ADPase CD39 Fused to Glycoprotein VI-Fc Boosts Local Antithrombotic Effects at Vascular Lesions

Abstract

Background: GPVI (Glycoprotein VI) is the essential platelet collagen receptor in atherothrombosis. Dimeric GPVI-Fc (Revacept) binds to GPVI binding sites on plaque collagen. As expected, it did not increase bleeding in clinical studies. GPVI-Fc is a potent inhibitor of atherosclerotic plaque-induced platelet aggregation at high shear flow, but its inhibition at low shear flow is limited. We sought to increase the platelet inhibitory potential by fusing GPVI-Fc to the ectonucleotidase CD39 (fusion protein GPVI-CD39), which inhibits local ADP accumulation at vascular plaques, and thus to create a lesion-directed dual antiplatelet therapy that is expected to lack systemic bleeding risks.

Methods and results: GPVI-CD39 effectively stimulated local ADP degradation and, compared with GPVI-Fc alone, led to significantly increased inhibition of ADP-, collagen-, and human plaque-induced platelet aggregation in Multiplate aggregometry and plaque-induced platelet thrombus formation under arterial flow conditions. GPVI-CD39 did not increase bleeding time in an in vitro assay simulating primary hemostasis. In a mouse model of ferric chloride-induced arterial thrombosis, GPVI-CD39 effectively delayed vascular thrombosis but did not increase tail bleeding time in vivo.

Conclusions: GPVI-CD39 is a novel approach to increase local antithrombotic activity at sites of atherosclerotic plaque rupture or injury. It enhances GPVI-Fc-mediated platelet inhibition and presents a potentially effective and safe molecule for the treatment of acute atherothrombotic events, with a favorable risk-benefit ratio.

Keywords: glycoprotein; platelet; thrombosis.

Figure 1

Structure scheme and biochemical characterization of bifunctional, recombinant GPVI‐CD39 compared to GPVI‐Fc. A, Structure (left panel) and putative 3D‐modeling (right panel) of bifunctional, recombinant GPVI‐CD39 compared with GPVI‐Fc. The 3D model shows the extracellular GPVI domains in blue, the Fc part in green, and the CD39 domain in light red. The N‐terminal leader peptides of each protein are cleaved before secretion of each protein. B, Coomassie staining of GPVI‐CD39 as purified from supernatants of GPVI‐CD39–expressing cells. C, Specific ADPase activity of recombinant GPVI‐CD39 (333 nmol/L) in comparison to that of commercially available soluble CD39 (666 nmol/L; n=3, mean±SEM). Both specific activities were determined at other enzyme molarities and did not differ, as expected, because substrate concentrations were not limiting. GPVI indicates glycoprotein VI; GPVI‐CD39, dimeric glycoprotein VI and CD39 fusion protein; GPVI‐Fc, dimeric glycoprotein VI; nr, nonreducing conditions; r, reducing conditions.

Figure 2

Effects of GPVI‐CD39 and GPVI‐Fc fusion proteins and of control proteins on static platelet aggregation in blood after stimulation with collagen or ADP. Platelet aggregation was determined by impedance aggregometry. Values are mean±SEM. A, Platelet aggregation after stimulation with 12 μg/mL collagen extracted from rabbit aorta. Preincubation with increasing concentrations of GPVI‐CD39 reduces platelet aggregation more strongly than GPVI‐Fc alone (n=5; **P<0.01 and ***P<0.001, compared with GPVI‐Fc). B, Platelet aggregation after stimulation with 103 μg/mL collagen from cultured human fibroblasts (VitroCol; Advanced BioMatrix), as determined by impedance aggregometry. Preincubation with increasing concentrations of GPVI‐CD39 reduces platelet aggregation more strongly than GPVI‐Fc alone (n=5; *P<0.05 and **P<0.01, compared with GPVI‐Fc). C, Platelet aggregation after stimulation with 6.5 μmol/L ADP. Preincubation with increasing concentrations of either GPVI‐CD39 (n=8) or of soluble CD39 markedly reduces ADP‐induced platelet aggregation, whereas GPVI‐Fc alone has no effect (**P<0.01 and ***P<0.001). AU indicates arbitrary unit; GPVI‐CD39, dimeric glycoprotein VI and CD39 fusion protein; GPVI‐Fc, dimeric glycoprotein VI; solCD39, soluble CD39.

Figure 3

GPVI‐CD39 inhibits static platelet aggregation in blood stimulated by human plaque more potently than GPVI‐Fc. Blood samples were preincubated for 3 min with increasing concentrations of GPVI‐CD39 or GPVI‐Fc before stimulation with plaque homogenate (333 μg/mL) for 10 min. Values are mean±SEM (n=8). ***P<0.001 by 2‐way ANOVA for factor concentration and drug and secondary pairwise comparisons of isomolar GPVI‐CD39 vs GPVI‐Fc by Fisher least significant difference. The asterisks indicate significant differences between the 2 drugs at isomolar concentrations. In addition, direct‐pair comparisons between isomolar drug concentrations by Student t testing resulted in the same significance levels. AU indicates augmented unit; GPVI‐CD39, dimeric glycoprotein VI and CD39 fusion protein; GPVI‐Fc, dimeric glycoprotein VI.

