Rhodocytin (Aggretin) Activates Platelets Lacking alpha 2beta 1 Integrin, Glycoprotein VI, and the Ligand-binding Domain of Glycoprotein Ibalpha *

Wolfgang BergmeierDagger , Daniel Bouvard§, Johannes A. Eble||, Rabée Mokhtari-NejadDagger , Valerie SchulteDagger , Hubert ZirngiblDagger , Cord Brakebusch§, Reinhard Fässler§, and Bernhard NieswandtDagger **

From the Dagger  Department of Molecular Oncology, General Surgery, Witten/Herdecke University, 42117 Wuppertal, Germany, the § Department of Experimental Pathology, Lund University, 22185 Lund, Sweden, and the || Institute for Physiological Chemistry and Pathobiochemistry, Universität Münster, 48149 Münster, Germany

Received for publication, May 1, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Although alpha 2beta 1 integrin (glycoprotein Ia/IIa) has been established as a platelet collagen receptor, its role in collagen-induced platelet activation has been controversial. Recently, it has been demonstrated that rhodocytin (also termed aggretin), a snake venom toxin purified from the venom of Calloselasma rhodostoma, induces platelet activation that can be blocked by monoclonal antibodies against alpha 2beta 1 integrin. This finding suggested that clustering of alpha 2beta 1 integrin by rhodocytin is sufficient to induce platelet activation and led to the hypothesis that collagen may activate platelets by a similar mechanism. In contrast to these findings, we provided evidence that rhodocytin does not bind to alpha 2beta 1 integrin. Here we show that the Cre/loxP-mediated loss of beta 1 integrin on mouse platelets has no effect on rhodocytin-induced platelet activation, excluding an essential role of alpha 2beta 1 integrin in this process. Furthermore, proteolytic cleavage of the 45-kDa N-terminal domain of glycoprotein (GP) Ibalpha either on normal or on beta 1-null platelets had no significant effect on rhodocytin-induced platelet activation. Moreover, mouse platelets lacking both alpha 2beta 1 integrin and the activating collagen receptor GPVI responded normally to rhodocytin. Finally, even after additional proteolytic removal of the 45-kDa N-terminal domain of GPIbalpha rhodocytin induced aggregation of these platelets. These results demonstrate that rhodocytin induces platelet activation by mechanisms that are fundamentally different from those induced by collagen.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Collagen is one of the major components of the vessel wall and contributes to platelet activation and adhesion at sites of vascular injury. The interaction between platelets and collagen can either occur indirectly via immobilized von Willebrand factor (vWf)1 binding to platelet receptors glycoprotein (GP) Ib-V-IX and/or activated alpha IIbbeta 3 integrin (1) or by direct recognition of collagen by specific receptors expressed on the platelet surface. Several receptors for collagen have been identified on platelets, most importantly the Ig-like receptor GPVI (2) and alpha 2beta 1 integrin (3). In contrast to earlier reports, we have recently shown with beta 1 integrin-null platelets that alpha 2beta 1 integrin is not essential for platelet adhesion to fibrillar collagen. GPVI, however, was found to be indispensable for this process (4).

Although GPVI has been established as the major activating platelet collagen receptor, the way alpha 2beta 1 integrin modulates the activation process is still unclear. Experimental evidence suggests that collagen contains two distinct epitopes contributing to activation of (murine) platelets. One of these epitopes specifically binds to GPVI, and this interaction is blocked by the anti-GPVI mAb JAQ1 (5). In contrast, activation through the second epitope is not blocked by JAQ1 and involves GPVI, alpha 2beta 1 integrin, and high concentrations of fibrillar collagen (4). The mechanisms underlying this activation pathway and the role of alpha 2beta 1 integrin are unclear. In addition to alpha 2beta 1 and GPVI, other receptors may be involved in this activation pathway. One candidate is GPIbalpha , because this receptor indirectly interacts with collagen via vWf (1).

