Human Platelet Glycoprotein VI: Identification of Residues Involved in the Binding to Collagen.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1550-1550
Author(s):  
Martine Jandrot-Perrus ◽  
Christelle Lecut ◽  
Veronique Arocas ◽  
Hans Ulrichts ◽  
Jean-Luc Villeval ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Platelet Glycoprotein ◽  
Directed Mutagenesis ◽  
Time Sharing ◽  
Binding Assays ◽  
Glycoprotein Vi ◽  
Linear Peptide ◽  
Relative Homology ◽  
Collagen Receptor ◽  
Competition Assays

Abstract Glycoprotein VI (GPVI), an essential platelet collagen receptor, belongs to the immunoglobulin (Ig) superfamily and presents two Ig-like loops in its extracellular domain (EC1, EC2). Little is known about the structures involved in GPVI binding to collagen and to its specific ligands, convulxin and collagen related peptides (CRP). Our aim was to characterize these sites using different approaches: competition assays, monoclonal antibodies (MoAb) directed against the GPVI extracellular par, phage display, molecular modelling and site-directed mutagenesis. In binding assays using soluble or cell-expressed human GPVI, we observed that: (i) collagen, CRP and convulxin, competed with one another for the binding to GPVI; (ii) GPVI interaction with either ligand is inhibited by at least two MoAb directed to different independent epitopes, and (iii) MoAb displayed selective inhibitory properties on GPVI interaction with each ligand. These observations indicate that the binding sites on GPVI for the diverse ligands seem to be distinct, exhibiting specific features but at the same time sharing common elements. MoAb 9O12 is a potent inhibitor of GPVI binding to collagen. Its epitope was mapped by screening a 15 mer linear peptide library. This allowed the identification of a motif of 7 residues of which 5 are conserved and that presented a relative homology with the GPVI 30–40 sequence. In a computer model, these residues appeared in close vicinity on a surface loop exposed at the surface of the molecule and accessible to ligands. Site-directed mutagenesis revealed that Val34 and Leu36 are critical for GPVI interaction with collagen and CRP. This study allowed the identification of residues within a loop on GPVI EC1 that might be part of a collagen/CRP-binding site.

scholarly journals Identification of Residues within Human Glycoprotein VI Involved in the Binding to Collagen

2004 ◽  
Vol 279 (50) ◽  
pp. 52293-52299 ◽  
Author(s):  
Christelle Lecut ◽  
Véronique Arocas ◽  
Hans Ulrichts ◽  
Anthony Elbaz ◽  
Jean-Luc Villeval ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Molecular Modeling ◽  
Binding Sites ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Binding Assays ◽  
Glycoprotein Vi ◽  
Ligand Binding Sites ◽  
Human Receptor

Glycoprotein VI (GPVI) has a crucial role in platelet responses to collagen. Still, little is known about its interaction with its ligands. In binding assays using soluble or cell-expressed human GPVI, we observed that (i) collagen, and the GPVI-specific ligands collagen-related peptides (CRP) and convulxin, competed with one another for the binding to GPVI and (ii) monoclonal antibodies directed against the extracellular part of the human receptor displayed selective inhibitory properties on GPVI interaction with its ligands. Monoclonal antibody 9E18 strongly reduced the binding of GPVI to collagen/CRP, 3F8 inhibited its interaction with convulxin, whereas 9O12 prevented all three interactions. These observations suggest that ligand-binding sites are distinct, exhibiting specific features but at the same time also sharing some common residues participating in the recognition of these ligands. The epitope of 9O12 was mapped by phage display, along with molecular modeling of human GPVI, which allowed the identification of residues within GPVI potentially involved in ligand recognition. Site-directed mutagenesis revealed that valine 34 and leucine 36 are critical for GPVI interaction with collagen and CRP. The loop might thus be part of a collagen/CRP-binding site.

