IIIa

THROMBOSIS RESEARCH 67; 711-719,1992 0049-3848192 $5.00 + .OOPrinted in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.

INTERACTIONS BETWEEN THROMBOLYTIC AGENTS AND PLATELETS: EFFECTS OF PLASMIN ON PLATELET GLYCOPROTEINS lb AND Ilblllla

J.J.M.L. Department

HOFFMANN

and W.C.M.

of Clinical Laboratories, Haematology P.O. Box 1350, 5602 ZA Eindhoven,

JANSSEN Division, Catharina the Netherlands

Hospital,

(Fieceived 6.4.1992; accepted in revised form 22.7.1992 by Editor W. Nieuwenhuizen)

ABSTRACT The mechanisms by which thrombolvtic agents affect platelet function are not vet elucidated. The aim of the present studv was to investigate the effects of plasmin, generated by thrombolytic agents in plasma, on platelet glycoproteins (GP) lb and Ilblllla. Platelet-rich plasma was incubated with pharmacological amounts of streptokinase, anistreplase and tissue-type plasminogen activator and the platelet surface GP’s were investigated with a panel of monoclonal antibodies using ffow cytometry. As assessed from the mean fluorescente intensity of incubated and control platelets, no significant changes in the binding of antibodies to GP lb and GP Ilb/llla were found. The functional integritv of these glycoproteins was severelv impaired by treatment with the thrombolytic agents, as shown by significant inhibition of ADP- and ristocetin-induced platelet aggregation. Experiments with purified plasmin and washed platelets indicated significant degradation of GP Ilblllla and upregulation of GP lb, which is in agreement with previous findings. In addition, platelet activation by plasmin was shown using two monoclonal antibodies activation-specific antigens. We conclude that degradation of platelet GP’s plasmin offers no likely explanation for the defect in platelet function, which induced

bv thrombolvtic

agents

in platelet-rich

to by is

plasma.

INTRODUCTION Plasminogen activators used clinically as thrombolytic drugs will convert plasminogen to plasmin and the dosages used generate so much plasmin that the inhibiting potential of az-antiplas,min and other protease inhibitors is surpassed. This results in the so-called systemic lytic state and platelets are known to be affected by the systemic lytic state. Plasmin can inhibit platelet aggregation in platelet rich plasma (PRP) in vifro (1,2) and recently we have shown that plasimin generated after the administration of anistreplase to patients caused a marked decrease in ex vivo platelet aggregation, which lasted for about 12 hours (3). The mechanism by which platelet function is inhibited is not precisely known. Three possible explanations can be propo$ed: extensive depletion of plasma fibrinogen, competition between fibrin(ogen) degradation prodwcts and intact fibrinogen for binding sites on the platelets and finally, degradation of platelet

I
anistreplase aggregation

- flow cytometry - streptokinase.

glycoprotein

711

plasminogen

activator

- platelet

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PLASMIN AND PLATELET RECEPTORS

712

membrane receptor proteins (4). Some of the glycoproteins on the platelet surface are necessary for adequate platelet function. Among them are glycoprotein lb (GP lb), the receptor for the von Willebrand factor and GP Ilb/llla, the fibrinogen receptor. It has been shown in vifro that plasmin can degrade GP lb on washed platelets (5-7), but it is questionable whether this plasmin-induced proteolysis occurs also in PRP (6,8). Recently, evidente has been presented that GP lb remains intact after in vivo administration of thrombolytic drugs (8.9). Concerning the effect of plasmin on GP Ilb/llla the available data are highly controversial: decrease, increase and no variation in this GP complex have all been reported (5,6,8,10-12). The aim of the present study was to investigate the effects of thrombolytic agents on platelet surface antigens as defined by different monoclonal antibodies. We used incubations of PRP with pharmacological concentrations of three thrombolytic drugs, as wel1 as treatment of washed platelets with plasmin. Binding of the monoclonal antibodies to the surface GPs was visualized using fluorescente flow cytometry.

