Platelets Interact with Soluble and Insoluble Collagens through Characteristically Different Reactions*

Stephanie M. JungDagger and Masaaki Moroi

From the Department of Protein Biochemistry, Institute of Life Science, Kurume University, Kurume-shi, Fukuoka-ken 839-8016, Japan

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Platelet interaction with soluble and insoluble collagens was characterized through binding studies. In contrast to resting platelets, cells reacted with activators, TS2/16 (integrin alpha 2beta 1-activating antibody), thrombin, collagen-related peptide, or ADP, exhibited specific soluble collagen binding that is Mg2+-dependent, but inhibited by prostaglandin I2, Ca2+, and Gi9 (anti-integrin alpha 2beta 1 antibody). Each platelet has 1500-3500 soluble collagen binding sites, with a dissociation constant of 3.5-9 × 10-8 M. This is the first study to show the specific binding of soluble collagen to platelets; our data strongly suggest that the receptor is integrin alpha 2beta 1 after it becomes activated upon platelet activation. These results suggest that activation of platelets transforms integrin alpha 2beta 1 to a state with higher affinity binding sites for soluble collagen. The soluble collagen-platelet interaction was compared with the platelet interaction with fibrillar collagen, which has until now not been demonstrated to bind specifically to platelets. Here, we demonstrated specific, biphasic fibrillar collagen binding. One phase is rapid and metal ion-independent, and accounts for most of the binding. The other phase is slow and Mg2+-dependent. The characteristic differences in the specific bindings of soluble and fibrous collagens demonstrate the different contributions of two different collagen receptors.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

When platelets react with collagen, they form aggregates after activation of their signal transduction systems through processes involving many reactions including calcium release, formation of phosphoinositides, and phosphorylation of proteins. Platelets can also adhere to an immobilized collagen surface. Collagen is one of the main components of the vascular subendothelium and becomes exposed to the blood stream when endothelial cells are damaged. These observations suggest that the interaction of platelets with collagen is one of the first, requisite reactions in thrombus formation under physiological conditions. The interaction of platelets with collagen has been studied under two conditions. One approach is to analyze platelet adhesion and aggregation onto a collagen-coated surface under flow conditions using whole blood (1-5). This method observes platelet adhesion under flow conditions that closely simulate physiological conditions, and the fact that platelets adhere and aggregate under these conditions shows that the interaction with collagen is involved in physiological thrombus formation. Alternatively, platelet adhesion on a collagen-coated surface can be analyzed under static conditions; this type of assay uses washed platelets instead of whole blood (6, 7). In both systems, two platelet glycoproteins, glycoprotein (GP)1 Ia/IIa, also known as integrin alpha 2beta 1, and GP VI were found to participate in platelet adhesion to the collagen surface.

However, the direct binding of collagen to platelets has not been studied in detail because soluble collagen did not bind to platelets. Resting platelets do not react with soluble collagen. They only react with insoluble collagen fibers or an immobilized collagen surface, both substrates having physical properties that made it very difficult to analyze the binding reaction of platelets and collagen. Gordon and Dingle (8) reported that collagen did not bind to platelets until it became fibrillar. Binding of platelets to radiolabeled insoluble collagen particles was indicated to be a very fast reaction and Mg2+-independent (9), but they could not find measurable specific binding of labeled collagen fibrils to platelets under their conditions. Thus, their binding data were not suitable for kinetic analysis of the binding reaction.

Our present study is the first to demonstrate the binding of soluble collagen to platelets and the kinetic analyses of this reaction. The soluble collagen binding is Mg2+-dependent, and the activation of integrin alpha 2beta 1 is involved in this reaction. In addition, we were also able to demonstrate the specific binding of fibrous collagen particles by platelets, finding that the major portion of this binding is Mg ion-independent. These results suggest that the two collagen receptors, integrin alpha 2beta 1 and a Mg2+-independent collagen receptor, probably GP VI, would be involved in the collagen-platelet interaction with different specificities.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Preparation of Soluble and Insoluble (Fibrillar) Collagen-- Bovine type III collagen and human type III collagen were obtained from Koken Co., Ltd. (Tokyo, Japan) and Fuji Chem. Ind. Co., Ltd. (Takaoka, Toyama, Japan), respectively. Both collagens were dialyzed against 136 mM NaCl, 2.7 mM KCl, 0.42 mM NaH2PO4, 12 mM NaHCO3, 5.5 mM glucose, and 5 mM HEPES, pH 7.4 (buffer A) at 4 °C. After dialysis, the collagen solution was incubated at 37 °C for 1 h. The resultant collagen gel was centrifuged at 8000 × g for 20 min to obtain the supernatant and the pellet. The supernatant was further centrifuged at 100,000 × g for 1 h at 25 °C, and the obtained supernatant solution was used as soluble collagen. This soluble collagen was stored at 4 °C until used in the experiments. The collagen pellet obtained after centrifugation was washed once with buffer A and then suspended with buffer A at the concentration of about 1 mg/ml. The suspended collagen was sonicated for 1 min three times with a sonicator attached with a cup horn (Heat Systems-Ultrasonics Inc., Farmingdale, NY), which was cooled by ice. The obtained microparticles of collagen suspensions were used as insoluble collagen.

Soluble collagen was radiolabeled with Na125I by using IODO-BEADS (Pierce) at room temperature according to the manufacturer's instructions. For the preparation of 125I-labeled insoluble collagen, collagen dialyzed against buffer A was labeled on ice by using IODO-BEADS and dialyzed against buffer A at 4 °C. 125I-Labeled collagen was polymerized at 37 °C and centrifuged, and the pellet of labeled collagen obtained was processed as described above to make labeled insoluble collagen microparticles (fibrillar collagen). The specific radioactivities ranged between 3 and 9 × 104 cpm/µg. The data presented in this paper are those obtained with bovine collagen. Similar data were obtained with human collagen, but its limited availability made it more feasible to employ bovine collagen for extensive studies.

