Biochemical properties of natural and recombinant staphylokinase

F,hnnolya,\ (lYY2)6,214225 01992 Longman Gmup UK Ltd

Biochemical Properties of Natural and Recombinant Staphylokinase

H. R. Lijnen,

B. Van Hoef,

L. Vandenbossche,

D. Collen

SUMMARY. The biochemical properties of natural staphylokinase

(STAN), purified from the culture broth of a selected Staphylococcus aureus strain, were compared with those of different molecular species of recombinant staphylokinase (STAR), produced by expression of a genomic DNA fragment of the Staphylococcus aureus strain in E. co/i. STAR was obtained from the supernatant (STAR-S) or periplasmic fraction (STAR-P) of the transformed E. coli either as a low molecular weight form lacking the 10 NHz-terminal amino acids (STA-AlO) (peak I following chromatography of STAR-S or STAR-P on CM-Sephadex, STA-CM-I) or as a mixture of mature STA (STA-M) and a proteolytic derivative lacking the 6 NH*-terminal amino acids (STA-A6) (peak II following chromatography of STAR-S or STAR-P on CM-Sephadex, STA-CM-II), or it was obtained from the cytosol fraction (STAR-C) as STA-AIO. STAN, STA-CM-I, STA-CM-II and STAR-C were found to be similar with respect to following properties: rate and extent of complex formation with plasminogen, plasminogen activating potential in the absence or the presence of a fibrin-like stimulator and inhibition rate of the plasminogen-STA complex by az-antiplasmin, and fibrinolytic and fibrinogenolytic potential in a human plasma milieu in vitro. In a human plasma milieu in vitro, STAN had a somewhat lower fibrinolytic activity versus platelet-rich plasma ((PRP); 4x 10’ platelets/pi) clots than versus platelet-poor plasma ((PPP); <5x103 platelets/PI) clots, with equi-effective concentrations (causing 50% clot lysis in 2h; C5,,) of 0.37+-0.02 Fg/ml or 0.58+0.07 kg/ml (mean+SEM; n=6), respectively (p=O.O2). In contrast, no significant lysis of PRP clots was obtained using up to 20 kg/ml of streptokinase, whereas its Cso for PPP clots was 2.lkO.13 pg/ml. Clot lysis was inhibited by platelets in a dose-dependent way: 50% inhibition occurred with 1.4X105/p1 platelets for streptokinase, and with 13x lO’/pl platelets for STAN. When clot retraction was inhibited with a synthetic platelet GPIIb/IIIa receptor antagonist, the thrombolytic potency of STAN towards a platelet-rich clot was unaffected, whereas that of streptokinase was increased to a level similar to that observed with PPP clots. Mechanically compressed human plasma clots, submerged in a human plasma milieu in vitro, were equally sensitive to lysis with STAN (CsO of 0.34kO.03 kg/ml) as PPP clots, but were very resistant to lysis with streptokinase (slO% lysis with 20 kg/ml). Thus, STAN, STA-CM-I, STA-CM-II and STAR-C are functionally indistinguishable, and in a human plasma milieu STA is relatively more potent towards retracted or compressed plasma clots than streptokinase.

Lys-Gly-Asp-Asp-Ala(STA-A6) and with NH2-terminal sequence Lys-Gly-Asp-Asp-Ala(STA-AlO). These forms could be partially separated by chromatography on CM-Sephadex, which yielded a first peak (STA-CM-I consisting of STA-AlO) and a second peak (STA-CM-II consisting of a mixture of STA-M and STA-A6). ’ It has previously been demonstrated that STA, like streptokinase is not an enzyme, but that it forms a stoichiometric complex with plasminogen that then activates other plasminogen molecules.“.4 In the absence of fibrin, the plasminogen-STA complex is very rapidly neutralised by c;u2-antiplasmin,“-’ thus reducing systemic plasminogen activation in a plasma milieu.6.7 In the presence of fibrin, the lysine-binding sites of the plasminogen-STA complex are occupied,

Methods for the large scale production of natural (STAN) and recombinant (STAR) staphylokinase were developed’ in order to allow systematic studies of the thrombolytic properties of staphylokinase (STA).2 Whereas STAN was obtained as a homogeneous species with NH2-terminal sequence LysGly-Asp-Asp-Ala-, three variants of STAR were obtained from the supernatant (STAR-S) or periplasmic space (STAR-P) of transformed E. coli, with NH2-terminal sequence Ser-Ser-Ser-Phe-Asp-LysGly-Lys-Tyr-Lys-Lys-Gly-Asp-Asp-Ala(STA-M), with NH*-terminal sequence Gly-Lys-Tyr-Lys-LysH. R. Lijnen, B. Van Hoef, L. Vandenbossche, D. Collen, Center for Thrombosis and Vascular Research, University of Leuven, Leuven, Belgium. 214

Fihrinolyais

and inhibition by a2-antiplasmin is delayed. thus allowing preferential plasminogen activation at the fibrin surface.‘,8 In the present study we have compared STAN and the different molecular species of STAR with streptokinase in terms of their biochemical characteristics, including plasminogen activating potential and fibrinolytic properties in a human plasma milieu in vitro. STAN and STAR appear to be more potent than streptokinase for the dissolution of platelet-enriched retracted plasma clots in vivo in a hamster pulmonary embolism model.’ Sabovic et al” have previously shown that, in a plasma milieu in vitro, retracted blood clots and mechanically compressed plasma clots are more sensitive to lysis with the fibrin-specific plasminogen activators tissue-type plasminogen activator (t-PA) and single chain urokinase-type plasminogen activator (scu-PA). than with the non-fibrin specific agents streptokinase and two chain urokinase-type plasminogen activator (tcu-PA). It was suggested that this differential sensitivity might result from alteration of the plasminogen/a2-antiplasmin ratio in the clot during retraction. Therefore, in the present study, we have investigated the potential of STA for lysis of platelet-rich and mechanically compressed plasma clots in the plasma clot lysis system.

