1880 Blood, Vol 73. No 7 (May 15). 1989: pp 1880-1887

The Effect of Peptides and Monoclonal Antibodies That Bind to Platelet

Glycoprotein lib-Illa Complex on the Development of Clot Tension

By Isaac Cohen, Dan L. Burk, and James G. White

The development of tension in platelet-rich clots is a

manifestation of fibrin polymer binding to platelets as well

as platelet contractile activity. Arg-Gly-Asp(RGD)-contain-

ing peptides of fibrinogen a-chain and y-400-41 1 of fibrino-

gen ‘y chain increased clot tension considerably. especially

when it developed under isometric conditions. Morphome-

try revealed increased confluence of oriented fibrin and

platelet aggregates. Monoclonal antibodies directed

against different epitopes on the glycoprotein lIb-Illa com-

plex had varying effects on clot tension development.

Monoclonal antibodies A2A� and 7E3 inhibited clot tension

P LATELET AGGREGATION is a consequence of

fibrinogen binding to platelets.’ The lIb-Illa mem-

brane glycoprotein heterodimer complex (GPIIb-IIIa) in

stimulated platelets is the site for the binding of fibrinogen.27

Monoclonal antibodies against the IIb-IIIa complex inhibit

platelet aggregation and fibrinogen binding to platelets.27

The recognition specificities of the IIb-IIIa binding site for

fibrinogen have been localized to the carboxyl terminal

dodecapeptide of the fibrinogen �y chain (y-400-4 1 1 ) and the

tripeptide Arg-Gly-Asp (RGD), which occurs twice in the

fibrinogen a-chain.8” In view of their inhibitory activity on

fibrinogen binding to platelets and platelet aggregation,

RGD- and ‘y-400-4l I-containing peptides have been consid-

ered as potential antithrombotic drugs.’2

It is still unclear whether fibrinogen and polymerizing

fibrin share the same receptor domain on stimulated plate-

lets. Thrombasthenic platelets that are deficient in the

llb-Illa complex do not develop tension and remain isolated

in a fibrin isometric clot system.’3 This indicates that the

lIb-Illa complex is essential to the binding of polymerizing

fibrin to platelets and/or to the intracellular anchoring of the

actin cytoskeleton to the platelet membrane.

Because of platelet contractile activity, the interaction

between activated platelets and polymerizing fibrin results in

clot tension.’4 Clot tension can be evaluated with accuracy by

directly measuring the force generated in an isometric sys-

tem,’5 and indirectly, in isotonic conditions, by measuring the

shortening ofa free-floating clot’6 or its retraction from glass

in a test-tube system.’7

From the Atherosclerosis Program. Rehabilitation Institute of

Chicago. Department of Molecular Biology. Northwestern Univer-

sity Medical School, Chicago. and the Department of Laboratory

Medicine and Pathology. Pediatrics, University ofMinnesota, Mm-

neapolis.

Submitted April 22. 1 988; accepted January 21, 1989.

Address reprint requests to Isaac Cohen, PhD. Atherosclerosis

Program, Northwestern University. 345 E Superior St. 1407R1C.

Chicago. IL 60611.

The publication costs ofthis article were defrayed in part by page

charge payment. This article must therefore be hereby marked

“advertisement” in accordance with 18 U.S.C. section 1 734 solely to

indicate this fact.

(� I 989 by Grune & Stratton. Inc.

0006-4971/89/7307-0028$3.OO/O

while T10 and bE5 increased it. Since neither peptides nor

antibodies affected the platelet actomyosin ATPase activi-ty. their effect on tension must reflect the interaction

between platelets and polymerizing fibrin. We conclude

that y-400-41 1 and RGD-peptides increase platelet-poly-

merizing fibrin interaction. This suggests that clot tensionrequires a platelet receptor for polymerizing fibrin. which isdifferent from the fibrinogen receptor domain required for

aggregation. The results with the monoclonal antibodies

support this hypothesis.

S 1989 by Grune & Stratton, Inc.

In this study, the effect of peptides and monoclonal

antibodies that bind to the lIb-Illa complex on tension and

ultrastructure of platelet-rich clots was evaluated. The

results suggest that clot tension requires a platelet receptor

for polymerizing fibrin distinct from the fibrinogen receptor

domain.

