THROMBOSIS RESEARCH 59; 509~520,199O 00493848/90 $3.00 + .OO Printed in the USA. Copyright (c) 1990 Pergamon Press pk. All rights reserved.

COLLAGEN-PLATELET INTERACTION: TYPE XI COLLAGEN-INDUCED PLATELET AGGREGATION T. M. Chiang, M. Cremer, and A. H. Kang From the V.A. Medical Center and the Departments of Medicine and Biochemistry, University of Tennessee, Memphis, TN 38104, U.S.A.

(Received

27.3.1990;

accepted

in revised form 22.51990

by Editor N.U. Bang)

ABSTRACT Type XI collagen in its native fibrillar but not in soluble monomeric form mediates human platelet aggregation and release of adenosine triphosphate in a dose-dependent manner. Its action is inhibited by aspirin. Type XI collagen also increased radiolabelled phosphate incorporation into protein bands with molecular weights of 42 KDa and 22 KDa, respectively. In contract, these events were not observed in platelets incubated with type IX collagen. These results suggest that the fibrillar type XI collagen has the same ability as other types of collagen to induce human platelet aggregation. ODUCTION Collagen exerts important biological effects on various cells including fibroblasts ( 1 ), macrophages ( 2 ), and platelets ( 3-8 ). Recent studies .. indicating the existence of heterogeneity in the collagen structure have made it necessary to examine the properties of each of the specific types of collagen in their interactions with these cellular elements. At least twelve Key Words:

collagen,

platelet

aggregation,

509

protein

phosphorylation,

aspirin

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genetically distinct types of collagen have been discovered. Of these, both soluble and fibrillar forms of types I, II, and III collagen have been demonstrated to mediate platelet aggregation and the release reaction in vitro ( 3-6 ). Collagen types IV, V, and VI can also aggregate platelets and induce the release reaction but only in fibrillar form ( 7-9 ). The ability of the remaining types of collagen to interact with platelets has not been investigated. Types XI and IX collagens have been isolated from cartilage and chemically chatacterized ( 10 ), but the ability of these collagens to interact with human platelets has not been studied. It has been reported that type XI collagen can cause arthritis in rats ( 11 ). Other biological properties of this type of collagen has not been identified. We have examined the ability of purified type XI but not type IX collagen to interact with human platelets. We report here that type XI collagen can induce platelet aggregation and the release reaction when added to human platelet-rich plasma in the native fibrillar form but not in soluble or amorphous forms. MATERIALS

AND METHODS

Preparations of Platelets: Human blood was collected from healty volunteers not ingesting any drugs after an overnight fast, and put into polyproylene tubes containing 0.1 volume of 3.8% sodium citrate as described previously ( 6 ). A two-syringe technique was employed, with the blood collected in second syringe being used for preparation of platelet-rich plasma (PRP). PRP was prepared by centrifuging the titrated blood at room temperature for 10 minutes at 226 x g. Whole blood and PRP were exposed to only plastic surfaces or siliconized vessels. The platelet counts of the PRP ranged from 200,000 to 350,000 per ~1. Equal volumes of PRP was mixed with 0.01 M Tris/0.14 M NaCl/O.OOl M EDTA, pH 7.4 (Tris-EDTA buffer) and centrifges at 1,500 x g for 5-10 mintes. The washing and centrifugation steps were repeated and the washed platelets were resuspended in the Tris-EDTA buffer at a concentration of 400,000 to 600,000 platelets&l. This preparation is referred to, hereafter, as “washed platelets”. Preparation of Collagens: Types II, XI, and IX collagens were isolated from fetal bovine cartilage by limited pepsin-digestion ( 10 ). Each type of collagen was recovered from the supernatant as a precipitate by a stepwise addition of NaCl to 0.86 M, 1.2 M, and 2.0 M, respectively. Types XI and IX collagens were further purified by dissolving the precipitates in 0.5 M acetic acid containing 0.8 M and 1.2 M NaCl, respectively. After centrifugation at 100,000 x g for 30 minutes in an ultracentrifuge (Model L-75, Beckman, Norcross, GA), types XI and IX were recovered from the supernatant by dialysis against 0.5 M acetic acid containing 1.2 M NaCl or

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3.0 M NaCl, respectively.

