Nicolas Receveur

Integrin α6β1 Is the Main Receptor for Vascular Laminins and Plays a Role in Platelet Adhesion, Activation, and Arterial Thrombosis

Background— Laminins are major components of basement membranes, well located to interact with platelets upon vascular injury. Laminin-111 (&agr;1&bgr;1&ggr;1) is known to support platelet adhesion but is absent from most blood vessels, which contain isoforms with the &agr;2, &agr;4, or &agr;5 chain. Whether vascular laminins support platelet adhesion and activation and the significance of these interactions in hemostasis and thrombosis remain unknown. Methods and Results— Using an in vitro flow assay, we show that laminin-411 (&agr;4&bgr;1&ggr;1), laminin-511 (&agr;5&bgr;1&ggr;1), and laminin-521 (&agr;5&bgr;2&ggr;1), but not laminin-211 (&agr;2&bgr;1&ggr;1), allow efficient platelet adhesion and activation across a wide range of arterial wall shear rates. Adhesion was critically dependent on integrin &agr;6&bgr;1 and the glycoprotein Ib-IX complex, which binds to plasmatic von Willebrand factor adsorbed on laminins. Glycoprotein VI did not participate in the adhesive process but mediated platelet activation induced by &agr;5-containing laminins. To address the significance of platelet/laminin interactions in vivo, we developed a platelet-specific knockout of integrin &agr;6. Platelets from these mice failed to adhere to laminin-411, laminin-511, and laminin-521 but responded normally to a series of agonists. &agr;6&bgr;1-Deficient mice presented a marked decrease in arterial thrombosis in 3 models of injury of the carotid, aorta, and mesenteric arterioles. The tail bleeding time and blood loss remained unaltered, indicating normal hemostasis. Conclusions— This study reveals an unsuspected important contribution of laminins to thrombus formation in vivo and suggests that targeting their main receptor, integrin &agr;6&bgr;1, could represent an alternative antithrombotic strategy with a potentially low bleeding risk.

Novel Function of Tenascin-C, a Matrix Protein Relevant to Atherosclerosis, in Platelet Recruitment and Activation Under Flow

Objective—The identification of platelet-reactive proteins exclusively present in atherosclerotic plaques could provide interesting targets for effective and safe antithrombotic strategies. In this context, we explored platelet adhesion and activation to tenascin-C (TN-C), a matrix protein preferentially found within atheroma. Methods and Results—We show that platelets efficiently adhere to TN-C under both static and flow conditions. Videomicroscopy revealed a unique behavior under flow, with platelets exhibiting stationary adhesion to TN-C; in contrast, platelets rolled over von Willebrand factor and detached from fibrinogen. Platelet interaction with TN-C was predominantly supported by integrin &agr;2&bgr;1 under static conditions, whereas under high shear, it was dependent on both the &agr;2&bgr;1 integrin and the glycoprotein Ib-IX complex. Integrin &agr;IIb&bgr;3 appeared to play a secondary role but only at low shear rates. The glycoprotein Ib-IX–dependent interaction was indirect, relying on von Willebrand factor, and increased as a function of wall shear rate. Von Willebrand factor bound directly to TN-C, as shown by ELISA and coimmunoprecipitation, suggesting that it acts as a bridge between TN-C and platelets. The adhesion of platelets to TN-C triggered their activation, as demonstrated by a shape change and increases in intracellular calcium level. Conclusion—This study provides evidence that TN-C serves as a novel adhesive matrix for platelets in a context that is relevant to atherothrombosis.

Identification of five novel 14-3-3 isoforms interacting with the GPIb-IX complex in platelets.

