Characterization of the Role of Integrin α5ß1 in Platelet Function, Hemostasis, and Experimental Thrombosis.

OBJECTIVE: Integrins are key regulators of various platelet functions. The pathophysiological importance of most platelet integrins has been investigated, with the exception of α5ß1, a receptor for fibronectin. The aim of this study was to characterize the role of α5ß1 in megakaryopoiesis, platelet function, and to determine its importance in hemostasis and arterial thrombosis. APPROACH AND RESULTS: We generated a mouse strain deficient for integrin α5ß1 on megakaryocytes and platelets (PF4Cre-α5-/-). PF4Cre-α5-/- mice were viable, fertile, and presented no apparent signs of abnormality. Megakaryopoiesis appears unaltered as evidence by a normal megakaryocyte morphology and development, which is in agreement with a normal platelet count. Expression of the main platelet receptors and the response of PF4Cre-α5-/- platelets to a series of agonists were all completely normal. Adhesion and aggregation of PF4Cre-α5-/- platelets under shear flow on fibrinogen, laminin, or von Willebrand factor were unimpaired. In contrast, PF4Cre-α5-/- platelets displayed a marked decrease in adhesion, activation, and aggregation on fibrillar cellular fibronectin and collagen. PF4Cre-α5-/- mice presented no defect in a tail-bleeding time assay and no increase in inflammatory bleeding in a reverse passive Arthus model and a lipopolysaccharide pulmonary inflammation model. Finally, no defects were observed in three distinct experimental models of arterial thrombosis based on ferric chloride-induced injury of the carotid artery, mechanical injury of the abdominal aorta, or laser-induced injury of mesenteric vessels. CONCLUSION: In summary, this study shows that platelet integrin α5ß1 is a key receptor for fibrillar cellular fibronectin but is dispensable in hemostasis and arterial thrombosis.

Respective roles of Glycoprotein VI and FcγRIIA in the regulation of αIIbß3-mediated platelet activation to fibrinogen, thrombus buildup, and stability.

BACKGROUND: The interplay between platelets and fibrinogen is the cornerstone of thrombus formation. Integrin αIIbß3 is the main platelet adhesion receptor for fibrinogen and mediates an outside-in signal upon ligand binding that reinforces platelet activation. In addition, FcγRIIA and glycoprotein VI (GPVI) contribute to platelet activation on fibrinogen, thereby participating in thrombus growth and stability. To date, the relative importance of these two immunoreceptor tyrosine-based activation motif-bearing receptors in these processes remains unknown. OBJECTIVE: The aim of this study was to evaluate the relative contributions of FcγRIIA and GPVI to platelet activation on fibrinogen and subsequent thrombus growth and stability. METHODS: We evaluated human and mouse platelet adhesion to fibrinogen in static assays and a flow-based approach to evaluate the contribution of FcγRIIA and GPVI to thrombus growth and stability. RESULTS: We first confirmed that integrin αIIbß3 is the key receptor supporting platelet adhesion and spreading on fibrinogen. Using human platelets treated with pharmacological blocking agents and transgenic mouse platelets expressing human receptors, data indicate that GPVI, but not FcγRIIA, plays a prominent role in platelet activation on fibrinogen. Moreover, using a flow-based assay, we observed that blockade of GPVI with 1G5, but not FcγRIIA with IV.3, prevents thrombus growth. Finally, we observed that 1G5, but not IV.3, promotes the disaggregation of thrombi formed on collagen in vitro. CONCLUSION: This study provides evidence that GPVI, but not FcγRIIA, induces platelet activation and spreading on fibrinogen, and promotes thrombus buildup and stability.

Shear rate gradients promote a bi-phasic thrombus formation on weak adhesive proteins, such as fibrinogen in a VWF-dependent manner.

Blood flow profoundly varies throughout the vascular tree due to its pulsatile nature and to the complex vessel geometry. While thrombus formation has been extensively studied in vitro under steady flow, and in vivo under normal blood flow conditions, the impact of complex hemodynamics such as flow acceleration found in stenosed arteries has gained increased appreciation. We investigated the effect of flow acceleration, characterized by shear rate gradients, on the function of platelets adhering to fibrinogen, a plasma protein which plays a key role in hemostais and thrombosis. While we confirmed that under steady flow, fibrinogen only supports single platelet adhesion, we observed that under shear rate gradients, this surface becomes highly thrombogenic, supporting efficient platelet aggregation leading to occlusive thrombus formation. This shear rate gradient-driven thrombosis is biphasic with an initial step of slow platelet recruitment supported by direct plasma VWF adsorption to immobilized fibrinogen and followed by a second phase of explosive thrombosis initiated by VWF fiber formation on platelet monolayers. In vivo experiments confirmed that shear rate gradients accelerate thrombosis in a VWF-dependent manner. Together, this study characterizes a process of plasma VWF-dependent accelerated thrombosis on immobilized fibrinogen in the presence of shear rate gradients.

