Glycoprotein VI is not a Functional Platelet Receptor for Fibrin Formed in Plasma or Blood

2020 ◽  
Vol 120 (06) ◽  
pp. 977-993 ◽  
Author(s):  
Danmei Zhang ◽  
Mariam Ebrahim ◽  
Kristin Adler ◽  
Xavier Blanchet ◽  
Janina Jamasbi ◽  
...  
Keyword(s):  
Platelet Adhesion ◽  
Aggregate Formation ◽  
Glycoprotein Vi ◽  
Platelet Receptor ◽  
Platelet Aggregate ◽  
Flowing Blood ◽  
Coated Surfaces ◽  
Long Fibers ◽  
Collagen Receptor

AbstractGlycoprotein VI (GPVI), a platelet collagen receptor, is crucial in mediating atherothrombosis. Besides collagen, injured plaques expose tissue factor (TF) that triggers fibrin formation. Previous studies reported that GPVI also is a platelet receptor for fibrinogen and fibrin. We studied the effect of anti-GPVI antibodies and inhibitors of GPVI signaling kinases (Syk and Btk) on platelet adhesion and aggregate formation onto immobilized fibrinogen and different types of fibrin under arterial flow conditions. Fibrin was prepared from isolated fibrinogen (“pure fibrin”), recombinant fibrinogen (“recombinant fibrin”), or generated more physiologically from endogenous fibrinogen in plasma (“plasma fibrin”) or by exposing TF-coated surfaces to flowing blood (“blood fibrin”). Inhibition of GPVI and Syk did not inhibit platelet adhesion and aggregate formation onto fibrinogen. In contrast anti-GPVI antibodies, inhibitors of Syk and Btk and the anti-GPIb antibody 6B4 inhibited platelet aggregate formation onto pure and recombinant fibrin. However, inhibition of GPVI and GPVI signaling did not significantly reduce platelet coverage of plasma fibrin and blood fibrin. Plasma fibrin contained many proteins incorporated during clot formation. Advanced optical imaging revealed plasma fibrin as a spongiform cushion with thicker, knotty, and long fibers and little activation of adhering platelets. Albumin intercalated in plasma fibrin fibers left only little space for platelet attachment. Pure fibrin was different showing a dense mesh of thin fibers with strongly activated platelets. We conclude that fibrin formed in plasma and blood contains plasma proteins shielding GPVI-activating epitopes. Our findings do not support a role of GPVI for platelet activation by physiologic fibrin.

Role of the PAR4 Thrombin Receptor in Stabilizing Platelet-platelet Aggregates as Revealed by a Patient with Hermansky-Pudlak Syndrome

2002 ◽  
Vol 87 (04) ◽  
pp. 722-727 ◽  
Author(s):  
Lidija Covic ◽  
Christopher Singh ◽  
Hedy Smith ◽  
Athan Kuliopulos
Keyword(s):  
Thrombin Receptor ◽  
Aggregate Formation ◽  
Dense Granules ◽  
Normal Individuals ◽  
Platelet Aggregate ◽  
Adp Release ◽  
Platelet Aggregates ◽  
Adp Receptor ◽  
Hermansky Pudlak Syndrome

SummaryIndividuals with Hermansky-Pudlak Syndrome (HPS) lack platelet dense granules and have no ADP-autocrine response. Despite these platelet deficiencies, HPS patients exhibit a surprisingly mild bleeding phenotype. We hypothesize that activation of the PAR4 thrombin receptor compensates for the lack of an ADP-autocrine response by the P2Y12 ADP receptor in individuals with HPS. Here, we determine that PAR4 activation by thrombin occurs well after ADP release from dense granules in normal individuals. However, the signal from PAR4 stabilizes platelet-platelet aggregate formation in the absence of P2Y12 activation by ADP. Thus, the strong signal emanating from PAR4 during platelet aggregation would provide an explanation for the mild bleeding diathesis of HPS.

scholarly journals Fibrin exposure triggers αIIbβ3‐independent platelet aggregate formation, ADAM10 activity and glycoprotein VI shedding in a charge‐dependent manner

