@article{PoppThielmanNieswandtetal.2015, author = {Popp, Michael and Thielman, Ina and Nieswandt, Bernhard and Stegner, David}, title = {Normal Platelet Integrin Function in Mice Lacking Hydrogen Peroxide-Induced Clone-5 (Hic-5)}, series = {PLoS One}, volume = {10}, journal = {PLoS One}, number = {7}, doi = {10.1371/journal.pone.0133429}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-125724}, pages = {e0133429}, year = {2015}, abstract = {Integrin αIIbβ3 plays a central role in the adhesion and aggregation of platelets and thus is essential for hemostasis and thrombosis. Integrin activation requires the transmission of a signal from the small cytoplasmic tails of the α or β subunit to the large extracellular domains resulting in conformational changes of the extracellular domains to enable ligand binding. Hydrogen peroxide-inducible clone-5 (Hic-5), a member of the paxillin family, serves as a focal adhesion adaptor protein associated with αIIbβ3 at its cytoplasmic tails. Previous studies suggested Hic-5 as a novel regulator of integrin αIIbβ3 activation and platelet aggregation in mice. To assess this in more detail, we generated Hic-5-null mice and analyzed activation and aggregation of their platelets in vitro and in vivo. Surprisingly, lack of Hic-5 had no detectable effect on platelet integrin activation and function in vitro and in vivo under all tested conditions. These results indicate that Hic-5 is dispensable for integrin αIIbβ3 activation and consequently for arterial thrombosis and hemostasis in mice.}, language = {en} } @phdthesis{Popp2018, author = {Popp, Michael}, title = {Mechanisms of platelet activation and receptor regulation in genetically modified mice}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-135494}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2018}, abstract = {This work summarizes the results of studies on several major aspects of platelet activation and platelet receptor regulation. Therefore, this thesis is divided into four parts. Platelet activation and aggregation at sites of vascular injury is critical to prevent excessive blood loss, but may also lead to life-threatening ischemic disease states, such as myocardial infarction and stroke. Agonist-induced elevation in cytosolic Ca2+ concentrations is essential for platelet activation in hemostasis and thrombosis. The principal route of Ca2+ influx in platelets is store-operated calcium entry (SOCE). The calcium sensor molecule stromal interaction molecule 1 (STIM1) regulates SOCE by activating the membrane calcium channel protein Orai1, but the exact mechanisms of this interaction are not fully understood. Using affinity chromatography to screen for STIM1 interacting proteins in platelets, bridging integrator 2 (BIN2), an adapter protein belonging to the family of BAR proteins that is mainly expressed in the hematopoietic system, was identified. Newly generated BIN2 KO mice were viable and fertile but their platelets displayed markedly impaired SOCE in response to thapsigargin (TG) as well as agonists acting on immunoreceptor tyrosine-based activation motif (ITAM) or G protein-coupled receptors. This SOCE defect resulted in impaired (hem)ITAM induced platelet activation, aggregate formation under flow and procoagulant activity. As a consequence, mice lacking BIN2 in platelets were protected from occlusive arterial thrombus formation and thrombo-inflammatory cerebral infarct progression in a model of experimental stroke. These results identify BIN2 as a critical regulator of platelet SOCE in thrombosis and thrombo-inflammatory disease. Integrin αIIbβ3 plays a central role in the adhesion and aggregation of platelets. Integrin activation requires the transmission of a signal from the small cytoplasmic tails of the α or β subunit to the large extracellular domains resulting in conformational changes of the extracellular domains to enable ligand binding. It was hypothesized that Hic-5 is a novel regulator of integrin αIIbβ3 activation in mice. As demonstrated in the second part of this thesis, lack of Hic-5 had no detectable effect on platelet integrin activation and function in vitro and in vivo under all tested conditions. These results indicate that Hic-5 is dispensable for integrin αIIbβ3 activation and consequently for arterial thrombosis and hemostasis in mice. The Rho GTPase family members RhoA and Rac1 play major roles in platelet activation at sites of vascular injury. Little is known about possible redundant functions of these Rho GTPases in regulating platelet function. To investigate functional redundancies of RhoA and Rac1 in platelet production and function, mice with MK- and platelet-specific double- deficiencies in RhoA and Rac1 were generated. RhoA/Rac1 double-deficiency phenocopied the respective single knockouts without any additional effects in the double-knockout animals, demonstrating for the first time a functional non-redundancy of RhoA and Rac1 in platelet function. Antibodies against platelet glycoproteins (GP) trigger platelet destruction in immune thrombocytopenia (ITP) by binding to Fcγ receptors (FcγRs) on immune cells. However, antibodies against the platelet collagen receptor GPVI exert powerful anti-thrombotic action in vivo by inducing ectodomain shedding of the receptor associated with a transient thrombocytopenia. As shown in the final part of this thesis, blockade or deficiency of the inhibitory FcγRIIB abolished sequestration of anti-GPVI opsonized platelets in the hepatic vasculature and GPVI shedding. This process was mediated by liver sinusoidal endothelial cells (LSEC), the major FcγRIIB expressing cell type in the body. Furthermore, LSEC FcγRIIB mediated hepatic platelet sequestration and contributed to thrombocytopenia in mice treated with antibodies against αIIbβ3, the major target antigen in human ITP. These results reveal a novel and unexpected function of hepatic FcγRIIB in the processing of antibody-opsonized platelets.}, subject = {H{\"a}mostase}, language = {en} } @article{DuettingGaitsIacovoniStegneretal.2017, author = {D{\"u}tting, Sebastian and Gaits-Iacovoni, Frederique and Stegner, David and Popp, Michael and Antkowiak, Adrien and van Eeuwijk, Judith M.M. and Nurden, Paquita and Stritt, Simon and Heib, Tobias and Aurbach, Katja and Angay, Oguzhan and Cherpokova, Deya and Heinz, Niels and Baig, Ayesha A. and Gorelashvili, Maximilian G. and Gerner, Frank and Heinze, Katrin G. and Ware, Jerry and Krohne, Georg and Ruggeri, Zaverio M. and Nurden, Alan T. and Schulze, Harald and Modlich, Ute and Pleines, Irina and Brakebusch, Cord and Nieswandt, Bernhard}, title = {A Cdc42/RhoA regulatory circuit downstream of glycoprotein Ib guides transendothelial platelet biogenesis}, series = {Nature Communications}, volume = {8}, journal = {Nature Communications}, number = {15838}, doi = {10.1038/ncomms15838}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170797}, year = {2017}, abstract = {Blood platelets are produced by large bone marrow (BM) precursor cells, megakaryocytes (MKs), which extend cytoplasmic protrusions (proplatelets) into BM sinusoids. The molecular cues that control MK polarization towards sinusoids and limit transendothelial crossing to proplatelets remain unknown. Here, we show that the small GTPases Cdc42 and RhoA act as a regulatory circuit downstream of the MK-specific mechanoreceptor GPIb to coordinate polarized transendothelial platelet biogenesis. Functional deficiency of either GPIb or Cdc42 impairs transendothelial proplatelet formation. In the absence of RhoA, increased Cdc42 activity and MK hyperpolarization triggers GPIb-dependent transmigration of entire MKs into BM sinusoids. These findings position Cdc42 (go-signal) and RhoA (stop-signal) at the centre of a molecular checkpoint downstream of GPIb that controls transendothelial platelet biogenesis. Our results may open new avenues for the treatment of platelet production disorders and help to explain the thrombocytopenia in patients with Bernard-Soulier syndrome, a bleeding disorder caused by defects in GPIb-IX-V.}, language = {en} }