MATERIALS

AND METHODS

Proteins and Reagents STAN and STAR moieties were purified and characterised as described.’ STAN was purified from culture broth of Staphylococcus aureus (strain no. 23); STAR was purified from the supernatant (STAR-S) or the periplasmic fraction (STAR-P) obtained from culture broth of E. cofi transformed with the recombinant pUC19 containing the 2.9 kb Hin dIII genomic DNA fragment of Staphylococcus aureus strain no. 23. STA-CM-l, lacking the 10 NH*-terminal amino acids (STA-A10 with NHz-terminal amino acid sequence Lys-Gly-Asp-Asp-Ala-) and STA-CM-II. a mixture of STA-M with NHZ-terminal sequence Ser-Ser-Ser-Phe-Asp-Lys-Gly-Lys-Tyr-Lys-Lys-GlySTA-A6 with NH?-terminal Asp-Asp-Ala and Gly-Lys-Tyr-Lys-Lys-Gly-Asp-Asp-Ala sequence were obtained by chromatography on CM-Sephadex C-50 of STAR-S or STAR-P; STAR-C was purified from the cytosol fraction of culture broth of transwere formed E. coli. Before use, the preparations dialysed extensively against 0.1 M phosphate buffer, pH 7.3, containing 0.01% Tween 80. Streptokinase was Streptase@ purchased from Hoechst (Brussels, Belgium). Its concentration was determined from the activity on the label, assuming a specific activity of 100000 units/mg. Streptokinase devoid of albumin was purchased from Boehringer Mannheim (Germany). Recombinant tissue-type plasminogen activator (rt-PA) was Actilyse&, a kind

1-15

gift from Boehringer lngelheim (Brussels, Belgium). tcu-PA was obtained from recombinant single chain urokinase-type plasminogen activator produced in Chinese hamster ovary cells,“’ by treatment with plasmin and isolation on benzamidine.Sepharose, as described elsewhere. ’’The specific activities of rt-PA and tcu-PA, determined on fibrin platesI by comparison with the International Reference Preparations, were 480000 lU/mg and 130000 lU/mg, respectively. The International Reference Preparations of t-PA (86/670) and u-PA (66/46) were obtained from the National Institute for Biological Standards and Control (London, UK). Human plasminogen (NH2-terminal glutamic acid), cwz-antiplasmin and CNBr-digested fibrinogen were prepared and characterised as described elsewhere.‘3-‘” The chromogenic substrate D-valyl-leucyl-lysine-p-nitroanilide (S-2251) was purchased from KabiVitrum (Brussels, Belgium), ‘251-labelled fibrinogen from Amersham (Buckinghamshire, UK), plasminogen-containing bovine fibrinogen (Povite quality) from Organon-Teknika (Oss, The Netherlands) and bovine thrombin from Sigma (St. Louis, MO). G-4120 (L-cysteine, N-(mercaptoacetyl)-Dtyrosyl-L-arginylglycyl-L-a-aspartyl-cyclic (1->5)sulphide,j-oxide), a synthetic pentapeptide with a high affinity for the platelet GPllb/llla receptor” was a gift from Genentech Inc., South San Francisco, CA (courtesy of Dr S. Bunting). Plasma was pooled fresh frozen human plasma obtained from blood collected on acid-citrate-dextrose, from at least 10 healthy blood donors.

Methods The fibrinolytic activities of STA solutions were determined with a clot lysis assay” and expressed in home units (HU) by comparison with an in-house standard of STAN (lot STAN.5) which was assigned an activity of 100000 HU per mg protein as determined by amino acid composition.’ The clot lysis assay consisted of 100 ~1 STA solution, 20 ~1 Glu-plasminogen solution (5 mgiml), 800 ~1 bovine fibrinogen solution (2mg/ml) and 80 ~1 thrombin (400 NIH units/ml) solution, all made up in 0.06M phosphate buffer. pH 7.40 containing 0.01% Tween 80. Protein concentrations were determined by amino acid analysis or by the method of Bradford,‘” using the STAN standard as the primary reference. With this assay, the specific activities of the STA preparations used were (mean+-SEM; n=3 with three independent preparations) 100000i5600 HU/mg for STAN, 14000017000 HU/mg for STA-CM-l or 160000+37000 HU/mg for STAR-C. For STA-CM11 only one preparation was used with a specific activity of 120000 HUimg. Human plasminogen and a?-antiplasmin were labelled with ‘2iI using the Iodogen method2” to specific radioactivities of 8~10~ cpm/pg and 3~10~ cpm/t*g, respectively.

216

Biochemical

Properties

of Natural

and Recombinant

Staphylokinase

Platelet-rich (PRP) and platelet-poor (PPP) human plasma was prepared from fresh blood, CO]lected in O.OllM citrate (final concentration), from volunteers who had not taken aspirin for at least 10 days. PRP was collected after centrifugation at room temperature for 15 min at 950 rpm (150x g) and PPP (platelet count 1 to 5 x lO”/l~l) was collected after an additional centrifugation for 10 min at 3000 rpm (1500xg). After determination of the platelet count (with a Cell-Dyn 16 platelet counter, Sequoia-Turner, Mountain View, CA), plasma samples with different platelet count (0.4~ 10’ to 6.0~ 105/ul) were reconstituted by dilution of PRP with PPP. Washed platelets were isolated from plasma by gel filtration of PRP on a column of Sepharose 2B, equilibrated with HEPES balanced salt buffer at a now rate of 2ml/min at room temperature. Fibrinogen levels in plasma samples were monitored with a clotting rate assay,2’ and a2-antiplasmin and plasminogen levels by chromogenic substrate assay. 22.23 Biochemical

Characterisation

of STAN and STAR

Complex Formation with Plasminogen The generation of an active site in complexes of plasminogen with STAN or STAR or in complexes of plasminogen with streptokinase was monitored as follows. Plasminogen (final concentration 0.4 ~.LM) was incubated with STA or streptokinase (final concentration 2kM) at 37°C in 0.1 M phosphate buffer, pH 7.4. At different time intervals (@lo min) generation of an active site in the plasminogen-STA or plasminogen-streptokinase complex was measured with S-2251 (final concentration 1 mM) after 25-fold dilution of samples in 0.05M Tris-HC1 buffer, pH 7.4, containing 0.038M NaCl and 0.01% Tween 80. The kinetic parameters for the hydrolysis of S-2251 (0. l1 .OmM) by plasminogen-STA or plasminogen-streptokinase complex (10nM each) were determined by Lineweaver-Burk analysis.

