CAMKKβ/AMPK-α1 pathway regulates phosphorylation of cytoskeletal targets in thrombin-stimulated human platelets
; Oury, Cécile ; et al
in Journal of Thrombosis and Haemostasis [=JTH] (2014), 12(6), 973-986
Background. Platelet activation requires sweeping morphological changes, supported by contraction and remodelling of platelet actin cytoskeleton. In various other cell types, AMP-activated protein kinase ... [more ▼]
Background. Platelet activation requires sweeping morphological changes, supported by contraction and remodelling of platelet actin cytoskeleton. In various other cell types, AMP-activated protein kinase (AMPK) controls the phosphorylation state of cytoskeletal targets. Objective. We hypothesized that AMPK is activated during platelet aggregation and contributes to the control of cytoskeletal targets. Results. We found that AMPK-α1 was mainly activated by thrombin and not by other platelet agonists in purified human platelets. Thrombin activated AMPK-α1 ex vivo via a Ca2+/calmodulin-dependent kinase kinase β (CAMKKβ)-dependent pathway. Pharmacological inhibition of CAMKKβ blocked thrombin-induced platelet aggregation and counteracted thrombin-induced phosphorylation of several cytoskeletal proteins, namely, regulatory myosin light chains (MLC), cofilin and vasodilator-stimulated phosphoprotein (VASP), three key elements involved in actin cytoskeleton contraction and polymerization. Platelets isolated from mice lacking AMPK-α1 exhibited reduced aggregation in response to thrombin, associated with a defect in MLC, cofilin and VASP phosphorylation and actin polymerization. More importantly, we show for the first time that AMPK pathway was activated in platelets of patients undergoing major cardiac surgery, in a heparin-sensitive manner. Conclusion. AMPK-α1 is activated by thrombin in human platelets. It controls phosphorylation of key cytoskeletal targets and actin cytoskeleton remodelling during platelet aggregation. [less ▲]Detailed reference viewed: 8 (0 ULg)
Connection between cardiac vascular permeability, myocardial oedema and inflammation during sepsis: role of the alpha1AMPK isoform
; ; et al
in Critical Care Medicine (2013), 41(12), 411-22
Objective: Since AMP-activated protein kinase (AMPK) both controls cytoskeletonorganization in endothelial cells (ECs) and exerts anti-inflammatory effects, we here postulated that it could influence ... [more ▼]
Objective: Since AMP-activated protein kinase (AMPK) both controls cytoskeletonorganization in endothelial cells (ECs) and exerts anti-inflammatory effects, we here postulated that it could influence vascular permeability and inflammation, thereby counteracting cardiac wall oedema during sepsis. Design: Controlled animal study Settings: University research laboratory Subjects: C57BL/6J, α1AMPK-/- and α1AMPK+/+ mice Intervention: Sepsis was triggered in vivo using a sub-lethal injection of lipopolysaccharide (LPS, O55B5, 10 mg.kg-1), inducing systolic left ventricular (LV) dysfunction. LV function, oedema, vascular permeability and inflammation were assessed in vivo in both wild type (WT) mice (α1AMPK+/+) and α1AMPK-deficient mice (α1AMPK-/-). 5-Aminoimidazole-4-carboxamide riboside (AICAr) served to study the impact of AMPK activation on vascular permeability in vivo. The integrity of EC monolayers was also examined in vitro after LPS challenge in the presence of AICAr and/or after α1AMPK silencing. Measurements and main results: α1AMPK-deficiency dramatically impaired tolerance to LPS challenge. Indeed, α1AMPK-/- exhibited heightened cardiac vascular permeability after LPS challenge compared to α1AMPK+/+. Consequently, an increase in LV mass corresponding to exaggerated wall oedema occurred in α1AMPK-/-, without any further decrease in systolic function. Mechanistically, the LPS-induced α1AMPK-/- cardiac phenotype could not be attributed to major changes in the systemic inflammatory response, but was due to an increased disruption of interendothelial tight junctions. Accordingly, AMPK activation by AICAr counteracted LPS-induced hyperpermeability in WT mice in vivo as well as in ECs in vitro. This effect was associated with a potent protection of ZO-1 linear border pattern in ECs. Conclusions: Our results demonstrate, for the first time the involvement of a signalling pathway in the control of LV wall oedema during sepsis. AMPK exerts a protective action through the preservation of interendothelial tight junctions. Interestingly, exaggerated LV wall oedema was not coupled with aggravated systolic dysfunction. However, it could contribute to diastolic dysfunction in septic patients. [less ▲]Detailed reference viewed: 31 (14 ULg)