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The last three steps were repeated twice again to Collagen samples assure the removal of contaminating types of collagens. were dissolved in 0.5 M acetic acid (1.2 mg/ml) and dialyzed in 20 mM phosphate/l30 mM NaCl, pH 7.2 (PBS) at 4OC. Under these conditions, type XI collagen formed native type fibrils (see Figure 3), in contrast to type II Purity of type XI collagen or type XI collagen which remained in solution. was confirmed by SDS gel electrophoresis and CM-cellulose chromatography which revealed 1: 1: 1 stochiometry of u -chains. Gel electrophoresis and immunoblots of type XI collagen showed the presence of high- and low-molecular weight bands which stained comparably; type II collagen was not seen. Both type XI and IX collagen preparations were free of type II collagen based on enzyme-linked immunosorbent assay ( 12-13 ) using monoclonal antibodies specific for native type II collagen. Platelet Aggrepation and Release Reaction: Platelet aggregation and the “release reaction” were studied by using a Chrono-Log Lumiaggregometer (Chrono-Log Corp., Havertown, PA) ( 14-15 ). An aliquot of 0.45 ml PRP and 25 pl of Luciferase Luciferin Reagent (Chrono-Log Corp.) were pipetted into the siliconized cuvette and stirred at a constant speed of 1,100 rpm at 37OC. The collagen sample was added in volumes of 25 pl. The changes in percent transmittance or luminescence increase were recorded continuously. The Eff of A irin n T fl d Pl el t A recation: Various amounts of aspirin (in volume of 50 pl) were added into cuvettes containing 0.45 ml of PRP for 20 minutes. At the end of incubation, aliquot of Luciferase Luciferin Reagent (25 pl) was added into the cuvette and then collagen (10 pl) was added as described in the above paragraph. Protein Phosnhorvlation: The washed platelets (10 ml) were incubated at 25oC for one hour in the presence of carrier-free 32PO4 (200 pCi/ml, DuPont Inc., Boston, MA). At the end of incubation, the platelets were collected by centrifugation at 1,500 x g for 5-10 minutes. The platelets were washed once with Tris-EDTA buffer and then resuspended in the same buffer (1 ml). For determining the effect of type XI collagen on platelet protein phosphorylation, an aliquot of 45 pl of the 32P04-labeled platelets was mixed with buffer, type XI collagen, type I collagen, thrombin, or phorbol ester for two minutes. At the end of incubation, an equal volume of sodium dodecyl sulfate (SDS) sample buffer was added to the suspension, boiled for 3 minutes and analyzed with 12% SDSpolyacrylamide gel electrophoresis (PAGE). The radioautographams were made by contacting the dried gel with Kodak XAR-5 film for two days. SDS Polyacrvlamide Electrophoresis (SDS-PAGE): Type XI collagen or platelet samples were dissolved in SDS sample buffer in the presence of B-mercaptoethanol and boiled for 3 minutes. The samples were analyzed with 5% or 12% gel for collagen or platelet, respectively. After an

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electrophoresis at 40 volts in Tris-glycine buffer, pH 8.3, the gels were stained with Coomassie Brilliant Blue (Eastman Kodak Co., Rochester, NY), and destained with methanol-acetic acid (lo%-7%) solution ( 16 ). Ultrastructure of TyDe XI colla=: Samples of type XI collagen were placed on a Formvar-coated grid, and the excess fluid was removed by gently touching the grid with filter paper and air-drying. The specimens were washed three times with water, stained with 0.15 M phosphotungstic acid for 3 minutes, and then washed with water. The grid was stained with 1% uranyl acetate for 15 minutes and washed with water. All specimens were examined with an AEI model EM 6B electron microscope ( 8 ). Collagen concentrations were Determination of Collijgen Concentration: determined by an amino acid analyzer (Applied Biosystems, Foster City, CA). RESULTS Platelet awtion and the release of ATP bv tvpe XI collagen. The purity of the type XI collagen preparation was examined with reduced

U

al

XI

a2

xl

a3 XI

Figure 1.

