THROMBOSIS RESEARCH Vol . 13, No. 2, pp . 267-277 . © Perpamon Press Ltd . 1978 . Printed in Great Britain .

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From the Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, and the Department of Surgery, Harvard Medical School and Beth Israel Hospital, Boston, Massachusetts 02115 USA

(Received 3 .3 .1978 ; in revised form 26 .6 .1978 . Accepted by Editor M .I . Barnhart)

ABSTRACT Chondroitin 6-sulfate (Ch6-S) a glycosaminoglycan (GAG), has been shown to inhibit collagen fibrillogenesis and collagen induced platelet aggregation . Complexation of soluble microfibrillar collagen with Ch6-S at low pH followed by saline dialysis results in the stabilization of 300K wide microfibrillar aggregates with no banding in the electron microscope . These structures, which may be intermediates in collagen fibrillogenesis, do not aggregate platelets or cause serotonin release, whereas fibrils formed from uncomplexed microfibrillar collagen induce platelet aggregation and serotonin release . Neutral complexation of microfibrillar collagen with Ch6-S does not inhibit fibril formation or platelet aggregation, Soluble Ch6-S does not interfere with platelet aggregation in response to soluble microfibrillar collagen, indicating that Ch6-S does not block sites on the platelet membrane or on collagen fibrils which may be specifically involved in the collagen-platelet interaction . These results imply that GAG complexes with collagen may be suitable as blood compatible materials .

INTRODUCTION The role of collagen in hemostasis and the collagen-platelet interaction have been the topics of extensive research . It is widely accepted that exposure of subendothelial collagen fibers to blood is a major event in initiation of hemostasis and in the genesis of thrombosis due to vascular injury [1] . The detailed mechanism of the collagen-platelet interaction is still unclear [2], but it is likely that the process involves two separate events :

a To whom correspondence should be addressed . 1 This work was taken in part from the Ph .D . thesis of F .H .S . submitted to the Department of Mechanical Engineering, M .I .T . on May S, 1977 . 267



adhesion of platelets to collagen and platelet

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- platelet aggregation .

Deykin and Jaffe [3] reported that soluble tropocollagen requires a lag phase of 180 seconds to initiate platelet aggregation in vitro, whereas microfibrillar (600A wide) and fibrillar collagen initiate platelet aggregation after a lag phase of only 60 seconds . The lag phase is probably composed of at least three separate events : (1) tropocollagen-tropocollagen aggregation to form microfibrils, (2) platelet adhesion to collagen microfibrils, and (3) release of platelet constituents and platelet aggregation . Steps (2) and (3) appear to occupy about 60 seconds of the lag phase . Inhibition of collagen assembly with glucosamine [4] increases the lag period and inhibits platelet release . These observations suggest that the rate controlling step in vitro is collagen aggregation and that inhibition of such collagen interactions should inhibit collagen induced platelet aggregation . In the following studies we examine the effect of chondroitin 6-sulfate complexation with microfibrillar collagen on collagen - collagen interactions and collagen induced platelet aggregation .