Summary.  Background: Binding of von Willebrand factor to the platelet glycoprotein (GP)Ib–IX complex initiates a signaling cascade leading to integrin αIIbβ3 activation, a key process in hemostasis and thrombosis. Interaction of 14‐3‐3ζ with the intracytoplasmic domain of GPIb appears to be a major effector of this activation pathway. Objective: The aim of our study was to determine whether other members of the 14‐3‐3 family bind to GPIb–IX. Results: In this study, western blot analyses showed that platelets also contain the 14‐3‐3β, 14‐3‐3γ, 14‐3‐3ε, 14‐3‐3η and 14‐3‐3θ isoforms, but lack 14‐3‐3σ. Coimmunoprecipitation studies in platelets and CHO transfectants demonstrated that all six 14‐3‐3 isoforms expressed in platelets, including, as previously reported, 14‐3‐3ζ, bind to GPIb–IX. In addition, their interaction was found to critically require the same GPIbα domains (580–590 and 605–610) already identified as essential for 14‐3‐3ζ binding, in agreement with the conservation of the sequence of the I‐helix among these different isoforms. Pull‐down experiments indicated that all six 14‐3‐3 isoforms present in platelets bind to GPIbβ. In contrast, deletion or mutation of the GPIbβ intracytoplasmic tail did not affect the interaction of GPIb–IX with the 14‐3‐3 isoforms, questioning the importance of this domain. Conclusions: Our study suggests that, to inhibit GPIb‐induced integrin αIIbβ3 activation, a more appropriate strategy than inhibiting individual 14‐3‐3 isoforms would be to target the 14‐3‐3‐binding motif on GPIb or, alternatively, the conserved 14‐3‐3 I‐helix.

Aryl hydrocarbon receptor–dependent enrichment of a megakaryocytic precursor with a high potential to produce proplatelets

The mechanisms regulating megakaryopoiesis and platelet production (thrombopoiesis) are still incompletely understood. Identification of a progenitor with enhanced thrombopoietic capacity would be useful to decipher these mechanisms and to improve our capacity to produce platelets in vitro. Differentiation of peripheral blood CD34(+) cells in the presence of bone marrow-human mesenchymal stromal cells (MSCs) enhanced the production of proplatelet-bearing megakaryocytes (MKs) and platelet-like elements. This was accompanied by enrichment in a MK precursor population exhibiting an intermediate level of CD41 positivity while maintaining its expression of CD34. Following sorting and subculture with MSCs, this CD34(+)CD41(low) population was able to efficiently generate proplatelet-bearing MKs and platelet-like particles. Similarly, StemRegenin 1 (SR1), an antagonist of the aryl hydrocarbon receptor (AhR) transcription factor known to maintain CD34 expression of progenitor cells, led to an enriched CD34(+)CD41(low) fraction and to an increased capacity to generate proplatelet-producing MKs and platelet-like elements ultrastructurally and functionally similar to circulating platelets. The effect of MSCs, like that of SR1, appeared to be mediated by an AhR-dependent mechanism because both culture conditions resulted in repression of its downstream effector CYP1B1. This newly described isolation of a precursor exhibiting strong MK potential could be exploited to study normal and abnormal thrombopoiesis and for in vitro platelet production.

Targeting Platelet GPIbβ Reduces Platelet Adhesion, GPIb Signaling and Thrombin Generation and Prevents Arterial Thrombosis

