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1.
Nat Commun ; 5: 3738, 2014 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-24769558

RESUMO

To enable leukocyte adhesion to activated endothelium, the leukocyte receptor P-selectin is released from Weibel-Palade bodies (WPB) to the endothelial cell surface where it is stabilized by CD63. Here we report that loss of annexin A8 (anxA8) in human umbilical vein endothelial cells (HUVEC) strongly decreases cell surface presentation of CD63 and P-selectin, with a concomitant reduction in leukocyte rolling and adhesion. We confirm the compromised leukocyte adhesiveness in inflammatory-activated endothelial venules of anxA8-deficient mice. We find that WPB of anxA8-deficient HUVEC contain less CD63, and that this is caused by improper transport of CD63 from late multivesicular endosomes to WPB, with CD63 being retained in intraluminal vesicles. Consequently, reduced CD63 cell surface levels are seen following WPB exocytosis, resulting in enhanced P-selectin re-internalization. Our data support a model in which anxA8 affects leukocyte recruitment to activated endothelial cells by supplying WPB with sufficient amounts of the P-selectin regulator CD63.


Assuntos
Anexinas/metabolismo , Adesão Celular/imunologia , Células Endoteliais/imunologia , Leucócitos/imunologia , Modelos Imunológicos , Tetraspanina 30/metabolismo , Corpos de Weibel-Palade/metabolismo , Análise de Variância , Animais , Anticorpos Monoclonais , Southern Blotting , Western Blotting , Primers do DNA/genética , Células Endoteliais/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imunofluorescência , Células Endoteliais da Veia Umbilical Humana , Humanos , Leucócitos/metabolismo , Camundongos , Microscopia de Força Atômica , Selectina-P/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética
2.
Blood ; 122(6): 1042-51, 2013 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-23757730

RESUMO

The large multimeric glyocoprotein von Willebrand factor (VWF) is a crucial component of both primary and secondary hemostasis. It is stored in secretory granules of vascular endothelial cells, the Weibel-Palade bodies (WPBs), and is released following stimulation by agonists that raise intracellular Ca(2+) or cyclic adenosine monophosphate (cAMP) levels. cAMP-induced exocytosis of WPBs requires protein kinase A activity, but downstream factors that are regulated by phosphorylation/dephosphorylation are not known. Here we identify the complex consisting of the lipid-binding protein annexin A2 (AnxA2) and S100A10 as such a factor. Knockdown and specific rescue approaches reveal that a functional AnxA2-S100A10 complex is required for the forskolin-induced, cAMP-dependent release of VWF. Forskolin triggers dephosphorylation of AnxA2 that is mediated by a calcineurin-like phosphatase and stabilizes the AnxA2-S100A10 complex, thereby promoting VWF release. Serine 11 of AnxA2 was identified as the target residue of this phosphorylation switch because a phosphomimicking mutation at this site prevents complex formation with S100A10 and, in contrast to wild-type or S11A-AnxA2, is unable to restore cAMP-dependent VWF secretion in AnxA2-depleted cells. Thus, complex formation of AnxA2 with S100A10 is a central regulatory mechanism in the acute release of VWF in response to cAMP-elevating agonists.


Assuntos
Anexina A2/metabolismo , AMP Cíclico/metabolismo , Endotélio/metabolismo , Fator de von Willebrand/metabolismo , Calcineurina/metabolismo , Cálcio/metabolismo , Colforsina/farmacologia , Exocitose , Células Endoteliais da Veia Umbilical Humana , Humanos , Fosforilação , Ligação Proteica , Transporte Proteico , Proteínas S100/metabolismo , Corpos de Weibel-Palade/metabolismo , Corpos de Weibel-Palade/patologia
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