METHODS Acid soluble rat tail tendon collagen (RTT, microfibrillar collagen) . Acid soluble rat tail tendon collagen was prepared using tails from 7 week old female albino rats (Pel-Freez, Biologicals, Inc ., Rogers, Arkansas) . Since the precise morphology of the precipitated collagen fibrils depends sensitively on the detailed sequence of preparatory steps, we outline our preparative procedure in some detail below as well as in Figure 1 . All steps were carried out at 4 ° C . The tails were first washed with distilled water, and the tendons were removed using a pair of wire strippers . Tendon fibers were allowed to swell and dissolve for at least 24 hours in 0 .05M acetic acid solution at 4 ° C . Figure 1 is a flow diagram for this process, which is based on the procedure of Michaeli [5] . The collagen dispersion was centrifuged for 1 hour at 50,000 g to separate soluble from insoluble collagen . After soluble collagen was precipitated by adding S% (w/v) solid NaCl, the solution was allowed to stand overnight at 4 ° C . Precipitated collagen was pelleted by centrifugation at 2,000 g for 10 minutes in the cold and redissolved in 0 .OSM acetic acid solution . Centrifugation at 50,000 g for 1 hour removed any insoluble material . Soluble collagen was twice reprecipitated, centrifuged at 2,000 g, redissolved, centrifuged at 50,000 g, and then dialyzed versus 0 .02M Na2HP04 . The precipitate was centrifuged at 2,000 g for 10 minutes, washed with distilled water, lyophilized, and stored at -10 ° C . Low pH complexes of microfibrillar collagen and chondroitin 6-sulfate . Acid soluble rat tail tendon collagen was dissolved 0,1% (w/v) in 0 .05M acetic acid (pH 3 .5) and centrifuged at 50,000 g for 1 hour in the cold to remove (Grade B, insoluble material . Chondroitin 6-sulfate from shark cartilage Calbiochem, San Diego, Calif .), was dissolved 0 .1% (w/v) in 0 .05M acetic acid rapid and slowly added (0 .04 ml/min) to the collagen solution followed agitation . The mixture was left to stand overnight at 4 ° C and then dialyzed at 4 ° C versus saline (0 .9% w/v NaCl) .


Neutral complexes of rat tail tendon collagen and chondroitin 6-sulfate . After a 1 hour centrifugation at 50,000 g, acid soluble microfibrillar collagen was dissolved 0 .1% (w/v) in O .OSM acetic acid and dialyzed 24 hours versus saline . Chondroitin 6-sulfate was dissolved 0 .1% (w/v) in saline (0 .9% w/v NaCl) and added slowly to saline dialyzed microfibrillar collagen .

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Purity . Acid soluble rat tail tendon collagen solutions were tested for purity by amino acid analysis, optical rotatory studies, intrinsic viscosity measurements, and solid state infrared spectroscopy . All tests indicated that this preparation was composed of 100% native collagen . According to Michaeli [5] this procedure isolates Type I collagen . Protein . Collagen concentrations were determined from amino acid analysis based on the amount of glycine and hydroxyproline in the sample . PlateletRich Plasma . Whole blood was collected by venipuncture from a male donor who had not ingested any drugs including aspirin for 2 weeks prior to donation . Blood was anticoagulated with 1/10 sodium citrate 3 .8% (w/v) after discarding the first 5 .0 ml . After a 20 minute room temperature centrifugation at 600 g, the platelet rich plasma (PRP), was transferred to a polypropylene tube using a siliconized glass pipet and was stored at room temperature . Using a plastic pipet, 0 .5 ml samples of PRP were incubated for two minutes at 37 ° C in a siliconized cell (Chronolog, Broomall, Pa .) stirred by a teflon flea magnet . The cell was then placed in an aggregometer (Chronolog, Broomall, Pa .), and the light transmittance was measured after the addition of collagen or glycosaminoglycan-modified collagen . PRP was used within 2 hours of venipuncture . Serotonin Release [6] . To each 40 ml of citrated whole blood used to make PRP was added 3 .0 microcuries of 2-C14-5-hydroxytryptamine (Amersham Searle, Arlington Heights, I11 .) . PRP from the platelet aggregation test was spun at 16,000 rpm in a Brinkmann Eppendorf centrifuge (Brinkmann Instruments, Westbury, N .Y .) for 2 minutes to pellet the platelets . To 5 .0 ml of AquasolR (New England Nuclear, Boston, Mass .) was added 100 microliters of each sample super natant . Each sample was counted for 10 minutes in a liquid scintillation counter (Mark I, Nuclear Chicago, Chicago, Illinois) . The percentage of serotonin incorporated into platelets that is subsequently released was calculated from the following formula : % = 100 [S-PPPI/[PRP-PPP] where S is the sample counts, PPP is platelet poor plasma counts, and PRP is platelet rich plasma counts . In most cases serotonin release tests were done after 10 minutes of incubation of PRP with the test material . TransmissionElectron Microscopy . Solutions or dispersions of rat tail tendon collagen and glycosaminoglycans were dropped from a Pasteur pipet on a carbon coated plastic reinforced copper grid (Ernest F . Fullam, Inc ., Schenectady, New York) . Excess dispersion was removed by gently applying filter paper to the edge of the grid . The sample was stained with aqueous phosphotungstic acid pH 7 .0 (Ernest F . Fullam, Inc ., Schenectady, New York) for several seconds . Excess stain was removed with filter paper, and the sample was viewed in a Jeolco 1000 transmission electron microscope (Jeolco U .S .A . Inc ., Medford, Mass .) . Laser Light Scattering Spectroscopy [7] . Measurements were made at a scattering angle of 90" and a temperature of 4"C using an argon ion laser . Scattered light was Fourier transformed using a phototube . From the photocurrent decay constant an average diffusion coefficient, D, was obtained . All samples were centrifuged 1 hr at 50,000 g in the cold before D was measured . RESULTS At 4 ° C and pH 3 .5 acid soluble rat tail tendon collagen, observed in the transmission electron microscope at magnifications of up to 165,000 X appeared to consist of highly swollen microfibrils . These structures, which were about 100A wide, could not be seen clearly even at high magnifications . In agreement with transmission electron microscopy, laser light scattering studies

