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Differential Inhibition of Human Atherosclerotic Plaque–Induced Platelet Activation by Dimeric GPVI-Fc and Anti-GPVI Antibodies

Background Glycoprotein VI (GPVI) is the essential platelet collagen receptor in atherothrombosis, but its inhibition causes only a mild bleeding tendency. Thus, targeting this receptor has selective antithrombotic potential. Objectives This study sought to compare compounds interfering with platelet GPVI–atherosclerotic plaque interaction to improve current antiatherothrombotic therapy. Methods Human atherosclerotic plaque–induced platelet aggregation was measured in anticoagulated blood under static and arterial flow conditions (550/s, 1,100/s, and 1,500/s). Inhibition by dimeric GPVI fragment crystallizable region of IgG (Fc) masking GPVI binding sites on collagen was compared with that of 3 anti-GPVI antibodies: BLO8-1, a human domain antibody; 5C4, a fragment antigen-binding (Fab fragment) of monoclonal rat immunoglobulin G; and m-Fab-F, a human recombinant sFab against GPVI dimers. Results GPVI-Fc reduced plaque-triggered platelet aggregation in static blood by 51%, BLO8-1 by 88%, and 5C4 by 93%. Under arterial flow conditions, BLO8-1 and 5C4 almost completely inhibited platelet aggregation while preserving platelet adhesion on plaque. Inhibition by GPVI-Fc, even at high concentrations, was less marked but increased with shear rate. Advanced optical imaging revealed rapid persistent GPVI-Fc binding to collagen under low and high shear flow, upstream and downstream of plaque fragments. At low shear particularly, platelets adhered in plaque flow niches to GPVI-Fc–free segments of collagen fibers and recruited other platelets onto aggregates via ADP and TxA2 release. Conclusions Anti-GPVI antibodies inhibit atherosclerotic plaque-induced platelet aggregation under static and flow conditions more effectively than GPVI-Fc. However, potent platelet inhibition by GPVI-Fc at a higher shear rate (1,500/s) suggests localized antithrombotic efficacy at denuded or fissured stenotic high-risk lesions without systemic bleeding. The compound-specific differences have relevance for clinical trials targeting GPVI-collagen interaction combined with established antiplatelet therapies in patients with spontaneous plaque rupture or intervention-associated plaque injury.

T he most common cause of acute myocardial infarction and ischemic stroke is arterial thrombosis at sites of erosion or rupture of atherosclerotic plaques that expose thrombogenic plaque material to circulating blood (1,2). We recently described a 2-step mechanism of arterial thrombus formation induced by human atherosclerotic plaques with rapid glycoprotein VI (GPVI)-mediated platelet adhesion and aggregation onto plaque collagen, followed by plaque tissue factor-mediated fibrin formation (3). Indeed, morphologically altered collagen type I and III structures present in atherosclerotic plaques (3)(4)(5)(6) are highly thrombogenic and induce platelet aggregation under static and flow conditions through binding to GPVI (3,5,7). In contrast to flow studies with isolated collagen fibers (8,9), the collagen receptor a 2 b 1 integrin is not involved in plaque-induced platelet aggregation (5,6). Therefore, targeting GPVI might preferentially inhibit atherosclerotic plaque-induced thrombosis.
GPVI, a 60 to 65 kDa type I transmembrane glycoprotein member of the immunoglobulin (Ig) superfamily, is a main platelet collagen receptor (10)(11)(12)(13). Its expression is restricted to platelets and megakaryocytes; thus, direct targeting of this receptor does not affect other cell types (14). The monomeric form of GPVI predominates on resting platelets, but when platelets are stimulated by von Willebrand factor (vWF), collagen-related peptide, or thrombin, dimeric GPVI expression increases on the platelet surface (15,16). Only the dimeric form of GPVI shows high affinity binding to collagen (17,18), recognizing tandem glycine-proline-hydroxyproline (GPO) motifs in collagen fibers (9,19). GPVI binds to collagen via its tandem Ig domains D1 and D2, which are held out from the platelet surface by an O-glycosylated mucinlike stalk (20).
GPVI deficiency causes only a limited bleeding tendency, reinforcing its potential as a selective and relatively safe drug target (10,14,21). The GPVIcollagen interaction can be inhibited either by occupation of GPO-binding sites on collagen using extracellular GPVI fused to the Fc region of human IgG (GPVI-Fc, Revacept, advanceCOR, Munich, Germany) or by antibodies directed against platelet GPVI. In phase I studies, GPVI-Fc was well tolerated without affecting systemic hemostasis in healthy human volunteers. It inhibited collagen-induced platelet aggregation ex vivo in a dose-dependent manner (22). A human recombinant Fab (m-Fab-F) specifically blocks GPVI dimers (18).
BLO8-1, a human anti-GPVI domain antibody consisting of a single Ig variable domain recognizes residue K59 in domain D1 on the apical surface of GPVI (23). 5C4, the Fab fragment of a monoclonal GPVI-blocking rat IgG, targets epitopes of GPVI at D1 and the intersection to domain D2 (24).
The aim of this study was to explore the plateletinhibiting potential of GPVI-Fc and anti-GPVI antibodies under both static and arterial flow conditions. Blood was stimulated with human atherosclerotic plaque material to mimic pathophysiological conditions of plaque rupture.

