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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 4, 2003 as doi:10.1096/fj.03-0464fje. |
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Klinikum rechts der Isar, 1. Medizinische Klinik, Technische Universität München, D-81675 München, Germany;
* Procorde GmbH, D-82152 Martinsried, Germany;
Institute of Molecular Immunology, GSF-National Research Center for Environment and Health, D-81377 München, Germany;
Institut für Pathologie, Klinikum rechts der Isar, Technische Universität München, D-80567 München, Germany; and
GSF National Research Center for Environment and Health, Institute of Pathology, D-85764 Neuherberg, Germany
2 Correspondence: S.M. or M.G., Deutsches Herzzentrum and 1. Medizinische Klinik, Technische Universität München, Lazarettstrasse 36, D-80636 München, Germany. E-mail: massberg{at}dhm.mhn.de or gawaz{at}dhm.mhn.de
SPECIFIC AIMS
The aim of our present study was to clone a soluble dimeric form of GPVI (GPVI-Fc) and to determine its effects on platelet adhesion to immobilized collagen in vitro and to the injured mouse carotid artery in vivo.
PRINCIPAL FINDINGS
1. Cloning, viral expression, and purification of soluble human and murine GPVI
To generate a soluble dimeric form of human GPVI, the extracellular domain of human GPVI was cloned and fused to the human immunoglobin Fc domain including a specific hinge region. Adenoviral constructs coding for the GPVI-Fc fusion protein or control Fc were prepared and GPVI-Fc or control Fc were expressed as secreted soluble proteins using the human HeLa cell line to prevent misfolding and nonglycosylation of the expressed proteins. The molecular mass of GPVI-Fc was 
80 kDa under reducing conditions in SDS-PAGE (as detected by Coomassie blue stain) by immunoblotting with peroxidase-conjugated goat anti-human Fc antibody or using the anti-GPVI mAb 5C4 generated by a described method. An 160 kDa protein was identified under nonreducing conditions, supporting the notion that GPVI-Fc is present as dimer.
2. Binding of soluble GPVI to immobilized collagen
We determined the binding of GPVI-Fc to immobilized collagen. Different concentrations of the GPVI-Fc fusion protein were incubated with collagen I-coated plates. Binding to collagen was determined using peroxidase-conjugated goat anti-human IgG antibody. GPVI-Fc, but not the control Fc protein lacking the external GPVI domain, showed a dose-dependent and saturable binding to immobilized collagen. GPVI-Fc did not interact with BSA or vWF, supporting the specificity of GPVI-Fc binding. We tested the ability of solubilized fibrillar collagen to compete with immobilized collagen for the association with GPVI-Fc. Soluble collagen inhibited GPVI-Fc binding to immobilized collagen in a dose-dependent manner. These data indicated that GPVI-Fc binding to collagen is specific and characterized by high affinity.
3. Effect of soluble GPVI-Fc on platelet adhesion to immobilized collagen under flow
We assessed the effect of GPVI-Fc on adhesion of washed human platelets to immobilized collagen under shear conditions in vitro. Platelets adhered firmly to immobilized collagen at low (500 s-1) and high (2000 s-1) shear rates forming thrombi. Soluble GPVI-Fc significantly attenuated platelet adhesion on immobilized collagen by 37 and 44% at shear rates of 500 s-1 and 2000 s-1, respectively. Inhibition was specific since GPVI-Fc did not affect platelet adhesion on immobilized vWF. We determined the effects of soluble GPVI-Fc on thrombus formation in flowing whole blood ex vivo. Immobilized collagen induced profound thrombus formation in flowing blood. An area of 40 x 103 µm2 was covered by adherent platelets in the presence of control Fc lacking the external GPVI domain. In the presence of GPVI-Fc, the surface area covered with adherent platelets was reduced to 6 x 103 µm, indicating that soluble GPVI-Fc inhibited thrombus formation in whole blood by 84% (P<0.05 vs. control Fc).
4. Effect of soluble GPVI-Fc on adhesion of GPVI-expressing GPVI-CHO transfectants to immobilized collagen
To further substantiate the specificity of GPVI-Fc-mediated inhibition of GPVI-collagen interactions, we generated the GPVI-expressing GPVI-Flp-InTM-CHO cell line. GPVI-transfectants but not control CHO cells avidly bound to immobilized collagen. Adhesion of GPVI-transfected CHO cells was 4.5-fold increased compared with nontransfected control cells (P<0.05). GPVI-Fc significantly reduced adhesion of GPVI-expressing CHO cells by 71% (P<0.05 vs. vehicle), but not of control CHO cells. Control Fc had no effect on the adhesion of GPVI-expressing or control CHO cells. This further supports the concept that GPVI-Fc inhibits cell adhesion by interfering with GPVI–collagen interaction.