2 70


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indicated that the average diffusion constant was quite small (0 .015 x 10- 7 cm 2 /sec) suggesting the presence of long thin collagen aggregates . Using the Stokes-Einstein relationship [8] and the Perrin equation [9] axial-diameter Rat Tails


+ O .OSM Acetic. Acid 4° C

Dispersion of Collagen Discard Pellet

Centrifuge 1 hr $ 50,000g Supernatant Soluble Collagen W + 5% NaCl (w/v) Precipitation

Discard Supernatant

Centrifuge ,000g, 10 min . Dissolve Pellet in 0 .05M Acetic Acid oluble R .T .T . Dial yyze Versus 0 .02M Na2HP04 Centrifuge Discard Supernatant 2,000g, y 10 min . Pel let Lyophilize

FIGURE 1 . Rat tail tendon collagen preparation procedure [5] . Rat tail tendon collagen is solubilized and purified by dissolution in 0 .05M acetic acid, high speed centrifugation, precipitation with 5% (w/v) sodium chloride, resolubilization in 0 .0SM _ acetic, and dialysis versus Na 2 HP04 . All steps are carried out at 4 ° C . ratios in excess of 10 3 are required to give a model aggregate with a diffusion constant of 0 .015 Ficks . Saline dialyzed (24 hours) acid soluble rat tail tendon collagen consisted of long thin (S0-10OX wide) microfibrils . Although these microfibrils appeared to possess some form of banding, it was impossible to determine if Above this a high degree of lattice only 6401 periodicity was present . imperfection caused the smearing out of bands, which are seen in Figure 2 . Hereafter, we will refer to acid soluble rat tail tendon collagen as microfibrillar collagen . Saline dialyzed microfibrillar collagen induced platelet aggregation after a lag time of about 3 .0 minutes as shown in Figure 2 . Serotonin release averaged 60+5% for saline dialyzed microfibrillar collagen as listed in Table 1 . Low pH complexes of microfibrillar collagen and chondroitin 6-sulfate, were composed of long thin microfibrillar aggregates ° as shown in Figures 3, 4, and S . These microfibrillar aggregates were 200-300A wide and lacked observ-

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able periodicity . Lateral associations resulting in widths above 30OX were not seen, but junction points where several microfibrillar aggregates cross were present . Addition of increased amounts of chondroitin° 6-sulfate (Figures 3, 4, and 5) did not significantly affect the basic 200-300A microfibrillar subunit, although the density of these aggregates increased with increased amounts of chondroitin 6-sulfate .