2020 ◽  
Vol 18 (6) ◽  
pp. 1447-1458 ◽  
Author(s):  
Samantha J. Montague ◽  
Sarah M. Hicks ◽  
Christine S‐M. Lee ◽  
Lucy A. Coupland ◽  
Christopher R. Parish ◽  
...  
Keyword(s):  
Dependent Manner ◽  
Aggregate Formation ◽  
Glycoprotein Vi ◽  
Platelet Aggregate ◽  
A Charge

scholarly journals Function of Platelet Glycosphingolipid Microdomains/Lipid Rafts

2020 ◽  
Vol 21 (15) ◽  
pp. 5539
Author(s):  
Keisuke Komatsuya ◽  
Kei Kaneko ◽  
Kohji Kasahara
Keyword(s):  
Lipid Rafts ◽  
Platelet Adhesion ◽  
G Protein Coupled Receptors ◽  
Clot Retraction ◽  
Functional Roles ◽  
Glycoprotein Vi ◽  
Cellular Processes ◽  
Specific Proteins ◽  
G Protein Coupled ◽  
Collagen Receptor

Lipid rafts are dynamic assemblies of glycosphingolipids, sphingomyelin, cholesterol, and specific proteins which are stabilized into platforms involved in the regulation of vital cellular processes. The rafts at the cell surface play important functions in signal transduction. Recent reports have demonstrated that lipid rafts are spatially and compositionally heterogeneous in the single-cell membrane. In this review, we summarize our recent data on living platelets using two specific probes of raft components: lysenin as a probe of sphingomyelin-rich rafts and BCθ as a probe of cholesterol-rich rafts. Sphingomyelin-rich rafts that are spatially and functionally distinct from the cholesterol-rich rafts were found at spreading platelets. Fibrin is translocated to sphingomyelin-rich rafts and platelet sphingomyelin-rich rafts act as platforms where extracellular fibrin and intracellular actomyosin join to promote clot retraction. On the other hand, the collagen receptor glycoprotein VI is known to be translocated to cholesterol-rich rafts during platelet adhesion to collagen. Furthermore, the functional roles of platelet glycosphingolipids and platelet raft-binding proteins including G protein-coupled receptors, stomatin, prohibitin, flotillin, and HflK/C-domain protein family, tetraspanin family, and calcium channels are discussed.

Role of Lipid Rafts in GPVI Agonist-Induced Platelet Signaling.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3576-3576
Author(s):  
Patricia G. Quinter ◽  
Todd M. Quinton ◽  
Carol A. Dangelmaier ◽  
Satya P. Kunapuli ◽  
James L. Daniel
Keyword(s):  
Platelet Aggregation ◽  
Lipid Rafts ◽  
Lipid Raft ◽  
Glycoprotein Vi ◽  
Activated Platelets ◽  
Low Concentrations ◽  
Multiple Receptors ◽  
Platelet Signaling ◽  
Collagen Receptor

Abstract The collagen receptor glycoprotein VI (GPVI), plays an essential role in platelet activation and the regulation of hemostasis. Microdomains within the plasma membrane, called lipid rafts, have been implicated in GPVI signaling. The GPVI receptor has been shown to associate with the lipid rafts in both resting and activated platelets. It has been reported that there is a reduction in GPVI signaling in raft-disrupted platelets following activation with various GPVI agonists, especially at low to moderate agonist concentrations. Since platelet aggregation is potentiated by secreted adenosine 5′-diphosphate (ADP) at low concentrations of convulxin and at all concentrations of collagen and collagen-related peptide (CRP), we wanted to determine whether the decrease in GPVI signaling found in platelets with disrupted rafts was due to the loss of agonist potentiation by ADP. We compared platelet aggregation, protein phosphorylation, and calcium mobilization in platelets with intact and disrupted lipid rafts following activation with the GPVI agonists, collagen, convulxin and CRP. We show that lipid raft disruption inhibits aggregation induced by collagen and convulxin, but this inhibition is no longer apparent in the presence of ADP feedback inhibitors. Furthermore, raft-disrupted platelets had the same level of phosphorylation of proteins involved in GPVI signaling (i.e. Syk, LAT, and PLCγ2) and the same ability to mobilize calcium following activation with collagen or convulxin. Therefore, the effects of lipid raft disruption on aggregation can be attributed to the loss of ADP feedback. Interestingly, however, raft disruption directly inhibited aggregation and Syk phosphorylation induced by CRP in the presence and absence of ADP feedback. We propose that these differences are due to the fact that CRP is a relatively small, synthesized peptide of 37 amino acids, while collagen and convulxin are large ligands. These agonists are all able to bind the GPVI receptor, but they may not have the same ability to simultaneously cluster multiple receptors due to their size differential. The lipid rafts may be important for CRP stimulation, but not for collagen or convulxin, because they may have a higher density of the GPVI receptor than nonraft membrane regions, allowing CRP to cluster multiple receptors and activate the GPVI signaling cascade. When we disrupt the lipid rafts, we are reducing the effective concentration of GPVI available for activation by CRP but not by collagen or convulxin.

