8.6 using a linear salt gradient in order conditions) at 230C, was found to be 2.5 X 104 M-1 s-'. 50 mM Tris-HCl, pH 8.6, from 0.14 M NaCl to 0.2 M NaCl. The resulting This was very striking, since normal a,-AT does not detectably protein, a single band on sodium dodecyl sulfate-polyacrylamide gel inactivate Factor XIIf (16). Furthermore, AT-P was calculated electrophoresis, was stored in I00-Ml aliquots at -70'C as a stock solution to be 470-fold more potent than antithrombin III in the inac- of 3.3 mg/ml (60 gM), as determined by radial immunodiffusion (17) tivation of Factor XIIf (k" for antithrombin III is 5.3 X 10' and 3.1 mg/ml, as determined by the BCA assay, in 20 mM Tris-Cl, pH M' s-') (16). 7.4, containing 0.15 M NaCl and 0.1% PEG. Comparison of second-order-inactivation rate constants of was isolated from the a,-Antitrypsin-Pittsburgh (AT-P) patient's A T-P with normal plasma protease inhibitors (in purified sys- plasma, as described by Carrell et al. ( 19). The concentration of the stock tems). The second-order inactivation rate constants for the con- solution was 1.25 mg/ml (23 AM), as determined by radial immunodif- fusion (I17), and 1.4 mg/ml, as determined by the BCA assay, in 20 mM tact enzymes and thrombin for four normal plasma protease Tris-Cl, pH 7.4, containing 0.15 M NaCl and 0.1% PEG. Aliquots of inhibitors and AT-P are presented in Table I (the k" is a mea- 100 ML were stored at -70°C. The good agreement between the two surement of inhibitor efficiency or affinity, independent of its methods of protein determination indicates that the antigenicity of the concentration). None of the normal plasma protease inhibitors variant antitrypsin is identical to that of normal a,-AT. are very potent for Factor XIa, but the mutant inhibitor, AT- Determination of inhibitory activity of purified plasma protease in- P, is an excellent inhibitor. The two major inhibitors of kallikrein, hibitors of the contact enzymes. Purified enzyme was incubated (sepa- Cl -inhibitor and a2-macroglobulin, are moderately good inhib- rately) with each inhibitor in the appropriate buffer (20-22) containing itors; however, AT-P is 5-7-fold more potent. This contrasts 0.1% PEG at 230C. At various times, a portion was removed and assayed with normal a,-AT, which is a very weak kallikrein inhibitor. for residual at Factor XIa amidolytic (enzymatic) activity 37°C. activity Cl-inhibitor is the protease inhibitor that was determined the substrate S-2366 kallikrein was only plasma efficiently using synthetic (20); inactivates Factor assayed with S-2302 (21); and Factor XIIf was measured with S-2302 XIIf (16); however, AT-P appears to be eight- (22), as previously described. All enzyme assays were performed using fold more potent. AT-P is 40-fold more potent than the major a Gilford 210 recording spectrophotometer with a thermostated cham- thrombin inhibitor, antithrombin III, 620-fold more potent than ber (37°C). a2-macroglobulin, and 6,000-fold more potent for the inacti- Calculation of second-order rate constants (k). The second-order vation ofthrombin than normal a I-AT. Furthermore, from these rate law is as follows (23): 1/(a - b)[ln(b/aXa - x)/(b - x)] = kt, where data we conclude that AT-P is a more potent anti-Factor XIa a = initial inhibitor concentration, b = initial enzyme concentration, x than it is an antithrombin. = product concentration at time "t" of the reaction, and k" is the apparent second-order rate constant. When the left side ofeach equation is plotted versus time, the slope = k". Table I. Comparison ofSecond-order Inactivation Rate Constants (230C) Results (k', M-' s-') Isoelectric focusing ofpurified a,-A T and A T-P. Purified a -AT (type M) and purified AT-P were run under the conditions in Factor XIa Kallikrein Factor XIIf Thrombin reference 7. Each band in the AT-P preparation migrated more than its normal CI-INH 250 (6) 17,000 (24) 3,000 (16) 0 cathodally counterpart, indicating that the pu- a2M rified preparation did not contain the M-subtype (data not 0 (6) 11,500 (24) 0 (16) 500 (37) shown). AT-III 170 (6) 300 (24) 53 (16) 7,200 (36) a,-AT 66 (6) 4.2 (24) 0 (16) 48 A T-P on Factor XIa. When Factor (27) Effect of plasma purified AT-P 510,000 89,000 25,000 XIa (0.035 gM) was incubated with AT-P (0.035-0.068 NM), at 310,000 (10) a loss of 23°C, rapid concentration-dependent amidolytic activity Inactivation rate constants, determined for