SDS-PAGE of type XI collagen. 15 pg of the precipitated type XI collagen was analyzed with 5% SDS-PAGE under reducing conditions.

5%

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SDS-PAGE and found to contain trace amounts of type IX collagen (c 3% in The type XI preparation did not react with a density tracing) (Figure 1). monoclonal antibody specific for type II collagen suggesting that the preparation was free of type II collagen contamination. An unidentified high molecular weight band (U) was noted to migrate slower than a 1(X1) chain. This band failed to stain on immunoblot with antibodies to types II and IX collagen suggesting that it may represent a D-chain component of U 1(X1) and a 2(X1) chains. When fibrillar type XI collagen was suspended in PBS and added to PRP in doses ranging from 10 to 50 pg , it induced platelet aggregation as measured by aggregometry and release of ATP ( Figure 2). However, when added into PRP in soluble form at a high concentration ( 50 pg ), no platelet aggregation was detected (data not shown). These results suggest that the quaternary structure of type XI collagen is essential in its interaction with platelet. Lastly, platelet aggregation was not detected when PBS soluble type IX collagen was substituted for type XI.

TIME,

Figure 2.

(

-

=

1 flin.

)

Type XI collagen-induced platelet aggregation and the release of ATP. The amounts of type XI collagen used are: panel A, buffer control., panel B, 50 p.g; panel C, 20 p.g; and panel D, 10 pg. The downward deflections are aggregation and the upward deflections are release of ATP (luminescence). At the end of buffer control, 1 p.M ATP was added at the of the experiment at n (panel A). For comparing the amount of ATP released from platelet by type XI collagen.

Phvsical nronerties of tvpe XI collagen and its relationship to platelet urezation. As examplified by its precipitation when dialyzed against

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cold PBS, the solubility of type XI collagen was found to differ significantly from those of the other interstitial collagens which remained in solution under the conditions used. This provided a convenient opportunity to investigate the structural requirement of type XI collagen important in its platelet aggregating property and to obtain additional evidence that platelet aggregation induced by type XI collagen was not attributable to possible contaminating of other types of collagen. As shown in Figure 3, dialysis of acetic acid soluble type XI collagen into PBS resulted in the spontaneous formation of a visible precipitate, which had a typical banding pattern of native-type fibrils (Figure 3B). Any contaminating interstitial collagens would have remained in solution. The PBS supernatant had no aggregating activity (data not shown). The acid soluble type XI collagen did not show any banding pattern (Figure 3A) and nor did type IX collagen ata not shown).

A

Figure 3.

Electron micrograph of type XI collagen preparation obtained by dialysis against PBS at 4oC. Panel A: acid soluble, panel B: fibrillar insoluble (x 30,000).

The e ffect of aspirin on type XI collagen-induced nlatelet aearegation and release of ATP. Aspirin has been shown to inhibit types I, II, and III collagen-induced platelet aggregation and the release of serotonin in vitro.

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In order to see whether aspirin also inhibited the type XI collagen-induced platelet aggregation and release reaction (ATP release in this study). various amounts of aspirin were added to PRP before adding collagen fibril suspension. Indeed, aspirin also inhibited type XI collagen-induced platelet aggregation and the release of ATP (Figure 4). These results suggest that similar mechanism existed in type XI and other types of collagen which interact with human platelets. The effect of tvpe XI collagen~r. It has been reported that aggregating agents can cause the phosphorylation of two platelet proteins with molecular weight of 42 KDa and 22 KDa. We have used the same approach to investigate whether type XI collagen plays the same role in platelet protein phosphorylation. Figure 5 shows

TIME,

Figure 4.

