Platelet Adhesion to Native Type I Collagen Fibrils
ROLE OF GPVI IN DIVALENT CATION-DEPENDENT AND -INDEPENDENT ADHESION AND THROMBOXANE A2 GENERATION*

Takashi NakamuraDagger , G. A. Jamieson§, Minoru Okuma, Jun-ichi KambayashiDagger , and Narendra N. TandonDagger par

From the Dagger  Otsuka America Pharmaceutical Inc., Rockville, Maryland 20850, the § American Red Cross, Rockville, Maryland 20855, and  Takashima General Hospital, Shiga, 520-11 Japan

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Three glycoproteins (GPs), namely GPIa-IIa, GPVI, and GPIV, have been recently implicated in platelet-collagen adhesive interactions. We have employed antibodies to these GPs to investigate further their role in platelet adhesion to immobilized monomeric and polymeric fibrillar collagen under static conditions in the presence and the absence of Mg2+. In the presence of Mg2+, each antibody inhibited platelet adhesion to fibrillar collagen from 70 to 85%, especially during the early phase (<15 min), but the inhibitory effects diminished dramatically to 25% or less by 60 min. Combination of anti-GPVI with anti-GPIa-IIa antibodies completely inhibited platelet adhesion at 60 min. Anti-GPIV and anti-GPIa-IIa or anti-GPVI antibodies in combinations were more effective in inhibiting adhesion than was anti-GPIa-IIa or anti-GPVI alone. In the absence of Mg2+, anti-GPVI completely inhibited adhesion at 60 min, while anti-GPIV antibody inhibited adhesion by about 50% and minimal effects were seen with anti-GPIa-IIa, suggesting that GPIa-IIa does not play a significant role in the divalent cation-independent platelet adhesion to immobilized fibrillar collagen. Under either divalent cation-dependent or -independent conditions, platelets adhered to fibrillar collagen were able to secrete contents of both alpha -granules and dense granules and generate thromboxane A2 (TXA2), but platelets adhering to acid soluble monomeric collagen neither secreted their granular contents nor generated TXA2. Although anti-GPVI antibodies were not able to inhibit Mg2+-dependent adhesion, they completely inhibited TXA2 generation under both divalent cation-dependent and -independent conditions. With the other antibodies, TXA2 generation corresponded with the amount of adhesion observed. These results suggest that GPVI is directly associated with the TXA2 generating system during platelet-collagen interaction.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Collagen has been identified to be the most thrombogenic of all the macromolecular constituents present in the extracellular matrix underlying the subendothelium. Besides supporting adhesion, it is also capable of inducing secretion and the subsequent platelet aggregation that is crucial in the maintenance of hemostatic function (1). Elucidation of the mechanism by which unactivated platelets initially recognize fibrillar collagen and initiate the subsequent platelet activation is essential for the understanding of hemostatic functions.

Both monomeric and fibrillar collagens effectively support platelet adhesion, whereas the polymerization of the monomeric collagen is required to activate platelets and induce secretion of their granule contents (2-6). Over the years, numerous candidates have been proposed for platelet-collagen receptors, probably reflecting the range of techniques that have been used to study this phenomenon (6). Seven criteria have been proposed to establish the role of putative collagen receptors for platelets (6, 7). Three glycoproteins (GPs)1 have been accepted as major collagen receptors on platelets that meet most if not all of the seven criteria proposed for a collagen receptor; these are alpha 2beta 1 integrin (GPIa-IIa), CD36 (GPIV, also known as GPIIIb), and GPVI. However, the role played by individual GPs in the overall adhesive interactions of platelets with collagen and their possible interdependence are not fully understood. Another collagen receptor has recently been identified in human platelets. A recombinant receptor protein (54 kDa), obtained by using a prokaryotic expression system, reacted specifically with type I collagen but not with type III collagen (8).

Four patients have been described with mild bleeding disorders whose platelets were found to lack GPIa and to be unresponsive to collagen but aggregated normally to other agonists (9-12). Subsequent studies in two patients revealed, however, that the defect in these platelets was not in their ability to adhere to collagen following contact but in their ability to spread on collagen following their initial adherence (10, 13). Further evidence for the participation of GPIa-IIa has been shown by the complete inhibition of Mg2+-dependent platelet adhesion to monomeric collagen by prior incubation of platelets with monoclonal antibodies directed against GPIa-IIa (14-16). On the other hand, adhesion to native collagen fibers has a considerable divalent cation-independent component (17) that is not inhibited by anti-GPIa-IIa antibodies (18).

