Activation of the GP IIb-IIIa Complex Induced by Platelet Adhesion to Collagen Is Mediated by Both alpha 2beta 1 Integrin and GP VI*

Takashi NakamuraDagger , Jun-ichi KambayashiDagger , Minoru Okuma§, and Narendra N. TandonDagger

From Dagger  Otsuka America Pharmaceutical Inc., Rockville, Maryland 20850 and the § Takashima General Hospital, Shiga, 520-1121, Japan

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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alpha 2beta 1 integrin, CD36, and GP VI have all been implicated in platelet-collagen adhesive interactions. We have investigated the role of these glycoproteins on activation of the GP IIb-IIIa complex induced by platelet adhesion to type I fibrillar and monomeric collagen under static conditions. In the presence of Mg2+, platelet adhesion to fibrillar collagen induced activation of the GP IIb-IIIa complex and complete spreading. Anti-alpha 2beta 1 integrin and anti-GP VI antibodies inhibited the activation of the GP IIb-IIIa complex by about 40 and 50%, respectively, at 60 min although minimal inhibitory effects on adhesion were seen. Platelet spreading was markedly reduced by anti-alpha 2beta 1 integrin antibody. The combination of anti-alpha 2beta 1 integrin with anti-GP VI antibody completely inhibited both platelet adhesion and activation of the GP IIb-IIIa complex. Anti-CD36 antibody had no significant effects on platelet adhesion, spreading, and the activation of the GP IIb-IIIa complex at 60 min. Aspirin and the thromboxane A2 receptor antagonist SQ29548 inhibited activation of the GP IIb-IIIa complex about 30% but had minimal inhibitory effect on adhesion. In the absence of Mg2+, there was significant activation of the GP IIb-IIIa complex but minimal spreading was observed. Anti-GP VI antibody completely inhibited adhesion whereas no effect was observed with anti-alpha 2beta 1 integrin antibody. Anti-CD36 antibody partially inhibited both adhesion and the activation of the GP IIb-IIIa complex. Platelet adhesion to monomeric collagen, which requires Mg2+ and is exclusively mediated by alpha 2beta 1 integrin, resulted in partial activation of the GPIIb-IIIa complex and spreading. No significant effects were observed by anti-CD36 and anti-GP VI antibodies. These results suggest that both alpha 2beta 1 integrin and GP VI are involved in inside-out signaling leading to activation of the GP IIb-IIIa complex after platelet adhesion to collagen and generation of thromboxane A2 may further enhance expression of activated GP IIb-IIIa complexes.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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When the integrity of the vascular endothelium is disrupted, various macromolecular components of the vascular subendothelium become exposed and accessible to platelets. Although several of these components, such as laminin, fibronectin, and von Willebrand factor, all provide a suitable substrate for platelet adhesion, fibrillar collagen is the most thrombogenic constituent of the vascular subendothelium since it supports not only adhesion but also causes platelet activation leading to platelet aggregation (1, 2). Because platelet-platelet interactions are primarily mediated by the simultaneous binding of the multivalent adhesive glycoproteins fibrinogen and/or von Willebrand factor to activated GP1 IIb-IIIa complexes on two different platelets, collagen must ultimately operate by effecting the activation of the GP IIb-IIIa complexes on an adhered platelet to either capture or serve as docking site for a circulating platelet.

Three GPs, namely alpha 2beta 1 integrin (GPIa-IIa), CD36 (GPIV, also known as GPIIIb), and GP VI have been implicated in platelet-collagen adhesive interactions, however, the roles of these GPs on the adhesion-induced expression of activated GP IIb-IIIa complexes are not fully understood.

Four patients have been described with mild bleeding diathesis attributable to deficient expression of alpha 2-integrin and platelets from these patients had impaired collagen-induced aggregation but aggregated normally to other agonists (3-6). Anti-CD36 antibodies have been shown to partly inhibit platelet adhesion to fibrillar collagen under both static and flow conditions (7-9). However, platelets from Naka-negative donors which constitutively lack CD36 have been shown to aggregate normally to collagen (10-12). Several Japanese patients with mild bleeding disorders have been described whose platelets failed to aggregate in response to collagen (13-16). Analysis of membrane glycoproteins in these patients revealed that their platelets either lacked GP VI or had very little of it. Thus far, lack of either alpha 2beta 1 integrin or GP VI in patients has been associated with impaired platelet aggregation in response to collagen. These results imply that both alpha 2beta 1 integrin and GP VI have definite roles in activation of the GP IIb-IIIa complex after platelet binding to collagen. Clinical studies also suggested that more than a single partial defect is required for a disorder of collagen platelet interaction to become clinically important (17). A recent study has shown that GP VI-deficient platelets bind some fibrinogen in response to collagen without aggregation, suggesting that collagen may induce some signaling via alpha 2beta 1 integrin, leading to the activation of the GP IIb-IIIa complex (18). In addition, 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 (19).

