Reelin Amplifies Glycoprotein VI Activation and AlphaIIb Beta3 Integrin Outside-In Signaling via PLC Gamma 2 and Rho GTPases.

OBJECTIVE: Reelin, a secreted glycoprotein, was originally identified in the central nervous system, where it plays an important role in brain development and maintenance. In the cardiovascular system, reelin plays a role in atherosclerosis by enhancing vascular inflammation and in arterial thrombosis by promoting platelet adhesion, activation, and thrombus formation via APP (amyloid precursor protein) and GP (glycoprotein) Ib. However, the role of reelin in hemostasis and arterial thrombosis is not fully understood to date. Approach and Results: In the present study, we analyzed the importance of reelin for cytoskeletal reorganization of platelets and thrombus formation in more detail. Platelets release reelin to amplify alphaIIb beta3 integrin outside-in signaling by promoting platelet adhesion, cytoskeletal reorganization, and clot retraction via activation of Rho GTPases RAC1 (Ras-related C3 botulinum toxin substrate) and RhoA (Ras homolog family member A). Reelin interacts with the collagen receptor GP (glycoprotein) VI with subnanomolar affinity, induces tyrosine phosphorylation in a GPVI-dependent manner, and supports platelet binding to collagen and GPVI-dependent RAC1 activation, PLC gamma 2 (1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase gamma-2) phosphorylation, platelet activation, and aggregation. When GPVI was deleted from the platelet surface by antibody treatment in reelin-deficient mice, thrombus formation was completely abolished after injury of the carotid artery while being only reduced in either GPVI-depleted or reelin-deficient mice. CONCLUSIONS: Our study identified a novel signaling pathway that involves reelin-induced GPVI activation and alphaIIb beta3 integrin outside-in signaling in platelets. Loss of both, GPVI and reelin, completely prevents stable arterial thrombus formation in vivo suggesting that inhibiting reelin-platelet-interaction might represent a novel strategy to avoid arterial thrombosis in cardiovascular disease.

The Proteoglycan Biglycan Modulates Platelet Adhesion and Thrombus Formation in a GPVI-Dependent Manner.

BACKGROUND: Vascular injury induces the exposure of subendothelial extracellular matrix (ECM) important to serve as substrate for platelets to adhere to the injured vessel wall to avoid massive blood loss. Different ECM proteins are known to initiate platelet adhesion and activation. In atherosclerotic mice, the small, leucine-rich proteoglycan biglycan is important for the regulation of thrombin activity via heparin cofactor II. However, nothing is known about the role of biglycan for hemostasis and thrombosis under nonatherosclerotic conditions. METHODS: The role of biglycan for platelet adhesion and thrombus formation was investigated using a recombinant protein and biglycan knockout mice. RESULTS: The present study identified biglycan as important ECM protein for the adhesion and activation of platelets, and the formation of three-dimensional thrombi under flow conditions. Platelet adhesion to immobilized biglycan induces the reorganization of the platelet cytoskeleton. Mechanistically, biglycan binds and activates the major collagen receptor glycoprotein (GP)VI, because reduced platelet adhesion to recombinant biglycan was observed when GPVI was blocked and enhanced tyrosine phosphorylation in a GPVI-dependent manner was observed when platelets were stimulated with biglycan. In vivo, the deficiency of biglycan resulted in reduced platelet adhesion to the injured carotid artery and prolonged bleeding times. CONCLUSIONS: Loss of biglycan in the vessel wall of mice but not in platelets led to reduced platelet adhesion at the injured carotid artery and prolonged bleeding times, suggesting a crucial role for biglycan as ECM protein that binds and activates platelets via GPVI upon vessel injury.

Molecular Drivers of Platelet Activation: Unraveling Novel Targets for Anti-Thrombotic and Anti-Thrombo-Inflammatory Therapy.

