Potentiation of platelet interaction with collagen substrates by heparin is insensitive to aspirin

THROMBOSIS RESEARCH 52; 573-585, 1988 0049-3848/88 $3.00 t .OO Printed in the USA. Copyright (c) 1988 Pergamon Press plc. All rights reserved.

POTENTIATION OF PLATELET INTERACTION WITH COLLAGENSUBSTRATES BY HEPARIN IS INSENSITIVE TO ASPIRIN.

A.V.Mazurov, USSR

Cardiology

V.E.Sinitsyn

Research Center, Cherepkovskaya 15a,

and V.S.Repin

Academy of Medical Moscow 121552, USSR

Sciences,

3rd

(Received 11.4.1988; accepted in revised form 4.10.1988 by Editor M. Verstraete)

ABSTRACT The effects of two standart, unfractionated heparin preparations on collagen-induced adhesion and aggregation of platelets were studied by Born and scanning electron aggregometry microscopy (SEM). Heparin from porcine intestinal mucosa (HI) and from bovine lung (HL) added to human PRP in the concentration of 2.5 to 5.0 U/ml (1) did not induce platelet aggregation in suspension by itself but stimulated it in combination with the subthreshold doses of fibrillar human collagen type III (CIII); (2) increased by 1.5-2.0 but did not affect platelet spreading on a fold the adhesion, surface coated with human collagen type IV (CIV); and (3) enlarged by 2.5-3.0 fold the area of the CIII-coated surface covered with increasing both the number and the size of surfaceaggregates, blocked platelet aggregation in Aspirin bound aggregates. near threshold doses, of fibrillar CIII suspension induced by low, heparin, and by subthreshold doses of CIII in combination with but had no effect on platelet aggregation induced by high ( > 10 CIII. Aspirin failed to decrease platelet threshold) doses of and formation of adhesion to and spreading on CIV substrate, well surface-bound aggregates on CIII substrate in the absence as as in the presence of heparin.

INTRODUCTION side-effects such as therapy causes hepar in some patients , In and hemorrhage [l-7]. These complications thrombosis thrombocytopenia, both direct [lresult from the activating effect of heparin on platelets, 31 and/or mediated by immune system [3-S]. _______________________-_______________-_-~-----___-----------~---------human aggregation, platelet platelet adhesion, Heparin, Key words : collagens, aspirin

573

574

HEPARIN AND PLATELET ACTIVATION

Heparin can cause platelet aggregation platelet aggregat.ion induced by different etc.) and prevents the action of platelet

Vol. 52, No. 6

by itself (8-16), potent iates agonists (ADP, co1 lagen, U4663 9 inhibitors [9,10,17-211.

Contraversial results were obtained in the st:udies of heparin effects upon adhesion and adhesion-induced platelet aggregation. Baumgartner [ 221 and Ession et a3 [23] observed no effects of heparin on the interaction of platelets with subendothelium. However thrombi formation was more marked when heparin was used as anticoagulant instead of citrate [221. Decrease adhesion of washed platelets in the presence of heparin was reported by Davies was registrated only in the and Menus [24] , though this effect absence of plasma. In several studies inhibition of platelet interacti.on with a damaged vessel wall was shown after heparin administration in vivo [25,26]. In the previous works we have demonstrated that the surfaces co1 lagens of different types offer convenient substrates for and formation of adhesion and spreading of platelets (CIV), bound aggregates (CI and CIII) [27-301.

coated with investigation of surface-

In the present study we have investigated the effect of two heparin preparations upon the interaction of platelets with CIVsubstrat:es, It was shown that heparin potentiated collageninduced adhesion and aggregation, that the effects of heparin and prevenl.ed by aspirin.