A large number of investigators have examined the collagen-induced platelet activation process including the expression of activated GP IIb-IIIa complex during collagen-induced aggregation of washed platelets in suspension or during perfusion of whole blood over collagen-coated coverslips. Three excellent reviews have appeared in the past year (20-22). However, time-dependent changes in the expression of activated GP IIb-IIIa complex during platelet adhesion to immobilized collagen under static conditions have not been studied in detail. We recently showed the role of GP VI in divalent cation-independent platelet adhesion to fibrillar collagen under static conditions and its direct association with the adhesion-induced thromboxane A2 (TXA2) generating system (9). Using our static adhesion assay, we have now investigated the role of alpha 2beta 1 integrin, CD36, and GP VI on the expression of activated GP IIb-IIIa complexes on platelets adhered to type I fibrillar and monomeric collagens. We simultaneously measured adhesion rate and the activation of the GP IIb-IIIa complexes by employing 125I-PAC-1 which binds only to the activated forms of the GP IIb-IIIa complexes (23). Furthermore, by using confocal laser scanning microscopy, we were able to visualize adhesion-induced changes in the morphology of platelets and distribution of activated GP IIb-IIIa complexes as detected by FITC-labeled PAC-1. Our results clearly demonstrate that platelet adhesion to immobilized collagen can directly induce the activation of the GP IIb-IIIa complex and this inside-out signaling is mediated by both alpha 2beta 1 integrin and GP VI.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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Reagents-- Acid-insoluble equine tendon fibrillar collagen (Chrono Par) was obtained from Chrono-Log Co. (Broomall, PA). Acid-soluble rat tail type I collagen was purchased from Collaborative Biomedical Products Research (Bedford, MA). 51Cr (Na251CrO4, 250 mCi/mg) and Na125I (1 mCi/10 µl) were purchased from Amersham Co. Prostaglandin E1 and SQ29548 were obtained from Cayman Chemicals (Ann Arbor, MI). Chelex 100 was from Sigma. Unlabeled PAC-1 and fluorescein isothiocyanate (FITC)-labeled PAC-1 were purchased from Becton Dickinson (San Jose, CA).

Antibodies-- Protein G-affinity purified mouse monoclonal antibody 6F1, directed against human platelet alpha 2beta 1 integrin and recognizing the alpha 2-subunit, was generously provided by Dr. Barry S. Coller (School of Medicine, State University of New York, Stony Brook, NY). An unrelated mouse monoclonal antibody of the IgG1 subclass (clone-MOPC 21) was purchased from Sigma. A monospecific polyclonal antibody to human platelet CD36 (number 916) was raised in New Zealand White rabbits as described previously (24). A monospecific antibody against GP VI was purified from the plasma of a patient with idiopathic thrombocytopenic purpura who had developed an autoantibody against GP VI (16). Fab fragments (Fabs) were prepared from the IgG fraction of the patient's plasma, from normal human plasma and from the rabbit anti-CD36 serum by digestion with agarose-coupled papain utilizing a ImmunoPure Fab preparation kit (Pierce Chemical Co., Rockford, IL) principally according to the manufacturer's instructions but with a slight modification in temperature and time of incubation (9). The final product in each case was dialyzed extensively against HEPES saline, pH 7.4. Fabs thus obtained retained their activity as judged by their ability to block aggregation of washed platelets induced by the corresponding intact IgG. In addition, 6F1 and Fabs from control IgG, rabbit anti-CD36 serum, and anti-GP VI serum did not induce any platelet aggregation and [14C]serotonin secretion, suggesting that these antibodies do not activate platelets. For antibody experiments, platelets were incubated with each antibody for 30 min at room temperature prior to their being allowed to adhere to collagen-coated wells.