(

-

= 1 Min. )

Effect of aspirin on type XI collagen-induced platelet aggregation and release of ATP. An aliquot of 0.45 ml of PRP was incubated with various amounts of aspirin (panel B, 0 pg; panel C, 40 ltg and panel D, 80 l.tg) and challenged with 15 pg of type XI collagen. Panel A challenged with buffer. The downward deflections represent platelet aggregation; the upward deflections, the release of ATP. As a control 0.5 p.M of ATP was added to the cuvette at for comparing the amount of ATP released from each experiment (panel A at n ).

the result of this study. The type XI collagen (lane 2) as well as type I collagen (lane 3) and phorbol ester (lane 4) enhanced the radiolabelled

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phosphate incorporation into two 42 KDa and 22 KDa, respectively and 6). The result indicates that increase protein phosphorylation

1 Figure 5.

2

INTERACTION

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protein bands with molecular weights of compared with buffer control (lanes 1, 5, type XI collagen has the same ability to as do other aggregating agents.

3

4

5

6

Protein phosphorylation induced by aggregating agents. Inorganic phosphate-labelled platelets were treated with PBS (lane l), type XI collagen (lane 2), type I collagen (lane 3), phorbol ester (lane 4), control for phorbol vehicle (lane 5) and dialysate control (lane 6). DISCUSSION

The results of the present studies show that the cartilage specific collagen (type XI) in native-type fibrillar form induces human platelets to In SDS-PAGE of our type XI collagen aggregate and release ATP. preparation, we see a high molecular band weight which migrated above We also see a lower molecular weight band which type II collagen.

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These results suggest type XI and type comigrated with type IX collagen. IX collagens might form a hybrid fibril as suggested by Smith et al. ( 16 ). However, purified type IX collagen did not form fibrils under the same conditions that resulted in the spontaneous formation of the type XI collagen fibrils, and did not induce platelet aggregation or ATP release at a concentration of 100 l.tg/ml. This is the first paper to describe the ability of type XI collagen to We and others have shown that fibrilar but induce platelet aggregation. not soluble form of type V collagen can cause platelets to aggregate ( 7, 8 ). This suggests that types XI and V collagen share common properties to interact with human platelets. This result suggests that specific quaternary structures must be present at the microfibrillar level to be It is interesting to note that the a 1(X1) and a2(XI) recognized by platelets. chains of type XI collagen share significant similarity with Q l(V) and a 2(V) of type V collagen ( 10 ). Aspirin has been known to inhibit the other type of collagens to interact with human plate1et.s ( 18-20 ). The inhibition was shown to block the cyclooxygenase ( 21 ). In this report, we observed that type XI collagen-induced platelet aggregation and release of ATP was inhibited by the addition of aspirin to PRP sugesting. the cyclooxygenase products play a role in type XI collagen-platelet interaction. It has been established that types I and III or other non-collagenous aggregating agents-thrombin or phorbol ester etc. increase platelet protein phosphorylation ( 22-24 ). The increases in protein phosphorylation are seen in two proteins with molecular weight of 42 KDa and 22 KDa, respectively. In this investigation, we also found that type XI collagen increases the radiolabelled phosphate incorporation of two proteins of similar molecular weight. From these studies, we conclude that the type XI collagen behaves like other types of collagen in interacting with platelets and shared a common pathway. The pathophysiological implications of platelet-type XI collagen interaction is not clear since type found only in cartilage and cartilage-like tissues which are XI collagen is avascular. Nevertheless, our findings underscore the importance of collagen quatenary structure in the genesis of collagen-induced platelet aggregation. ACKNOWLEDGEMENTS We wish to thank Ms. M. Akin-Deko and MS V. Woo for their expert technical assistant, Mrs. L. Hatmarker for electron micrograph of collagen This study was supported and MS K. Pearson for typing this manuscript. by research funds from Veterans Administration.

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Collagen-platelet interaction: type XI collagen-induced platelet aggregation.

Type XI collagen in its native fibrillar but not in soluble monomeric form mediates human platelet aggregation and release of adenosine triphosphate i...
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