Fab fragments of polyclonal antibodies against GPIV have been shown to partly inhibit platelet adhesion to fibrillar collagen (7). The role of GPIV in adhesive interactions of platelets with collagen was further confirmed (19) by the use of platelets of the Naka-negative phenotype, which constitutively lack GPIV (20). In comparison with control platelets, Naka-negative platelets showed significantly reduced platelet adhesion to acid-insoluble type I fibrillar collagen, especially at early time points (19, 21). Platelets from Naka-negative donors also showed reduced adhesion to type IV collagen under both flow and static conditions but responded normally with collagens type I and III (22).

Several Japanese patients with mild bleeding disorders have been described whose platelets failed to aggregate in response to collagen. Analysis of the membrane glycoproteins in these patients revealed that their platelets either lacked GPVI or had very little of it (23-25). One patient developed antibodies to GPVI after platelet transfusion. The intact IgG from the patient serum induced aggregation of normal platelets but not her own platelets, and the Fab fragments obtained from the patient's IgG inhibited collagen-induced platelet aggregation (26).

We have utilized antibodies to these GPs to obtain more precise information about their roles in the adhesive interactions of platelets with acid-insoluble type I fibrillar collagen under static conditions. We have studied the effects of blocking two receptors simultaneously on divalent cation-dependent and -independent platelet adhesion as well as on the secretion of serotonin, platelet factor 4 (PF4), and beta -thromboglobulin (beta -TG) and the generation of TXA2. Our results suggest a direct association of GPVI with the TXA2 generation system.

    EXPERIMENTAL PROCEDURES
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Introduction
Procedures
Results
Discussion
References

Reagents-- Acid-insoluble equine tendon fibrillar collagen (Chrono Par) was obtained from Chrono-Log Corp. (Broomall, PA). Acid-soluble rat tail type I collagen was purchased from Collaborative Biomedical Products Research (Bedford, MA). [2-14C]Serotonin (5-hydroxytryptamine creatinine sulfate; 58 mCi/mmol) and 51Cr (Na251CrO4; 250 mCi/mg) were purchased from Amersham Corp. Prostaglandin E1 was from Cayman Chemicals (Ann Arbor, MI). Enzyme immunoassay kits for thromboxane B2 were from PerSeptive Diagnostic, Inc. Kits for PF4 and beta -TG were obtained from Diagnostica Stago (France). Bovine serum albumin and other chemicals were purchased from Sigma.

Antibodies-- Protein G affinity-purified mouse monoclonal antibody 6F1, directed against human platelet GPIa-IIa and recognizing the GPIa subunit, was generously provided by Dr. Barry S. Coller (Mount Sinai Medical School, New York). An unrelated mouse monoclonal antibody of the IgG1 subclass (clone MOPC 21) was purchased from Sigma. A monospecific polyclonal antibody to human platelet GPIV (antibody 916) was raised in New Zealand White rabbits as described previously (19). A monospecific antibody against GPVI was purified from the plasma of a patient with idiopathic thrombocytopenic purpura who had developed an autoantibody against GPVI (26). Fab fragments (Fabs) were prepared from the IgG fraction of the patient's plasma, from normal human plasma, and from the rabbit anti-GPIV serum by digestion with agarose-coupled papain utilizing an ImmunoPure Fab preparation kit (Pierce), principally according to the manufacturer's instructions but with slight modifications in the digestion temperature and the mode of shaking. In preliminary experiments using conditions suggested by the manufacturer, the resulting Fabs from the patient's IgG failed to block the aggregation of washed platelets by the intact IgG. In subsequent digestions, therefore, the IgG samples were digested at 35 °C in an oven with gentle rotation. The final product in each case was dialyzed extensively against HEPES-saline, pH 7.4. Fab fragments thus obtained retained their activity as judged by their ability to block aggregation of washed platelets induced by the corresponding intact IgG.

