Yuping Yuan

PI 3-kinase p110β : a new target for antithrombotic therapy.

Platelet activation at sites of vascular injury is essential for the arrest of bleeding; however, excessive platelet accumulation at regions of atherosclerotic plaque rupture can result in the development of arterial thrombi, precipitating diseases such as acute myocardial infarction and ischemic stroke. Rheological disturbances (high shear stress) have an important role in promoting arterial thrombosis by enhancing the adhesive and signaling function of platelet integrin αIIbβ3 (GPIIb-IIIa). In this study we have defined a key role for the Type Ia phosphoinositide 3-kinase (PI3K) p110β isoform in regulating the formation and stability of integrin αIIbβ3 adhesion bonds, necessary for shear activation of platelets. Isoform-selective PI3K p110β inhibitors have been developed which prevent formation of stable integrin αIIbβ3 adhesion contacts, leading to defective platelet thrombus formation. In vivo, these inhibitors eliminate occlusive thrombus formation but do not prolong bleeding time. These studies define PI3K p110β as an important new target for antithrombotic therapy.

Two distinct pathways regulate platelet phosphatidylserine exposure and procoagulant function

Procoagulant platelets exhibit hallmark features of apoptotic cells, including membrane blebbing, microvesiculation, and phosphatidylserine (PS) exposure. Although platelets possess many well-known apoptotic regulators, their role in regulating the procoagulant function of platelets is unclear. To clarify this, we investigated the consequence of removing the essential mediators of apoptosis, Bak and Bax, or directly inducing apoptosis with the BH3 mimetic compound ABT-737. Treatment of platelets with ABT-737 triggered PS exposure and a marked increase in thrombin generation in vitro. This increase in procoagulant function was Bak/Bax- and caspase-dependent, but it was unaffected by inhibitors of platelet activation or by chelating extracellular calcium. In contrast, agonist-induced platelet procoagulant function was unchanged in Bak(-/-)Bax(-/-) or caspase inhibitor-treated platelets, but it was completely eliminated by extracellular calcium chelators or inhibitors of platelet activation. These studies show the existence of 2 distinct pathways regulating the procoagulant function of platelets.

Calpain Cleavage of Focal Adhesion Proteins Regulates the Cytoskeletal Attachment of Integrin αIIbβ3 (Platelet Glycoprotein IIb/IIIa) and the Cellular Retraction of Fibrin Clots

The intracellular thiol protease calpain catalyzes the limited proteolysis of various focal adhesion structural proteins and signaling enzymes in adherent cells. In human platelets, calpain activation is dependent on fibrinogen binding to integrin αIIbβ3 and subsequent platelet aggregation, suggesting a potential role for this protease in the regulation of postaggregation responses. In this study, we have examined the effects of calpain activation on several postaggregation events in human platelets, including the cytoskeletal attachment of integrin αIIbβ3, the tyrosine phosphorylation of cytoskeletal proteins, and the cellular retraction of fibrin clots. We demonstrate that calpain activation in either washed platelets or platelet-rich plasma is associated with a marked reduction in platelet-mediated fibrin clot retraction. This relaxation of clot retraction was observed in both thrombin and ionophore A23187-stimulated platelets. Calcium dose-response studies (extracellular calcium concentrations between 0.1 μM and 1 M) revealed a strong correlation between calpain activation and relaxed clot retraction. Furthermore, pretreating platelets with the calpain inhibitors calpeptin and calpain inhibitor I prevented the calpain-mediated reduction in clot retraction. Relaxed fibrin clot retraction was associated with the cleavage of several platelet focal adhesion structural proteins and signaling enzymes, resulting in the dissociation of talin, pp60c-src, and integrin αIIbβ3 from the contractile cytoskeleton and the tyrosine dephosphorylation of multiple cytoskeletal proteins. These studies suggest an important role for calpain in the regulation of multiple postaggregation events in human platelets. The ability of calpain to inhibit clot retraction is likely to be due to the cleavage of both structural and signaling proteins involved in modulating integrin-cytoskeletal interactions.

