Page 230 - 2021_07-Haematologica-web

Letters to the Editor
Glenzocimab does not impact glycoprotein
VI-dependent inflammatory hemostasis
Glycoprotein VI (GPVI), the main platelet receptor for collagen, has emerged as a new target for antithrombotic therapy because its genetic deficiency or pharmacological blocking inhibits platelet aggregation and experimental thrombosis without increasing bleeding time.1–5 While these data have stimulated the development of new antiplatelet drugs targeting GPVI, recent findings have indicated that GPVI is essential for repair of neutrophil- induced vascular injury in various inflamed organs and tissues.6–9 It thus appears important to assess and antici- pate the yet uninvestigated risk of inflammation-induced bleeding under GPVI antagonists, especially considering that inflammation is a component of various thrombotic diseases. In that respect, it is worth noting that neu- trophil mobilization is a predictor of hemorrhagic trans- formation of ischemic stroke10 and contributes to intraplaque hemorrhage,11 which is known to precipitate plaque rupture and the clinical expression of atheroscle- rosis.
Among the newly developed drugs targeting GPVI, ACT017 (Glenzocimab, Acticor Biotech) is a humanized antibody fragment (Fab) that has already completed its phase I clinical trial in healthy volunteers12 and has just entered a phase II trial in stroke patients (Acute Ischemic Stroke Interventional Study “ACTIMIS”, clinicaltrials gov. Identifier: NCT03803007). ACT017 binds to human GPVI and inhibits the procoagulant activity and aggrega- tion of collagen-stimulated platelets, as well as platelet adhesion and thrombus formation onto collagen surfaces under arterial flow conditions.1,13,14 The inhibitory action of ACT017 occurs without causing thrombocytopenia or depletion of GPVI, and is not associated with sponta- neous bleeding events or increased bleeding time.14 Nevertheless, whereas preclinical bleeding time tests can help evaluate the risk of bleeding associated with trauma or surgery, they may not predict the risk of bleeding asso- ciated with inflammation.15 Here, using the cutaneous reverse passive Arthus reaction (rpA) as a model situation where GPVI plays a major role in inflammatory hemosta- sis, we investigated whether ACT017 increases the risk of inflammation-induced bleeding.
We first assessed the contribution of GPVI to the pre- vention of inflammation-induced bleeding by platelets in the brain and lungs. In agreement with previous results obtained with an antibody causing depletion of mouse Gpvi,16,17 there was no cerebral hemorrhage in any of the Gpvi-/- mice subjected to transient (90 minutes) middle cerebral artery occlusion (Figure 1A). In contrast, cerebral hemorrhage occurred in all mice that had been rendered severely thrombocytopenic by the mean of a platelet- depleting antibody (Figure 1A). Genetic deficiency in GPVI was not associated with an increased bleeding risk in the model of acute lung injury induced by inhalation of Pseudomonas aeroginosa endotoxin either (Figure 1B). In the cutaneous rpA, as predicted by previous reports,6,7,9 GPVI-/- mice developed skin bleeding at the inflammatory reaction site, a bleeding phenotype that was seen neither in GPVI+/+ mice nor in GPVI+/- mice, which expressed half of normal GPVI surface levels (Figure 1C and D). Taken together, these results are consistent with evidence that GPVI is dispensable for hemostasis in the inflamed brain and lungs6,16–18 but primarily involved in the prevention of bleeding in the rpA-inflamed skin. Notably, they further indicate that 50% of normal GPVI surface levels are suf- ficient for hemostasis during the cutaneous rpA.
The ability of ACT017 to inhibit collagen/GPVI inter- actions and their functional consequences has been pre- viously demonstrated in humans and in nonhuman pri- mates.14 However, it has not been tested in hGPVI mice. We thus verified the activity of ACT017 against GPVI from hGPVI mice. Like its murine precursor Fab 9O121, ACT017 added to whole blood from hGPVI mice caused a drastic reduction in platelet adhesion and aggregation onto collagen fibers under arterial and venous flow con- ditions (Figure 2A and B; Online Supplementary Movie). We next tested whether hGPVI mice treated with therapeutic (16 mg/kg) or higher doses of ACT017 (32 and 64 mg/kg) were sensitized to inflammation-induced bleeding during the cutaneous rpA. No bleeding occurred in ACT017- treated hGPVI mice, whatever the dose of ACT017 used (data not shown, Figure 2C and D). There was no bleeding either when ACT017 at the highest dose tested (64 mg/kg) was given through a continuous infusion over the 4 hours of rpA (data not shown). The absence of bleeding in hGPVI mice treated with ACT017 was in contrast to the petechial bleeding observed in GPVI-/- mice (Figure 1) and in platelet-depleted GPVI+/+ and hGPVI mice (Figures 1C and D, and 2C and D), which is known to be a con- sequence of neutrophil recruitment.7 Absence of bleeding in rpA-challenged hGPVI mice treated with ACT017 was not due to altered neutrophil recruitment as this was comparable to that in hGPVI mice (Figure 2E). Interestingly, the latter result indicates that ACT017 does not impact neutrophil recruitment, at least in this model. Importantly, ACT017 did not alter platelet recruitment to the inflamed skin either (Figure 2F). The latter result underscores a major difference between the impact of genetic deficiency in GPVI and that of GPVI blocking by ACT017. In fact, bleeding in rpA-challenged Gpvi-/- mice was previously shown to be associated with reduced platelet recruitment at the reaction site.7
Solid tumors represent another inflammatory situation in which platelets were shown to continuously prevent leukocyte-induced bleeding and recent data have sug- gested that GPVI could be central to this function.19 Like in the cutaneous rpA, acute treatment of hGPVI mice bearing skin tumors with ACT017 did not cause tumor bleeding. The absence of effect of ACT017 on tumor ves- sel stability was in contrast to the effect of acute deple- tion of platelets, which caused tumor bleeding (Figure 2F).
In conclusion, in addition to confirming that GPVI is not required for inflammation-associated hemostasis in the brain and lungs, our results show that pharmacologi- cal blockade of GPVI by ACT017 does not impair GPVI- dependent inflammatory hemostasis. There are several non-exclusive reasons that could explain why pharmaco- logical inhibition of GPVI by ACT017 does not impair the vasculoprotective recruitment of platelets during the cutaneous rpA. First, it was shown previously that GPVI can co-operate with other platelet receptors like integrin a2β1 to provide residual collagen-dependent platelet activation when its collagen binding site is blocked phar- macologically.20 Furthermore, while ACT017 blocks the interactions between GPVI and collagen, it remains unknown whether ACT017 has similar blocking effects towards the other ligands of GPVI. Besides collagen, fib- rin(ogen) and a number of adhesive proteins of the vessel wall have been reported as GPVI ligands (e.g., laminin, fibronectin and vitronectin) and could thus provide redundant binding mechanisms. Moreover, were colla- gen to be one of the ligands supporting the adhesion of platelets to inflamed skin vessels, it is interesting to note that despite a drastic reduction in platelet adhesion and
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haematologica | 2021; 106(7)