Determination of Collagen Concentrations-- The concentrations of soluble and fibrillar collagen were determined by assaying the protein by the bicinchoninic protein assay (10), with collagen as the standard protein. The molar concentration was calculated with 3 × 105 as the molecular weight of collagen.

Platelet Preparation-- Whole blood was drawn from the cubital vein of healthy volunteers into 0.1 volume of 3.8% sodium citrate. After platelet-rich plasma (PRP) was obtained, platelets were sedimented from PRP by centrifugation at 900 × g for 12 min after the addition of sodium prostaglandin I2 (PGI2) (Funakoshi, Tokyo, Japan) at the final concentration of 0.1 µg/ml. The centrifuged platelets were washed once with 6.85 mM citrate, 130 mM NaCl, 4 mM KCl, and 5.5 mM glucose, pH 6.5; and the washed platelets were finally suspended with buffer A containing 2% bovine serum albumin (Sigma) at concentrations of 1-3 × 109 cells/ml.

Binding of Soluble Collagen-- To ensure that the 125I-labeled soluble collagen mixture consisted of only soluble collagen, just prior to use in each experiment, an aliquot of the preparation was warmed at 37 °C for 30 min, followed by centrifugation at 56,000 × g for 60 min at 25 °C (Beckman TL-100 Ultracentrifuge with a TLA 100.3 rotor; Beckman, Inc., Palo Alto, CA). The supernatant was removed and used as the soluble collagen preparation for the experiments. Routinely, an aliquot of the supernatant was retained for subsequent determination of protein concentration.

The selection of the optimum reaction conditions and separation procedures was based on the data described under "Results." For the soluble collagen binding experiments, non-siliconized tubes were used. Washed platelet suspensions were used for all the experiments. The complete mixture (final volume of 50-70 µl) for determination of total binding consisted of 2 mM MgCl2, 125I-labeled soluble collagen, any desired reagents such as inhibitors or antibodies, 0.2% BSA, and 4 × 108 platelets/ml (unless otherwise stated) in buffer A. 125I-Labeled soluble collagen was used at a concentration of about 75-100 nM in most experiments, except for the kinetic studies for determination of binding parameters where the stated range of concentrations was employed. Nonspecific binding was determined by adding an approximately 100-fold excess of unlabeled soluble bovine collagen or 5 mM EDTA (further described under "Results") to reaction mixtures containing the other components of the total binding mixture. Platelets were usually added last to the reaction tubes containing the remaining reagents to initiate the binding reaction. Four to eight replicates for each binding condition were used. Binding was allowed to occur for 80 min at room temperature, without agitating the reaction mixtures. Then, each reaction mixture was processed by a procedure based on the method described by Mazurov et al. (9), which was modified to optimize the separation of platelet-bound soluble collagen from the unbound soluble collagen; each reaction mixture was layered over a 250-µl pad of 20% sucrose in buffer A containing 0.2% BSA in a needle-tipped 0.3-ml narrow-tipped microcentrifuge tube (Assist, Tokyo, Japan) and centrifuged at 10,000 × g in a microcentrifuge (model MRX-150, MRX-150 rotor; Tomy, Tokyo, Japan) for 5 min at 20 °C. The tubes were placed in a -80 °C freezer for at least 30 min. The tips of the frozen tubes containing the platelets/platelet-bound soluble collagen were then cut off by scissors, individually placed in counting tubes, and the radioactivity determined in an Aloka Auto Well gamma  system (model ARC-1000 M; Aloka, Tokyo, Japan).

Binding of Insoluble Collagen-- Just prior to use in each experiment, the 125I-labeled collagen microparticles (prepared as described above) were processed by the following procedure to sort out collagen microparticles of an appropriate size range, i.e. would bind well to resting platelets, but also not large enough to be pelleted under the conditions of the centrifugation assay (see "Results" for description of how the optimum conditions were chosen). An aliquot of the labeled microparticular collagen was sonicated for 1 min at 60% power, three times with a 1-min interval between each, by a sonicator attached with a cup horn under cooling with ice. The sonicated suspension was then centrifuged at 2000 rpm (250 × g) for 3 min in the MRX-150 high speed micro-refrigerated centrifuge; the supernatant obtained was used for the binding studies. For the binding experiments of insoluble collagen, siliconized tubes and tips were used. The total binding mixture (total volume of 50 µl) contained 5 mM EDTA or 2 mM Mg2+, any agents to be tested, microparticular suspension of 125I-labeled fibrillar collagen at the desired concentrations, and 0.4-4 × 108 platelets/ml in buffer A containing 0.2% BSA. In the experiments where Mg2+ was employed, the washed platelet suspensions were added with 0.1 µg/ml PGI2 and 10 µM indomethacin, to prevent aggregation, and incubated at room temperature for 10 min before aliquots of them were added to the binding mixtures. When binding was performed in the presence of EDTA, PGI2 and indomethacin were not added unless the EDTA-containing binding experiment was performed to compare the results with those in the Mg2+-containing one. Nonspecific binding was determined in the presence of an 80-100-fold excess, as estimated by protein concentration, of unlabeled bovine fibrillar collagen, microparticle suspension prepared in a manner similar to the labeled material. Platelets were added last to tubes containing the remaining binding mixture components to initiate the binding reaction, and incubation was carried out for 60 min at room temperature with gentle shaking on a shaking table, to prevent the settling of the microparticles of collagen. Each sample was then layered over a 250-µl pad of 20% sucrose and 2% BSA, centrifuged at 12,000 rpm for 10 min at room temperature, and processed for counting as described above for soluble collagen binding.

Data Analyses-- In the soluble collagen binding experiments, specific binding was calculated by subtracting the nonspecific binding obtained in the presence of excess unlabeled collagen or 5 mM EDTA.