commercial and CIIIplatelet were not

METHODS

Reagents. Heparin from porcine intestinal mucosa, grade I, 176 lJ/mg, was obtained from Sigma Chemical Co, MO (N H3125) and hoparin from St I,ouis, bovine lung (HL) - from Moscow endocrine factory, 140 H/ag . Hcpar i ns were dissolved to t.he required concentration in PBS, pH 7,4, and stored in this form at 4’ for not more than one week. JJ46619 was a gent!rous gift Of IJpjtrhn co. ) Kalamazoo, MI. Aspirin and ADP were from Sigma and other reagents were purchased from Merck, Darmst adt , FRG. Co1 lagens * III (CIII)_ and IV (CIV) were kindly provided by S.P.Domogatsky (USSRardiology Research Center). Collagens were extracted from human placenta by pepsin/acetic acid and then separat.cd by sequential precipitations in acidic and neutral NaCl solutions of different concentrations [31] with subsequent chromatography on DEAR celllllosf!. The pur i ty of collagen preparations was ascertained by electrophoresis and immuru~logically (for details see [32]). Lyophilized CITI and CIV were d i SW 1ved in PBS, pH 2.5, in the concentration of l--2 mg/ml and stored at 4” for not more than a week. For f.ibrills formation the CTTI solution was neutralized with NaOH to pH 7.4 and incubated for 2 hours at 37’. The kinetic of f ibrillogenesis was registrated spectrophotometrically by the increasf! ill 1 i ght scattering at 405 nm. For preparation of roll agen coated surfacr:: 10 ~tg/ml solutions of CIII and CIV were prepared in carbonate buffer, pH 9.6, immediat,ely prior to use. These solutions were added by 0.5 ml into 36.4 mm wells of Multiwell culture plates (Falcon Div. Recton, Dickinson & Co, Oxnard, KA) and incubated for 12.- 18 hours at 4’. Refore platelet addition the wells were washed 3 times with PRS, pH 7.4.

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575

HEPARIN AND PLATELET ACTIVATION

Preparation of PRP. Blood was drawn from healthy donors in a 3.8% sodium citrate (blood:anticoagulant ratio 9:l). All donors did not take any drugs for two weeks and never received heparin. PRP was obtained by centrifuging blood at 180 g for 10 min at 22’, and platelet poor plasma - by centrifuging the remaining cell pellet at 1000 g for 20 min. Platelets were counted in an automatic PL-100 Platelet Coqter (TOA Medical Electronics, Sysmex) and diluted with plasma to 2x10 platelets per ml. In the experiments with aspirinized platelets, aspirin at the final concentration of 0.5 mMwas added to a portion of PRP. This dose completely inhibited the aggregation induced by 1 mM arachidonic acid. Aq;gregation of platelet k suspension was registrated in PRP in a dual channel Payton Aggregometer Module (Payton Associates Ltd.) at 37’ with stirring at the rate of 900 rpm. Heparin was added 2-3 min prior the inducers and the respective volume of PBS was added to the control samples. The threshold doses of ADP and U46619 were determined by adding the agonists to the several portions of PRP at the doses differed by 0.5 JIM and The level of aggregation induced by 0.1 JIM for ADP and U46619 respectively. fibrillar CIII was measured as alteration in light transmission 5 min after the addition of collagen. While testing the aggregation activity of heparins themselves they were incubated with PRP in a cuvette of the aggregometer for 10 min. Adhesion and aggregation _of platelets on collagen substrates. PRP (250 pl) was added to the wells previously coated with CT11 and CIV. For investigation of platelet adhesion and spreading platelets were incubated with CIV-substrate. The incubation with CIV was performed for 20 min at 37’ without stirring. In our previous experiments with gel-filtered platelets we have shown that platelet spreading is more pronounced when incubation is performed without stirring [33]. The time of incubation was chose after the analysis of the kinetic curves of adhesion and spreading on CIV which reached plateau within 20 min. Incubation of PRP with CIII-substrate was carried out for 40 min at 37’ stirring the suspension in the horisontal plane at 36 rpm in a Lab-Line Orbit Incubator Shaker (Lab-Line Instruments Inc.). Preliminary we determined that the number of surface-bound aggregates, mean area covered with one aggregate and the total area of the substrate coated with aggregates stabilized within 30 to 40 min. After incubation, unattached platelets were washed 3 times with PBS, and adherent platelets were fixed with a 2.5% glutardialdehyde in a 100 mM phosphate buffer, pH 7.4, for 1 hour at 37’. Samples earlier

for scanning [34].