Activation of Plasminogen Activation of plasminogen (final concentration 1.5 ~.LM)with STA or streptokinase (final concentration 2 nM) was monitored at 37°C in 0.1 M phosphate buffer, pH 7.4, containing 0.01% Tween 80. Therefore, at different time intervals (&30 min), generated plasmin was measured with S-2251 (final concentration 1 mM) after 50-fold dilution of samples. For kinetic analysis of plasminogen activation, equimolar plasminogen-STA or plasminogen-streptokinase complexes (final concentration approximately 5l.~M each with a 5-10% excess of plasminogen) were prepared by incubation of plasminogen with STAN, STA-CM-I, STA-CM-II, STAR-C and streptokinase at 37°C for 3 min in 0.1 M phosphate buffer, pH 7.4, containing 25% glycerol;

the mixture was then stored on ice. PlasminogenSTA or plasminogen-streptokinase complex (final concentration 2nM) was incubated with plasminogen (final concentration 1.620 FM for STA moieties and 0.1-2.0 PM for streptokinase) at 37°C in 0.1 M phosphate buffer, pH 7.4. Generated plasmin was measured at different time intervals (&4 min) with S-2251 (final concentration 1 mM) after 25-fold dilution of the sample. Initial activation rates were obtained from plots of the concentration of generated plasmin versus time.

Effect of CNBr-digested Fibrinogen on Plasmirlogen Activation by STAN and STAR Moieties Plasminogen (final concentration 1 .OpM) in 0.1 M phosphate buffer, pH 7.4, was activated at 37°C with equimolar plasminogen-STA complex prepared with STAN, STA-CM-I, STA-CM-II or STAR-C, as described above (final concentration 1.2nM) in the presence of different concentrations of CNBr-digested fibrinogen (final concentration 0-l FM). At different time points (&4 min), samples were removed from the incubation mixtures and generated plasmin was quantitated with S-2251 (final concentration 1mM) after 25-fold dilution. Control experiments were performed with 1.2nM plasminogenstreptokinase complex. Initial activation rates in the absence or the presence of CNBr-digested fibrinogen were determined from plots of the concentration of generated plasmin versus time.

Inhibition of Plasminogen-STA antiplasmin

Complexes by (Ye-

Plasminogen (final concentration 0.5 ~J,M) was complexed with STAN, STA-CM-I, STA-CM-II or STAR-C (final concentration 2.5 FM) by incubation for 3 min at 37°C in 0.1 M phosphate buffer, pH 7.4. The complex was kept on ice in the presence of 25% glycerol. Inhibition of the plasminogen-STA complexes (final concentration 5 nM) by a2-antiplasmin (final concentration 25 nM) was monitored continuously in the presence of S-2251 (final concentration 1 .OmM) as described previously.’ Residual complex was determined at different time intervals (t&2 min) and the apparent second order rate constant (ki,,,,) was calculated using the formula ki,,,,=ln2/(ti/2.[1]), where [I] = [cw2-antiplasmin].

Fibrinolytic and Fibrinogenolytic Properties of STAN and STAR Moieties in a Human Plasma Milieu In vitro 12’I-fibrin labelled plasma clots were prepared from pooled normal human plasma, following addition of 500 000 cpm/ml of ‘251-1abelled fibrinogen, and coagulation with CaC12 (final concentration 35mM) and

Fihrinolysis 217

thrombin (final concentration 2NIH U/ml). Lysis of 12”1-fibrin labelled plasma clots (0.12ml volume) by addition of different concentrations of STAN, ST,4CM-I, STA-CM-II or STAR-C (final concentration 0.05-l .6 &ml) or of streptokinase (final concentration 0.3-9.6 pg/ml) in 0.5ml normal human plasma, was measured over 4 h as previously described.‘” The concentration of fibrinolytic agent required to obtain 50% clot lysis in 2 h (Cso), was determined from plots of percent lysis versus the concentration of test compound. Residual fibrinogen and a2-antiplasmin levels at Cso were determined graphically from plots of residual reactive protein at 2h (expressed in percent of the baseline value) versus the concentration of test compound. Systemic activation of the fibrinolytic system by STAN, STA-CM-I, STA-CM-II or STAR-C (final concentration O-20 IJ-g/ml) or by streptokinase (final concentration t&1.0 p&ml) in normal human plasma in the absence of fibrin, was determined at 2h, by measuring residual fibrinogen levels. The concentration of plasminogen activator required to obtain 50’% fibrinogen degradation within 2h, was determined graphically from dose-response curves representing the concentration of test compound versus residual reactive protein (expressed in percent of the baseline value).

Effect of Platelet Count, Clot Retraction and Mechanical Compression on the Lysis of ‘*%-fibrin Labelled Human Plasma Clots by STA Moieties in a Plasma Milieu In vitro PPP and PRP clots were prepared by mixing either PPP (<5x 10” platelets/$) or PRP (4x 10’ platelets/ pl), containing approximately 500000 cpm/ml of ‘251-1abe11ed fibrinogen with CaCl2 (final concentration 35 mM) and thrombin (final concentration 2NIH U/ml). The solution was immediately drawn into Silastic tubing (inner diameter 4mm) and incubated for 1 h at 37°C. The tubing containing the clotted plasma was then cut into 0.5cm sections, yielding clots of about 0.06ml. The clots were removed from the tubing and washed in 0.05M Tris-HC1 buffer, pH 7.4, containing 0.038M NaCl and 0.01% Tween SO. Lysis of 0.06ml PPP or PRP clots, submerged in 0.5 ml titrated normal human plasma, with different concentrations of STAN (final concentration 0.075 ‘to 10 P&ml), streptokinase (final concentration 0.16 ‘to 20 &ml), rt-PA (final concentration 0.02-10 kg/ml) or tcu-PA (final concentration 0.12-20 p.g/ml) was measured after 2h incubation at 37°C from the release of radioactivity in the surrounding plasma, as described elsewhere. ” In order to study the effect of clot retraction on lysis, PPP and PRP clots were prepared after addition of the platelet GPIIb/IIIa inhibitor G-4120 to a final concentration of 10 ug/ml. The effect of platelets on the lysis of human plasma clots by STAN or streptokinase was quantitated using clots prepared ;as

