Neutrophil, NETs and Behçet's disease: A review.

Behçet's disease (BD) is a chronic systemic vasculitis characterized by recurrent oral and genital ulcers, skin lesions, articular, neurological, vascular and sight-threatening ocular inflammation. BD is thought to share both autoimmune and autoinflammatory disease features. BD is triggered by environmental factors such as infectious agents in genetically predisposed subjects. Neutrophils seem to play an instrumental role in BD and recent works regarding the role of neutrophils extracellular traps (NETs) provides new insight in the pathophysiology of BD and the mechanisms involved in immune thrombosis. This review provides a recent overview on the role of neutrophils and NETs in the pathogenesis of BD.

Targeting protease nexin-1, a natural anticoagulant serpin, to control bleeding and improve hemostasis in hemophilia.

Hemophilia A and B, diseases caused by the lack of factor VIII (FVIII) and factor IX (FIX) respectively, lead to insufficient thrombin production, and therefore to bleeding. New therapeutic strategies for hemophilia treatment that do not rely on clotting factor replacement, but imply the neutralization of natural anticoagulant proteins, have recently emerged. We propose an innovative approach consisting of targeting a natural and potent thrombin inhibitor, expressed by platelets, called protease nexin-1 (PN-1). By using the calibrated automated thrombin generation assay, we showed that a PN-1-neutralizing antibody could significantly shorten the thrombin burst in response to tissue factor in platelet-rich plasma (PRP) from patients with mild or moderate hemophilia. In contrast, in PRP from patients with severe hemophilia, PN-1 neutralization did not improve thrombin generation. However, after collagen-induced platelet activation, PN-1 deficiency in F8-/-mice or PN-1 blocking in patients with severe disease led to a significantly improved thrombin production in PRP, underlining the regulatory role of PN-1 released from platelet granules. In various bleeding models, F8-/-/PN-1-/- mice displayed significantly reduced blood loss and bleeding time compared with F8-/-mice. Moreover, platelet recruitment and fibrin(ogen) accumulation were significantly higher in F8-/-/PN-1-/- mice than in F8-/-mice in the ferric chloride-induced mesenteric vessel injury model. Thromboelastometry studies showed enhanced clot stability and lengthened clot lysis time in blood from F8-/-/PN-1-/- and from patients with hemophilia A incubated with a PN-1-neutralizing antibody compared with their respective controls. Our study thus provides proof of concept that PN-1 neutralization can be a novel approach for future clinical care in hemophilia.

Platelets and vascular integrity: how platelets prevent bleeding in inflammation.

Platelets play a central role in primary hemostasis by forming aggregates that plug holes in injured vessels. Half a century ago, detailed studies of the microvasculature by electron microscopy revealed that under inflammatory conditions that do not induce major disruption to vascular structure, individual platelets are mobilized to the vessel wall, where they interact with leukocytes and appear to seal gaps that arise between endothelial cells. Recent developments in genetic engineering and intravital microscopy have allowed further molecular and temporal characterization of these events. Surprisingly, it turns out that platelets support the recruitment of leukocytes to sites of inflammation. In parallel, however, they exercise their hemostatic function by securing the integrity of inflamed blood vessels to prevent bleeding from sites of leukocyte infiltration. It thus appears that platelets not only serve in concert as building blocks of the hemostatic plug but also act individually as gatekeepers of the vascular wall to help preserve vascular integrity while coordinating host defense. Variants of this recently appreciated hemostatic function of platelets that we refer to as "inflammation-associated hemostasis" are engaged in different contexts in which the endothelium is challenged or dysfunctional. Although the distinguishing characteristics of these variants and the underlying mechanisms of inflammation-associated hemostasis remain to be fully elucidated, they can differ notably from those supporting thrombosis, thus presenting therapeutic opportunities.

Functional redundancy between RAP1 isoforms in murine platelet production and function.