Abstract 391: Platelet CD40L Affects Thrombus Formation via Phosphatidylinositol 3-kinase ß, But Not Via CD40 or IKKα

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Marijke J Kuijpers ◽  
Nadine J Mattheij ◽  
Lina Cipolla ◽  
Johanna P van Geffen ◽  
Toby Lawrence ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Thrombus Formation ◽  
Aggregate Formation ◽  
Phosphatidylinositol 3 Kinase ◽  
Akt Phosphorylation ◽  
Soluble Cd40l ◽  
Granule Secretion ◽  
Collagen Receptor

Objective: To investigate the roles and signaling pathways of CD40L and CD40 in platelet activation and thrombus formation under atherothrombotic conditions. Approach and Results: Mouse platelets lacking CD40L (Cd40lg -/- Apoe -/- ) showed diminished αIIbβ3 activation and α-granule secretion in response to collagen receptor (GPVI) stimulation, while CD40 deficient platelets (Cd40 -/- Apoe -/- ) showed increased responses. ADP- or thrombin-evoked activation was unaffected. In both Cd40lg -/- Apoe -/- and Cd40 -/- Apoe -/- mice, formation of multi-layered thrombi was decreased on both atherosclerotic plaque material and collagen, in comparison to controls. Addition of CD40L prior to perfusion over collagen or plaque material enhanced dense aggregate formation in Apoe -/- , Cd40lg -/- Apoe -/- and Cd40 -/- Apoe -/- blood. CD40L or low GPVI stimulation separately did not cause platelet aggregation. But when combined, aggregation was potentiated, even in the absence of CD40. This potentiation was antagonized by inhibiting PI3Kβ, as well as in platelets from Pik3cb R/R mice. CD40L enhanced Akt phosphorylation at low GPVI stimulation, which was again antagonized by PI3Kβ inhibition and absent in platelets from Pik3cb R/R mice. Finally, Chuk1 A/A Apoe -/- mice, deficient in IKKα, displayed no differences in platelet aggregation - with or without CD40L - nor in thrombus formation in whole blood, indicating that these effects are not mediated via IKKα/NFkB. Conclusions: Under atherothrombotic conditions, CD40L enforces collagen-dependent platelet activation, by supporting integrin αIIbβ3 activation, secretion and dense thrombus formation via PI3Kβ, but not IKKα. Since shedding of CD40L starts minutes after activation, these results point to a joint role of both platelet-bound and soluble CD40L in controlling the size of rapidly formed thrombi.

scholarly journals Differential role of glycolipid-enriched membrane domains in glycoprotein VI- and integrin-mediated phospholipase Cγ2 regulation in platelets

2002 ◽  
Vol 364 (3) ◽  
pp. 755-765 ◽  
Author(s):  
Peter WONEROW ◽  
Achim OBERGFELL ◽  
Jonathan I. WILDE ◽  
Régis BOBE ◽  
Naoki ASAZUMA ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Membrane Domains ◽  
Adapter Protein ◽  
Glycoprotein Vi ◽  
Receptor Signalling ◽  
Fibrinogen Receptor ◽  
Fibrinogen Binding ◽  
Collagen Receptor ◽  
Lines Of Evidence

