Inherent hepatocytic heterogeneity determines expression and retention of edited F9 alleles post-AAV/CRISPR infusion.

Infusing CRISPR/donor-loaded adeno-associated viral vectors (AAV/CRISPR) could enable in vivo hepatic gene editing to remedy hemophilia B (HB) with inherited deficiency of clotting factor IX (FIX). Yet, current regimens focus on correcting HB with simple mutations in the coding region of the F9, overlooking those carrying complicated mutations involving the regulatory region. Moreover, a possible adverse effect of treatment-related inflammation remains unaddressed. Here we report that a single DNA cutting-mediated long-range replacement restored the FIX-encoding function of a mutant F9 (mF9) carrying both regulatory and coding defects in a severe mouse HB model, wherein incorporation of a synthetic Alb enhancer/promoter-mimic (P2) ensured FIX elevation to clinically meaningful levels. Through single-cell RNA sequencing (scRNA-seq) of liver tissues, we revealed that a subclinical hepatic inflammation post-AAV/CRISPR administration regulated the vulnerability of the edited mF9-harboring host cells to cytotoxic T lymphocytes (CTLs) and the P2 activity in a hepatocytic subset-dependent manner via modulating specific sets of liver-enriched transcription factors (LETFs). Collectively, our study establishes an AAV/CRISPR-mediated gene-editing protocol applicable to complicated monogenetic disorders, underscoring the potentiality of improving therapeutic benefits through managing inflammation.

RIG-I modulates Src-mediated AKT activation to restrain leukemic stemness.

Retinoic acid (RA)-inducible gene I (RIG-I) is highly upregulated and functionally implicated in the RA-induced maturation of acute myeloid leukemia (AML) blasts. However, the underlying mechanism and the biological relevance of RIG-I expression to the maintenance of leukemogenic potential are poorly understood. Here, we show that RIG-I, without priming by foreign RNA, inhibits the Src-facilitated activation of AKT-mTOR in AML cells. Moreover, in a group of primary human AML blasts, RIG-I reduction renders the Src family kinases hyperactive in promoting AKT activation. Mechanistically, a PxxP motif in RIG-I, upon the N-terminal CARDs' association with the Src SH1 domain, competes with the AKT PxxP motif for recognizing the Src SH3 domain. In accordance, mutating PxxP motif prevents Rig-I from inhibiting AKT activation, cytokine-stimulated myeloid progenitor proliferation, and in vivo repopulating capacity of leukemia cells. Collectively, our data suggest an antileukemia activity of RIG-I via competitively inhibiting Src/AKT association.

Homoharringtonine deregulates MYC transcriptional expression by directly binding NF-κB repressing factor.

Homoharringtonine (HHT), a known protein synthesis inhibitor, has an anti-myeloid leukemia effect and potentiates the therapeutic efficacy of anthracycline/cytarabine induction regimens for acute myelogenous leukemia (AML) with favorable and intermediate prognoses, especially in the t(8;21) subtype. Here we provide evidence showing that HHT inhibits the activity of leukemia-initiating cells (Lin-/Sca-1-/c-kit+; LICs) in a t(8;21) murine leukemia model and exerts a down-regulating effect on MYC pathway genes in human t(8;21) leukemia cells (Kasumi-1). We discovered that NF-κB repressing factor (NKRF) is bound directly by HHT via the second double-strand RNA-binding motif (DSRM2) domain, which is the nuclear localization signal of NKRF. A series of deletion and mutagenesis experiments mapped HHT direct binding sites to K479 and C480 amino acids in the DSRM2 domain. HHT treatment shifts NKRF from the nucleus (including nucleoli) to the cytoplasm by occupying the DSRM2 domain, strengthens the p65-NKRF interaction, and interferes with p65-p50 complex formation, thereby attenuating the transactivation activity of p65 on the MYC gene. Moreover, HHT significantly decreases the expression of KIT, a frequently mutated and/or highly expressed gene in t(8;21) AML, in concert with MYC down-regulation. Our work thus identifies a mechanism of action of HHT that is different from, but acts in concert with, the known mode of action of this compound. These results justify further clinical testing of HHT in AML.

PALLD Regulates Phagocytosis by Enabling Timely Actin Polymerization and Depolymerization.