MATERIALS

AND

METHODS

Materials Purified human plasmin was purchased from Sigma, St.Louis, MO, USA. The thrombolytic drugs were clinical grade: streptokinase (Kabikinase”) from KabiVitrum, Amsterdam, the Netherlands; anistreplase (APSAC; Eminase’) was a gift from SmithKline Beecham Farma, Rijswijk, the Netherlands and rtPA (Actilyse@) was donated by Dr. Karl Thomae GmbH, Biberach, FRG. Aprotinin (Trasylo?; 10,000 KIU/ml) was from Bayer AG, Leverkusen, FRG. The monoclonal antibodies were obtained from the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam (thromb/l, thromb/7, thromb/8, gran/l2), from Dako AS, Glostrup, Denmark (Y2/51 and AN51), and Immunotech SA, Marseille, France (thromblô). The specificity and references of these antibodies are given in Table 1. With the exception of AN51 and thromb/6 antibodies, which were directly labelled with fluorescein isothiocyanate (FITC), the antibodies were in the purified, unconjugated form. We used Ffab’), fragments of goat anti-mouse IgG, FITC-conjugated, as the second antibody (Caltag Laboratories, South San Francisco, CA, USA). For platelet aggregation we used adenosine diphosphate (ADP; Sigma)

and ristocetin

Blood collection Blood was drawn antecubital

vein

(Lundbeck,

between after

short,

Copenhagen,

Denmark).

8 and 10 a.m. from minimal

stasis.

healthy

The first

volunteers few

through

millilitres

were

a clean puncture discarded.

The

of an blood

was collected into plastic syringes or siliconized glass tubes containing trisodium citrate (0.01 M final concentration). Platelet rich plasma was prepared by low-speed centrifugation (180 g for 5 min at 21 “Cl and platelet poor plasma bv centrifugation at 3000 p for 15 min. The platelet count of PRP was set to 200/nl with platelet poor plasma. Platelets were washed in phosphate buffered saline (PBS; 150 mM NaCI and 0.01 M phosphate, pH 7.4), containing 20 mM EDTA and resuspended in the same buffer. When using the AN51 monoclonal antibody, an additional aliquot was washed and resuspended in PBS without EDTA. Incubations Platelets in PRP were treated with the thrombolytic drugs, using concentrations equivalent to those attained therapeutically: SI< 300 Ulml, APSAC 10 mU/ml (about 70 nM) and tPA 5 pg/ml (al1 final concentrations). Solutions of drug were added to PRP (200 platelets /nl) and incubated for 90 min at 37 “C. At the end of the incubation, aprotinin was added (final concentration 250 KIU/ml) in order to inhibit ongoing plasmin activity. In parallel to the experiments with active drugs, control incubations were carried out using saline. In some experiments, samples of whole blood anticoagulated with citrate or heparin (10 Ulml) or PRP prepared from the latter were tested. Washed platelets (200/nl), suspended in PBS-EDTA, were treated with plasmin for 90 min at 37 ‘C and the incubation terminated by adding aprotinin (1000 KIU/ml). Then the platelets were washed and stained as described below. The generation of plasmin by SI< in PRP was

Vol.

67, No. 6

measured using purified plasmin.

PLASMIN

the

chromogenic

AND

substrate

PLATELET

S-2251

TABLE

RECEPTORS

and compared

713

to a calibration

(clone)

AN51

CD number

using

1

Specificity of the Monoclonal Antibodies with Designation of Clusters Differentiation (CD Number) according to the International Workshop on Human Leukocyte Differentiation Antigens (13). Antibody

curve

of

Antigen

Reference

42b

GP lb

61

GP Illa in the GP ilb/llla

61

GP Illa (nat complex-specific)

16

41

GP llb/llla

complex

17

thrombi6

62

GMP-140

on activated

platelets

gram1 2

63

lysosomal

p53 antigen

on activated

thromb/8

9

thromb/l

(Cl 7)

Y215l thromb/7

(6C9)

14 complex

15

(PADGEM) platelets

p24 antigen

18 19 20

lmmuno fluorescente Incubated platelets were washed twice and divided into 50 ul aliquots containing 10’ platelets each. Each aliquot was incubated with 50 vl of a saturating amount of monoclonal antibody for 30 min at 4 “C. In order to prevent platelet activation by the antibodies, a mixture of citrate, theophylline, adenosine and dipyridamole (CTAD) was added together with the antibody. For indirect staining, the platelets were washed twice and incubated with 50 ~1 of a 1 :30 dilut,ed solution of FITC-labelled goat anti-mouse IgG, also for 30 min at 4 ‘C and then suspended into 0.5 ml PBS for measuring. In experiments with directly labelled monoclonal antibodies, the platelets were suspended in 0.5 ml PBS immediately after the incubation. For measuring platelet-bound fluorescente, we used a FACScan flow cytometer (BectonDickinson, Mountain View, CA, USA), which was operated according to the instructions of the manufacturer. The optica1 system was calibrated with fluorescent microbeads (Calibrite beads; Becton-Dickinson)

and checked

daily.