FIGURE 2 . Transmission electron micrograph and aggregometer curve (redrawn from actual tracings) for saline dialyzed microfibrillar collagen . Samples were stained with 0 .5% (w/v) phosphotungstic acid (PTA) adjusted to pH 7 and viewed at 33,000 X . Aggregometry for saline dialyzed microfibrillar collagen is at a concentration of 16 mg/ml at 37 ° C . Low pH complexation of chondroitin 6-sulfate with microfibrillar collagen resulted in two fractions : an insoluble precipitate phase composed of chondroitin 6-sulfate complexed with microfibrillar collagen and a soluble phase composed primarily of soluble microfibrillar collagen . Platelet aggregation by unfractionated microfibrillar collagen treated with 5% (w/w) chondroitin 6-sulfate was preceded by a lag phase of about 4 .5 minutes as seen in Figure 3 . Fractionation by high speed centrifugation (16,000 rpm for 2 minutes in a Brinkmann Ependorf centrifuge) separated soluble microfibrillar collagen from insoluble complexes of chondroitin 6-sulfate and microfibrillar collagen . Platelet aggregating activity of soluble microfibrillar collagen was similar to the untreated control (Figure 2) . In contrast saline washed insoluble collagen - chondroitin 6-sulfate complexes did not aggregate platelets, and did not cause serotonin release (within experimental error, + 5%), Microfibrillar collagen treated with 10% (w/w) chondroitin 6-sulfate at low pH initiated platelet aggregation after a lag phase of about 5 .0 minutes . Serotonin release was 15+5% for this unfractionated mixture . Treatment with 20% (w/w) chondroitin 6-sulfate at low pH resulted in an unfractionated mixture of precipitate (see Figure 5) and soluble microfibrillar collagen that did not aggregate platelets or cause serotonin release . Neutral precipitation of acid soluble rat tail tendon collagen with 22% (w/w) chondroitin 6-sulfate produced hybrid SLS/FLS crystallites 1500-3000A in width . Platelet aggregation in response to these hybrid crystallites occurred after a lag phase of 3 .0 minutes (Fig . 6) . Addition of soluble chondroitin 6-sulfate to PRP did not affect platelet



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aggregation in response to microfibrillar collagen (Figure 7) . TABLE 1 Serotonin Release Data for Low pH Precipitated Microfibrillar Collagen Sample

% Serotonin Released

Saline dialyzed microfibrillar collagen


Microfibrillar collagen treated with 5% (w/w)


chondroitin 6-sulfate soluble fraction insoluble fraction Microfibrillar collagen treated with 10% (w/w)

58+6 0 15+5

chondroitin 6-sulfate Microfibrillar collagen treated with 20% (w/w)


chondroitin 6-sulfate

FIGURE 3 . Transmission electron micrograph and aggregometer curve for microfibrillar collagen treated with 5% (w/w) chondroitin 6-sulfate at low pH . Samples were stained with 0 .5% PTA and viewed at 25,000 X . Aggregometry was done at a concentration of 20 mg/ml and 37 ° C for mixture soluble, and insoluble fractions .


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9 c


Cn E-S



FIGURE 4 . Transmission electron micrograph and aggregometry curve for microfibrillar collagen treated with 10% (w/w) chondroitin 6-sulfate at low pH . Samples were stained with 0 .5% (w/v) PTA Aggregometry was done at and viewed at 25,000 X . a concentration of 22 mg/ml at 37°C .

FIGURE 5 . Transmission electron micrograph and aggregometry curve for microfibrillar collagen treated with 20% (w/w) chondroitin 6-sulfate at low pH . Samples were stained with 0 .5% (w/v) PTA and viewed at 50,000 X . Aggregometry was run using 19 mg/ml at 37 ° C .




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9 E



Y 9-


TI E 1


Transmission electron micrograph and FIGURE 6 . aggregometry curve for microfibrillar collagen treated with 20% (w/w) chondroitin 6-sulfate at neutral pH . Samples were stained with 0 .5% (w/v) PTA and viewed at 12,000 X . Aggregometry was run using 22 mg/ml at 37 °C .