described above, from plasma with different platelet counts. In order to study the effect of mechanical compression on clot lysis, serum was expressed from ‘*‘Ifibrin labelled PPP clots: prepared from pooled normal human plasma, as described elsewhere.” Mechanically compressed human plasma clots were washed as described above, and then submerged in human plasma. Clot lysis with STAN (final concentration 0.1610 kg/ml) or with streptokinase (final concentration 0.32-20 pg/ml) was measured as described above. Residual fibrinogen levels after incubation for 2h at 37°C were determined as described above. Binding of Plasminogen Plasma Clots

and clr2-antiplasmin to

In separate experiments, “‘I-labelled plasminogen (about 500000 cpm/ml) or ‘251-labe11ed cw2-antiplasmin (about 250000 cpm/ml) were added to pooled human plasma or to PPP or PRP from individual donors, before clotting (without addition of 12’1labelled fibrinogen). After incubation for 1 h at 37°C and extensive washing of the clots, the amount of ‘2sI-plasminogen or ““I-cwz-antiplasmin associated with the clots was determined by radioisotope counting. Binding of ‘2’I-labe11ed plasminogen or cr2-antiplasmin to PPP or PRP clots made in the presence of G-4120 was also quantitated. RESULTS Complex Formation with Plasminogen

of STAN and STAR Moieties

Figure 1 shows that in mixtures of plasminogen with a 5-fold molar excess of either streptokinase or STA moieties, the active site as monitored with the chromogenic substrate S-2251, was rapidly exposed. Under the experimental conditions used, plasminogen, STAN, STA-CM-I, STA-CM-II, STAR-C or streptokinase alone did not react with S-2251. The 5-fold lower amidolytic activity observed with the plasminogen-STA complexes than with the plasminogen-streptokinase complex, is mainly due to a lower reactivity of the former with S-2251 (data not shown), as reported previously.7.8 Activation

of Plasminogen

Figure 2 shows that time-dependent activation of plasminogen (final concentration 1.5 ~.LM)by STAN, STA-CM-I, STA-CM-II, STAR-C and streptokinase (final concentration 2nM), as monitored by quantitation of generated plasmin with S-2251 was very similar. Under the conditions used, and in the absence of glycerol, about 50% of the plasminogen is activated in 30 min with all plasminogen activator moieties tested.

218

Biochemical

Properties

of Natural

and Recombinant

Staphylokinase

vation rate of plasminogen by plasminogen-STA complexes as well as by plasminogen-streptokinase complex (Fig. 3). At saturating concentrations of CNBr-digested fibrinogen, stimulation of the initial activation rate of plasminogen was 2- to 2.5-fold for the plasminogen-STA complexes and 1.3-fold for the plasminogen-streptokinase complex.

400

300

200

Inhibition of Plasminogen-STA antiplasmin

Complexes

by (YZ-

Semilogarithmic plots of residual plasminogen-STA complex activity as a function of time, following incubation of preformed complexes with a2-antiplasmin under pseudo first-order kinetic conditions, were linear (not shown). The apparent second-order rate constant (k’ .;,np) for the inhibition of 5 nM plasminogen-STA complex by 25 nM cuz-antiplasmin (meantSD of n determinations) was 2.1+0.26x 10” M-’ s-’ (n=lO) for STAN, 1.8*0.14x10” M-’ s-’ (n=lO)forSTA-CM-I,1.9~0.16~10”M-’s-’(n=7) forSTA-CM-IIand2.0~0.16~10~M-‘s~’(n=8)for STAR-C.

60

40

20

0 0

2

4

Time

6

8

10

12

(min)

Fig. 1 Generation of active sites in mixtures of plasminogen with a 5-fold molar excess of STAN (O), STA-CM-I (V), STA-CMII (+), STAR-C (m) or streptokinase (A). Amidolytic activity was measured wth S-2251 (final concentration 1 mM) after 25. fold dilution of the samples. The data represent meanfSEM of three independent determinations.

Kinetic analysis revealed that activation of plasminogen to plasmin by plasminogen-STA complexes as well as by plasminogen-streptokinase complex follows Michaelis-Menten kinetics, as revealed by linear double reciprocal plots of the initial activation rates versus the plasminogen concentration (not shown). The kinetic constants, obtained by linear regression analysis, were (mean*SEM of 3 determinations with r>O.99) K,=8.1+-0.10 FM and k2=1.4?0.14s-’ for plasminogen-STAN, K,=7.7*1.2pM and k2=2.4&0.27s-’ for plasminogen-STA-CM-I K,,=6.2?1.OpM and kz= 1.7-tO. 15 s- ’ for plasminogen-STA-CM-II and K,=7.3+0.20kM and k2=1.5&0.21 s-’ for plasminogen-STAR-C, as compared to K,=0.69?0.12kM and k2=0.69+0.17s-’ for plasminogen-streptokinase. The catalytic efficiency (kdK,) of the plasminogen-streptokinase complex thus is about 3- to 5-fold higher than that of the plasminogen-STA complexes (1.0 FM-’ ss’ and 0.17-0.31 FM-’ s-’ respectively).

Fibrinolytic and Fibrinogenolytic Properties of STAN and STAR Moieties in a Human Plasma Milieu In vitro Dose- and time-dependent lysis of “‘I-fibrin labelled human plasma clots submerged in human plasma was obtained in all experiments with STAN, STA-CM-I, STA-CM-II, STAR-C and streptokinase (Fig. 4). Spontaneous clot lysis during the experimental period was always <5% (not shown). Equi-effective concentrations of test compound (causing 50% clot lysis in 2 h; Cs,,), determined graphically from plots of clot lysis at 2h versus the concentration of plasminogen activator (not shown), were 0.19-0.35 Fg/rnl for the STA moieties as compared to 2.7 trg/rnl for

0

5

10

15

20

25

30

35

Time (min) Effect of CNBr-digested Fibrinogen on Plasminogen Activation with STAN and STAR Moieties Addition of CNBr-digested fibrinogen resulted concentration-dependent increase of the initial

in a acti-

Fig. 2 Activation of plasminogen (final concentration I .S FM) as a function of time by STAN (0). STA-CM-I (V), STA-CM-II (+), STAR-C (m), and streptokinase (A) (final concentration 2nM each). The data represent mean*SEM of three independent determinations.

