Involvement of the Glycoproteic Ib-V-IX Complex in Nickel-Induced Platelet Activation.
The mechanism of action of nickel compounds in the pathogenesis of several diseases linked to occupational exposure has been analyzed, but the toxicity, uptake, and mutagenicity of nickel are not fully understood. Nickel compounds exhibit differential activities at the level of cell surface; consequently, physicochemical surface interactions might contribute to cell injury in nickel-induced cytotoxicity (1). Moreover, nickel can induce in several human and rodent cell lines the expression of the receptor system Cap43, which is involved in a [Ca.sup.2+]-dependent process of signal transduction (2-4). Nickel induces CD69 expression in lymphocyte subpopulations of allergic patients with contact dermatitis (5). CD69 is constitutively expressed on human platelet surface where it can be involved in signal transduction (6). Hypernickelemia has been found in patients with acute myocardial infarction and has been thus related to the pathogenesis of myocardial ischemic injuries (7), in which platelet interactions with the exposed collagen fibers play a major role.
In previous studies we showed that Ni[Cl.sub.2] can enhance platelet aggregation induced by collagen in the presence of fibrinogen via a rapid cytoskeletal reorganization consequent to tyrosine phosphorylation of [pp60.sup.src], a signaling molecule that has been detected in a submembraneous location (8). However, nickel-induced platelet activation, expressed as [p60.sup.src] phosphorylation, occurs even in the absence of fibrinogen, whereas aggregation requires fibrinogen binding to its receptor, integrin [Alpha]IIb[Beta]3 (8).
The [pp60.sup.src] protein is not the only protein involved in regulating the formation of platelet cytoskeletal signaling complexes. Calpain, a thiol protease, is responsible for the cleavage of several adhesion structural proteins (talin and integrin [Alpha]II[Beta]3) (9,10) and signaling enzymes [focal adhesion kinase (FAK); phosphatidyl inositol 3-kinase (PIK)] (11,12) in spreading and aggregating platelets.
Von Willebrand factor (vWf)--induced platelet stimulation requires the coactivation of two different glycoproteins, the GpIb-V-IX complex and GpIIb-IIIa, or integrin [Alpha]IIb[Beta]3, the former being responsible for the initial contact and the latter leading to spreading and irreversible adhesion (13). However, vWf binding to GpIb-V-IX complex induces the activation and cytoskeletal association of [pp60.sup.src] and PIK (14); this behavior resembles that observed in Ni[Cl.sub.2]-treated platelets.
Our aim was thus to verify whether the GpIb-V-IX complex has a role in Ni[Cl.sub.2]-induced platelet activation and to investigate the molecules involved in the signal transduction cascade that follows Ni[Cl.sub.2] binding to platelet membrane.
Materials and Methods
Materials. Luciferin and luciferase were obtained from Chrono-Log (Havertown, PA, USA). HEPES, fibrinogen, Arg-Gly-Asp-Ser (RGDS) peptide, bovine serum albumin (V-BSA), glucose, wortmannin, ADP, and U46619 were obtained from Sigma (St. Louis, MO, USA). Calpeptin and Ro 31-8220 were obtained from Calbiochem-Novabiochem (La Jolla, CA, USA). Mocarhagin, lyophilized venom from Naja mossambica mossambica, was obtained from Latoxan (Rosans, France). And anti-PIK, p85 (rabbit polyclonal IgG), was obtained from Upstate Biotechnology (Lake Placid, NY, USA).
Platelet isolation. We collected human platelets from drug-free, healthy donors in acid/citrate/dextrose (ACD)-containing tubes. Platelet-rich plasma (PRP) was obtained after centrifugation at 180 x g for 15 min and concentrated at 800 x g for 20 min. The platelet pellet was resuspended in one-third volume of autologous platelet-poor plasma (PPP) and incubated with 1 mM aspirin for 15 min at 37 [degrees] C. The platelets were then washed twice in Tyrode's buffer (137 mM NaCl, 2.68 mM KCl, 0.42 mM Na[H.sub.2][PO.sub.4], 1.7 mM Mg[Cl.sub.2]) containing 10 mM HEPES (pH 6.5) and resuspended in Tyrode's buffer containing 0.2% BSA, 0.1% glucose, and 10 mM HEPES (pH 7.35). The final platelet suspension was adjusted to 2.5 x [10.sup.8] cells/mL.