In the fibrillar collagen binding experiments, total binding and nonspecific binding were first corrected by subtracting the experimentally determined value for pelleted labeled fibrillar collagen in the absence and presence of excess unlabeled fibrillar collagen, respectively (see "Results" for further description). Specific binding was then calculated by subtracting the corrected nonspecific binding from the corrected total binding.

Kinetic analyses of the data to obtain the binding parameters were performed by non-linear regression analyses by the software Prism (Version 2.01; GraphPad Software, Inc., La Jolla, CA). Other data analyses were also performed by the same program.

Other Materials-- Collagen-related peptide (CRP) was synthesized by the method of Morton et al.(11). The peptide Tyr-Gly-Lys-Hyp-(Gly-Pro-Hyp)10-Gly-Lys-Hyp-Gly, was synthesized by Tana Laboratories, L.C. (Houston, TX) using standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) solid-phase chemistry. The peptide was dissolved in phosphate buffer, pH 7.6, and then it was cross-linked with 0.25% glutaraldehyde for 1 h on ice (11). The obtained CRP was dialyzed against buffer A and used for the experiments. The obtained CRP aggregated platelets in PRP at the concentrations of 0.2 µg/ml or higher. Monoclonal antibodies, P2 (anti-GP IIb/IIIa), SZ2 (anti-GP Ib), and Gi-9 (anti-integrin alpha 2beta 1), were purchased from Immunotech (Luminy, France). The anti-GP Ib monoclonal antibody NNKY5-5 (12) and the integrin alpha 2beta 1-activating antibody, TS2/16 (13) were kindly given to us by Dr. Shosaku Nomura, Kansai Medical University, Osaka, Japan and by Drs. Yuji Saito and Junichi Takagi, Tokyo Institute of Technology, Tokyo, Japan, respectively. Ristocetin and ADP were obtained from Chrono-Log Corp., Havertown, PA. Peptides Gly-Arg-Gly-Asp-Ser (GRGDS) and Gly-Arg-Gly-Glu-Ser (GRGES) were synthesized in our laboratory with a model 431A peptide synthesizer (Applied Biosystems, Foster City, CA) and the FastMoc method according to the company's protocol (5). Thrombin and the thrombin receptor agonist TRAP (SFLLRNPNDKYEPF) were obtained from Sigma and Calbiochem (San Diego, CA), respectively.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Conditions for Optimum Separation of Samples-- The centrifugation conditions for maximizing platelet sedimentation with the minimum of labeled collagen sedimentation was established by using 51Cr-labeled platelets and 125I-labeled soluble or microparticular fibrillar collagen. As shown in Fig. 1, platelet sedimentation was not affected by the presence of an excess of unlabeled soluble collagen in either the presence or absence of 2 mM Mg2+; greater than 96% platelet pelleting was obtained in about 4-5 min at 12,000 rpm as judged by the amount of sedimented 51Cr. Addition of 5 mM EDTA to the platelet mixtures containing excess collagen and 2 mM MgCl2 did not affect the sedimentation properties (data not shown). There was very little pelleting of the precentrifuged labeled soluble collagen at centrifugation times as high as 10 min; the amount sedimented varied among different soluble collagen preparations, but usually was less than 1% of the added 25I counts, and the sedimented amount plateaued before 5 min of centrifugation.


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Fig. 1.   Platelet sedimentation time in the presence of an excess of soluble collagen. The following mixtures were incubated for 80 min at room temperature without agitation: 51Cr-labeled platelets (4 × 108 cells/ml) + 2 mM Mg + 300 µg/ml unlabeled soluble collagen (bullet ), 51Cr-labeled platelets (4 × 108 cells/ml) only (open circle ), 125I-labeled soluble collagen + 2 mM Mg2+ (black-square), and 125I-labeled soluble collagen only (square ). Then 50-µl aliquots of each mixture were individually centrifuged over a pad of 250-µl 20% sucrose (in HEPES-Tyrode's buffer, pH 7.4) in a needle-tipped centrifuge tube for various times at 12,000 rpm, and the amount of sedimented radioactivity (51Cr or 125I) was determined as described under "Materials and Methods." The pelleted radioactivity is reported as a percent of the total label (51Cr or 125I) in the sample. Each data point and error bar is the mean ± S.D. of quadruplicate determinations; where error bars are not visible, the S.D. is smaller or equal to the size of the symbol. The recovery of counts (pellet counts + supernatant counts) was greater than 98% in all cases.

In contrast, the situation with microparticles of labeled fibrillar collagen was quite different. The presence of the fibrillar collagen substantially slowed the rate of platelet sedimentation (Fig. 2, left graph). Furthermore, the presence of excess unlabeled fibrillar collagen affected the sedimentation of labeled collagen fibrils (Fig. 2, right graph). Although some labeled collagen was sedimented in the absence of excess unlabeled collagen, its inclusion resulted in 1.5-3-fold higher levels of labeled collagen sedimentation at all centrifugation times and speeds. The conditions of 10-min centrifugation time at 12,000 rpm were chosen to optimize the pelleting of platelets (>95%), and the effect of high concentrations of unlabeled collagen fibrils, which were required for the establishment of nonspecific binding, was corrected for by determining how much 125I-labeled fibrillar collagen is sedimented in the presence or absence of excess unlabeled fibrillar collagen at each concentration of labeled fibrillar collagen employed in an experiment. This had to be done for each experimental session because the preparation of collagen fibrils would be expected to be different on each occasion, even though the size of the fibrils may lie within a similar range.