The platelet collagen receptor glycoprotein VI (GPVI) and the fibrinogen receptor integrin αIIbβ3 trigger intracellular signalling cascades involving the tyrosine kinase Syk, the adapter SLP-76 and phospholipase Cγ2 (PLCγ2). Similar pathways are activated downstream of immune receptors in lymphocytes, where they have been localized in part to glycolipid-enriched membrane domains (GEMs). Here we provide several lines of evidence that GPVI-mediated tyrosine phosphorylation of PLCγ2 in platelets is dependent on GEM-organized signalling and utilizes the GEM resident adapter protein LAT (linker for activation of T cells). In sharp contrast, although fibrinogen binding to platelets stimulates αIIbβ3-dependent activation of Syk and tyrosine phosphorylation of SLP-76 and PLCγ2, it does not utilize GEMs to promote these responses or to support platelet aggregation. These results establish that GPVI and αIIbβ3 trigger distinct patterns of receptor signalling in platelets, leading to tyrosine phosphorylation of PLCγ2, and they highlight the role of GEMs in compartmentalizing signalling reactions involved in haemostasis.

Genetic evidence for a predominant role of PI3Kβ catalytic activity in ITAM- and integrin-mediated signaling in platelets

Blood ◽  
2009 ◽  
Vol 114 (10) ◽  
pp. 2193-2196 ◽  
Author(s):  
Ilaria Canobbio ◽  
Lucia Stefanini ◽  
Lina Cipolla ◽  
Elisa Ciraolo ◽  
Cristian Gruppi ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Adhesion ◽  
Small Gtpase ◽  
Genetic Evidence ◽  
Integrin Αiibβ3 ◽  
Akt Phosphorylation ◽  
Glycoprotein Vi ◽  
Relative Contribution ◽  
Inactive Form

Abstract Phosphatidylinositol 3-kinase (PI3K) isoforms PI3Kβ and PI3Kγ are implicated in platelet adhesion, activation, and aggregation, but their relative contribution is still unclear or controversial. Here, we report the first comparative functional analysis of platelets from mice expressing a catalytically inactive form of PI3Kβ or PI3Kγ. We demonstrate that both isoforms were similarly required for maximal activation of the small GTPase Rap1b and for complete platelet aggregation upon stimulation of G protein–coupled receptors for adenosine 5′-diphosphate (ADP) or U46619. Their contribution to these events, however, was largely redundant and dispensable. However, PI3Kβ, but not PI3Kγ, enzymatic activity was absolutely required for Akt phosphorylation, Rap1 activation, and platelet aggregation downstream of the immunoreceptor tyrosine-based activation motif (ITAM)–bearing receptor glycoprotein VI (GPVI). Moreover, PI3Kβ was a major essential regulator of platelet adhesion to fibrinogen and of integrin αIIbβ3-mediated spreading. These results provide genetic evidence for a crucial and selective role of PI3Kβ in signaling through GPVI and integrin αIIbβ3.

scholarly journals A collagen-related peptide regulates phospholipase Cγ2 via phosphatidylinositol 3-kinase in human platelets

1999 ◽  
Vol 342 (1) ◽  
pp. 171-177 ◽  
Author(s):  
Jean-Max PASQUET ◽  
Régis BOBE ◽  
Barbara GROSS ◽  
Marie-Pierre GRATACAP ◽  
Michael G. TOMLINSON ◽  
...  
Keyword(s):  
Inositol Phosphates ◽  
Human Platelets ◽  
Ph Domain ◽  
Phosphatidylinositol 3 Kinase ◽  
Glycoprotein Vi ◽  
Related Peptide ◽  
Lipid Species ◽  
Collagen Receptor ◽  
Phosphatidylinositol 3

The collagen receptor glycoprotein VI (GPVI) induces platelet activation through a similar pathway to that used by immune receptors. In the present study we have investigated the role of phosphatidylinositol 3-kinase (PI 3-kinase) in GPVI signalling. Our results show that collagen-related peptide {CRP: [GCP*(GPP*)10GCP*G]n; P* = hydroxyproline}, which is selective to GPVI, induces formation of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] and phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] in platelets. The increase in the two 3-phosphorylated lipids is inhibited completely by wortmannin and by LY294002, two structurally unrelated inhibitors of PI 3-kinase. The formation of inositol phosphates and phosphatidic acid (PA), two markers of phospholipase C (PLC) activation, by CRP are inhibited by between 50 and 85% in the presence of wortmannin and LY294002. This is associated with inhibition of elevation of intracellular Ca2+ ([Ca2+]i) and aggregation. Wortmannin and LY294002 also partially inhibit elevation of Ca2+ by CRP in murine megakaryocytes. Microinjection of the pleckstrin-homology PH domain of Bruton's tyrosine kinase, which binds selectively to PI(3,4,5)P3, but not the R28C (Arg28 → Cys) mutant which binds to PI(3,4,5)P3 with low affinity, also inhibits elevation of [Ca2+]i in megakaryocytes, suggesting that it is this lipid species which mediates the action of the PI 3-kinase pathway. Studies in platelets show that the action of wortmannin and LY294002 is not mediated through an alteration in tyrosine phosphorylation of PLCγ2. These results demonstrate that PI 3-kinase is required for full activation of PLCγ2 by GPVI in platelets and megakaryocytes.