RAP GTPases, important regulators of cellular adhesion, are abundant signaling molecules in the platelet/megakaryocytic lineage. However, mice lacking the predominant isoform, RAP1B, display a partial platelet integrin activation defect and have a normal platelet count, suggesting the existence of a RAP1-independent pathway to integrin activation in platelets and a negligible role for RAP GTPases in megakaryocyte biology. To determine the importance of individual RAP isoforms on platelet production and on platelet activation at sites of mechanical injury or vascular leakage, we generated mice with megakaryocyte-specific deletion (mKO) of Rap1a and/or Rap1b Interestingly, Rap1a/b-mKO mice displayed a marked macrothrombocytopenia due to impaired proplatelet formation by megakaryocytes. In platelets, RAP isoforms had redundant and isoform-specific functions. Deletion of RAP1B, but not RAP1A, significantly reduced α-granule secretion and activation of the cytoskeleton regulator RAC1. Both isoforms significantly contributed to thromboxane A2 generation and the inside-out activation of platelet integrins. Combined deficiency of RAP1A and RAP1B markedly impaired platelet aggregation, spreading, and clot retraction. Consistently, thrombus formation in physiological flow conditions was abolished in Rap1a/b-mKO, but not Rap1a-mKO or Rap1b-mKO, platelets. Rap1a/b-mKO mice were strongly protected from experimental thrombosis and exhibited a severe defect in hemostasis after mechanical injury. Surprisingly, Rap1a/b-mKO platelets were indistinguishable from controls in their ability to prevent blood-lymphatic mixing during development and hemorrhage at sites of inflammation. In summary, our studies demonstrate an essential role for RAP1 signaling in platelet integrin activation and a critical role in platelet production. Although important for hemostatic/thrombotic plug formation, platelet RAP1 signaling is dispensable for vascular integrity during development and inflammation.

The reversed passive Arthus reaction as a model for investigating the mechanisms of inflammation-associated hemostasis.

In recent years, accumulating evidence has indicated that platelets continuously repair vascular damage at sites of inflammation and/or infection. Studies in mouse models of inflammation have highlighted the fact that the mechanisms underlying bleeding prevention by platelets in inflamed organs can substantially differ from those supporting primary hemostasis following tail tip transection or thrombus formation in models of thrombosis. As a consequence, exploration of the hemostatic function of platelets in inflammation, as well as assessment of the risk of inflammation-induced bleeding associated with a platelet deficit and/or the use of anti-thrombotic drugs, require the use of dedicated experimental models. In the present review, we present the pros and cons of the cutaneous reversed passive Arthus reaction, a model of inflammation which has been instrumental in studying how inflammation causes vascular injury and how platelets continuously intervene to repair it. The limitations and common issues encountered when working with mouse models of inflammation for investigating platelet functions in inflammation are also discussed.

Critical role of neutrophil extracellular traps (NETs) in patients with Behcet's disease.

OBJECTIVES: Behçet's disease (BD) is a chronic systemic vasculitis. Thrombosis is a frequent and life-threatening complication. The pathogenesis of BD is poorly understood and evidence supporting a role for primed neutrophils in BD-associated thrombotic risk is scant. To respond to inflammatory insults, neutrophils release web-like structures, known as neutrophil extracellular traps (NETs), which are prothrombotic. We evaluated the role of NETs and markers of NETs in BD. METHODS: Blood samples were collected from patients with BD, according to the International Study Group Criteria for Behçet's disease, and healthy donors (HD). NET components, including cell-free DNA (CfDNA) and neutrophil enzymes myeloperoxidase (MPO), were assessed in serum or in purified neutrophils from patients with BD and HD. RESULTS: Patients with active BD had elevated serum cfDNA levels and MPO-DNA complexes compared with patients with inactive BD and to HD. In addition, levels of cfDNA and MPO-DNA complexes were significantly higher in patients with BD with vascular involvement compared with those without vascular symptoms. Purified neutrophils from patients with BD exhibited spontaneous NETosis compared with HD. Thrombin generation in BD plasma was significantly increased and positively correlated with the levels of MPO-DNA complexes and cfDNA. Importantly, DNAse treatment significantly decreased thrombin generation in BD plasma but not in HD plasma. In addition, biopsy materials obtained from patients with BD showed NETs production in areas of vasculitic inflammation and thrombosis. CONCLUSIONS: Our data show that NETs and markers of NETS levels are elevated in patients with BD and contribute to the procoagulant state. Targeting NETs may represent a potential therapeutic target for the reduction or prevention of BD-associated thrombotic risk.