Platelet Preparation-- Human blood was collected from healthy individuals who had not ingested any medication for at least 14 days prior to phlebotomy by the two-syringe method of venipuncture using a 19-gauge butterfly needle. Whole blood was collected directly into a syringe containing 3.8% sodium citrate as anticoagulant (9:1 whole blood/anticoagulant, v/v). Platelet-rich plasma was obtained by centrifugation at 150 × g for 20 min at room temperature. Washed platelets were prepared by the citrate wash method (19) with minor modifications. Briefly, citrate-washed platelets were suspended in Tyrode-HEPES buffer (136.7 mM NaCl, 5.5 mM glucose, 2.6 mM KCl, 13.8 mM, NaHCO3, 1.0 mM MgCl2·6H2O, 0.36 mM NaH2PO4·H2O, 0.25% bovine serum albumin, pH 7.4) at a concentration of 2 × 109 platelets/ml. When required, platelets (1 × 109) were labeled with Na251CrO4 (50 µCi/ml) for 1 h at room temperature followed by washing twice with citrate wash buffer containing 0.5% bovine serum albumin (19). To avoid platelet activation during washing and aggregation during adhesion assays, prostaglandin E1 (250 ng/ml) was included in all buffers used to prepare washed platelets and in subsequent operations.

Adhesion Assay-- Microtiter wells were coated with type I acid-insoluble equine tendon fibrillar collagen or with acid-soluble rat tail type I collagen maintained under acid conditions to ensure maintenance of monomer structure. Divalent cation-free adhesion buffer was made by replacing Mg2+ (1 mM) in the Tyrode-HEPES buffer with 50 µM EDTA (19). 51Cr-Labeled platelets were suspended in divalent cation-free adhesion buffer or in Tyrode-HEPES at a cell concentration of 3 × 108/ml, and adhesion assays were carried out as described previously (19).

Serotonin Release Reaction-- For adhesion-induced secretion studies, washed platelets from a single donor were divided into two equal aliquots: one aliquot was labeled with 51Cr, while the other aliquot was labeled in parallel with [14C]serotonin. Washed platelets (1 × 109 cells/ml) were incubated with 14C-labeled serotonin (0.1 µCi/ml, 1 µM) for 60 min at room temperature. The unincorporated radioactivity was removed by washing the platelets twice with platelet wash buffer (19). Platelets were finally suspended in Tyrode-HEPES buffer containing Mg2+ (1 mM) or EDTA (50 µM) and imipramine (1 µM) to stop the reuptake of released serotonin.

An aliquot (50 µl) of serotonin-loaded platelets was added to individual collagen-coated wells. At the desired times, adhesion was stopped by removing nonadhered platelets by washing each well six times by decantation using 200-µl aliquots of Tyrode-HEPES buffer containing 1 mM MgCl2 or 50 µM EDTA (19). The adhered platelets were solubilized in SDS (2%) for 30 min, and their serotonin content was quantitated by counting the lysates in a beta -counter (C). At the end of the incubation, an aliquot (50 µl) of unused platelet suspension was solubilized with an equal volume of SDS (2%) and counted (T). An aliquot of the supernatant obtained from the unused platelet suspension was also counted to determine the background radioactivity (B). The adhesion rate (R = percentage of adhesion × 0.01) was quantitated in parallel experiments using 51Cr-labeled platelets. The adhesion-induced serotonin release was calculated by the following equation.
<UP>Secretion</UP> (%)=<FENCE><FR><NU>(T−B)×R−C</NU><DE>(T−B)×R</DE></FR></FENCE>×100 (Eq. 1)

PF4 and beta -TG Release-- Levels of both PF4 and beta -TG were measured in the supernatants of the adhered platelets by commercially available kits based on enzyme-linked immunosorbent assay. At the desired times, nonadherent platelets were removed, and the wells were washed twice with wash buffer (200 µl). Suspensions of the nonadhered platelets and the two washes were combined and centrifuged at 6000 × g for 2 min to sediment the platelets, and the supernatants were immediately frozen. At the same time, an aliquot (50 µl) of unused platelet suspension was also frozen. Subsequently, all samples were thawed on ice and made 1% with respect to Triton X-100 by adding an equal volume of chilled Triton X-100 (2%) in Tyrode-HEPES buffer. After 1 h on ice, samples were centrifuged at 15,000 × g for 15 min at 4 °C to sediment Triton X-100-insoluble components. PF4 and beta -TG levels were measured in the supernatants according to the manufacturer's instructions.