Pharmacological Blockade of Glycoprotein VI Promotes Thrombus Disaggregation in the Absence of Thrombin.

OBJECTIVE: Atherothrombosis occurs upon rupture of an atherosclerotic plaque and leads to the formation of a mural thrombus. Computational fluid dynamics and numerical models indicated that the mechanical stress applied to a thrombus increases dramatically as a thrombus grows, and that strong inter-platelet interactions are essential to maintain its stability. We investigated whether GPVI (glycoprotein VI)-mediated platelet activation helps to maintain thrombus stability by using real-time video-microscopy. Approach and Results: We showed that GPVI blockade with 2 distinct Fab fragments promoted efficient disaggregation of human thrombi preformed on collagen or on human atherosclerotic plaque material in the absence of thrombin. ACT017-induced disaggregation was achieved under arterial blood flow conditions, and its effect increased with wall shear rate. GPVI regulated platelet activation within a growing thrombus as evidenced by the loss in thrombus contraction when GPVI was blocked, and the absence of the disaggregating effect of an anti-GPVI agent when the thrombi were fully activated with soluble agonists. The GPVI-dependent thrombus stabilizing effect was further supported by the fact that inhibition of any of the 4 key immunoreceptor tyrosine-based motif signalling molecules, src-kinases, Syk, PI3Kß, or phospholipase C, resulted in kinetics of thrombus disaggregation similar to ACT017. The absence of ACT017-induced disaggregation of thrombi from 2 afibrinogenemic patients suggests that the role of GPVI requires interaction with fibrinogen. Finally, platelet disaggregation of fibrin-rich thrombi was also promoted by ACT017 in combination with r-tPA (recombinant tissue plasminogen activator). CONCLUSIONS: This work identifies an unrecognized role for GPVI in maintaining thrombus stability and suggests that targeting GPVI could dissolve platelet aggregates with a poor fibrin content.

Megakaryocytes use in vivo podosome-like structures working collectively to penetrate the endothelial barrier of bone marrow sinusoids.

BACKGROUND: Blood platelets are anucleate cell fragments that prevent bleeding and minimize blood vessel injury. They are formed from the cytoplasm of megakaryocytes located in the bone marrow. For successful platelet production, megakaryocyte fragments must pass through the sinusoid endothelial barrier by a cell biology process unique to these giant cells as compared with erythrocytes and leukocytes. Currently, the mechanisms by which megakaryocytes interact and progress through the endothelial cells are not understood, resulting in a significant gap in our knowledge of platelet production. OBJECTIVE: The aim of this study was to investigate how megakaryocytes interact and progress through the endothelial cells of mouse bone marrow sinusoids. METHODS: We used a combination of fluorescence, electron, and three-dimensional microscopy to characterize the cellular events between megakaryocytes and endothelial cells. RESULTS: We identified protrusive, F-actin-based podosome-like structures, called in vivo-MK podosomes, which initiate the formation of pores through endothelial cells. These structures present a collective and spatial organization through their interconnection via a contractile network of actomyosin, essential to regulate the endothelial openings. This ensures proper passage of megakaryocyte-derived processes into the blood circulation to promote thrombopoiesis. CONCLUSION: This study provides novel insight into the in vivo function of podosomes of megakaryocytes with critical importance to platelet production.

Clot Contraction Drives the Translocation of Procoagulant Platelets to Thrombus Surface.

Objective- After activation at the site of vascular injury, platelets differentiate into 2 subpopulations, exhibiting either proaggregatory or procoagulant phenotype. Although the functional role of proaggregatory platelets is well established, the physiological significance of procoagulant platelets, the dynamics of their formation, and spatial distribution in thrombus remain elusive. Approach and Results- Using transmission electron microscopy and fluorescence microscopy of arterial thrombi formed in vivo after ferric chloride-induced injury of carotid artery or mechanical injury of abdominal aorta in mice, we demonstrate that procoagulant platelets are located at the periphery of the formed thrombi. Real-time cell tracking during thrombus formation ex vivo revealed that procoagulant platelets originate from different locations within the thrombus and subsequently translocate towards its periphery. Such redistribution of procoagulant platelets was followed by generation of fibrin at thrombus surface. Using in silico model, we show that the outward translocation of procoagulant platelets can be driven by the contraction of the forming thrombi, which mechanically expels these nonaggregating cells to thrombus periphery. In line with the suggested mechanism, procoagulant platelets failed to translocate and remained inside the thrombi formed ex vivo in blood derived from nonmuscle myosin ( MYH9)-deficient mice. Ring-like distribution of procoagulant platelets and fibrin around the thrombus observed with blood of humans and wild-type mice was not present in thrombi of MYH9-knockout mice, confirming a major role of thrombus contraction in this phenomenon. Conclusions- Contraction of arterial thrombus is responsible for the mechanical extrusion of procoagulant platelets to its periphery, leading to heterogeneous structure of thrombus exterior.

Imaging thrombosis with 99mTc-labeled RAM.1-antibody in vivo.