Platelet Preparation-- Human platelet-rich plasma was prepared as described previously (9). Washed platelets were prepared by the citrate wash method with minor modifications. Briefly citrate-washed platelets were suspended in Chelex 100-pretreated modified Tyrode-HEPES buffer (136.7 mM NaCl, 5.5 mM glucose, 2.6 mM KCl, 13.8 mM, NaHCO3, 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. Finally washed platelets were resuspended in Chelex 100-treated modified Tyrode-HEPES buffer containing 10 µM Ca2+ with or without further supplementation with 1 mM Mg2+. 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 and Binding of 125I-PAC-1 to Adhered Platelets-- Monoclonal antibody PAC-1 was radiolabeled with Na125I using IODO-BEADS as suggested by the manufacturer (Pierce). Labeled antibody was separated from free Na125I by gel filtration on a PD-10 column (Pharmacia, Piscataway, NJ) which was equilibrated with Tyrode-HEPES buffer. The specific activity of 125I-PAC-1 was approximately 200,000 cpm/µg protein. Microtiter wells were coated with type I acid-insoluble equine tendon fibrillar collagen or with type I acid-soluble rat tail collagen maintained under acid conditions to ensure maintenance of monomer structure. Platelet adhesion assays were performed as described previously (9), but with a slight modification in preparation of the suspending medium. Since maximal PAC-1 binding to the activated form of the GP IIb-IIIa complex is observed at 3-10 µM free Ca2+ (23), Tyrode-HEPES buffer was first treated with Chelex 100 to remove any divalent cations and later supplemented with 1 mM Mg2+ and 10 µM Ca2+ for Mg2+-dependent adhesion and with 10 µM Ca2+ alone for Mg2+-independent adhesion. Platelets from a single donor were aliquoted into two portions. One aliquot was converted to a 51Cr-labeled platelet suspension containing unlabeled PAC-1 (1 µg/ml) while the other aliquot was converted to a nonlabeled platelet suspension containing 125I-PAC-1 (1 µg/ml). A portion (50 µl) of the unlabeled platelet preparation was added to collagen-coated wells. At the desired time adhesion was stopped by removing nonadhered platelets by washing each well six times by decantation with 200-µl aliquots of suspending Tyrode-HEPES buffer. The adhered platelets were solubilized in SDS (2%) for 30 min and 125I-PAC-1 binding was quantitated by counting the lysates in a gamma -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 for total count (T). Another aliquot of unused platelet suspension was centrifuged at 13,000 rpm in a microcentrifuge through a 20% sucrose and the pellet was solubilized in 50 µl of SDS (2%) and counted to obtain nonspecific binding (NS). Adhesion rate (R = % adhesion × 0.01) was quantitated in parallel experiments using 51Cr-labeled platelets. The adhesion-induced 125I-PAC-1 binding was calculated by the following equation,
<SUP>125</SUP><UP>I-PAC-1 binding </UP>(%)=<FENCE><FR><NU>(C−NS×R)</NU><DE>T−NS</DE></FR></FENCE>×100 (Eq. 1)

Confocal Laser Scanning Microscopy-- Microwells were prepared by attaching a Flexiperm chamber (Heraeus Instruments, Osterode, Germany) on a cover glass. Collagen coating and bovine serum albumin blocking were performed as described above. Washed platelets containing FITC-labeled PAC-1 (1 µg/ml) were added to the microwells and incubated for the indicated times. Unbound antibodies and platelets were removed by washing six times with Tyrode-HEPES buffer and then adhered platelets were fixed with 1% paraformaldehyde for 30 min at room temperature. Differential interference contrast images and fluorescent confocal images of 0.5-µm optical sections were simultaneously obtained using a Carl Zeiss 510 confocal laser scanning microscope (Thornwood, NY) with a Zeiss Plan-Apo 100 × 1.40 NA oil immersion objective. FITC fluorescence was detected at an excitation wavelength of 488 nm with a barrier filter at 500 nm.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Platelet Adhesion to Collagen and Adhesion-induced Activation of the GP IIb-IIIa Complex-- We previously used Mg2+ or EDTA-containing Tyrode-HEPES buffer to characterize divalent cation-dependent or -independent platelet adhesion to fibrillar and monomeric collagens (9). To detect activation of the GP IIb-IIIa complex in the present work, we employed 125I-PAC-1 which requires 3-10 µM free Ca2+ to fully bind to the activated form of the GP IIb-IIIa complex (23). We therefore, modified the suspension medium as described under "Experimental Procedures." Adhesion rate was determined using 51Cr-labeled platelets and the activation of the GP IIb-IIIa complex was simultaneously determined by measuring 125I-PAC-1 binding to adhered platelets. Typical patterns of time-dependent platelet adhesion to type I acid-insoluble fibrillar collagen and acid-soluble monomeric collagen and concomitant activation of the GP IIb-IIIa complexes both under Mg2+-dependent and -independent conditions are shown in Fig. 1, a and b, respectively. Under these conditions, PAC-1 had a negligible effect on platelet adhesion (data not shown). Both in the presence and absence of extracellular Mg2+, platelets adhered to fibrillar collagen showed time-dependent PAC-1 binding and the Mg2+-independent platelet adhesion was about half of the adhesion observed in the presence of Mg2+. As shown in Fig. 1b, platelets adhered to monomeric collagen also showed PAC-1 binding, however, this adhesion was exclusively Mg2+-dependent and the extent of PAC-1 binding was always less than that of platelets adhered to fibrillar collagen. At the 60-min time point, PAC-1 binding was about 60% of that seen with fibrillar collagen even though the same extent of adhesion was seen in both cases.