Vascular endothelial cell expression of JAK2V617F is sufficient to promote a pro-thrombotic state due to increased P-selectin expression.

Thrombosis is the main cause of morbidity and mortality in patients with JAK2V617F myeloproliferative neoplasms. Recent studies have reported the presence of JAK2V617F in endothelial cells of some patients with myeloproliferative neoplasms. We investigated the role of endothelial cells that express JAK2V617F in thrombus formation using an in vitro model of human endothelial cells overexpressing JAK2V617F and an in vivo model of mice with endothelial-specific JAK2V617F expression. Interestingly, these mice displayed a higher propensity for thrombus. When deciphering the mechanisms by which JAK2V617F-expressing endothelial cells promote thrombosis, we observed that they have a pro-adhesive phenotype associated with increased endothelial P-selectin exposure, secondary to degranulation of Weibel-Palade bodies. We demonstrated that P-selectin blockade was sufficient to reduce the increased propensity of thrombosis. Moreover, treatment with hydroxyurea also reduced thrombosis and decreased the pathological interaction between leukocytes and JAK2V617F-expressing endothelial cells through direct reduction of endothelial P-selectin expression. Taken together, our data provide evidence that JAK2V617F-expressing endothelial cells promote thrombosis through induction of endothelial P-selectin expression, which can be reversed by hydroxyurea. Our findings increase our understanding of thrombosis in patients with myeloproliferative neoplasms, at least those with JAK2V617F-positive endothelial cells, and highlight a new role for hydroxyurea. This novel finding provides the proof of concept that an acquired genetic mutation can affect the pro-thrombotic nature of endothelial cells, suggesting that other mutations in endothelial cells could be causal in thrombotic disorders of unknown cause, which account for 50% of recurrent venous thromboses.

Microfluidic Modeling of Thrombolysis.

Objective- Despite the high clinical relevance of thrombolysis, models for its study in human flowing blood are lacking. Our objective was to develop a microfluidic model for comparative evaluation of thrombolytic therapeutic strategies. Approach and Results- Citrated human blood was supplemented with 3,3'-dihexyloxacarbocyanine iodide and Alexa Fluor 647 fibrinogen conjugate, recalcified, and perfused for 3 to 4 minutes at venous or arterial wall shear rate in microfluidic flow chambers coated with collagen and tissue factor to generate nonocclusive fluorescent thrombi. A second perfusion was performed for 10 minutes with rhodamine-6G-labeled citrated whole blood, supplemented or not with r-tPA (recombinant tissue-type plasminogen activator), fluorescein isothiocyanate-conjugated r-tPA, and Alexa Fluor 568 plasminogen conjugate. Plasminogen and r-tPA bound to preformed thrombi and r-tPA caused a concentration-dependent decrease in thrombus fibrin content (up to 50% reduction at 15 µg/mL r-tPA) as assessed by fluorescence microscopy. Fibrinolysis was confirmed by measurement of D-dimers in the output flow. Remarkably, despite ongoing fibrinolysis, new platelets continued to be recruited to the thrombus under lysis. Under the arterial condition, combining r-tPA with hirudin enhanced fibrinolysis but did not prevent the recruitment of new platelets, which was, however, prevented by antiplatelet agents (ticagrelor or the GPVI [glycoprotein VI]-blocking antigen-binding fragment 9O12). Conclusions- Our microfluidic thrombolysis model is suitable for studying thrombolysis and testing the efficacy of drugs used in combination with r-tPA. Real-time analysis of fibrin and platelets during r-tPA-mediated fibrinolysis at arterial or venous flow conditions showed that platelets continue to accumulate during fibrinolysis. Such platelet accumulation may impair r-tPA-mediated recanalization.

CalDAG-GEFI Deficiency Reduces Atherosclerotic Lesion Development in Mice.