The fluorescente

measurement

was

confined

to platelets

by means of electronic gating on their forward and side scatter signals. This procedure had been validated before and indicated that over 99% of the events within this gate reacted with plateletspecific antibodies and were negative with a red celi specific monoclonal antibody. In each sample at least 10,000 platelets were counted and their fluorescente signal was recorded with logarithmic amplification. Signal analysis was accomplished with the use of the Facscan Research software (Becton-Dickinson), The fluorescente signal was expressed as the mean fluorescente intensity (MFI; in Channel number units). Platelet aggregation PRP was treated with each of the thrombolvtic drugs and a saline control, as described above, and aggregation measured in an optica1 aggregometer (PAP-4; Biodata, Hatboro, PA, USA). The minimum and maximum levels of light transmission were set using PRP and platelet poor plasma, respectively. To 0.45 ml of treated PRP we added 50 vl of inducer solution, giving final concentrations of 3.6 PM ADP or 1.5 mg/ml ristocetin.

714

Vol. 67, No. 6

PLASMIN AND PLATELET RECEPTORS

Statistics Results are given as mean values < 0.05 significant.

* SD. The paired

t-test

was

used for comparing

data,

considering

p

RESULTS Effects of thrombolytic drugs in PRP Platelets from healthy subjects were treated in PRP with pharmacological concentrations of the thrombolytic agents and then stained with monoclonal antibodies to GP lb, GP Ilblllla and some other surface antigens (Table 1). None of the three thrombolytic drugs caused significant changes in the density, expressed as MFI, of the epitopes recognized by the antibodies, as shown in Table 2. Subpopulations of platelets with higher or lower fluorescente intensity than the main population never emerged after treatment with the thrombolytic drugs. When testing platelets every 15 min during the incubation period of 90 min, no changes in MFI were observed, either. Under these conditions 0.9 2 0.05 CU/ml plasmin was generated (n =6). Even when PRP was treated with a 25,000 Ulml SK, which is about lOO-fold the maximum therapeutic concentration, or with plasmin (up to 2.0 CUlml) there were no significant changes in MFI (data not shown). The expression of the activation markers thrombl6 and gran/l2 did not increase during incubation with any of the thrombolytics (Table 2). indicating no substantial activation by these drugs. Finally, there was no change in the intensity of the thromb/8 which was used as a control.

300

platelet antigen,

\ .,.-.__ __._......_.

1 __.._._______.___.......~-

. . &trol

2004 0

0.01

0.05 0.1

0.5 1.0 2.0 Plasmin (CU/ml)

FIGURE Effect of increasing concentrations GP Ilb/llla epitopes (thromb/7; irrelevant monoclonal antibody.

1

of plasmin on the MFI of GP lb (AN 511 and two Y2/5ll; n = 2-4. Dotted line: control using an

Effects of thrombolytic drugs in whole blood In order to investigate whether the presence of erythrocytes or calcium ions had any influence on the effect of thrombolytic agents on platelet antigens, incubations were performed using citrated whole blood, heparinized whole blood and heparinized PRP as substitutes for citrated PRP. The results were identical to those presented above for citrated PRP (data not shown). Effects of plasmin on washed platelets Washed platelets were treated with increasing concentrations of plasmin for 90 min and then stained with monoclonal antibodies. Typical examples are given in Fig 1 for anti-GP Ilblllla (thrombl7 and Y2l51) and anti-GP lb (AN51). lt appeared that significant changes were only

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PLASMIN AND PLATELET RECEPTORS

715

obtained when using 2 1 .O CU/rnl plasmin (P
the

subsequent

Figure 2 shows that the GP Ilb/llla epitopes recognized by the antibodies thromb/l and thromb/7 nearly completely disappeared, while the Y2/51 epitope on the same molecule remained intact. The expression of the GP lb epitope AN51 did not significantly change. The activation antigens thromb/ô

and gram1 2 were

clearly

discovered

(Fig 2).

