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Effects of aprotinin on coagulation and fibrinolysis enzymes
Fibrinolysis & Proteolysis (1997) 11(4), 209-214 © PearsonProfessionalLtd 1997
Effects of aprotinin on c o a g u l a t i o n and fibrinolysis e n z y m e s U. Christensen, J. Schiodt Kemisk Laboratodum IV, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen a, Denmark.
Summary Large quantities of aprotinin, the tight-binding inhibitor of trypsin and kallikrein, are used with success to reduce bleeding, e.g. in cardiac pulmonary surgery. The effect has been attributed to inhibition of fibrinolysis, which immediately seems plausible since aprotinin is a known effective inhibitor of plasmin. However, fast kinetic studies reveal that the rate of the plasmin-aprotinin reaction may be too slow to compete effectively with (z2-antiplasmin, even when present at subphysiological concentrations. Fibrinolysis experiments in microtiter plates confirm that the effect of aprotinin on plasmin may be negligible in the presence of c~2-antiplasmin. Therefore, the effects of high concentrations of aprotinin on a number of coagulation and fibrinolysis enzymes previously assumed not to be affected by the inhibitor were also investigated using steady state kinetic-rate methods. It was found that aprotinin is a multi-functional inhibitor which inhibits urokinase, activated protein C (APC) and thrombin sufficiently well, if present in gM-concentrations, to significantly affect their activity. A kinetic analysis showed simple competitive inhibition in all these cases, corresponding with binding of aprotinin to the active site of the enzyme and thus a reduction in the amount of enzyme available for substrate interaction. However, Factor Xa, Factor Vlla and tissue-plasminogen activator were not affected. All in all, the results indicate that the effects of high concentrations of aprotinin may also stem from enzymes other than plasmin. Known kinetic data on various physiological inhibitors show that the effects of aprotinin on urokinase and APC may be more important than has been hitherto anticipated.
INTRODUCTION
Aprotinin is used to reduce bleeding and the need for donor-blood transfusion in cardiac surgical patients and has been shown to be effective in complex surgical procedures in which there is a high risk of postoperative bleeding (see Royston 1 for a recent review). Administration of aprotinin in a high-dose regimen (2 x 10 ~ KIU loading dose, 2 x 10 ~ KIU into the cardiac pulmonary surgery (CPB) circuit, and 5x 105 KIU/h continuous infusion during surgery) has been shown to reduce blood loss by 40%-50% and to reduce the need for donor-blood transfusion by 40%-80% in various surgical procedures. 2-~ Aprotinin (Basic Pancreatic Trypsin Inhibitor) was first discovered as a kallikrein inactivator 7 and later isolated from bovine pancreas 8 as a trypsin inhibitor. It is a small, well-characterized protein recognized as an inhibitor of a Received 31 December 1996 Accepted after revision 25 June 1997 Correspondence to: U. Christensen, Tel. +(45)35320266; Fax. +(45)35320299; E-mail: u.christensen @pop.ki.ku.dk
number of serine proteinases, e.g. chymotrypsin and plasmin, as well as trypsin and kallikrein, and weak inhibition of urokinase has also been described 9. The reducing effect of aprotinin on bleeding has been attributed to inhibition of fibrinolysis; 1-6'1° first of all of plasmin. This is plausible as plasmin is strongly inhibited by aprotinin. 11'12 However, low-dose aprotinin infusion that should be sufficient to inhibit plasmin in the clinical situation was not found to be clinically useful.l° In accordance with this, the kinetic results presented here reveal that the plasmin-aprotinin reaction may be too slow to compete effectively with that of a2-antiplasmin. The aprotinin effect may also stem from other enzymes. The effects of high concentrations of aprotinin on a number of other coagulation and fibrinolysis enzymes previously assumed not to be affected by the inhibitor were investigated using steady state kinetic-rate methods. It is found that aprotinin inhibits urokinase, activated protein C (APC) and thrombin sufficiently well, if present in gM-concentrations, to significantly affect their activity. A kinetic analysis showed simple competitive inhibition in all these cases, corresponding with 209
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Christensen & Schiodt
binding of aprotinin to the active site of the enzyme and thus a reduction in the amount of enzyme available for substrate interaction. Factor Xa, Factor VIIa and tissueplasminogen activator (tPA) were not affected. MATERIALS A N D METHODS Proteins and chemicals
Aprotinin (Trasylol) was kindly provided as a gift from Birte Petersen, Bayer Danmark A/S; its concentration was determined from titrations with trypsin 13 and showed 106 KIU = 22 gmol. Fibrinogen was kindly provided as a gift from Jorgen Jespersen, Esbjerg, Denmark. Human plasmin was prepared and titrated as previously described. ~3-14 Human urokinase was Trombolysin from IMMUNO DANMARK A/S. Two-chain tPA was from Biopool Sweden. APC was from Chromogenix AB, Sweden. Trypsin, h u m a n thrombin and bovine Factor Xa were from Sigma, St. Louis, MO. Factor VIIa (human) was a gift from Lars Christian Petersen, Novo Nordisk A/S, Denmark. Tripeptide-p-nitroanilide substrates were from Chromogenix AB, Sweden. All other chemicals were analytical grade from Sigma or Merck. Fluorescence titration
Intrinsic fluorescence emission spectra of aprotinin, plasmin and the aprotinin-plasmin complex were obtained using a Perkin-Elmer LS-50 fluofimeter equipped with FLDM software. Exitation was at 280 nm with a slit width of 5 nm, the emission spectra were recorded in the range 300-500nm, also with a slit width of 5nm. Titration experiments were performed at 25 °C in 50 mM Tris-HC1, 0.1 M NaCl-buffer, pH 7.6, by mixing aprotinin (1.5 ml, O; 0.5; 1; 1.5; 2; 2.5; 3 and 4gM) and plasmin (1.5ml, 2gM) or reference buffer (1.5ml). After 5 min incubation for equilibration, the emission spectrum was recorded three times. The fluorescence intensities were each taken as the integrated signal and then averaged. The relative fuorescence change, AF, was calculated as (F - Fo) x 100%/Fo, where F is the averaged fluorescence intensity of the sample solution minus that of the corresponding reference solution, and Fo is F for plasmin in the absence of aprotinin. As defined, AF is a negative quantity. Stopped-flow kinetic experiments
Fast kinetic experiments were performed on the interaction of aprotinin and plasmin in a Hi-Tech Scientific PQ/SF-53 spectrofluorimeter equipped with a high-intensity Xenon arc lamp. The reaction resulted in a decrease of the total protein fluorescence, similar to that observed for the reaction of plasmin with c~2-antiplasmin.15 Time Fibrinolysis & Proteolysis (1997) 11(4), 209-214
