The effect of chemical or enzymatic modifications upon the ability of collagen to form multimers and to initiate platelet aggregation

THROXBOSIS RESE.AHCH Printed in the Unired

1-01.

Srates

7, PP. 113-122, Pergamon Press,

1975 Inc.

THE EFFECT OF CHEMICAL OR ENZYMATIC MODIFICATIONS UPON THE ABILITY OF COLLAGEN TO FORM MULTIMERS AND TO INITIATE PLATELET AGGREGATION

ELVIN HARPER, ELIZABETH R. SIMONS, CAROLYN MCI. CHESNEY,* and ROBERT W. COLMAN* Department of Chemistry, University of California, La Jolla, Cal.; Department of Biochemistry, Boston University School of Medicine, Boston, Ma.; Department of Medicine, Harvard Medical School, Boston, Ma.

in revised form 15.5.1975. (Received 7.12.1975; Accepted by Editor S. Niewiarowski)

ABSTRACT The previous paper (1) presented some correlations between collagen multimer formation and platelet aggregation. The present study investigates the effect of chemical or enzymic modification upon its melting temperature and rate of multimerization, and therefore upon platelet aggregation. Digestion of collagen with tadpole or bacterial collagenase led to a decreased melting point of the collagen fragments and prevented their initiation of platelet aggregation. In contrast, the absence of interchain crosslinks, in lathyritic collagen, had no effect on these parameters and thus these crosslinks are not required for collagen multimer formation and induction of platelet aggregation. Oxidation of the galactoses alone with galactose oxidase, or of all the sugars by periodate, led to a perturbed melting curve featuring two melting points,- 28' and 37'. These treated collagen preparations were thus unable to form multimers at platelet aggregation (>29O) temperatures, and they could not initiate the aggregation of platelets. Reduction with sodium borohydride regenerated a normal melting curve, multimerization, and platelet aggregation initiation. Thus the carbohydrate residues, and a majority of the intact tropocollagen chains, are required for the maintenance of native structure and of multimerization at 37' which, in turn, are necessary for initiation of platelet aggregation by guinea pig skin collagen.

*Present address: Department of Medicine, University of Tennessee Center for Health Sciences, Memphis, Tenn. 38103 *Present address: Hematology Division, Hospital of the University of Pennsylvania, Philadelphia, Pa. 19104

INTRCDUCTIC!S The data presented in the preceding paper (1) led us to ccnclude that the presence of collagen multimers at the temperature of the experiment was necessary and sufficient for the _in vitro initiation of platelet aggregation by soluble guinea pig skin collagen.

Such multimers could not form unless

the collagen was in its native conformation at the temperature in question. We therefore undertook an investigation of the effect of chemical

modifi-

cation of collagen upon these properties. It had been reported earlier that digestion of collagen with bacterial (2) or tadpole (3) collagenase abolished its ability to initiate the aggregation of platelets, but no partial collagenolysis or correlation with other collagen properties had been attempted.

Some Previous

studies have

also investigated whether the carbohydrate moieties of collagen were involved in its platelet aggregating function, (3-10) although these results remain in dispute (11).

It therefore seemed important to investigate.the role of

the carbohydrates in the thermal stability, the multimerization capabilities and the platelet aggregation potential of collagen.

MATERIALS AND METHODS Tadpole collagenase:

The enzyme was prepared and the digestion performed

as described by Harper (12).

These fragments were then isolated (13,14).

The fragments obtained after completion of the reaction correspond to the C-terminal one-quarter (TCB) and the N-terminal three-quarters (TCA) of the collagen molecule, each fragment still maintaining the native triple helical structure but with lower T,. Bacterial collapenase from Clostridium histolyticum:

This enzyme was puri-

fied by the method of Harper and Kang (15), and has been shown to be free of non-specific proteolytic activity.

The reactions were stopped at the

115

Ci3LLAGET YODIFICATIOS

desired time by adjusting the pH to 4.0 with acetic acid. bacteria? collagenase is irreversibly inactivated.

At such a low pH

An aliquot of the mix-

ture, diluted 1:lO with Tris buffer and adjusted to pH 7.6, was then used for the Tm and aggregation studied. Periodate oxidation:

Three ml of 0.04 >! potassium metaperiodate dissolved

in 0.05 M sodium acetate buffer were added to an equal volume of 0.4% guinea pig skin collagen in 0.4 M NaCl.

The reaction was carried out in the dark

at 4O with intermittent stirring for 24 hours.