Fihrinolysis

0.00

[CNBr-digested Fig. 3 Effect of CNBr-digested

fibrinogen] fibrinogen

streptokinase (3- to 4-fold lower on a molar basis) (Table 1). Fibrinogen and al-antiplasmin levels during the 4h observation period are also shown in Figure 4. More extensive systemic fibrinolytic activation is observed with streptokinase than with the STA preparations. Residual fibrinogen and cw2-antiplasmin levels after 2h at Cso (determined graphically from dose-response curves, not shown), were not significantly decreased with the STA preparations (>70%), but were virtually depleted with streptokinase (Table 1). No significant differences were observed between the different STA moieties. The concentrations of test compound causing 50% fibrinogen degradation in 2h in normal human plasma in the absence of fibrin were determined graphically from dose-response curves (not shown). These values were (mean+SEM of 3 independent experiments) 8.1fl.5 pg/ml for STAN, 6.1k1.2 kg/ml for STA-CM-I, 6.7kO.2 kg/ml for STA-CM-II and 9.5k1.6 kg/ml for STAR-C, as compared to 0.16-tO.01 kg/ml for streptokinase. Thus, lOO- to 150-fold (on a molar basis) higher concentrations of the STA moieties as compared to streptokinase, are

Qn l.CU

0.80

0.40

(PM)

on plasminogen

I .OPM) by equimolar complexes activation (final concentration of plasminogen with STAN (O), STA-CM-I (v). STA-CM-II (+). STAR-C (W) or streptokinase (A) (1.2 nM final concentration each). The increase over the control value in the initial activation rate of plasminogcn (v) is plotted versus the concentration of CNBr-digested fibrinogen. The data represent meanfSEM of three independent determinations.

III

Time (hour*)

Tlmr

(hour*)

Tlmr

219

IV

(hours)

Time

V

(hourr)

Tlmr

(houra)

Fig. 4 Lysis of human “I-fibrin lahelled plasma clots submerged in human plasma following addition of different concentrations of STAN (panel I), STA-CM-I (panel II). STA-CM-IT (panel III), STAR-C (panel IV) or streptokinase (panel V). Clot lysis was monitored over 4 h from the release of ‘Z’I-lahelled fibrin degradation products, and is expressed in percent. The evolution of fibrinogen and cr>-antiplasmin levels over 4h. expressed in percent of the hascline value, is also shown. Concentrations of plasminogen activator used are: (O), 0.05 &ml; (O), 0.10 (*g/ml; (v). 0.20 &ml; (+). 0.40 &ml; (B), 0.80 &ml or (A), 1.6 pg/ml for the STA preparations and (0). 0.30 &ml: (0). 0.60 &ml; (r). 1.2 &ml; (+), 2.4 p&‘/ml; (D). 4.8 kg/ml or (A) 9.6 pg/ml for streptokinase. The data represent mean+SEM of three independent experiments.

220

Biochemical

Properties

of Natural

and Recombinant

Staphylokinase

IIA

-

op ,.,,..,, ~,,.I.. ,.,,,.,, 1

0.1

CONCENTRATION

10

0.1

10

(pg/ml)

IIB

IB 100

1 CONCENTRATION

(pg/ml)

-

v-‘=‘pp-o

‘$1

7 I V

0 \

\

\2

’

?? -.-.-&L-.-.-.--r 1

0.1

.

.

,

.

0.1

10

CONCENTRATION

CONCENTRATION

(&ml)

I

.

1

,

P

.

.

I

,

L

10 (,,g/ml)

Fig. 5 Iysis of ‘251-fibrin labelled PPP (panels A) or PRP (panels B) clots in human plasma by STAN (0). streptokinase (O), rt-PA (V) or tcu-PA (v). In Panels IA and IB, percent clot lysis after 2h is plotted against the concentration of test compound. In panels IIA and IIB, residual fibrinogen levels after 2h, expressed in percent of the baseline value, are plotted against the concentration of test compound. The results represent mean+SEM of three independent experiments with plasma from different donors.

required to cause 50% fibrinogen degradation. significant differences were observed between different STA moieties.

No the

Effect of Clot Retraction on the Lysability of “‘1. fibrin Labelled Human Plasma Clots with STAN in a Plasma Milieu In vitro STAN, streptokinase, rt-PA and tcu-PA induced dose-dependent lysis of PPP clots in normal human plasma (Fig. 5, panel IA). The concentrations required for 50% clot lysis in 2 h (C&r) (Table 2) were

Table 1 Comparative in vitro

fibrinolytic

properties

of STAN,

STA-CM-I,

0.37 pg/ml (24nM) for STAN, 2.1 &ml (47nM) for streptokinase, 0.18 pg/ml(2.6nM) for rt-PA and 0.90 pg/rnl (17 nM) for tcu-PA. Residual fibrinogen levels after 2h at these equi-effective concentrations (Fig. 5, panel IIA) were 95,
STA-CM-II,

STAR-C

and streptokinase

in a human

plasma

milieu

Test compound

Cw (&ml)

Residual fibrinogen (percent)

Residual az-antiplasmin (percent)

STAN STA-CM-I STA-CM-II STAR-C Streptokinase

0.29f0.02 0.19f0.04 0.2.5f0.03 0.35f0.03 2.750.32

82+2 70*1 82fl 81f2 1322

8923 7324 81?6 89f5 2826

C5,, represents a,-antiplasmin determinations.

the concentration of test compound levels after 2 h at C
required to obtain 50% clot lysis within 2h. The residual fibrinogen and in percent of the baseline value. The results are meanfSEM of three independent

Fibrinolysis

Table 2 Lysis of ‘251-tihrin labelled plasma clots 5ubmergcd

in human plaam,r bv dittcrent

221

test compounds

CW”

Fibrinogen” (percent)

(tq$nl) Test compound

PPP clots

PRP clots

PPP clots

PRP clots

STAN Streptokinase rt-PA tcu-PA

0.37z!c0.02 2. I fO.13 0.18to.01 0.90i0.08

0.5x-to.07 >20 0.22~0.02 >20

951-3
92*2 92+3

The data are mean*SEM of 3-6 determinations for clot lysis and of 3 determinations for fibrinogen levels. usrng plasma from different donors. ” C5r,: Concentration of test compound required to obtain 50% clot Iysts within 2h of PPP clots or of PRP clots submerged in normal human plasma. ” Residual fibrinogen levels after 2h. at C.v,. expressed as percent of the baseline value.

