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The influence of N-linked glycosylation on the function of platelet glycoprotein VI.

ABSTRACT: Using recombinant human glycoprotein VI (GPVI), we evaluated the effect of N-linked glycosylation at the consensus site Asparagine92-Glycine-Serine94 (N92GS94) on binding of this platelet-specific receptor to its ligands, human type I collagen, collagen-related peptide (CRP), and the snake venom C-type lectin convulxin (CVX). 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. In stably transfected Dami cells, the substitutions N92A or S94A, but not L95H, resulted in a 30% to 40% decrease in adhesion to CVX, but a 90% or greater decrease in adhesion to CRP and a 65% to 70% decrease in adhesion to type I collagen. Treatment with PNGase F, but not Endoglycosidase H (Endo H) (specific for high-mannose N-linked glycans), produced an equivalent decrease in molecular weight. Neither N92A nor S94A affected the expression of GPVI, based on the direct binding of murine anti-human GPVI monoclonal antibody 204-11 to transfected Dami cells. These findings indicate that N-linked glycosylation at N92 in human GPVI is not required for surface expression, but contributes to maximal adhesion to type I collagen, CRP and, to a lesser extent, CVX.

SUBMITTER: Kunicki TJ

PROVIDER: S-EPMC1895313 | biostudies-literature | 2005 Oct

REPOSITORIES: biostudies-literature

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The influence of N-linked glycosylation on the function of platelet glycoprotein VI.

Kunicki Thomas J TJ Cheli Yann Y Moroi Masaaki M Furihata Kenichi K

Blood 20050712 8

Using recombinant human glycoprotein VI (GPVI), we evaluated the effect of N-linked glycosylation at the consensus site Asparagine92-Glycine-Serine94 (N92GS94) on binding of this platelet-specific receptor to its ligands, human type I collagen, collagen-related peptide (CRP), and the snake venom C-type lectin convulxin (CVX). 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 th ...[more]

PMID: 16014566

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Project description:Essentials RAS proteins are expressed in platelets but their functions are largely uncharacterized. TC21/RRas2 is required for glycoprotein VI-induced platelet responses and for thrombus stability in vivo. TC21 regulates platelet aggregation by control of ?IIb ?3 integrin activation, via crosstalk with Rap1b. This is the first indication of functional importance of a proto-oncogenic RAS protein in platelets. SUMMARY:Background Many RAS family small GTPases are expressed in platelets, including RAC, RHOA, RAP, and HRAS/NRAS/RRAS1, but most of their signaling and cellular functions remain poorly understood. Like RRAS1, TC21/RRAS2 reverses HRAS-induced suppression of integrin activation in CHO cells. However, a role for TC21 in platelets has not been explored. Objectives To determine TC21 expression in platelets, TC21 activation in response to platelet agonists, and roles of TC21 in platelet function in in vitro and in vivo thrombosis. Results We demonstrate that TC21 is expressed in human and murine platelets, and is activated in response to agonists for the glycoprotein (GP) VI-FcR? immunoreceptor tyrosine-based activation motif (ITAM)-containing collagen receptor, in an Src-dependent manner. GPVI-induced platelet aggregation, integrin ?IIb ?3 activation, and ?-granule and dense granule secretion, as well as phosphorylation of Syk, phospholipase C?2, AKT, and extracellular signal-regulated kinase, were inhibited in TC21-deficient platelets ex vivo. In contrast, these responses were normal in TC21-deficient platelets following stimulation with P2Y, protease-activated receptor 4 and C-type lectin receptor 2 receptor agonists, indicating that the function of TC21 in platelets is GPVI-FcR?-ITAM-specific. TC21 was required for GPVI-induced activation of Rap1b. TC21-deficient mice did not show a significant delay in injury-induced thrombosis as compared with wild-type controls; however, thrombi were unstable. Hemostatic responses showed similar effects. Conclusions TC21 is essential for GPVI-FcR?-mediated platelet activation and for thrombus stability in vivo via control of Rap1b and integrins.

Project description:Essentials Collagen and thrombin when used simultaneously generate highly activated platelets. The effect of thrombin stimulation on subsequent glycoprotein VI (GPVI) function was observed. Soluble fibrin, but not protease activated receptor (PAR) activation, prevented GPVI activation. Circulating soluble fibrin in coagulopathic blood may cause an acquired GPVI signaling defect. SUMMARY:Background In coagulopathic blood, circulating thrombin may drive platelet dysfunction. Methods/Results Using calcium dye-loaded platelets, the effect of thrombin exposure and soluble fibrin generation on subsequent platelet GPVI function was investigated. Exposure of apixaban-treated platelet-rich plasma (12% PRP) to thrombin (1-10 nm), but not ADP or thromboxane mimetic U46619 exposure, dramatically blocked subsequent GPVI activation by convulxin, collagen-related peptide or fibrillar collagen. Consistent with soluble fibrin multimerizing and binding GPVI, the onset of convulxin insensitivity required 200-500 s of thrombin exposure, was not mimicked by exposure to PAR-1/4 activating peptides, was not observed with washed platelets, and was blocked by fibrin polymerization inhibitor (GPRP) or factor XIIIa inhibitor (T101). PAR-1 signaling through G?q was not required because vorapaxar blocked thrombin-induced calcium mobilization but had no effect on the ability of thrombin to impair GPVI-signaling. Convulxin insensitivity was unaffected by the metalloprotease inhibitor GM6001 or the ?IIb ?3 antagonist GR144053, indicating negligible roles for GPVI shedding or ?IIb ?3 binding of fibrin. Thrombin treatment of washed platelets resuspended in purified fibrinogen also produced convulxin insensitivity that was prevented by GPRP. Exposure of apixaban/PPACK-treated whole blood to thrombin-treated fibrinogen resulted in > 50% decrease in platelet deposition in a collagen microfluidic assay that required soluble fibrin assembly. Conclusions Conversion of only 1% plasma fibrinogen in coagulopathic blood would generate 90 nm soluble fibrin, far exceeding ~1 nmGPVI in blood. Soluble fibrin, rather than thrombin-induced platelet activation throuh PAR-1 and PAR-4, downregulated GPVI-signaling in response to stimuli, and may lead to subsequent hypofunction of endogenous or transfused platelets.

Dataset Information

The influence of N-linked glycosylation on the function of platelet glycoprotein VI.

Publications

The influence of N-linked glycosylation on the function of platelet glycoprotein VI.

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