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Fig. 1.   Time-dependent platelet adhesion to plastic-immobilized type I collagen and 125I-PAC-1 binding. 51Cr-labeled washed platelets containing 1 µg/ml unlabeled PAC-1 or unlabeled washed platelets containing 1 µg/ml 125I-PAC-1 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 and corresponding 125I-PAC-1 binding were measured as described under "Experimental Procedures." Platelet adhesion and 125I-PAC-1 binding have been expressed as percentage of total number of platelets and total count of 125I-PAC-1 added. Solid bars represent platelet adhesion while hatched bars represent 125I-PAC-1 binding. Values given are the mean ± S.D. of at least five experiments using platelets from different individuals each run in replicate of three.

Effects of Anti-alpha 2beta 1 Integrin, Anti-CD36, and Anti-GP VI Antibodies and Aspirin on Platelet Adhesion to Type I Fibrillar Collagen and Activation of the GP IIb-IIIa Complex-- We next examined the effect of antibodies against three collagen receptors, namely anti-alpha 2beta 1 integrin IgG (6F1; 20 µg/ml), anti-CD36 Fabs (916; 300 µg/ml), and anti-GP VI Fabs (300 µg/ml), on platelet adhesion to fibrillar collagen and activation of the GP IIb-IIIa complex at the 60-min time point. These concentrations of antibodies have been shown to be optimal in previous studies (9). We also examined the effect of aspirin (1 mM) since we previously showed that platelets adhered to fibrillar collagen generated considerable amounts of TXA2 both in the presence and absence of Mg2+ (9). As shown in Fig. 2a, in the presence of Mg2+, anti-alpha 2beta 1 integrin, anti-CD36, anti-GP VI antibody, and aspirin showed minimal inhibition of platelet adhesion to fibrillar collagen (5-15%) at the 60-min time point. In contrast, anti-alpha 2beta 1 integrin, anti-GP VI antibody, and aspirin significantly inhibited PAC-1 binding by about 40, 50, and 30%, respectively: the TXA2 receptor antagonist SQ29548 (10 µM) showed similar effects to aspirin (data not shown). Anti-CD36 antibody had no effect on adhesion or on PAC-1 binding. The combination of anti-alpha 2beta 1 integrin and anti-GP VI antibody inhibited platelet adhesion completely as reported earlier (9) and no activation of the GP IIb-IIIa complex was detected. We then examined the combination of each antibody and aspirin. No apparent additive or synergistic inhibitory effects were observed. As shown in Fig. 2b, in the absence of Mg2+ anti-GP VI antibody inhibited completely both adhesion and PAC-1 binding whereas no effect was seen with anti-alpha 2beta 1 integrin antibody which had shown significant inhibition of PAC-1 binding in the presence of Mg2+. Anti-CD36 antibody inhibited platelet adhesion to collagen about 30% which correlated with the inhibition of PAC-1 binding. Aspirin showed minor inhibitory effects on both platelet adhesion and PAC-1 binding. A combination of anti-alpha 2beta 1 integrin or anti-CD36 antibody with aspirin did not show significantly additive or synergistic effects on either adhesion or PAC-1 binding. Under each set of conditions, unrelated mouse IgG1 and control Fabs prepared from rabbit and human normal IgG used as negative controls had negligible effects on adhesion and PAC-1 binding (data not shown). Platelets adhered to fibrillar collagen secreted their alpha  and dense granule contents even in the absence of Mg2+ (9). Therefore we examined the possible role of released ADP and serotonin on activation of the GP IIb-IIIa complex. Neither ADP removal by creatine phosphate/creatine phosphokinase nor the presence of serotonin receptor antagonist, ketanserin, had any significant effect on the activation of the GP IIb-IIIa complex (data not shown).