The von Willebrand Factor-Glycoprotein Ib/V/IX Interaction Induces Actin Polymerization and Cytoskeletal Reorganization in Rolling Platelets and Glycoprotein Ib/V/IX-transfected Cells

Platelet adhesion to sites of vascular injury is initiated by the binding of the platelet glycoprotein (GP) Ib-V-IX complex to matrix-bound von Willebrand factor (vWf). This receptor-ligand interaction is characterized by a rapid on-off rate that enables efficient platelet tethering and rolling under conditions of rapid blood flow. We demonstrate here that platelets adhering to immobilized vWf under flow conditions undergo rapid morphological conversion from flat discs to spiny spheres during surface translocation. Studies of Glanzmann thrombasthenic platelets (lacking integrin αIIbβ3) and Chinese hamster ovary (CHO) cells transfected with GPIb/IX (CHO-Ib/IX) confirmed that vWf binding to GPIb/IX was sufficient to induce actin polymerization and cytoskeletal reorganization independent of integrin αIIbβ3. vWf-induced cytoskeletal reorganization occurred independently of several well characterized signaling processes linked to platelet activation, including calcium influx, prostaglandin metabolism, protein tyrosine phosphorylation, activation of protein kinase C or phosphatidylinositol 3-kinase but was critically dependent on the mobilization of intracellular calcium. Studies of Oregon Green 488 1,2-bis(o-amino-5-fluorophenoxy)ethane-N,N,N′,N-tetraacetic acid tetraacetoxymethyl ester-loaded platelets and CHO-Ib/IX cells demonstrated that these cells mobilize intracellular calcium in a shear-dependent manner during surface translocation on vWf. Taken together, these studies suggest that the vWf-GPIb interaction stimulates actin polymerization and cytoskeletal reorganization in rolling platelets via a shear-sensitive signaling pathway linked to intracellular calcium mobilization.

Signaling Role for Phospholipase Cγ2 in Platelet Glycoprotein Ibα Calcium Flux and Cytoskeletal Reorganization INVOLVEMENT OF A PATHWAY DISTINCT FROM FcRγ CHAIN AND FcγRIIA

Interaction of the platelet GPIb-V-IX complex with surface immobilized von Willebrand factor (vWf) is required for the capture of circulating platelets and their ensuing activation. In previous work, it was found that GPIb/vWf-mediated platelet adhesion triggers Ca2+ release from intracellular stores, leading to cytoskeletal reorganization and filopodia extension. Despite the potential functional importance of GPIb-induced cytoskeletal changes, the signaling mechanisms regulating this process have remained ill-defined. The studies presented here demonstrate an important role for phospholipase C (PLC)-dependent phosphoinositide turnover for GPIb-dependent cytoskeletal remodeling. This is supported by the findings that the vWf-GPIb interaction induced a small increase in inositol 1,4,5-triphosphate (IP3) and that treating platelets with the IP3 receptor antagonist APB-2 or the PLC inhibitor U73122 blocked cytosolic Ca2+ flux and platelet shape change. Normal shape change was observed in Gαq–/– mouse platelets, excluding a role for PLCβ isoforms in this process. However, decreased shape change and Ca2+ mobilization were observed in mice lacking PLCγ2, demonstrating that this isotype played an important, albeit incomplete, role in GPIb signaling. The signaling pathways utilized by GPIb involved one or more members of the Src kinase family as platelet shape change and Ca2+ flux were inhibited by the Src kinase inhibitors PP1 and PP2. Strikingly, shape change and Ca2+ release occurred independently of immunoreceptor tyrosine-based activation motif (ITAM)-containing receptors, because these platelet responses were normal in human platelets treated with the anti-FcγRIIA blocking monoclonal antibody IV.3 and in mouse platelets deficient in the FcRγ chain. Taken together, these studies define an important role for PLCγ2 in GPIb signaling linked to platelet shape change. Moreover, they demonstrate that GPIb-dependent calcium flux and cytoskeletal reorganization involves a signaling pathway distinct from that utilized by ITAM-containing receptors.

Synergistic Adhesive Interactions and Signaling Mechanisms Operating between Platelet Glycoprotein Ib/IX and Integrin αIIbβ3 STUDIES IN HUMAN PLATELETS AND TRANSFECTED CHINESE HAMSTER OVARY CELLS