Objective—The glycoprotein (GP) Ib-V-IX complex regulates the adhesion, activation, and procoagulant activity of platelets. We previously reported that RAM.1, a rat monoclonal antibody directed against the extracellular domain of mouse GPIb&bgr;, diminished adhesion of platelets and chinese hamster ovary cells transfected with the human GPIb-IX complex to von Willebrand factor under flow conditions. Here, we further evaluated the functional importance of GPIb&bgr; by studying the impact of RAM.1 on GPIb-mediated platelet responses and in vitro and in vivo thrombus formation. Approach and Results—We show that RAM.1 dramatically reduced GPIb-mediated filopodia extension of chinese hamster ovary GPIb-IX cells after adhesion to von Willebrand factor. RAM.1 also reduced filopodia extension and GPIb-mediated Ca2+ signaling after adhesion of mouse platelets to von Willebrand factor. RAM.1 inhibited thrombin generation in platelet-rich plasma without impairing phosphatidylserine exposure. In addition, RAM.1 reduced thrombus formation after perfusion of mouse whole blood over collagen in a shear-dependent manner. This effect was confirmed in vivo, because injection of F(ab)′2 fragments of RAM.1 diminished thrombus formation induced by laser beam injury of mesenteric arterioles and forceps injury of the abdominal aorta. In contrast, RAM.1 F(ab)′2 did not prolong the tail-bleeding time or increase the volume of blood lost. Conclusions—These findings are the first evidence that targeting a subunit other than GPIb&agr; can lead to an antithrombotic effect via the GPIb-V-IX complex. This could represent an alternative way to reduce thrombus formation with a minor impact on hemostasis.

Fibrillar cellular fibronectin supports efficient platelet aggregation and procoagulant activity.

The ability of cellular fibronectin, found in the vessel wall in a fibrillar conformation, to regulate platelet functions and trigger thrombus formation remains largely unknown. In this study, we evaluated how parietal cellular fibronectin can modulate platelet responses under flow conditions. A fibrillar network was formed by mechanically stretching immobilised dimeric cellular fibronectin. Perfusion of anticoagulated whole blood over this surface resulted in efficient platelet adhesion and thrombus growth. The initial steps of platelet adhesion and activation, as evidenced by filopodia extension and an increase in intracellular calcium levels (419 ± 29 nmol/l), were dependent on integrins α5β1 and αIIbβ3. Subsequent thrombus growth was mediated by these integrins together with the GPIb-V-IX complex, GPVI and Toll-like receptor 4. The involvement of Toll-like receptor 4 could be conveyed via its binding to the EDA region of cellular fibronectin. Upon thrombus formation, the platelets became procoagulant and generated fibrin as revealed by video-microscopy. This work provides evidence that fibrillar cellular fibronectin is a strong thrombogenic surface which supports efficient platelet adhesion, activation, aggregation and procoagulant activity through the interplay of a series of receptors including integrins α5β1 and αIIbβ3, the GPIb-V-IX complex, GPVI and Toll-like receptor 4.

β-arrestin-1 participates in thrombosis and regulates integrin aIIbβ3 signalling without affecting P2Y receptors desensitisation and function

β-arrestin-1 (β-arr1) and β-arrestin-2 (β-arr2) are cytosolic proteins well-known to participate in G protein-coupled receptor desensitisation and signalling. We used genetically-inactivated mice to evaluate the role of β-arr1 or β-arr2 in platelet function, P2Y receptor desensitisation, haemostasis and thrombosis. Platelet aggregation, soluble fibrinogen binding and P-selectin exposure induced by various agonists were near normal in β-arr1-/- and β-arr2-/- platelets. In addition, deficiency in β-arr1 or β-arr2 was not critical for P2Y receptors desensitisation. A functional redundancy between β-arr1 and β-arr2 may explain these unchanged platelet responses. Interestingly, β-arr1-/- but not β-arr2-/- mice were protected against laser- and FeCl3-induced thrombosis. The tail bleeding times, number of rebleeds and volume of blood loss were unchanged in β-arr1-/- and β-arr2-/- mice, suggesting no defect in haemostasis. β-arr1-/- platelet activation upon adhesion to immobilised fibrinogen was inhibited, as attested by a 37 ± 5% (n = 3, p<0.0001) decrease in filopodia extension, suggesting defective signalling through integrin αIIbβ3. β-arr1 appeared to be located downstream of Src family kinases and to regulate αIIbβ3 signalling by increasing Akt phosphorylation. Overall, this study supports a role for β-arr1 in promoting thrombus formation, in part through its participation in αIIbβ3 signalling, and no role of β-arr1 and β-arr2 in agonist-induced platelet activation and P2Y receptors desensitisation.