The final concentration

ranged from 2 to 3 mg collagen/ml 0.4 M NaCl,

No residual galactose was

detected by Galactostat, as used according to manufacturer's directions. (Worthington Biochemicals, Freehold, X.J.) Galactose oxidase:

Galactose oxidase (2.2 mg) in 0.03 M Tris pH 7.0 was

added to 12 mg of guinea pig skin collagen in the same buffer. volume of the reaction mixture was 3.4 ml. 24 hours at 24'.

The total

The mixture was incubated for

A control sample of the same collagen was incubated under

identical conditions without the enzyme.

The extent of oxidation was mea-

sured by means of the Galactostat test. Sodium Borohydride Reduction: Preparations:

Performed as in Reference 3.

Platelet poor plasma (PPP), platelet rich plasma (PRP) and

acid soluble guinea pig skin collagen were prepared as described in the accompanying paper Platelet aggregometry: the accompanying paper.

Platelet aggregometry was performed as described in The time from the addition of collagen to the be-

ginning of the increase in light transmission is defined as lag time. Circular dichroism:

Thermal denaturation spectra for collagen were obtained

as described in the accompanying paper. as T . m AC?

The midpoint of the curve is defined

The circular dichroism is reported in terms of recorder deflection,

measured directly from the Cary 61 circular dichroism spectrum.

The

circular express

dichroism is a parameter denaturation

of

confornation;

2~2 caz

therefore

data in terms of the remaining fraction of

al.Sc?

native

con-

(where 6 is the actual formation, defined asEc- 6 fully denatured native- dfully denatured recorder-reading,cnative and Edenatured the readings at T-20' and T-48' respectively). Collagen multimerization:

The kinetics of collagen multimerization were

obtained by the techniques described in the accompanying paper (1).

If

the light intensity measured perpendicularly to the entering beam is plotted as a function of time, the initial velocity v0 is defined as the slope of such a curve at t.-0. RESULTS A)

Collagen fragments produced by partial enzymic cleavage were prepared

and examined.

Although tadpole collagenase fragments exhibit normal helical

content at room temperature, we found their melting points to be lower (35.0' 36.4' for TCB and TCA, respectively) than soluble tropo collagen, so that no native conformation and therefore no multimerization could be observed at Bacterial collagenase from Clostridium histolyticum yields a larger

37O.

number of products of lower molecular weight (16).

After fifteen minutes of

incubation (see Methods), the highest melting point exhibited by these fragments was 36'; they were unable to form multimers or to aggregate platelets. B)

Our investigation of the extent and nature of the collagen structure

required for platelet aggregation led us to study collagens whose carbohydrate moieties had been modified.

The accessible galactose residues were

oxidized by exhaustive treatment with galactose oxidase (17); a second approach involved the oxidation of glucosyl and galactosyl residues, as well as probably some of the amino acid side chains, by potassium m-periodate, (18). As shown in Figure 1 in comparison to normal collagen, the

Vo1.7,3o.l

COLLAGEN

MODIFICATION

11;

!

-0

I I

t

I

1

40

30 TEMPfRarLJRE

50 "C

FIG. 1 Thermal denaturation of guinea pig skin collagen: A: native; B: Treated with galactose oxidase; C: Treated with sodium periodate. (see Methods). melting curves of these oxidized collagens are similar although the stability after periodate treatment is clearly lower than that subsequent to galactose oxidase action.

The curves were definitely biphasic, exhibiting Tm near 28O

and near 37' as well as less sharp melting and thus less cooperativity in the melting process.

No residual accessible galactose residues could be

detected by the Galactostat method.

Both of these carbohydrate-modified

collagens failed to form multimers at 37' and were unable to initiate the aggregation of platelets even after 30 minutes' observation in the aggregometer. It has been recently shown (19) that certain galactose oxidase preparations are contaminated with proteolytic enzymes.

Since we knew that collagen

digestion leads to loss of fibril formation and of platelet aggregation

TABLE 1 EFFECT OF COLLAGEN MODIFICATION ON ITS H.VLTINERIZATIOSMD AGGREGATIKG PROPERTIES Sample

ye(a)

Trn 4?.0°

Control Collagen

PWTELET

Lag Time(b) 4 min

75

Galactose oxidase treated

28.8,37.0

> 20

Periodate treated

27.6,37.6

> 20

Galactose oxidase + borohydride

38.1

66

7.5

Sodium borohydride treated

37.3

61

5.6

Lathyritic Collagen

39.0

105

5.0

(a) (b)

Rate of multimerization at 35' expressed as A 1900/min.(~f.$lethods (1) Lag time at equivalent collagen concentrations (50Pg/0.5ml PRP)

0.1

c

ot-0.1 t

I

! I /

!

