Human platelet glycoprotein VI function is antagonized by monoclonal antibody-derived Fab fragments.
Lecut, Christelle ; ; et al
in Journal of Thrombosis and Haemostasis [=JTH] (2003), 1(12), 2653-62
Platelet interactions with adhesive ligands exposed at sites of vascular injury initiate the normal hemostatic response but may also lead to arterial thrombosis. Platelet membrane glycoprotein (GP)VI is a ... [more ▼]
Platelet interactions with adhesive ligands exposed at sites of vascular injury initiate the normal hemostatic response but may also lead to arterial thrombosis. Platelet membrane glycoprotein (GP)VI is a key receptor for collagen. Impairment of GPVI function in mice results in a long-term antithrombotic protection and prevents neointimal hyperplasia following arterial injury. On the other hand, GPVI deficiency in humans or mice does not result in serious bleeding tendencies. Blocking GPVI function may thus represent a new and safe antithrombotic approach, but no specific, potent anti-GPVI directed at the human receptor is yet available. Our aim was to produce accessible antagonists of human GPVI to evaluate the consequences of GPVI blockade. Amongst several monoclonal antibodies to the extracellular domain of human GPVI, one, 9O12.2, was selected for its capacity to disrupt the interaction of GPVI with collagen in a purified system and to prevent the adhesion of cells expressing recombinant GPVI to collagen and collagen-related peptides (CRP). While 9O12.2 IgGs induced platelet activation by a mechanism involving GPVI and Fc gamma RIIA, 9O12.2 Fab fragments completely blocked collagen-induced platelet aggregation and secretion from 5 microg mL-1 and fully prevented CRP-induced activation from 1.5 microg mL-1. 9O12.2 Fabs also inhibited the procoagulant activity of collagen-stimulated platelets and platelet adhesion to collagen in static conditions. Furthermore, 9O12.2 Fabs impaired platelet adhesion, and prevented thrombi formation under arterial flow conditions. We thus describe here for the first time a functional monoclonal antibody to human GPVI and demonstrate its effect on collagen-induced platelet aggregation and procoagulant activity, and on thrombus growth. [less ▲]Detailed reference viewed: 25 (4 ULg)
Expression and function of the collagen receptor GPVI during megakaryocyte maturation.
; ; et al
in Journal of Biological Chemistry (2001), 276(18), 15316-25
In this report, the expression and function of the platelet collagen receptor glycoprotein VI (GPVI) were studied in human megakaryocytes during differentiation and maturation of mobilized blood and cord ... [more ▼]
In this report, the expression and function of the platelet collagen receptor glycoprotein VI (GPVI) were studied in human megakaryocytes during differentiation and maturation of mobilized blood and cord blood derived CD34(+) cells. By flow cytometry, using an anti-GPVI monoclonal antibody or convulxin, a GPVI-specific ligand, GPVI was detected only on CD41(+) cells including some CD41(+)/CD34(+) cells, suggesting expression at a stage of differentiation similar to CD41. These results were confirmed at the mRNA level using reverse transcription-polymerase chain reaction. GPVI expression was low during megakaryocytic differentiation but increased in the more mature megakaryocytes (CD41(high)). As in platelets, megakaryocyte GPVI associates with the Fc receptor gamma chain (FcRgamma). The FcR gamma chain was detected at the RNA and protein level at all stages of megakaryocyte maturation preceding the expression of GPVI. The other collagen receptor, alpha(2)beta(1) integrin (CD49b/CD29), had a pattern of expression similar to GPVI. Megakaryocytic GPVI was recognized as a 55-kDa protein by immunoblotting and ligand blotting, and thus it presented a slightly lower apparent molecular mass than platelet GPVI (58 kDa). Megakaryocytes began to adhere to immobilized convulxin via GPVI after only 8-10 days of culture, at a time when megakaryocytes were maturing. At this stage of maturation, they also adhered to immobilized collagen by alpha(2)beta(1) integrin-dependent and -independent mechanisms. Convulxin induced a very similar pattern of protein tyrosine phosphorylation in megakaryocytes and platelets including Syk, FcRgamma, and PLC(gamma)2. Our results showed that GPVI is expressed early during megakaryocytic differentiation but functionally allows megakaryocyte adherence to collagen only at late stages of differentiation when its expression increases. [less ▲]Detailed reference viewed: 64 (2 ULg)