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Fig. 2.   Effects of anti-alpha 2beta 1 integrin, anti-CD36, anti-GP VI antibodies, and aspirin (ASA) on platelet adhesion to type I acid-insoluble fibrillar collagen and 125I-PAC-1 binding. 51Cr-labeled platelets were incubated with anti-alpha 2beta 1 integrin (6F1; 20 µg/ml), anti-CD36 (number 916 Fabs; 300 µg/ml), anti-GP VI (anti-p62 Fabs; 300 µg/ml) antibodies, and aspirin (1 mM) for 30 min at room temperature prior to their addition to the microtiter wells. Adhesion and 125I-PAC-1 binding were measured as described under "Experimental Procedures." Nonimmune IgG subclass-matched antibodies, used as a negative control, had no effect on platelet adhesion and 125I-PAC-1 binding. Platelet adhesion and 125I-PAC-1 binding are expressed as percentage of the corresponding control value. Adhesion and 125I-PAC-1 binding were studied in the presence of Mg2+ (a) or in its absence (b). Values shown are the mean ± S.D. of four experiments each run in replicate of three.

Effects of Anti-alpha 2beta 1 Integrin, Anti-CD36, and Anti-GP VI Antibodies on Platelet Adhesion to Type I Monomeric Collagen and Activation of the GP IIb-IIIa Complex-- As reported earlier, platelet adhesion to type I monomeric collagen is exclusively Mg2+-dependent and solely mediated by alpha 2beta 1 integrin (9). This interaction does not cause any release reaction and generation of TXA2. As shown in Fig. 3, anti-alpha 2beta 1 integrin antibody inhibited both platelet adhesion and PAC-1 binding completely at the 60-min time point. However, anti-CD36 and anti-GP VI antibody alone or in combination had minimal effects on both platelet adhesion and PAC-1 binding.


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Fig. 3.   Effects of anti-alpha 2beta 1 integrin, anti-CD36, and anti-GP VI antibodies on Mg2+-dependent platelet adhesion to monomeric type I rat tail collagen and 125I-PAC-1 binding. Conditions were similar to those used in Fig. 2a except that acid-soluble rat tail collagen was used. Platelet adhesion and 125I-PAC-1 binding are expressed as percentages relative to the corresponding control values. Values shown are the mean ± S.D. of four experiments each run in replicate of three.

Visualization of Adhesion-induced Morphological Changes and the Expression of the Activated GP IIb-IIIa Complex-- To visualize time-dependent platelet adhesion to collagen and changes in PAC-1 binding, FITC-labeled PAC-1 (1 µg/ml) was added to washed platelet suspensions and adhesion assays were performed as described under "Experimental Procedures." Fig. 4, a-i, shows the time course of differential interference contrast images of platelets adhered to type I fibrillar and monomeric collagen and the fluorescent confocal images of the surface activation of the GP IIb-IIIa complexes as detected by FITC-labeled PAC-1. FITC-labeled PAC-1 binding was specific since FITC-labeled control antibody showed no binding to adherent platelets and prostaglandin E1-treated resting platelet showed negligible FITC-labeled PAC-1 binding (data not shown). These series of photomicrographs depict direct induction of the activated GP IIb-IIIa complexes and their distribution after platelet adhesion to fibrillar and monomeric collagen. At the 15-min time point, in the presence of Mg2+, platelets in contact with fibrillar collagen showed pseudopodia followed by broad lamellae formation (Fig. 4a). PAC-1 binding was evident on partially spread platelets. It should be noted that some platelets showed blebbing (Fig. 4a, arrow). This formation is further evident at the 30-min time point (Fig. 4b). Platelets adhered to fibrillar collagen spread out with time and the surface was almost covered with fully spread out platelets at the 60-min time point, however, no apparent aggregates were seen (Fig. 4c). To evaluate possible platelet aggregate formation, adhered platelets at the 60-min time point were fixed with 1% paraformaldehyde and incubated with mouse monoclonal anti-fibrinogen antibody and further incubated with FITC-labeled anti-mouse antibody. Anti-fibrinogen antibody binding was found to be localized at the boundary of adjacent platelets that had already spread onto collagen. However, no anti-fibrinogen antibody binding was observed on the platelet surface, confirming that no aggregates were formed in this assay (data not shown). In the absence of Mg2+, platelets adhered to fibrillar collagen and also showed PAC-1 binding, however, spreading was minimal, suggesting that Mg2+ is required for platelet spreading (Fig. 4, d-f). Fig. 4, g-i, shows the time course of platelet adhesion to type I monomeric collagen in the presence of Mg2+. Platelets adhered to monomeric collagen also showed some spreading and a lesser extent of activation of the GP IIb-IIIa complex as compared with fibrillar collagen. Fig. 4, j-l, shows the effects of anti-alpha 2beta 1 integrin, anti-GP VI antibody, and aspirin on platelet adhesion to fibrillar collagen and PAC-1 binding in the presence of Mg2+ at the 60-min time point. In the presence of anti-alpha 2beta 1 integrin antibody, platelet spreading was strongly reduced but not completely inhibited, suggesting that some integrin other than alpha 2beta 1 integrin, possibly GP IIb-IIIa, may be involved in platelet spreading. As for PAC-1, anti-alpha 2beta 1 integrin antibody slightly inhibited its binding and redistribution was minimal as compared with control (Fig. 4c). In the presence of anti-GP VI antibody, PAC-1 binding was remarkably reduced and platelet spreading was slightly inhibited. Aspirin had minimal effects on platelet adhesion and spreading, however, a significant decrease of PAC-1 binding was observed confirming our 125I-PAC-1 binding data.


