Disease relevance of GP6

• This review discusses these recent developments and proposes possible mechanisms for how GPVI acts in concert with other receptors and signaling pathways to initiate hemostasis and arterial thrombosis [1].
• BACKGROUND: By site-directed mutagenesis of recombinant receptor fragments, we have previously identified residue lysine59 of the platelet collagen receptor glycoprotein VI (GPVI) as being critical for its interaction with the synthetic ligand collagen-related peptide (CRP) and the inhibitory phage antibody 10B12 [2].
• We studied the association of the GPVI T13254C with fatal myocardial infarction (MI) and coronary artery disease among the 300 men of the Helsinki Sudden Death Study (HSDS) [3].
• Our findings support previous results on the role of this GPVI polymorphism, or another linked polymorphism, as a possible predictor of the risk of coronary thrombosis [3].
• Patients with acute coronary syndrome (ACS) showed a significantly enhanced GPVI expression on admission when compared with patients with stable angina pectoris (SAP) (ACS: 21.4+/-9.7; SAP: 18.6+/-7.1 mean fluorescence intensity+/-SD; P=0.004) [4].

High impact information on GP6

• We have previously shown that uncharacterized glycoprotein VI (GPVI), which is constitutively associated and coexpressed with Fc receptor gamma chain (FcRgamma) in human platelets, is essential for collagen-stimulated tyrosine phosphorylation of FcRgamma, Syk, and phospholipase Cgamma2 (PLCgamma2), leading to platelet activation [5].
• However, there were significantly decreased incorporations of both radioactivities into a 61-kD membrane glycoprotein (GP), which was identified as GPVI from its mobility on unreduced-reduced, two-dimensional SDS-PAGE [6].
• These included a loss of normal CI receptor (alpha2beta1 integrin)-mediated inhibition of proplatelet formation, a marked abrogation of SDF-1-induced chemotactic migration of CD41+ MKs adherent to CI, and an almost complete lack of actin-rich podosomes, normally induced by interaction between CI and its receptors GPVI or alpha2beta1 integrin [7].
• Furthermore, when UT-7/EPO-Mpl cells, which stably express human C-myeloproliferative leukemia virus ligand (c-Mpl), were treated with TPO, demethylation of the GP6 promoter was induced [8].
• Thrombopoietin initiates demethylation-based transcription of GP6 during megakaryocyte differentiation [8].

Chemical compound and disease context of GP6

• The platelet collagen receptor glycoprotein (GP)(1) VI plays a crucial role in platelet activation and thrombus formation and decreased levels or defective GPVI may lead to excessive bleeding [9].
• The platelet glycoproteins (GPs) Ib, integrin alpha(2)beta(1), and GPVI are considered central to thrombus formation [10].
• Together, these results indicate that in advanced plaques collagen type I is a major trigger of thrombus formation and PS exposure, acting via GPVI and ADP release, while tissue factor directly enhances coagulation [11].
• Furthermore, emerging evidence supports a topographical co-association of these receptors of the leucine-rich repeat family (GPIb-IX-V) and immunoglobulin superfamily (GPVI) in an adhesive cluster or 'adhesosome'. This arrangement may underlie common mechanisms of initiating thrombus formation in haemostasis or thrombotic disease [12].

Biological context of GP6

• GP6 haplotype b (Pro(219)) was also associated with increased scores (P = .03) after adjustment for donor age [13].
• The platelet collagen receptor, glycoprotein VI (GPVI), and GPIb-IX-V, which binds von Willebrand factor, initiate platelet aggregation at low or high shear stress, respectively [14].
• These results establish that genetic differences in the adhesion receptor subunits alpha(2), alpha(IIb,) and GPVI can influence the phenotype of VWD type 1 [13].
• Stimulation of platelets with collagen or convulxin (a selective GPVI agonist) resulted in PI3K-dependent, and aggregation independent, Ser(473) and Thr(308) phosphorylation of PKBalpha, which results in PKB activation [15].
• To identify the respective role of these domains in GPVI, we have substituted D1 and D2 by their FcalphaRI homologue in a soluble GPVI fusion protein (GPVI-Fc) and have modified the linker motif by mutagenesis [16].