5. Effect of soluble GPVI-Fc on platelet counts, bleeding time, and platelet adhesion to the injured carotid artery in vivo
Next, we addressed the effect of a soluble form of GPVI on platelet function in the mouse in vivo. Animals were treated with 1 or 2 mg/kg GPVI-Fc or equimolar doses of control Fc lacking the external GPVI domain. Infusion of GPVI-Fc or control Fc even at the highest dose of 2 mg/kg had no significant effect on peripheral platelet counts (534±88, 894±155, and 894±223x103 plts/µL after infusion of control Fc or 1 and 2 mg/kg GPVI-Fc, respectively). By contrast, the GPVI-Fc fusion protein (but not the control Fc) induced a moderate prolongation of tail bleeding times by 11% (1 mg/kg) and 21% (2 mg/kg) compared with control animals (Fig. 1
a). Absolute bleeding times were 6.9 ± 0.2, 4.8 ± 0.9 min in PBS and control Fc-treated mice, respectively, and 7.2 ± 0.1 and 9.7 ± 0.2 min in mice treated with 1 or 2 mg/kg GPVI-Fc. Bleeding times were consistently higher than 10 min in ASA-treated animals (5 mg·kg-1·day-1 orally).
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Finally, we addressed the effects of GPVI-Fc on platelet recruitment in a mouse model of carotid injury using intravital fluorescence microscopy. Animals were treated with 1 or 2 mg/kg GPVI-Fc or an equimolar amount of control Fc lacking the external GPVI domain. After infusion of GPVI-Fc or control Fc, endothelial denudation of the mouse carotid artery was induced by vigorous ligation. Ligation of the carotid artery consistently caused complete loss of the endothelial cell layer as confirmed by scanning electron microscopy (SEM). Platelet adhesion was directly visualized and quantified using in vivo fluorescence microscopy. In control (Fc-treated) mice, numerous platelets were tethered to the vascular wall within the first minutes after endothelial denudation. Platelets establishing contact with the subendothelium initially exhibited a slow surface translocation, often followed by firm platelet adhesion and platelet aggregation (5.494±874 adherent platelets/mm2 and 114±17 platelet thrombi/mm2). In the presence of GPVI-Fc platelet, recruitment to the site of vascular injury was dramatically attenuated. Platelet tethering was reduced by 65 and 71% compared with Fc-treated animals after pretreatment with 1 or 2 mg/kg GPVI-Fc (P<0.05 vs. control Fc). Firm platelet adhesion was reduced by 49 and 65% after administration of 1 or 2 mg/kg GPVI-Fc, respectively (P<0.05 vs. control). Likewise, aggregation of adherent platelets was virtually absent in animals treated with 2 mg/kg GPVI-Fc fusion protein (P<0.05 vs. control Fc, Fig. 1b-c
). SEM also clearly demonstrated that platelet adhesion and aggregation after endothelial denudation of the common carotid artery were dramatically reduced in GPVI-Fc-treated, but not in Fc-pretreated mice. The presence of GPVI-Fc at the site of injury was confirmed using immunohistochemistry. Similarly, ex vivo incubation of injured carotid arteries showed specific binding of GPVI-Fc but not of control Fc to the site of injury. These data demonstrate that GPVI-Fc specifically binds to sites of vascular injury in vivo and prevents subsequent platelet recruitment.
CONCLUSIONS AND SIGNIFICANCE
After rupture of the atherosclerotic plaque, exposure of subendothelial collagen is the major trigger that initiates platelet adhesion and aggregation at the site of injury, followed by arterial thrombosis. The platelet glycoprotein GPVI, recently cloned, has been identified as the major platelet collagen receptor.
In the present study we generated a soluble form of GPVI and tested its inhibitory activities on platelet adhesion in vitro and in vivo. Soluble GPVI-Fc specifically binds to immobilized collagen in a saturable manner with high affinity. Soluble GPVI-Fc, but not control Fc lacking the external GPVI domain, inhibited adhesion of washed human platelets to collagen under both low and high shear conditions in vitro. We addressed the in vivo effects of soluble GPVI-Fc on platelet recruitment after injury of the mouse carotid artery. We demonstrate for the first time in vivo that soluble GPVI-Fc attenuates stable platelet recruitment after endothelial denudation in a dose-dependent manner (see Fig. 2
).
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Apart from preventing stable arrest of platelets, GPVI-Fc significantly reduced initial platelet tethering/slow surface translocation at sites of endothelial denudation. We demonstrated earlier that inhibition of GPIb
or of GPVI attenuate platelet tethering to a similar extent, supporting that GPVI and GPIb interaction need to act in concert to promote platelet tethering to subendothelial collagen. The high "on" and "off" rates reported for the GPVI–ligand interaction are consistent with the role of GPVI as a tethering receptor.
The profound anti-thrombotic protection achieved by GPVI-Fc identifies GPVI-Fc as a promising tool to control the onset and progression of pathological arterial thrombosis, e.g., during acute coronary syndrome. This concept is further supported by the observation that GPVI-Fc is targeted to the exposed subendothelium at the site of vascular injury. This implicates that inhibition of GPVI-collagen interactions by soluble GPVI-Fc are likely to be restricted to the site of vascular injury whereas a prolonged systemic inhibition of platelet function is limited by the expected short half-life of unbound GPVI-Fc. Targeting of platelet membrane proteins inevitably leads to systemic inhibition of GPVI or GPIIb-IIIa on all circulating platelets. It is appealing to speculate that the GPVI-Fc therapy might be associated with a lower risk of clinical hemorrhage than with anti-GPVI mAb-based strategies. We show that at doses sufficient to reduce platelet adhesion, the soluble form of GPVI prolonged tail bleeding times only moderately.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0464fje 
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