PALLD is an actin cross-linker supporting cellular mechanical tension. However, its involvement in the regulation of phagocytosis, a cellular activity essential for innate immunity and physiological tissue turnover, is unclear. We report that PALLD is highly induced along with all-trans-retinoic acid-induced maturation of myeloid leukemia cells, to promote Ig- or complement-opsonized phagocytosis. PALLD mechanistically facilitates phagocytic receptor clustering by regulating actin polymerization and c-Src dynamic activation during particle binding and early phagosome formation. PALLD is also required at the nascent phagosome to recruit phosphatase oculocerebrorenal syndrome of Lowe, which regulates phosphatidylinositol-4,5-bisphosphate hydrolysis and actin depolymerization to complete phagosome closure. Collectively, our results show a new function for PALLD as a crucial regulator of the early phase of phagocytosis by elaborating dynamic actin polymerization and depolymerization.

Arsenic trioxide at conventional dosage does not aggravate hemorrhage in the first-line treatment of adult acute promyelocytic leukemia.

OBJECTIVES: The arsenic trioxide (ATO) plus all-trans retinoic acid (ATRA) therapy has demonstrated a tremendous success in the first-line treatment of acute promyelocytic leukemia (APL). Actually, early death (ED) is currently thought as a major challenge in APL. ATO has been reported to inhibit platelet function in vitro, and whether it increases the ED rate by exacerbating the hemorrhagic symptoms remains to be investigated. METHODS: Effects of ATO on platelet aggregation and adhesion were evaluated in vitro and in thirty-two complete remission (CR) and four newly diagnosed APL patients. Furthermore, concentrations of plasma total arsenic were monitored in APL patients via ICP-MS. RESULTS: The inhibition of platelet function, either aggregation or adhesion, did occur in vitro when the concentration of ATO reached 2 µmol/L. However, in CR APL patients receiving ATO with normal platelet count, the platelets responded normally when being activated and so did those in the newly diagnosed patients with thrombocytopenia. Our data further showed that the conventional dosage of ATO reached a plasma concentration substantially below the required concentration to inhibit platelets. CONCLUSIONS: In the first-line treatment of APL, the use of ATO is safe and effective and does not compromise the hemostatic potential that may eventually increase ED rate.

Preferential eradication of acute myelogenous leukemia stem cells by fenretinide.

Leukemia stem cells (LSCs) play important roles in leukemia initiation, progression, and relapse, and thus represent a critical target for therapeutic intervention. However, relatively few agents have been shown to target LSCs, slowing progress in the treatment of acute myelogenous leukemia (AML). Based on in vitro and in vivo evidence, we report here that fenretinide, a well-tolerated vitamin A derivative, is capable of eradicating LSCs but not normal hematopoietic progenitor/stem cells at physiologically achievable concentrations. Fenretinide exerted a selective cytotoxic effect on primary AML CD34(+) cells, especially the LSC-enriched CD34(+)CD38(-) subpopulation, whereas no significant effect was observed on normal counterparts. Methylcellulose colony formation assays further showed that fenretinide significantly suppressed the formation of colonies derived from AML CD34(+) cells but not those from normal CD34(+) cells. Moreover, fenretinide significantly reduced the in vivo engraftment of AML stem cells but not normal hematopoietic stem cells in a nonobese diabetic/SCID mouse xenotransplantation model. Mechanistic studies revealed that fenretinide-induced cell death was linked to a series of characteristic events, including the rapid generation of reactive oxygen species, induction of genes associated with stress responses and apoptosis, and repression of genes involved in NF-κB and Wnt signaling. Further bioinformatic analysis revealed that the fenretinide-down-regulated genes were significantly correlated with the existing poor-prognosis signatures in AML patients. Based on these findings, we propose that fenretinide is a potent agent that selectively targets LSCs, and may be of value in the treatment of AML.

Eriocalyxin B ameliorates experimental autoimmune encephalomyelitis by suppressing Th1 and Th17 cells.