courses of the intrinsic fluorescence changes of the plasmin-aprotinin reaction were recorded for an appropriate time (1, 2 or 10 s) after stopped-flow mixing (mixing time approx. 10-3 s) of plasmin- and aprotinin-solutions in 50 mM Tris-HC1, 0.1 M NaCl-buffer pH 7.6, 25°C, at second order reaction conditions, i.e. the same concentration of the enzyme and of the inhibitor. The final concentrations were: 5, 2.5, 1.25 and 0.67gM. The exitation wavelength was 280 nm, slit width 5 nm. The light emitted from the reaction mixture passed a WG 320 filter before detection in the photomultiplier. Thus, an integrated fluorescence signal was obtained. In each experiment, 400 data points were recorded and sets of data from three to four experiments were averaged and analysed with the Hi-Tech HS-1 Data Pro Software. In all cases, the best fitted curve was that corresponding to second order reaction kinetics. The regression analysis used is based on the Gauss-Newton procedure. After recording 1, 2 or 10 s of the reaction, control measurements were made on the aged solution in the stoppedflow cell, the last one after 10 rain. Such measurements showed no further change of the fluorescence after the initial recording. Thus, only one fast step giving rise to changes of the fluorescence is apparent when plasmin and aprotinin interact. Simulations of plasmin inhibition in the presence of aprotinin and a2- antiplasmin were made with the Mathcad PLUS 6.0 software (MathSoft Inc). Steady state kinetic measurements
The steady state kinetic parameters of the hydrolysis of a reasonably specific tripeptide-p-nitroanilide substrate catalysed by the coagulation or fibrinolysis enzyme in question were determined from initial rate measurdments in the presence and absence of aprotinin, essentially as previously described, ~3,14,16,17 using a Perkin-Elmer 17 spectrophotometer equipped with the PECSS-software, which provides calculations of initial rates. The substrates used were: S-2444 for urokinase; 16 S-2288 for tPA, Factor Xa, 17'18 APC 19 and Factor VIIa; 2° S-2266 for thrombin.17 The experiments were designed to optimally reveal competitive as well as noncompetitive inhibition, i.e. substrates with low Kin-values were avoided so that precise rate measurements could be made at substrate concentrations less than Kin. The final concentrations of aprotinin used were 64, 10.7, 5.3 and 2.7 gM. The nonlinear least-squares fitting program GraFit (Erithacus Software Ltd.) was used for data analysis. Fibrinolysis experiments
Measurements of inhibition of fibrinolysis were performed in microtiter plates using optimized fibrin gels as described by Sidelmann et al.2o 30gl test samples all © Pearson Professional Ltd 1997
Effects of aprotinin on coagulation and fibrinolysis enzymes
containing 5 nM of tissue plasminogen activator and various concentrations of a2-antiplasmin (0, 0.2, 0.5 and 1 gM), in the presence or absence of 1 gM aprotinin, were placed on top of preformed fibrin gels and the A4o5 was measured in a Ceres 900C, Bio-Tek Instruments Inc. microtiter plate reader every 10 min for 20 h. The continuous decrease in the A4o5, obtained after a lag period and calculated using the KC3 Kineticalc software, was used as a measure of the fibrinolysis rate.
211
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03
U.
RESULTS
AND
DISCUSSION
Figure 1 shows a fluorescence titration curve of 1 gM (final concentration) of plasmin with aprotinin. The 1 : 1stoichiometry of the plasmin-aprotinin-complex is obvious. The relative fluorescence change, zXF, is negative and proportional to the amount of complex formed. The intrinsic fluorescence of the complex is found to be 13% smaller than the sum of that of plasmin and of aprotinin at the same concentrations. Given this large AF-value resulting from the formation of the plasmin-aprotinin-complex, it was possible to follow the reaction between plasmin and aprotinin directly using stopped-flow fluorescence kinetic measurements. Series of such experiments were performed in which various equal concentrations of plasmin and aprotinin were mixed. Stopped-flow mixing is obtained within 10-3 s so that fast reactions can be followed. An example of the averaged stopped-flow traces obtained is shown in Fig. 2. Figure 2 also shows the curve resulting from the fit of a second order rate equation to the data. No other rate equation showed a good fit. Series of such experiments all showed second order reaction
14 12 10 8 IJ _ .<
6 4 2 0
qF
0
,
I
i
0.5
l
,
1
I
1.5
i
I
2
Aprotinin (P.M) Fig. 1 Equilibrium fluorescence titration of 1 gM plasmin with aprotinin. The relative fluorescence decrease is plotted as a function of the concentration of aprotinin added.
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0.2 '
o14 time
0.6 '
0'.a
1.0 '
(s)
Fig. 2 Typical time course of the stopped-flow fluorescence signal resulting from the inhibition of human plasmin with aprotinin. From series of such experiments performed at second order reaction conditions, the second order nature of the reaction and the value of the rate constant (Table 1) k = 1.5 10 6 M-1 s-1 were determined.
kinetics with rate constant k = 1.5 106 M -1 s -1 for the formation of the plasmin-aprotinin complex (Table 1). Similar experiments on the plasmin-(z2-antiplasmin reaction 15 have shown the rate constant of this reaction to be k = 2.25 107 M -1 S-1 for the formation of the plasmin-a2-antiplasmin complex. For the plasmin-a2macroglobulin reaction, the value of the association rate constant i s 22 k = 5 105 M -1 s -1 . The time courses of inhibition of plasmin by c~2-antiplasrain and aprotinin in competition at physiological concentrations of a2-AP (0.5-1.5 gM) and high concentration of aprotinin (1 gM) were then simulated. An example is illustrated in Figure 3. It is generally accepted that the concentration of free plasmin is always very low, 9 so that pseudo first order inhibition is occurring. Here it was further assumed that depletion of a2-antiplasmin did not o c c u r . 2 3 ' 2 4 Apparently a valid assumption, Aasen et a123 conclude in their open heart surgery study that 'a2antiplasmin activities did not fall below 20% of preoperation values' and that 'a functional reserve of antiplasmin was present throughout the duration of CPB.' The result is the formation of at least 95% a2-antiplasmin-plasmin and maximally 50/0 aprotinin-plasmin-complexes (Fig. 3A). The competing effect of a2-antiplasmin on the inhibition of plasmin by aprotinin is shown in Figure 3B, where the amount of plasmin-aprotinin in the presence of a2antiplasmin is compared with that obtained in the absence of a2-antiplasmin. Thus, the effect of aprotinin on plasmin in the absence of physiological inhibitors is quite different from that in their presence. Finally, Figure 3C illustrates the effect of aprotinin on the total inhibition of plasmin. It is seen that the presence of aprotinin does not at any time Fibrinolysis & Proteolysis (1997) 11(4), 209-214
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Christensen & Schiedt
Table I Values of the second order rate constant of the association of plasmin and aprotinin obtained from stopped-flow kinetic measurements Protein concentrations ([aM)
Rate constant, k (M -1 s -1)
5.0 2.5 1.25 0.67
1.53 1.48 1.51 1.47
100
,
~
x x x x
106 106 106 106
~. . . . . . , . . . . . . . . . .