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Fig. 4.   Confocal microscopic images showing fluorescent PAC-1 binding and differential interference contrast images showing morphology of platelets adhered to type I collagen in the presence of Mg2+. Washed platelets containing 1 µg/ml FITC-labeled PAC-1 were added to microwells coated with type I acid-insoluble fibrillar collagen or acid-soluble monomeric collagen. At the desired time points, unbound antibodies were washed and adhered platelets were fixed with 1% paraformaldehyde for 30 min at room temperature. Samples were analyzed by using confocal laser scanning microscopy (LSM 510) as described under "Experimental Procedures." Typical patterns of FITC-PAC-1 binding (left panel), differential interference contrast imaging (middle panel), and overlapped imaging (right panel) are shown. Platelet adhesion to fibrillar collagen in the presence of Mg2+ at 15- (a), 30- (b), and 60-min (c) time points and in the absence of Mg2+ at 15- (d), 30- (e), and 60-min (f) time points are shown. Arrows indicate bleb formation. Platelet adhesion to monomeric collagen at 15- (g), 30- (h), and 60-min (i) time points are shown. Platelets were incubated with anti-alpha 2beta 1 integrin (6F1; 20 µg/ml) (j), anti-GP VI (anti-p62 Fabs; 300 µg/ml) (k), antibodies and aspirin (1 mM) (l) for 30 min at room temperature and platelet adhesion at 60-min time points are shown. Scale bar = 5 µm.


    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The accessibility of activated GP IIb-IIIa complexes on the surface of platelets adhered to a damaged vessel plays a crucial role in subsequent platelet recruitment to a thrombus. In this study, we have investigated the role of collagen receptors, namely alpha 2beta 1 integrin, CD36, and GP VI, on the expression of activated GP IIb-IIIa complexes induced by platelet adhesion to type I fibrillar and monomeric collagen under static conditions.

Platelet adhesion to monomeric collagen requires Mg2+ and is exclusively mediated by alpha 2beta 1 integrin and there is no significant role of CD36 or GP VI in this interaction. Platelets adhered to monomeric collagen neither secrete their granule content nor generate TXA2 (9). However, these platelets show significant spreading and induce activation of the GP IIb-IIIa complex although this expression is about 40% less than adhesion to fibrillar collagen. This is consistent with the earlier report which showed the binding of thromboerythrocytes to platelets adhered to type I rat skin collagen (25). Taken together, these results suggest that alpha 2beta 1 integrin-mediated inside-out signaling is able to induce partial platelet spreading and partial activation of the GP IIb-IIIa complex. Monomeric collagen effectively supports platelet adhesion, whereas polymerization of monomeric collagen is required to induce platelet aggregation and secretion (26-30). These observations may be explained by the fact that the number of activated GP IIb-IIIa complexes and their localization induced by monomeric collagen is not sufficient to elicit a full aggregatory response.