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Fig. 2.   Effects of a high concentration of unlabeled fibrillar collagen on the sedimentation of platelets and 125I-labeled fibrillar collagen. Left graph, the following mixtures (all in HEPES-Tyrode's buffer, pH 7.4 containing 2% BSA) were incubated in siliconized tubes for about 40 min at room temperature, with agitation of the tubes: 51Cr-labeled platelets + 5 mM EDTA (bullet ), 51Cr-labeled platelets + unlabeled fibrillar collagen (350 µg/ml) + 5 mM EDTA (black-square), 125I-labeled fibrillar collagen + 5 mM EDTA (open circle ), and 125I-labeled fibrillar collagen + excess of unlabeled fibrillar collagen (350 µg/ml) + 5 mM EDTA (black-square). After incubation, 50-µl aliquots of each mixture were individually centrifuged over 250 µl of 20% sucrose for various times at 12,000 rpm for various times, and the amounts of radioactivities of the obtained pellets and supernatants were counted. The ordinate shows the percentage of counts relative to the total counts added of either 51Cr (radiolabeled platelets) or 125I (radiolabeled fibrillar collagen). Right graph, mixtures containing a fixed concentration of 125I-labeled fibrillar collagen (about 2 µg/ml), 5 mM EDTA, and varying amounts of unlabeled fibrillar collagen were centrifuged over 250-µl of 20% sucrose for 10 min at 12,000 rpm, and the radioactivities of the pellet and supernatant were measured. The ordinate shows the percentage of counts relative to the total counts of 125I-labeled fibrillar collagen added. In both graphs, each data point is the mean ± S.D. of 10 determinations.

Preincubation Time with Activation Agents and the Specific Binding of Soluble Collagen-- Platelets were preincubated with TS2/16 (5 µg/ml), thrombin (0.5 units/ml), or CRP (0.5 µg/ml) for 0-30 min to determine if preincubation of platelets with the agents affected the activation of their soluble collagen binding. As shown in Fig. 3, the bindings of the platelets preincubated with TS2/16, thrombin, or CRP for 5, 10, 20, and 30 min were similar to that observed when the respective agent was added simultaneously (0 min) with the 125I-labeled collagen (about 100 nM) to initiate the binding reaction. Platelets not activated by the addition of one of the agents showed little or no binding. Therefore, we performed the following binding experiments without preincubation.


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Fig. 3.   Effect on soluble collagen binding of preincubating platelets with activating agents for varying times. Platelets were incubated with TS2/16 (5 µg/ml) (black-square), thrombin (0.5 units/ml) (bullet ), or CRP (0.5 µg/ml) (black-triangle) for various times, and then the binding was initiated by adding 125I-labeled soluble collagen and the reaction allowed to occur for 80 min at room temperature. For the zero time point, the activating agent was added simultaneously with the ligand. All samples were processed by the centrifugation method, and the radioactivities in the platelet pellet were determined and reported as femtomoles/106 platelets. Each data point is the mean ± S.D. of four determinations.

Time Course of Soluble Collagen Binding to Activated Platelets-- Soluble collagen binding to agonist-activated platelets reached equilibrium within about 70-80 min. Typical of the time courses observed are those for TS2/16- and thrombin-activated platelets shown in Fig. 4. Control mixtures that contained no activating agents showed a very low level of counts sedimenting along with the platelet pellet; this might be due to a small amount of sedimentable soluble collagen, the amount which varied with the collagen preparation, or platelets may have become slightly activated during the platelet preparation procedures or during the incubation period.


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Fig. 4.   Time course of soluble collagen binding to activated platelets. A platelet suspension (4 × 108 cells/ml, Hepes-Tyrode's buffer plus 2% BSA) was incubated with TS2/16 (5 µg/ml, black-triangle), thrombin (0.5 units/ml, black-square), or buffer (control unactivated platelets, ×); 2 mM Mg2+; and 125I-labeled soluble collagen for varying times and the binding assessed by the centrifugation procedure. Mixtures for determining nonspecific binding contained 5 mM EDTA in addition to the other components. Each data point is the mean ± S.D. of four determinations.

In this figure, TS2/16-activated platelets bound more soluble collagen than the thrombin-activated platelets. Although we observed a similar phenomena numerous times in the course of our experiments, in other instances, thrombin induced soluble collagen binding to levels comparable to those by TS2/16. This could be explained by the instability of platelet activation with thrombin. Platelets apparently have an optimum concentration of thrombin for their maximum activation, which varied from individual to individual. In contrast, TS2/16-activated platelets showed stable binding activity with high capacity.

Effects of Metal Ions on Soluble Collagen Binding to Platelets Activated by Various Agonists-- The effects of metal ions on soluble collagen binding to activated platelets activated by ADP, thrombin, TS2/16, or CRP are shown in Table I. The actual amounts of binding and the extent of metal ion effect differed among individuals. For platelets activated by any of the agents, soluble collagen binding was Mg2+-dependent. More specific binding was observed in the presence of 2 mM Mg2+ than 1 mM; and in each experiment, specific binding induced by the agonists in the presence of either concentration of Mg2+ was higher than that observed in the control platelets to which no metal ions had been added. The control platelets in the 2 mM Mg2+ experiment showed a significant amount of soluble collagen binding in the absence of added activator, suggesting that in some cases, there may have been some activation during the platelet isolation procedures. Because of this, the percent increase in soluble collagen binding relative to the control was less prominent, especially in the 2 mM Mg2+ experiments (see explanation in the next paragraph). Compared with the binding in the presence of 2 mM Mg2+, that in the presence of Mn2+ was higher.

                              
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Table I
Effect of metal ions on the specific binding of soluble collagen to activated platelets
Each experiment was performed on a different day with platelets from a different individual. Because platelets from different individuals showed variable sensitivities to the activating agents, only values from the same person can be validly compared with each other. Nonspecific binding was determined in the presence of a 100-fold excess of unlabeled soluble collagen in all experiments except for experiment 4, in which 5 mM EDTA was used. Specific binding is reported as femtomoles/106 platelets.

As illustrated in Fig. 5, a more detailed examination of the Mg2+ concentration dependence in the cases of two activators, TS2/16 (5 µg/ml) and thrombin (0.1 units/ml), indicates that saturation with Mg2+ occurs around 2 mM for the bindings induced by both activators, since the specific binding of soluble collagen plateaus after this level of metal ion. As seen from the control curve (no added activators), there is indeed some increase in soluble collagen binding as higher concentrations of this divalent ion are included in the reaction mixture; because the saturation level for this low level of binding (a maximum of about 10%) occurs at about the same concentration as the TS2/16 and thrombin curves, it is likely due to some activation of the platelets during the isolation procedures or incubation, instead of an independent activation by Mg2+ itself which would likely show a different Mg2+ ion dependence.