,
~ 50
Aprotinin inhibiton of the hydrolysis of specific chromogenic substrates b y coagulation and fibrinolysis enzymes using series of experiments at various concentrations of aprotinin and the substrates are illustrated in Figure 4. Urokinase, APC and thrombin are obviously affected at concentrations of aprotinin in the range of interest (1-5 gM), whereas the effects on t-PA, Factor Xa and Factor VIIa are each negligible, even in this high concentration range. The curves shown stem from non-linear least squares fits of the Michaelis equation to the experimental data. The resulting kinetic parameters show no or simple competitive inhibitions, i.e. values of Km,app increasing linearly with the concentration of aprotinin, i, and unaffected Vm~-Values, corresponding to the rate equation:
a-
vi = Vma~/[1 + Km,app/S ] = Vmax/[1 + Kin(1 +i/Ki)/S ] o.1
0.2
0.3
0.4
0.5
time(s) lO0
LE --.c
50
/ I
r
[
l
0.1
0.2
0.3
0.4
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a
0.5
time (s)
I00
/ o q
[
I
1
0.1
0.2
0,3
0.4
0.5
time (s)
Fig. 3 Simulations of plasmin-inhibition kinetics. A. Plasmin inhibition in the presence of aprotinin and o~2-antiplasmin (1 [aM each). Line: Total plasmin inhibited (plasmin-aprotinin plus plasmin-o~2-antiplasmin). Dots: Plasmin-o~2-antiplasmin-complex formed. Broken line: Plasmin-aprotinin-complex formed. B. Plasmin-aprotinin complex formed in the presence (broken line) and absence (line) of o~2-antiplasmin (1 [aM). C. Total plasmin inhibited with 1 [aM o~2-antiplasmin in the presence (line) and absence (dots) of aprotinin (1 [aM).
add significantly to the amount of plasmin inhibited. In the absence of aprotinin, a little more a2-antiplasminplasmin complex will be formed than in its presence, but the total inhibition is practically unchanged. In vitro fibrinolysis experiments confirmed this (Table 2). Table 2 shows that the fibrinolysis rates 21 obtained in the presence of both c~2-antiplasmin and aprotinin are comparable with those obtained in the presence of only a2-antiplasmin. Fibrinolysis & Proteolysis (1997) 11(4), 209-214
The values of the inhibition constants (Ki-values) obtained from the fits are listed in Table 3 and the lower limits of Kcvalues obtained for the enzymes not inhibited in the actual concentration range are given. As is shown for plasmin, the effect of aprotinin m a y be of no importance if 'irreversible' physiological inhibition is kinetically very fast. The actual rate of such an enzymeinhibitor reaction is determined b y the product of the rate constant and the concentration of the inhibitor. The physiological inhibitor of urokinase is PAI-1. The reaction of urokinase with PAI-1 is governed by a very fast association rate constant of the order 107 M -1 s-1.25,26 However, PAI-1 is present in plasma only in very low concentrations 27 (
No aprotinin
1 [aM aprotinin
0 0.2 0.5 1.0
1.9±0.1 0.25±0.05 0.17±0.04 0.05±0.02
0.2±0.06 0.24±0.05 0.16±0.05 0.05±0.02
The units of the fibrinolysis rates are zXOD x 103/min. The standard errors of the estimates given are each based on six identical experiments. It is seen that within experimental error aprotinin does not add to the inhibition in the presence of c~2-antiplasmin.
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Effects of aprotinin on coagulation and fibrinolysis enzymes
Activated
Urokinase I
I
1
I
I
I
80
20
50
15
40
10
protein
I
213
Thrombin
C L
k
1.5
2
> 5
20
0 0.05
0.1
0.15
[S-2444]
0,2
0.25
0.5
(mM)
[S-22881
3O
20
> 10
I
I
I
0.1
0,2
0.3
[S-2288]
I
I
0.1
0.2
[S-2266]
(mMI
/F
t-PA 40
E
1
0.3
(raM)
Factor Vlla
60
40
20
0 0,4
0.1
(mMI
0.2 [S-22881
0.3
0.4
(mM)
0.2
0.4 [S-2288]
0.6
0.8
{raM)
Fig. 4 Steady state kinetic velocities obtained in the presence and absence of aprotinin calculated from the initial slope of A41o recorded. Concentrations of aprotinin: (©): no aprotinin; (V): 64#M; (A): 2.7 #M; (V): 5.3 #M; (+): 10.7 #M. The curves shown stem from fits of Eqn. 1 to the data.
catalysis, which point out the advantages of weak binding of substrates 29 (viz high Kin-values relative to physiological concentrations of substrates), the competitive effect of the urokinase substrate, plasminogen, on the urokinaseaprotinin interaction is small. That effect is determined by the ratio of the plasminogen concentration (2 gM) and the urokinase-plasminogen Km-Value (approx. 40 ~M), 3° thus amounting to only approx. 5%. Thus, all things considered, effective urokinase inhibition by aprotinin in the pM-concentration range in plasma is highly likely. The physiological inhibitor of APC is PCI (PAI-3). As for PAI-1, the physiological concentration of PCI is very low 3~ (10 nM) compared with that of c%-antiplasmin (1-1.5 M). Again, the reaction rate of APC and PCI in plasma is most probably significantly less than that of APC and aprotinin at high dose. The rate constant of APC inhibition with PCI has not been measured, but it must, of course, be less than the value determined by the Table 3 Values of aprotinin inhibition constants, K~, obtained from steady state kinetics Enzyme
Ki (#M)
t-Plasminogen activator Urokinase Activated protein C Thrombin Factor Vlla Factor Xa
>600 4.8 2.6 27 > 100 >100
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diffusion control-limit (10s-109 M -1 S-1) and therefore a value similar to that of the reactions of urokinase-PAI-I and of plasmin-a2-antiplasmin is a realistic maximal value. This means that the effective rate of the reaction of APC with PCI at plasma concentrations is at least 10 -2 times as slow as that of plasmin-a2-antiplasmin and that aprotinin at high dose in plasma may be an important inhibitor of APC. The reaction of APC and (zl-antitrypsin 32 with an effective rate of only 0.0004 M -~ s-~ is of no importance in this context. The aprotinin inhibition effect on thrombin is probably of no importance, antithrombin is present in 2-4 gM concentrations in normal plasma and the thrombinantithrombin reaction is fast, particularly in the presence of heparin, 33 whereas urokinase and APC are each candidates for the aprotinin-effect. At the concentration used in surgery (1-5 gM), immediate inhibitions of uroMnase up to 50% and of APC up to 6 6 % are likely. Finally, it sems w o ~ h noting that kallikrein could have profibrinolytic activity and inhibition of kallikrein may contribute to the clinical effect. In conclusion, the inhibition of urokinase and APC might represent additional mechanisms explaining the clinical effects of aprotinin. REFERENCES