Thromboxane A2 Generation-- TXA2 was measured as TXB2, a stable metabolite of TXA2, by a commercially available kit. At the desired times, unadhered platelets and suspending medium were transferred to ice-cold tubes and centrifuged at 6000 × g for 2 min to sediment platelets. Clear supernatants were kept frozen until used. Eicosanoids were extracted from the supernatants in ethyl acetate (27) prior to their quantitation by the immunosorbent assay kit. Briefly, the supernatants (100 µl) were acidified to a pH of 3-3.5 with formic acid (15 µl, 2.5%) and made 0.5 g/ml with respect to NaCl by the addition of 50 mg of solid NaCl to each tube, and eicosanoids were extracted twice with ethyl acetate (2.2 ml/extraction). Organic phases from two extractions were combined and vacuum-dried. Samples were reconstituted in kit buffer for assay and processed further according to the manufacturer's instructions. In preliminary experiments, extraction efficiency was determined by lacing the supernatants with known amounts of tritium-labeled TXB2 before extraction with ethyl acetate; 90-98% of the radioactivity was found in the organic phase.

    RESULTS
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Procedures
Results
Discussion
References

Platelet Adhesion to Monomeric and Fibrillar Type I Collagen-- Platelets can interact with collagen by both divalent cation-dependent and -independent mechanisms (6, 18, 19). Adhesion assays were carried out by a previously published method (7, 28) except that Tris-based buffer previously used to minimize platelet aggregation was replaced with Tyrode-HEPES buffer, since Tris slightly affects both the morphology (29) and responses of platelets (30). HEPES-based adhesion buffer has been used successfully to study platelet adhesion to collagen and Matrigel-coated plastic wells (31). Divalent cation-dependent platelet adhesion was measured in the presence of Mg2+ (1 mM), while the divalent cation-independent adhesion was measured in its absence but in the presence of a small amount of EDTA (50 µM) to chelate minute amounts of divalent cations present in the water and the chemicals used to prepare buffers (19). Typical patterns of time-dependent platelet adhesion to type I acid-insoluble fibrillar collagen and acid-soluble monomeric collagen both under Mg2+-dependent and -independent conditions are shown in Fig. 1, a and b, respectively. Under these conditions, at the 60-min time point, the divalent cation-independent platelet adhesion was about one-fourth of the adhesion observed in the presence of Mg2+ and was similar to that obtained using Tris-based buffers (19). However, platelet adhesion to monomeric collagen was exclusively divalent cation-dependent, since no adhesion was observed in the absence of Mg2+ (Fig. 1b).


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Fig. 1.   Time-dependent platelet adhesion to plastic-immobilized type I collagen. 51Cr-Labeled washed platelets were added to microtiter wells coated with type I acid-insoluble equine tendon fibrillar collagen (a) or type I acid-soluble rat tail collagen (b). Mg2+-dependent and -independent adhesion was measured as described under "Experimental Procedures." Platelet adhesion has been expressed as percentage of platelets adhered in relation to the total number of platelets added. Solid bars represent Mg2+-dependent adhesion, while the open bars represent Mg2+-independent adhesion. Values given are the means ± S.D. of at least six experiments using platelets from different individuals each run in replicate of five.

Effects of Anti-GPIa-IIa, Anti-GPIV, and Anti-GPVI Antibodies on Platelet Adhesion-- In preliminary studies, we determined the dose-dependent effect of each antibody on platelet adhesion to both the acid-insoluble fibrillar type I collagen from equine tendon and to acid-soluble monomeric type I collagen from rat tail. Adhesion was studied for 30 min both in the presence and absence of Mg2+. Since IgG fractions of both anti-GPIV and anti-GPVI antibodies induced aggregation of the washed platelets, Fab fragments were used in both cases. Maximal inhibition of divalent cation-independent platelet adhesion to acid-insoluble fibrillar type I collagen was obtained at 300 µg/ml Fab fragments of anti-GPIV and anti-GPVI. Mg2+-dependent adhesion was not affected by higher concentrations of these Fabs. Maximal inhibition of Mg2+-dependent adhesion to rat tail monomeric collagen was obtained at a concentration of 20 µg/ml of anti-GPIa-IIa IgG (6F1). In subsequent experiments, therefore, we used 6F1, anti-GPIV (916 Fab), and anti-GPVI (anti-p62 Fab) at 20, 300, and 300 µg/ml, respectively.