Snake venom-derived proteins are frequently used to study mechanisms of platelet activation and aggregation because many of them specifically bind to platelet surface glycoprotein receptors and interfere with their function. Rhodocytin (also termed aggretin (6)), purified from the venom of Calloselasma rhodostoma belongs to the family of C-type lectins and induces aggregation of human as well as mouse platelets (7). Recent studies gave rise to conflicting results on the mechanisms underlying this activation process. Several experiments suggested that rhodocytin activates platelets in a collagen-like manner. First, both processes are sensitive to inhibition of thromboxane A2 formation by treatment with acetylsalicylic acid, and, second, both can be inhibited by mAbs against alpha 2beta 1 integrin (7, 8). Based on these results, the authors concluded that rhodocytin activates platelets by interacting with alpha 2beta 1 integrin (8). Others reported that rhodocytin activates platelets through alpha 2beta 1 integrin and GPIbalpha (9), results that were also based on experiments with inhibitory antibodies against both receptors.

Both hypotheses, however, were challenged by our finding that rhodocytin does not bind recombinant, soluble alpha 2beta 1 (10), and the same result has meanwhile been obtained with wild-type alpha 2beta 1 integrin isolated from human platelets.2 Additionally, rhodocytin activates platelets from FcRgamma chain-deficient mice (7, 9), which lack GPVI (11) and do not respond to collagen (4, 12), suggesting that rhodocytin uses other mechanisms than collagen to induce aggregation. Moreover, we have recently shown that beta 1-null platelets fail to express alpha 2beta 1 integrin but display no reduced response to fibrillar collagen, indicating that alpha 2beta 1 integrin is not a major signaling collagen receptor on platelets (4).

To directly test whether rhodocytin induces platelet activation by mechanisms similar to those induced by collagen, we now examined the effects of this agonist on platelets lacking alpha 2beta 1 integrin, GPVI, the ligand-binding domain on GPIbalpha , or all three of them. The results of these studies demonstrate that none of these receptors is required for platelet activation by rhodocytin.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Animals: Generation of Mice with beta 1-null Platelets-- Mice carrying the beta 1-null allele in megakaryocytes were generated as described previously (4). Briefly, beta 1(fl/fl) mice (13) were crossed with transgenic mice carrying the Mx-cre transgene (mx-cre+) (14). Deletion of the beta 1 gene was induced in 4-5-week-old (beta 1(fl/fl)/Mx-cre+) mice by three intraperitoneal injections of 250 µg of polyinosinic-polycytidylic acid at 2-day intervals. Control mice beta 1(fl/fl) received the same treatment and were derived from same litters. For experiments, mice were used at least 2 weeks after polyinosinic-polycytidylic acid injection. The absence of the alpha 2 and beta 1 integrin subunits on the platelets from these mice was always confirmed by flow cytometry and Western blotting as described (4). C57Bl/6 mice deficient in the FcRgamma chain (15) were obtained from Taconics (Germantown, NY). C57Bl/6 × SV129 mice deficient in GPV were kindly provided by F. Lanza (Strasbourg, France).

Depletion of Platelet GPVI-- Mice were injected with 100 µg of JAQ1 intraperitoneally, and platelets were isolated on day 7. As reported previously, GPVI was not detectable on those platelets by flow cytometry and Western blotting (16).

Reagents-- High molecular weight heparin (Sigma), alpha -thrombin (Roche Molecular Biochemicals), collagen (Nycomed GmbH, Munich, Germany), and O-sialoglycoprotein endopeptidase (Cedarlane, Hornby, Canada) were purchased. Apyrase was purified from potatoes as described previously (17). Acetylsalicylic acid was from Sanofi-Synthelabo (Paris, France). Botrocetin and purified human vWf were kindly provided by F. Lanza (Strasbourg, France) and G. Dickneite (Marburg, Germany), respectively.

Purification of Rhodocytin-- C. rhodostoma (Malayain pit viper) venom was purchased from Sigma. Dissolved in 50 mM Tris/HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.02% sodium azide, the venom was separated by gel filtration chromatography on a Superose 6 column. The eluate fractions of respective size were pooled. After dilution with Mono S-buffer A (20 mM MES/NaOH, pH 6.5), the pooled fractions were applied to a Mono S HR5/5 column (Amersham Pharmacia Biotech). Not binding to Mono S resin under these conditions, the rhodocytin in the flow-through was dialyzed against Mono Q buffer A (20 mM Tris/HCl, pH 8.0, 0.02% sodium azide) and applied to a Mono Q HR5/5 column (Amersham Pharmacia Biotech). Rhodocytin was eluted from the Mono Q column in a linear NaCl gradient at sodium chloride concentrations above 300 mM. The rhodocytin-containing eluate fraction was concentrated by centrifugal ultrafiltration in a Centricon 10 tube. Finally, the concentrated solution of rhodocytin was purified by gel filtration on a tandem array of TSK G3000 SWXL and TSK G2000 SWXL (TosoHass, Stuttgart, Germany). Purity was determined by SDS-polyacrylamide gel electrophoresis in a 12-18% acrylamide separating gel. Protein concentration was assayed by the BCA method according to the manufacturer's protocol (Pierce).

