Andrew Mack

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.

A transfer matrix method is described for the conformational spread (CS) model of allosteric cooperativity within a one-dimensional arrangement of four-state binding sites. Each such binding site can realize one of two possible conformational states. Each of these states can either bind ligand or not bind ligand. Thus, analytical expressions that are exact within the context of the CS model are derived for the grand partition function, for the mean fraction of binding sites occupied by ligand versus ligand concentration, and for the mean fraction of binding sites in a given allosteric state versus ligand concentration. The utility of our analytical results is demonstrated by least-mean-square fitting of prior experimental results obtained on the bacterial flagellar motor for the fraction of FliM/FliG/FliN complexes with CheY-P bound [V. Sourjik and H. C. Berg, Proc. Natl. Acad. Sci. U.S.A. 99, 12669 (2002)] and for the cw bias [P. Cluzel et al., Science 287, 1652 (2000)], which plausibly may be identified as the fraction of protomers realizing state 2. Finally, the relationships between our analytical results and the classical Monod-Wyman-Changeaux, Koshland-Nemethy-Filmer, and McGhee-Von Hippel treatments of allosteric cooperativity are elucidated, as is the connection to an earlier approximate analytical treatment of the CS model. DOI: http://dx.doi.org/10.1103/PhysRevE.82.031913 © 2010 The American Physical Society