In the presence of Mg2+, all three antibodies effectively inhibited platelet adhesion to acid-insoluble fibrillar collagen at the 15-min time point (Fig. 2a). Both anti-GPIa-IIa and anti-GPVI inhibited adhesion by about 85%, while anti-GPIV inhibited adhesion by 70-75%. However, these inhibitory effects diminished with time. At the 60-min time point, anti-GPIa-IIa showed inhibition of ~25% and anti-GPVI showed ~15%, while anti-GPIV showed inhibition of only 5%. In the absence of Mg2+ (Fig. 2b), anti-GPIa-IIa had minimal effects, while both anti-GPVI and anti-GPIV Fabs effectively inhibited platelet adhesion to fibrillar collagen at all time points examined. At 300 µg/ml, anti-GPVI Fabs inhibited adhesion completely, while anti-GPIV Fabs showed an inhibition of about 50%. Under each set of conditions, unrelated mouse IgG1 (clone MOPC 21) and control Fabs prepared from rabbit and human normal IgG used as negative controls were without effect (data not shown).


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Fig. 2.   Effects of anti-GPIa-IIa, anti-GPIV, and anti-GPVI antibodies on platelet adhesion to type I acid-insoluble fibrillar collagen. 51Cr-Labeled platelets were incubated with anti-GPIa-IIa (6F1; 20 µg/ml), anti-GPIV (916 Fabs; 300 µg/ml), and anti-GPVI (anti p62Fabs; 300 µg/ml) antibodies for 30 min at room temperature prior to their addition to the microtiter wells. Adhesion was measured as described under "Experimental Procedures." Nonimmune antibodies, used as a negative control, in each case had no effect on platelet adhesion. Platelet adhesion is expressed as a percentage of the corresponding control value. Adhesion was studied in the presence of Mg2+ (Fig. 2a) or its absence (Fig. 2b). Values shown are the means ± S.D. of four experiments each run in replicate of five.

We next examined the combined effects of these antibodies both under Mg2+-dependent and -independent conditions. In the presence of Mg2+, combinations of antibodies were significantly more effective in inhibiting platelet adhesion as compared with the individual antibody alone. Notably, the combination of anti-GPIa-IIa and anti-GPVI completely inhibited adhesion even at 60 min in the presence of Mg2+ (Fig. 2a). Combination of anti-GPIV with anti-GPIa-IIa or anti-GPVI also enhanced inhibition, but these effects decreased with time, and only ~50% inhibition was observed at 60 min. In the absence of Mg2+, preincubation of platelets with anti-GPVI resulted in a complete inhibition of adhesion at all time points (Fig. 2b). Interestingly, anti-GPIV, which had minimal effect on Mg2+-dependent platelet adhesion at the 60-min time point, significantly inhibited platelet adhesion at all time points (35-50%) in the absence of Mg2+. Contrary to its inhibitory effect in the presence of Mg2+, anti-GPIa-IIa had a minimal effect on time-dependent adhesion in the absence of Mg2+. To conserve the human anti-GPVI antibody, and since it completely inhibited platelet adhesion to acid-insoluble fibrillar collagen under Mg2+-free conditions, we did not study the effects of combinations of anti-GPVI Fabs with either anti-GPIa-IIa IgG or anti-GPIV Fabs, but we did examine the combined effects of anti-GPIa-IIa IgG and anti-GPIV Fabs. The combination of anti-GPIa-IIa with anti-GPIV was slightly more effective in inhibiting adhesion than was anti-GPIV alone, but substantial adhesion was seen at all time points, suggesting that in a Mg2+-free system GPVI alone can overcome the loss of two major receptors. Under each set of conditions, unrelated mouse IgG1 and control Fabs prepared from rabbit and human normal IgG used as negative controls were without effect (data not shown).

Effects of Anti-GPIa-IIa, Anti-GPIV, and Anti-GPVI Antibodies on Platelet Adhesion to Monomeric Type I Collagen-- Rat tail acid-soluble monomeric collagen was coated onto microtiter wells as described for acid-insoluble fibrillar collagen under "Experimental Procedures." Since no adhesion could be seen in the absence of Mg2+ (Fig. 1b), we examined first the effect of the three antibodies individually in the presence of Mg2+ (Fig. 3). Preincubation of platelets with anti-GPIa-IIa IgG resulted in a complete inhibition of adhesion. Anti-GPVI Fabs, which significantly inhibited adhesion to acid-insoluble type fibrillar collagen at early time points (Fig. 2a), had little or no effect on platelet adhesion to rat tail monomeric collagen. Anti-GPIV Fabs by themselves inhibited adhesion significantly at early time points. The addition of both anti-GPVI Fabs and anti-GPIV Fabs showed no further inhibition, confirming again that GPVI has no role in platelet adhesion to acid-soluble monomeric collagen under static conditions. These results support earlier observations that GPIa-IIa is an important adhesion receptor for monomeric collagen (14-16) and that GPIV may also participate in the adhesive interactions of platelets with monomeric collagen.