OBJECTIVE: Platelets are important for the development and progression of atherosclerotic lesions. However, relatively little is known about the contribution of platelet signaling to this pathological process. Our recent work identified 2 independent, yet synergistic, signaling pathways that lead to the activation of the small GTPase Rap1; one mediated by the guanine nucleotide exchange factor, CalDAG-GEFI (CDGI), the other by P2Y12, a platelet receptor for adenosine diphosphate and the target of antiplatelet drugs. In this study, we evaluated lesion formation in atherosclerosis-prone low-density lipoprotein receptor deficient (Ldlr(-/-)) mice lacking CDGI or P2Y12 in hematopoietic cells. APPROACH AND RESULTS: Lethally irradiated Ldlr(-/-) mice were reconstituted with bone marrow from wild-type (WT), Caldaggef1(-/-) (cdgI(-/-)), p2y12(-/-), or cdgI(-/-)p2y12(-/-) (double knockout [DKO]) mice and fed a high-fat diet for 12 weeks. Ldlr(-/-) chimeras deficient for CDGI or P2Y12 developed significantly smaller atherosclerotic lesions in the aortic sinus and in aortas when compared with the Ldlr(-/-)/WT controls. We also observed a significant reduction in platelet-leukocyte aggregates in blood from hypercholesterolemic Ldlr(-/-)/cdgI(-/-) and Ldlr(-/-)/p2y12(-/-) chimeras. Consistently, fewer macrophages and neutrophils were detected in the aortic sinus of Ldlr(-/-)/cdgI(-/-) and Ldlr(-/-)/ p2y12(-/-) chimeras. Compared with controls, the plaque collagen content was significantly higher in Ldlr(-/-) chimeras lacking CDGI. Interestingly, no statistically significant additive effects were seen in Ldlr(-/-)/DKO chimeras when compared with chimeras lacking only CDGI. CONCLUSIONS: Our findings suggest that CDGI is critical for atherosclerotic plaque development in hypercholesterolemic Ldlr(-/-) mice because of its contribution to platelet-leukocyte aggregate formation and leukocyte recruitment to the lesion area.

Platelet immunoreceptor tyrosine-based activation motif (ITAM) signaling and vascular integrity.

Platelets are well-known for their critical role in hemostasis, that is, the prevention of blood loss at sites of mechanical vessel injury. Inappropriate platelet activation and adhesion, however, can lead to thrombotic complications, such as myocardial infarction and stroke. To fulfill its role in hemostasis, the platelet is equipped with various G protein-coupled receptors that mediate the response to soluble agonists such as thrombin, ADP, and thromboxane A2. In addition to G protein-coupled receptors, platelets express 3 glycoproteins that belong to the family of immunoreceptor tyrosine-based activation motif receptors: Fc receptor γ chain, which is noncovalently associated with the glycoprotein VI collagen receptor, C-type lectin 2, the receptor for podoplanin, and Fc receptor γII A, a low-affinity receptor for immune complexes. Although both genetic and chemical approaches have documented a critical role for platelet G protein-coupled receptors in hemostasis, the contribution of immunoreceptor tyrosine-based activation motif receptors to this process is less defined. Studies performed during the past decade, however, have identified new roles for platelet immunoreceptor tyrosine-based activation motif signaling in vascular integrity in utero and at sites of inflammation. The purpose of this review is to summarize recent findings on how platelet immunoreceptor tyrosine-based activation motif signaling controls vascular integrity, both in the presence and absence of mechanical injury.

Platelet Inhibitors Reduce Rupture in a Mouse Model of Established Abdominal Aortic Aneurysm.

OBJECTIVE: Rupture of abdominal aortic aneurysms causes a high morbidity and mortality in the elderly population. Platelet-rich thrombi form on the surface of aneurysms and may contribute to disease progression. In this study, we used a pharmacological approach to examine a role of platelets in established aneurysms induced by angiotensin II infusion into hypercholesterolemic mice. APPROACH AND RESULTS: Administration of the platelet inhibitors aspirin or clopidogrel bisulfate to established abdominal aortic aneurysms dramatically reduced rupture. These platelet inhibitors reduced abdominal aortic platelet and macrophage recruitment resulting in decreased active matrix metalloproteinase-2 and matrix metalloproteinase-9. Platelet inhibitors also resulted in reduced plasma concentrations of platelet factor 4, cytokines, and components of the plasminogen activation system in mice. To determine the validity of these findings in human subjects, a cohort of aneurysm patients were retrospectively analyzed using developed and validated algorithms in the electronic medical record database at Vanderbilt University. Similar to mice, administration of aspirin or P2Y12 inhibitors was associated with reduced death among patients with abdominal aortic aneurysm. CONCLUSIONS: These results suggest that platelets contribute to abdominal aortic aneurysm progression and rupture.