MATERIALS AND METHODS

Platelet suspensions. Venous blood was collected from the

antecubital vein of normal donors after obtaining informed consent

and mixed with I volume of93 mmol/L Na citrate, 7 mmol/L citric

acid, 140 mmol/L dextrose for 9 volumes of blood. Platelet-richplasma (PRP) and gel-filtered platelets were prepared and main-tamed at room temperature and used within three hours of blood

collection. PRP was obtained after centrifugation at 125 g for 15

minutes. It was centrifuged once more at I 25 g for five minutes andthe occasional pellet oferythrocytes discarded. Platelet-poor plasma

(PPP) was obtained by centrifugation for I 5 minutes at 1 ,000 g.

When gel-filtered platelets were needed, venous blood was anticoag-

ulated with I volume of 84 mmol/L Na citrate, 64.7 mmol/L citric

acid, I I I mmol/L dextrose for 6 volumes of blood.’8 Ccntrifugation

at 125 g for 15 minutes yielded PRP with a pH of 6.5. This was

incubated five minutes at 37#{176}Cwith I U/mL apyrase (Sigma, St

Louis, grade I) and centrifuged for five minutes at 125 g. The

erythrocyte-poor PRP was then centrifuged at 1,000 g for ten

minutes. The platelet pellet was resuspended in Tyrode buffer, pH

6.5 (0.136 mol/L NaCI, 0.05 mol/L KCI, 8 mmol/L NaH2PO4, I

mmol/L MgCl2, I I .9 mmol/L NaHCO3, 5.5 mmol/L dextrose, 5

mmol/L HEPES) containing 0.35g/dL bovine serum albumin and I

U/mL apyrase. Following incubation for five minutes at 37#{176}C,theplatelet suspension was gel-filtered over Sepharose CL-4B, equili-

brated, and eluted with Tyrode buffer, pH 7.4, containing 0.35 g/dL

bovine serum albumin. The platelet count was adjusted to 8 x l0�

ML’. The gel-filtered platelets did not aggregate in the presence of

ADP unless fibrinogen was added.

Preparation and assay of platelet actomyosin. Platelet acto-

myosin was prepared according to Adelstein et al.’9 The assayconditions for the ATPase activity were 0.05 mol/L KCI, 2 mmol/L

MgCI2, 0.1 mmol/L CaCI2, 1 mmol/L ATP, 10 mmol/L Tris-l-ICl,pH 7.5, and I .4 mg platelet actomyosin in a total volume of 2 mL and

at 37#{176}C.After addition of I mL I 5% trichloroacetic acid, inorganicphosphate was determined according to Fiske-Subbarow.2#{176}

Fibrinogen peptides and monoclonal antibodies. Arg-Gly-Asp-

5cr (RGDS), Arg-Gly-Asp-Val (RGDV), Lys-Gly-Asp-Ser(KGDS), Arg-Gly-Glu-Ser (RGES), and Ala-Gly-Asp-Val

(AGDV) were obtained from Peninsula Lab (Belmont, CA). The‘y-400-4l I peptide was kindly provided by F. Hoffman-LaRoche

(Basle, Switzerland). The potency of the peptides was confirmed by

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PEPTIDES AND ANTIBODIES AFFECT CLOT TENSION 1881

fourfold increase of maximal tension and rate of tension

analyzing their inhibitory effect on platelet aggregation and fibrino-gen binding to platelets by the method of Kunicki et al.2’ The 1D50(concentration causing 50% inhibition of fibrinogen binding toplatelets) was 20 zmol/L and I 5 ,�mol/L respectively, for RGDS

and RGDV and >500 �mol/L for KGDS, RGES, and AGDV. The

7-400-41 1 peptide was four times less potent than RGDS and

RGDV in inhibiting platelet aggregation and fibrinogen-binding to

platelets as previously described.22

Monoclonal antibodies 10E5 and 7E3 were obtained from Dr BarryColler (SUNY, Stony Brook), T,0 from Dr Rodger P. McEver(University of Oklahoma, Oklahoma City), and A2A9 from Dr JoelS. Bennett (University of Pennsylvania, Philadelphia). They allbound to lIb-Illa and prevented platelet aggregation and fibrinogen-binding to platelets.2’356 Before use, the potency of the antibodieswas confirmed by their inhibitory effect on platelet aggregation

induced by ADP and a-thrombin. Monoclonal antibody SSA6,which bound to GPIIIa, was obtained from Dr Joel S. Bennett.

