Tetraspanin CD9 Regulates GPIIb-IIIa-Mediated Platelet Function: Identification of Unique Protein Interactomes
ISTH Academy. Kotha J. 07/11/20; 296839; PB1734
Topic: Platelet Receptors
Abstract
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Main Topic: Platelets and Megakaryocytes
Category: Platelet Receptors
PB1734
Tetraspanin CD9 Regulates GPIIb-IIIa-Mediated Platelet Function: Identification of Unique Protein Interactomes
J. Kotha1, X. Zhai1, M. Herr1,2, S. Hill3, L. Jennings1
1CirQuest Labs, Memphis, United States, 2University of Tennessee Health Science Center, Anatomy and Neurobiology, Memphis, United States, 3University of Tennessee Health Science Center, Biochemistry, Immunology, and Biochemistry, Memphis, United States
Background: Background. Tetraspanins are transmembrane proteins involved in cellular events such as adhesion, motility, and proliferation. CD9 (Tspan-29) is highly expressed on the platelet surface. Previously, it was determined that CD9 and the platelet integrin, GPIIb-IIIa form noncovalent complexes that are dependent upon the GPIIb-IIIa conformation state.
Aims: The aim of this study was to further define how CD9 and its integrin partner, GPIIb-IIIa, regulate platelet function via common and unique protein interactomes.
Methods: We characterized CD9-dependent platelet phenotypes using Fab fragments of a well-characterized anti-CD9 mAb (mFab7). Platelet adhesion and spreading were examined on matrices fibrinogen (FG), fibronectin (FN), fibrin (Fb) or Fb-FN. Platelet activation, GPIIb-IIIa -mediated aggregation response and aggregate stability (LTA) were measured after mFab7 ligation or addition of a recombinant CD9 protein fragment. Protein interactomes were characterized by immunoprecipitation (IP) by 10E5 (anti-GPIIb-IIIa) or mAb7 (n=3) followed by shotgun mass spectrometry.
Results: Confocal microscopy showed that CD9 was highly localized in platelet-platelet contact regions. mFab7 pre-treatment increased platelet spreading and LTA responses to threshold levels of agonists ADP or collagen by 40% (p< 0.01) and 20% (p< 0.05), respectively. Consistent with platelet activation, fibrinogen binding, CD62p and CD63 expression were significantly increased
(p< 0.01). Interactome analyses identified 24 proteins common to both CD9 and GPIIb-IIIa. Six unique protein interaction partners for CD9 included tetraspanin-9, CD63 and Rho family GTPase-activating proteins (p ≤ 0.001) whereas eight unique GPIIb-IIIa partners included cytoskeleton organization regulators (p ≤ 0.01) and complexes involved in early stage hemostasis (p ≤ 0.001).
Conclusions: CD9 localizes to platelet-platelet contacts, modulates GPIIb-IIIa functional status and facilitates platelet responses to ligands upon platelet activation. Such interactions are likely controlled by co-complex members that serve as hubs for the crosstalk of different signaling events. Our findings provide new molecular insights contributing toward a better understanding of platelet signaling and function that might improve therapeutic strategies.
Category: Platelet Receptors
PB1734
Tetraspanin CD9 Regulates GPIIb-IIIa-Mediated Platelet Function: Identification of Unique Protein Interactomes
J. Kotha1, X. Zhai1, M. Herr1,2, S. Hill3, L. Jennings1
1CirQuest Labs, Memphis, United States, 2University of Tennessee Health Science Center, Anatomy and Neurobiology, Memphis, United States, 3University of Tennessee Health Science Center, Biochemistry, Immunology, and Biochemistry, Memphis, United States
Background: Background. Tetraspanins are transmembrane proteins involved in cellular events such as adhesion, motility, and proliferation. CD9 (Tspan-29) is highly expressed on the platelet surface. Previously, it was determined that CD9 and the platelet integrin, GPIIb-IIIa form noncovalent complexes that are dependent upon the GPIIb-IIIa conformation state.
Aims: The aim of this study was to further define how CD9 and its integrin partner, GPIIb-IIIa, regulate platelet function via common and unique protein interactomes.
Methods: We characterized CD9-dependent platelet phenotypes using Fab fragments of a well-characterized anti-CD9 mAb (mFab7). Platelet adhesion and spreading were examined on matrices fibrinogen (FG), fibronectin (FN), fibrin (Fb) or Fb-FN. Platelet activation, GPIIb-IIIa -mediated aggregation response and aggregate stability (LTA) were measured after mFab7 ligation or addition of a recombinant CD9 protein fragment. Protein interactomes were characterized by immunoprecipitation (IP) by 10E5 (anti-GPIIb-IIIa) or mAb7 (n=3) followed by shotgun mass spectrometry.
Results: Confocal microscopy showed that CD9 was highly localized in platelet-platelet contact regions. mFab7 pre-treatment increased platelet spreading and LTA responses to threshold levels of agonists ADP or collagen by 40% (p< 0.01) and 20% (p< 0.05), respectively. Consistent with platelet activation, fibrinogen binding, CD62p and CD63 expression were significantly increased
(p< 0.01). Interactome analyses identified 24 proteins common to both CD9 and GPIIb-IIIa. Six unique protein interaction partners for CD9 included tetraspanin-9, CD63 and Rho family GTPase-activating proteins (p ≤ 0.001) whereas eight unique GPIIb-IIIa partners included cytoskeleton organization regulators (p ≤ 0.01) and complexes involved in early stage hemostasis (p ≤ 0.001).
Conclusions: CD9 localizes to platelet-platelet contacts, modulates GPIIb-IIIa functional status and facilitates platelet responses to ligands upon platelet activation. Such interactions are likely controlled by co-complex members that serve as hubs for the crosstalk of different signaling events. Our findings provide new molecular insights contributing toward a better understanding of platelet signaling and function that might improve therapeutic strategies.