Protease nexin-1 regulates retinal vascular development.

We recently identified protease nexin-1 (PN-1) or serpinE2, as a possibly underestimated player in maintaining angiogenic balance. Here, we used the well-characterized postnatal vascular development of newborn mouse retina to further investigate the role and the mechanism of action of PN-1 in physiological angiogenesis. The development of retinal vasculature was analysed by endothelial cell staining with isolectin B4. PN-1-deficient (PN-1(-/-)) retina displayed increased vascularization in the postnatal period, with elevated capillary thickness and density, compared to their wild-type littermate (WT). Moreover, PN-1(-/-) retina presented more veins/arteries than WT retina. The kinetics of retinal vasculature development, retinal VEGF expression and overall retinal structure were similar in WT and PN-1(-/-) mice, but we observed a hyperproliferation of vascular cells in PN-1(-/-) retina. Expression of PN-1 was analysed by immunoblotting and X-Gal staining of retinas from mice expressing beta-galactosidase under a PN-1 promoter. PN-1 was highly expressed in the first week following birth and then progressively decreased to a low level in adult retina where it localized on the retinal arteries. PCR arrays performed on mouse retinal RNA identified two angiogenesis-related factors, midkine and Smad5, that were overexpressed in PN-1(-/-) newborn mice and this was confirmed by RT-PCR. Both the higher vascularization and the overexpression of midkine and Smad5 mRNA were also observed in gastrocnemius muscle of PN-1(-/-) mice, suggesting that PN-1 interferes with these pathways. Together, our results demonstrate that PN-1 strongly limits physiological angiogenesis and suggest that modulation of PN-1 expression could represent a new way to regulate angiogenesis.

Emerging role of serpinE2/protease nexin-1 in hemostasis and vascular biology.

Serine protease inhibitors, termed serpins, are key regulators in many biologic events. Protease nexin-1 (PN-1) is a serpin that is barely detectable in plasma but found in many organs and produced by most cell types, including monocytes, platelets, and vascular cells. It has a large inhibition spectrum because it is the most efficient tissue inhibitor of thrombin but also a powerful inhibitor of plasminogen activators and plasmin. It has a high affinity for glycosaminoglycans, such as heparan sulfates, which potentiate its activity toward thrombin and target it to the pericellular space. PN-1 has been previously largely described as a crucial regulator of the proteolytic activity in nerves and of central and peripheral nervous system function. In contrast, little was known about its involvement in hemostasis and vascular biology. This article reviews recent data underlining its emerging role as a key factor in the responses of vessels to injury. Indeed, studies of PN-1-deficient mice have established important antithrombotic and antifibrinolytic properties of this serpin that have heretofore gone unrecognized. The roles of PN-1 in the areas of hemostasis and thrombosis summarized here provide insights that may allow the development of drugs and treatment strategies to prevent or limit thrombotic disorders.

Endothelial protease nexin-1 is a novel regulator of A disintegrin and metalloproteinase 17 maturation and endothelial protein C receptor shedding via furin inhibition.

OBJECTIVE: Human protein C is a plasma serine protease that plays a key role in hemostasis, and activated protein C (aPC) is known to elicit protective responses in vascular endothelial cells. This cytoprotective activity requires the interaction of the protease with its cell membrane receptor, endothelial protein C receptor. However, the mechanisms regulating the beneficial cellular effects of aPC are not well known. We aimed to determine whether a serine protease inhibitor called protease nexin-1 (PN-1) or serpinE2, expressed by vascular cells, can modulate the effect of aPC on endothelial cells. APPROACH AND RESULTS: We found that vascular barrier protective and antiapoptotic activities of aPC were reduced both in endothelial cells underexpressing PN-1 and in endothelial cells whose PN-1 function was blocked by a neutralizing antibody. Our in vitro data were further confirmed in vivo. Indeed, we found that vascular endothelial growth factor-mediated hyperpermeability in the skin of mice was markedly reduced by local intradermal injection of aPC in wild-type mice but not in PN-1-deficient mice. Furthermore, we demonstrated a previously unknown protective role of endothelial PN-1 on endothelial protein C receptor shedding. We provided evidence that PN-1 inhibits furin, a serine protease that activates a disintegrin and metalloproteinase 17 involved in the shedding of endothelial protein C receptor. We indeed evidenced a direct interaction between PN-1 and furin in endothelial cells. CONCLUSIONS: Our results thus demonstrate an original role of PN-1 as a furin convertase inhibitor, providing new insights for understanding the regulation of endothelial protein C receptor-dependent aPC endothelial protective effects.