Coagulation-induced shedding of platelet glycoprotein VI mediated by factor Xa

Blood ◽  
2011 ◽  
Vol 117 (14) ◽  
pp. 3912-3920 ◽  
Author(s):  
Mohammad Al-Tamimi ◽  
George Grigoriadis ◽  
Huy Tran ◽  
Eldho Paul ◽  
Patricia Servadei ◽  
...  
Keyword(s):  
Confidence Interval ◽  
Tissue Factor ◽  
Platelet Rich Plasma ◽  
Factor Xa ◽  
Selective Inhibitor ◽  
Thrombin Inhibitors ◽  
Platelet Glycoprotein ◽  
Ectodomain Shedding ◽  
Glycoprotein Vi ◽  
Collagen Receptor

Abstract This study evaluated shedding of the platelet collagen receptor, glycoprotein VI (GPVI) in human plasma. Collagen or other ligands induce metalloproteinase-mediated GPVI ectodomain shedding, generating approximately 55-kDa soluble GPVI (sGPVI) and approximately 10-kDa platelet-associated fragments. In the absence of GPVI ligands, coagulation of platelet-rich plasma from healthy persons induced GPVI shedding, independent of added tissue factor, but inhibitable by metalloproteinase inhibitor, GM6001. Factor Xa (FXa) common to intrinsic and tissue factor-mediated coagulation pathways was critical for sGPVI release because (1) shedding was strongly blocked by the FXa-selective inhibitor rivaroxaban but not FIIa (thrombin) inhibitors dabigatran or hirudin; (2) Russell viper venom that directly activates FX generated sGPVI, with complete inhibition by enoxaparin (inhibits FXa and FIIa) but not hirudin; (3) impaired GPVI shedding during coagulation of washed platelets resuspended in FX-depleted plasma was restored by adding purified FX; and (4) purified FXa induced GM6001-inhibitable GPVI shedding from washed platelets. In 29 patients with disseminated intravascular coagulation, mean plasma sGPVI was 53.9 ng/mL (95% confidence interval, 39.9-72.8 ng/mL) compared with 12.5 ng/mL (95% confidence interval, 9.0-17.3 ng/mL) in thrombocytopenic controls (n = 36, P < .0001), and 14.6 ng/mL (95% confidence interval, 7.9-27.1 ng/mL) in healthy subjects (n = 25, P = .002). In conclusion, coagulation-induced GPVI shedding via FXa down-regulates GPVI under procoagulant conditions. FXa inhibitors have an unexpected role in preventing GPVI down-regulation.

scholarly journals Long-Term Antithrombotic Protection by in Vivo Depletion of Platelet Glycoprotein VI in Mice

2001 ◽  
Vol 193 (4) ◽  
pp. 459-470 ◽  
Author(s):  
Bernhard Nieswandt ◽  
Valerie Schulte ◽  
Wolfgang Bergmeier ◽  
Rabée Mokhtari-Nejad ◽  
Kirsten Rackebrandt ◽  
...  
Keyword(s):  
Matrix Protein ◽  
Adenosine Diphosphate ◽  
Selective Inhibition ◽  
Extracellular Matrix Protein ◽  
Platelet Glycoprotein ◽  
Glycoprotein Vi ◽  
Coronary Artery Thrombosis ◽  
Collagen Receptor

Coronary artery thrombosis is often initiated by abrupt disruption of the atherosclerotic plaque and activation of platelets on the subendothelial layers in the disrupted plaque. The extracellular matrix protein collagen is the most thrombogenic constituent of the subendothelial layer; therefore, a selective inhibition of the collagen activation pathway in platelets may provide strong antithrombotic protection while preserving other platelet functions. Here we demonstrate that treatment of mice with a monoclonal antibody against the activating platelet collagen receptor glycoprotein VI (GPVI; JAQ1) results in specific depletion of the receptor from circulating platelets and abolished responses of these cells to collagen and collagen-related peptides (CRPs). JAQ1-treated mice were completely protected for at least 2 wk against lethal thromboembolism induced by infusion of a mixture of collagen (0.8 mg/kg) and epinephrine (60 μg/ml). The tail bleeding times in JAQ1-treated mice were only moderately increased compared with control mice probably because the treatment did not affect platelet activation by other agonists such as adenosine diphosphate or phorbol myristate acetate. These results suggest that GPVI might become a target for long-term prophylaxis of ischemic cardiovascular diseases and provide the first evidence that it is possible to specifically deplete an activating glycoprotein receptor from circulating platelets in vivo.