(Fig. 5, panel IB), and depletion of fibrinogen occurred in the absence of significant clot lysis (Fig. 5, panel IIB). G-4120, which prevented clot retraction, did not significantly affect lysis of PPP (<5X 101 platelets/p]) or PRP (4x 10’platelets/k]) clots by STAN (Fig. 6A). The concentrations required for 50% clot lysis in 2h (Cso) were (mean+SEM; n=3 with the same 3 individual blood donors) 0.46-tO.05 kg/ml and 0.41~0.03 pg/ml (p=O.42) for PPP clots with and without addition of G-4120 respectively, as compared to 0.66+0.09 kg/ml and 0.6OkO.16 pg/ml (p=O.76) for non-retracted (with G-4120) or retracted PRP clots. In agreement with the results described above, streptokinase was very inefficient towards retracted PRP clots (Fig. 6B). However, Cso values for streptokinase were not significantly different (mean+SEM of 3 experiments with the same individual donors) for PPP clots without or with G-4120 (1.9kO.22 or l.SkO.26 )*g/ml, respectively, p=O.85), or for nonretracted (with G-4120) PRP clots (3.1kO.63 pg/ml; p=O.13 versus PPP clots with G-4120). At high concentrations of STAN (3 10 times its Cso value), lysis of PRP clots (4x10” platelets/lJ) decreased with increasing concentration of the compound (Fig. 5B). A similar phenomenon was observed with rt-PA. With tcu-PA, lysis of PRP clots did not exceed a maximum of 40% at 1 l+jrnl and also decreased at higher concentrations. Doubling of the plasminogen concentration in normal plasma by addition of purified human plasminogen (200 kg/ml plasma) before addition of the PRP clot, resulted in enhanced lysis of PRP clots with lOpg/ml streptokinase (33+3 vs 18&3%; p=O.O2) but not with lOl.~g/ml STAN (41*2 vs 41*5%). Addition of plasminogen (200 t_@ml) to the PRP before clotting, resulted in PRP clots enriched in plasminogen (10.4 vs 5.Op.g plasminogen per 0.06ml clot). Lysis of such plasminogen-enriched PRP clots in normal human plasma was significantly enhanced both with STAN (89+2%) and with streptokinase (92&2%) (both p
Binding of Plasminogen Plasma Clots

and cu2-antiplasmin to

In separate experiments, the amount of “‘I-1abelled plasminogen or ‘251-labelled cY*-antiplasmin was determined which remained associated with plasma

A

loo

t?

40

2

I

,,,I

0.1

1

CONCENTRATION

loot

(,q/mi)

1

B

CONkENTRATION (pi/ml)

10

Fig. 6 Effect of clot retraction on the lysis with STAN (panel A) or streptokinase (panel B) of tLI-fibrin labelled human plasma clots in a plasma milieu in vitro. Percent clot lysis after 2h, is plotted against the concentration of test compound, for PPP clots without (0) or with (0) addition of G-4120 and for PRP clots (4x IO’ platelets/PJ) without (V) or with (V) addition of G-4120. The results represent meanfSEM of three independent experiments with plasma from different donors.

222

Biochemical

Properties

of Natural

and Recombinant

Staphylokinase

1.5~ 10” to 6.0~ lo5 platelets/tJ), whereas clot lysis by streptokinase is progressively impaired at higher platelet counts (from 0.4X105-3.0X lo5 platelets/kl). When residual clot lysis is plotted as a function of the platelet count (Fig. SC), the efficacy for clot lysis decreases to 50% at a platelet count of 1.4~ 105/pl for

100

1

60

40

L

20

0

-

//

STAPHYLOKINASE 10

kg/ml

”

0.1

STREPTOKINASE 10 &ml

Fig. 7 Contribution of the plasminogen content of the plasma or of the PRP clot to clot lysis with STAN or streptokinase. The open bars represent lysis after 2 h of PRP clots in normal plasma with lOug/ml STAN or streptokinase (mean+SEM, n=6). The filled bars represent lysis of PRP clots in normal plasma supplemented with 200 &ml plasminogen (meanfSEM, n=3). The hatched bars represent lysis of PRP clots prepared from PRP plasma supplemented with 200 &ml plasminogen and submerged in normal plasma (meanfSEM. n=6). The data are meanfSEM of 3 or 6 determinations with PRP plasma from different donors. (*) or (**), ~~0.02 or ptO.OO1 versus lysis of PRP clots in normal plasma.

1

CONCENTRATION

OF

STAPHYLOKINASE

(fig/ml)

100

OC 0.1

clots prepared in the same way as for the clot lysis experiments. These values were (mean?SEM of 3-5 determinations, using plasma from different donors) for plasminogen 411!13 and 44+1.5% for PPP clots without and with G-4120, respectively (p=OSO), as compared to 29&3 and 45&3% for PRP clots without (retracted) and with (non-retracted) G-4120, respectively (p=O.Ol). Thus, in the absence of G-4120, binding of plasminogen to PPP clots was higher than to PRP clots (p=O.O2), whereas binding in the presence of G-4120 was not different (p=O.78). The amount of a2-antiplasmin associated with PPP clots was not significantly different from that associated with PRP clots (45+3 and 40+2%, respectively, p=O.19). Effect of Platelet Count on the Lysability of “‘Ifibrin Labelled Human Plasma Clots in a Plasma Milieu In vitro Both STAN (Fig. 8A) and streptokinase (Fig. 8B) induced dose-dependent lysis of 12’I-fibrin labelled plasma clots with different platelet counts, submerged in normal human plasma in vitro. The data show that clot lysis by STAN is not significantly affected by increasing platelet count in the plasma clot (from

10

1 CONCENTRATION

OF

STREPTOKINASE

(fig/ml)

r

10

1 PLATELET

COUNT

(x105/,d)

Fig. 8 Effect of platelet count on the lysis of “‘l-fibrin labelled human plasma clots in a normal plasma milieu in vitro, with STAN (panel A) or streptokinase (panel B). Percent clot lysis after 2 h is plotted against the concentration of test compound, for plasma clots with different platelet count. Panel A: (0), PPP; (O), 1.5~ IO’ platelets/kI; (V). 3.0x 10’ platelets/kI; (v), 4.5X105 plateletsipl; (n), 6.0X105 plateletslyl. Panel B: (0), PPP: (O), 0.4~10’ platelets/ul; (V), 0.75~10~ platelets/PI; (v), 1.5X10s platelets/uI; (O), 3.0X105 platelets/ul. Panel C represents residual clot lysis at 2 h as a function of the platelet count using an equi-effective concentration (causing 50% lysis of PPP clots in 2 h) of STAN (0) or streptokinase (0). The results represent mean*SEM of three independent experiments with plasma from different donors.

