Publications (21) View all
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Article: What is vinculin needed for in platelets?
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ABSTRACT: Background: Vinculin links integrins to the cell cytoskeleton by virtue of its binding to proteins such as talin and F-actin. It has been implicated in the transmission of mechanical forces from the extracellular matrix to the cytoskeleton of migrating cells. Vinculin's function in platelets is unknown. Objective: To determine whether vinculin is required for the functions of platelets and their major integrin, α(IIb) β(3) . Methods: The murine vinculin gene (Vcl) was deleted in the megakaryocyte/platelet lineage by breeding Vcl fl/fl mice with Pf4-Cre mice. Platelet and integrin functions were studied in vivo and ex vivo. Results: Vinculin was undetectable in platelets from Vcl fl/fl Cre(+) mice, as determined by immunoblotting and fluorescence microscopy. Vinculin-deficient megakaryocytes exhibited increased membrane tethers in response to mechanical pulling on α(IIb) β(3) with laser tweezers, suggesting that vinculin helps to maintain membrane cytoskeleton integrity. Surprisingly, vinculin-deficient platelets displayed normal agonist-induced fibrinogen binding to α(IIb) β(3) , aggregation, spreading, actin polymerization/organization, clot retraction and the ability to form a procoagulant surface. Furthermore, vinculin-deficient platelets adhered to immobilized fibrinogen or collagen normally, under both static and flow conditions. Tail bleeding times were prolonged in 59% of vinculin-deficient mice. However, these mice exhibited no spontaneous bleeding and they formed occlusive platelet thrombi comparable to those in wild-type littermates in response to carotid artery injury with FeCl(3) . Conclusion: Despite promoting membrane cytoskeleton integrity when mechanical force is applied to α(IIb) β(3) , vinculin is not required for the traditional functions of α(IIb) β(3) or the platelet actin cytoskeleton.Journal of Thrombosis and Haemostasis 10/2010; 8(10):2294-304. · 5.73 Impact Factor -
Article: Glycoprotein Ib-IX-mediated activation of integrin alpha(IIb)beta(3): effects of receptor clustering and von Willebrand factor adhesion.
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ABSTRACT: The interaction between the platelet glycoprotein (GP) Ib-IX complex and von Willebrand factor (VWF) initiates both hemostasis and pathological thrombosis. This interaction is not only the first adhesive event of platelets at sites of vessel injury, but also facilitates fibrinogen binding to alpha(IIb)beta(3), which subsequently results in platelet aggregation. Since it has been suggested that GP Ib-IX clustering may promote platelet activation, we investigated the effect of such clustering on both VWF-GP Ib-IX and fibrinogen-alpha(IIb)beta(3) bonds using optical tweezers. In our system, fusion of tandem repeats of FK506-binding protein (FKBP) to the cytoplasmic tail of the GP IX subunit of the GP Ib-IX complex allowed subsequent receptor clustering within the plasma membrane by the bivalent, cell-permeant small molecule ligand AP20187. We measured binding forces between polystyrene beads coated with either plasma-derived VWF or the VWF A1 domain and GP Ib-IX(FKBP)2, and those between fibrinogen-coated beads and alpha(IIb)beta(3) expressed on Chinese hamster ovary cells. The minimal detachment force between GP Ib-IX(FKBP)(2) and A1 or plasma-derived VWF doubled after AP20187 was added. The binding force between immobilized fibrinogen and alpha(IIb)beta(3) was not changed by the clustering agent; however, the strength of single fibrinogen-alpha(IIb)beta(3) bonds increased significantly after ligation of GP Ib-IX(FKBP)(2) by A1. These results demonstrate that GP Ib-IX clustering increases the overall strength of its interaction with VWF. Furthermore, signals from GP Ib-IX can activate alpha(IIb)beta(3), thereby increasing the strength of its interaction with fibrinogen.Journal of Thrombosis and Haemostasis 07/2003; 1(6):1150-7. · 5.73 Impact Factor -
Article: Glycoprotein Ib–IX‐mediated activation of integrin αIIbβ3: effects of receptor clustering and von Willebrand factor adhesion