METHODS
Atherosclerotic plaques were obtained from patients undergoing endarterectomy for high-grade carotid artery stenosis. Patient informed consent was ob- The carotid plaque tissue was endarterectomized, processed, and preserved as described (3,25). Plaque homogenates from 5 patients were mixed to obtain plaque pools that were kept in aliquots at À80 C.
Plaque homogenates were used for platelet aggregation studies or coated onto glass coverslips for flow studies (3,26,27 with GPVI-Fc and subsequent plaque stimulation, we performed experiments with plaque pre-incubated with 35-fold or 50-fold higher concentrations of GPVI-Fc than finally present in blood to maximally saturate the collagen-binding sites for GPVI.
For additional details, see the Online Appendix.

RESULTS
Aggregation measurements showed that GPVI-Fc, but not Fc lacking the external GPVI domain, delayed plaque-and collagen-stimulated platelet aggregation in blood ( Figure 1A). In plaque-stimulated samples, the lag time until the start of aggregation increased from 65 AE 19 s with Fc control protein to 119 AE 23 s (n ¼ 6; p < 0.001) with GPVI-Fc (50 mg/ml, 300 nM), when the proteins were pre-incubated with blood and from 72 AE 20 s with Fc control protein to 134 AE 25 s (n ¼ 6; p < 0.001) with GPVI-Fc, when the proteins were mixed with plaque before blood stimulation.
Inhibition was specific because GPVI-Fc did not affect platelet aggregation when stimulated with adenosine diphosphate (ADP) and thrombin receptor-activating peptide ( Figure 1B). Collagen-stimulated platelet aggregation was dose dependently reduced (maximally by À45%) ( Figure 1C) as was plaque-induced platelet aggregation by blood pre-treatment with GPVI-Fc (maximally by À51%) ( Figure 1D). Surprisingly, inhibition was not significantly enhanced if plaque was pre-incubated with a 35-fold higher GPVI-Fc concentration before stimulation of the blood samples ( Figure 1E), even if pre-incubation time was extended to 30 min (Online Figure 1).
The anti-GPVI antibodies BLO8-1 (10 mg/ml, 833 nM) and 5C4 (1.25 mg/ml, 25 nM) almost completely inhibited plaque-and collagen-induced platelet aggregation in a concentration-dependent manner (Online Figures 2A and 2B, and not shown). The highest concentration of BLO8-1 decreased aggregation to 12% of control (n ¼ 9) after plaque stimulation and to 16% (n ¼ 8) after collagen stimulation. Residual aggregation after pre-incubation with the highest 5C4 concentration was 7% on plaque stimulation (n ¼ 5) and 18% on collagen stimulation (n ¼ 5). Inhibition was specific because neither BLO8-1 nor 5C4 affected platelet aggregation when stimulated by ADP and thrombin receptor-activating peptide (Online Figure 2C).    previously (17). In line with the high affinity of GPVI-Fc to collagen, fluorescent-labeled GPVI-Fc added to flowing blood rapidly bound to plaque. This binding was stable, indicating a very low off rate of GPVI-Fc.
Differences between soluble GPVI-Fc and platelet GPVI dimer must be due to factors other than affinity.
First, GPVI dimers are recruited from monomers during platelet activation and cluster in lipid rafts leading to a high local GPVI-dimer density on the platelet surface (15,16,31). Second, spacing of the 2 collagen binding sites on dimeric GPVI-Fc lacking the mucinlike stems of the platelet GPVI dimer might be less flexible and may not always fit with the GPO repeats exposed on plaque collagen (Central Illustration A, left). This might explain why even pre-incubation of plaque with very high GPVI-Fc concentrations did not enhance platelet inhibition under static and low shear rate flow conditions (Figure 1, Online Figures 1 and 4).  anti-GPIV-antibodies might provide superior antithrombotic efficacy, but might be burdened with a higher bleeding risk than GPVI-Fc, especially when tested in combination with established dual-antiplatelet therapy.