Fibrinolysis

D-

I

/

/ CONCENTRATION

-

10

1

0.1

-0

223

(pg/ml)

(v, V). compressed human plasma clots in human plasma by SI‘AN (0. 0) or strcptokinase Percent clot lysis after 2h (closed symbols) or residual fihrinogen Icvel~ after 2h. exprcsaed in percent of the baseline value (open symbols) is plotted versus the concentration of test compound. The results represent mean?SEM of 3 independent determinations for clot lqsis and mean values of 2 or 3 determinations for fibrinogen levels.

Fig. 9 Lysis of mechanically

streptokinase and at an extrapolated value of 13 x lo’/ f~.l for STAN (mean of 3 independent determinations).

Lysis of Mechanically Compressed Clots in a Plasma Milieu In vitro

Human Plasma

lysis of mechanically STAN induced dose-dependent compressed human plasma clots. submerged in normal human plasma (Fig. 9). 50% clot lysis in 2h (Cso) was obtained with (mean?SEM of 3 independent determinations) 0.34*0.03 &ml; the corresponding Cso value for normal PPP clots (prepared from pooled plasma) was 0.32 fJg/rnl (not shown). Residual fibrinogen levels after 2h at Csc, in the systems with mechanically compressed plasma clots or with PPP clots were >95%. At concentrations of STAN above 1.5 kg/ml, lysis of mechanically compressed clots decreased with increasing STAN concentration (Fig. 9). At a STAN concentration of lO~g/ml, lysis of mechanically compressed clots in human plasma was only 3211% , with residual fibrinogen levels of ~15% (Fig. 9) and residual plasminogen levels of approximately 35% (not shown). Streptokinase did not induce significant lysis of mechanically compressed plasma clots in human plasma (~10% lysis at 20&m]), whereas fibrinogen levels dropped to ~15% with a streptokinase concentration of 0.16 kg/ml (Fig. 9), and plasminogen levels to
DISCUSSION In order

to permit

investigation

of the thrombolytic

properties of STA we have isolated and characterised STAN and three molecular forms of STAR.’ STAN. STA-CM-I. STA-CM-II and STAR-C were indistinguishable in terms of their rate and extent of complex formation with plasminogen, and their plasminogen activating potential in the absence or the presence of a fibrin-like stimulator. The catalytic efficiencies (k,/K,,) for plasminogen activation were comparable for the four preparations of STA but were 3- to .5-fold lower than for streptokinase, mainly as a result of a higher K, value (K,,=fXyM for the STA moieties as compared to K,,=O.69kM for streptokinase). The inhibition rates of the plasminogen-STA complexes by cwz-antiplasmin were comparable for the four STA preparations, whereas the plasminogen-streptokinase complex is virtually not inhibited by cu2-antiplasmin.“4 In a human plasma milieu in vitro, equi-effective concentrations (Cjo; causing 50% clot lysis in 2 h) were, on a molar basis. 3- to 4-fold lower for STA than for streptokinase. whereas residual fibrinogen and (YZantiplasmin levels after 2h at Csc, were significantly higher. Furthermore, in the absence of fibrin, the STA moieties induced less systemic fibrinolytic activation in plasma than streptokinase, as evidenced by the lOO- to 150-fold higher concentrations of STA which were required to cause 50% consumption of fibrinogen. These functional properties of STAN and STAR moieties are comparable to those reported previously with STAR and confirm and extend the similarities and/or differences observed with streptokinase in previous studies.5-7 In the present study the fibrinolytic potency of STAN was also compared with that of streptokinase in an in vitro system consisting of 12”1-fibrin labelled PPP or PRP clots submerged in human plasma. It

224

Biochemical

Properties

of Natural

and Recombinant

Staphylokinase

that STAN has a comparable fibrinolytic appears activity versus PRP and PPP clots, whereas streptokinase is very inefficient towards PRP clots. When retraction of PRP clots was prevented by addition of G-4120, a GPIIb/IIIa antagonist,” PPP and PRP clots became comparably sensitive to lysis with streptokinase. A similar resistance towards streptokinase and tcu-PA, but not to rt-PA and scu-PA, was shown previously using retracted whole blood clots as well as This clots.” compressed plasma mechanically phenomenon was explained by an enhanced systemic plasminogen activation with the non-fibrin-specific agents, which precluded recruitment of plasminogen from the surrounding plasma and inhibition of clot lysis as a result of ‘plasminogen steal’.y~2” In agreement with this hypothesis, mechanically compressed plasma clots, prepared as described by Sabovic et al,” were found to be resistant to lysis by streptokinase but not by STAN. At concentrations causing 50% clot lysis in 2h, streptokinase caused extensive fibrinogen degradation and plasminogen consumption, whereas STAN was not associated with significant systemic fibrinolytic activation. The amount of plasminogen associated with PRP clots was significantly lower than with PPP clots, but was comparable in the presence of G-4120, whereas the amount of a2-antiplasmin associated with PRP clots (possibly covalently bound via Factor XIII crosslinking) was not significantly different from that associated with PPP clots. These findings suggest that the differential response of STAN and streptokinase toward platelet-mediated clot retraction may be related to extrusion of plasminogen from the clot, with alteration of the plasminogen/a2-antiplasmin ratio. Interestingly, at high concentrations of the fibrinspecific agent STAN, lysability of PRP clots decreased again with increasing concentration of plasminogen activator. A similar phenomenon was observed with PRP clots and t-PA (Fig. 5, panel IB) and with mechanically compressed plasma clots and STAN (Fig. 9). As shown in Figure 7, this phenomenon could be reversed by doubling of the plasminogen concentration in the PRP clot, but not by doubling of the plasminogen concentration in the surrounding plasma. Possibly at high concentrations of STAN (10 kg/ml equals 670nM) insufficient fibrinbound plasminogen is available to produce clot lysis. This phenomenon, however, was only observed at STAN concentrations which were 310 times the concentration required for complete lysis of PRP clots in 2h, and thus may not be relevant for thrombolysis at therapeutic doses of STA. In conclusion, the plasminogen activating potential and fibrinolytic properties of STAN and STAR were found to be indistinguishable. STAR thus appears to be a suitable and convenient source for large scale production of the protein, and for more detailed evaluation of its thrombolytic potency in vivo. Furthermore, the findings of the present study pro-

vide a mechanism for the differential sensitivity PRP clots to lysis with STA and streptokinase human plasma in vitro.

