Overexpression of CD39 in mouse airways promotes bacteria induced inflammation

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 ▲]

Lack of P2X1 ion channels increases endotoxemia associated coagulation and organ damage through neutrophil hyperresponsiveness.

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 ▲]

Contribution of ATP-gated P2X1 ion channels to the control of neutrophil chemotaxis.

in Purinergic Signalling (2008), 4

New role for ATP P2X1 receptors in the control of neutrophil apoptosis and respiratory burst activity.

in Blood (2006), 108(11, Part 1), 1647

Fibrillar type I collagens enhance platelet-dependent thrombin generation via glycoprotein VI with direct support of alpha2beta1 but not alphaIIbbeta3 integrin.

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 ▲]

Principal role of glycoprotein VI in alpha2beta1 and alphaIIbbeta3 activation during collagen-induced thrombus formation.

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 ▲]

Human platelet glycoprotein VI function is antagonized by monoclonal antibody-derived Fab fragments.

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 ▲]

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.

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 ▲]