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ABSTRACT: The interaction between the platelet glycoprotein (GP) Ib–IX complex and von Willebrand factor (VWF) initiates both hemostasis and pathological thrombosis. This interaction is not only the first adhesive event of platelets at sites of vessel injury, but also facilitates fibrinogen binding to αIIbβ3, which subsequently results in platelet aggregation. Since it has been suggested that GP Ib–IX clustering may promote platelet activation, we investigated the effect of such clustering on both VWF–GP Ib–IX and fibrinogen–αIIbβ3 bonds using optical tweezers. In our system, fusion of tandem repeats of FK506-binding protein (FKBP) to the cytoplasmic tail of the GP IX subunit of the GP Ib–IX complex allowed subsequent receptor clustering within the plasma membrane by the bivalent, cell-permeant small molecule ligand AP20187. We measured binding forces between polystyrene beads coated with either plasma-derived VWF or the VWF A1 domain and GP Ib–IX(FKBP)2, and those between fibrinogen-coated beads and αIIbβ3 expressed on Chinese hamster ovary cells. The minimal detachment force between GP Ib–IX(FKBP)2 and A1 or plasma-derived VWF doubled after AP20187 was added. The binding force between immobilized fibrinogen and αIIbβ3 was not changed by the clustering agent; however, the strength of single fibrinogen–αIIbβ3 bonds increased significantly after ligation of GP Ib–IX(FKBP)2 by A1. These results demonstrate that GP Ib–IX clustering increases the overall strength of its interaction with VWF. Furthermore, signals from GP Ib–IX can activate αIIbβ3, thereby increasing the strength of its interaction with fibrinogen.Journal of Thrombosis and Haemostasis 05/2003; 1(6):1150 - 1157. · 5.73 Impact Factor -
Conference Proceeding: Measurement of the bond strength between VWF A1 domain and clustered platelet glycoprotein Ib-IX using optical tweezers
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ABSTRACT: Adhesion between the A1 domain of the plasma protein von Willebrand factor (VWF) and the platelet glycoprotein (GP) Ib-IX complex is the first stage in thrombus formation and ultimately leads to firm platelet adhesion. Studies have suggested that GP Ib-IX receptor clustering may modulate platelet adhesion and signaling. Therefore, we have investigated the importance of GP Ib-IX clustering in the bond strength between Al and GP Ib-IX. After immobilizing A1 on polystyrene beads, we optically trapped the bead using a laser tuned to 830 nm. The bead was then allowed to adhere for 10 seconds to a transfected Chinese hamster ovary cell which expressed GP Ib-IX receptors that could be conditionally clustered with a small molecule dimerizer. We obtained the force to detach A1 from GP Ib-IX by determining the minimum laser power required to separate the bead from the cell. When pulling the bound bead away from the cell, we found that the lowest strength between GP Ib-IX and A1 effectively doubled when the dimerizer was added. These results indicate that GP Ib-IX clustering can increase the bond strength between the receptor and its cognate ligand, suggesting a role for receptor clustering in thrombus formation.Engineering in Medicine and Biology, 2002. 24th Annual Conference and the Annual Fall Meeting of the Biomedical Engineering Society EMBS/BMES Conference, 2002. Proceedings of the Second Joint; 11/2002 -
Article: Role for ADAP in shear flow-induced platelet mechanotransduction.
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ABSTRACT: Binding of platelets to fibrinogen via integrin alphaIIbbeta3 stimulates cytoskeletal reorganization and spreading. These responses depend on tyrosine phosphorylation of multiple proteins by Src family members and Syk. Among Src substrates in platelets is adhesion- and degranulation-promoting adapter protein (ADAP), an adapter with potential binding partners: SLP-76, VASP, and SKAP-HOM. During studies of platelet function under shear flow, we discovered that ADAP(-/-) mouse platelets, unlike ADAP+/+ platelets, formed unstable thrombi in response to carotid artery injury. Moreover, fibrinogen-adherent ADAP(-/-) platelets in shear flow ex vivo showed reduced spreading and smaller zones of contact with the matrix. These abnormalities were not observed under static conditions, and they could not be rescued by stimulating platelets with a PAR4 receptor agonist or by direct alphaIIbbeta3 activation with MnCl2, consistent with a defect in outside-in alphaIIbbeta3 signaling. ADAP+/+ platelets subjected to shear flow assembled F-actin-rich structures that colocalized with SLP-76 and the Rac1 exchange factor, phospho-Vav1. In contrast, platelets deficient in ADAP, but not those deficient in VASP or SKAP-HOM, failed to form these structures. These results establish that ADAP is an essential component of alphaIIbbeta3-mediated platelet mechanotransduction that promotes F-actin assembly and enables platelet spreading and thrombus stabilization under fluid shear stress.Blood 12/2009; 115(11):2274-82. · 9.90 Impact Factor