Cardiovascular diseases (CVDs) are the leading cause of death globally-partly a consequence of increased population size and ageing-and are major contributors to reduced quality of life. Platelets play a major role in hemostasis and thrombosis. While platelet activation and aggregation are essential for hemostasis at sites of vascular injury, uncontrolled platelet activation leads to pathological thrombus formation and provokes thrombosis leading to myocardial infarction or stroke. Platelet activation and thrombus formation is a multistage process with different signaling pathways involved to trigger platelet shape change, integrin activation, stable platelet adhesion, aggregation, and degranulation. Apart from thrombotic events, thrombo-inflammation contributes to organ damage and dysfunction in CVDs and is mediated by platelets and inflammatory cells. Therefore, in the past, many efforts have been made to investigate specific signaling pathways in platelets to identify innovative and promising approaches for novel antithrombotic and anti-thrombo-inflammatory strategies that do not interfere with hemostasis. In this review, we focus on some of the most recent data reported on different platelet receptors, including GPIb-vWF interactions, GPVI activation, platelet chemokine receptors, regulation of integrin signaling, and channel homeostasis of NMDAR and PANX1.

The NMDA receptor regulates integrin activation, ATP release and arterial thrombosis through store-operated Ca2+ entry in platelets.

Introduction: Platelet activation and thrombus formation is crucial for hemostasis, but also trigger arterial thrombosis. Calcium mobilization plays an important role in platelet activation, because many cellular processes depend on the level of intracellular Ca2+ ([Ca2+](i)), such as integrin activation, degranulation, cytoskeletal reorganization. Different modulators of Ca2+ signaling have been implied, such as STIM1, Orai1, CyPA, SGK1, etc. Also, the N-methyl-D-aspartate receptor (NMDAR) was identified to contribute to Ca2+ signaling in platelets. However, the role of the NMDAR in thrombus formation is not well defined. Methods: In vitro and in vivo analysis of platelet-specific NMDAR knock-out mice. Results: In this study, we analyzed Grin1fl/fl-Pf4-Cre+ mice with a platelet-specific knock-out of the essential GluN1 subunit of the NMDAR. We found reduced store-operated Ca2+ entry (SOCE), but unaltered store release in GluN1-deficient platelets. Defective SOCE resulted in reduced Src and PKC substrate phosphorylation following stimulation of glycoprotein (GP)VI or the thrombin receptor PAR4 followed by decreased integrin activation but unaltered degranulation. Consequently, thrombus formation on collagen under flow conditions was reduced ex vivo, and Grin1fl/fl-Pf4-Cre+ mice were protected against arterial thrombosis. Results from human platelets treated with the NMDAR antagonist MK-801 revealed a crucial role of the NMDAR in integrin activation and Ca2+ homeostasis in human platelets as well. Conclusion: NMDAR signaling is important for SOCE in platelets and contributes to platelet activation and arterial thrombosis. Thus, the NMDAR represents a novel target for anti-platelet therapy in cardiovascular disease (CVD).

Minimal Collagen-Binding Epitope of Glycoprotein VI in Human and Mouse Platelets.

Glycoprotein VI (GPVI) is a platelet-specific receptor for collagen and fibrin, regulating important platelet functions such as platelet adhesion and thrombus growth. Although the blockade of GPVI function is widely recognized as a potent anti-thrombotic approach, there are limited studies focused on site-specific targeting of GPVI. Using computational modeling and bioinformatics, we analyzed collagen- and CRP-binding surfaces of GPVI monomers and dimers, and compared the interacting surfaces with other mammalian GPVI isoforms. We could predict a minimal collagen-binding epitope of GPVI dimer and designed an EA-20 antibody that recognizes a linear epitope of this surface. Using platelets and whole blood samples donated from wild-type and humanized GPVI transgenic mice and also humans, our experimental results show that the EA-20 antibody inhibits platelet adhesion and aggregation in response to collagen and CRP, but not to fibrin. The EA-20 antibody also prevents thrombus formation in whole blood, on the collagen-coated surface, in arterial flow conditions. We also show that EA-20 does not influence GPVI clustering or receptor shedding. Therefore, we propose that blockade of this minimal collagen-binding epitope of GPVI with the EA-20 antibody could represent a new anti-thrombotic approach by inhibiting specific interactions between GPVI and the collagen matrix.