of in

ABBREVIATIONS PPP: platelet-poor plasma PRP: platelet-rich plasma t-PA: tissue-type plasminogen activator rt-PA: recombinant t-PA u-PA: urokinase-type plasminogen activator scu-PA: single chain u-PA tcu-PA: two chain u-PA, urokinase Other abbreviations, see accompanying paper

(ref. 1)

REFERENCES I

2

3

4

5

6 7

8

9

IO.

Il.

12.

13.

14. 15.

16.

Collen D. Silence K. Demarsin E, De Mol M, Lijnen H R. Isolation and characterization of natural and recombinant staphylokinase. Fibrinolysis 1992; 6: OOt~OOO. Collen D, De Cock F, Vanlinthout I, Declerck P J. Lijnen H R, Stassen J M. Comparative thromholytic and immunogenic properties of staphylokinase and streptokinase. Fibrinolysis 1992; 6: OO(~OOO. Kowalska-Loth B. Zakrzewski K. The activation bv staphylokinase of human plasminogen. Acta Biochym Pol 1975: 22: 327-339. Ericson R. The mechanism of the activation of plasminogen by staphylokinase. Chem Abstr 1977; 86: 202 (abstract 673862). Sakai M, Watanuki M, Matsuo 0. Mechanism of fibrinspecific fibrinolysis by staphylokinase: participation of cyzplasmin inhibitor. Biochem Biophys Res Commun 1989; 162: 83(&S37. Matsuo 0, Okada K, Fukao H et al. Thrombolytic properties of staphylokinase. Blood 1990; 76: 925-929. Lijnen H R, Van Hoef B, De Cock F et al. On the mechanism of fibrin-specific plasminogen activation by staphylokinase. J Biol Chem 1991; 266: 11826l1832. Lijnen H R, Van Hoef B, Matsuo 0. Collen D. On the molecular interactions between plasminogen-staphylokinase. cxz-antiplasmin and fibrin. Biochim Biophys Acta 1992; 1118: 144-148. Sabovic M, Lijnen H R, Keber D, Collen D. Effect of retraction on the lysis of human clots with fibrin specific and non-fibrin specific plasminogen activators. Thromb Haemost 1989; 62: 1083-1087. Nelles L, Lijnen H R, Collen D, Holmes W E. Characterization of recombinant human single chain urokinase-type plasminogen activator mutants produced by site-specific mutagenesis of Lys”‘. J Biol Chem 1987; 262: 5682-5689. Lijnen H R, Van Hoef B. Collen D. Comparative kinetic analysis of the activation of human plasminogen by natural and recombinant single chain urokinase-type plasminogen activator. Biochim Biophys Acta 1986; 884: 402-408. Astrup T, Muellertz S. Fibrin plate method for estimating fibrinolytic activity. Arch Biochem Biophys 1952; 40: 346 351. Deutsch D G, Mertz E T. Plasminogen: purification from human plasma by affinity chromatography. Science 1970; 170: 1095-1096. Wiman B. Affinity-chromatographic purification of human alpha-2-antiplasmin. Biochem J 1980; 191: 229-232. Verheijen J, Mullaert E, Change G T G, Kluft C, Wijngaards G. A simple, sensitive spectrophotometric assay for extrinsic (tissue-type) plasminogen activator applicable to measurement in plasma. Thromb Haemost 1982; 48: 266-269. Zamarron C, Lijnen H R, Collen D. Kinetics of the activation of plasminogen by natural and recombinant

Fibrinolysis

17.

1x.

19.

20.

21.

tissue-type plasmlnogen activator. J Biol Chem 1984; 259: 2080-2083. Barker P L, Burnier J P. Gadeh T, Thorsett E D. Small cyclic peptide aggregation inhibitors. PCT Int Appl WO91. 01331 (7 Feb. 1991). Gaffney P J. Curtis A D. A collaborative study of a proposed international standard for tissue plasminogen activator (t-PA). Thromh Haemost 1985; 53: 134136. Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Bochem 1976; 72: 248-254. Fraker P .I. Speck Jr J C. Protein and cell membrane iodinations with a sparingly soluble chloroamide 1,3.4,6tetrachloro-3a,ha-diphenylglycoluril. Biochem Biophys Res Comm 197X; 80: 849-857. Clauss A. Gerinnungsphysiologische Schnellmethode zur Bestimmung des Fibrinogens. Acta Haemat 1957; 17: 237246.

Received: 23 March 1992 Accepted after revision: 15 May 1992 Offprint orders to: D. Collen, Center for Thrombosis and Vascular Research. University of Leuven, Campus Gasthuisberg, 0 & N. Herestraat 49, B-3000 Leuven. Belgium. Tel: 32-16-215772. Fax: 32-16-215990.

225

22. Edy J. Collen D. Verstraete M. Quantification of the plasma protease inhibitor antiplasmin with the chromogenic substrate S-2251 In: Davidson J F et al. eds. Progress in chemical fibrinolysis and thrombolysis. Vol. 3. New York: Raven Press, 197X; 315-322. 23. Friberger P, Knos M. Plasminogen determination in human plasma. In: Scully M F, Kakkar V V. eds. Chromogenic peptide substrates. Edinburgh: Churchill Livingstone, 1979; 128-140. 24. Cederholm-Williams S A. De Cock F. Lijnen H R. Collen D. Kinetics of the reactions between streptokinase, plasmin and a,-antiplasmin. Eur J Biochem 1979; 100: 125-132. 25. Sobel B E, Nachowiak D A, Fry ET A, Bergmann S R. Torr S R. Paradoxical attenuation of fibrinolysis attributable to ‘plasminogen steal’ and its implications for coronary thrombolysis. Coron Art Dis l9YO; 1: 11 I-119.