Tension measurements. The fibrinogen peptides were incubatedwith gel-filtered platelets for four minutes at 37#{176}Cin the presence of

l0�mol/L ADPand 0.5 mmol/L CaC12 in a final volumeofO.5 mL.Platelet concentration was 8 x I0� ML’. Following addition of 0.5mL PPP and a-thrombin (0.5 U/mL), the mixture was transferredinto glass cylinders (7 x 0.5 cm ID) plugged at one end with

parafilm. After eight minutes, the cylindrical clot was poured into a

Petri dish containing Tyrode solution, pH 7.4, at room temperature.

The clot was then tied at both extremities and the isometric tensionmeasured at 37#{176}Cas described.’3 In some experiments, peptides

were added to the Tyrode solution too.Monoclonal antibodies were incubated for five minutes at 37#{176}Cin

PRP. Isometric tension was measured in clots following a-thrombin

addition. Under these conditions, 7E3, which binds faster to stimu-lated than to unstimulated platelets, still shows substantial binding

to unstimulated platelets.6Since tension development is a function of platelet and fibrin

concentration,’6 controls were always included within each set ofassays and studied concurrently. Analyses were carried out in

triplicate, and differences between samples analyzed by the Studentt test.

Contraction in some experiments was measured in the absence ofan external load using the floating clot technique’6 whereby the rateofshortening ofa cylindrical clot freely suspended in Tyrode solution

at 37#{176}Cwas measured.Clot retraction from a glass surface was measured at 37#{176}Cin

aggregometer cuvettes (4.5 x 0.6 cm ID) as described by Gartnerand Ogilvie.23 The cuvettes were heated to “red” on a Bunsen burnerbefore use. a-thrombin at 0.5 U/mL final concentration was used.The platelet concentration in PRP was 4 x l0� iLL’. In some

experiments the mixture before clotting was rapidly layered over I 00

�zL of 20% sucrose in 0. 15 mol/L NaCI, I 0 mmol/L HEPES buffer,pH 7.4, and the clot loosened from the cuvette wall with a fine

needle.

Amino acid analysis. Fifty nanomoles of peptide was hydro-lyzed with 6N HCI for 22 hours at 106#{176}C.Following evaporation todryness, the residue was dissolved in I mL 0.01 N HCI; 0.8 mL was

then submitted to analysis in a JEOL Model JLC-6AH amino acidanalyzer. A single column system with four citrate elution bufferswas used. The analysis of peptides showed equimolar ratios for the

amino acids, confirming the specifications of the manufacturer

(Peninsula Lab, Belmont, CA). When RGDS was analyzed forevidence of thrombin-induced cleavage, 0. 1 mL (50 nmol RGDS) ofthe mixture (nonhydrolyzed) was diluted with 0.9 mL 0.01 N HCI

and 0.8 mL was then submitted for amino acid analysis.Electron microscopy. Clots fixed under isometric tension were

processed as described previously.’3 Briefly, the suspension buffer

was drained and replaced by 20 mL of a solution containing 10 mL

of the original Tyrode solution and 10 mL of 2% glutaraldehyde in

White’s saline.24 After five minutes at 37#{176}C,this solution was

drained and replaced by a mixture of 10 mL ofTyrode solution and10 mL of6% glutaraldehyde in White’s saline. After 30 minutes the

clot was removed and cut into small fragments so that the original

orientation of the clot was preserved. Clot fragments were then fixed

an additional 30 minutes in 3% glutaraldehyde in White’s saline.

The fragments were subsequently transferred to a solution of I %osmium tetroxide in a I 5 mg/mL solution of potassium ferrocyanidein distilled water (pH 7.4) for 90 minutes at 2#{176}C.After osmium

fixation, the samples were dehydrated in a graded series of ethanol

concentrations, then treated with propylene oxide and embedded inEpon. Sections were cut with the samples oriented along the axis oftension. The sections were then stained with uranyl acetate and lead

citrate to enhance contrast, and examined in a Philips 301 electron

microscope.Image analysis. Morphometric analysis was carried out using a

fully equipped International Systems Model 75 (Digital Processor,Milpitas, CA) interfaced to a mini-computer, Masscomp MC 535,

which runs the 5575 software under the Unix operating system. Thin

sections of all the clots were photographed on the same day and atthe same microscope settings. Random images of the clots wererecorded on 35-mm film at an on scope magnification of 720x . After

development the negatives were loaded into the morphometric

analysis system using a video input device connected to the Model

75. Images were projected on the videometer with a resolution of

5 1 2 x 5 1 2 pixels. The gray level corresponding to the fibrin clot was

pseudocolored and the total number of pixels associated with fibrinand platelets were representative of the percentage of the totalnumber of screen pixels. Results were calculated and represented asthe mean ± I SD of the sample mean. The statistical differences

were determined using the Student t test.