Platelet aggregation. In vitro platelet aggregation was performed in a PACKS-4 aggregometer (Helena Laboratories, Beaumont, TX, USA) using siliconized glass cuvettes at 37 [degrees] C under continuous stirring. Ni[Cl.sub.2] (1 mM and 5 mM), ADP (5 [micro]M), and the thromboxane A2 analog U46619 (1 [micro]M) were used as platelet agonists. Fibrinogen (1 mg/mL) was added before the agonists.
ATP release. Platelet activation was stopped after 2 min by adding formaldehyde/EDTA according to Costa and Murphy (15). After centrifugation at 10,000 x g for 30 sec, we measured the ATP concentration in the supernatant in an LKB 1251 luminometer (LKB, Turku, Finland) after adding luciferin (40 mg/mL) and luciferase (880 U/mL). The results were expressed as the percentage of ATP released relative to the total ATP present in cells lysed by means of digitonin (50 [micro]M) (16).
Mocarhagin purification. We purified mocarhagin from the crude venom of the snake Naja mossambica mossambica for its heparin binding properties according to De Luca et al. (17). Briefly, crude lyophilized venom (0.5 g) was dissolved in water (10 mL) and loaded onto a heparin-sepharose CL-6B column (1.5 x 40 cm; Pharmacia, Uppsala, Sweden) at 25 mL/hr. After washing with the column buffer containing 0.01 M Tris, 0.15 M sodium chloride, pH 7.4, bound protein was eluted with a linear 250-mL, 0.15-1.0 M sodium chloride gradient in 0.01 M Tris, pH 7.4. Fractions containing mocarhagin were identified by HPLC gel-filtration using a G2000SWXL column (15 mm, 30 cm x 7.8 mm; Supelco Inc., Bellefonte, PA, USA), pooled, ultraconcentrated, and then loaded at 25 mL/hr onto a sepharose CL-6B column (1.5 x 70 cm; Pharmacia). Peak eluted fractions were dialyzed against the column-washing buffer. Mocarhagin (10 [micro]g/mL) was added to platelets 15 min (at 37 [degrees] C) before agonist stimulation.
Cytoskeleton studies. Aliquots of 500 [micro]L of aspirinated peptide RGDS-treated platelet suspensions (2.5 x [10.sup.8] cells/mL) were stimulated with 5 mM nickel. After 1, 3, and 5 min the reactions were stopped by the addition of an equal volume of ice-cold Triton extraction buffer [2% Triton X-100, 10 mM EGTA, 0.1 mM Tris, 1 mg/mL leupeptin, 20 mM pepstatin A, 2 mM phenylmethylsulphonyl fluoride (PMSF)]. Lysates were then centrifuged at 1,500 x g for 10 min to remove intact platelets. Triton-insoluble proteins were isolated by centrifugation at 15,600 x g for 15 min at 4 [degrees] C. Cytoskeletal proteins were separated by 10% SDS-polyacrylamide gel electrophoresis, under denaturating conditions, and then transferred to Immobilion-P (Millipore, Bedford, MA, USA) membranes. The nonspecific bindings were saturated with 3% BSA. Proteins were identified with anti-p-85 (subunit of phosphatidyl inositol 3-kinase) polyclonal antibody, followed by horseradish peroxidase-conjugated secondary antibody and visualized with ECL chemiluminescence reaction reagent (Amersham, Buckinghamshire, England) and Kodak X-ray film (X-OMAT AR; Sigma).
In the presence of fibrinogen (1 mg/mL), Ni[Cl.sub.2] induced platelet aggregation (1 mM: 46.7 [+ or -] 9.6%; 5 mM: 78 [+ or -] 8.7%) (Figure 1) and released ATP from the internal stores (25.4 [+ or -] 4.5% of the total ATP present in lysed cells vs. 3.7 [+ or -] 1.2% in the absence of fibrinogen). Control, nonstimulated platelets produced an ATP release of 2.5 [+ or -] 0.6% (Figure 2).