Eriocalyxin B (EriB), a diterpenoid isolated from Isodon eriocalyx, was previously reported to have antitumor effects via multiple pathways, and these pathways are related to immune responses. In this study, we demonstrated that EriB was efficacious in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. Treatment with EriB led to amelioration of EAE, which correlated with reduced spinal cord inflammation and demyelination. EriB treatment abolished encephalitogenic T-cell responses to myelin oligodendrocyte glycoprotein in an adoptive transfer EAE model. The underlying mechanism of EriB-induced effects involved inhibition of T helper (Th) 1 and Th17 cell differentiation through Janus Kinase/Signal Transducer and Activator Of Transcription and Nuclear factor-κB signaling pathways as well as elevation of reactive oxygen species. These findings indicate that EriB exerts potent antiinflammatory effects through selective modulation of pathogenic Th1 and Th17 cells by targeting critical signaling pathways. The study provides insights into the role of EriB as a unique therapeutic agent for the treatment of autoimmune diseases.

Structural framework of c-Src activation by integrin ß3.

The integrin ß3-mediated c-Src priming and activation, via the SH3 domain, is consistently associated with diseases, such as the formation of thrombosis and the migration of tumor cells. Conventionally, activation of c-Src is often induced by the binding of proline-rich sequences to its SH3 domain. Instead, integrin ß3 uses R(760)GT(762) for priming and activation. Because of the lack of structural information, it is not clear where RGT will bind to SH3, and under what mechanism this interaction can prime/activate c-Src. In this study, we present a 2.0-Å x-ray crystal structure in which SH3 is complexed with the RGT peptide. The binding site lies in the "N"-Src loop of the SH3 domain. Structure-based site-directed mutagenesis showed that perturbation on the "N"-Src loop disrupts the interaction between the SH3 domain and the RGT peptide. Furthermore, the simulated c-Src:ß3 complex based on the crystal structure of SH3:RGT suggests that the binding of the RGT peptide might disrupt the intramolecular interaction between the SH3 and linker domains, leading to the disengagement of Trp260:"C"-helix and further activation of c-Src.

Molecular basis of type I antithrombin deficiency in two women with recurrent venous thromboembolism in the first trimester of pregnancy.

Inherited antithrombin (AT) deficiency carries a 50% risk of venous thromboembolism (VTE) during pregnancy. Here, we investigated the molecular basis of type I AT deficiency in two women with recurrent VTE in the first trimester of pregnancy. Phenotype analysis showed both probands had almost 50% of normal AT levels. Two novel heterozygous AT mutations were identified: g.7920C>T resulting in a Trp225Cys mutation in case 1 and g.13863C>A causing an Ala404Asp mutation in case 2. Transient expression of either wild-type (WT) or mutant AT expression vectors in HEK293T and CHO cells showed impaired secretion of both AT mutant proteins. Immunofluorescence analysis revealed that the staining of AT-Trp225Cys in both endoplasmic reticulum (ER) and Golgi apparatus was similar to that of AT-WT, and the staining of AT-Ala404Asp was mainly present in ER but was weaker than that of AT-WT. These results revealed that the type I AT deficiency in two patients was caused by impaired secretion of the AT-Trp225Cys and AT-Ala404Asp mutant proteins, respectively. The two mutations are associated with a high risk of thrombotic onset and women with these AT mutations are prone to VTE in early pregnancy.

[Phenotype and genotype analysis of two Chinese pedigrees with type 3 von Willebrand diseases].

OBJECTIVE: To analyze the phenotype and genotype of two Chinese pedigrees with von Willebrand diseases, and to investigate the molecular pathogenesis. METHODS: Bleeding time (BT), activated partial thromboplastin time (APTT), ristocetin-induced platelet aggregation (RIPA), von Willebrand factor-ristocetin cofactor (vWF:Rco), von Willebrand factor antigen (vWF:Ag), von Willebrand factor activity (vWF:A), von Willebrand factor collagen binding assay (vWF:CB) and multimer analysis were used for phenotype diagnosis. DNA was extracted. All of the 52 exons and exon-intron bounda ries of the VWF gene were amplified with polymerase chain reaction(PCR) and analyzed by direct sequencing. RESULTS: APTT and BT were prolonged. Plasma RIPA, vWF:Rco, vWF:Ag, vWF:A and vWF:CB was significantly decreased. No VWF multimer can be found by plasma VWF multimer analysis. Homozygous insertional mutation g.82888_82889insCATG in exon 17 was found in proband A. Compound heterozygous mutations g.94865 G to A (Trp856stop) in exon 20 and g.110698_110699delinsG in exon 28 were found in proband B. CONCLUSION: Homozygous insertional mutation g.82888_82889insCATG and compound heterozygous mutations g.94865G to A(Trp856X) and g.110698_110699delinsG probably have respectively induced type 3 von Willebrand diseases in the two probands.