TABLE

2

Effect of Treatment of Platelets in PRP with SI< (300 U/ml), APSAC (10 mU/ml) and tPA (5 pg/ml) on the Fluorescente Intensity (MFI) of Antibodies Binding to Platelets. PRP incubated with Saline served as a Control. Mean * SD is given In = 8). MFI (Channel) Antibody

control

AN51

SI<

APSAC

tPA

256

î 17

258

i 18

252

i 17

266

zt 24

438

* 15

427

* 17

426

* 13

433

t 26

thrombil

489

* 18

495

zt 25

492

* 22

506

f 38

Y2151

506

* 22

503

zk 21

504

* 22

507

-t 27

thrombi7

554

* 12

551

+ 12

553

* 14

555

* 19

thrombiô

196

* 12

203

+ 12

200

f 14

201

i 29

gram1 2

199

f 15

207

i 14

202

k 15

209

t 28

thrombi8

437

* 27

436

t 25

441

* 21

443

* 28

AN51,

no EDTA

FIGURE 2 Fluorescente histograms of washed platelets treated with plasmin with the antibodies indicated. Solid lines: plasmin treatment; dotted

12.0 CU/mIl lines: saline

and stained control.

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PLASMIN AND PLATELET RECEPTORS

716

Platelet aggregation After incubating PRP with therapeutic concentrations of SI<, APSAC and tPA, the aggregation of platelets, induced by ADP and ristocetin, was progressively inhibited. A typical profile of ADPinduced aggregation of normal PRP treated with SI< is shown in Fig 3. The effects of tPA and APSAC on aggregation were similar, but the degree of inhibition showed some variation. APSAC caused about the same grade of inhibition as SK did, whereas tPA had a more moderate effect (data not shown).

ADP 3.6 pM f0 5

60

FIGURE 3 Tracings of platelet aggregation induced with 3.6 PM ADP, in PRP which was treated for the times indicated (min) with 300 Wml SK. Light transmission (y) versus time (xl.

DISCUSSION In an attempt to elucidate the mechanisms involved in the platelet dysfunction arising during thrombolvtic therapy (31, we have focused on the integrity of platelet surface glycoprotein receptors known to play a role in platelet aggregation. In order to imitate the conditions occurring in vivo as closely as possible, we used platelets in PRP and pharmacological concentrations of thrombolytic drugs. Under these circumstances, platelet aggregation defects are found which are very similar to those seen in patients (Fig 3). The effect of plasmin generated in PRP on GP lb expression has not been described in detail before. Balduini et al., using a monoclonal antibody to GP lb and flow cytometry, reported briefly that treatment of PRP with SI< caused no modification in platelet composition (8). This is in accordance with the lack of effect of SI<, APSAC and tPA on platelets in PRP found here (Table 21, but not with previous findings in washed platelets (5). We showed that plasmin had no effect on MFI in PRP, but 1 .O CUlml affected GP lb on washed platelets (Fig 1); this excludes the possibility of ineffective plasrnin doses. In theory, replenishment of surface GP lb from intracellular stores (7,s) could obscure GP lb degradation, but our finding of no change in GP lb expression for 90 min rejects this possibility. The reported failure to find significant differences in GP lb density after in vivo thrombolysis in patients (8.9) despite a reduction in ristocetin-induced platelet agglutination during such therapy (3,8), is fully compatible with our in vitro findings. Therefore it is more likelv that this defect is caused by proteolysis of plasma von Willebrand factor (21), rather than of platelet GP Ib.