Overexpression of CD39 in mouse airways promotes bacteria induced inflammation
Theatre, Emilie ; ; et al
in Journal of Immunology (2012), 189(4), 1966-1974
In airways, the ecto-nucleoside triphosphate diphosphohydrolase CD39 plays a central role in the regulation of physiological mucosal nucleotide concentrations and likely contributes to the control of ... [more ▼]
In airways, the ecto-nucleoside triphosphate diphosphohydrolase CD39 plays a central role in the regulation of physiological mucosal nucleotide concentrations and likely contributes to the control of inflammation because accelerated ATP metabolism occurs in chronic inflammatory lung diseases.We sought to determine whether constant elevated CD39 activity in lung epithelia is sufficient to cause inflammation and whether this affects the response to acute LPS or Pseudomonas aeruginosa exposure. We generated transgenic mice overexpressing human CD39 under the control of the airway-specific Clara cell 10-kDa protein gene promoter. Transgenic mice did not develop any spontaneous lung inflammation. However, intratracheal instillation of LPS resulted in accelerated recruitment of neutrophils to the airways of transgenic mice. Macrophage clearance was delayed, and the amounts of CD8+ T and B cells were augmented. Increased levels of keratinocyte chemoattractant, IL-6, and RANTES were produced in transgenic lungs. Similarly, higher numbers of neutrophils and macrophages were found in the lungs of transgenic mice infected with P. aeruginosa, which correlated with improved bacteria clearance. The transgenic phenotype was partially and differentially restored by coinstillation of P2X1 or P2X7 receptor antagonists or of caffeine with LPS. Thus, a chronic increase of epithelial CD39 expression and activity promotes airway inflammation in response to bacterial challenge by enhancing P1 and P2 receptor activation. [less ▲]Detailed reference viewed: 23 (8 ULg)
AMP-activated protein kinase controls liposaccharide-induced hyperpermeability
; ; et al
in Critical Care (2012, March), 16(suppl 1), 17
Organ dysfunction determines the severity of sepsis and is correlated to mortality. Endothelial increased permeability contributes to the development of organ failure. AMP-activated protein kinase (AMPK ... [more ▼]
Organ dysfunction determines the severity of sepsis and is correlated to mortality. Endothelial increased permeability contributes to the development of organ failure. AMP-activated protein kinase (AMPK) has been shown to modulate cytoskeleton and could mediate endothelial permeability. Our hypothesis is that AMPK controls sepsis-induced hyperpermeability in the heart and is involved in septic cardiomyopathy. Sepsis was induced by intraperitoneal injection of liposaccharide, 10 mg/kg (LPS). Alpha-1 AMPK knockout mice (α1KO) were compared with wild-type. Vascular permeability was characterized by Evans blue extravasation. Inflammatory cytokine mRNA expression was determined by qPCR analysis. Left ventricular mass was assessed by echocardiography. In addition, to emphasize the beneficial role of AMPK on heart vascular permeability, AMPK activator (acadesine) was administered to C57Bl6 mice before LPS injection. The ANOVA test with Bonferroni's post hoc test and the log-rank test were used. P < 0.05 was considered as significant. Increased cardiac vascular permeability was observed in the LPS group in comparison to untreated animals (2.5% vs. 16%; P < 0.05). The α1KO mice exhibited an increase vascular permeability after LPS injection in comparison to wild-type mice (41.5% vs. 16%; P < 0.05). α1KO animals had a significant mortality increase after LPS injection (70% vs. 10%; P < 0.05). LPS markedly induced the production of proinflammatory cytokines (TNFα, IL-1β, IL-6) that were significantly higher in the α1KO animals. More importantly, LPS treatment leads to an increased left ventricular mass in the α1KO mice within 24 hours, suggesting the onset of edema. Finally LPS-induced vascular hyperpermeability was greatly reduced after AMPK activation by acadesine (13.2% vs. 40%; P < 0.05). AMPK importantly regulates cardiac vascular permeability and could control the sepsis-induced cardiomyopathy. AMPK could represent a new pharmacological target of sepsis. [less ▲]Detailed reference viewed: 13 (1 ULg)
ATP-gated P2X1 ion channels protect from endotoxemia by dampening neutrophil activation
Lecut, Christelle ; Faccinetto, Céline ; Delierneux, Céline et al
in Journal of Thrombosis and Haemostasis [=JTH] (2012), 10(3), 453-65
Background: In sepsis, extracellular ATP, secreted by activated platelets and leukocytes, may contribute to the crosstalk between hemostasis and inflammation. Previously, we showed that, in addition to ... [more ▼]
Background: In sepsis, extracellular ATP, secreted by activated platelets and leukocytes, may contribute to the crosstalk between hemostasis and inflammation. Previously, we showed that, in addition to their role in platelet activation, ATP-gated P2X1 ion channels are involved in promoting neutrophil chemotaxis. <br />Objectives: To elucidate the contribution of P2X1 ion channels to sepsis and associated disturbance of hemostasis. <br />Methods: We used P2X1-/- mice in a model of lipopolysaccharide (LPS)-induced sepsis. Hemostasis and inflammation parameters were analysed together with outcome. Mechanisms were further studied ex vivo using mouse and human blood or isolated neutrophils and monocytes. <br />Results: P2X1-/- mice were more susceptible to LPS-induced shock than wild-type mice despite normal cytokine production. Plasma levels of thrombin-antithrombin complexes were higher, thrombocytopenia was worsened and whole blood coagulation time was markedly reduced, pointing to aggravated hemostasis disturbance in the absence of P2X1. However, whole blood platelet aggregation occurred normally and P2X1-/- macrophages displayed normal levels of total tissue factor activity. We found that P2X1-/- neutrophils produced higher amounts of reactive oxygen species. Increased amounts of myeloperoxidase were released in the blood of LPS-treated P2X1-/- mice, and circulating neutrophils and monocytes expressed higher levels of CD11b. Neutrophil accumulation into the lungs was also significantly augmented, as was lipid peroxidation in the liver. Desensitization of P2X1 ion channels led to increased activation of human neutrophils and enhanced formation of platelet-leukocyte aggregates. [less ▲]Detailed reference viewed: 37 (19 ULg)
Lack of P2X1 ion channels increases endotoxemia associated coagulation and organ damage through neutrophil hyperresponsiveness.
Lecut, Christelle ; Faccinetto, Céline ; et al
in Journal of Thrombosis and Haemostasis [=JTH] (2011), 9(suppl S2),
ATP-gated P2X1 ion channels contribute to arterial thrombosis by amplifying platelet activation. In the search for novel anti-platelet strategies, targeting P2X1 ion channels is appealing. However, in ... [more ▼]
ATP-gated P2X1 ion channels contribute to arterial thrombosis by amplifying platelet activation. In the search for novel anti-platelet strategies, targeting P2X1 ion channels is appealing. However, in this study we found that lack or inhibition of P2X1 channels enhanced neutrophil respiratory burst activity ex vivo. <br />To study the consequence of P2X1 deficiency on neutrophil function in vivo, P2X1-/- mice were used in a model of endotoxin-induced sepsis. Upon injection of lipopolysaccharides (LPS), plasma myeloperoxidase (MPO) concentrations reached higher levels in the P2X1-/- mice, and circulating neutrophils expressed higher levels of surface CD11b compared to wild-type mice. Neutrophil relocalization into the lungs of LPS-treated P2X1-/- mice was also significantly augmented, reflecting a higher activation state of P2X1-/- neutrophils under conditions of sepsis. Accordingly, more