The highly specific platelet glycoprotein (GP) VI agonist trowaglerix impaired collagen-induced platelet aggregation ex vivo through matrix metalloproteinase-dependent GPVI shedding.
BACKGROUND
C-type lectin proteins (CLPs) have diverse targets including platelet GPIb, GPVI and integrin alpha(2)beta(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 alpha(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 alpha(IIb)beta(3) and platelet aggregation, and also induces MMP-dependent GPVI shedding from platelets.
Chang
C-H
CH
Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan.
Chung
C-H
CH
Kuo
H-L
HL
Hsu
C-C
CC
Huang
T-F
TF
D000818:Animals; D001760:Bleeding Time; D003094:Collagen / Q000037:antagonists & inhibitors / Q000494:pharmacology; D003435:Crotalid Venoms / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D004151:Dipeptides / Q000494:pharmacology; D006801:Humans; D037181:Lectins, C-Type / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D061965:Matrix Metalloproteinase Inhibitors; D020782:Matrix Metalloproteinases / Q000502:physiology*; D008970:Molecular Weight; D010766:Phosphorylation / Q000187:drug effects; D015539:Platelet Activation / Q000187:drug effects; D010974:Platelet Aggregation / Q000187:drug effects*; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D010980:Platelet Membrane Glycoproteins / Q000819:agonists*; D053657:Platelet-Rich Plasma; D011485:Protein Binding; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D021122:Protein Subunits; D015398:Signal Transduction / Q000187:drug effects; D017819:Viperidae / Q000378:metabolism
D003435:Crotalid Venoms; D004151:Dipeptides; D037181:Lectins, C-Type; D061965:Matrix Metalloproteinase Inhibitors; C078131:N-(2(R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl)-L-tryptophan methylamide; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D010980:Platelet Membrane Glycoproteins; D021122:Protein Subunits; C098341:platelet membrane glycoprotein VI; C528917:trowaglerix protein, Tropidolaemus wagleri; C028890:convulxin; D003094:Collagen; D020782:Matrix Metalloproteinases