Effect of metabolic inhibitors on platelet attachment, spreading and aggregation on collagen-coated surfaces .

THROMBOSIS RESEARCH 46; 233-240, 1987 0049-3848/87 $3.00 t .OO Printed in the USA. Copyright

(c)

OF

EFFECT SPREADING

Central Sciences *USSR of

1987

Pergamon

Journals

Ltd.

INHIBITORS METABOLIC AND AGGREGATION ON

All

rights

ON

(Received

3d

Research Center, Cherepkovskaya,

PLATELET

ATTACHMENT, SURFACES

COLLAGEN-COATED

Misselwitz, V. L. Leytin*, F. Institute for Cardiovascular of the GDR, Wiltbergstr. 50,

Cardiology the USSR,

reserved.

and V. S. Research, Berlin-Buch,

Academy 15a,

of

Medical

Moscow,

12.11.1986; Accepted in revised by Editor J. Stilrzebecher)

Repin* Academy 1115,

form

121552,

.

.of GDR,

Sciences USSR.

28.1.1987

ABSTRACT The interaction of human gel-filtered platelets (GFP) with surfaces coated with fibrillar calf skin collagen (CSC) or monomeric human type I, III, IV, and V collagen CIV, CV) includes both energy dependent and (CI, CIII, independent stages. Incubation of platelets with a collagen-coated surface at 4OC versus 37'C reduces only shape change and the spreading response of adhering platelets, but does not affect the initial attachment. Additionally, the energy dependence was evident from the reduction of platelet spreading and platelet aggregate formation in the presence of 2-Deoxy-D-glucose (ZDG). Antimycin A or 2,4-Dinitrophenol (DNP) did (AMA), Oligomycin (OM), not abolish the adhesion-induced platelet activation, indicating that the energy is supplied by glycolysis rather than by oxydative phosphorylation. In contrast, neither inhibition of glycolysis, nor inhibition of the respiratory chain did affect the initial attachment of nonactivated platelets to the collagen-coated surface. The present data suggest (i) that during the interaction of platelets with collagenous substrates there exists an initial energy independent attachment stage, and (ii) that the following stages of adhesion-induced platelet activation require metabolic energy supported mainly by anaerobic glycolysis.

Key

words:

Platelet-collagen platelet energy, spreading, mural

233

interaction, metabolic attachment, platelet thrombi formation.

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PLATELET ADHESION

Vol.

46,

No.

1NTRODUCTION

The initial ing a loss telets to attachment ing platelets accompanied

event in of vascular the exposed

hemostasis integrity subendothelial

of

platelets, undergo a by degranulation

which shape

and

arterial

thrombosis followof blood plaAfter an initial

is the adhesion collagen.

may be receptor-mediated, change and a spreading (release reaction) and

adherprocess mural throm-

bi formation / l-3 /. The mechanism of platelet interaction with the collagenous components of the extracellular matrix of the vessel wall and the subsequent series of events are of great importance for understanding the pathogenesis of vascular disea-

se. sion

the are

However, process

biochemical not yet

fully

changes associated understood.

with

the

adhe-

Aggregation of platelets in suspension induced by physiologic inductors and release reaction are energy-dependent processes / 4-6 /, but there are contradictory findings concerning the adhesion of platelets to collagen / 7-11 /. There is some evidence that the adhesion process includes energy-dependent and independent phases to determine two

/

8-10 what

/; however, morphological

no sufficient phenomena

data correspond

are

available to these

phases.

Previously, titative ing to a

we described a morphometric technique based on quanscanning electron microscopy (SEM) of platelets adhercollagen-coated surface, which allows to determine successive stages of the platelet-collagen interaction: (i) initial attachment of discoid and spheroid platelets, (ii) spreading of attached platelets, and (iii) formation of thrombus-like platelet aggregates bound to a collagen-coated surface / 12, 13 /. This model system was used to determine the effect of metabolic inhibitors on the initial attachment and spreading of platelets and mural thrombi formation.

MATERIALS

AND

METHODS

serum albumin (BSA), Hepes, D-glucose, 2-Deoxy-D-glucose, Antimycin A, Oligomycin, 2,4_Dinitrophenol, Glutardialdehyde, Calf skin collagen (grade III) were from Sigma Chemical Co., 28 - from Pharmacia St Louis, MO, USA. Sepharose Fine Chemicals, Uppsala, Sweden.