RESULTS

Effect offibrinogen peptides on clot tension under isomet-

nc conditions. The tension developed in platelet-rich clots

under isometric conditions is a function of platelet and fibrin

concentrations as well as initial cross-sectional area of the

clot.’5’6 Clots obtained from mixed gel-filtered platelets and

PPP, containing a final concentration of 4 x l0� platelets/

mL and 1.4 mg fibrin/mL, developed tension at a rate of 0.06

g/min/cm2 and reached a maximal tension of 0.85 g/cm2

after 20 minutes, (Fig I, Table 1). Clots obtained from

platelet-rich plasma of different donors developed tension at

rates between 0.08 and 0. 1 5 g/min/cm2 and maximal tension

varying between 1.1 and 1.72 g/cm2 (Table 2).

When RGDS or RGDV in a concentration of62.5 �smol/L

was included in the reaction mixture, there was a two-to

threefold increase in the rate of tension development under

isometric conditions as compared with the buffer controls,

and a five-fold increase when the concentration of the

peptides was 250 zmol/L (Fig 1, Table I). The maximal

tension was increased in the presence of RGDV, similarly to

the effects of RGDS (Fig 1). Addition of the nonspecific

peptides KGDS, RGES, or AGDV had no effect on isometric

tension. RGDV appears to be somewhat more potent than

RGDS in increasing the tension rate (Table I ). This effect of

RGDV is interesting in view of its increased potency in

inhibiting fibrinogen binding to isolated platelets as com-

pared with RGDS.” The �y-400-4I I peptide induced a

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Table 1 . Effect of Peptides on Rate of Isometric Tension Development

Peptide.

Concentration

(�unol/L)

. . . 2Peptides Used (Tension rate in g/cm 1mm)

RGDS RGDV KGDS AGES AGDV y-400-41 1

0 0.06 ± 0.004 0.06 ± 0.004 0.06 ± 0.004 0.06 ± 0.004 0.06 ± 0.004 0.06 ± 0.004

31.25 0.09 ± 0.109 0.19 ± 0.015 0.06 0.06 0.06 0.06

62.5 0.15 ± 0.013 0.20 ± 0.01 0.06 0.06 0.06 0.06

125 0.18 ± 0.014 0.22 ± 0.015 0.06 0.06 0.06 0.1 ± 0.013

250 0.29 ± 0.027 0.31 ± 0.018 0.06 ± 0.008 0.07 ± 0.007 0.05 ± 0.004 0.12 ± 0.08

500 - - - - - 0.15 ± 0.012

1.000 - - - - - 0.25 ± 0.015

1882 COHEN, BURK. AND WHITE

Initial tension rate was measured. Values are mean ± i SD (n = 3). Using the Student t test, P values for all concentrations of RGDS. RGDV, and

�y-40O-4 1 1 as compared with control are < .0 1 . P values for 250 �mol/L KGDS, RGES, and AGDV as compared with control are >6.

Time, mm

Fig 1 . Effect of RGDS on isometric tension development. Final

concentrations are indicated. One of three representative tracingsis shown.

development when the concentration of the peptide was I

mmol/L (Fig 2, Table I). Similar results were obtained

when peptides were included in the Tyrode solution sur-

rounding the clot. In these experiments, using gel-filtered

platelets and ADP, the fibrinogen concentration was half

that present in normal plasma. When the peptides were

added to PRP, which was then clotted with a-thrombin, the

tension was enhanced threefold when the concentration of

RGDS or RGDV was 250 zmol/L.