1. Royston D. High-dose aprot/nin therapy: a review of the first five years' experience.J CardiothoracVascAnesth 1992; 6: 76-100. Fibrinolysis & Proteolysis (1997) 11(4), 209-214
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2. Dietrich W, Barankay A, Hahnel C, Richter JA. High-dose aprotinin in cardiac surgery: three years' experience in 1784 patients. J Cardiothorac Vasc Anesth 1992; 6:324-327. 3. Cosgrove DM, Hetic B, Lytle BW, et al. Aprotinin therapy for reoperative myocardial revascularization. Ann Thorac Surg 1992; 54: 1031-1038. 4. Bidstrup BP, Harrison J, Royston D, Taylor KM, Treasure T. Aprotinin therapy in cardiac operations: a report on use in 41 cardiac centers in the United Kingdom. Ann Thorac Surg 1993; 55: 971-976. 5. Bidstrup BP, Underwood SR, Sapsford RN. Effect of aprotinin (Trasylol) on aorta-coronary bypass graft patency. J Thorac Cardiovasc Surg 1993; 105: 147-153. 6. Levy JH, Pifarre R, SchaffHV, et al. A multicenter, double-blind, placebo-controlled trial of aprotinin for reducing blood loss and the requirement for donor-blood transfusion in patients undergoing coronary artery bypass grafting. Circulation 1995; 92: 2236-2244. 7. Kraut H, Frey EK, Werle E. Uber die inactivierung des Kallikreins. Hoppe-Seyler's Z Physiol Chem 1930; 192:1-21. 8. Kunitz M, Northrop JH. Isolatinn frombeefpancreas of crystalline trypsinogen, trypsin, trypsin inhibitor and an inhibitor-trypsin compound. J Gen Physiol 1936; 19: 991-1007. 9. Fritz H, Wunderer G. Biochemistry and applications of aprotinin, the kallicrein inhibitor from bovine organs. ArzneimForsch/Drug Res 1983; 33: 479-494. 10. Hardy J-F, Desroches J, Belisle S, Perranlt J, Carrier M, Robitaille D. Low- dose aprotinin infusion is not clinically useful to reduce bleeding and transfusion of homologous blood products in high-risk cardiac surgical patients. Can J Anaesth 1993; 40: 625-631. 11. Wiman B. On the reaction of plasmin or plasmin-streptokinase complex with aprotinin or c~a-antiplasmin. Thromb Res 1980; 17: 143-152. 12. Petersen LC, Clemmensen I. Kinetics of plasmin inhibition in the presence of synthetic tripeptide substrate. Biochem J 1981; 199: 121-127. 13. Christensen U, Ipsen H-H. Steady-state kinetics of plasmin and trypsin-catalysed hydrolysis of a number of tripeptide-pnitroanilides. Biochim Biophys Acta 1979; 569:177-183. 14. Christensen U. pH effects in plasmin-catalysed hydrolysis of a-N-benzoyl-L-arginine compounds. Biochim Biophys Acta 1975; 397: 459-467. 15. Christensen U, Bangert K, Thorsen S. Reaction of c~2-antiplasmin and plasmin. Stopped-flow fluorescence kinetics. FEBS Lett 1996; 387: 58-62. 16. Ipsen H-H, Christensen U. Kinetic studies of urokinasecatalysed hydrolysis of 5-oxo-L-prolyl-l-arginine 4-nitroanilide. Biochim Biophys Acta 1980; 613: 476-481. 17. Lottenberg R, Christensen U, Jackson CM, Coleman PL. Assay of coagulation proteases using peptide chromogenic and fluorogenic substrates. Meth Enzymol 1981; 80: 341-361. 18. Lottenberg R, Hall JA, Paufler E, Zupan A, Christensen U,
Fibrinolysis & Proteolysis (1997) 11(4), 209-214
19. 20. 21.
22. 23.
24. 25.
26.
27. 28.
29. 30. 31. 32.
Jackson CM. The action of Factor Xa on peptide p-nitroanilide substrates: substrate selectivity and examination of hydrolysis with different reaction conditions. Biochim Biophys Acta 1986; 874: 326-336. Stone SR, HofsteengeJ. Specificity of activated Protein C. Biochem J 1985; 230: 497-502. Pedersen AH, Nordfang O, Norris F, et al. Recombinant human extrinsic pathway inhibitor. J Biol Chem 1990; 265:16786-16793. Sidelmann J, Jespersen J, Gram J. Measurements of plasminogen activator activity and plasma-coagulum lysis measured by use of optimized fibrin gel structure preformed in microtiter plates. Clin Chem 1995; 41: 979-85. ChristensenU, Sottmp-JensenL. Mechanism of c~2-macroglobulinproteinase interactions. Studies with trypsin and plasmin. Biochemistry 1984; 23: 6619-6626. Aasen AO, Gallimore MJ, Lilleaasen P, Lyngaas K, Larsbraaten M, Amundsen E. Changes in antiplasmin and plasmin activities during extreme hemodilution and open heart surgery in dogs. Eur Surg Res 1979; 11: 145-153. Btiller HR, Henny Ch P, Ritzen M, Kahl6 LH, ten Care JW. Intraoperative antithrombin III, plasminogen and c~2-antiplasmin behaviour. Thromb Haemos 1981 ; 46: 300. Coleman PL, Patel PD, Cwikel BJ, Rafferty UM, Sznycer-Laszuk R, Gelehrter TD. Characterization of the dexamethasoneinduced inhibitor of plasminogen activator in HTC hepatoma cells. J Biol Chem 1986; 261: 4352-4357. Hekman CM, LoskntoffDJ. Kinetic analysis of the interaction between plasminogen activator inhibitor 1 and both urokinase and tissue plasminogen activator. Arch Biochem Biophys 1988; 262: 199-210. Chmielewska J, R~nby M, Wiman B. Evidence for a rapid inhibitor to tissue plasminogen activator in plasma. Thromb Res 1983; 31: 427-436. Juhan-Vague I, Moerman B, DeCock F, Aillaud MF, Collen D. Plasma levels of a specific inhibitor of tissue-type plasminogen activator (and urokinase) in normal and pathological conditions. Thromb Res 1984; 33: 523- 530. Pauling L. Chemical achievements and hope for the future. Amer Scientist, 1948; 36: 51- 58. Christensen U. Urokinase-catalysed plasminogen activation. Effects of ligands binding to the AH-site of plasminogen. Biochim. Biophys Acta 1988; 957; 258-265. Stieff T, Radtke KP, Heimburger N. Evidence for identity of PCI and plasminogen activator inhibitor 3. Biol Chem Hoppe-Seyler, 1987; 368: 1427-1433. Heeb MJ, Griffin JH. Physiological inhibition of human activated protein C by c~l-antitrypsin. J Biol Chem 1988; 263: 11613-11616.
33. Feinman RD. Kinetics and mechanism ofthe antithrombinprotease reaction. In: Collen D, Wiman B, Verstraete M, eds. The physiological inhibitors of blood coagulation and fibrinolysis. Amsterdam: Elsevier/North-Holland Biomedical Press, 1979; 55-66.