Anatomical context of GP6

• The GP6 promoter is highly methylated in cord blood mononuclear cells (progenitors) but not in CD41+-enriched cells obtained after TPO differentiation [8].
• In this study we investigate the regulation of GPVI expression and show that thrombopoietin induces its expression in the megakaryocytic cell line UT-7/TPO [9].
• Because CD40L is a potent platelet-derived cytokine that is involved in thrombosis and atherosclerosis, we evaluated the effect of GPVI-mediated release of CD40L on activation of endothelial cells [17].
• We next subcloned the 5' regulatory region of GP6 into pGL3-basic [pGL3(-1576)] and used deletion mutagenesis to identify important regulatory regions, comparing the activity of transiently expressed promoter-luciferase constructs in Dami and HeLa cells [18].
• In COS-7 cells transiently transfected with GPVI, deglycosylation with peptide-N-glycosidase F (PNGase F; specific for complex N-linked glycans) or tunicamycin decreases the molecular weight of GPVI and reduces transfected COS-7 cell binding to both CRP and CVX [19].

Associations of GP6 with chemical compounds

• Besides glycoprotein Ib (GPIb) and alphaIIbbeta3 integrin, which indirectly interact with collagen via von Willebrand factor (VWF), several collagen receptors have been identified on platelets, most notably alpha2beta1 integrin and the immunoglobulin (Ig) superfamily member GPVI [1].
• It is now recognized that platelet adhesion to collagen requires prior activation of integrins through "inside-out" signals generated by GPVI and reinforced by released second-wave mediators adenosine diphosphate (ADP) and thromboxane A2 [1].
• Immunoprecipitation of GPIb-IX with anti-GPIb alpha from the 1% (v/v) Triton-soluble fraction of unstimulated platelets and immunoblotting with anti-GPVI demonstrated association between GPIb-IX and GPVI [20].
• Remarkably, the mutation of residues arginine60 in domain one and arginine166 in domain two, individually to alanine, which had no significant affect on CRP binding, reduced binding of recombinant GPVI to collagen [2].
• In addition, vWF is also able to potentiate 5-HT secretion responses to collagen-related peptide, confirming that GPVI is able to support synergy with vWF [21].
• We have characterized cAMP-dependent endocytosis of GPVI mediated by a human GPVI-specific mAb as what we believe to be a novel antiplatelet therapy [22].

Physical interactions of GP6

• Platelet glycoprotein (GP)Ib-IX-V and GPVI are unique platelet receptors that bind von Willebrand factor or collagen, respectively, and control the initial interaction of circulating platelets with the blood vessel wall in physiology (hemostasis) or pathology (heart attack or stroke) [23].
• Adherence was unaffected by monospecific polyclonal antibodies to glycoprotein (GP) Ib and GPIV, and was normal with platelets from two patients with Glanzmann's thrombasthaenia, indicating that GPIb, the GPIIb/IIIa complex and GPIV are not involved in platelet-laminin interaction [24].

Regulatory relationships of GP6

• Stimulation of GPVI through a specific anti-GPVI monoclonal antibody significantly enhanced surface expression of CD40L (P = 0.006) [17].

Other interactions of GP6

• The levels of platelet GPVI were similar in symptomatic and asymptomatic VWD patients (109.6 +/- 58.4 vs 114.1 +/- 52.5, respectively; p: 0.77) [25].
• To investigate the possibility that GPIb-IX-V and GPVI are associated on the platelet surface, we first ascertained that aggregation induced by a GPVI-specific agonist, collagen-related peptide, like collagen, is markedly cross-blocked by a GPIb alpha-specific monoclonal antibody, SZ2 [20].
• Convulxin, a GPVI-selective agonist, also induced PMP formation at the magnitude which far exceeds those of other agonists, such as thrombin receptor-activating peptide, ADP or epinephrine [26].
• Here we provide several lines of evidence that GPVI-mediated tyrosine phosphorylation of PLCgamma2 in platelets is dependent on GEM-organized signalling and utilizes the GEM resident adapter protein LAT (linker for activation of T cells) [27].
• The platelet collagen receptor glycoprotein VI (GPVI) and the fibrinogen receptor integrin alphaIIbbeta3 trigger intracellular signalling cascades involving the tyrosine kinase Syk, the adapter SLP-76 and phospholipase Cgamma2 (PLCgamma2) [27].

Analytical, diagnostic and therapeutic context of GP6

• Within the last few years, major advances have been made in understanding platelet-collagen interactions including the molecular cloning of GPVI, the generation of mouse strains lacking individual collagen receptors, and the development of collagen receptor-specific antibodies and synthetic peptides [1].
• Deletion analyses and site-directed mutagenesis identified Sp1(227), GATA(177), and Ets(48) sites as essential for GPVI expression [9].
• GPVI content was measured by western blotting [25].
• Surface plasmon resonance demonstrated that W7 specifically blocked the association of calmodulin with an immobilized synthetic peptide corresponding to the calmodulin-binding sequence of GPVI [28].
• This surface was confirmed as critical for collagen binding by epitope mapping of an inhibitory phage antibody against GPVI [29].

References