Platelet adhesion to fibrillar collagen is composed of Mg2+-dependent and -independent adhesion (9, 31). In the presence of Mg2+, platelets adhered to fibrillar collagen showed full spreading and increased activation of the GP IIb-IIIa complex than that of platelets adhered to monomeric collagen. Resting platelets have been shown to have approximately 50,000 molecules of GP IIb-IIIa complex on their surface. Upon activation with strong agonist such as thrombin, this number increases to 100,000 sites per platelet, probably reflecting the recruitment from the internal pool of GP IIb-IIIa complex (23, 32). However, the maximum number of PAC-1-binding sites on activated platelets under saturating conditions has been reported to be only 25,000 sites per platelet (23). In the presence of Mg2+, platelets adherent to fibrillar collagen at the 60-min time point bound about 12,000 125I-PAC-1 molecules per platelet. This number is about half of the total PAC-1-binding sites as determined by thrombin stimulation (23). In our confocal images, PAC-1 binding was observed only on the nonadhered surface of the platelets; no apparent PAC-1 binding was observed by Z-sectioning or vertical images (X-Z sectioning) on the side of the cell adhering to collagen fibers (data not shown). Thus our PAC-1 binding data may reflect only one side surface expression of the activated GP IIb-IIIa complex. In our previous report (9), we measured both alpha - and dense granule secretion during platelet adhesion and found that only fibrillar collagen can induce these granules secretion (50% in the presence of Mg2+). Therefore, the increased number of PAC-1-binding sites on platelets adherent to fibrillar collagen may partly reflect the recruitment of the GP IIb-IIIa complex originating from internal pools.

Anti-alpha 2beta 1 integrin antibody inhibited activation of the GP IIb-IIIa complex about 40% and significantly inhibited but did not eliminate platelet spreading. These results confirm that alpha 2beta 1 integrin-mediated signaling is partly responsible for the activation of the GP IIb-IIIa complex and platelet spreading once platelets have adhered to fibrillar collagen. The mechanism of platelet spreading which is not attributable to alpha 2beta 1 integrin remains to be elucidated. It has been reported that alpha 2beta 1 integrin-deficient platelets were not able to spread on collagen and collagen failed to induce platelet aggregation (3, 4, 6). In the presence of anti-alpha 2beta 1 integrin antibody, collagen-induced platelet aggregation was also abolished (5, 33). Accordingly, it could be concluded that platelet spreading on collagen fibers is mainly mediated by alpha 2beta 1 integrin and that nearly full activation of the GP IIb-IIIa is required for platelet aggregation induced by collagen. It is also possible that alpha 2beta 1 integrin may be necessary for an initial tethering of floating platelets to the collagen fiber (22, 34-37).

In our previous study, Mg2+-independent adhesion was studied in the presence of EDTA to chelate any Mg2+ arising from the impurities of the reagents used to make Tyrode-HEPES buffer (9). In order to remove Mg2+ and other divalent cations from the buffer, we used Chelex 100. Divalent cations were added as needed. The results of our previous study indicated that divalent cation-independent adhesion amounts to about one-fourth of the adhesion observed in the presence of Mg2+. Using the chelation method we found that Mg2+-independent adhesion was about half that seen in the presence of Mg2+ suggesting that EDTA, even at only 50 µM concentration, can affect platelet adhesion. The results of this study, as well as our earlier study, clearly show that in the absence of Mg2+ platelets adhered to fibrillar collagen and this interaction is mainly dependent on GP VI and there seems to be no role of alpha 2beta 1 integrin. Platelets adhered to fibrillar collagen in the absence of Mg2+ do secrete and generate TXA2 (9). Although this interaction does not induce platelet spreading, apparent activation of the GP IIb-IIIa complex was observed. In the presence of Mg2+, anti-GP VI Fabs also inhibited the activation of the GP IIb-IIIa complex by about 50% and platelet spreading was slightly inhibited. These results suggest that GP VI-mediated signaling also plays an important role in adhesion-induced activation of the GP IIb-IIIa complex. GP VI-deficient platelets failed to aggregate in response to collagen (13-16) and anti-GP VI Fabs also abolished collagen-induced platelet aggregation (16). In addition, a recent study under flow conditions has shown that GP VI-deficient platelets exhibit almost normal primary adhesion to collagen fibers but that platelet aggregates formation is abrogated (38). These observations are consistent with the role of GP VI and the activation of the GP IIb-IIIa complex.

