The platelet receptor for von Willebrand factor (VWF), glycoprotein (GP) Ib-IX, mediates platelet adhesion and activation. The cytoplasmic domains of the GPIb ␣ and ␤ subunits contain binding sites for the phosphorylation-dependent signaling molecule, 14-3-3. Here we show that a novel membrane-permeable inhibitor of 14-3-3-GPIb␣ interaction, MP␣C, potently inhibited VWF binding to platelets and VWF-mediated platelet adhesion under flow conditions. MP␣C also inhibited VWF-dependent platelet agglutination induced by ristocetin. Furthermore, activation of the VWF binding function of GPIb-IX induced by GPIb␤ dephosphorylation is diminished by mutagenic disruption of the 14-3-3 binding site in the C-terminal domain of GPIb␣, mimicking MP␣C-induced inhibition, indicating that the inhibitory effect of MP␣C is likely to be caused by disruption of 14-3-3 binding to GPIb␣. These data suggest a novel 14-3-3-dependent regulatory mechanism that controls the VWF binding function of GPIb-IX, and also suggest a new type of antiplatelet agent that may be potentially useful in preventing or treating thrombosis. ( IntroductionPlatelet adhesion plays an important role in hemostasis and thrombosis. Under high shear rate flow conditions such as in arteries and capillaries, platelet adhesion to the subendothelium is dependent on interaction between subendothelial-bound von Willebrand factor (VWF) and its receptor, the platelet glycoprotein Ib-IX-V complex (GPIb-IX-V). 1 VWF-GPIb-IX-V interaction also mediates the formation of platelet microaggregates in patients with abnormally large VWF multimers, 2 resulting in thrombotic thrombocytopenic purpura (TTP). Interaction of VWF with GPIb-IX-V triggers intracellular signaling events such as elevation of intracellular calcium 3 and cyclic guanosine monophosphate (cGMP) levels, 4 activation of phosphoinositide 3-kinase, 5 and activation of multiple protein kinase pathways. 4,[6][7][8] These events lead to the activation of the ligand binding function of the integrin ␣ IIb ␤ 3 , 9-11 which mediates platelet spreading and aggregation.GPIb-IX-V consists of 4 different transmembrane subunits. GPIb, which consists of disulfide-linked GPIb␣ and GPIb␤ subunits, forms a 1:1 complex with GPIX. The GPIb-IX complex (GPIb-IX) is sufficient for ligand binding and signaling function. 11,12 GPIb-IX forms a 2:1 complex with GPV. 12 The N-terminal region of GPIb␣ contains binding sites for VWF and thrombin. Binding of VWF to GPIb-IX is tightly controlled, and normally occurs only at sites of vascular injury or under pathologically high shear stress. Shear stress may induce changes in both VWF and platelets. In vitro, ristocetin and botrocetin are used to induce VWF binding to GPIb-IX by mimicking the effects of the subendothelial matrix and shear stress on VWF and/or GPIb-IX. Increasing evidence indicates that VWF binding to GPIb-IX is regulated by intraplatelet signals such as cyclic adenosine monophosphate (cAMP) levels. [13][14][15] Elevation of intracellular cAMP activates the cAMP-dependent protein kin...

Rapid cut‐off of blood supply in diseases involving thrombosis is a major cause of morbidity and mortality worldwide. However, the current thrombolysis strategies offer limited results due to the therapeutics' short half‐lives, low targeting ability, and unexpected bleeding complications. Inspired by the innate roles of platelets in hemostasis and pathological thrombus, platelet membrane‐camouflaged polymeric nanoparticles (nanoplatelets) are developed for targeting delivery of the thrombolytic drug, recombinant tissue plasminogen activator (rt‐PA), to local thrombus sites. The tailor‐designed nanoplatelets efficiently accumulate at the thrombi in pulmonary embolism and mesenteric arterial thrombosis model mice, eliciting a significantly enhanced thrombolysis activity compared to free rt‐PA. In addition, the nanoplatelets exhibit improved therapeutic efficacy over free rt‐PA in an ischemic stroke model. Analysis of in vivo coagulation indicators suggests the nanoplatelets might possess a low risk of bleeding complications. The hybrid biomimetic nanoplatelets described offer a promising solution to improve the efficacy and reduce the bleeding risk of thrombolytic therapy in a broad spectrum of thrombosis diseases.

Summary. Background: The interaction of glycoprotein (GP) Iba with von Willebrand factor (VWF) initiates platelet adhesion, and simultaneously triggers intracellular signaling cascades leading to platelet aggregation and thrombus formation. Some of the signaling events are similar to those occurring during apoptosis, however, it is still unclear whether platelet apoptosis is induced by the GPIba-VWF interaction. Objectives: To investigate whether the GPIba-VWF interaction induces platelet apoptosis and the role of 14-3-3f in apoptotic signaling. Methods: Apoptotic events were assessed in platelets or Chinese hamster ovary (CHO) cells expressing wild-type (1b9) or mutant GPIb-IX interacting with VWF by flow cytometry or western blotting. Results: Ristocetin-induced GPIba-VWF interaction elicited apoptotic events in platelets, including phosphatidylserine exposure, elevations of Bax and Bak, gelsolin cleavage, and depolarization of mitochondrial inner transmembrane potential. Apoptotic events were also elicited in platelets exposed to pathologic shear stresses in the presence of VWF; however, the shear-induced apoptosis was eliminated by the anti-GPIba antibody AK2. Furthermore, apoptotic events occurred in 1b9 cells stimulated with VWF and ristocetin, but were significantly diminished in two CHO cell lines expressing mutant GPIb-IX with GPIba truncated at residue 551 or a serine-to-alanine mutation at the 14-3-3f-binding site in GPIba. Conclusions: This study demonstrates that the GPIba-VWF interaction induces apoptotic events in platelets, and that the association of 14-3-3f with the cytoplasmic domain of GPIba is essential for apoptotic signaling. This finding may suggest a novel mechanism for platelet clearance or some thrombocytopenic diseases.

Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by low platelet count which can cause fatal hemorrhage. ITP patients with antiplatelet glycoprotein (GP) Ib-IX autoantibodies appear refractory to conventional treatments, and the mechanism remains elusive. Here we show that the platelets undergo apoptosis in ITP patients with anti-GPIbα autoantibodies. Consistent with these findings, the anti-GPIbα monoclonal antibodies AN51 and SZ2 induce platelet apoptosis in vitro. We demonstrate that anti-GPIbα antibody binding activates Akt, which elicits platelet apoptosis through activation of phosphodiesterase (PDE3A) and PDE3A-mediated PKA inhibition. Genetic ablation or chemical inhibition of Akt or blocking of Akt signaling abolishes anti-GPIbα antibody-induced platelet apoptosis. We further demonstrate that the antibody-bound platelets are removed in vivo through an apoptosis-dependent manner. Phosphatidylserine (PS) exposure on apoptotic platelets results in phagocytosis of platelets by macrophages in the liver. Notably, inhibition or genetic ablation of Akt or Akt-regulated apoptotic signaling or blockage of PS exposure protects the platelets from clearance. Therefore, our findings reveal pathogenic mechanisms of ITP with anti-GPIbα autoantibodies and, more importantly, suggest therapeutic strategies for thrombocytopenia caused by autoantibodies or other pathogenic factors.