electron

microscopy

(SEM) were prepared

as

described

SEM analysis of platelet interaction with collagen substrates was performed The Netherlands. scanning electron microscope PSEM 500, Philips, iian:ification of platelet adhesion, spreading and aggregation on collagen by SEM was partially described in our previous papers substrates Twenty fields at the magnification of 2500 were examined in [27,28,33,34]. from the edge to the center of a well with a each preparation moving The number of unspread and spread platelets adherent to spacing of 0.4 mm. was counted. On the basis of these values, we CIV (U and S , respectively) of spread calculgted thg adhesion on CIV (A = UC + SC) and the percentage The A values among the adhersnt one: (P = s xlOO%/(U + s )). platelets Sn CIFI the nukber of per 1 mm of the CIQ-subgtrate. were calculated

HEPARIN AND PLATELET ACTIVATION

Vol. 52, No. 6

aggregates and the total area of the substrate coated with aggregates was counted. Multilayer aggregates containing not less than 10 platelets were counted. On the basis of these data, a mean area occupied by one ggregate 9 was calculated. The number of aggregates was calculated per 1 mm of the CIII-substrate; the total area of the s’ubstrate coated with aggregates was expressed as percentage of ths total substrate area, and the mean area covered with one aggregate in p . Statistics. differences

of

Means + SEM are given in the tables. controiwas calculated by t-test for means.

Significance

of

RESULTS Platelet aggregation in suspension. The effects of heparin on the aggregation in suspension induced by fibrillar CIII. ADP and U46619 were investigated. Both heparins themselves added to PRP in doses up to 5 U/ml without any other agonists did not stimulate the aggregation of platelets in 5 donors tested. At the same time, heparin in the concentration of 2.5 U/ml sharply increased platelet response to the subthreshold doses of CIII. These doses of collagen (0.1-l pg/ml in different experiments), by themselves, fail to cause or stimulate only a slow, low amplitude aggregation of platelets, but induce intensive aggregation in combination with heparins (Fig. la). Both preparations of heparin have approximately the same effects on CIII-induced aggregation (Table 1). HI and HL also increased the sensitivity of platelets to ADP and lJ46619, reducing by nearly 2-fold the threshold doses of these inducers causing the irreversible aggregation (Table 1).

Potentiation

of

Platelet

TABLE 1. Aggregation

in Suspension

-----______---_-______.-----..-___--------____ light Control

CIII transmission, 8.353.0

HL

40.2L9.8

HI

41.728.2

**

**

by Heparin.

-__.____ __.__-.__________._ ADP threshold

%

U46619 dose, ).rM

2.61~0.27

0.72~0.07

1.40+0.19*

0.38~_0.03

1.

12

**

0.

**

.06*

HL and HI were added to PRP in the ConcentratioD - 2.5 U/ml*t,CIII was used in the threshold doses (< 1 /Jg/ml). p < 0.05; p < 0.01; without asterisks - non-significant (n = 4-9). of aspirin on CIII-induced platelet aggregation in suspension were Effects dose of CIII. depended on the Aspirin prevented platelet aggregation induced by low (approximating to the threshold) doses of CIII and subthreshold doses of CIII in combination with heparin. However, it was practically ineffective at high CIII concentrations (> IO-fold higher than the threshold) (Fig. 1). Adhesion and spreading of platelets z CIV-substrate. to 2 foldplatelet adhesion on CIV in a dose-dependent affect the level of spreading (Fig. 2A,B. Fig. 3

Heparin increased manner, but did and Table 2).

up not The

lmin

FIG

c(

I min

1.

L,.ASPyI(w

J

1

0.4 )rg/ml

-

A

c Ill.

lmin

M

Effects of heparin and aspirin on aggregation of platelets in suspension induced by different concentrations of fibrillar CIII. (A) Buffer or 2.5 U/ml HL were added to native and aspirinized PRP and 0.2 )Ig/ml CIII (B) 0.4 pg/al CIII (threshold dose) was added (subthreshold dose for ethis experiment) was added 3 rain later; to native and aspirinixed PRP; (C) lOpg/ml CIIJ was added to native and aspirinized PRP. The results of one of 4 representative experiments are shown. Both praparations of heparin, HL and HI, were used.

5 i

I

578

HEPARIN AND PLATELET ACTIVATION

Vol. 52, No. 6

HEPARIN AND PLATELET ACTIVATION

Vol. 52, No. 6

Platelet.

Adhesion

and Spreading of Heparin

TARLE 2 on CIV Subst.rate and Aspirin.