INTRODUCTION: Platelets play a major role in thrombo-embolic diseases, notably by forming a thrombus that can ultimately occlude a vessel. This may provoke ischemic pathologies such as myocardial infarction, stroke or peripheral artery diseases, which represent the major causes of death worldwide. The aim of this study was to evaluate the specificity of radiolabeled Rat-Anti-Mouse antibody (RAM.1). METHODS: We describe a method to detect platelets by using a RAM.1 coupled with the chelating agent hydrazinonicotinic acid (HYNIC) conjugated to 99mTc, for Single Photon Emission Computed Tomography (SPECT). To induce platelet accumulation at a site of interest, we used a mouse model of FeCl3 induced injury of the carotid artery. 90 min after i.v. injection of [99mTc][Tc(HYNIC)-RAM.1], biodistribution of the radiolabeled RAM.1 was assessed, SPECT imaging and histological analysis were performed on the mice that underwent FeCl3-induced vessel damage. RESULTS: We demonstrated a quick and strong affinity of the radiolabeled RAM.1 for the platelet thrombus. Results clearly demonstrated the ability of this radioimmunoconjugate for detecting thrombi from 10 min post injection with an exceptional thrombi uptake. Using FeCl3, the median ratio between the thrombus and the background was 12.4 (range 9.3-42.3) as compared to 1.0 (range: 0.86-2.7) p < 0.05 when using 0.9% NaCl. CONCLUSION: Thanks to the high sensitivity of SPECT, we provided evidence that [99mTc][Tc(HYNIC)-RAM.1] represents a powerful tool to detect localized platelet thrombi which could potentially be used in humans. Because of the relative low cost and high sensitivity, these results encourage further study like the detection of non-induced thrombus and further developments toward clinical application. This is further supported by the fact that RAM.1 recognizes human platelets.

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.

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.

Coagulation factors bound to procoagulant platelets concentrate in cap structures to promote clotting.

Binding of coagulation factors to phosphatidylserine (PS)-exposing procoagulant-activated platelets followed by formation of the membrane-dependent enzyme complexes is critical for blood coagulation. Procoagulant platelets formed upon strong platelet stimulation, usually with thrombin plus collagen, are large "balloons" with a small (∼1 µm radius) "cap"-like convex region that is enriched with adhesive proteins. Spatial distribution of blood coagulation factors on the surface of procoagulant platelets was investigated using confocal microscopy. All of them, including factors IXa (FIXa), FXa/FX, FVa, FVIII, prothrombin, and PS-sensitive marker Annexin V were distributed nonhomogeneously: they were primarily localized in the "cap," where their mean concentration was by at least an order of magnitude, higher than on the "balloon." Assembly of intrinsic tenase on liposomes with various PS densities while keeping the PS content constant demonstrated that such enrichment can accelerate this reaction by 2 orders of magnitude. The mechanisms of such acceleration were investigated using a 3-dimensional computer simulation model of intrinsic tenase based on these data. Transmission electron microscopy and focal ion beam-scanning electron microscopy with Annexin V immunogold-labeling revealed a complex organization of the "caps." In platelet thrombi formed in whole blood on collagen under arterial shear conditions, ubiquitous "caps" with increased Annexin V, FX, and FXa binding were observed, indicating relevance of this mechanism for surface-attached platelets under physiological flow. These results reveal an essential heterogeneity in the surface distribution of major coagulation factors on the surface of procoagulant platelets and suggest its importance in promoting membrane-dependent coagulation reactions.

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.

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 α5ß1 and αIIbß3. 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 α5ß1 and αIIbß3, the GPIb-V-IX complex, GPVI and Toll-like receptor 4.

Integrin α6ß1 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 (α1ß1γ1) is known to support platelet adhesion but is absent from most blood vessels, which contain isoforms with the α2, α4, or α5 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 (α4ß1γ1), laminin-511 (α5ß1γ1), and laminin-521 (α5ß2γ1), but not laminin-211 (α2ß1γ1), allow efficient platelet adhesion and activation across a wide range of arterial wall shear rates. Adhesion was critically dependent on integrin α6ß1 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 α5-containing laminins. To address the significance of platelet/laminin interactions in vivo, we developed a platelet-specific knockout of integrin α6. Platelets from these mice failed to adhere to laminin-411, laminin-511, and laminin-521 but responded normally to a series of agonists. α6ß1-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 α6ß1, could represent an alternative antithrombotic strategy with a potentially low bleeding risk.

Targeting platelet GPIbß 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 GPIbß, 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 GPIbß 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 Ca(2+) 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 GPIbα 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.

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 α(2)ß(1) under static conditions, whereas under high shear, it was dependent on both the α(2)ß(1) integrin and the glycoprotein Ib-IX complex. Integrin α(IIb)ß(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. CONCLUSIONS: This study provides evidence that TN-C serves as a novel adhesive matrix for platelets in a context that is relevant to atherothrombosis.