Influence of Platelet Collagen Receptor Polymorphisms on Risk for Arterial Thrombosis

2002 ◽  
Vol 126 (3) ◽  
pp. 305-309 ◽  
Author(s):  
Kenichi Furihata ◽  
Diane J. Nugent ◽  
Thomas J. Kunicki
Keyword(s):  
Risk Factors ◽  
Genetic Polymorphisms ◽  
Platelet Glycoprotein ◽  
Glycoprotein Vi ◽  
Collagen Receptors ◽  
Normal Individuals ◽  
Increased Risk ◽  
Normal Hemostasis ◽  
Thrombotic Diseases ◽  
Collagen Receptor

Abstract Context.—Collagens are major components of the vascular subendothelium, and the interaction of platelets with collagens initiates normal hemostasis or pathologic arteriothrombosis. Genetic factors that affect the interaction of platelets with collagens could represent risk factors for either arteriothrombosis or excessive hemorrhage. In this regard, we first found that platelet levels of one of the major platelet collagen receptors, integrin α2β1, vary up to 10-fold in normal healthy individuals and that the higher-level phenotype is associated with allele 1 (807T) of the integrin α2 gene. More recently, we found that there is roughly a fivefold range in platelet glycoprotein VI content among normal individuals, which may also influence risk for thromboembolism. Objective.—To determine if genetic polymorphisms of platelet glycoproteins involved in collagen-related function are associated with higher risk for thrombotic disorders, such as coronary heart disease, myocardial infarction, or stroke. Methods.—We examined the genetic mechanisms responsible for variation in expression levels of the collagen receptor integrin α2β1 and the potential influence of this variation on risk for thrombotic diseases. Results.—We found that patients with arteriothrombotic diseases have a higher frequency of α2 allele 1 (associated with higher levels of platelet integrin α2β1). We further found that platelet glycoprotein VI content directly correlates with platelet prothrombinase activity, suggesting that a higher phenotype of platelet glycoprotein VI also may contribute to increased risk of arteriothrombotic diseases. Conclusion.—Genetic polymorphisms that influence the level or function of platelet collagen receptors need to be seriously considered as genetic risk factors for arteriothrombotic diseases.

Site-directed mutagenesis of platelet glycoprotein Ibα demonstrating residues involved in the sulfation of tyrosines 276, 278, and 279

Blood ◽  
2002 ◽  
Vol 99 (12) ◽  
pp. 4422-4427 ◽  
Author(s):  
A. Sasha Tait ◽  
Jing-Fei Dong ◽  
José A. López ◽  
Ian W. Dawes ◽  
Beng H. Chong
Keyword(s):  
Posttranslational Modification ◽  
Site Directed Mutagenesis ◽  
Platelet Glycoprotein ◽  
Directed Mutagenesis ◽  
Novel Mutations ◽  
Tyrosine Residues ◽  
Lysine Residues ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Acidic Region

The interaction between platelet glycoprotein (GP) Ibα and von Willebrand factor (VWF) is essential for initiation of hemostasis. The sulfation of the 3 tyrosine residues 276, 278, and 279 in GPIbα is an important posttranslational modification that seems to promote the interaction with VWF. The environment where sulfation of tyrosines occurs has been proposed to contain highly acidic residues. This investigation has examined the highly acidic region from Asp249 to Asp287 in the mature GPIbα protein. Changes to most of the carboxylic acids in this region resulted in decreased reactivity to VWF. Only 3 mutants (Glu270Gln, Asp283Asn, Asp283Asn/Glu285Gln/Asp287Asn) resulted in the abolition of sulfation. Two novel mutations were also created. First, a deletion of the 7 amino acids from Tyr276 to Glu282 led to a loss of sulfation and totally abolished VWF binding in the presence of botrocetin. This confirms that it is these 3 tyrosines that undergo sulfation and that this region is crucial for botrocetin-mediated VWF binding. The second mutation involves changing the lysine residues at 253, 258, and 262 to alanine. This also led to distinct changes in VWF binding and abolition of sulfation.

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