Recent advances in the understanding of the molecular mechanisms regulating platelet integrin αIIbβ3 activation

Integrins are allosteric cell adhesion receptors that cycle from a low to a high affinity ligand binding state,a complex process of receptor activation that is of particular importance in blood cells such as platelets or leukocytes.Here we highlight recent progress in the understanding of the molecular pathways that regulate integrin activation in platelets and leukocytes,with a special focus on the structural changes in platelet integrinαIIbβ3 brought about by key intracellular proteins,namely talin and kindlins,that are of crucial importance in the regulation of integrin function.Evidence that the small GTPase Rap1 and its guanine exchange factor CalDAG-GEF1,together with RIAM,a Rap1GTP adaptor protein,promote the interaction of talin with the integrinβsubunit,has greatly contributed to fill the gap in our understanding of the signaling pathway from G-coupled agonist receptors and their phospholipase C-dependant second messengers,to integrin activation.Studies of patients with the rare blood cell disorder LAD-III have contributed to the identification of kindlins as new co-regulators of the talin-dependent integrin activation process in platelets and leukocytes,underlining the relevance for the in-depth investigation of patients with rare genetic blood cell disorders.

Long-term correction of hemorrhagic diathesis in hemophilia A mice by an AAV-delivered hybrid FⅧcomposed of the human heavy chain and the rat light chain

Conventional therapies for hemophilia A(HA)are prophylactic or on-demand intravenous FⅧinfusions.However,they are expensive and inconvenient to perform.Thus,better strategies for HA treatment must be developed.In this study,a recombinant FⅧcDNA encoding a human/rat hybrid FⅧwith an enhanced procoagulant potential for adeno-associated virus(AAV)-delivered gene therapy was developed.Plasmids containing human FⅧheavy chain(hHC),human light chain(hLC),and rat light chain(rLC)were transfected into cells and hydrodynamically injected into HA mice.Purified AAV viruses were intravenously injected into HA mice at two doses.Results showed that the hHC+rLC protein had a higher activity than the hHC+hLC protein at comparable expression levels.The specific activity of hHC+rLC was about 4-to 8-fold higher than that of their counterparts.Hydrodynamic injection experiments obtained consistent results.Notably,the HA mice undergoing the AAV-delivered hHC+rLC treatment exhibited a visibly higher activity than those treated with hHC+hLC,and the therapeutic effects lasted for up to 40 weeks.In conclusion,the application of the hybrid FⅧ(hHC+rLC)via an AAV-delivered gene therapy substantially improved the hemorrhagic diathesis of the HA mice.These data might be of help to the development of optimized FⅧexpression cassette for HA gene therapy.

A rare case of B-lymphoproliferative disorder with villous lymphocytes harboring t（8;14）（q24;q32） translocation

Splenic lymphoma with villous lymphocytes （SLVL） or splenic marginal zone lymphoma with circulating villous lymphocytes is rare, and prolymphocytic transformation of SLVL is rarer. At present, only one case of SLVL with t（8;14）（q24;q32） translocation has been reported. In this study, we report a case of B- lymphoproliferative disorder with villous lymphocytes harboring t（8;14）（q24;q32） chromosome translocation that we inclined to SLVL with a prolymphocytic transformation. A 73-year-old female showed marked hepatosplenomegaly and high lymphocytosis （lymphocytes 〉 200 × 10^9/L）. The abnormal lymphocytes had short coarse villi and round nuclei with prominent nucleoli. The immunophenotypes showed CD19^＋, CD20^＋, HLA- DR^＋, CD22^＋, CD5^＋, Kappa^＋, CD25^dim, CD71^dim, Lambda, CD7, CD10-, CD23 , CD34, CD33, CD13 , CD14, CDll7, CD64, CD103, and CD11c-. The karyotype showed complex abnormality： 46XX,＋ 3,-10, t（8;14）（q24; q32）lll]/46XX[9]. The cytoplasmic projection, immunological characteristics, and trisomy 3 chromosome abnormality supported the diagnosis of SLVL. However, the presence of prominent nucleoli and high lymphocytosis suggested prolymphocytic transformation, probably as a result of t（8,14） chromosome translocation. In this report, we described an unusual case of B-lymphoproliferative disorder with villous lymphocytes harboring t（8;14）（q24;q32） translocation, which could provide help in the diagnosis and differential diagnosis of B-lymphocytic proliferative diseases.

Roles of integrin β3 cytoplasmic tail in bidirectional signal transduction in a trans-dominant inhibition model

We evaluated the roles of calpain cleavage-related mutations of the integrin β3 cytoplasmic tail in integrin αIIbβ3 bidirectional signaling using a trans-dominant inhibition model. Chimeric Tac-β3 proteins （i.e., Tac-β3, Tac-β3△741, Tac-β3△747, Tac-β3△754, Tac-β3△759, and Tac-β3ANITY） consisting of the extracellular and transmembrane domains of human IL-2 receptor （Tac） and the human integrin β3 cytoplasmic domain were stably expressed in the 123 CHO cells harboring human glycoprotein Ib-IX and wild-type integrin uIIbβ3. The different cells were assayed for stable adhesion and spreading on immobilized fibrinogen, and for binding soluble fibrinogen representing outside-in and inside-out signaling events, respectively. The chimeric protein Tac-β3 inhibited, and Tac-β3△NITY partially attenuated stable adhesion and spreading. Tac-β3, Tac-β3△759, Tac-β3ANITY, and Tac-β3△754, but not Tac-β3△747 or Tac-β3△741, impaired the soluble fibrinogen binding. Results indicated that the bidirectional signaling was significantly inhibited by Tac-β3△ and Tac-β3ANITY, albeit to a much lesser extent. Moreover, only inside-out signaling was impaired in the 123/Tac-β3△759 and 123/Tac-β3△754 cells in contrast to an intact bidirectional signaling in the 123/Tac-β3△747 and 123/Tac-β3△741 cells. In conclusion, the calpain cleavage of integrin β3 resulted in the regulatory effects on signaling by interrupting its interaction with cytoplasmic proteins rather than altering its conformation, and may thus regulate platelet function.