scholarly journals Intercellular calcium communication regulates platelet aggregation and thrombus growth

2003 ◽  
Vol 160 (7) ◽  
pp. 1151-1161 ◽  
Author(s):  
Warwick S. Nesbitt ◽  
Simon Giuliano ◽  
Suhasini Kulkarni ◽  
Sacha M. Dopheide ◽  
Ian S. Harper ◽  
...  
Keyword(s):  
Platelet Adhesion ◽  
Purinergic Receptor ◽  
Cytosolic Calcium ◽  
Calcium Flux ◽  
Aggregate Formation ◽  
Signaling Mechanism ◽  
Activated Platelets ◽  
Platelet Aggregate ◽  
Platelet Aggregates

The ability of platelets to form stable adhesion contacts with other activated platelets (platelet cohesion or aggregation) at sites of vascular injury is essential for hemostasis and thrombosis. In this study, we have examined the mechanisms regulating cytosolic calcium flux during the development of platelet–platelet adhesion contacts under the influence of flow. An examination of platelet calcium flux during platelet aggregate formation in vitro demonstrated a key role for intercellular calcium communication (ICC) in regulating the recruitment of translocating platelets into developing aggregates. We demonstrate that ICC is primarily mediated by a signaling mechanism operating between integrin αIIbβ3 and the recently cloned ADP purinergic receptor P2Y12. Furthermore, we demonstrate that the efficiency by which calcium signals are propagated within platelet aggregates plays an important role in dictating the rate and extent of thrombus growth.

Dimeric Glycoprotein VI Binds to Collagen but Not to Fibrin

2018 ◽  
Vol 118 (02) ◽  
pp. 351-361 ◽  
Author(s):  
Mariam Ebrahim ◽  
Janina Jamasbi ◽  
Kristin Adler ◽  
Remco Megens ◽  
Yacine M'Bengue ◽  
...  
Keyword(s):  
Atherosclerotic Plaque ◽  
Fusion Proteins ◽  
Thrombus Formation ◽  
Platelet Glycoprotein ◽  
Binding Studies ◽  
Glycoprotein Vi ◽  
Fibrin Formation ◽  
Platelet Thrombus ◽  
Flowing Blood ◽  
Coated Surfaces

AbstractPlatelet glycoprotein VI (GPVI) acts as a decisive collagen receptor in atherothrombosis. Besides collagen, injured atherosclerotic plaques expose tissue factor (TF) that triggers fibrin formation. Two recent studies reported that platelet GPVI also functions as fibrin receptor, which would importantly widen the mode of action of GPVI-targeted antithrombotic drugs. We studied the binding of two GPVI fusion proteins to fibrin under static and arterial flow conditions. Fibrin was prepared from purified fibrinogen or generated more physiologically from endogenous fibrinogen by coagulating plasma with thrombin. Fibrin formation was also triggered by exposing TF-coated surfaces or human atherosclerotic plaque slices to arterially flowing blood. By binding studies and advanced optical imaging, we found that recombinant dimeric GPVI-Fc fusion proteins with Fc from either IgG1 (GPVI-Fc1) or IgG2 (GPVI-Fc2) bound to collagen fibres, but neither to fibrin prepared from purified fibrinogen obtained from three suppliers, nor to physiological fibrin formed by thrombin in plasma or triggered by exposing TF or atherosclerotic plaque slices to arterially flowing blood. Our findings do not support a role of dimeric platelet GPVI as receptor for fibrin. This is important for the understanding of plaque-triggered platelet thrombus formation and is clinically relevant for future GPVI-targeting therapies with recombinant GPVI-Fc and anti-GPVI antibodies.

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