Yanyan Bai

BACKGROUND: Thrombosis often starts with platelet adhesion via the interaction between VWF (von Willebrand factor) and its platelet receptor, the GPIb-IX (glycoprotein Ib-IX) complex. GPIb-IX also induces intracellular signals, stimulating VWF-, thrombin- and collagen-induced platelet activation. GPIb-IX signaling requires SFK (Src family kinase) Lyn. However, it remains unclear how Lyn mediates GPIb-IX signaling, whether Lyn directly binds to GPIb-IX, and if it is possible to target this signaling node for developing novel antithrombotics. METHODS: Direct binding of recombinant Lyn to glycoprotein Ibβ (GPIbβ) fragments was performed to map the Lyn-binding site. An inhibitory peptide mPLβ was then synthesized and tested for its interference with Lyn-GPIb-IX co-immunoprecipitation, platelet adhesion, ristocetin-, thrombin- or collagen-induced platelet aggregation/secretion, FeCl3-induced mouse carotid artery thrombosis and tail bleeding time. Biomembrane force probe was used to determine VWF-GPIb molecular bonding and consequent signaling. RESULTS: Lyn directly binds to GPIbβ (V144-A161) at the transmembrane/cytoplasmic domain interface, which is critical for not only the extracellular GPIb-IX ligand–induced intracellular signaling but also transmits outbound signals enhancing VWF-GPIb-IX interaction. The peptide mPLβ inhibited VWF-induced or α-thrombin-induced GPIbβ-Lyn interaction, Lyn/SFK activation, stable platelet adhesion and aggregation, and, notably, reduced GPIb-mediated platelet adhesion to VWF even in the presence of integrin inhibitors. BFP study further shows that mPLβ reduces the VWF-A1 domain-GPIb adhesion frequency and inhibits force-induced A1-mediated intraplatelet calcium elevation. Importantly, mPLβ formulated as a high-loading peptide nanoparticle inhibited platelet adhesion/aggregation induced by collagen and in vivo arterial thrombosis with a mild effect in prolonging bleeding time. CONCLUSIONS: Direct binding of Lyn to GPIbβ mediates 2-way GPIb-IX signaling to activate platelets and modulate VWF-GPIb interaction. Lyn-mediated GPIb-IX signaling is critical for platelet adhesion and aggregation induced by VWF and collagen and in arterial thrombosis. Targeting the Lyn-GPIbβ interaction has the potential for treating GPIb-IX-dependent thrombosis.

The integrins and G protein-coupled receptors are both fundamental in cell biology. The cross talk between these two, however, is unclear. Here we show that β₃ integrins negatively regulate G protein-coupled signaling by directly inhibiting the Gα₁₃-p115RhoGEF interaction. Furthermore, whereas β₃ deficiency or integrin antagonists inhibit integrin-dependent platelet aggregation and exocytosis (granule secretion), they enhance G protein-coupled RhoA activation and integrin-independent secretion. In contrast, a β₃-derived Gα₁₃-binding peptide or Gα₁₃ knockout inhibits G protein-coupled RhoA activation and both integrin-independent and dependent platelet secretion without affecting primary platelet aggregation. In a mouse model of myocardial ischemia/reperfusion injury in vivo, the β₃-derived Gα₁₃-binding peptide inhibits platelet secretion of granule constituents, which exacerbates inflammation and ischemia/reperfusion injury. These data establish crucial integrin-G protein crosstalk, providing a rationale for therapeutic approaches that inhibit exocytosis in platelets and possibly other cells without adverse effects associated with loss of cell adhesion.

Background: Platelet adhesion and aggregation play a crucial role in arterial thrombosis and ischemic stroke. Here, we identify platelet ERO1α (endoplasmic reticulum oxidoreductase 1α) as a novel regulator of Ca ²⁺ signaling and a potential pharmacological target for treating thrombotic diseases. Methods: Intravital microscopy, animal disease models, and a wide range of cell biological studies were utilized to demonstrate the pathophysiological role of ERO1α in arteriolar and arterial thrombosis and to prove the importance of platelet ERO1α in platelet activation and aggregation. Mass spectrometry, electron microscopy, and biochemical studies were used to investigate the molecular mechanism. We used novel blocking antibodies and small-molecule inhibitors to study whether ERO1α can be targeted to attenuate thrombotic conditions. Results: Megakaryocyte-specific or global deletion of Ero1α in mice similarly reduced platelet thrombus formation in arteriolar and arterial thrombosis without affecting tail bleeding times and blood loss following vascular injury. We observed that platelet ERO1α localized exclusively in the dense tubular system and promoted Ca ²⁺ mobilization, platelet activation, and aggregation. Platelet ERO1α directly interacted with STIM1 (stromal interaction molecule 1) and SERCA2 (sarco/endoplasmic reticulum Ca ²⁺ -ATPase 2) and regulated their functions. Such interactions were impaired in mutant STIM1-Cys49/56Ser and mutant SERCA2-Cys875/887Ser. We found that ERO1α modified an allosteric Cys49-Cys56 disulfide bond in STIM1 and a Cys875-Cys887 disulfide bond in SERCA2, contributing to Ca ²⁺ store content and increasing cytosolic Ca ²⁺ levels during platelet activation. Inhibition of Ero1α with small-molecule inhibitors but not blocking antibodies attenuated arteriolar and arterial thrombosis and reduced infarct volume following focal brain ischemia in mice. Conclusions: Our results suggest that ERO1α acts as a thiol oxidase for Ca ²⁺ signaling molecules, STIM1 and SERCA2, and enhances cytosolic Ca ²⁺ levels, promoting platelet activation and aggregation. Our study provides evidence that ERO1α may be a potential target to reduce thrombotic events.

Systemic inflammation as manifested in sepsis is an excessive, life-threatening inflammatory response to severe bacterial or viral infection or extensive injury. It is also a thrombo-inflammatory condition associated with vascular leakage/hemorrhage and thrombosis that is not effectively treated by current anti-inflammatory or anti-thrombotic drugs. Here, we show that MB2mP6 peptide nanoparticles, targeting the Gα₁₃-mediated integrin “outside-in” signaling in leukocytes and platelets, inhibited both inflammation and thrombosis without causing hemorrhage/vascular leakage. MB2mP6 improved mouse survival when infused immediately or hours after onset of severe sepsis. Furthermore, platelet Gα₁₃ knockout inhibited septic thrombosis whereas leukocyte Gα₁₃ knockout diminished septic inflammation, each moderately improving survival. Dual platelet/leukocyte Gα₁₃ knockout inhibited septic thrombosis and inflammation, further improving survival similar to MB2mP6. These results demonstrate that inflammation and thrombosis independently contribute to poor outcomes and exacerbate each other in systemic inflammation, and reveal a concept of dual anti-inflammatory/anti-thrombotic therapy without exacerbating vascular leakage.