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PLASMIN

AND

PLATELET

RECEPTORS

717

Data on the effect of thrombolYtic agents or plasmin on GP Ilb/llla are highly controversial, but most results are from studies with washed platelets (5,6,10,12). Two preliminary studies carried out in a plasma milieu showed concordant results, namely no or at most subtle changes in GP Ilb/llla expression, in spite of significant reduction in GP Ilb/llla-mediated fibrinogen binding and platelet aggregation (8,l 1). While we demonstrated significant effects of plasmin on washed platelets (Figs 1 and 2), no such effects were found in platelets in PRP treated with the same concentration of plasmin or with any of the thrombolytic drugs investigated (Table 2). This of agrees with the results of other investigators (8,l 1). The same holds true for the appearance activation antigens after plasmin treatment of washed platelets. The conspicuous differente between effects in PRP and in purified systems is almost certainly due to the presence in plasma of plasmin inhibitors and large quantities of other suitable plasmin substrates like fibrinogen and some clotting factors. Moreover, even after complete activation of all available plasminogen in plasma, the concentration of plasmin is stil1 not sufficient to overcome in normal

the threshold for an effect on platelet GPs. We could generate only 0.9 CUlml plasmin PRP by exhaustive activation with SI<, a value similar to previous data (5,22), whereas

Fig 1 indicates that at least 1 .O CU/ml is necessary for a detectable decrease in GP Ilblllla epitopes. Thus, studies on the effects of plasmin using washed platelets should be interpreted with reserve and the results not be extrapolated to more physiological conditions, like some authors seem to suggest (5). It is conceivable that the effects of thrombolytic agents on platelets are different in PRP and in whole blood (23), but from the lack of changes in surface GPs we infer that neither red blood cells nor calcium ions play a significant role in the interaction between plasmin and platelets. The significant decreases in surface GP Ilb/llla of washed platelets treated with plasmin, as found with the antibodies thromb/l and thromb/7 and, to a more limited extent, with Y2/51 (Figs 1 and 2) disagree with the activation and upregulation of the GP Ilbillla complex found by others (10,22,24,251. Apparently, the incubation time used here was long enough to degrade even upregulated GP Ilb/llla. On the other hand, Iow plasmin concentrations failed to induce any notable increase in the epitopes reacting with thromb/7 and Y2151 (Fig 1). A smal1 but significant increase was present, however, using the anti-GP lb antibody (AN51) if a relatively high concentration of plasmin, 1.0 CU/ml, was used (Fig 1). This supports the concept of GP lb redistribution (7,9,12). Finally, the main limitations of the current investigations must be considered. Firstly, it is an in vitro study, the results of which can not be directly extrapolated to the in vivo situation. In the second place it should be kept in mind that the immunological technique used here can only provide information on the structure of the GP receptors, rather than on their function. Wheteas the anti-GP

lb and anti-GP

are necessary

for

adequate

Ilb/llla

monoclonals

platelet

function

employed (14-17),

are known there

to react

remains

the

with

epitopes

possibility

that

which ether

functionally critical epitopes on the receptors could be degraded by plasmin and stil1 lead to impaired platelet function. However, recent findings by others that fibrinogen receptors on activated platelets are resistant to plasmin digestion (26) and that there remains sufficient functional GP Ilb/llla to support fibrinogen-dependent aggregation after treatment of platalets with tPA (251, sustain our findings. Furthermore, we could not demonstrate any effect of the which has no known receptor function in thrombolytic agents on the thromb/8 antigen, haemostasis and therefore served as an indifferent control. In conclusion, we have shown that plasmin degradation of the platelet surface receptors G,P lb and GP Ilbillla plays no relevant role in the development of the platelet dysfunction, which is induced by thrombolytic drugs in platelet rich plasma. Therefore this platelet defect must be explained by other mechanisms and this is currently under investigation in our laboratory.

ACKNOWLEDGEMENTS The authors thank Mrs. M. Smulders for her excellent technical assistance, the volunteers who repeatedly gave blood for this study, Mr. N. Vreeswijk (Central Laboratory of the Netherlands Blood Transfusion Service) for donating monoclonal antibodies, dr. J.A. van Mourik for his advice and A.L. Hoffmann for his help with the figures. The thrombolytic drugs were kindly provideU by

PLASMIN AND PLATELET RECEPTORS

718

Vol. 67, No. 6

SmithKline Beecham Farma, Rijswijk, the Netherlands (anistreplase) and Dr. Kar1 Thomae GmbH, Biberach an der Riss, FRG (recombinant tPA). Financial support was received from the Scientific Foundation of the Catharina Hospital.

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