Bovine

Highly

purified human placental generous gift from Dr. Cardiology Research Center. V

were

a

Isolation tained

from

healthy oc.

GFP

collagens

of qel-filtered

platelets,

acid-citrate-dextrose

-

male donors was isolated

by centrifugation on

Sepharose

type

I,

S. P. Domogatsky

28

III, from

Platelet-rich anticoagulated

at 200 g for equilibrated

IV, the

plasma blood

was

ob-

from

10 min with

and USSR

a

at 20 . modified

2

Vol. 46, No. 2

PLATELETADHESION

235

Tyrode's solution containing 3.5 mg/ml BSA and 10 mM Hepes / 12, 14 /. Platelet count was adjusted to 1.2 x lo8 platelets/ml. Uniess otherwise indicated, the Tyrode's solution contained 10 mM D-glucose. In some experiments D-glucose was omitted and 10 mM 2DG was added to the equilibration and elution buffer. eeparation of collaqen-coated surfaces. 16.4 mm-wells of Multiwell tissue culture wells (Falcon, Oxnard, USA) were coated with fibrillar CSC, or monomeric CI, CIII, CIV, or CV. After preforming fibrils / 15, 16 / CSC was immobilized to the well bottom / 13 /. Monomeric human collagen isotypes were noncovalently adsorbed on the surface of the culture plates in a monolayer fashion / 17 /. Adhesion of platelets. 250 ~1 GFP containing 3 x 10' platelets were added into the collagen-coated wells and incubated 40 min at 37OC with gentle agitation / 12, 13 /. After removing the nonadherent platelets, the adherent ones were fixed with 2.5 % glutardialdehyde and morphometrically analyzed with a Philips scanning electron microscope (PSEM 500x, Holland) / 12, 13 /. The morphometric analysis includes determination of the number of unspread and spread platelets and thrombus-like platelet aggregates per 1 mm2.

RESULTS -In the present study the temperature dependence as well as the effect of inhibitors of glycolytic or respiratory metabolic energy supply on the adhesion of platelets to collagen-coated surfaces were investigated. The experiments were carried out using GFP containing divalent cations (1 mM MgC12, 2 mM CaC12), and 3.5 mg/ml BSA, the latter in order to reduce nonspecific bind, ing. It was demonstrated that incubation of platelets with fibrillar CSC-substrate at 4OC versus 37'C greatly reduces the spreading of adhering platelets (88 % inhibition), whereas attachment remains unaffected (Fig. 1). These temperature effects may reflect the need for active cellular metabolism to realize cytoskeletal re-arrangements associated with shape change, spreading and release reaction. Therefore, we have investigated the effect of ZDG, a competitive substrate blocking the entry of sugars into the glycolytic pathway, AMA and OM, inhibiting the electron transfer through the respiratory chain, as well as DNP, a de-coupling agent, on subsequent stages of the interaction of platelets with collagen-coated surfaces. It was shown that only an inhibition of glycolysis by 10 mM 2DG reduces platelet spreading on a CIV-substrate and thrombus-like platelet aggregate formation on CI- or CIII-substrates (Table 1). Inhibitors of oxydative phosphorylation (0.1 mM DNP, 2 pg/ml AMA, 2 ug/ml OM), in contrast, do not abolish the spreading response and the thrombus formation (Table 1). The initial attachment of nonactivated platelets to a CV-substrate was entirely independent of the supply of metabolic energy:

236

Vol.

PLATELET ADHESION

Neither resulted the combination of inhibitors and oxydative phosphorylation (Fig. 2) nor its addition in a decrease of initia attachment.

46, No. 2

of glycolysis separate

-

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.~.‘.‘.‘.‘.‘.. ::::::: ::::..:.. .>:.:.:.:.>: ::::::. ::::::. ::::::. ::::::. ::::::. ::::::. ::::::. :::..:... ::::::. :::::... ::::::. ::::::. .‘:::::. ::::..... ..:::::. . . . . . . :.:.:.:.:.:.: ..::::.*.

-

I -

l-

ATTACHMENT

SPREADING FIG.