30

50

40 TEMPERA

TUffE

“C

FIG. 2 Therstaldenaturation of guinea pig skin collagen: Treated with A: borohydride; B: galactose oxidase followed by sodium borohydride.

Sodium

\- 0 1 . ; ,

s0

COLLAGE3

1

3

potential,

we verified

collagen

was

intact.

collagenolysis out

These

of

on

the

less

if

the

collagen

thermal

galactose the

was

Borohydride perties

of

indeed

reversible

of

was

This

material

Figure

dent

and slightly

collagen ble

samples

of

obtained

that

the

is

fully

with

and

but

were

then

be

not

be

by any cleavage

shorter,

temperature.

sodium

multi-

by reduction

would

accompanied

would

(Figure

and with-

on the

reversible

involved,

a lower

with

no

contaminants

initiation

had been

at

were

that

employed.

be

alone

products

treated

multimerization

time

the

the

have

Treatment

borohydride

proved

of

the

the

pro-

that

2).

non-modified

collagen

carbohydrates,

stabilization

like

lag

should

showed

treated

did

not

affect

here.

the

collagen.

as

Fxidase

electrophoresis

conditions

procedure

and melt

The involvement in

galactose

aggregation

collagen

treatment

tested

1 inks,

platelet

oxidative

stability,

oxidase

the

carbohydrate

backbone

oxidation

C)

the

of

our

indicating

under

of

the modification

of

gel

purification,

capabilities

reversible

backbone

The same results

collagenolysis

effects

merization if

occurred.

oxidase

of

the

SDS acrylamide

had

galactose

incapable

that

119

MODIFICATIOS

1,

curve

of further

A.

higher

previously

Its

of

rate for

for

an equal

of

internal

and

the

by experiments

is

platelet

aggregation

concentration

of

facilitation

of

lathyritic

“normal”

temperature

This

collagen.

cross-

with

39O and a curve

multimerization

normal

(3)

of

than

molecule

substantiated a T,

than

to

collagen

exhibits

observed

when compared

the

rather

explains by

normal

shape depen-

the

shorter

lathyritic

collagen

(cf

Ta-

I>.

DISCUSSION While maintain aggregation shorter,do

native their of not

and triple

platelets exhibit

lathyritic helical at

collagen, native

37’,

any of

Tm

structure,

collagenolytic

these

properties.

42’

and 390

form

multimers,

reaction Such

respectively,

products, fragments

and

do initiate

which have

less

are

thermal stability, therefore melt at temperatures lower than that of the intact molecule, and they do not support multimerization aggregation.

and platelet

This holds both for the tadpole collagenase fragments TCA

and TCB , which are known to be helical at room temperature but have Tm's around 36.4 and 35.0 (14), respectively, and for the bacterial collagenase fragments whose highest melting fragment exhibited a Tm = 36'. These results demonstrate that, while modification of the galactose moiety of guinea pig skin collagen prevents collagen induced platelet aggregation (3), the effect may be indirect.

While our earlier report (3) invoked

the role of.glucosyl transferase (4,5), which remains in contention, our data are equally consistent with the effect on tertiary structure and thence on multimerization, as suggested by Muggli and Baumgartner (11). Since galactose oxidase modified collagen exhibits such a low Tm, and hence cannot form multimers at 37', the non-aggregation of platelets exhibited by galactose oxidase treated collagen may be due to impaired formation of tropocollagen microfibrils

by any residual undenatured monomers, if present.

et al (20) has shown that the carbohydrate reRecent work by Aguilar, -sidues are located predominantly in the amino terminal portion of the collagen molecule.

Selective modification of these carbohydrates might result in

unstable multimers which cannot promote platelet aggregation due to lack of tertiary structure at the aggregation temperature.

Since it is possible to

reverse these effects completely by reducing with borohydride, it may be inferred that modification of the collagen galactose residues alone can be sufficient to impair thermal stability and hence multimerization and platelet aggregation. remain

The details of the effect of such modifications on collagen

incomplete. Carbohydrates also seem to be involved in the effect of

other proteins on platelet aggregation, as shown recently (21) for a factor VIII

glycoprotein which aggregates platelets only if its carbohydrates are

first freed of terminal sialic acids.

i-01

.;,so.

COLLXGEX

1

121

YODIFICdTIOS

While the specific role of carbohydrate in the collagen mediated initiation of platelet aggregation is thus either an indirect effect by destabilization of native structure and hence multimerization, or a direct specific carbohydrate effect, neither our studies to date nor any others in the literature allow a definitive choice between these alternatives. ACKNOWLEDGEMENTS We gratefully acknowledge the support, in part, of the Medical Foundation, Boston, Ma.