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Fig. 3.   Effects of anti-GPIa-IIa, anti-GPIV, and anti-GPVI antibodies on Mg2+-dependent platelet adhesion to monomeric type I rat tail collagen. Conditions were similar to those used in Fig. 2a except that acid-soluble rat tail collagen was used. Platelet adhesion is expressed as percentage relative to the corresponding control values. Values shown are the means ± S.D. of three experiments each run in replicate of five.

Adhesion-induced Platelet Release Reaction-- The extent of the platelet release reaction was determined by measuring the amount of [14C]serotonin secreted into the medium from the dense granules and of PF4 and beta -TG from the alpha -granules following adhesion to fibrillar collagen. In the presence of Mg2+, adhered platelets secreted ~60% of their dense granule contents into the medium (Table I). In the absence of Mg2+, the amount of adhesion was reduced to about one-fourth, but the adhered platelets secreted ~80% of their [14C]serotonin content. These results suggest that Mg2+ is not required for the collagen-induced release reaction. Adhesion to monomeric collagen did not induce a significant release reaction (data not shown). The combination of anti-GPIa-IIa and anti-GPVI Fabs that completely inhibited Mg2+-dependent adhesion also completely inhibited serotonin secretion (Table II). Similarly, anti-GPVI Fabs which inhibited platelet adhesion in Mg2+-free buffer also inhibited secretion of serotonin from platelets. Like serotonin secretion, the secretion of PF4 and beta -TG was also inhibited by anti-GPVI Fabs by themselves alone and in combination with anti-GPIa-IIa (data not shown).

                              
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Table I
Divalent cation-dependent and -independent adhesion of platelets to fibrillar type I collagen and [14C]serotonin secretion
Adhesion assays were terminated at the desired time, and the amount of serotonin release from the adhered platelets was measured by counting 14C content of the adhered platelets in a beta -counter as described under "Experimental Procedures." The values given are means ± S.D. from six experiments each run in replicate of five.

                              
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Table II
Effects of anti-GPIa-IIa, anti-GPIV and anti-GPVI on platelet adhesion to fibrillar type I collagen and [14C]serotonin secretion
Platelets were incubated with anti-GPIa-IIa (6F1; 20 µg of IgG/ml), anti-GPIV (916 Fabs; 300 µg/ml), and anti-GPVI (anti-p62 Fabs; 300 µg/ml) for 30 min prior to their addition to microtiter wells. Adhesion and release reactions were measured as described under "Experimental Procedures." In each case, nonimmune mouse IgG or normal rabbit or human Fabs were run as controls. The data represent means ± S.D. of three experiments using platelets from different individuals, and each samples was run in triplicate. The interassay variability was within a 2-6% range.

Adhesion-induced TXA2 Generation-- TXB2, a stable metabolite of TXA2, was measured by immunosorbent assay in the platelet suspending medium after the adhesion as described under "Experimental Procedures." Both in the presence and absence of Mg2+, adhered platelets generated TXA2 in a time-dependent manner (Table III). The absolute amounts of TXA2 generated in each assay during early time points were greater in the presence of Mg2+ than in its absence. However, when TXA2 generation was correlated with the amount of adhesion, there was relatively 3-4 times more TXA2 generation in the absence of Mg2+ than in its presence. We then examined the effects of the three antibodies individually and in combination on adhesion-induced TXA2 generation (Fig. 4). In the presence of Mg2+, preincubation of platelets with anti-GPVI Fabs resulted in only a small decrease (15%) in adhesion but completely inhibited TXA2 generation. Anti-GPIa-IIa antibody inhibited platelet adhesion by ~25% but had no effect on TXA2, generation and anti-GPIV Fab had minimal effects on both platelet adhesion and TXA2 generation. In the absence of Mg2+, anti-GPVI totally suppressed adhesion and TXA2 generation (Fig. 4a). Modest inhibition of both was seen with anti-GPIa-IIa (~25%) and with anti-GPIV (~50%), and their inhibitory effects were essentially additive when added in combination. To exclude the possible role of newly formed TXA2 in enhancing platelet adhesion and TXA2 generation, we examined the effect of aspirin and SQ29548, a TXA2 receptor antagonist, on both platelet adhesion to type I acid-insoluble collagen and TXA2 generation. Preincubation of platelets with aspirin (1 mM) and SQ29548 (10 µM) individually had minimal effect on platelet adhesion to collagen, suggesting that TXA2 by itself does not influence the platelet adhesion process in our assay system. Furthermore, SQ29548 had no effect on adhesion-induced TXA2 generation, suggesting that generated TXA2 does not induce further TXA2 generation (data not shown).