extensive lipid peroxidation was observed in the liver of LPS-treated P2X1-/- mice, indicative of exaggerated oxidative damage. Concomitantly, the levels of thrombin-antithrombin complexes were higher in the plasma of LPS-treated P2X1-/- mice and thrombocytopenia was worsened as compared to wild type mice. Elevated numbers of microthrombi were also found in the lungs of these mice. These observations coincided with a higher susceptibility of P2X1-/- mice to LPS-induced septic shock than wild type animals. <br />Our results strongly suggest that P2X1 ion channels play a protective role in sepsis by negatively regulating systemic neutrophil activation, thereby limiting oxidative damage, activation of coagulation and platelet accumulation into the lungs. Therefore, since antagonists of P2X1 ion channels may not only target platelets but also affect neutrophils, inhibiting these channels in the highly inflammatory environment of severe sepsis or of acute coronary syndromes might be detrimental. [less ▲]Detailed reference viewed: 26 (10 ULg)
P2X1 Ion Channels Promote Neutrophil Chemotaxis through Rho Kinase Activation
Lecut, Christelle ; Frederix, Kim ; et al
in Journal of Immunology (2009)
This study shows that activation of P2X1 ion channels by ATP promotes neutrophil chemotaxis, a process involving Rho kinase-dependent actomyosin-mediated contraction at the cell rear. These ion channels ... [more ▼]
This study shows that activation of P2X1 ion channels by ATP promotes neutrophil chemotaxis, a process involving Rho kinase-dependent actomyosin-mediated contraction at the cell rear. These ion channels may therefore play a significant role in host defense and inflammation. [less ▲]Detailed reference viewed: 58 (20 ULg)
Contribution of ATP-gated P2X1 ion channels to the control of neutrophil chemotaxis.
Lecut, Christelle ; Frederix, Kim ; et al
in Purinergic Signalling (2008), 4Detailed reference viewed: 15 (5 ULg)
P2X1 receptors as new regulators of neutrophil life span
Faccinetto, Céline ; Lecut, Christelle ; Jacobs, Nathalie et al
in Journal of Thrombosis and Haemostasis [=JTH] (2007)Detailed reference viewed: 26 (4 ULg)
New role for ATP P2X1 receptors in the control of neutrophil apoptosis and respiratory burst activity.
Faccinetto, Céline ; Lecut, Christelle ; Greimers, Roland et al
in Blood (2006), 108(11, Part 1), 1647Detailed reference viewed: 28 (11 ULg)
Contribution of platelet glycoprotein VI to the thrombogenic effect of collagens in fibrous atherosclerotic lesions.
; ; Lecut, Christelle et al
in Atherosclerosis (2005), 181(1), 19-27
Collagens (types I and III) are among the strongest thrombus-forming components of the vascular subendothelium. We compared the thrombogenic effects of four collagen-containing advanced atherosclerotic ... [more ▼]
Collagens (types I and III) are among the strongest thrombus-forming components of the vascular subendothelium. We compared the thrombogenic effects of four collagen-containing advanced atherosclerotic lesions with those of purified types I and III collagen fibers. Cell-free homogenates from the human plaques effectively promoted platelet adhesion and aggregate formation under high-shear flow conditions, as well as exposure of procoagulant phosphatidylserine (PS) on platelets. With all plaques, blocking of the glycoprotein VI (GPVI) receptor for collagen abolished aggregation and PS exposure. Blocking of platelet ADP receptors resulted in similar, but less complete inhibitory effects. Type I collagen was more potent than type III collagen in inducing aggregation and PS exposure under flow, via stimulation of GPVI and ADP receptors. Type I collagen also more strongly enhanced thrombin generation with platelets and tissue factor, again via GPVI activation and PS exposure. The plaque material enhanced thrombin generation, partly due to the presence of tissue factor and partly via GPVI and ADP receptors. 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. [less ▲]Detailed reference viewed: 39 (4 ULg)
Fibrillar type I collagens enhance platelet-dependent thrombin generation via glycoprotein VI with direct support of alpha2beta1 but not alphaIIbbeta3 integrin.
