Current and future antiplatelet therapies: emphasis on preserving haemostasis.

Antiplatelet drugs, such as aspirin, P2Y12 antagonists, and glycoprotein (GP) IIb/IIIa inhibitors, have proved to be successful in reducing the morbidity and mortality associated with arterial thrombosis. These agents are, therefore, the cornerstone of therapy for patients with acute coronary syndromes. However, these drugs all carry an inherent risk of bleeding, which is associated with adverse cardiovascular outcomes and mortality. Thus, the potential benefits of more potent, conventional antiplatelet drugs are likely be offset by the increased risk of bleeding. Data from experiments in vivo have highlighted potentially important differences between haemostasis and thrombosis, raising the prospect of developing new antiplatelet drugs that are not associated with bleeding. Indeed, in preclinical studies, several novel antiplatelet therapies that seem to inhibit thrombosis while maintaining haemostasis have been identified. These agents include inhibitors of phosphatidylinositol 3-kinase-ß (PI3Kß), protein disulfide-isomerase, activated GPIIb/IIIa, GPIIb/IIIa outside-in signalling, protease-activated receptors, and platelet GPVI-mediated adhesion pathways. In this Review, we discuss how a therapeutic ceiling has been reached with existing antiplatelet drugs, whereby increased potency is offset by elevated bleeding risk. The latest advances in our understanding of thrombus formation have informed the development of new antiplatelet drugs that are potentially safer than currently available therapies.

Platelet Integrins in Tumor Metastasis: Do They Represent a Therapeutic Target?

Platelets are small anucleated cell fragments that ensure the arrest of bleeding after a vessel wall injury. They are also involved in non-hemostatic function such as development, immunity, inflammation, and in the hematogeneous phase of metastasis. While the role of platelets in tumor metastasis has been recognized for 60 years, the molecular mechanism underlying this process remains largely unclear. Platelets physically and functionally interact with various tumor cells through surface receptors including integrins. Platelets express five integrins at their surface, namely α2ß1, α5ß1, α6ß1, αvß3, and αIIbß3, which bind preferentially to collagen, fibronectin, laminin, vitronectin, and fibrinogen, respectively. The main role of platelet integrins is to ensure platelet adhesion and aggregation at sites of vascular injury. Two of these, α6ß1 and αIIbß3, were proposed to participate in platelet-tumor cell interaction and in tumor metastasis. It has also been reported that pharmacological agents targeting both integrins efficiently reduce experimental metastasis, suggesting that platelet integrins may represent new anti-metastatic targets. This review focuses on the role of platelet integrins in tumor metastasis and discusses whether these receptors may represent new potential targets for novel anti-metastatic approaches.

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.

Platelet glycoprotein VI binds to polymerized fibrin and promotes thrombin generation.

Fibrin, the coagulation end product, consolidates the platelet plug at sites of vascular injury and supports the recruitment of circulating platelets. In addition to integrin αIIbß3, another as-yet-unidentified receptor is thought to mediate platelet interaction with fibrin. Platelet glycoprotein VI (GPVI) interacts with collagen and several other adhesive macromolecules. We evaluated the hypothesis that GPVI could be a functional platelet receptor for fibrin. Calibrated thrombin assays using platelet-rich plasma (PRP) showed that tissue factor-triggered thrombin generation was impaired in GPVI-deficient patients and reduced by the anti-GPVI Fab 9O12. Assays on reconstituted PRP and PRP from fibrinogen-deficient patients revealed a fibrinogen-dependent enhancement of thrombin generation, which relied on functional GPVI. The effect of GPVI was found to depend on fibrin polymerization. A binding assay showed a specific interaction between GPVI-Fc and fibrin, inhibited by the Fab 9O12. This Fab also reduced platelet adhesion to fibrin at low (300 s(-1)) and high (1500 s(-1)) wall shear rates. Platelets adherent to fibrin displayed shape change, exposure of procoagulant phospholipids, and the formation of small clots. When hirudinated blood was perfused at 1500 s(-1) over preformed fibrin-rich clots, the Fab 9O12 decreased the recruitment of platelets by up to 85%. This study identifies GPVI as a platelet receptor for polymerized fibrin with 2 major functions: (1) amplification of thrombin generation and (2) recruitment of circulating platelets to clots. These so-far-unrecognized properties of GPVI confer on it a key role in thrombus growth and stabilization.

[Anti-platelets without a bleeding risk: novel targets and strategies]. / Les anti-plaquettaires sans risque de saignement : nouvelles cibles et stratégies.

Anti-platelet agents such as aspirin, clopidogrel and antagonists of integrin αIIbß3 allowed to efficiently reduce morbidity and mortality associated with arterial thrombosis. A major limit of these drugs is that they increase the risk of bleeding. During the last few years, several innovative anti-thrombotic strategies with a potentially low bleeding risk were proposed. These approaches target the collagen receptor glycoprotein (GP) VI, the GPIb/von Willebrand factor axis, the thrombin receptor PAR-1, the activated form of integrin αIIbß3 or the ADP receptor P2Y1. While an antagonist of PAR-1 was recently marketed, the clinical proofs of the efficiency and safety of the other agents remain to be established. This review evaluates these new anti-platelet approaches toward safer anti-thrombotic therapies.

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.