Antibodies-- The rat anti-mouse P-selectin mAb RB40.34 was kindly provided by D. Vestweber (Münster, Germany) and modified in our laboratories. FITC hamster anti-beta 1 integrin (Ha31/8), FITC hamster anti-alpha 2 integrin, and rat anti-beta 1 integrin (9EG7) were from BD Pharmingen. Horseradish peroxidase-labeled rabbit anti-FITC, polyclonal rabbit anti-fibrinogen, and polyclonal rabbit anti-vWf were purchased from DAKO. All other antibodies were generated, produced, and modified in our laboratories: JAQ1 (anti-GPVI) (11), JON1 (anti-GPIIb/IIIa) (18), p0p4 (anti-GPIbalpha ) (18), DOM1 (anti-GPV) (19), and ULF1 (anti-CD9) (19).

Platelet Preparation-- Mice were bled under ether anesthesia from the retroorbital plexus. The blood was collected in a tube containing 10% (v/v) 7.5 units/ml heparin, and platelet-rich plasma was obtained by centrifugation at 300 × g for 10 min at room temperature. The platelets were washed twice in Tyrode's buffer (137 mM NaCl, 2 mM KCl, 12 mM NaHCO3, 0.3 mM NaH2PO4, 1 mM MgCl2, 2 mM CaCl2, 5.5 mM glucose, 5 mM Hepes, pH 7.3) containing 0.35% bovine serum albumin and finally resuspended at a density of 2 × 105 platelets/µl in the same buffer in the presence of 0.02 unit/ml of the ADP scavenger apyrase, a concentration sufficient to prevent desensitization of platelet ADP receptors during storage. Platelets were kept at 37 °C throughout all experiments.

Immunoblotting-- Platelets (108) were solubilized in 1 ml of lysis buffer (Tris-buffered saline containing 20 mM Tris/HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 0.5 µg/ml leupeptin, and 0.5% Nonidet P-40; all from Roche Molecular Biochemicals). After lysis, whole cell extract was run on a 9% SDS-polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane. The membrane was first incubated with 5 µg/ml FITC-labeled mAb followed by rabbit anti-FITC-horseradish peroxidase (1 µg/ml). The proteins were visualized by ECL.

Flow Cytometry-- Washed platelets (2 × 106) were incubated with the indicated amounts of agonists for 5 min followed by staining with fluorophore-conjugated Abs (5 µg/ml) for 10 min at 37 °C and immediately analyzed on a FACScalibur (Becton Dickinson). Platelets were gated by forward scatter/side scatter characteristics.

Treatment of Platelets with O-Sialoglycoprotein Endopetidase-- The washed platelets (2 × 109/ml) were resuspended in Tyrode's buffer (1 mM MgCl2, 1 mM CaCl2) and incubated at 37 °C for 30 min with 100 µg/ml O-sialoglycoprotein endopeptidase. Aliquots of the platelet suspensions were analyzed in flow cytometry and Western blotting to estimate markers of platelet activation and alterations in platelet glycoproteins.

Aggregometry-- To determine platelet aggregation, light transmission was measured using washed platelets (200 µl with 0.5 × 106 platelets/µl). Transmission was recorded on a Fibrintimer 4 channel aggregometer (LAbor, Hamburg, Germany) over 10 min and was expressed as arbitrary units with 100% transmission adjusted with Tyrode's buffer.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

It has been reported that rhodocytin induces platelet aggregation by interacting with the collagen receptor alpha 2beta 1 integrin. Based on these results, it was suggested that collagen might also activate platelets via alpha 2beta 1 integrin (7). These findings were challenged by our result showing that rhodocytin does not bind to a recombinant soluble form of alpha 2beta 1 integrin (10).