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Fig. 5.   Mg 2+ dependence of soluble collagen binding to TS2/16- and thrombin-activated platelets. The binding of soluble collagen to platelets activated with TS2/16 (5 µg/ml) (bullet ) or thrombin (0.1 unit/ml) (black-down-triangle ) was determined in the presence of various concentrations of Mg2+. Control mixtures (open circle ) contained no activating agent. Nonspecific binding was determined in the presence of 5 mM EDTA (0, 0.5, 1, and 2 mM Mg2+) or 10 mM EDTA (5 and 10 mM Mg2+). Samples were processed by the centrifugation procedure, and the pellets were counted. The specific binding is reported as femtomoles/106 platelets, and each value is the mean ± S.D. of quadruplicate determinations.

For all the activators, little binding occurred in the presence of 5 mM EDTA. The level observed in the presence of 5 mM EDTA was similar to that observed in the presence of an excess of unlabeled soluble collagen (about 350 µg/ml), which we added to assess nonspecific binding in the preliminary experiments. Because of this, we employed 5 mM EDTA to assess nonspecific binding; this allowed us to determine nonspecific binding at higher concentrations of labeled soluble collagen where it would otherwise not have been possible to obtain the 100-fold excess of unlabeled ligand required because of the limitation of how high a concentration of soluble collagen in solution can be achieved.

For the ADP-, thrombin-, TS2/16-, or CRP-activated platelets, little binding was seen in the presence of 2 mM Ca2+; when both 1 mM Ca2+ and 1 mM Mg2+ were present, specific binding was lower than that seen in the presence of 1 mM Mg2+ alone. These observations indicate the inhibitory effect of calcium ion as reported by Santoro (6).

A similar study performed with thrombin receptor agonist peptide (TRAP) (20-95 µM) indicated that it could also activate platelets to bind soluble collagen, although it was a weaker activator than thrombin because the maximum amount of binding induced by this agent was less than that obtained with thrombin (data not shown). The specific binding of soluble collagen to TRAP-activated platelets showed the same ion dependence as the bindings induced by the above agents (data not shown).

Effect of PGI2 on Soluble Collagen Binding to Activated Platelets-- As shown in Fig. 6, the presence of PGI2 markedly decreased the specific binding in the presence of 2 mM Mg2+ in thrombin- or CRP-activated platelets. On the other hand, it did not inhibit the specific binding of TS2/16-activated platelets. In the ADP-induced platelets, soluble collagen binding was low, so it was difficult to assess the effect of PGI2 accurately.


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Fig. 6.   Effect of PGI2 on soluble collagen binding to activated platelets. The binding of soluble collagen to platelets were determined in the presence of one of the following activating agents: ADP (20 µM), TS2/16 (5 µg/ml), thrombin (0.1 units/ml), and CRP (0.5 µg/ml), with 2 mM Mg2+ (empty bars) or 2 mM Mg2+ + PGI2 (solid bars) in the binding mixture. To determine the effect on non-activated platelets, platelets were incubated with PGI2 (0.1 µg/ml) (dotted bars) or without PGI2 in the absence of Mg2+ (diagonally shaded bars). The samples were processed by the centrifugation procedure and the pellets were counted. Each bar represents the mean ± S.D. from eight determinations.

Effect of Inhibitors on Soluble Collagen Binding to Activated Platelets-- The results illustrated in Fig. 7 show typical data observed in four separate experiments with different platelets and soluble bovine collagen preparations, with 0.1 units/ml thrombin as the platelet activating agent. Compared with the specific binding in the absence of any inhibitor, taken as 100% in the figure, the specific binding of soluble collagen was inhibited by the anti-GP Ia/IIa antibody Gi9 (10 µg/ml) to a level similar to that observed with PGI2 (0.1 µg/ml), but antibodies P2 (anti-GP IIb/IIIa, 10 µg/ml), NNKY5-5 (anti-GP Ib, 10 µg/ml), GRGDS (1 mM), and GRGES (negative control, data not shown) were not inhibitory.


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Fig. 7.   Effect of inhibitors on soluble collagen binding to activated platelets. Soluble collagen binding to platelets activated by thrombin (0.1 units/ml) was determined with the presence of no added inhibitor or one of the following agents: PGI2 (0.1 µg/ml), P2 (10 µg/ml), NNKY5-5 (10 µg/ml), Gi9 (10 µg/ml), and GRGDS (1 mM). Each bar shows the mean ± S.D. of six determinations. The data shown here is representative of four experiments performed with different platelets and different soluble collagen preparations.

Activating Agent Concentration Dependence of Soluble Collagen Binding to Platelets-- Fig. 8 shows the concentration dependence curve for CRP, which shows an apparent optimum concentration for activation of soluble collagen binding. To determine if this is due to CRP competing with soluble collagen for binding to activated platelets, the effect of high CRP concentrations on the binding of soluble collagen to TS2/16-activated platelets was assessed. The data shown in Fig. 9 shows that this is the case; CRP inhibited the specific binding of soluble collagen to TS2/16-activated platelets in a concentration-dependent manner, with the inhibitory concentrations of CRP corresponding to those at which soluble collagen binding to CRP-activated platelets were decreased in Fig. 8.


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Fig. 8.   CRP concentration dependence of soluble collagen binding to platelets. Binding of soluble collagen to platelets was determined in the presence of various concentrations of CRP and 2 mM Mg2. Each data point is the mean ± S.D. of six determinations.