1

Temperature dependence of platelet spreading and attachment on a calf skin collagen-coated surface. GFP was incubated 40 min with a surface coated with fibrillar CSC at 37OC (toned columns) or 4OC (black columns). Number of adhering platelets per square millimeter and percent of spread platelets were determined using SEM as described previously / 13 /. Data represented as means 2 standard error of means, n=5. The asterisk indicates a statistically significant difference (P < 0.05, U-test, Mann and Whitney). DISCUSSION Previous investigations into the role of metabolic energy in platelet-collagen interactions have failed to take into account the influence of divalent cations, due to the addition of EDTA to prevent platelet aggregation. Furthermore, models which allow the platelets to interact with particulate collagen in suspension, or fibrillar collagen bound to Sepharose, are not well suited for the determination of early morphological phenomena of platelet adhesion (i.e. spreading), as well as thrombus formation / 7-11 /. For this reason we used different genetic types of

237

PLATELET ADHESION

Vol. 46, No. 2

TABLE 1 Effect of Metabolic Inhibitors on the Spreading of Platelets and the Formation of Thrombus-like Aggregates on Collagen-coated Surfaces.

Percent of Inhibition Platelet Spreading on CIV 2-Deoxy-D-glucose 10 mM 2,4_Dinitrophenol 0.1 mM Antimycin

73 + 27' (5)

Thrombus Formation on CI/CIII 100 * 0 x (5)

329 (5)

14 f 23 (5)

5 24 (6)

20 + 9 (6)

A (4)

Oligi";;;;l

(2)

GFP

was incubated 40 min at 37'C with CIV-substrate (spreading) or CI, CIII-substrate (Thrombus formation) in the presence of metabolic inhibitors or the appropriate vehicle alone (control). Percent of spread platelets and number of platelet thrombi were determined using SEM. Results (means + standard error of means) were expressed as percent inhibition referring to control values. The asterisk indicates a statistically significant inhibition (P < 0.05, U-test, Mann and Whitney).

FIG. 2

1oa

5

s

6 fa

0

Effect of metabolic inhibitors on attachment of platelets to a collagen type V - substrate, CV-coated surfaces were incubated 40 min at 37OC with GFP alone (A), GFP plus 2DG, 10 mM and DNP, 0.1 mM (B), or with GFP plus ZDG, 10 mM and AMA, 2 pg/ml (C), Results are expressed as percent of the control (A). Means obtained from 5 experiments.

238

PLATELET ADHESION

Vol. 46, No. 2

collagen immobilized on a surface to study energy requirements of subsequent stages of the platelet-collagen interaction. Due to the pronounced differences in platelet reactivity of the difdeterferent collagenoua substrates / 12 /, one can in parallel mine the role of metabolic energy in initial platelet attachment and thrombus formation (CI and (CV), platelet spreading (CIV), The absence of platelet aggregation during the interacCIII). tion of platelets with CV and CIV allows the experiments to be carried out in the presence of physiological concentrations of which play an essential role in platelet-colladivalent cations, In accordance with other authors gen interaction / 18, 19 /. / 8, 11 / we demonstrated that platelet adhesion was nearly unaffected by lowering the temperature. Metabolic ATP-depleted platelets adhere to collagen as well as metabolically active suggesting that initial attachment of platelets (Fig, 2) / 10 /, platelets to a collagenous substrate does not depend on metabodue to the existence of pre-exposed collagen-binding lic energy, We demonstrated that inhibition of sites on the plasma membrane. oxydative phosphorylation does not affect subsequent activation of platelets adhered on the collagen substrate (Table l), indicating that glycolysis alone can provide metabolic energy sufficient for platelet activation / 4, 7, 8 /. Only the inhibition of anaerobic glycolysis by suspending the platelets in a glucose-free Tyrode’s solution containing 10 mM 2DG, or by adding 30 mM 2DG to a GFP, normally containing 10 mM D-glucose, drastically abolish adhesion-induced platelet activation (Table Besides the effect of 2DG on platelet aggregation, reported 1). previously / 4, 10 /, we demonstrated its effect on platelet spreading. one can conclude that the first step of plateletIn summary, collagen interaction, the initial attachment, does not depend on metabolic energy. This attachment is, perhaps, accounted for by specific collagen binding sites on the plasma membrane of the platelet / 20 /. Since the binding per se does not depend on one can speculate that no unmasking, exposure, metabolic energy, or clustering of these receptors is necessary. Activation of adhered platelets (spreading, degranulation, mural thrombus requires metabolic energy supplied formation), in contrast, mainly by anaerobic glycolysis.

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