To directly investigate whether rhodocytin activates platelets through mechanisms similar to those induced by collagen, we compared the effects of rhodocytin and fibrillar collagen on mouse platelets lacking the collagen receptors alpha 2beta 1 integrin and GPVI. As reported previously, FcRgamma chain-deficient platelets lack GPVI but express normal amounts of alpha 2beta 1 integrin (Fig. 1a) and do not respond to collagen (11, 12) (Fig. 1b). In contrast, platelets from mice with a Cre/loxP-mediated deletion of the beta 1 gene in megakaryocytes lack all beta 1 integrins but express normal amounts of GPVI (Fig. 1a). beta 1-null platelets display delayed but not reduced aggregation to fibrillar collagen (4) (Fig. 1b).


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Fig. 1.   Characterization of collagen receptor-deficient platelets. a, platelets from the indicated mice were stained with fluorophore-conjugated mAbs against GPVI or beta 1 integrin at a final concentration of 5 µg/ml for 10 min at 37 °C and analyzed directly. b, washed platelets from the indicated mice were stimulated by the addition of fibrillar collagen (2 µg/ml), and light transmission was recorded on a standard aggregometer. wt, wild type.

The effects of rhodocytin on mouse platelets were similar to those reported for human platelets (7-9). Aggregation occurred with a dose-dependent lag phase (Fig. 2a) and was sensitive to inhibitors of the thromboxane A2-producing system (not shown). Platelet activation by rhodocytin occurred independently of GPVI/FcRgamma (Fig. 2a) and was accompanied by marked degranulation (as shown by P-selectin expression) and strong fibrinogen binding (Fig. 2b).


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Fig. 2.   Rhodocytin activates platelets independently of alpha 2beta 1 integrin. a, washed platelets from the indicated mice were stimulated with rhodocytin (5 or 50 nM), and light transmission was recorded on a standard aggregometer. b, washed platelets from the indicated mice were left untreated (lower panel) or stimulated with rhodocytin (10 nM, upper panel) and subsequently incubated with anti-fibrinogen-FITC and anti-P-selectin-PE Abs for 10 min at 37 °C and analyzed directly. The platelets were gated by forward scatter/side scatter characteristics and Fl3 positivity (anti-mouse GPIbalpha -PE/Cy5). The results shown are representative of six individual experiments.

Strikingly, rhodocytin also induced aggregation of beta 1-null platelets. The extent of degranulation and fibrinogen binding was indistinguishable between beta 1-null and control platelets (Fig. 2b). Furthermore, no differences were found in the dose-response characteristics between normal and beta 1-null platelets (not shown). These results demonstrate that beta 1 integrins, including alpha 2beta 1, are not essential for rhodocytin-induced platelet activation.

It has been shown that snake venom toxins may induce platelet activation by interacting with multiple receptors (20). Thus, blocking only one of them would not inhibit platelet activation/aggregation. Therefore, we examined the effects of rhodocytin on platelets lacking both alpha 2beta 1 and GPVI. To generate such platelets, GPVI was depleted in mice with beta 1-null platelets by injection of JAQ1 (100 µg/mouse). This treatment induces a virtually complete internalization and proteolytic degradation of GPVI in circulating platelets but does not affect other receptors, including alpha IIbbeta 3, GPIb-V-IX, and CD9 (16). GPVI-depleted platelets do not respond to collagen, whereas activation by other agonists is not affected (16). As shown in Fig. 3a, platelets from JAQ1-treated beta 1-null mice lacked both collagen receptors. However, these beta 1/GPVI-deficient platelets responded normally to rhodocytin as demonstrated by aggregometry and flow cytometric analysis (Fig. 3, b and c). This finding excludes an essential role of alpha 2beta 1 and GPVI in rhodocytin-induced platelet activation.


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Fig. 3.   GPVI-depleted beta 1-null platelets respond normally to rhodocytin. Mice with beta 1-null platelets were injected with 100 µg of JAQ1 intraperitoneally, and the platelets were isolated on day 7. a, flow cytometric detection of beta 1 integrin and GPVI in normal (shaded area) and GPVI-depleted beta 1-null platelets (solid line). b, washed GPVI-depleted beta 1-null platelets were stimulated with rhodocytin (10 nM), and light transmission was recorded on a standard aggregometer. c, washed platelets from these mice were stimulated with rhodocytin (10 nM) and subsequently incubated with anti-fibrinogen-FITC and anti-P-selectin-PE Abs for 10 min at 37 °C and analyzed directly. The experiment was repeated three times with comparable results.