Lecut, Christelle ; ; et al
in Thrombosis and Haemostasis (2005), 94(1), 107-14
The role of collagens and collagen receptors was investigated in stimulating platelet-dependent thrombin generation. Fibrillar type-I collagens, including collagen from human heart, were most potent in ... [more ▼]
The role of collagens and collagen receptors was investigated in stimulating platelet-dependent thrombin generation. Fibrillar type-I collagens, including collagen from human heart, were most potent in enhancing thrombin generation, in a way dependent on exposure of phosphatidylserine (PS) at the platelet surface. Soluble, non-fibrillar type-I collagen required pre-activation of integrin alpha2beta1 with Mn2+ for enhancement of thrombin generation. With all preparations, blocking of glycoprotein VI (GPVI) with 9O12 antibody abrogated the collagen-enhanced thrombin generation, regardless of the alpha2beta 1 activation state. Blockade of alpha2beta1 alone or antagonism of autocrine thromboxane A2 and ADP were less effective. Blockade of alphaIIbbeta3 with abciximab suppressed thrombin generation in platelet-rich plasma, but this did not abolish the enhancing effect of collagens. The high activity of type-I fibrillar collagens in stimulating GPVI-dependent procoagulant activity was confirmed in whole-blood flow studies, showing that these collagens induced relatively high expression of PS. Together, these results indicate that: i) fibrillar type-I collagen greatly enhances thrombin generation, ii) GPVI-induced platelet activation is principally responsible for the procoagulant activity of fibrillar and non-fibrillar collagens, iii) alpha2beta1 and signaling via autocrine mediators facilitate and amplify this GPVI activity, and iv) alphaIIbbeta3 is not directly involved in the collagen effect. [less ▲]Detailed reference viewed: 16 (2 ULg)
Severe deficiency of glycoprotein VI in a patient with gray platelet syndrome.
; ; et al
in Blood (2004), 104(1), 107-14
We report a novel case of gray platelet syndrome (GPS) where a severe deficiency of the platelet collagen receptor, glycoprotein (GP) VI, accompanies classical symptoms of a low platelet count and ... [more ▼]
We report a novel case of gray platelet syndrome (GPS) where a severe deficiency of the platelet collagen receptor, glycoprotein (GP) VI, accompanies classical symptoms of a low platelet count and platelets lacking alpha-granules. Dense granules were normally present. Platelet aggregation with collagen was severely decreased, as was the response to convulxin (Cvx), a GPVI agonist. Quantitative analysis of GPVI using fluorescein isothiocyanate (FITC)-Cvx in flow cytometry showed its virtual absence on the patient's platelets. The GPVI deficiency was confirmed using monoclonal antibodies in Western blotting and in immunogold labeling on frozen thin sections where internal pools of GPVI were confirmed for normal platelets. The Fc receptor gamma-chain, constitutively associated with GPVI in normal platelets, was present in subnormal amounts, and the phospholipase C gamma 2-dependent activation pathway appeared to function normally. No autoantibodies to GPVI were found in the patient's serum using monoclonal antibody immobilization of platelet antigen (MAIPA). Sequencing of coding regions of the GPVI gene failed to show abnormalities, and mRNA for GPVI was present in the patient's platelets, pointing to a probable acquired defect in GPVI expression. Our results may provide a molecular explanation for the subgroup of patients with severely deficient collagen-induced platelet aggregation as previously described for GPS in the literature. [less ▲]Detailed reference viewed: 38 (2 ULg)
Principal role of glycoprotein VI in alpha2beta1 and alphaIIbbeta3 activation during collagen-induced thrombus formation.