Effect of fibrinogen peptides on clot shortening under

isotonic conditions. When the free-floating clot technique

was used, the rate of shortening in the presence of 250

�mol/L RGDS was 40% more rapid than that obtained with

the control (Fig 3). Similar results were obtained with

RGDV (250 �tmol/L) or ‘y-400-41l (I mmol/L) and no

effect on clot shortening was observed with AGDV, KGDS,

or RGES. Consistent with the observations made under

isometric conditions, the rate of shortening was not

Table 2. Effect f Monoclonal Antibodies

on Tension Developm

Directed Against llb/Illa

ent

Agents Tension Rate Maximal Tension(jig/mL) (g/min/cm2) (g/cm2)

A2A9

0 0.15 ± 0.014 1.72 ± 0.08

50 0.037 ± 0.002 0.54 ± 0.04

100 0.022 ± 0.001 0.325 ± 0.01

200 0 0

7E3

0 0.087 ± 0.008 1.12 ± 0.08

25 0.06 ± 0.004 0.97 ± 0.015

50 0 0

1 0E5

0 0.087 ± 0.008 1.12 ± 0.08

100 0.12 ± 0.01 1.60 ± 0.035

200 0.12 ± 0.004 1.65 ± 0.037

T10

0 0.097 ± 0.01 1.4 ± 0.11

100 0.15 ± 0.007 2.12 ± 0.1

200 0.15 ± 0.009 2.4 ± 0.13

Control experiments carried out for each monoclonal antibody tested

at beginning and end of experiment. Since tension is a function of both

platelet and fibrin concentration, ie experiments carried out with controls

from a given donor are grouped together. Platelet concentration varied

between 3 x 10� �sL’ and 4 x 10� �L’ in the different groups. Initial

tension rate was measured. Maximal tension measured after 20 minutes.

Values are mean ± SD (n = 3). Using the Students t test, P values for all

concentrations of antibodies as compared with control are < .0 1 except

for 7E3, 25 M9 (P - .036).

influenced by the presence of peptides in the Tyrode solution

surrounding the clot.

When the glass tube clot retraction technique was used,

the rate of retraction from the glass surface during the first14 minutes following thrombin addition was retarded by 500

Mmol/L RGDS or RGDV or 2 mmol/L �y-400-4l I . Inaddition, increased adhesion to glass was observed in the

presence of these peptides. No such effect was observed in

the presence of AGDV, KGDS, or RGES. After I 4 minutes,

however, the clots containing RGDS, RGDV, or ‘y-400-41 I

detached from the glass and the rate of shortening increased.

When the clotting mixture was layered over sucrose and the

clots loosened from the glass surface immediately after

clotting, the rate of shortening was faster in the presence of

RGDS than in the control, and no delay was observed (Fig

4). Using the sucrose overlay technique, similar results were

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2.5

1.5

0.5

0

125

12

Time, mm

Fig 2. Effect of ‘y-400-41 1 on isometric tension development.Final concentrations are indicated. One of three representativetracings is shown.

0.7

0.6

0.5

0

Dl0

-I

5 10 15 20

Time, mm.

25 30

Fig 3. Effect of RGDS on rate of shortening of free-floating

clots. Log10 of clot length (I) was plotted as a function of time. #{149}.control; #{149}.250 Mmol/L RGDS. This pattern is representative ofthree experiments. In each experiment. RGDS enhanced the rateof shortening by approximately 40%.

PEPTIDES AND ANTIBODIES AFFECT CLOT TENSION 1883

obtained with RGDV and -y-400-41l, and no effect of

AGDV, KGDS, or RGES was observed.

�uM Effect of monoclonal antibodies on clot tension. Mono-clonal antibodies A2A9 and 7E3 decreased the maximal

1 ,000 tension as well as the rate of tension development, underisometric conditions, with total inhibition reached at concen-

trations of 200 zg/mL and 50 jsg/mL, respectively (Table

2). On the other hand, monoclonal antibodies T,0 and 10E5

increased the rate of tension development by 37% and 54%,

respectively, when used at a concentration of 100 �zg/mL

(Table 2). Monoclonal antibody 55A6, which binds to

GPIIIa, did not affect clot tension. The highest concentration

of antibody used was 200 zg/mL, enough to saturate all the

platelet receptor sites for fibrinogen, taking into account the

parameters of binding kinetics of all monoclonal antibodies

used.235’6

0 The monoclonal antibodies A2A9 (100 j�g/mL) and 7E3

(50 �tg/mL) inhibited clot shortening as measured by either

the floating clot procedure or the retraction technique.

However, the rate of shortening, using these techniques was

20 not affected by either T,0 or 10E5 used at concentrations of100 �tg/mL.