A very recent report showed that rhodocytin (aggretin)-induced platelet activation was inhibited by a mAb against the 45-kDa N-terminal domain on GPIbalpha (9). This domain contains the binding sites for all known ligands, including vWf, thrombin (21), P-selectin (22), and MAC-1 (23), as well as snake venom-derived C-type lectins like jararaca GPIb-BP (24), alboaggregin A (20), and echicetin (25). Based on their results, Navdaev et al. (9) concluded that GPIbalpha plays an essential role in rhodocytin-induced platelet activation. To directly test this hypothesis, we treated platelets with O-sialoglycoprotein endopeptidase (26). This treatment resulted in complete proteolytic removal of the 45-kDa N-terminal domain of GPIbalpha as demonstrated by flow cytometric analysis (Fig. 4a). Interestingly, Western blot analysis revealed that, in addition to cleavage of the 45-kDa N-terminal domain, the truncated remainder of GPIbalpha (105 kDa) was further cleaved in close vicinity to the transmembrane region of GPIbalpha , resulting in the release of glycocalicin lacking the 45-kDa N-terminal region (~85 kDa) (Fig. 4b). Because of the complete lack of the 45-kDa N-terminal domain of GPIbalpha , botrocetin-induced vWf binding was abolished in O-sialoglycoprotein endopeptidase-treated platelets (Fig. 4c). However, these platelets responded normally to rhodocytin (Fig. 4, d and e), demonstrating that the ligand-binding domain on GPIbalpha is not essential for this activation process.


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Fig. 4.   The 45-kDa N-terminal domain of GPIbalpha is not required for rhodocytin-induced activation of mouse platelets. Washed wild-type platelets were incubated with O-sialoglycoprotein endopeptidase (0.1 mg/ml) for 30 min at 37 °C. a, flow cytometric analysis of control (shaded area) or O-sialoglycoprotein endopeptidase-treated (solid line) platelets stained for the N-terminal domain of GPIbalpha (5 µg/ml p0p4-PE). b, Western blot analysis of GPIbalpha in control and O-sialoglycoprotein endopeptidase (OSE)-treated platelets. Whole cell lysates were separated on an 8% SDS-polyacrylamide gel-PE under reducing conditions and immunoblotted with FITC-labeled p0p5 (anti-GPIbalpha ) followed by horseradish peroxidase-labeled anti-FITC antibodies/ECL. p0p5 binds to an epitope on the glycocalicin portion on GPIbalpha that is not located in the 45-kDa N-terminal domain. On the right side, the positions of intact GPIbalpha , the truncated form lacking the 45-kDa N-terminal domain (t-GPIbalpha ), and the corresponding truncated form of glycocalicin (t-GC) are indicated. c, control (shaded area) and O-sialoglycoprotein endopeptidase-treated (solid line) platelets were incubated with human vWf (5 µg/ml) in the presence of botrocetin (2 µg/ml). Bound vWf was detected by FITC-labeled anti-vWf Abs (10 µg/ml). d, O-sialoglycoprotein endopeptidase-treated platelets were stimulated with 10 nM rhodocytin and light transmission was recorded on a standard aggregometer. e, O-sialoglycoprotein endopeptidase-treated platelets were stimulated with rhodocytin (10 nM) and subsequently incubated with anti-fibrinogen-FITC and anti-P-selectin-PE Abs for 10 min at 37 °C and analyzed directly. The results shown are representative of six individual experiments.

Although our results demonstrated that neither the collagen receptors alpha 2beta 1 integrin and GPVI nor GPIbalpha are essential for rhodocytin-induced platelet activation, it could not be excluded that rhodocytin binds to all three receptors that independently elicit aggregation. Therefore, we removed the 45-kDa N-terminal region of GPIbalpha from beta 1- and beta 1/GPVI-deficient platelets and examined their response to rhodocytin. As shown in Fig. 5, even the absences of alpha 2beta 1, GPVI, and the 45-kDa N-terminal domain of GPIbalpha had no significant effect on rhodocytin-induced platelet activation and aggregation.