(ERS), The Boston University School of Medicine

Faculty Grants Committee (ERS) and the National Institutes of Health Grants HL 15335 Awards

HL 48075

(CMcIC).

(ERS),

HL 11519

(RWC),

(RWC) and AM 34222

AM 15367

(EH), and

(EH), Career Development

Special Fellowship

HL 48190

We thank Alan Goldman for technical assistance.

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SIMONS, E. R., C. MCI. CHESNEY, R.W. COLMAN, E. HARPER and E. SAMBERG. The effect of the conformation of collagen on its ability to aggregate platelets. This journal, preceeding paper.

2.

ZUCKER, M.B. and J. BORELLI. Platelet clumping produced by connective tissue suspensions and by collagen. Proc. Sot. Expt. Med. 109, 779, 1962.

3.

CHESNEY,

4.

JAMIESON, G.A., C.L. URBAN and A.J. BARBER. Enzymatic basis for platelet collagen adhesion as the primary step in hemostasis. Nature New 234, 5, 1971. w.

5.

BARBER, A.J. and G.A. JAMIESON. Platelet collagen and lesion characterization of collagen glucosyltransferase of plasma membranes of human platelets. Biochim Biophys. Acta . 252, 533, 1971. .

6.

BOSMANN, H.B. Platelet adhesiveness and aggregation. Commun. 43, 1118, 1971.

7.

BOSMANN, H.B. and E.H. EYLAR. Attachment of carbohydrate to collagen. Isolation, purification and properties of the glucosyl transferase. ibid. 30, 89, 1968.

Critical role of the C. MCI., E. HARPER and R.W. COLMAN. carbohydrate side chains of collagen in platelet aggregation. J. Clin. Invest. 5l, 2693, 1971.

Biochem. Res.

8.

PUETT, D., B.K. WASSERM.., J.D. FORD and L.:;.CUXX.KGttQI. Colla3 tzil mediated platelet aggregation: effects of collagen modification involving the protein and carbohydrate moieties. J. Clin. invest. 52, 2495, 1973.

9.

KANG, A.H., E.W. BEACHEY and B.L. KATZMAN.

Interaction of an active glycopep ide from chick skin collagen (CrjCB5)with human platelets. J. Biol. Chem. 249, 1054, 1974.

10.

KATZMAN, R.L., A.H. KANG and E.H. BEACHEY. Collagen induced platelet aggregation: involvement of an active glyco-peptide fragment (6) CBS). Science, l&, 670, 1973.

11.

MUGGLI, R. and H.R. BAUMGARTNER. Collagen induced platelet aggregation: requirement for tropocollagen multimers. Thrombosis Research, 3, 715, 1973.

12.

HARPER, E. and J. GROSS. Separation of collagenase and pephdase activities of tadpole tissues in culture. Biochem Biophys. Acta., 198, 286, 1970.

13.

GROSS, J., and Y. NAGAI. Specific Degradation of the collagen molecule by tadpole collagenolytic enzyme. Proc. Nat. Acad. Sci. (USA). 54, 1197, 1965.

14.

SAKAI, R. and J. GROSS. Some properties of the products of the reaction of tadpole collagenase with collagen. Biochemistry, 6, 578, 1967.

15.

HARPER, E. and A.H. KANG. Studies on the specificity of bacterial collagenase. Biochem. Biophys. Res. Commun., 5, 482, 1970.

16.

STARK, M. and K. KUHN. The properties of molecular fragments obtained in treating calf skin collagen with collagenase from Clostridium histolyticum. Eur. J. Biochem., 5, 534, 542, 1968.

17.

BLUMENFELD, O.O., M.A. PAZ, P.M. GALLOP and S. SEIFTER. The nature, quantity and mode of attachment of hexoses in Ichthyocol. J. Biol. 3835, 1963. Chem., 238,

18.

SPIRO, R.C. Characterization and quantitative determination of the hydroxylysine - linked carbohydrate units of several collagens. JBiol. Chem., 244, 602, 1969.

19.

SARASWATHI, S., and R.W. COLMAN, Role of galactose in bovine Factor V. Circulation, 2, Suppl. III, 292, 1974.

20.

AGUILAR, J.H., H.G. JACOBS, W.J. BUTLER and L.W. CUNNINGHAM. The distribution of carbohydrate groups in rat skin collagen. J. Biol. Chem., 248, 5106, 1973.

21.

VERMYLEN, J., M.B. DONATI, G. DeGABTANO and M. VERSTRAETE. Aggregation of human platelets by bovine or human factor VIII: role of carbohydrate side chains. Nature, 244, 167 1973.