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Fig. 9.   CRP competes with binding of soluble collagen to TS2/16-activated platelets. Platelets (4 × 108 cells/ml) were activated by incubation with 5 µg/ml TS2/16 in the presence of 2 mM Mg2+ for 20 min. Then binding was initiated by adding a mixture containing various concentrations of CRP and 125I-labeled soluble collagen in HEPES-Tyrode's buffer plus 2% BSA and after 80 min at room temperature, the samples were processed by the centrifugation method. The data are expressed relative to the specific soluble collagen binding in TS2/16-activated platelets in the absence of CRP (100%). Each data point is the mean ± S.D. of six determinations.

Kinetic Analyses of Soluble Collagen Binding by Activated Platelets-- The binding parameters for soluble collagen binding to platelets activated with TS2/16 (5 µg/ml) and thrombin (0.1 units/ml) were determined by measuring the specific binding of soluble collagen as a function of soluble collagen in the range of 4-456 nM soluble collagen. Fig. 10 shows the binding curve obtained for TS2/16. Data were analyzed by non-linear regression of the binding curve, fitting the data to either a one-site or two-site model. Both the TS2/16 and thrombin data showed a better fit to the one-site model, although it should be noted that the Scatchard plots in all experiments (see Fig. 10, inset, for the Scatchard plot in one experiment with TS2/16) appear to fit better to a curve than a straight line. The binding parameters (Km and Vmax) obtained are summarized in Table II; within the experimental error for the parameters, TS2/16-activated platelets show binding constants similar to those of thrombin-activated ones.


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Fig. 10.   Binding curve of soluble collagen to TS2/16-activated platelets. The binding of soluble collagen to platelets activated with TS2/16 (5 µg/ml) in the presence of 2 mM Mg2+ was determined at various concentrations of 125I-labeled soluble collagen. Nonspecific binding was measured in the presence of 5 mM EDTA. The binding data were analyzed by non-linear regression analysis; the data best fit to a one-site binding equation. The inset shows the Scatchard plot of the data. These data correspond to experiment 1 in Table II, which summarizes the binding parameters.

                              
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Table II
Binding parameters for soluble collagen binding to activated platelets

Time Course of Fibrillar Collagen Binding to Platelets-- In contrast to soluble collagen binding by activated platelets, the collagen fibrils bound to platelets even in the presence of 5 mM EDTA. Furthermore, the time courses of fibrillar collagen binding in either the presence of Mg2+ or 5 mM EDTA was very rapid (Fig. 11). The specific binding in the presence of Mg2+ reached a level that was about 30% higher than the binding in the presence of EDTA, which would suggest that the fibrillar collagen binding consists of a Mg2+-dependent component and a Mg2+-independent component, which accounts for most of the binding.


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Fig. 11.   Time course of fibrillar collagen binding to platelets. Fibrillar collagen binding to platelets (7.5 × 107 cells/ml, HEPES-Tyrode's buffer plus 2% BSA; pretreated by the addition of PGI2 and indomethacin as indicated under "Materials and Methods") was assessed in the presence of 5 mM EDTA (black-square) or 2 mM Mg2+ (bullet ). Nonspecific binding was determined in the presence of about a 100-fold excess of unlabeled fibrillar collagen (about 350 µg/ml). The reaction mixtures were incubated for various times at room temperature, with slow agitation on a shaking table. Processing of the samples by the centrifugation assay and calculation of specific binding were performed as described in the text. Each data point is the mean ± S.D. of five determinations.

Other Characteristics of Microparticular Fibrillar Collagen Binding-- Platelets were able to specifically bind fibrillar collagen in either the presence of Mg2+ (2 mM) or EDTA (5 mM), as described above. The specific binding was concentration-dependent; a typical example is shown in Fig. 12. However, it was not possible to obtain concentration data in a sufficient range to permit an analyses of the binding curve because collagen is a macromolecule whose solution properties (e.g. viscosity and stickiness) limit its workable concentration to a maximum of about 500 µg/ml; it was therefore impossible to determine nonspecific binding at the higher concentrations of ligand because a 80-100-fold excess (from preliminary experiments, data not shown) of unlabeled fibrous collagen would be necessary. Furthermore, the heterogeneity of fibrillar collagen makes it far from an ideal defined ligand, so binding analyses techniques are not applicable. In this figure, the concentration of collagen was calculated using 300,000 as the molecular mass of collagen. However, we measured the binding of fibrous collagen aggregates, so the actual molar concentration of collagen should be much less. Thus, this figure shows the concentration dependence at a very low concentration range of fibrous collagen (compare this with Fig. 10).


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Fig. 12.   Binding of fibrillar collagen to platelets. The binding of fibrillar collagen to platelets (7.5 × 107 cells/ml) in the presence of 5 mM EDTA was determined at various concentrations of 125I-labeled fibrillar collagen. Nonspecific binding was measured in the presence of an excess of unlabeled fibrillar collagen. Details about the method and calculation of specific binding are given in the text. The inset shows the data for total binding (open circle ---open circle ) and nonspecific binding (× - - ×) from which the specific binding was calculated. Each point is a single determination (150 in total for each curve). Each curve was obtained by fitting the data by non-linear regression analysis using a second degree polynomial.

The effect of several anti-platelet glycoprotein antibodies on the specific binding of fibrous collagen was tested. The anti-GP Ia/IIa antibody Gi-9 inhibited the specific binding of fibrous collagen by 25% relative to the control (100%) that did not contain any antibody, whereas antibodies P2 (anti-GP IIb/IIIa) and SZ-2 (anti-GP Ib) had little effect (Fig. 13).


