The highly specific platelet glycoprotein (GP) VI agonist trowaglerix impaired collagen‐induced platelet aggregation ex vivo through matrix metalloproteinase‐dependent GPVI shedding

Abstract

impaired collagen-induced platelet aggregation ex vivo through matrix metalloproteinase-dependent GPVI shedding. J Thromb Haemost 2008; 6: 669-76.Summary. Background: C-type lectin proteins (CLPs) have diverse targets including platelet GPIb, GPVI and integrin a 2 b 1 , and affect platelet function in a various way. In this study, we characterized a huge, heterodimeric venom protein, trowaglerix, which belongs to the CLP family. Methods: We purified a potent platelet-aggregation inducer, trowaglerix, from the crude venom of Tropidolaemus wagleri. Biotinylated trowaglerix was used for binding assays, and immunoblotting was used to investigate the signal transduction involved. Results: Two distinct subunits of trowaglerix with similar masses of around 16 kDa were eluted by high-performance liquid chromatography after reduction and alkylation. Trowaglerix induced platelet aggregation of washed human platelets and platelet-rich plasma (PRP) in a concentration-dependent manner. Biotinylated trowaglerix specifically bound to platelet membrane GPVI, but not to GPIb or a 2 integrin. Treatment with trowaglerix induced GPVI loss in human platelets in vitro and impaired the platelet aggregation of mouse PRP ex vivo in response to collagen but not in response to adenosine diphosphate (ADP). However, GM6001, a matrix metalloproteinase (MMP) inhibitor, inhibited trowaglerix-induced GPVI cleavage and restored the platelet responsiveness of PRP to collagen. Conclusions: Trowaglerix activates platelets through specific binding to GPVI, leading to kinases-dependent exposure of functional a IIb b 3 and platelet aggregation, and also induces MMPdependent GPVI shedding from platelets.

Characterization of the platelet transcriptome by RNA sequencing in patients with acute myocardial infarction

“…PZP inhibits human tissue kallikrein and type IV collagenases MMP2 and MMP9 [43]. Inhibition of platelet metalloproteinases prevents clearance of GP6 and retains collagen reactivity, evoking a model consistent with our observations: ↑PZP > ↓MMP > ↑GP6 > ↑collagen reactivity [44,45]. In concert with evidence from prior Mmp2−/− results, lower PZP expression and loss of MMP2 inhibition in human STEMI platelets could promote pro-thrombotic outcomes [46].…”

Novel Therapeutic Agent against Platelet Activation In Vitro and Arterial Thrombosis In Vivo by Morin Hydrate

“…Among the platelet receptors known to interact directly with collagen, integrin α 2 β 1 and glycoprotein (GP) VI [15] appear to play a key role and have recently gained academic attention. GP VI is widely recognized as a requisite factor for formation of platelet aggregates on collagen surfaces under blood flow [16]. Integrin α 2 β 1 is another major collagen receptor on endothelial cells and platelets.…”

Trowaglerix Venom Polypeptides As a Novel Antithrombotic Agent by Targeting Immunoglobulin-Like Domains of Glycoprotein VI in Platelet

“…17 We have also predicted the structure of decapeptide and analyzed its possible binding sites at the inner surface of the area between D1 and D2 domains of GPVI, which is novel and probably different from well-known collagen-binding site. Most importantly, we found that the hexa-/decapeptide of trowaglerix α subunit (Troα6/Troα10) specifically exhibited inhibitory activity toward collagen-induced aggregation via interacting with platelet GPVI receptor and displayed antithrombotic effect in vivo.…”

“…17 Here, we determined the partial amino acid sequences of trowaglerix and synthesized the small-mass peptides derived from these snaclecs C terminal accordingly. We found that the hexa-/decapeptides of trowaglerix α subunit specifically exhibited marked inhibitory activity on collagen-induced platelet aggregation by interacting with GPVI receptor in vitro and effectively exerted antithrombotic activity in mice model without causing bleeding tendency.…”