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Effects of aprotinin on c o a g u l a t i o n and fibrinolysis e n z y m e s U. Christensen, J. Schiodt Kemisk Laboratodum IV, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen a, Denmark.
Summary Large quantities of aprotinin, the tight-binding inhibitor of trypsin and kallikrein, are used with success to reduce bleeding, e.g. in cardiac pulmonary surgery. The effect has been attributed to inhibition of fibrinolysis, which immediately seems plausible since aprotinin is a known effective inhibitor of plasmin. However, fast kinetic studies reveal that the rate of the plasmin-aprotinin reaction may be too slow to compete effectively with (z2-antiplasmin, even when present at subphysiological concentrations. Fibrinolysis experiments in microtiter plates confirm that the effect of aprotinin on plasmin may be negligible in the presence of c~2-antiplasmin. Therefore, the effects of high concentrations of aprotinin on a number of coagulation and fibrinolysis enzymes previously assumed not to be affected by the inhibitor were also investigated using steady state kinetic-rate methods. It was found that aprotinin is a multi-functional inhibitor which inhibits urokinase, activated protein C (APC) and thrombin sufficiently well, if present in gM-concentrations, to significantly affect their activity. A kinetic analysis showed simple competitive inhibition in all these cases, corresponding with binding of aprotinin to the active site of the enzyme and thus a reduction in the amount of enzyme available for substrate interaction. However, Factor Xa, Factor Vlla and tissue-plasminogen activator were not affected. All in all, the results indicate that the effects of high concentrations of aprotinin may also stem from enzymes other than plasmin. Known kinetic data on various physiological inhibitors show that the effects of aprotinin on urokinase and APC may be more important than has been hitherto anticipated.
INTRODUCTION
Aprotinin is used to reduce bleeding and the need for donor-blood transfusion in cardiac surgical patients and has been shown to be effective in complex surgical procedures in which there is a high risk of postoperative bleeding (see Royston 1 for a recent review). Administration of aprotinin in a high-dose regimen (2 x 10 ~ KIU loading dose, 2 x 10 ~ KIU into the cardiac pulmonary surgery (CPB) circuit, and 5x 105 KIU/h continuous infusion during surgery) has been shown to reduce blood loss by 40%-50% and to reduce the need for donor-blood transfusion by 40%-80% in various surgical procedures. 2-~ Aprotinin (Basic Pancreatic Trypsin Inhibitor) was first discovered as a kallikrein inactivator 7 and later isolated from bovine pancreas 8 as a trypsin inhibitor. It is a small, well-characterized protein recognized as an inhibitor of a Received 31 December 1996 Accepted after revision 25 June 1997 Correspondence to: U. Christensen, Tel. +(45)35320266; Fax. +(45)35320299; E-mail: u.christensen @pop.ki.ku.dk
number of serine proteinases, e.g. chymotrypsin and plasmin, as well as trypsin and kallikrein, and weak inhibition of urokinase has also been described 9. The reducing effect of aprotinin on bleeding has been attributed to inhibition of fibrinolysis; 1-6'1° first of all of plasmin. This is plausible as plasmin is strongly inhibited by aprotinin. 11'12 However, low-dose aprotinin infusion that should be sufficient to inhibit plasmin in the clinical situation was not found to be clinically useful.l° In accordance with this, the kinetic results presented here reveal that the plasmin-aprotinin reaction may be too slow to compete effectively with that of a2-antiplasmin. The aprotinin effect may also stem from other enzymes. The effects of high concentrations of aprotinin on a number of other coagulation and fibrinolysis enzymes previously assumed not to be affected by the inhibitor were investigated using steady state kinetic-rate methods. It is found that aprotinin inhibits urokinase, activated protein C (APC) and thrombin sufficiently well, if present in gM-concentrations, to significantly affect their activity. A kinetic analysis showed simple competitive inhibition in all these cases, corresponding with 209
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Christensen & Schiodt
binding of aprotinin to the active site of the enzyme and thus a reduction in the amount of enzyme available for substrate interaction. Factor Xa, Factor VIIa and tissueplasminogen activator (tPA) were not affected. MATERIALS A N D METHODS Proteins and chemicals
Aprotinin (Trasylol) was kindly provided as a gift from Birte Petersen, Bayer Danmark A/S; its concentration was determined from titrations with trypsin 13 and showed 106 KIU = 22 gmol. Fibrinogen was kindly provided as a gift from Jorgen Jespersen, Esbjerg, Denmark. Human plasmin was prepared and titrated as previously described. ~3-14 Human urokinase was Trombolysin from IMMUNO DANMARK A/S. Two-chain tPA was from Biopool Sweden. APC was from Chromogenix AB, Sweden. Trypsin, h u m a n thrombin and bovine Factor Xa were from Sigma, St. Louis, MO. Factor VIIa (human) was a gift from Lars Christian Petersen, Novo Nordisk A/S, Denmark. Tripeptide-p-nitroanilide substrates were from Chromogenix AB, Sweden. All other chemicals were analytical grade from Sigma or Merck. Fluorescence titration
Intrinsic fluorescence emission spectra of aprotinin, plasmin and the aprotinin-plasmin complex were obtained using a Perkin-Elmer LS-50 fluofimeter equipped with FLDM software. Exitation was at 280 nm with a slit width of 5 nm, the emission spectra were recorded in the range 300-500nm, also with a slit width of 5nm. Titration experiments were performed at 25 °C in 50 mM Tris-HC1, 0.1 M NaCl-buffer, pH 7.6, by mixing aprotinin (1.5 ml, O; 0.5; 1; 1.5; 2; 2.5; 3 and 4gM) and plasmin (1.5ml, 2gM) or reference buffer (1.5ml). After 5 min incubation for equilibration, the emission spectrum was recorded three times. The fluorescence intensities were each taken as the integrated signal and then averaged. The relative fuorescence change, AF, was calculated as (F - Fo) x 100%/Fo, where F is the averaged fluorescence intensity of the sample solution minus that of the corresponding reference solution, and Fo is F for plasmin in the absence of aprotinin. As defined, AF is a negative quantity. Stopped-flow kinetic experiments
Fast kinetic experiments were performed on the interaction of aprotinin and plasmin in a Hi-Tech Scientific PQ/SF-53 spectrofluorimeter equipped with a high-intensity Xenon arc lamp. The reaction resulted in a decrease of the total protein fluorescence, similar to that observed for the reaction of plasmin with c~2-antiplasmin.15 Time Fibrinolysis & Proteolysis (1997) 11(4), 209-214