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Fig. 13.   Effect of antibodies against specific platelet glycoproteins on fibrillar collagen binding to platelets. The binding mixtures contained platelets (7 × 107 cells/ml, pretreated with PGI2 and indomethacin as indicated under "Materials and Methods"), HEPES-Tyrode's buffer, 2% BSA, 1 mM Mg2+, 125I-labeled fibrillar collagen and one of the following antibodies: Gi9 (10 µg/ml), P2 (10 µg/ml), and SZ-2 (10 µg/ml), or contained an equal volume aliquot of buffer in the control experiment (no added antibody). Nonspecific binding was determined in the presence of 350 µg/ml unlabeled fibrillar collagen. The mixtures were incubated for 60 min, with agitation, at room temperature and then processed by the centrifugation procedure. The graph is a plot of the specific binding in the presence of each of the antibodies relative to the control (no added antibody, 100%). Each bar is the mean ± S.D. of 10 determinations.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Platelet-collagen binding has yet to be thoroughly investigated, even though an analysis of this interaction would be prerequisite to understanding the mechanism of platelet adhesion to collagen and subsequent aggregation, the important initial steps of thrombus formation in vivo. This is primarily due to the properties of fibrous collagen that make it a far from an ideal, homogeneous ligand. Several reports indicated that only fibrous collagen can bind to platelets, and no binding of soluble collagen could be demonstrated (8, 9). Two collagen receptors have been identified on the platelet surface, integrin alpha 2beta 1 (also known as GP Ia/IIa) (6, 14) and GP VI (15, 16). However, the interrelationship of these two collagen receptors in the adhesion and aggregation of platelets with collagen has not been elucidated. In this paper, we showed the specific binding of soluble and fibrous collagens to platelets and obtained data demonstrating the different contributions of these collagen receptors to the bindings of these two types of collagens.

As indicated by other researchers, we could not detect any specific binding of soluble collagen to resting platelets. However, we found that platelets reacted with a monoclonal anti-integrin alpha 2beta 1 antibody, TS2/16, exhibited significant specific binding of soluble collagen. TS2/16 is an activating antibody that binds to the beta 1-subunit of integrin alpha 2beta 1 (17) and indicated to increase the binding of fibronectin and collagen to the corresponding integrin receptors (17, 18). Therefore, in our experiments, TS2/16 would induce the activation of integrin alpha 2beta 1 molecules, enabling them to bind to soluble collagen; this indicates that the activated form of integrin alpha 2beta 1 would have high affinity to bind soluble collagen. We could also detect the specific soluble collagen binding to platelets that were treated with other platelet stimulants such as thrombin, ADP, and CRP (11). The effect of ADP was weak compared with that of thrombin and also the amount of specific binding was variable from experiment to experiment. Soluble collagen binding to platelets in response to any of these stimulants, with the exception of TS2/16, was inhibited by PGI2 (Fig. 6). These results strongly suggest that integrin alpha 2beta 1 became activated when platelets were reacted with these stimulants. A similar mechanism was found for the binding of fibrinogen to GP IIb/IIIa (integrin alpha IIbbeta 3) (19, 20). Thus, both GP IIb/IIIa and integrin alpha 2beta 1 would become activated when platelets are reacted with various types of stimulants, gaining the ability to bind fibrinogen and soluble collagen, respectively.

The binding of soluble collagen to platelets required the presence of Mg2+ ions, but Ca2+ ions were inhibitory. This metal ion dependence profile is similar to the one reported for the interaction of integrin alpha 2beta 1 and collagen (6), supporting the above hypothesis that integrin alpha 2beta 1 mainly contributes to the binding of soluble collagen. We also found that manganese ions had a stimulatory effect. They not only induced the activations of integrin alpha 2beta 1 in the absence of other activators, but also stimulated the collagen binding of platelets activated by thrombin or TS2/16. This result suggests that Mn2+ ion would activate integrin alpha 2beta 1 through a mechanism different from that responsible for the TS2/16- or thrombin-induced activation Other beta 1- and beta 2-integrins were shown to have a similar metal ion dependence: stimulation by Mn2+ and inhibition by Ca2+ (21-23). In addition, Mn2+-induced activation of beta 1-integrin, was suggested to be different from the activation induced by TS2/16 in the presence of Mg2+ (24). In contrast to Mn2+, the stimulatory effect of Mg2+ would be attributed to the partial activation of platelets that had occurred during the preparation of washed platelets, because the extent of the platelet activation was low even in the presence of a high concentration of Mg2+ (Fig. 5).

The kinetic analysis of soluble collagen binding showed that the binding is saturable and fit to a one-site binding model. The dissociation constant and the number of binding sites per cell were calculated to be 3.5-9 × 10-8 M and 1500-3500 sites per cell, respectively (Table II). Platelets activated with thrombin and those activated by TS2/16 showed similar binding parameters, indicating that the integrin activated by TS2/16 through direct binding and that activated by thrombin through inside-out signaling would have a similar active conformation. By using monoclonal antibodies, the number of integrin alpha 2beta 1 molecules on platelets was calculated to be 1000-2800 per platelet (25, 26). The number of soluble collagen binding sites determined in the present experiments is well consistent with those reported numbers of integrin alpha 2beta 1. We used 300 kDa as the molecular mass of collagen to calculate the number of binding sites, which assumes that the soluble collagen is present in the monomeric form. Thus these binding data suggest that the collagen bound to integrin alpha 2beta 1 is mainly the monomeric form. Our present study is the first to determine the dissociation constant for the interaction of integrin alpha 2beta 1 with collagen.

The finding that soluble collagen can bind to CRP-activated platelets should provide some clues about the relationship between GP VI and integrin alpha 2beta 1 because CRP was reported to bind to GP VI and aggregate platelets (11, 27). Soluble collagen binding was inhibited by PGI2, which suggests that platelets were first activated by CRP and then integrin alpha 2beta 1 becomes activated through the inside-out signaling induced by CRP. These results suggest the relationship between the GP VI and integrin alpha 2beta 1.