The treatment of washed platelets with a protein kinase C inhibitor, Ro 31-8220 (10 [micro]M), did not modify the aggregometrical response to 1 mM Ni[Cl.sub.2] (data not shown) and only slightly reduced 5 mM-induced response (56.2 [+ or -] 2.5 vs. 78 [+ or -] 8.7 maximum % of aggregation) (Figure 3).
When washed platelets were treated with a calpain selective inhibitor, calpeptin (50 [micro]g/mL), platelet aggregation in response to the thromboxane synthetic agonist U46619 (1 [micro]M) was completely abolished (Figure 4, trace a), whereas platelet response to Ni[Cl.sub.2] was unmodified (Figure 4, trace b).
Figure 5 shows that when washed platelets were treated with mocarhagin (10 [micro]g/mD, a cobra venom metalloprotease that cleaves GpIb[Alpha], the platelet response to the specific agonist of GpIb[Alpha] (Ristocetin) was completely inhibited (data not shown), whereas platelet aggregation induced by 5 mM Ni[Cl.sub.2] in the presence of fibrinogen was significantly reduced (37.8 [+ or -] 18.6 vs. 78 [+ or -] 8.7 maximum % of aggregation), and the response to ADP, whose action is not exerted through GpIb-V-IX, was unaffected (70.8 [+ or -] 12.3 vs. 71.4 [+ or -] 10.6 maximum % of aggregation; Figure 5).
When washed platelets were treated with a PIK inhibitor, wortmannin (10 [micro]M), platelet aggregation induced by 1 mM Ni[Cl.sub.2] was completely abolished, and the aggregation induced by 5 mM Ni[Cl.sub.2] was strongly decreased (36.6 [+ or -] 15.9 maximum % of aggregation; Figure 6).
Figure 7 shows the immunoblot analysis of the low-speed Triton insoluble cytoskeletal fraction of nickel-stimulated platelets using a polyclonal antibody directed against the p85 subunit of PIK, closely correlated with PtdIns 3-kinase enzymatic activity in platelets (18). The figure shows that a faint band, corresponding to p-85, already detectable after 3 min Ni[Cl.sub.2] stimulation, became marked after 5 min stimulation of aspirinated, RGDS-treated platelets.
In the present study we provided evidence that nickel can cause platelet aggregation and ATP release from the internal stores. Ni[Cl.sub.2] exerts these actions without entering the cell; in fact, in unpublished observations, we found that the addition of Ni[Cl.sub.2] to Fura-2--treated platelets did not quench Fura-2 fluorescence, but an evident reduction was obtained after cellular lysis. This result was obtained comparing the reduction of Fura-2 fluorescence induced by Ni[Cl.sub.2] to the capability of [Mn.sup.2+] to totally quench the Fura-2 fluorescence after the addition of digitonin (50 taM), according to the method described by Sage (19).
Moreover, although fibrinogen is essential for Ni[Cl.sub.2]-induced platelet aggregation, it is not required for Ni[Cl.sub.2]-induced platelet activation, expressed as [p60.sup.src] phosphorylation, as demonstrated by the fact that such response is not inhibited by the incubation with the tetrapeptide RGDS (120 [micro]g/mL), which prevents fibrinogen binding to its receptor, the integrin [Alpha]IIb[Beta]3 (8).
Many authors suggested that fibrinogen receptor exposure involves protein kinase C (20). However, Ni[Cl.sub.2]-induced platelet aggregation does not seem to depend on protein kinase C activation, as demonstrated by the observation that the treatment with a protein kinase C inhibitor, Ro 31-8220, only slightly modified the aggregometrical response to Ni[Cl.sub.2].
In a previous study (8), we demonstrated that Ni[Cl.sub.2] induces a rapid cytoskeletal reorganization consequent to tyrosine phosphorylation of the signaling molecule [p60.sup.src]. Among the key proteins regulating the formation of platelet cytoskeletal signaling process, calpain is not involved in Ni[Cl.sub.2]-induced triggering of signal transduction. In fact, the treatment with the calpain inhibitor calpeptin did not reduce Ni[Cl.sub.2]-induced response.