Platelets and neutrophils cooperate to induce increased neutrophil extracellular trap formation in JAK2V617F myeloproliferative neoplasms.

BACKGROUND: Neutrophils participate in the pathogenesis of thrombosis through the formation of neutrophil extracellular traps (NETs). Thrombosis is the main cause of morbidity and mortality in patients with myeloproliferative neoplasms (MPNs). Recent studies have shown an increase in NET formation (NETosis) both in patients with JAK2V617F neutrophils and in mouse models, and reported the participation of NETosis in the pathophysiology of thrombosis in mice. OBJECTIVES: This study investigated whether JAK2V617F neutrophils are sufficient to promote thrombosis or whether their cooperation with other blood cell types is necessary. METHODS: NETosis was studied in PF4iCre;Jak2V617F/WT mice expressing JAK2V617F in all hematopoietic lineages, as occurs in MPNs, and in MRP8Cre;Jak2V617F/WT mice in which JAK2V617F is expressed only in leukocytes. RESULTS: In PF4iCre;Jak2V617F/WT mice, an increase in NETosis and spontaneous lung thrombosis abrogated by DNAse administration were observed. The absence of spontaneous NETosis or lung thrombosis in MRP8Cre;Jak2V617F/WT mice suggested that mutated neutrophils alone are not sufficient to induce thrombosis. Ex vivo experiments demonstrated that JAK2V617F-mutated platelets trigger NETosis by JAK2V617F-mutated neutrophils. Aspirin treatment in PF4iCre;Jak2V617F/WT mice reduced NETosis and reduced lung thrombosis. In cytoreductive-therapy-free patients with MPN treated with aspirin, plasma NET marker concentrations were lower than that in patients with MPN not treated with aspirin. CONCLUSION: Our study demonstrates that JAK2V617F neutrophils alone are not sufficient to promote thrombosis; rather, platelets cooperate with neutrophils to promote NETosis in vivo. A new role for aspirin in thrombosis prevention in MPNs was also identified.

High factor VIII concentrations interfere with glycoprotein VI-mediated platelet activation in vitro.

BACKGROUND: The recruitment of activated factor VIII (FVIII) at the surface of activated platelets is a key step toward the burst of thrombin and fibrin generation during thrombus formation at the site of vascular injury. It involves binding to phosphatidylserine and, possibly, to fibrin-bound αIIbß3. Seminal work had shown the binding of FVIII to resting platelets, yet without a clear understanding of a putative physiological relevance. OBJECTIVES: To characterize the effects of FVIII-platelet interaction and its potential modulation of platelet function. METHODS: FVIII was incubated with washed platelets. The effects on platelet activation (spontaneously or triggered by collagen and thrombin) were studied by flow cytometry and light transmission aggregometry. We explored the involvement of downstream pathways by studying phosphorylation profiles (Western blot). The FVIII-glycoprotein (GP) VI interaction was investigated by ELISA, confocal microscopy, and proximity ligation assay. RESULTS: FVIII bound to the surface of resting and activated platelets in a dose-dependent manner. FVIII at supraphysiological concentrations did not induce platelet activation but rather specifically inhibited collagen-induced platelet aggregation and altered glycoprotein VI (GPVI)-dependent phosphorylation. FVIII, freed of its chaperone protein von Willebrand factor (VWF), interacted in close proximity with GPVI at the platelet surface. CONCLUSION: We showed that VWF-free FVIII binding to, or close to, GPVI modulates platelet activation in vitro. This may represent an uncharacterized negative feedback loop to control overt platelet activation. Whether locally activated FVIII concentrations achieved during platelet accumulation and thrombus formation at the site of vascular injury in vivo are compatible with such a function remains to be determined.