                              
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Table III
Time-dependent TXA2 generation by platelets adhered to type I fibrillar collagen under static conditions
Adhesion assays were terminated at the desired time, and levels of TXA2 released from the adhered platelets were measured by enzyme immunoassay as described under "Experimental Procedures." The values given are means ± S.D. from three experiments each run in duplicate. The interassay values varied 2-9%.


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Fig. 4.   Effects of anti-GPIa-IIa, anti-GPIV, and anti-GPVI antibodies on TXA2 generation by the adhered platelets. 51Cr-Loaded platelets were suspended either in Mg2+-containing buffer (Fig. 4a) or Mg2+-free buffer (Fig. 4b) and incubated with the various antibodies for 30 min at room temperature prior to their addition to the microtiter wells as described in the legend of Fig. 2. After a 60-min incubation, TXA2 generation was measured in the supernatant as described under "Experimental Procedures." Solid bars represent percentage of adhesion relative to corresponding control values, and hatched bars show adhesion-induced TXA2 formation. Values given are the means ± S.D. of three experiments each run a duplicate.

    DISCUSSION
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Procedures
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Discussion
References

The purpose of this study was to investigate in detail the roles of GPIa-IIa, GPIV, and GPVI in platelet adhesion to collagen under static conditions. Type I monomeric collagen interacts primarily with platelet membrane GPIa-IIa in a Mg2+-dependent fashion without release of dense granule contents (6). On the other hand, interaction with polymeric collagen has a strong divalent cation-independent element that is not inhibitable by anti-GPIa-IIa antibodies (18). We have evaluated, therefore, both acid-insoluble polymeric type I collagen and acid-soluble monomeric type I collagen as substrates to study platelet-collagen adhesive interactions.

In our adhesion assay, 1) platelet adhesion to acid-soluble monomeric collagen was Mg2+-dependent, since no adhesion was seen in the absence of Mg2+; 2) it was completely inhibited by preincubation of platelets with anti-GPIa-IIa antibody; and 3) an insignificant release reaction from the adhered platelets was seen. These results confirm earlier observations made by Santoro (6). The lack of effect of anti-GPVI Fabs alone on platelet adhesion to monomeric collagen suggests that GPVI does not play a significant role in these interactions. Anti-GPIV Fabs by themselves were able to significantly inhibit adhesion only during early time points.

Platelets adhere to polymeric fibrillar collagen via both Mg2+-dependent and -independent mechanisms (18). In fact, our results suggest that one-fourth of the total adhesion is Mg2+-independent. Mg2+-independent adhesion was not inhibited by anti-GPIa-IIa antibody, but it was completely inhibited by anti-GPVI Fabs and partially (~50%) inhibited by anti-GPIV Fabs. Moroi et al. (23) described a GPVI-deficient patient whose platelets failed to adhere to collagen fibrils in the presence of EDTA, which is consistent with our results and further confirms that GPVI is a primary receptor in divalent cation-independent platelet adhesion to collagen. In addition, 50% inhibition seen with anti-GPIV Fabs confirms our earlier observation that GPIV-mediated adhesion is Mg2+-independent (28). These results suggest that both GPVI and GPIV interact with the immobilized collagen in a Mg2+-independent fashion. It is, of course, important to emphasize that these Mg2+-independent reactions go forward in the presence of Mg2+ but are not dependent on it.