Although G proteins, intracellular calcium, protein kinases, and many other proteins are thought to be involved in the expression of activated GP IIb-IIIa complex, the exact mechanisms that control the expression of activated GP IIb-IIIa complex are still obscure (39, 40). Several lines of evidence suggest that activation of platelets stimulated by collagen is mediated through a tyrosine kinase pathway that involves the Fc receptor gamma  chain, Syk, and phospholipase Cgamma 2 (41-43). Phosphorylated phospholipase Cgamma 2 becomes activated leading to the generation of inositol triphosphate and elevation of intracellular Ca2+. These phosphorylation events seem to be mediated by both alpha 2beta 1 integrin and GP VI (44-47). In other words, alpha 2beta 1 integrin- and GP VI-mediated signaling converge on intracellular Ca2+ elevation. In fact, it has been shown that collagen directly induces a rise in cytosolic calcium in single human platelets (48). The intracellular Ca2+ chelator, BAPTA, inhibited 125PAC-1 binding in a dose-dependent manner although it does not itself affect platelet adhesion,2 suggesting that intracellular Ca2+ seems to be a key second messenger for the inside-out signaling leading to the activation of the GP IIb-IIIa complex. Further experiments are currently underway to elucidate the relationship between intracellular calcium ion and the expression of activated GP IIb-IIIa complexes.

We have previously reported that platelets adhered to fibrillar collagen are able to secrete dense and alpha  granule contents and generate TXA2: in particular TXA2 generation was found to be directly associated with GP VI (9). TXA2, ADP, and serotonin are considered as positive feedback agonists in that they bind back to platelets and synergistically potentiate platelet activation processes leading to large aggregate formation. Therefore, we estimated the contribution of TXA2, ADP, and serotonin to the activation of the GP IIb-IIIa complex. In the presence of Mg2+, aspirin, and the TXA2 receptor antagonist, SQ29548, showed about 30% inhibition of activation of the GP IIb-IIIa complex induced by platelet adhesion to fibrillar collagen, but only minimal effects on adhesion and spreading of platelets were observed. These results suggest that generated TXA2 binds to adhered platelets and induces activation of the GP IIb-IIIa complex. In the presence of Mg2+, a combination of aspirin with anti-alpha 2beta 1 integrin did not show any additive inhibitory effects on the activation of the GP IIb-IIIa complex. Furthermore, in the absence of Mg2+, aspirin did not inhibit activation of the GP IIb-IIIa complex. These results suggest that TXA2-mediated activation of the GP IIb-IIIa complex may require platelet spreading. In contrast, removal of secreted ADP by the creatine phosphate/creatine phosphokinase system or the presence of serotonin receptor antagonist, ketanserin, did not show any significant inhibition of activation of the GP IIb-IIIa complex, suggesting that ADP and serotonin do not have a significant effect on platelets already adhered to collagen. Platelets deficient in GP VI failed to aggregate and bind fibrinogen upon challenge with collagen-related peptide (CRP-X) suggesting a role of GP VI in the activation process and subsequent fibrinogen binding and aggregation. Although a reduced but significant amount of fibrinogen bound to GP VI-deficient platelets when they were challenged with fibrillar collagen (18). Convulxin, a minor component of tropical rattle snake venom, has been shown to activate platelets by interacting exclusively with GP VI (49). However, in a related study, an antibody to alpha 2beta 1 integrin inhibited convulxin-induced platelet aggregation by about 60%. The authors have proposed that during later stages of convulxin-induced platelet activation alpha 2beta 1 integrin may be partially responsible for the expression of activated GP IIb-IIIa complex (50). These observations and our own results further support the view that both GP VI and alpha 2beta 1 integrin play important roles in the expression of the activated GP IIb-IIIa complex induced by platelet-collagen interactions.

In summary, platelet adhesion to collagen can directly induce activation of the GP IIb-IIIa complex. Fibrillar collagen induced increased expression than monomeric collagen. This activation process is mediated by both alpha 2beta 1 integrin and GP VI and enhanced by TXA2. Although our static adhesion assay system was able to dissect the individual role of collagen receptors on activation of the GP IIb-IIIa complex, it is imperative to elucidate their exact roles under more physiological conditions such as whole blood under flow conditions. Finally, considering the role of GP VI in adhesion-induced TXA2 generation and the activation of the GP IIb-IIIa complex, blocking collagen-GP VI interaction may prove to be an alternative therapeutic approach to the treatment of thrombotic disease.

    ACKNOWLEDGEMENTS

We are grateful to Dr. Barry S. Coller for the generous donation of valuable antibody. We thank Dr. G. A. Jamieson for critical review and valuable suggestions.

    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.

To whom correspondence 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.

2 T. Nakamura, J-i. Kambayashi, M. Okuma, and N. N. Tandon, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: GP, glycoprotein; TXA2, thromboxane A2; FITC, fluorescein isothiocyanate; BAPTA, 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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