Adhes ion’3 platelets, 10 /mm2

Cont.rol

579

in the

% of

Presence

Spreading, adherent. platelets

1.73+0.27

39.L5 u*

39:6

III.

3.47+0.45

RI

2.70~0.24~

4226

Aspirin

1.71~0.36

4717

HL/HJ i Aspirin

2.8510.50*

:53+6

PRP was incubatccl with CIV-substrate in the ahsencc: addi.tions (Control) and in the presence of 2.5 U/ml HI., O.sr mM aspirin, 2.5 U/ml JiL or HJ + 0.5 mM aspirin. RI, 0.05; p < 0.01; without asterisks non sip;nif.icant.. 12).

Platelet.

Aggregate Presence

TARLE 3. Formation on CJII-substrate of Heparin and Aspirin.

Total surface covered with aggregates, “6

in

of

any

2.5,U/ml p < (n = 6

the

Area covered with one aggregate, p2

Number of aSgre0 tes, -9 per mm

142~14

322240

____._________...__~_.____________.__~.._~_.~._.~___~.____~__..___~______._

Control HI, HI Aspirin HL/HI + Aspirin

4.61_0.8 11.2:2.3*

255~20

13.1+3.2*

237231

3.0+0.7

136-15 -

11.8’2.0

_______-__--_------__--_--._~.---.

**

245525

** *

.487~8rl 51R+70* 215230

**

470+40*

_ ._- ._.__~__--_._--_ --_-- -- _---..- -

absence of incubated with CIII -subst.rate in the PRP was any in the presence of 2.5 U/ml HL, 2,5 U/ml HI, additions (Control), 0.05; 2.5U/mlJII,orHI -t 0.5mMaspirin. p< (&_” mM aspirin, (n : (1.6). without asterisks -- non-significant. p < 0.03;

HEPARIN AND PLATELETACTIVATION

580

%

Vol. 52, No. 6

4-

E

m’

‘0

3-

-30$

2I-

- IO

O-L I , 0 0.5 I.0 HEPARIN.

FIG

I 2.5 U/ml

,-0 50

t p’ % K 5

3.

heparin Platelet adhesion and spreading on CIV-substrate under various PRP was incubated with CIV in the presence of O-5.0 U/ml concentrations. The number of platelets adherent to CIV (H) and the percentage of HI. were determined by SEM. spread platelets (M)

HEPARIN.

FIG

U/ml

4.

Dose-responce curves of heparin stimulating effects on the formation of surface-bound aggregates on CIII-substrate. PRP was incubated with CIII in the presence of O-5.0 U/ml HI. The percentage of the total substrate surface covered with aggregates w), number of aggregates (o---o), and the mean area covered with one aggregate (C--r) were determined by SEM.

Vol. 52, No. 6 difference adhesion Aspirin in the induced

HEPARIN AND PLATELET ACTIVATION

between the was nonsignificant

effects of two heparin (Table 2).

preparations

did not affect the adhesion and spreading of presence and absence of heparin and failed increase of platelet adhesion (Table 2).

581 on

platelet

platelets on CIV both to prevent heparin-

Aggregate formation on CIII--substrate. Heparin increased platelet aggregation on the surface of CIII in a dose-dependent way. Taken in the concentration of 2.5 to 5.0 U/ml, it enlarges the area of the substrate coated with aggregates several times. This effect was achieved due both to increased number and size of surface-bound aggregates (Fig. Fig. 4, 2C,D, Table 3). HL and HI were equally effective in stimulation of platelet aggregation on CIII-substrate (Table 3). Aspirin failed to reduce aggregate formation on the CIII-substrate both in the absence and in the presence of heparin though in the absence of heparin some insignificant reduction of total surface covered with aggregates and the number of aggregates can be noticed (Table 3).