courses of the intrinsic fluorescence changes of the plasmin-aprotinin reaction were recorded for an appropriate time (1, 2 or 10 s) after stopped-flow mixing (mixing time approx. 10-3 s) of plasmin- and aprotinin-solutions in 50 mM Tris-HC1, 0.1 M NaCl-buffer pH 7.6, 25°C, at second order reaction conditions, i.e. the same concentration of the enzyme and of the inhibitor. The final concentrations were: 5, 2.5, 1.25 and 0.67gM. The exitation wavelength was 280 nm, slit width 5 nm. The light emitted from the reaction mixture passed a WG 320 filter before detection in the photomultiplier. Thus, an integrated fluorescence signal was obtained. In each experiment, 400 data points were recorded and sets of data from three to four experiments were averaged and analysed with the Hi-Tech HS-1 Data Pro Software. In all cases, the best fitted curve was that corresponding to second order reaction kinetics. The regression analysis used is based on the Gauss-Newton procedure. After recording 1, 2 or 10 s of the reaction, control measurements were made on the aged solution in the stoppedflow cell, the last one after 10 rain. Such measurements showed no further change of the fluorescence after the initial recording. Thus, only one fast step giving rise to changes of the fluorescence is apparent when plasmin and aprotinin interact. Simulations of plasmin inhibition in the presence of aprotinin and a2- antiplasmin were made with the Mathcad PLUS 6.0 software (MathSoft Inc). Steady state kinetic measurements
The steady state kinetic parameters of the hydrolysis of a reasonably specific tripeptide-p-nitroanilide substrate catalysed by the coagulation or fibrinolysis enzyme in question were determined from initial rate measurdments in the presence and absence of aprotinin, essentially as previously described, ~3,14,16,17 using a Perkin-Elmer 17 spectrophotometer equipped with the PECSS-software, which provides calculations of initial rates. The substrates used were: S-2444 for urokinase; 16 S-2288 for tPA, Factor Xa, 17'18 APC 19 and Factor VIIa; 2° S-2266 for thrombin.17 The experiments were designed to optimally reveal competitive as well as noncompetitive inhibition, i.e. substrates with low Kin-values were avoided so that precise rate measurements could be made at substrate concentrations less than Kin. The final concentrations of aprotinin used were 64, 10.7, 5.3 and 2.7 gM. The nonlinear least-squares fitting program GraFit (Erithacus Software Ltd.) was used for data analysis. Fibrinolysis experiments
Measurements of inhibition of fibrinolysis were performed in microtiter plates using optimized fibrin gels as described by Sidelmann et al.2o 30gl test samples all © Pearson Professional Ltd 1997
Effects of aprotinin on coagulation and fibrinolysis enzymes
containing 5 nM of tissue plasminogen activator and various concentrations of a2-antiplasmin (0, 0.2, 0.5 and 1 gM), in the presence or absence of 1 gM aprotinin, were placed on top of preformed fibrin gels and the A4o5 was measured in a Ceres 900C, Bio-Tek Instruments Inc. microtiter plate reader every 10 min for 20 h. The continuous decrease in the A4o5, obtained after a lag period and calculated using the KC3 Kineticalc software, was used as a measure of the fibrinolysis rate.
211
2
03
U.
RESULTS
AND
DISCUSSION
Figure 1 shows a fluorescence titration curve of 1 gM (final concentration) of plasmin with aprotinin. The 1 : 1stoichiometry of the plasmin-aprotinin-complex is obvious. The relative fluorescence change, zXF, is negative and proportional to the amount of complex formed. The intrinsic fluorescence of the complex is found to be 13% smaller than the sum of that of plasmin and of aprotinin at the same concentrations. Given this large AF-value resulting from the formation of the plasmin-aprotinin-complex, it was possible to follow the reaction between plasmin and aprotinin directly using stopped-flow fluorescence kinetic measurements. Series of such experiments were performed in which various equal concentrations of plasmin and aprotinin were mixed. Stopped-flow mixing is obtained within 10-3 s so that fast reactions can be followed. An example of the averaged stopped-flow traces obtained is shown in Fig. 2. Figure 2 also shows the curve resulting from the fit of a second order rate equation to the data. No other rate equation showed a good fit. Series of such experiments all showed second order reaction
14 12 10 8 IJ _ .<
6 4 2 0
qF
0
,
I
i
0.5
l
,
1
I
1.5
i
I
2
Aprotinin (P.M) Fig. 1 Equilibrium fluorescence titration of 1 gM plasmin with aprotinin. The relative fluorescence decrease is plotted as a function of the concentration of aprotinin added.
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0.2 '
o14 time
0.6 '
0'.a
1.0 '
(s)
Fig. 2 Typical time course of the stopped-flow fluorescence signal resulting from the inhibition of human plasmin with aprotinin. From series of such experiments performed at second order reaction conditions, the second order nature of the reaction and the value of the rate constant (Table 1) k = 1.5 10 6 M-1 s-1 were determined.
kinetics with rate constant k = 1.5 106 M -1 s -1 for the formation of the plasmin-aprotinin complex (Table 1). Similar experiments on the plasmin-(z2-antiplasmin reaction 15 have shown the rate constant of this reaction to be k = 2.25 107 M -1 S-1 for the formation of the plasmin-a2-antiplasmin complex. For the plasmin-a2macroglobulin reaction, the value of the association rate constant i s 22 k = 5 105 M -1 s -1 . The time courses of inhibition of plasmin by c~2-antiplasrain and aprotinin in competition at physiological concentrations of a2-AP (0.5-1.5 gM) and high concentration of aprotinin (1 gM) were then simulated. An example is illustrated in Figure 3. It is generally accepted that the concentration of free plasmin is always very low, 9 so that pseudo first order inhibition is occurring. Here it was further assumed that depletion of a2-antiplasmin did not o c c u r . 2 3 ' 2 4 Apparently a valid assumption, Aasen et a123 conclude in their open heart surgery study that 'a2antiplasmin activities did not fall below 20% of preoperation values' and that 'a functional reserve of antiplasmin was present throughout the duration of CPB.' The result is the formation of at least 95% a2-antiplasmin-plasmin and maximally 50/0 aprotinin-plasmin-complexes (Fig. 3A). The competing effect of a2-antiplasmin on the inhibition of plasmin by aprotinin is shown in Figure 3B, where the amount of plasmin-aprotinin in the presence of a2antiplasmin is compared with that obtained in the absence of a2-antiplasmin. Thus, the effect of aprotinin on plasmin in the absence of physiological inhibitors is quite different from that in their presence. Finally, Figure 3C illustrates the effect of aprotinin on the total inhibition of plasmin. It is seen that the presence of aprotinin does not at any time Fibrinolysis & Proteolysis (1997) 11(4), 209-214
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Christensen & Schiedt
Table I Values of the second order rate constant of the association of plasmin and aprotinin obtained from stopped-flow kinetic measurements Protein concentrations ([aM)
Rate constant, k (M -1 s -1)
5.0 2.5 1.25 0.67
1.53 1.48 1.51 1.47
100
,
~
x x x x
106 106 106 106
~. . . . . . , . . . . . . . . . .