In contrast to the binding of soluble collagen, which behaves more like a classical ligand, it is very difficult to measure the specific binding of fibrous collagen to platelets. Previous studies could not demonstrate the specific binding of fibrous collagen to platelets and reported only the total binding of the fibrous collagen (8, 9). Our binding studies indicated that one of the reasons for this is the fact that a higher amount of labeled collagen sedimented in the presence of excess cold collagen (Fig. 2), the condition usually employed to determine nonspecific binding; this would be due to the formation of bigger collagen fibrils in the presence of a high concentration of unlabeled collagen. Furthermore, a small portion of the labeled collagen precipitated even in the absence of excess cold collagen. Because of this, we performed control experiments that measured the sedimentation of labeled fibrous collagen in the presence and absence of excess unlabeled fibrous collagen; these values were subtracted from those of total binding and those of nonspecific binding, respectively, to obtain the actual value for the specific binding. Without these corrections, the specific binding appears to be very low; this would be the reason that the specific binding of fibrous collagen could not be detected by previous workers. Other precautions were also necessary to optimize the fibrous collagen binding assay and increase reproducibility. Since fibrous collagen readily sticks to laboratory ware, we used siliconized pipette tips and tubes, ran at least six replicates for each data point, and measured both the pellet and supernatant (in the binding tube) after removing the tips to obtain the total radioactivity we actually added to the tube. The specific fibrous collagen binding increased almost linearly as a function of added labeled fibrous collagen (Fig. 12), consistent with specific binding observed in the very low ligand concentration range, much lower than the dissociation constant. We could not perform a kinetic analysis of the fibrous collagen binding because fibrous collagen is not homogeneous and we could not measure the specific binding of fibrous collagen at the higher concentrations, which was precluded by the inability to add unlabeled collagen at the necessary 100-fold excess at high concentrations of labeled ligand.

We found that the binding of fibrous collagen had characteristics very different from those of soluble collagen binding. The initial binding of fibrous collagen is very rapid, accounts for most of the observed specific binding, and most notably is not dependent on the presence of metal ions. In the presence of Mg2+, there is a slow gradual increase in fibrous collagen binding after the initial rapid binding (Fig. 11). The amount of the Mg2+-dependent portion of the specific binding is similar to the percent decrease (about 30%) in specific binding observed in the presence of the anti-integrin alpha 2beta 1 antibody Gi9 (Fig. 13). These results suggest that the slow and Mg2+-dependent binding of fibrous collagen would be due to the contribution of integrin alpha 2beta 1. Considering the previously reported metal ion independence of the bindings of GP VI with collagen and CRP (11, 15), the main portion of the fibrous collagen binding observed in our present study, fast and also metal ion-independent, may be attributable to GP VI.

Soluble collagen binding was decreased when platelets were activated with high concentrations of CRP (Fig. 8). This is because soluble collagen binding is inhibited by CRP, as seen in Fig. 9, which illustrates the CRP concentration-dependent inhibition of soluble collagen binding to TS2/16 activated platelets. Since CRP is a synthetic polypeptide composed mainly of repeating tripeptide (Gly-Pro-Hyp) units, these results suggest that the basic triple helical structure of collagen would also contribute to the binding of soluble collagen to integrin alpha 2beta 1. The same conclusion was also deduced by Morton et al. (28) from their study of a synthetic polypeptide specifically recognized by integrin alpha 2beta 1. They determined the specific peptide sequence recognized by integrin alpha 2beta 1 by using overlapping peptide sequences of collagen type III covalently linked to a homotrimeric triple-helical peptide. Residues 522-528 of the collagen alpha 1(III) chain were found to be the minimum structure required for the recognition of integrin alpha 2beta 1. Although a linear DGEA sequence of collagen alpha 1(I) was assigned to the binding site of collagen (29), the effect of DGEA is questionable and attempts to synthesize a linear peptide that can react with integrin alpha 2beta 1 have not been successful (30).

Recent work from a number of laboratories have indicated the participation of two different signaling pathways in the platelet activation induced by collagen through experiments using GP VI-deficient platelets and GP VI-activating reagents, CRP and convulxin. CRP and convulxin induced tyrosine phosphorylation of platelet proteins, especially, tyrosine kinase Syk, Fc receptor gamma  chain, and phospholipase (PL)Cgamma 2 independently of integrin alpha 2beta 1 (27, 31-34). In the GP VI-deficient platelets, the tyrosine phosphorylation of Syk, Fc receptor gamma  chain and PLCgamma 2 was decreased and the phosphorylation of c-Src of the patient's platelets was inhibited by an anti-integrin alpha 2beta 1 antibody (33), suggesting that integrin alpha 2beta 1 would activate platelets through the phosphorylation of c-Src. Our data indicate that platelets have at least two different collagen receptors: integrin alpha 2beta 1, which binds with high affinity to soluble collagen, and another receptor, which may be GP VI, that rapidly binds to fibrous collagen. Since the signaling by the binding of fibrous collagen to GP VI was indicated by the above mentioned reports, it would be meaningful to analyze the effect of the binding of soluble collagen to integrin alpha 2beta 1 on the signaling system in platelets. The binding of soluble collagen to platelets should be a useful tool for analyzing the reaction of platelets with collagen.

    ACKNOWLEDGEMENTS

We thank Drs. Yuji Saito and Junichi Takagi, Tokyo Institute of Technology, Tokyo, Japan for giving us the antibody TS2/16 and Dr. Shosaku Nomura, Kansai Medical University, Osaka, Japan, for the antibody NNKY5-5. We also thank Dr. Yoshiki Miura of our department for the preparation of CRP.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Protein Biochemistry, Institute of Life Science, Kurume University, 2432-3 Aikawa-machi, Kurume-shi, Fukuoka-ken 839-8016, Japan. Tel.: 81-942-37-6315; Fax: 81-942-37-6319; E-mail: smj{at}ktarn.or.jp.

1 The abbreviations used are: GP, glycoprotein; PGI2, prostaglandin I2; BSA, bovine serum albumin; CRP, collagen-related peptide; GRGDS, Gly-Arg-Gly-Asp-Ser; GRGES, Gly-Arg-Gly-Glu-Ser; TRAP, thrombin receptor agonist peptide (SFLLRNPNDKYEPF); PRP, platelet-rich plasma.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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