9 c 7 0 0 ='



cN 5 0

Fr c P


Collagen Ch6-S

2 0


I I 1 1 7 9 II 5

Time (min .) FIGURE 7 . Inhibition studies . Aggregometer curve for saline dialyzed microfibrillar collagen (20 mg/ml) in the presence of soluble chondroitin 6-sulfate (20 mg/ml) at 37 ° C .

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DISCUSSION The smallest fibrous component of tendon is believed to be a microfibril [12] which is composed of many pentamers (S quarter-staggered collagen molecules) lined up end to end . Microfibrils are about 36A wide [13,14] and may extend the entire length of the fibril . Saline dialyzed acid soluble rat tail tendon collagen as described in the Methods section appears to be a mixture of microfibrils and microfibrillar aggregates . Microfibrillar collagen initiates platelet aggregation only after a 3 .0 minute lag phase (Figure 2) . Laser light scattering data do not support the presence of a significant fraction of isolated single collagen molecules in this preparation . A lag phase in excess of 1 .0 minute [3,4] reflects the time required for collagen to collagen interactions which precede collagen induced platelet aggregation . It appears that microfibrillar collagen is unable to initiate platelet aggregation . This result suggests that inhibition of fibrillogenesis of microfibrillar collagen should also prevent the induction of platelet aggregation . Complexation of microfibrillar collagen with chondroitin 6-sulfate at low inhibits collagen fibrillogenesis even after the complex is dialyzed versus 1% sodium chloride . These complexes are similar in size to intermediates seen Platelet aggregation actin in vitro studies of collagen aggregation [15] . ivity of these modified collagens cannot be detected by either aggregometry


(Figs . 3 and 5) or serotonin release ; its absence is not due to the presence of soluble chondroitin 6-sulfate (Fig . 7) . Chondroitin 6-sulfate inhibition of collagen fibrillogenesis is related to its role in stabilizing microfibrillar collagen at neutral pH . Monosaccharides such as glucose and galactose also inhibit collagen fibrillogenesis [16] possibly by stabilizing water structures around collagen . Chondroitin 6-sulfate may bind to positively charged residues on collagen [17] decreasing intermolecular interactions and also stabilizing water structure . Aggregates greater than 300A wide are therefore necessary to initiate platelet aggregation . Uncomplexed microfibrillar collagen (Fig . 3-soluble fraction) at concentrations of 20 mg per ml is capable of initiating platelet aggregation . In the presence of chondroitin 6-sulfate, as complexes form to reduce the amount of soluble microfibrillar collagen, the lag time increases . If collagen to collagen interactions are necessary for collagen induced platelet aggregation - unless aggregation is a concentration independent, zero order reaction - it is expected that the reaction rate (and lag time) is proportional to the collagen concentration raised to some power . Another consideration is that addition of increasing amounts of chondroitin 6-sulfate at low pH fractionates microfibrillar collagen by size . Smaller aggregates require larger amounts of chondroitin 6-sulfate to cause coprecipitation and have longer lag times in platelet aggregation .

In contrast to low pH complexation, neutral complexation With chondroitin 6-sulfate (Figure 6) results in hybrid crystallites 1500-3000A wide . These crystallites aggregate platelets in a similar manner as uncomplexed microfibrillar collagen (Figure 2) . Recent evidence [11] indicates that all fibrillar forms of collagen in which the molecules are organized parallel too each other are active in platelet aggregation . This implies that platelet aggregation activity is net associated with a single lattice structure .



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The observation that low pH complexation of chondroitin 6-sulfate with collagen blocks collagen induced platelet aggregation suggests that GAG modified collagen may be useful as a vascular prosthesis or in devices that come in contact with blood .


We wish to acknowledge the assistance and helpful advice of Dr . W . J . Landis and Miss Margaret Hammett of the Department of Orthopaedic Surgery, Children's Hospital Medical Center, Boston, Massachusetts relating to the electron microscopic aspect of this work . We thank Drs . G .N . Wogan and D . F . Waugh for providing us with access to centrifugation facilities and to Dr . D . Michaeli for several useful discussions . This work was supported by the National Heart, Lung, and Blood Institute, Contract No . NIH N01-HV-4-2969 to I .V .Y . and grants nos . HL11414, HL14322, and HL2D079 to E .W .S .