Factor XIa and kallikrein resulted. Since the inhibition was too rapid to perform kinetic by a,-AT and antithrombin III (AT-III), agreed well with our previous analyses under pseudo first-order conditions (> 10-fold excess of determinations (6, 24). Therefore, we are presenting a comparison of inhibitor), we employed second-order conditions (inhibitor/en- the inactivation rate constants of the enzymes by AT-P with the previ- zyme ratios = 1.0-1.9). From the data, we calculated a k" for ously published data. Numbers in parentheses denote references. C 1- the inactivation of Factor XIa by AT-P of 5.1 X 105 M-l s-'. INH, CI-esterase inhibitor; a2M, a2 macroglobulin. 632 Scott, Carrell, Glaser, Kueppers, Lewis, and Colman Comparison oftheoretical half-lives ofeach enzyme with each antithrombin III for the inactivation of the enzymes of the con- inhibitor at normalplasma concentration andA T-Pplasma con- tact-activated plasma protease system-a pathway that is in- centration. When the value of k is multiplied by its concentration volved in coagulation, , and the inflammatory re- in plasma, (7, 25, 26), the constant, k' (pseudo-first-order re- sponse. The most profound effect of AT-P is on the enzyme, action rate constant), can be calculated, which is an estimate Factor XIa-the only contact enzyme whose deficiency can lead measure of the total inhibitory capacity of that inhibitor at to a hemorrhagic disorder. The major plasma protease inhibitor plasma concentration. The reciprocal of k' (pseudo-first-order of Factor XIa is a,-AT (5), and accounts for 51% (6) of the total reaction rate constant) would then be the more familiar half-life plasma inhibitory activity when calculated using 25 AM (7) as (t1/2) of the enzyme at plasma concentrations (Table II) of each the concentration of aI-AT in plasma. However, under normal inhibitor. The t1/2 of Factor XIa, in the presence of plasma con- circumstances, this inhibition is slow, when compared with the centrations of its most effective inhibitor, a,-AT, is 600 s (6), inhibition of other plasma by their major inhibitors. compared with 0.21 s by AT-P at its concentration in the plasma For example, a,-AT is also the major inhibitor of human neu- of the patient during the quiescent phase, (9.2 jM [9]), and 0.05 trophil elastase, with a k" of 6.5 X 107 M-i s-' (27), compared s during the acute phase (40 ,M [9]). This increase in inhibitor with the k" for Factor XIa of 6.6 X 10' M' s-' (5). It is interesting activity represents a decrease of 2,800-12,000-fold in the t1/2 of to note that the mutant form of a,-AT, AT-P, has decreased Factor XIa in AT-P plasma. Similarly, the t/2 of kallikrein in inhibitory activity towards human (k" = 2.2 the presence of plasma Cl-inhibitor concentrations is 35 s (24), X 103 M-i s-') (10), while it has increased inhibitory activity and a2-macroglobulin is 25 s (24), whereas AT-P at the concen- towards Factor XIa (k" = 5.1 X 105 M-' si'). This functional tration in the plasma of the patient would result in a t1/2 for transformation is a consequence of a single substitution in the kallikrein of 1.22 s in the quiescent phase, and 0.28 s in the reactive center of the inhibitor molecule. Since Factor XIa prefers acute phase, which is an average decrease of 25- to 100-fold in arginine bonds (12) in the P1 position, it was inhibited more the t1/2, as compared with the two naturally occurring inhibitors. efficiently by the mutant protein than by normal aI-AT, in con- For Factor XIIf, the major inhibitor, C1 inhibitor, yields at t1/2 trast to human neutrophil elastase, which exhibits a preference of 190 s (16), compared with 4.3 s for AT-P at the concentration for methionine and valine residues (28), and is, therefore, in- in the patient's plasma during the quiescent phase, and 1.0 s hibited more efficiently by normal a,-AT. Kallikrein and Factor during the acute phase, which is a decrease of 44-190-fold in XIIf also cleave at arginyl residues (13, 14), which explains their the t1/2. Therefore, the total plasma inhibitory activity in the preference for AT-P over normal aI-AT. The importance of the AT-P patient's plasma was at least 1-4 orders of magnitude PI residue, which contributes to the carboxyl moiety of the greater than the inhibitory activity of the naturally occurring cleaved , has been emphasized recently for the contact enzyme inhibitors in normal plasma. Furthermore, the plasma proteinase