Immobilized fibrinogen activates human platelets through glycoprotein VI

Glycoprotein VI, a major platelet activation receptor for collagen and fibrin, is considered a particularly promising, safe antithrombotic target. In this study, we show that human glycoprotein VI signals upon platelet adhesion to fibrinogen. Full spreading of human platelets on fibrinogen was abolished in platelets from glycoprotein VI- deficient patients suggesting that fibrinogen activates platelets through glycoprotein VI. While mouse platelets failed to spread on fibrinogen, human-glycoprotein VI-transgenic mouse platelets showed full spreading and increased Ca2+ signaling through the tyrosine kinase Syk. Direct binding of fibrinogen to human glycoprotein VI was shown by surface plasmon resonance and by increased adhesion to fibrinogen of human glycoprotein VI-transfected RBL-2H3 cells relative to mock-transfected cells. Blockade of human glycoprotein VI with the Fab of the monoclonal antibody 9O12 impaired platelet aggregation on preformed platelet aggregates in flowing blood independent of collagen and fibrin exposure. These results demonstrate that human glycoprotein VI binds to immobilized fibrinogen and show that this contributes to platelet spreading and platelet aggregation under flow.

Bioreactivity of stent material: Activation of platelets, coagulation, leukocytes and endothelial cell dysfunction in vitro.

Abstract Outcome of patients with coronary artery disease has been significantly improved by percutaneous coronary interventions with stent implantation. However, despite progress made on devices and antithrombotic treatments, stent thrombosis remains an important issue because of serious adverse consequences. Several mechanisms are assumed to favor stent thrombosis as platelet aggregation, fibrin formation, defective healing and local inflammation. The objective of this study was to evaluate in vitro the thrombogenicity, proinflammatory properties and healing capacities of cobalt–chromium (CoCr), an alloy commonly used for cardiovascular implants. Platelet adhesion was quantified in static and flow conditions. Thrombin generation was performed using the calibrated automated thrombogram. Neutrophil adhesion and formation of extracellular traps were visualized by scanning electron microscopy and by immunofluorescence. The phenotype of endothelial cells grown on CoCr was analyzed using specific antibodies, whereas the procoagulant potential was analyzed by measuring thrombin generation and protein C activation. Our results show that human blood platelets adhere to and are activated on CoCr in static and flow conditions. Overall, CoCr significantly induced thrombin generation in the presence or absence of platelets by 1.5- and 4.8-fold, respectively, involving activation of the contact pathway and activation of platelets. CoCr triggered leukocyte adhesion and behaved as a scaffold for the formation of neutrophil extracellular traps in the presence of platelets. Endothelial cells adhered and formed a monolayer covering CoCr. However, they switched from an anticoagulant phenotype to a procoagulant one with a significant 2.2-fold increase in thrombin generation due to a combined 30% reduced capacity to trigger protein C activation and 30% increased expression of tissue factor. Moreover, endothelial cells grown on CoCr acquired an inflammatory phenotype as indicated by the increased expression of ICAM-1 and VCAM-1. These data show that bare CoCr is prothrombotic and proinflammatory due to its capacity to activate platelets and coagulation and to induce leukocyte adhesion and activation. More importantly, even if endothelialization is achievable, the switch in endothelial phenotype prevents effective healing. Furthermore, we propose our methodology for future preclinical in vitro evaluation of the thrombogenicity of stent materials.

Lentiviral gene rescue of a Bernard-Soulier mouse model to study platelet glycoprotein Ibβ function.

Essentials A signaling role of glycoprotein (GP)Ibβ is postulated but not formally demonstrated in platelets. Lentiviral‐mediated rescue in knock‐out mice can be used to evaluate GPIbβ function in vivo. Transduction of the native subunit corrected the main defects associated with GPIb‐IX deficiency Deletion of intracellular 159–170 segment increased thrombosis, 150–160 removal increased bleeding.