Main Topic: Platelets and Megakaryocytes
Category: Platelet Receptors
PB1734
Tetraspanin CD9 Regulates GPIIb-IIIa-Mediated Platelet Function: Identification of Unique Protein Interactomes
J. Kotha1, X. Zhai1, M. Herr1,2, S. Hill3, L. Jennings1
1CirQuest Labs, Memphis, United States, 2University of Tennessee Health Science Center, Anatomy and Neurobiology, Memphis, United States, 3University of Tennessee Health Science Center, Biochemistry, Immunology, and Biochemistry, Memphis, United States
Background: Background. Tetraspanins are transmembrane proteins involved in cellular events such as adhesion, motility, and proliferation. CD9 (Tspan-29) is highly expressed on the platelet surface. Previously, it was determined that CD9 and the platelet integrin, GPIIb-IIIa form noncovalent complexes that are dependent upon the GPIIb-IIIa conformation state.
Aims: The aim of this study was to further define how CD9 and its integrin partner, GPIIb-IIIa, regulate platelet function via common and unique protein interactomes.
Methods: We characterized CD9-dependent platelet phenotypes using Fab fragments of a well-characterized anti-CD9 mAb (mFab7). Platelet adhesion and spreading were examined on matrices fibrinogen (FG), fibronectin (FN), fibrin (Fb) or Fb-FN. Platelet activation, GPIIb-IIIa -mediated aggregation response and aggregate stability (LTA) were measured after mFab7 ligation or addition of a recombinant CD9 protein fragment. Protein interactomes were characterized by immunoprecipitation (IP) by 10E5 (anti-GPIIb-IIIa) or mAb7 (n=3) followed by shotgun mass spectrometry.
Results: Confocal microscopy showed that CD9 was highly localized in platelet-platelet contact regions. mFab7 pre-treatment increased platelet spreading and LTA responses to threshold levels of agonists ADP or collagen by 40% (p< 0.01) and 20% (p< 0.05), respectively. Consistent with platelet activation, fibrinogen binding, CD62p and CD63 expression were significantly increased
(p< 0.01). Interactome analyses identified 24 proteins common to both CD9 and GPIIb-IIIa. Six unique protein interaction partners for CD9 included tetraspanin-9, CD63 and Rho family GTPase-activating proteins (p ≤ 0.001) whereas eight unique GPIIb-IIIa partners included cytoskeleton organization regulators (p ≤ 0.01) and complexes involved in early stage hemostasis (p ≤ 0.001).
Conclusions: CD9 localizes to platelet-platelet contacts, modulates GPIIb-IIIa functional status and facilitates platelet responses to ligands upon platelet activation. Such interactions are likely controlled by co-complex members that serve as hubs for the crosstalk of different signaling events. Our findings provide new molecular insights contributing toward a better understanding of platelet signaling and function that might improve therapeutic strategies.
Category: Platelet Receptors
PB1734
Tetraspanin CD9 Regulates GPIIb-IIIa-Mediated Platelet Function: Identification of Unique Protein Interactomes
J. Kotha1, X. Zhai1, M. Herr1,2, S. Hill3, L. Jennings1
1CirQuest Labs, Memphis, United States, 2University of Tennessee Health Science Center, Anatomy and Neurobiology, Memphis, United States, 3University of Tennessee Health Science Center, Biochemistry, Immunology, and Biochemistry, Memphis, United States
Background: Background. Tetraspanins are transmembrane proteins involved in cellular events such as adhesion, motility, and proliferation. CD9 (Tspan-29) is highly expressed on the platelet surface. Previously, it was determined that CD9 and the platelet integrin, GPIIb-IIIa form noncovalent complexes that are dependent upon the GPIIb-IIIa conformation state.
Aims: The aim of this study was to further define how CD9 and its integrin partner, GPIIb-IIIa, regulate platelet function via common and unique protein interactomes.
Methods: We characterized CD9-dependent platelet phenotypes using Fab fragments of a well-characterized anti-CD9 mAb (mFab7). Platelet adhesion and spreading were examined on matrices fibrinogen (FG), fibronectin (FN), fibrin (Fb) or Fb-FN. Platelet activation, GPIIb-IIIa -mediated aggregation response and aggregate stability (LTA) were measured after mFab7 ligation or addition of a recombinant CD9 protein fragment. Protein interactomes were characterized by immunoprecipitation (IP) by 10E5 (anti-GPIIb-IIIa) or mAb7 (n=3) followed by shotgun mass spectrometry.
Results: Confocal microscopy showed that CD9 was highly localized in platelet-platelet contact regions. mFab7 pre-treatment increased platelet spreading and LTA responses to threshold levels of agonists ADP or collagen by 40% (p< 0.01) and 20% (p< 0.05), respectively. Consistent with platelet activation, fibrinogen binding, CD62p and CD63 expression were significantly increased
(p< 0.01). Interactome analyses identified 24 proteins common to both CD9 and GPIIb-IIIa. Six unique protein interaction partners for CD9 included tetraspanin-9, CD63 and Rho family GTPase-activating proteins (p ≤ 0.001) whereas eight unique GPIIb-IIIa partners included cytoskeleton organization regulators (p ≤ 0.01) and complexes involved in early stage hemostasis (p ≤ 0.001).
Conclusions: CD9 localizes to platelet-platelet contacts, modulates GPIIb-IIIa functional status and facilitates platelet responses to ligands upon platelet activation. Such interactions are likely controlled by co-complex members that serve as hubs for the crosstalk of different signaling events. Our findings provide new molecular insights contributing toward a better understanding of platelet signaling and function that might improve therapeutic strategies.
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