DISCUSSION The effects of two standart, unfractionated heparin preparations on the interaction of platelets with human collagens were investigated. Two heparin preparations, HL and HI. were used. Addition of heparin to PRP stimulated aggregation of platelets in suspension activated by subthreshold concentration of fibrillar CIII. Taken alone heparin and CIII in the doses used failed to induce aggregation. These results correspond to the data obtained in [10,17,20,21]. In these papers a rise of the collagen-induced aggregation in the presence of heparin was also registrated. Inhibitory effect of heparin on the collagen-induced aggregation in a suspension of washed platelets in the presence of plasma observed in [35] is, apparently, related to the formation of thrombin traces following addition of plasma to washed platelets. Heparin effects on collagen-induced platelet adhesion, spreading and surface-bound aggregate formation were studied using CIV- and CIII-coated surfaces. These substrates induced platelet spreading and aggregation respectively [27-301. It was shown that HL and HI increased 1.5- to 2.0fold platelet adhesion, but did not affect platelet spreading on a CIVcoated surface. Both heparin preparations sharply increased platelet aggregation on the CIII-substrate. Heparin increases both the number and size of surface-bound aggregates, enlarging approximately 2.5to 3-fold the total area of the substrate coated with aggregates. The method used in this study to investigate the formation of aggregates on CIII-substrate permitted to quantitate heparin effects on the background of a marked irreversible platelet aggregation, which cannot be done while analyzing aggregation in suspension according to the method of Born. The stimulative effects of heparin on adhesion and aggregation of platelets seen in this study manifested themselves at relatively low concentrations (l-5 U/ml) comparable with the in vivo concentrations achieved at heparin therapy. The heparin preparations used in this study (HL and HI) had the same activating effect on platelets in adhesion and aggregation tests.

582

HEPARIN AND PLATELET ACTIVATION

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Apparently stimulation of platelet interaction with CIII and CIV in the presence of heparin may be explained not only by direct action of heparin on platelets but also by increased sensitivety of platelets to secondary inducers, ADP and thromboxane A released from platelets during their activation. It was demonstrated t i+’ at heparin lowered the threshold doses of ADP and U46619, which agreed with the data of other authors [9,18-201. The effect of heparin on the adhesion to collagen was earlier studied by Davies and Menus [24] According to the brief report of these authors, inhibited platelet adhesion, but only in the absence of plasma. heparin These results contradict multiple observations indicating that the presence of plasma is requisite for the manifestation of heparin effects on the absence of plasma components was also the reason platelets. Probably, for Essien et al [23] to find out no effect of heparin on the adhesion of washed platelets to subendothelium. Using the perfusion chamber Baumgartner [22] failed to find a difference in the adhesion and aggregation of platelets to subendothelium between native and heparinized blood or blood It is possible that high containing only citrate and citrate with heparin. and thrombi formation in control samples of platelet adhesion degree (without heparin) does not allow to notice stimulative effects of heparin under such background. The possibility of inhibiting by aspirin of the collagen-dependent adhesion heparin and aggregation of platelets in the absence and in the presence of aggregation in Aspirin completely blocked platelet have been studied. but not high doses of fibrillar CIII which suspension induced by low, to the results of Kinlough-Hathbone et al [36] and Weiss et al correspond r371. Aspirjn had no effect on the adhesion and spreading of platelets on CIVaggregation on CIII-substrate. The absence of substrate and platelet aspirin effects on platelet adhesion to collagen agrees with the data obtained in other model systems [22,36-391. However, earlier, it had been reported about a small but significant decrease of platelet spreading on CIV in the presence of indomethacin [28], but these results were obtained using gel-filtered platelets. The inefficacy of aspirin to reduce the CIIIsubstrate-induced aggregation indicates that the activation of platelets by a CIII-coated surface is comparable in its strength with the activation by high doses of fibrillar CIII in suspension. Multiple studies demonstrated the ability of aspirin to inhibit the heparin-induced platelet aggregation [5,8,13-151. In the present study it was established that aspirin is much less effective in the case of combined activation of platelets by collagens and heparin. Aspirin completely blocked only the aggregation of platelets in suspension induced by the subthreshold doses of CIII in combination with heparin, but did not affect heparin stimulation of platelet adhesion to CIV, and aggregation on CIIIsubstrate. ACKNOWLEDGEMENTS The authors express sincere gratitude with collagens and to G.K.Abramov for microscopy studies.

to Dr S.P.Domogatsky technical assistance

for in

providing electron

HEPARIN AND.PLATELETACTIVATION

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583

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Platelet factors

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activation in hemostasis. Role in platelet-collagen interaction.

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PACKHAMM.A., MUSTARD F.J. pathway on the release of adherent to collagen. 2Lab