Effect ofpeptides and monoclonal antibodies on platelet

actomyosin A TPase activity. The effect of fibrinogen pep-

tides on platelet actomyosin ATPase activity was investi-

gated in order to exclude an effect on actin-myosin interac-

tion. The Mg�-stimulated activity of platelet actomyosin (50

nmol Pi/mg/min) was not affected by ‘y-400-4l 1, RGDS,

RGDV, KGDS, RGES, or AGDV at concentrations as high

as 500 �tmol/L. The monoclonal antibodies A2A9, 7E3, lOE9,

and T,0 also did not affect the ATPase.

Effect of thrombin on R-G bond of RGDS. Since an

Arg-Gly bond in fibrinogen a and �3 chains is susceptible to

thrombin, the effect of this enzyme on the R-G bond of

RGDS was investigated. A mixture of 500 �tmol/L RGDS

and 0.5 U a-thrombin/mL, preincubated for 30 minutes at

37#{176}C,was analyzed. As expected, no trace of free Arg was

detected, indicating that thrombin does not cleave RGDS.

Ultrastructure ofclots containingfibrinogen peptides and

monoclonal antibodies. Electron micrographs of control

clots fixed for ultrastructural study at intervals during

development of isometric tension revealed the progressive

orientation of fibrin strands and interacting platelet aggre-

gates in the long axis of force generation as previously

described’3 (Fig 5A). Thin sections of clots treated with

RGDS (Fig SB), ‘y-400-41 I (Fig SC), or RGDV (Fig SD)

revealed more platelet aggregates, confluence ofsmall aggre-

gates, and increased numbers of fibrin strands in the long

axis compared with control clots (Fig 5A). Groups of aggre-gated platelets and fibrin strands appeared to have moved

closer together to form a confluent, muscle-like mass during

exercise of increased contractile force. The compacted

appearance produced by treatment with RGD-containing

peptides was not observed in clots exposed to KGDS, RGES,

or AGDV. Evaluation of the control, RGDS-, RGDV-, or

‘y-400-41 1-treated platelet clots by image analysis reveal

several clear differences. The area of the photomicrograph

occupied by fibrin and platelet aggregates was significantly

greater at all RGDS, RGDV, and -y-400-4l I concentrations

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1884 COHEN. BURK. AND WHITE

C R C R C R CR

10mm 22mm 34mm 46mmFig 4. Effect of RGDS on clot retraction in glass tubes using the sucrose overlay technique. C. control; R. 500 �tmoI/L RGDS.

Photographs taken at different time intervals, as indicated. This pattern is representative of three experiments. Enhanced shortening was

also observed with 250 zmol/L RGDS.

Fig 5. Thin sections ofplatelet-fibrin clots fixed afterdevelopment of maximum ten-sion under isometric condi-

tions. (A) Linear orientation offibrin strands and platelet ag-

gregates in the long axis of the

clot fixed under isometric ten-sion. Clots exposed to 250�imol/L RGDS (B). 1 mmol/L

‘y-400-411 (C) or 250 pmol/LRGDV (D). demonstrated a

larger size and closer proximity

of platelet aggregates andincreased numbers of fibrin

strands. Together. the fibrinand platelets in peptide-

treated clots occupied a largerpercentage of the standardelectron micrograph frame

taken at the same magnifica-tion. compared with the same

structures in untreated clots orthose that had been exposed to

peptides that did not promoteincreased tension develop-

ment. Platelets were also incu-

bated with the monoclonal

antibodies A2A, (200 pg/mI)and 7E3 (50 pg/mI) (E and F.

respectively) before clot for-

mation and exposure to iso-metric conditions. Both anti-bodies inhibited platelet aggre-gation and platelet-fibrin inter-action. As a result. only singleplatelets. damaged cells orsmall aggregates consisting oftwo to three platelets were

apparent in the clots. and fibrinstrands were randomly dis-persed. Magnifications: A-D. x1.100; E and F. x 2.300. = 2pm.

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PEPTIDES AND ANTIBODIES AFFECT CLOT TENSION 1885

as compared with the control. Image analysis of RGDS-

treated clots shows a significant trend toward greater com-

paction with increasing concentrations of peptides. The

differences between the different concentrations of peptides

were, however, not statistically significant.

Clots obtained in the presence of A2A9 and 7E3 and fixed

under tension showed few platelets and platelet clumps and

less fibrin orientation in the direction of tension (Fig SE and

F). The monoclonal antibodies T,0 and IOE5 affected neither

the size of platelet clumps nor the orientation of fibrin

fibers.