REFERENCES 1 . FUGUES, J . Accolement des plaques aux structures conjonctives perivasculaires . Thromb . Diath . Haemorrah . 8, 241, 1962, 2 . MICHAELI, D ., and K .G . ORLOFF, Molecular considerations of platelet adhesion . In Progress in Hemostasis and Thrombosis . T . H . Spaet, Editor . Gune and Stratton, New York . 3, 29, 1976 . 3 . JAFFEE, R . and D . DEYKIN . Evidence for a structural requirement for the aggregation of platelets by collagen . J . Clin . Invest . 53, 875, 1974 . 4 . BRASS, L .F . and H .B . BENSUSAN . The role of collagen quaternary structure in the platelet :collagen interaction . J . Clin . Invest . 54, 1480, 1974 . 5 . MICHAELI, D . Studies on the molecular basis of collagen-platelet interaction and synthesis of peptides that will inhibit this interaction . NIH-NOl-HB-4-2984-1 . Available from National Technical Information Service, 5285 Port Royal Road, Springfield, Virginia 22151, 1975 . 6 . BUCKINGHAM, S ., and E .W . MAYNERT . The release of 5-hydroxytryptamine, potassium and amino acids from platelets . J . Pharm . Exp . Ther . 143, 332, 1964 . 7 . COHEN, R . J ., JEDZINIAK, J .A ., and BENEDEK, G .B . Study of the aggregation and allosteric control of bovine glutamate dehydrogenase by means of quasi-elastic light scattering spectroscopy . Proc . R . Soc . A345, 73, 1 .975 . 8 . BUECHE, F . 73, 1962 .

Physical Properties of Polymers .

Interscience, New York .

9 . PERRIN, F . Mouvement brownien d'un ellipsoide 7, 1, 1936 .

(II) .

J . Phys . Rad .

10 . SCHMITT, F .O . Macromolecular interaction patterns in biological systems . Proc . Amer . Phil . Soc . 100,476, 1956 . 11 . MUGGLI, R ., and BAUMGARTNER, H . R . Kollagen-induzierte plattchen aggregation : aktivitat von kollagen-polymorphem . Schweiz . med Wschr . 105, 505, 1975 .

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12 .



BAER, E ., GATHERCOLE, L .J ., and KELLER, A . Structural hierarchies in tendon collagen : an interim summary . Proceedings Colston Conference . 26 :189, 1974 .

13 . SMITH, J .W . Molecular pattern in native collagen . Nature . 219, 157, 1968 . 14 . MILLER, A ., and WRAY, J .S . Molecular packing in collagen . Nature . 230, 437, 1971 . 15 .

TRELSTAD, R .L ., HAYASHI, K ., and GROSS, J . Collagen fibrillogenesis : intermediate aggregates and suprafibrillar order . Proc . Natl . Acad . Sci . U .S .A . 73, 4027, 1976 .

16 . HAYASHI, T ., and NAGAI, Y . Factors affecting the interactions of collagen molecules as observed by in vitro fibril formation . J . of Biochem . 72, 749, 1972 . 17 . STEVEN, F .S ., KNOTT J ., JACKSON, D .S ., and PODRAZSKY, V . Collagenprotein-polysaccharide interactions in human intervertebral disc . Biochem . Biophys . Acta . 1,8, 1969 . 18 . WOOD, G .C ., and KEECH, M .K . The formation of fibrils from collagen solutions . Biochem . J . 75, 588, 1960 .

Glycosaminoglycan inhibition of collagen induced platelet aggregation.

THROMBOSIS RESEARCH Vol . 13, No. 2, pp . 267-277 . © Perpamon Press Ltd . 1978 . Printed in Great Britain . 149-3S4S/ 78/OW I 0721 S0 _ 2 .00/0 GLY...
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