Figure 4

Effects of GPVI‐CD39 and GPVI‐Fc on plaque‐induced platelet deposition from flowing blood at 2 arterial shear rates. A, Representative micrographs display platelet coverage of pooled plaque homogenate at different times after start of blood flow at 600/s. Blood was preincubated for 10 min with DiOC6 for platelet visualization in the absence (control) or presence of GPVI‐CD39 (150 nmol/L) or GPVI‐Fc (150 nmol/L). B, Effects of GPVI‐Fc and GPVI‐CD39 on the kinetics of platelet deposition onto human plaques from flowing blood at constant (shear rate 600/s) or pulsatile flow (60 pulses/min, mean shear rate 600/s). The binary fluorescent area fraction (1.0=total area) was quantified, as detailed in Methods. Values are mean±SEM of 8 experiments performed in parallel with the same blood donors. *P<0.05, by repeated‐measures ANOVA at 300 s and secondary pairwise comparison by Fisher least significant difference. Repeated measures refer to the comparison of aliquots from samples of each donor under different concurrent experimental conditions at the same time. C, Comparison of the effects of either 75 or 150 nmol/L GPVI‐CD39 on plaque‐ induced platelet deposition from flowing blood at low and high arterial shear rates. Blood was preincubated with DiOC6 for platelet visualization in the absence (control) or presence of GPVI‐CD39 (75 or 150 nmol/L) for 10 min at 37°C before start of flow at shear rates of 600/s or 1500/s. Values are mean±SEM (n=6). *P<0.05 and **P<0.01 by repeated‐measures ANOVA at 300 s and secondary pairwise comparison by Fisher least significant difference. DiOC6 indicates 3,3′‐dihexyloxacarbocyanine iodide; GPVI‐CD39, dimeric glycoprotein VI and CD39 fusion protein; GPVI‐Fc, dimeric glycoprotein VI.

Figure 5

Effect on thrombus formation after ferric chloride injury: mean times to occlusion after administration of vehicle (NaCl), GPVI‐Fc, or GPVI‐CD39. Administration of 3 mg/kg (10 nmol/kg) GPVI‐CD39 strongly delayed ferric chloride–induced thrombus formation in vivo compared with administration of 1.5 mg/kg (10 nmol/kg) GPVI‐Fc or vehicle only. Mean values of 7 independent experiments are shown with SEM. **P<0.01 by ANOVA. GPVI‐CD39 indicates dimeric glycoprotein VI and CD39 fusion protein; GPVI‐Fc, dimeric glycoprotein VI.

Figure 6

Effects on Innovance PFA‐200 closure times of human blood ex vivo. A, Effects of ticagrelor, GPVI‐CD39, GPVI‐Fc, or ASA at the indicated concentrations on closure times in collagen/ADP cartridges (n=8 samples from independent donors). No significant differences between groups occurred. B, Effects of ticagrelor, GPVI‐CD39, GPVI‐Fc, or ASA at the indicated concentrations on closure times in collagen/epinephrine cartridges (n=8 samples from independent donors). No significant differences between results for GPVI‐CD39 and GPVI‐Fc occurred. Closure time was significantly (P=0.04) prolonged after addition of ASA compared with PBS only. C, Effects of ticagrelor, GPVI‐CD39, GPVI‐Fc at the indicated concentrations on closure times in specific P2Y cartridges (n=8 samples from independent donors; **P<0.01 and ***P<0.001 vs PBS only). ASA indicates acetylsalicylic acid; GPVI‐CD39, dimeric glycoprotein VI and CD39 fusion protein; GPVI‐Fc, dimeric glycoprotein VI.

Figure 7

Pharmacokinetic and pharmacodynamic evaluation in mice in vivo; bleeding times in vivo. A, Plasma concentrations in mice up to 48 hours after administration of GPVI‐CD39 or Fc control protein. Blood samples were taken at the indicated times after IV administration of 4 mg/kg (13 nmol/kg) GPVI‐CD39, and plasma levels were detected by ELISA. Mean±SEM is shown.(n=3 animals). B, ADPase activities in mice up to 48 hours after administration of GPVI‐CD39 or Fc controls. Blood samples were taken at the indicated times after IV administration of either 4 mg/kg GPVI‐CD39 (13 nmol/kg, corresponding to 26 nmol/kg ADPase moieties) or 26 nmol/kg solCD39, and ADP turnover (mean±SEM) was measured by using a Malachite Green phosphate detection kit. Time is shown at a logarithmic scale to visualize decrease in activity during early time points (n=3 animals). C, Tail bleedings times. Tails were incised 15 minutes after IV administration of the indicated doses of GPVI‐CD39, GPVI‐Fc, or buffer, and tail bleeding times were determined. Mean values of 8 independent experiments are shown with SEM. No significant differences between groups occurred. GPVI‐CD39 indicates dimeric glycoprotein VI and CD39 fusion protein; GPVI‐Fc, dimeric glycoprotein VI; IV, intravenous; solCD39, soluble CD39.

Figure 8

Mode of action of GPVI‐CD39 at arterial plaques. GPVI indicates glycoprotein VI; GPVI‐CD39 indicates dimeric glycoprotein VI and CD39 fusion protein; Pi, inorganic phosphate.