Lecut, Christelle ; ; et al
in Arteriosclerosis, Thrombosis, and Vascular Biology (2004), 24(9), 1727-33
OBJECTIVE: High-shear perfusion of blood over collagen results in rapid platelet adhesion, aggregation, and procoagulant activity. We studied regulation of alpha2beta1 and alphaIIbbeta3 integrin ... [more ▼]
OBJECTIVE: High-shear perfusion of blood over collagen results in rapid platelet adhesion, aggregation, and procoagulant activity. We studied regulation of alpha2beta1 and alphaIIbbeta3 integrin activation during thrombus formation on collagen. METHODS AND RESULTS: Blockade of glycoprotein (GP) VI by 9O12 antibody or of P2Y purinergic receptors permitted platelet adhesion but reduced aggregate formation, fibrinogen binding, and activation of alpha2beta1 and alphaIIbbeta3, as detected with antibodies IAC-1 and PAC1 directed against activation-dependent epitopes of these integrins. Combined blockade of GPVI and P2Y receptors and thromboxane formation abolished integrin activation but still allowed adhesion of morphologically unstimulated, nonprocoagulant platelets. Exogenous ADP partly restored the suppressive effect of GPVI blockade on integrin alpha2beta1 and alphaIIbbeta3 activation. Adhesion was fully inhibited only with simultaneous blocking of GPVI and alpha2beta1, indicating that the integrin can support platelet-collagen binding in the absence of its activation. Blockade or absence of GPIbalpha only moderately influenced integrin activation and adhesion unless GPVI was inhibited. CONCLUSIONS: GPVI- and autocrine-released ADP induce affinity changes of alpha2beta1 and alphaIIbbeta3 during thrombus formation on collagen under flow. These integrin changes are dispensable for adhesion but strengthen platelet-collagen interactions and thereby collagen-induced platelet activation. Integrin activation during thrombus formation on collagen was studied using fluorescent-labeled antibodies IAC-1 and PAC1 directed against activation-dependent epitopes of alpha2beta1 and alphaIIbbeta3 integrin, respectively. Glycoprotein VI blockade by 9O12 antibody or P2Y ADP receptors reduced integrin activation along with aggregate formation and fibrinogen binding but not alpha2beta1-dependent adhesion. [less ▲]Detailed reference viewed: 29 (2 ULg)
Identification of residues within human glycoprotein VI involved in the binding to collagen: evidence for the existence of distinct binding sites.
Lecut, Christelle ; ; et al
in Journal of Biological Chemistry (2004), 279(50), 52293-9
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 ... [more ▼]
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. [less ▲]Detailed reference viewed: 15 (6 ULg)
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: 13 (3 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: 34 (2 ULg)
Basic residues of human group IIA phospholipase A2 are important for binding to factor Xa and prothrombinase inhibition comparison with other mammalian secreted phospholipases A2.
; ; Lecut, Christelle et al
in European Journal of Biochemistry (2000), 267(16), 4960-9
Human secreted group IIA phospholipase A2 (hGIIA) was reported to inhibit prothrombinase activity because of binding to factor Xa. This study further shows that hGIIA and its catalytically inactive H48Q ... [more ▼]
Human secreted group IIA phospholipase A2 (hGIIA) was reported to inhibit prothrombinase activity because of binding to factor Xa. This study further shows that hGIIA and its catalytically inactive H48Q mutant prolong the lag time of thrombin generation in human platelet-rich plasma with similar efficiency, indicating that hGIIA exerts an anticoagulant effect independently of phospholipid hydrolysis under ex vivo conditions. Charge reversal of basic residues on the interfacial binding surface (IBS) of hGIIA leads to decreased ability to inhibit prothrombinase activity, which correlates with a reduced affinity for factor Xa, as determined by surface plasmon resonance. Mutation of other surface-exposed basic residues, hydrophobic residues on the IBS, and His48, does not affect the ability of hGIIA to inhibit prothrombinase activity and bind to factor Xa. Other basic, but not neutral or acidic, mammalian secreted phospholipases A2 (sPLA2s) exert a phospholipid-independent inhibitory effect on prothrombinase activity, suggesting that these basic sPLA2s also bind to factor Xa. In conclusion, this study demonstrates that the anticoagulant effect of hGIIA is independent of phospholipid hydrolysis and is based on its interaction with factor Xa, leading to prothrombinase inhibition, even under ex vivo conditions. This study also shows that such an interaction involves basic residues located on the IBS of hGIIA, and suggests that other basic mammalian sPLA2s may also inhibit blood coagulation by a similar mechanism to that described for hGIIA. [less ▲]Detailed reference viewed: 21 (4 ULg)