,
~ 50
Aprotinin inhibiton of the hydrolysis of specific chromogenic substrates b y coagulation and fibrinolysis enzymes using series of experiments at various concentrations of aprotinin and the substrates are illustrated in Figure 4. Urokinase, APC and thrombin are obviously affected at concentrations of aprotinin in the range of interest (1-5 gM), whereas the effects on t-PA, Factor Xa and Factor VIIa are each negligible, even in this high concentration range. The curves shown stem from non-linear least squares fits of the Michaelis equation to the experimental data. The resulting kinetic parameters show no or simple competitive inhibitions, i.e. values of Km,app increasing linearly with the concentration of aprotinin, i, and unaffected Vm~-Values, corresponding to the rate equation:
a-
vi = Vma~/[1 + Km,app/S ] = Vmax/[1 + Kin(1 +i/Ki)/S ] o.1
0.2
0.3
0.4
0.5
time(s) lO0
LE --.c
50
/ I
r
[
l
0.1
0.2
0.3
0.4
J
a
0.5
time (s)
I00
/ o q
[
I
1
0.1
0.2
0,3
0.4
0.5
time (s)
Fig. 3 Simulations of plasmin-inhibition kinetics. A. Plasmin inhibition in the presence of aprotinin and o~2-antiplasmin (1 [aM each). Line: Total plasmin inhibited (plasmin-aprotinin plus plasmin-o~2-antiplasmin). Dots: Plasmin-o~2-antiplasmin-complex formed. Broken line: Plasmin-aprotinin-complex formed. B. Plasmin-aprotinin complex formed in the presence (broken line) and absence (line) of o~2-antiplasmin (1 [aM). C. Total plasmin inhibited with 1 [aM o~2-antiplasmin in the presence (line) and absence (dots) of aprotinin (1 [aM).
add significantly to the amount of plasmin inhibited. In the absence of aprotinin, a little more a2-antiplasminplasmin complex will be formed than in its presence, but the total inhibition is practically unchanged. In vitro fibrinolysis experiments confirmed this (Table 2). Table 2 shows that the fibrinolysis rates 21 obtained in the presence of both c~2-antiplasmin and aprotinin are comparable with those obtained in the presence of only a2-antiplasmin. Fibrinolysis & Proteolysis (1997) 11(4), 209-214
The values of the inhibition constants (Ki-values) obtained from the fits are listed in Table 3 and the lower limits of Kcvalues obtained for the enzymes not inhibited in the actual concentration range are given. As is shown for plasmin, the effect of aprotinin m a y be of no importance if 'irreversible' physiological inhibition is kinetically very fast. The actual rate of such an enzymeinhibitor reaction is determined b y the product of the rate constant and the concentration of the inhibitor. The physiological inhibitor of urokinase is PAI-1. The reaction of urokinase with PAI-1 is governed by a very fast association rate constant of the order 107 M -1 s-1.25,26 However, PAI-1 is present in plasma only in very low concentrations 27 (
No aprotinin
1 [aM aprotinin
0 0.2 0.5 1.0
1.9±0.1 0.25±0.05 0.17±0.04 0.05±0.02
0.2±0.06 0.24±0.05 0.16±0.05 0.05±0.02
The units of the fibrinolysis rates are zXOD x 103/min. The standard errors of the estimates given are each based on six identical experiments. It is seen that within experimental error aprotinin does not add to the inhibition in the presence of c~2-antiplasmin.
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Effects of aprotinin on coagulation and fibrinolysis enzymes
Activated
Urokinase I
I
1
I
I
I
80
20
50
15
40
10
protein
I
213
Thrombin
C L
k
1.5
2
> 5
20
0 0.05
0.1
0.15
[S-2444]
0,2
0.25
0.5
(mM)
[S-22881
3O
20
> 10
I
I
I
0.1
0,2
0.3
[S-2288]
I
I
0.1
0.2
[S-2266]
(mMI
/F
t-PA 40
E
1
0.3
(raM)
Factor Vlla
60
40
20
0 0,4
0.1
(mMI
0.2 [S-22881
0.3
0.4
(mM)
0.2
0.4 [S-2288]
0.6
0.8
{raM)
Fig. 4 Steady state kinetic velocities obtained in the presence and absence of aprotinin calculated from the initial slope of A41o recorded. Concentrations of aprotinin: (©): no aprotinin; (V): 64#M; (A): 2.7 #M; (V): 5.3 #M; (+): 10.7 #M. The curves shown stem from fits of Eqn. 1 to the data.
catalysis, which point out the advantages of weak binding of substrates 29 (viz high Kin-values relative to physiological concentrations of substrates), the competitive effect of the urokinase substrate, plasminogen, on the urokinaseaprotinin interaction is small. That effect is determined by the ratio of the plasminogen concentration (2 gM) and the urokinase-plasminogen Km-Value (approx. 40 ~M), 3° thus amounting to only approx. 5%. Thus, all things considered, effective urokinase inhibition by aprotinin in the pM-concentration range in plasma is highly likely. The physiological inhibitor of APC is PCI (PAI-3). As for PAI-1, the physiological concentration of PCI is very low 3~ (10 nM) compared with that of c%-antiplasmin (1-1.5 M). Again, the reaction rate of APC and PCI in plasma is most probably significantly less than that of APC and aprotinin at high dose. The rate constant of APC inhibition with PCI has not been measured, but it must, of course, be less than the value determined by the Table 3 Values of aprotinin inhibition constants, K~, obtained from steady state kinetics Enzyme
Ki (#M)
t-Plasminogen activator Urokinase Activated protein C Thrombin Factor Vlla Factor Xa
>600 4.8 2.6 27 > 100 >100
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diffusion control-limit (10s-109 M -1 S-1) and therefore a value similar to that of the reactions of urokinase-PAI-I and of plasmin-a2-antiplasmin is a realistic maximal value. This means that the effective rate of the reaction of APC with PCI at plasma concentrations is at least 10 -2 times as slow as that of plasmin-a2-antiplasmin and that aprotinin at high dose in plasma may be an important inhibitor of APC. The reaction of APC and (zl-antitrypsin 32 with an effective rate of only 0.0004 M -~ s-~ is of no importance in this context. The aprotinin inhibition effect on thrombin is probably of no importance, antithrombin is present in 2-4 gM concentrations in normal plasma and the thrombinantithrombin reaction is fast, particularly in the presence of heparin, 33 whereas urokinase and APC are each candidates for the aprotinin-effect. At the concentration used in surgery (1-5 gM), immediate inhibitions of uroMnase up to 50% and of APC up to 6 6 % are likely. Finally, it sems w o ~ h noting that kallikrein could have profibrinolytic activity and inhibition of kallikrein may contribute to the clinical effect. In conclusion, the inhibition of urokinase and APC might represent additional mechanisms explaining the clinical effects of aprotinin. REFERENCES