Because it has been demonstrated that pp60src translocation to the cytoskeleton is triggered by vWf binding to GpIb-V-IX complex, independently of ligand binding to [Alpha]IIb[Beta]3 (14), and that neither vWf nor [Alpha]IIb[Beta]3 requires calpain activation, it was conceivable that a direct involvement of GpIb-V-IX complex in [Ni.sup.2+] evoked platelet activation. To verify this hypothesis, we performed a platelet treatment with the cobra venom metalloprotease mocarhagin, which cleaves GpIba. The observation of a significant reduction of platelet aggregation in response to Ni[Cl.sub.2] led us to hypothesize that Ni[Cl.sub.2] exerts its action mainly through GpIb-V-IX. Moreover, this seems confirmed by the fact that the cytoskeletal translocation of activated [pp60.sub.src], similar to what was observed after vWf stimulation (14), was demonstrated in Ni[Cl.sub.2]-treated platelets (8). Because the association of [p60.sup.src] and PIK to cytoskeleton occurs once platelets aggregate (14), we tested whether the activation of PIK was required in Ni[Cl.sub.2]-induced response. The finding that the treatment with PIK inhibitor wortmannin strongly decreased platelet aggregation in response to Ni[Cl.sub.2] indicated a role for PIK in Ni[Cl.sub.2]-induced response.
PtdIns 3-kinase is known to translocate from the cytosol to the membrane cytoskeleton in the absence of fibrinogen only after vWf stimulation of platelets, with the ensuing activation of GpIb-V-IX complex, whereas stimulation with agonists such as ADP, epinephrine, or collagen has no such effect (14). In fact, vWf-induced cytoskeletal association of PIK and [pp60.sup.src] occurs despite treatment with RGDS or EDTA, which disrupts the ligand-binding capacity of [Alpha]IIb[Beta]3 but does not affect, the ability of vWf to bind GpIb-V-IX (21,22). Moreover, this last binding is specifically blocked by an anti-GpIb monoclonal antibody, and it is not observed in platelets lacking the glycoprotein Ib/IX complex (Bernard Soulier syndrome) (14). To verify whether Ni[Cl.sub.2]-induced platelet activation proceeds through its binding to platelet GpIb-V-IX, we studied the cytoskeletal localization of PIK after Ni[Cl.sub.2] addition in the absence of exogenous fibrinogen. The results demonstrated that nickel was able to cause PIK translocation in a time-dependent manner.
The fact that RGDS had no inhibitory effect on Ni[Cl.sub.2]-induced cytoskeletal association of PtdIns 3-kinase strongly supports the idea that Ni[Cl.sub.2] acts in a vWf-like manner. Our data confirm that this step, far from being dependent on [Alpha]IIb[Beta]3 binding to fibrinogen, involves other integrins, possibly GpIb.
Taken together, these observations suggest the existence of two phases in platelet response to Ni[Cl.sub.2] stimulation: a first step represented by the initial contact and binding to GpIb-V-IX complex, associated with cytoskeletal reorganization and translocation of PIK, and a second leading to the conversion of GpIIb-IIIa to an activated state necessary to support platelet aggregation. To the best of our knowledge, this is the first evidence of a direct role of Ni[Cl.sub.2] in inducing receptor activation in platelets.
REFERENCES AND NOTES
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Silvia Riondino, Fabio Maria Pulcinelli, Pasquale Pignatelli, and Pier Paolo Gazzaniga
Department of Experimental Medicine and Pathology, University of Rome La Sapienza, Rome, Italy
Address correspondence to P.P. Gazzaniga, Department of Experimental Medicine and Pathology, University of Rome La Sapienza, Viale Regina Elena 324, 00161, Rome, Italy. Telephone: +39-064452955. Fax: +39-064454820. E-mail: firstname.lastname@example.org
We thank L. Lenti for her collaboration and for helpful, stimulating discussions.
This work was supported partially by grant Ateneo 1998.
Received 19 September 2000; accepted 24 October 2000.