All three antibodies individually inhibited adhesion (70-85%) during the early phases of Mg2+-dependent adhesion, but at the 60 min time point only very limited inhibition was observed in each case. This loss of inhibition by the 60-min time point suggests that all three receptors may be involved during the early phases, but in the absence of one receptor the other two are ultimately sufficient to overcome the absence of the third. This may explain why patients with deficiency of either GPIa or GPVI do not exhibit a severe bleeding tendency. However, when two receptors were blocked simultaneously, the combination of anti-GPVI Fabs and anti-GPIa-IIa antibody completely abolished adhesion, while the other two combinations, namely anti-GPIa-IIa/anti-GPIV and anti-GPVI/anti-GPIV, were more effective than each individual antibody alone. These results suggest that, under static conditions and in the presence of extracellular Mg2+, all three receptors participate in adhesive interactions of platelets with polymeric type I collagen but that GPIa-IIa and GPVI play the major roles in these interactions.

Upon binding to collagen fibers, platelets spread, secrete their granular contents, and generate TXA2. Some of the secreted products, including TXA2, are themselves proaggregatory in that they bind back to platelets and induce aggregation resulting in the growth of thrombus size. The role of individual collagen receptors in various activation processes has not so far been elucidated. We have attempted to clarify the role of each receptor by measuring the adhesion-induced release reaction from dense granules and alpha -granules and the generation of TXA2. Minimal serotonin, PF4, and beta -TG release (about 15%) and no TXA2 generation was observed when monomeric type I collagen was used as a substrate (data not shown). In contrast, once adhered to fibrillar type I collagen, platelets were able to secrete 60-80% of their dense and alpha -granule contents and generate TXA2 under both divalent cation-dependent and -independent conditions. It is interesting to note that relatively more TXA2 was generated by the adhered platelets in the absence of Mg2+. To the best of our knowledge, this is the first report to evaluate Mg2+ effects on adhesion-induced TXA2 generation. These results further confirm that only native collagen fibers can induce the release reaction and demonstrate for the first time that polymeric fibrillar collagen is essential for TXA2 generation.

To explore further the role of the various collagen receptors in TXA2 generation, we carried out antibody blocking experiments. TXA2 generation paralleled the amount of adhesion when GPIa-IIa and GPIV were blocked with their respective antibodies alone or in combination (Fig. 4), suggesting that these receptors are not directly involved in TXA2 generation during adhesion to collagen under static conditions. On the other hand, anti-GPVI Fabs totally abolished TXA2 generation in the presence of Mg2+, but adhesion was inhibited by only ~15%. TXA2 generation was not seen in all cases where GPVI was blocked. These results are in agreement with those of Ryo et al. (25) and Sugiyama et al. (26), who showed that platelets lacking GPVI did not generate TXA2 upon stimulation with collagen under stirring conditions. The failure to detect TXA2 generation in GPIa-deficient platelets (9) probably reflects the absence of collagen-induced aggregation in these patients or a combined deficiency of other receptors such as GPIV and GPVI, which could affect platelet responses (11). These results strongly suggest a direct association between GPVI and TXA2 generation during platelet-collagen adhesive interactions.

In summary, platelets have both divalent cation-dependent (integrin; GPIa-IIa) and -independent (non-integrin; GPIV and GPVI) receptors for fibrillar type I collagen. GPVI and GPIa-IIa seem to be the primary receptors for immobilized fibrillar type I collagen under static conditions, and GPIV may accelerate the rate of adhesion once platelets establish contact with collagen fibers via GPVI and GPIa-IIa. Adhered platelets secrete dense and alpha -granule contents and induce the generation of TXA2 independent of divalent cations. GPVI-mediated signaling seems to play an important role in TXA2 generation. The steps involved in the interaction of collagen with GPVI and the formation of TXA2 are not known and require further study.

    ACKNOWLEDGEMENTS

We are grateful to Guertin Moe and Bob Dunn for performing TXB2 assays.

    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.

par To whom correspondence and reprint requests should be addressed: Maryland Research Laboratories, Otsuka America Pharmaceutical Inc., 9900 Medical Center Dr., Rockville, MD 20850. Tel.: 301-424-9055 (ext. 2301); Fax: 301-424-9054; E-mail: narendrt{at}mrl.oapi.com.

1 The abbreviations used are: GP, glycoprotein; TXA2 and TXB2, thromboxane A2 and B2, respectively; PF4, platelet factor 4; beta -TG, beta -thromboglobulin; Fabs, Fab fragments.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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