1. Royston D. High-dose aprot/nin therapy: a review of the first five years' experience.J CardiothoracVascAnesth 1992; 6: 76-100. Fibrinolysis & Proteolysis (1997) 11(4), 209-214
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2. Dietrich W, Barankay A, Hahnel C, Richter JA. High-dose aprotinin in cardiac surgery: three years' experience in 1784 patients. J Cardiothorac Vasc Anesth 1992; 6:324-327. 3. Cosgrove DM, Hetic B, Lytle BW, et al. Aprotinin therapy for reoperative myocardial revascularization. Ann Thorac Surg 1992; 54: 1031-1038. 4. Bidstrup BP, Harrison J, Royston D, Taylor KM, Treasure T. Aprotinin therapy in cardiac operations: a report on use in 41 cardiac centers in the United Kingdom. Ann Thorac Surg 1993; 55: 971-976. 5. Bidstrup BP, Underwood SR, Sapsford RN. Effect of aprotinin (Trasylol) on aorta-coronary bypass graft patency. J Thorac Cardiovasc Surg 1993; 105: 147-153. 6. Levy JH, Pifarre R, SchaffHV, et al. A multicenter, double-blind, placebo-controlled trial of aprotinin for reducing blood loss and the requirement for donor-blood transfusion in patients undergoing coronary artery bypass grafting. Circulation 1995; 92: 2236-2244. 7. Kraut H, Frey EK, Werle E. Uber die inactivierung des Kallikreins. Hoppe-Seyler's Z Physiol Chem 1930; 192:1-21. 8. Kunitz M, Northrop JH. Isolatinn frombeefpancreas of crystalline trypsinogen, trypsin, trypsin inhibitor and an inhibitor-trypsin compound. J Gen Physiol 1936; 19: 991-1007. 9. Fritz H, Wunderer G. Biochemistry and applications of aprotinin, the kallicrein inhibitor from bovine organs. ArzneimForsch/Drug Res 1983; 33: 479-494. 10. Hardy J-F, Desroches J, Belisle S, Perranlt J, Carrier M, Robitaille D. Low- dose aprotinin infusion is not clinically useful to reduce bleeding and transfusion of homologous blood products in high-risk cardiac surgical patients. Can J Anaesth 1993; 40: 625-631. 11. Wiman B. On the reaction of plasmin or plasmin-streptokinase complex with aprotinin or c~a-antiplasmin. Thromb Res 1980; 17: 143-152. 12. Petersen LC, Clemmensen I. Kinetics of plasmin inhibition in the presence of synthetic tripeptide substrate. Biochem J 1981; 199: 121-127. 13. Christensen U, Ipsen H-H. Steady-state kinetics of plasmin and trypsin-catalysed hydrolysis of a number of tripeptide-pnitroanilides. Biochim Biophys Acta 1979; 569:177-183. 14. Christensen U. pH effects in plasmin-catalysed hydrolysis of a-N-benzoyl-L-arginine compounds. Biochim Biophys Acta 1975; 397: 459-467. 15. Christensen U, Bangert K, Thorsen S. Reaction of c~2-antiplasmin and plasmin. Stopped-flow fluorescence kinetics. FEBS Lett 1996; 387: 58-62. 16. Ipsen H-H, Christensen U. Kinetic studies of urokinasecatalysed hydrolysis of 5-oxo-L-prolyl-l-arginine 4-nitroanilide. Biochim Biophys Acta 1980; 613: 476-481. 17. Lottenberg R, Christensen U, Jackson CM, Coleman PL. Assay of coagulation proteases using peptide chromogenic and fluorogenic substrates. Meth Enzymol 1981; 80: 341-361. 18. Lottenberg R, Hall JA, Paufler E, Zupan A, Christensen U,
Fibrinolysis & Proteolysis (1997) 11(4), 209-214
19. 20. 21.
22. 23.
24. 25.
26.
27. 28.
29. 30. 31. 32.
Jackson CM. The action of Factor Xa on peptide p-nitroanilide substrates: substrate selectivity and examination of hydrolysis with different reaction conditions. Biochim Biophys Acta 1986; 874: 326-336. Stone SR, HofsteengeJ. Specificity of activated Protein C. Biochem J 1985; 230: 497-502. Pedersen AH, Nordfang O, Norris F, et al. Recombinant human extrinsic pathway inhibitor. J Biol Chem 1990; 265:16786-16793. Sidelmann J, Jespersen J, Gram J. Measurements of plasminogen activator activity and plasma-coagulum lysis measured by use of optimized fibrin gel structure preformed in microtiter plates. Clin Chem 1995; 41: 979-85. ChristensenU, Sottmp-JensenL. Mechanism of c~2-macroglobulinproteinase interactions. Studies with trypsin and plasmin. Biochemistry 1984; 23: 6619-6626. Aasen AO, Gallimore MJ, Lilleaasen P, Lyngaas K, Larsbraaten M, Amundsen E. Changes in antiplasmin and plasmin activities during extreme hemodilution and open heart surgery in dogs. Eur Surg Res 1979; 11: 145-153. Btiller HR, Henny Ch P, Ritzen M, Kahl6 LH, ten Care JW. Intraoperative antithrombin III, plasminogen and c~2-antiplasmin behaviour. Thromb Haemos 1981 ; 46: 300. Coleman PL, Patel PD, Cwikel BJ, Rafferty UM, Sznycer-Laszuk R, Gelehrter TD. Characterization of the dexamethasoneinduced inhibitor of plasminogen activator in HTC hepatoma cells. J Biol Chem 1986; 261: 4352-4357. Hekman CM, LoskntoffDJ. Kinetic analysis of the interaction between plasminogen activator inhibitor 1 and both urokinase and tissue plasminogen activator. Arch Biochem Biophys 1988; 262: 199-210. Chmielewska J, R~nby M, Wiman B. Evidence for a rapid inhibitor to tissue plasminogen activator in plasma. Thromb Res 1983; 31: 427-436. Juhan-Vague I, Moerman B, DeCock F, Aillaud MF, Collen D. Plasma levels of a specific inhibitor of tissue-type plasminogen activator (and urokinase) in normal and pathological conditions. Thromb Res 1984; 33: 523- 530. Pauling L. Chemical achievements and hope for the future. Amer Scientist, 1948; 36: 51- 58. Christensen U. Urokinase-catalysed plasminogen activation. Effects of ligands binding to the AH-site of plasminogen. Biochim. Biophys Acta 1988; 957; 258-265. Stieff T, Radtke KP, Heimburger N. Evidence for identity of PCI and plasminogen activator inhibitor 3. Biol Chem Hoppe-Seyler, 1987; 368: 1427-1433. Heeb MJ, Griffin JH. Physiological inhibition of human activated protein C by c~l-antitrypsin. J Biol Chem 1988; 263: 11613-11616.
33. Feinman RD. Kinetics and mechanism ofthe antithrombinprotease reaction. In: Collen D, Wiman B, Verstraete M, eds. The physiological inhibitors of blood coagulation and fibrinolysis. Amsterdam: Elsevier/North-Holland Biomedical Press, 1979; 55-66.
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