Targeting of C-type lectin-like receptor 2 or P2Y12 for the prevention of platelet activation by immunotherapeutic CpG oligodeoxynucleotides.

Essentials CpG oligodeoxynucleotide (ODN) immuotherapeutics cause undesired platelet activating effects. It is crucial to understand the mechanisms of these effects to identify protective strategies. CpG ODN-induced platelet activation depends on C-type lectin-like receptor 2 (CLEC-2) and P2Y12. Targeting CLEC-2 or P2Y12 fully prevents CpG ODN-induced platelet activation and thrombosis. SUMMARY: Background Synthetic phosphorothioate-modified CpG oligodeoxynucleotides (ODNs) show potent immunostimulatory properties that are widely exploited in clinical trials of anticancer treatment. Unexpectedly, a recent study indicated that CpG ODNs activate human platelets via the immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptor glycoprotein VI. Objective To further analyze the mechanisms of CpG ODN-induced platelet activation and identify potential inhibitory strategies. Methods In vitro analyses were performed on human and mouse platelets, and on cell lines expressing platelet ITAM receptors. CpG ODN platelet-activating effects were evaluated in a mouse model of thrombosis. Results We demonstrated platelet uptake of CpG ODNs, resulting in platelet activation and aggregation. C-type lectin-like receptor 2 (CLEC-2) expressed in DT40 cells bound CpG ODNs. CpG ODN uptake did not occur in CLEC-2-deficient mouse platelets. Inhibition of human CLEC-2 with a blocking antibody inhibited CpG ODN-induced platelet aggregation. CpG ODNs caused CLEC-2 dimerization, and provoked its internalization. They induced dense granule release before the onset of aggregation. Accordingly, pretreating platelets with apyrase, or inhibiting P2Y12 with cangrelor or clopidogrel, prevented CpG ODN platelet-activating effect. In vivo, intravenously injected CpG ODN interacted with platelets adhered to mouse injured endothelium, and promoted thrombus growth, which was inhibited by CLEC-2 deficiency or by clopidogrel. Conclusions CLEC-2 and P2Y12 are required for CpG ODN-induced platelet activation and thrombosis, and might be targeted to prevent adverse events in patients at risk.

The Ca(2+) /calmodulin-dependent kinase kinase ß-AMP-activated protein kinase-α1 pathway regulates phosphorylation of cytoskeletal targets in thrombin-stimulated human platelets.

BACKGROUND: Platelet activation requires sweeping morphologic changes, supported by contraction and remodeling of the platelet actin cytoskeleton. In various other cell types, AMP-activated protein kinase (AMPK) controls the phosphorylation state of cytoskeletal targets. OBJECTIVE: To determine whether 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 Ca(2+) /calmodulin-dependent kinase kinase ß (CaMKKß)-dependent pathway. Pharmacologic inhibition of CaMKKß blocked thrombin-induced platelet aggregation and counteracted thrombin-induced phosphorylation of several cytoskeletal proteins, namely, regulatory myosin light chains (MLCs), cofilin, and vasodilator-stimulated phosphoprotein (VASP), three key elements involved in actin cytoskeletal contraction and polymerization. Platelets isolated from mice lacking AMPK-α1 showed reduced aggregation in response to thrombin, and this was associated with defects in MLC, cofilin and VASP phosphorylation and actin polymerization. More importantly, we show, for the first time, that the AMPK pathway is 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 the phosphorylation of key cytoskeletal targets and actin cytoskeletal remodeling during platelet aggregation.

ATP-gated P2X1 ion channels protect against endotoxemia by dampening neutrophil activation.

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 P2X(1) ion channels are involved in promoting neutrophil chemotaxis. OBJECTIVES: To elucidate the contribution of P2X(1) ion channels to sepsis and the associated disturbance of hemostasis. METHODS: We used P2X(1) (-/-) mice in a model of lipopolysaccharide (LPS)-induced sepsis. Hemostasis and inflammation parameters were analyzed together with outcome. Mechanisms were further studied ex vivo with mouse and human blood or isolated neutrophils and monocytes. RESULTS: P2X(1) (-/-) 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 P2X(1). However, whole blood platelet aggregation occurred normally, and P2X(1) (-/-) macrophages displayed normal levels of total tissue factor activity. We found that P2X(1) (-/-) neutrophils produced higher amounts of reactive oxygen species. Increased amounts of myeloperoxidase were released in the blood of LPS-treated P2X(1) (-/-) mice, and circulating neutrophils and monocytes expressed higher levels of CD11b. Neutrophil accumulation in the lungs was also significantly augmented, as was lipid peroxidation in the liver. Desensitization of P2X(1) ion channels led to increased activation of human neutrophils and enhanced formation of platelet-leukocyte aggregates. CONCLUSIONS: P2X(1) ion channels play a protective role in endotoxemia by negatively regulating systemic neutrophil activation, thereby limiting the oxidative response, coagulation, and organ damage.

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

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.

Expression and function of the collagen receptor GPVI during megakaryocyte maturation.

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.

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.

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.