Pharmacological Inhibition of Glycoprotein VI- and Integrin α2β1-Induced Thrombus Formation Modulated by the Collagen Type

Abstract

Background In secondary cardiovascular disease prevention, treatments blocking platelet-derived secondary mediators pose a risk of bleeding. Pharmacological interference of the interaction of platelets with exposed vascular collagens is an attractive alternative, with clinical trials ongoing. Antagonists of the collagen receptors, glycoprotein VI (GPVI), and integrin α2β1, include recombinant GPVI-Fc dimer construct Revacept, 9O12 mAb based on the GPVI-blocking reagent Glenzocimab, Syk tyrosine-kinase inhibitor PRT-060318, and anti-α2β1 mAb 6F1. No direct comparison has been made of the antithrombic potential of these drugs. Methods Using a multiparameter whole-blood microfluidic assay, we compared the effects of Revacept, 9O12-Fab, PRT-060318, or 6F1 mAb intervention with vascular collagens and collagen-related substrates with varying dependencies on GPVI and α2β1. To inform on Revacept binding to collagen, we used fluorescent-labelled anti-GPVI nanobody-28. Results and Conclusion In this first comparison of four inhibitors of platelet–collagen interactions with antithrombotic potential, we find that at arterial shear rate: (1) the thrombus-inhibiting effect of Revacept was restricted to highly GPVI-activating surfaces; (2) 9O12-Fab consistently but partly inhibited thrombus size on all surfaces; (3) effects of GPVI-directed interventions were surpassed by Syk inhibition; and (4) α2β1-directed intervention with 6F1 mAb was strongest for collagens where Revacept and 9O12-Fab were limitedly effective. Our data hence reveal a distinct pharmacological profile for GPVI-binding competition (Revacept), GPVI receptor blockage (9O12-Fab), GPVI signaling (PRT-060318), and α2β1 blockage (6F1 mAb) in flow-dependent thrombus formation, depending on the platelet-activating potential of the collagen substrate. This work thus points to additive antithrombotic action mechanisms of the investigated drugs.

Qualitative classification of thrombus images as a way to improve quantitative analysis of thrombus formation in flow chamber assays

“…The model can be trained to distinguish thrombi not only on the basis of their morphology, like in the presented paper, but also on the basis of staining of specific markers of platelet activation. Such an approach has been recently used to distinguish platelet aggregates varying in terms of the density of GPVI clusters [ 12 ]. The same solution has proved useful for classifying of adhered platelets with respect to different degree of spreading [ 13 ], or to detect platelets characterized by compromised spreading in a cohort of patients with bleeding disorders [ 14 ].…”

Platelet biology and function: plaque erosion vs. rupture

“… 17 In terms of thrombogenic properties, the different types of collagen support platelet adhesion and activation to a different extent depending on the differential contribution of the platelet collagen receptors glycoprotein VI (GPVI) and α2β1. 66 However, in vitro whole blood perfusion experiments over human atherosclerotic plaque homogenate showed a great dependency on GPVI as blockage of GPVI effectively inhibited thrombus formation. Inhibition of α2β1, however, was without effects.…”

Modelling arterial thrombus formation in vitro

“…A potential limitation of this model is priming of platelets as they flow over one spot, therefore affecting responses to downstream spots, however control experiments demonstrated no differences in thrombus formation based on the order of coated proteins [37]. The flexibility of this model has resulted in its extensive use over the past few years in a diverse range of experiments including assessment of variations in thrombus formation between healthy individuals [39], assessment of coagulation under flow [41,42], phenotyping patients with platelet disorders [40,43 ▪ ], assessment of pharmacological GPVI and integrin α2β1 inhibitors [44], characterization of calcium entry pathways in platelets [45] and assessment of the platelet inhibitory effects of different cancer treatments [46].…”