This study investigates three aspects of the adhesive interaction operating between platelet glycoprotein Ib/IX and integrin αIIbβ3. These include the following: 1) examining the sufficiency of GPIb/IX and integrin αIIbβ3 to mediate irreversible cell adhesion on immobilized von Willebrand factor (vWf) under flow; 2) the ability of the vWf-GPIb interaction to induce integrin αIIbβ3 activation independent of endogenous platelet stimuli; and 3) the identification of key second messengers linking the vWf-GPIb/IX interaction to integrin αIIbβ3 activation. By using Chinese hamster ovary cells transfected with GPIb/IX and integrin αIIbβ3, we demonstrate that these receptors are both necessary and sufficient to mediate irreversible cell adhesion under flow, wherein GPIb/IX mediates cell tethering and rolling on immobilized vWf, and integrin αIIbβ3mediates cell arrest. Moreover, we demonstrate direct signaling between GPIb/IX and integrin αIIbβ3. Studies on human platelets demonstrated that vWf binding to GPIb/IX is able to induce integrin αIIbβ3 activation independent of endogenous platelet stimuli under both static and physiological flow conditions (150–1800 s− 1). Analysis of the key second messengers linking the vWf-GPIb interaction to integrin αIIbβ3 activation demonstrated that the first step in the activation process involves calcium release from internal stores, whereas transmembrane calcium influx is a secondary event potentiating integrin αIIbβ3 activation.

Calpain Regulation of Cytoskeletal Signaling Complexes in Von Willebrand Factor-stimulated Platelets DISTINCT ROLES FOR GLYCOPROTEIN Ib-V-IX AND GLYCOPROTEIN IIb-IIIa (INTEGRIN αIIbβ3) IN VON WILLEBRAND FACTOR-INDUCED SIGNAL TRANSDUCTION

The adhesion of platelets to sites of vascular injury is critically dependent on the binding of subendothelial bound von Willebrand factor (vWf) to the platelet surface glycoprotein complexes, GP Ib-V-IX and GP IIb-IIIa (integrin αIIbβ3). There is growing evidence that the binding of vWf to these receptors is not only essential for stable platelet adhesion but is also important for the transduction of activation signals required for changes in platelet morphology, granule secretion, and platelet aggregation. In this study we have investigated signaling events induced by vWf binding to GP Ib-V-IX in both spreading and aggregated platelets. The adhesion of platelets to vWf resulted in dramatic actin filament reorganization, as assessed by immunofluorescence with fluorescein isothiocyanate-conjugated phalloidin, and the cytoskeletal recruitment of various structural proteins (talin and integrin αIIbβ3) and signaling enzymes (pp60c- src , focal adhesion kinase (FAK), phosphatidylinositol 3-kinase (PI 3-kinase), and protein-tyrosine phosphatase (PTP)-1B). Time course experiments in both spreading and aggregated platelets revealed that talin, FAK, and PTP-1B were proteolyzed after translocation to the cytoskeleton. The proteolysis of these proteins was dependent on the presence of extracellular calcium and was specifically inhibited by pretreating platelets with the membrane-permeable calpain inhibitors calpeptin, E64d, and MDL 28,170, but not with the membrane-impermeable inhibitors leupeptin, E64, and calpastatin. The cytoskeletal translocation of signaling enzymes in vWf-stimulated platelets was abolished by pretreating platelets with an anti-GP Ib-V-IX antibody but was unaffected by blocking ligand binding to integrin αIIbβ3. In contrast, calpain activation in vWf-stimulated platelets required ligand binding to both GP Ib-V-IX and integrin αIIbβ3. The activation of calpain in both spreading and aggregated platelets resulted in a substantial decrease in the level of tyrosine phosphorylation of multiple platelet proteins and was associated with a 50–80% reduction in the amount of cytoskeletal associated talin, integrin αIIbβ3, PI 3-kinase, FAK, pp60c- src , and PTP-1B. These studies suggest a potentially important role for calpain in regulating the formation and/or stability of cytoskeletal signaling complexes in vWf-stimulated platelets. Furthermore, they demonstrate distinct roles for GP Ib-V-IX and integrin αIIbβ3 in vWf-induced signal transduction.

Integrin αIIbβ3-dependent calcium signals regulate platelet-fibrinogen interactions under flow: Involvement of phospholipase Cγ2