inhibitors of proteases (10, 29). inhibition of the contact enzymes by AT-P was comparable with When present at plasma concentrations (as in the patient or greater than the inhibition ofthrombin by AT-P (157- to 687- with AT-P), the inhibitor not only controls thrombin activity, fold decrease of t1/2 as compared with antithrombin III). but to an even greater extent, Factor XIa (Tables I and II). Al- though Factor XI-deficient individuals usually have mild bleed- Discussion ing episodes, as compared with patients who are severely deficient in Factor VIII or Factor IX, severe bleeding has been reported The variant protein, AT-P, has been described as antitrypsin in a Factor XI-deficient individual who developed a potent an- that mutated to "antithrombin" (9). However, kinetic analyses tibody to Factor XI (30). This patient, early in life, bled exces- have revealed that AT-P is actually 40-fold more potent than sively after and also, as an adult, had several episodes naturally occurring antithrombin III for the inactivation of of spontaneous intramuscular hematomas. It is possible that an thrombin (11), as well as 300-3,000-fold more potent than abnormal inhibitor of a coagulation factor would result in a different clinical picture from a simple deficiency of the factor. If this is true, then AT-P also may have been responsible for a Table IL Calculated Theoretical Half-lives (s) ofEnzymes at Plasma Inhibitor Concentrations (230C) "pseudo-Factor XI deficiency" that could have further contrib- uted to the hemostatic defect in that patient. Inhibitor Factor XIa Kallikrein Factor XIIf Thrombin The fact that AT-P is a more efficient inhibitor of the contact proteases than any of their naturally occurring plasma protease AM inhibitors (Table I), suggests the possibility for its clinical use. CI-INH (1.7)* 2,500 [35] [190] 0 The combination of the recombinant DNA technologies of a2-M (3.5)* 0 [25] 0 700 cloning in bacteria (31) and site-directed mutagenesis in yeast AT-Ill (2.5)* 2,300 1,300 7,500 [55] (32, 33) that have allowed substitution of valine in the P1 position a,-AT (25)t [600] 9,500 0 830 of a I-antitrypsin to produce an elastase inhibitor that is resistant AT-P a (9.2)§ 0.21 1.22 4.3 0.35 to oxidation, have also allowed replacement of methionine with AT-P b (40)§ 0.05 0.28 1.0 0.08 arginine to produce an inhibitor of coagulation proteinases that is identical to AT-P (33a). Therefore, in gram-negative , where activation of contact proteases and consumption of plasma * Plasma protease inhibitor concentrations were based on references 25 and 26. protease inhibitors occurs (34), or during contact activation in * Reference 7. the absence of Cl-inhibitor (hereditary angioedema) (35), in- § Reference 9; a, quiescent; b, acute. fusion of genetically engineered AT-P may increase the survival [ J, Major inhibitor for each enzyme. rate of these critically ill patients by rapidly inhibiting the pro- t1/2, l/k' (I), when I equals inhibitor concentration; a2M, a2 macro- teases of the contact system, while concurrently blocking throm- ; AT-Ill, antithrombin III. bin action, which may prevent the development of disseminated Alpha-l-antitrypsin-Pittsburgh Inhibits Contact Enzymes 633 intravascular coagulation. Experiments are currently underway, a unique procedure designed for industrial application. Anal. Biochem. with an animal model, to test this hypothesis. 124:364-371. 19. Carrell, R. W., J. 0. Jeppson, C. B. Laurell, S. 0. Brennan, M. C. Owen, L. Vaughan, and D. R. Boswell. 1982. Structure and vari- Acknowledgments ation of human al-antitrypsin. Nature (Lond.). 298:329-334. 20. Scott, C. F., D. Sinha, F. S. Seaman, P. N. Walsh, and R. W. We wish to thank Dr. Robin A. Pixley for preparing purified Factor XIIf Colman. 1984. Amidolytic assay of human factor XI in plasma: com- and Lisa Procino for preparing purified kallikrein. parison with a coagulant assay and a new rapid radioimmunoassay. Blood. The work was supported, in part, by National Institutes of Health 63:42-50. grant HL24365, Council for Tobacco Research grant 1420, and by the 21. Kluft, C. 1978. Determination of in human plasma: Ben Franklin Partnership. optimal conditions for activating prekallikrein. J. Lab. Clin. Med. 91: 83-95. 22. Pixley, R. A., M. Schapira, and R. W. Colman. 1985. 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634 Scott, Carrell, Glaser, Kueppers, Lewis, and Colman