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Fig. 5.   Mouse platelets lacking alpha 2beta 1 integrin, GPVI, and the 45-kDa N-terminal domain of GPIbalpha respond normally to rhodocytin. Washed GPVI-depleted beta 1-null platelets were treated with O-sialoglycoprotein endopeptidase for 30 min at 37 °C. a, flow cytometric detection of beta 1 integrin, GPVI, the 45-kDa N-terminal domain of GPIbalpha , and GPIIb/IIIa. Control (shaded area) and O-sialoglycoprotein endopeptidase-treated (solid line) platelets were stained with fluorophore-labeled mAbs for 10 min at 37 °C and analyzed directly. Normal aggregation (b) and degranulation/fibrinogen binding (c) of those platelets in response to rhodocytin (10 nM) are shown. The experiment was repeated three times with comparable results.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The mechanism underlying platelet activation by rhodocytin (aggretin) has been debated. Several investigators suggested that rhodocytin activates platelets in a collagen-like manner by interacting with alpha 2beta 1 integrin or alpha 2beta 1 integrin and GPIbalpha (7, 20). In contrast to these hypotheses, we reported that rhodocytin does not bind to alpha 2beta 1 integrin (10).

In the present study we demonstrate that rhodocytin induces activation of murine platelets in the absence of alpha 2beta 1 integrin, GPVI, and the ligand-binding domain of GPIbalpha . These are the three major receptors that directly or indirectly interact with collagen. Our findings exclude an essential role for these receptors in rhodocytin-induced activation and demonstrate that the activation process follows mechanisms that are fundamentally different from those induced by collagen. Our present findings are in contrast to those reported by others (7, 9), who showed that mAbs against alpha 2beta 1 blocked rhodocytin-induced platelet aggregation and concluded that the integrin plays an essential role in the activation process. An explanation for this discrepancy could be that treatment of platelets with mAbs against alpha 2beta 1 may have different effects than the absence of the receptor. Antibodies may exert steric effects on other cell surface proteins or may elicit inhibitory signals. Previously, results from inhibition studies with some antibodies against alpha 2beta 1 could not be confirmed in a genetic model in which the beta 1 gene is deleted in all hematopoietic cells, including megakaryocytes. It was shown that antibodies against alpha 2beta 1 integrin markedly reduced or abolished platelet adhesion and aggregate formation on collagen in stasis and flow (27) as well as collagen-induced platelet aggregation (7). Using the Cre/loxP technology, we ablated the beta 1 gene in megakaryocytes and showed that alpha 2beta 1 integrin is not required for platelet adhesion and thrombus formation on fibrillar collagen under static as well as low and high shear flow conditions. beta 1-null platelets display a delayed but not reduced aggregation in response to collagen, demonstrating that alpha 2beta 1 integrin is not a major signaling collagen receptor on platelets (4). The investigations with convulxin provide another example in which antibody inhibition and gene deletion studies showed conflicting results. Whereas the action of convulxin can be inhibited with some antibodies against alpha 2beta 1 (28), Cre/loxP-mediated ablation of beta 1 integrin on platelets revealed no detectable role of alpha 2beta 1 for platelet activation by this agonist (4). Together, these findings strongly suggest that certain antibodies against alpha 2beta 1 integrin induce inhibitory effects that are not based on blockage of the integrin. Therefore, treatment of platelets with anti-alpha 2beta 1 antibodies may not be suitable for determining dependence on alpha 2beta 1 integrin.

Another striking difference between rhodocytin- and collagen-mediated platelet aggregation is the central role of GPVI for collagen but not for rhodocytin. We showed recently that GPVI is the major collagen receptor for platelet activation and that GPVI is essential for collagen-induced platelet aggregation (16). Therefore, GPVI-independent aggregation processes are different from collagen-induced aggregation. Both FcRgamma -null platelets, which lack GPVI (11), and GPVI-depleted platelets (16) fail to activate beta 1 and beta 3 integrins in response to collagen. Consequently, these platelets neither adhere to collagen nor do they bind adhesive ligands or aggregate in response to this agonist (4, 12, 16). From these data, we conclude that rhodocytin activates platelets by mechanisms that are different from those induced by collagen.