The localization, origin, and impact of platelets in the tumor microenvironment are tumor type-dependent.

BACKGROUND: How platelets interact with and influence the tumor microenvironment (TME) remains poorly characterized. METHODS: We compared the presence and participation of platelets in the TME of two tumors characterized by highly different TME, PyMT AT-3 mammary tumors and B16F1 melanoma. RESULTS: We show that whereas firmly adherent platelets continuously line tumor vessels of both AT-3 and B16F1 tumors, abundant extravascular stromal clusters of platelets from thrombopoietin-independent origin were present only in AT-3 mammary tumors. We further show that platelets influence the angiogenic and inflammatory profiles of AT-3 and B16F1 tumors, though with very different outcomes according to tumor type. Whereas thrombocytopenia increased bleeding in both tumor types, it further caused severe endothelial degeneration associated with massive vascular leakage, tumor swelling, and increased infiltration of cytotoxic cells, only in AT-3 tumors. CONCLUSIONS: These results indicate that while platelets are integral components of solid tumors, their localization and origin in the TME, as well as their impact on its shaping, are tumor type-dependent.

Platelet glycoprotein VI dimerization, an active process inducing receptor competence, is an indicator of platelet reactivity.

OBJECTIVE: The immune receptor homologue glycoprotein VI (GPVI)/FcR receptor γ chain complex is primarily responsible for platelet activation by collagen. There is growing evidence that optimal binding of GPVI to collagen depends on the assembly of GPVI dimers. The valence of GPVI on resting platelets needs to be clearly established because platelet avidity for collagen would be greater if GPVI is constitutively expressed as a dimer than as a monomer. METHODS AND RESULTS: Using a monoclonal antibody (9E18) that preferentially binds to GPVI dimers, we found that GPVI was maintained in a monomeric form on human resting platelets under the control of intraplatelet cAMP concentration. Activation by soluble agonists or von Willebrand factor induced a shift toward GPVI dimerization related to increased platelet adhesion to collagen. A correlation between platelet binding of 9E18 and P-selectin exposure was observed in patients experiencing coronary artery disease, and antagonists of the ADP receptor P2Y12 limited ADP-induced GPVI dimerization. CONCLUSION: The rapid assembly of highly competent dimers of GPVI at sites of vascular lesion represents an important step in the sequence of events leading to platelet activation by collagen. GPVI dimers could represent a new marker to analyze platelet reactivity.

Rap1-Rac1 circuits potentiate platelet activation.

OBJECTIVE: The goal of this study was to investigate the potential crosstalk between Rap1 and Rac1, 2 small GTPases central to platelet activation, particularly downstream of the collagen receptor GPVI. METHODS AND RESULTS: We compared the activation response of platelets with impaired Rap signaling (double knock-out; deficient in both the guanine nucleotide exchange factor, CalDAG-GEFI, and the Gi-coupled receptor for ADP, P2Y12), to that of wild-type platelets treated with a small-molecule Rac inhibitor, EHT 1864 (wild-type /EHT). We found that Rac1 is sequentially activated downstream of Rap1 on stimulation via GPVI. In return, Rac1 provides important feedback for both CalDAG-GEFI- and P2Y12-dependent activation of Rap1. When analyzing platelet responses controlled by Rac1, we observed (1) impaired lamellipodia formation, clot retraction, and granule release in both double knock-out and EHT 1864-treated wild-type platelets; and (2) reduced calcium store release in EHT 1864-treated wild-type but not double knock-out platelets. Consistent with the latter finding, we identified 2 pools of Rac1, one activated immediately downstream of GPVI and 1 activated downstream of Rap1. CONCLUSIONS: We demonstrate important crosstalk between Rap1 and Rac1 downstream of GPVI. Whereas Rap1 signaling directly controls sustained Rac1 activation, Rac1 affects CalDAG-GEFI- and P2Y12-dependent Rap1 activation via its role in calcium mobilization and granule/ADP release, respectively.