DISCUSSION

The development of tension in platelet rich fibrin clots is

due to the platelet contractile ability and the interaction

between platelets and polymerizing fibrin. Our results show

that ‘y-400-41 1 and RGD-peptides increase the isometric

tension of platelet-rich clots, reflecting a more effective

platelet-polymerizing fibrin interaction, as confirmed by

ultrastructural studies. The decreased potency of the y-

400-41 1 peptide in enhancing tension development may be

related to its decreased potency in inhibiting platelet aggre-

gation and fibrinogen binding to platelets, as compared with

RGDS.22 A fivefold increase in the rate of tension develop-

ment under isometric conditions was observed in the pres-

ence of 250 �zmol/L RODS or RGDV. This effect was due to

the tetrapeptide and not to a thrombin-induced cleavage of

the peptide. The concentration of peptides, which enhances

tension, is of the same order of magnitude as that which

inhibits platelet aggregation in a plasma system.’#{176}Since the

clot is immersed in Tyrode solution during tension develop-

ment, it is possible that peptides diffusing from the clot could

affect the tension.23 This appeared not to be the case since the

tension developed was similar when the peptides were present

or absent in the Tyrode solution. It is unlikely that they alter

the actin-myosin interaction, since they had no effect on the

Mg�-stimulated activity of platelet actomyosin.

Image analysis of photomicrographs taken on thin sections

of control and peptide-treated platelet clots, fixed under

tension, supports the data obtained by isometric measure-

ments. Prior treatment with ‘y-400-4l I or RGD-peptides

resulting in increased tension caused fibrin strands and

platelet aggregates in the clots to be closer together. This

results in a larger area of the photomicrograph occupied by

these elements compared to the control. A direct correlation

between peptide concentrations and platelet-fibrin aggre-

gates could not be established perhaps because increasing

platelet-fibrin polymer interaction does not necessarily fol-

low a linear increase of platelet-fibrin clumps.

Contraction measurements were also carried out by mea-

suring the rate of shortening of free floating clots. Since no

unidirectional orientation of fibrin fibers is apparent in this

isotonic contraction sy5tem,’3 steric hindrance prevents

excessive shortening of the fibrin clots. Although lacking the

accuracy and sensitivity of isometric tension measurements,

the contraction measurements using the floating clot tech-

nique still resulted in 40% shortening in the presence of

-y-400-4l I and RGD-peptides. Similar results were obtained

when the peptides were present in the Tyrode solution

surrounding the clot.

When clot contraction was measured in glass tubes, a

different pattern was observed. The rate of retraction from

glass in the presence of ‘y-400-41 1 and RGD-peptides was

diminished during the first 14 minutes of clotting while it

increased after detachment from glass. There was no delay in

the rate of clot shortening in the presence of the peptides

when the clotting mixture was layered over sucrose and the

clot loosened from the glass surface. The control peptides

KGDS, ROES, and AGDV had no effect on clot retraction.

Gartner and Ogilvie23 and Hantgan25 observed a transiently

decreased rate of clot retraction in the presence of ROD-

peptides. Their results are probably due to increased adhe-

sion to glass. This effect was enhanced due to the strong

adherence of the clots to glass in tubes of small diameter on

account ofsurface tension. It is possible that RODS, RODV,

and ‘y-400-41 1 , by interacting with platelets, change the

physical characteristics of the clot, which then adheres

strongly to glass. This effect, when added to the relatively

random orientation of fibrin fibers under isotonic condi-

tions,’3 renders the clot retraction technique in glass tubes

unsuitable when accurate determinations of tension are

required. Platelet-rich clots under isometric conditions, on

the other hand, exhibit an orientation of all fibrin fibers in

the direction of tension,’3 avoiding the hindrance of randomly

oriented fibers and rendering this technique ideally suited for

assessing increase or decrease of clot tension. For example,

cycles of increased and decreased tension as a function of

Ca� concentration are easily observed under isometric

‘4,5

Studies of mutual competition inhibition show that

numerous monoclonal antibodies directed against the llb-

lila complex bind to different epitopes on the heterodimer

complex.26 The differential effects of some monoclonal anti-

bodies on fibrinogen-binding to platelets and platelet aggre-

gation on the one hand and on isometric tension development

of platelet-rich fibrin clots on the other hand, confirm the

unique topography of the epitopes on the lIb-Illa complex

and provide evidence for distinct receptor domains for fibrin-

ogen and polymerizing fibrin. Since tension development

depends on fibrin binding, and because the monoclonal

antibodies A2A9 and 7E3 inhibit isometric tension develop-

ment, the epitopes for A2A9 and 7E3 must be closer to the

receptor for polymerizing fibrin than those for T,0 and bE5.