Navdaev and co-workers (9) proposed a mechanism for rhodocytin (aggretin)-induced platelet activation that involves two platelet receptors, alpha 2beta 1 integrin and GPIbalpha . The importance of GPIbalpha for rhodocytin-induced aggregation was concluded from a dose-dependent inhibitory effect of a mAb directed against the thrombin-binding site on GPIbalpha , which is located in the 45-kDa N-terminal region of the receptor (29). This finding was in contrast to observations made by other investigators who found no role of GPIb in rhodocytin-induced activation (30, 31) or no binding of rhodocytin to GPIb (7). The discrepancies in the binding studies are difficult to explain. They may be related to different experimental conditions used in these studies. In our present study we show that platelets lacking the 45-kDa N-terminal domain on GPIbalpha respond normally to rhodocytin (Fig. 5). This finding excludes an essential role for the ligand-binding region of GPIbalpha in the rhodocytin-induced activation process and is in clear contrast to the finding by Navdaev and co-workers (9). Steric hindrance or elicitation of inhibitory signals by the GPIbalpha -specific antibody could be an explanation for the conflicting results. It is known that occupancy of GPIbalpha induces tyrosine phosphorylation of different signaling molecules in vitro (32-34) and that dimerization of GPIbalpha by certain mAbs affects platelet function by yet undefined mechanisms in vitro and in vivo (18, 35, 36).

Our results do not exclude the possibility that rhodocytin interacts with an epitope on the GPIb-V-IX complex that is distinct from the 45-kDa N-terminal region of GPIbalpha . However, in studies using mAbs against different epitopes on either GPIX, GPV, or GPIbalpha /beta , we were unable to alter rhodocytin-induced activation/aggregation. In addition, GPV-deficient mouse platelets respond normally to rhodocytin (not shown). These results suggest that the GPIb-V-IX complex has no essential role in rhodocytin-induced platelet activation.

Using genetic ablation of alpha 2beta 1, antibody-mediated depletion of GPVI, and proteolytic digestion of GPIb, we show that platelets lacking all three major receptors that directly or indirectly interact with collagen (alpha 2beta 1, GPVI, and GPIb) respond normally to rhodocytin. Because rhodocytin-induced aggregation of human and mouse platelets occurs with a dose-dependent lag time and independently of the FcRgamma chain and both processes are sensitive to acetylsalicylic acid, it is unlikely that species-specific differences explain the contrasting results presented here and in other studies (7, 9, 20, 31). Furthermore, other snake venom-derived toxins like convulxin (16, 37), botrocetin (38), and alboaggregin A (20)3 also show similar activities on human and mouse platelets. Therefore, it appears that receptor(s) other than alpha 2beta 1, GPVI, and GPIb mediate rhodocytin-induced platelet activation. Rhodocytin induced marked degranulation, suggesting that it stimulates signaling pathways normally used by strong platelet agonists (e.g. thrombin). Alternatively, the strong effects of rhodocytin could be explained by the synergism between two or more platelet receptors. Such synergistic effects have previously been shown for a variety of agonists that stimulate different G-protein-coupled receptors (39-41) and Ig-like receptors and Gi-coupled receptors (42-44). Possible target receptors for rhodocytin on the platelet membrane include Ig-like receptors like the recently cloned F11 receptor (45), G-protein coupled receptors, as well as CD9, CD36, CD47 (integrin-associated protein), or alpha IIbbeta 3 integrin, all of which may transduce activation signals when stimulated appropriately. Further studies will be needed to reveal how rhodocytin induces platelet aggregation.

    ACKNOWLEDGEMENTS

We thank Kirsten Rackebrandt and Alison Pirro for excellent technical assistance and U. Barnfred for constant support throughout the study. We also thank F. Lanza for providing GPV-deficient mice.

    FOOTNOTES

* This work was supported by grants from the Deutsche Forschungsgemeinschaft (to B. N.) and the Swedish Research Foundation (to C. B. and R. F).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.

Wenner Gren Fellow.

** To whom correspondence should be addressed: Dept. of Molecular Oncology, Ferdinand Sauerbruch Klinikum Elberfeld, Arrenbergerstr. 20, Haus 10, 42117 Wuppertal, Germany. Tel.: 49-202-896-5280; Fax: 49-202-896-5283; E-mail: nieswand@klinikum-wuppertal.de.

Published, JBC Papers in Press, May 14, 2001, DOI 10.1074/jbc.M103892200

2 J. A. Eble, unpublished results.

3 B. Nieswandt, unpublished results.

    ABBREVIATIONS

The abbreviations used are: vWf, von Willebrand factor; FcR, Fc receptor; FITC, fluoresceine isothiocyanate; GP, glycoprotein; Ab, antibody; mAb, monoclonal antibody; PE, R-phycoerythrin; MES, 4-morpholineethanesulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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