Xin Huang

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.

BACKGROUND: The protein ZPI (Z-dependent protease inhibitor) binds to PZ (protein Z), which enables ZPI to inhibit membrane-bound FXa (activated factor X). ZPI also inhibits FXIa (activated factor XI) independently of PZ. METHODS: To study the PZ-independent ZPI function, we tested the in vitro and in vivo effect of disrupting the ZPI-PZ interaction by mutating ZPI Asp 293 to Ala (D293A). RESULTS: D293A mutation reduced PZ-dependent FXa inhibition without affecting FXIa inhibition. D293A also diminished FXIIa (activated FXII)-induced thrombin generation but reduced TF (tissue factor)-induced thrombin generation only at low TF concentrations. This suggests that D293A selectively inhibits the intrinsic pathway and the thrombin-FXI (factor XI) feedback loop that enhances low-dose TF-initiated coagulation. Wild-type and D293A ZPI both showed selectivity in inhibiting activated partial thromboplastin time but not prothrombin time. Increasing PZ in plasma enhances activated partial thromboplastin time inhibition and enables prothrombin time inhibition by wild-type but not D293A ZPI, further indicating that D293A ZPI selectively inhibits the intrinsic pathway independently of PZ. In mouse models, D293A inhibited FeCl ₃ -induced occlusive carotid artery thrombosis and venous thrombosis in the inferior vena cava. Thus, PZ-independent ZPI function plays a major role in ZPI inhibition of occlusive thrombosis, and D293A ZPI is an effective antithrombotic. Importantly, administering D293A ZPI did not affect tail bleeding time and showed improved hemostasis in a saphenous vein hemostasis model as compared with wild-type ZPI. CONCLUSIONS: The PZ-binding defective variant of ZPI, D293A, selectively inhibits the intrinsic coagulation pathway and is a new anticoagulant with reduced bleeding risk.

The anticoagulant serpin, protein Z-dependent protease inhibitor (ZPI), is catalytically activated by its cofactor, protein Z (PZ), to regulate the function of blood coagulation factor Xa on membrane surfaces. The X-ray structure of the ZPI-PZ complex has shown that PZ binds to a unique site on ZPI centered on helix G. In the present study, we show by Ala-scanning mutagenesis of the ZPI-binding interface, together with native PAGE and kinetic analyses of PZ binding to ZPI, that Tyr240 and Asp293 of ZPI are crucial hot spots for PZ binding. Complementary studies with protein Z–protein C chimeras show the importance of both pseudocatalytic and EGF2 domains of PZ for the critical ZPI interactions. To understand how PZ acts catalytically, we analyzed the interaction of reactive loop–cleaved ZPI (cZPI) with PZ and determined the cZPI X-ray structure. The cZPI structure revealed changes in helices A and G of the PZ-binding site relative to native ZPI that rationalized an observed 6-fold loss in PZ affinity and PZ catalytic action. These findings identify the key determinants of catalytic activation of ZPI by PZ and suggest novel strategies for ameliorating hemophilic states through drugs that disrupt the ZPI-PZ interaction.