The relative small increase in tension obtained with the

monoclonal antibodies T,0 and IOE5 may point to an

increased availability of fibrin receptor sites when the fibrin-

ogen sites are blocked. The presence of A2A9 and 7E3 in

platelet-rich clots results in sparse platelet-fibrin clumps.

The absence of increased confluence of platelet-fibrin clumps

in the presence of T,0 and bE5 may be associated to the

modest increase in tension produced by these monoclonal

antibodies.

The tension increase by y-400-4b I and ROD-peptides

may suggest an allosteric mechanism whereby occupancy of

the fibrinogen receptor site induces a positive cooperativity in

relation to the binding of polymerizing fibrin to activated

platelets. Since thrombasthenic platelets, which are deficient

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1886 COHEN. BURK. AND WHITE

in the lIb-Illa complex, do not develop tension in a fibrin

clot,’3 lIb-Illa appears to contain domains for the binding of

polymerizing fibrin. Another possibility is that clustered

glycoproteins enhance extracellular fibrin polymer binding

to platelets, thus extending the model proposed by Isenberg

et a122 whereby OPIIb-Illa clustering induced by receptor

occupancy promotes the intracellular association with the

actin cytoskeleton. An effect of the peptides or antibodies in

the fibrin polymerization process resulting in altered tension

cannot be ruled out. This is unlikely, however, since no

difference in strands width, length or appearance could be

observed in thin sections by electron microscopy.

Fibrin monomer and soluble oligomeric fibrin appear to

bind to the platelet receptor for fibrinogen,25’27 but polymer-

izing fibrin, resulting in a clot, probably binds to different

receptors. We considered the possibility that fibrinogen

binds to thrombin or ADP-stimulated platelets before it is

converted to fibrin and polymerizes with other fibrin mole-

cules. This is unlikely since in our experiments ‘y-400-4I I

and ROD-peptides were added to activated platelets before

the addition of fibrinogen and, at the concentrations used,

would have prevented fibrinogen binding to the GPIIb-IIIa.

Niewiarowski et al2t showed that unstimulated normal or

thrombasthenic platelets bind to polymerizing fibrin while

Tuszynski et a127 failed to show binding of fibrin monomer to

either the membrane or cytoskeleton of activated throm-

basthenic platelets. Hantgan et a129 reported that platelets

interact with polymerizing fibrin only after activation. These

conflicting reports may indicate that polymerizing fibrin

binds to more than one platelet receptor. Binding to a specific

receptor(s) may be crucial for tension development in plate-

let-rich clots. An effective tension requires adherence of

polymerizing fibrin to platelet surface receptor on the one

hand and anchoring of a contractile cytoskeleton to receptors

on the cytoplasmic side of the membrane on the other.

Overwhelming evidence shows that OPIIb-IIIa of acti-

vated platelets is involved in the binding of actin to the

cytoplasmic membrane either directly or through another

protein.�#{176}’32 The receptor for extracellular polymerizing

fibrin, necessary for tension development, may be a domain

of OPlIb-Illa not involved in fibrinogen binding and/or

another glycoprotein. Binding of polymerizing fibrin to the

von Willebrand protein, inducing binding of the latter to

platelet glycoprotein lb is another possibility.33

ACKNOWLEDGMENT

We thank Dr David Green (Northwestern University) for carefulreview of the manuscript; Dr Arther Veis (Northwestern University)

for fruitful discussions; Ruth Fullerton (Northwestern University)for the amino acid analysis; Dr John W. Fenton (New York State

Department of Health, Albany) for providing the purified a-

thrombin; Dr Joel S. Bennett (University of Pennsylvania), Dr Barry

Coller (SUNY, Stony Brook) and Dr Rodger McEver (University ofOklahoma) for providing the monoclonal antibodies; and F. Hoff-

man-La Roche (Baste, Switzerland) for providing the -y-400-41 I

peptide.

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1989 73: 1880-1887  

I Cohen, DL Burk and JG White glycoprotein IIb-IIIa complex on the development of clot tensionThe effect of peptides and monoclonal antibodies that bind to platelet 

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