Platelet adhesion to fibrinogen is important for platelet aggregation and thrombus growth. In this study we have examined the mechanisms regulating platelet adhesion on immobilized fibrinogen under static and shear conditions. We demonstrate that integrin alpha IIb beta 3 engagement of immobilized fibrinogen is sufficient to induce an oscillatory calcium response, necessary for lamellipodial formation and platelet spreading. Released ADP increases the proportion of platelets exhibiting a cytosolic calcium response but is not essential for calcium signaling or lamellipodial extension. Pretreating platelets with the Src kinase inhibitor PP2, the inositol 1,4,5-trisphosphate (IP3) receptor antagonist 2-aminoethoxydiphenyl borate (APB-2), or the phospholipase C (PLC) inhibitor U73122 abolished calcium signaling and platelet spreading, suggesting a major role for Src kinase-regulated PLC isoforms in these processes. Analysis of PLC gamma 2-/- mouse platelets revealed a major role for this isoform in regulating cytosolic calcium flux and platelet spreading on fibrinogen. Under flow conditions, platelets derived from PLC gamma 2-/- mice formed less stable adhesive interactions with fibrinogen, particularly in the presence of ADP antagonists. Our studies define an important role for PLC gamma 2 in integrin alpha IIb beta 3-dependent calcium flux, necessary for stable platelet adhesion and spreading on fibrinogen. Furthermore, they establish an important cooperative signaling role for PLC gamma 2 and ADP in regulating platelet adhesion efficiency on fibrinogen.

Integrin αβ-dependent calcium signals regulate platelet-fibrinogen interactions under flow: Involvement of PLCγ2

Platelet adhesion to fibrinogen is important for platelet aggregation and thrombus growth. In this study we have examined the mechanisms regulating platelet adhesion on immobilized fibrinogen under static and shear conditions. We demonstrate that integrin alpha IIb beta 3 engagement of immobilized fibrinogen is sufficient to induce an oscillatory calcium response, necessary for lamellipodial formation and platelet spreading. Released ADP increases the proportion of platelets exhibiting a cytosolic calcium response but is not essential for calcium signaling or lamellipodial extension. Pretreating platelets with the Src kinase inhibitor PP2, the inositol 1,4,5-trisphosphate (IP3) receptor antagonist 2-aminoethoxydiphenyl borate (APB-2), or the phospholipase C (PLC) inhibitor U73122 abolished calcium signaling and platelet spreading, suggesting a major role for Src kinase-regulated PLC isoforms in these processes. Analysis of PLC gamma 2-/- mouse platelets revealed a major role for this isoform in regulating cytosolic calcium flux and platelet spreading on fibrinogen. Under flow conditions, platelets derived from PLC gamma 2-/- mice formed less stable adhesive interactions with fibrinogen, particularly in the presence of ADP antagonists. Our studies define an important role for PLC gamma 2 in integrin alpha IIb beta 3-dependent calcium flux, necessary for stable platelet adhesion and spreading on fibrinogen. Furthermore, they establish an important cooperative signaling role for PLC gamma 2 and ADP in regulating platelet adhesion efficiency on fibrinogen.

A Mechanistic Model for Paradoxical Platelet Activation by Ligand-Mimetic αIIbβ3 (GPIIb/IIIa) Antagonists

Objective—Integrins are attractive therapeutic targets. Inhibition of integrin &agr;IIb&bgr;3 effectively blocks platelet aggregation. However, limitations with intravenous &agr;IIb&bgr;3 antagonists and failure of oral &agr;IIb&bgr;3 antagonists prompted doubts on the current concept of ligand-mimetic integrin blockade. Methods and Results—Evaluating P-selectin expression on platelets by flow cytometry, we report a mechanism of paradoxical platelet activation by ligand-mimetic &agr;IIb&bgr;3 antagonists and define three requirements: (1) Induction of ligand-bound conformation of &agr;IIb&bgr;3, (2) receptor clustering, (3) prestimulation of platelets. Conformational change is inducible by clinically used ligand-mimetic &agr;IIb&bgr;3 antagonists, RGD-peptides, and anti-LIBS antibodies. In a mechanistic experimental model, clustering is achieved by crosslinking integrins via antibodies, and preactivation is induced by low-dose ADP. Finally, we demonstrate that platelet adhesion on collagen represents an in vivo correlate of platelet prestimulation and receptor clustering, in which the presence of ligand-mimetic &agr;IIb&bgr;3 antagonists results in platelet activation as detected by P-selectin, CD63, and CD40L expression as well as by measuring Ca2+-signaling. Blockade of the ADP receptor P2Y12 by AR-C69931MX and clopidogrel inhibits &agr;IIb&bgr;3 antagonist-induced platelet activation. Conclusion—These findings can explain limitations of ligand-mimetic anti-&agr;IIb&bgr;3 therapy. They describe potential benefits of concomitant ADP receptor blockade and support a shift in drug development from ligand-mimetic toward allosteric or activation-specific integrin antagonists.