[The phenotypic and genotypic diagnosis of three Chinese patients with von Willebrand disease].

OBJECTIVE: To analyze the phenotype and genotype of three patients with von Willebrand disease (vWD), and to explore its molecular pathogenesis. METHODS: Bleeding time (BT), APTT, ristocetin induced platelet aggregation (RIPA), von Willebrand factor (vWF):ristocetin cofactor (Rco) (vWF:Rco), vWF antigen (vWF:Ag), vWF activity (vWF:A) test, vWF collagen binding assay (vWF:CB) and multimer analysis were detected for phenotype diagnosis. The dynamic process of blood coagulation was evaluated by using the thrombelastography. Genomic DNA was extracted from the peripheral blood. The vWF gene mutation was detected by sequencing. RESULTS: APTT, BT were prolonged in the three probands. Plasma vWF:Rco, vWF:Ag, vWF:A and vWF:CB were decreased in different degrees. RIPA was reduced in probands B and C. vWF multimer analysis found the lost of the large molecular weight multimers in proband B, while basically normal in probands A and C. The dynamic process of blood coagulation of proband C presented obvious hypocoagulability by using the thrombelastography. Heterozygous missense mutation g.106782G > T resulting in Cys1130Phe in exon 26, g.110988G > A resulting in Gly1579Arg in exon 28 and g.110373C > T resulting in Arg1374Cys in exon 28 were found in the probands A, B and C, respectively. CONCLUSION: Three probands were diagnosed as type 1, type 2A or type 2M vWD by phenotype detection. Heterozygous missense mutation Cys1130Phe, Gly1579Arg and Arg1374Cys induced vWD of three probands, respectively.

[Promoting Effect of Rat FVIII Light Chain Delivered by Adeno-Associated Virus Vector Serotype 8 on the Activity of Human FVIII Heavy Chain with Different Length].

OBJECTIVE: To explore the effects of the rat FVIII light chain (rLC) on the activity of human FVIII heavy chain hHC745 and hHC1690. METHODS: hHC745, hHC1690, human FVIII light chain (hLC) and rLC were cloned into adeno-associated virus serotype 8 (AAV8) expression vectors with CB promoter (ubiquitous expression), respectively, and transfected into 293T cells using a dual-chain strategy of co-expression of HC and LC. The cultured cell supernatant was collected at 48 hours after transfection. The plasmids (pAAV8-CB-hHC745, pAAV8-CB-hHC1690, pAAV8-CB-hLC and pAAV8-CB-rLC) were hydrodynamically injected into hemophilia A (HA) mice via lateral tail vein. Forty-eight hours after injection, the peripheral blood of the mice was collected through retroorbital venous plexus and the plasma was obtained by centrifugation. The activity of FVIII was detected by activated partial thromboplastin time (aPTT) assay, and the antigen expression level of FVIII was determined by enzyme-linked immunosorbent assay (ELISA). The specific activity of FVIII was calculated based on the activity and the antigen expression level. RESULTS: In 293T cells, the coagulation activity of FVIII was significantly enhanced when hHC745 and hHC1690 combined with rLC compared with them combined with hLC. The FVIII activity of hHC745+rLC was about 4.6 times higher than that of hHC745+hLC, while hHC1690+rLC was about 2.9 times higher than that of hHC1690+hLC. The data from ELISA showed that there was no significant difference in FVIII antigen expression when hHC745 and hHC1690 combined with rLC and hLC. The specific activity of hHC745+rLC increased to about 4.1 times compared with hHC745+hLC, while that of hHC1690+rLC increased to about 2.8 times compared with hHC1690+hLC. In HA mice administrated with hydrodynamic injection, the FVIII activity of hHC745+rLC and hHC1960+rLC was significantly higher than that of hHC745+hLC and hHC1690+hLC at comparable expression level. The FVIII activity of hHC745+rLC was significantly higher than that of hHC745+hLC, increasing to about 5.1 times, while, that of hHC1690+rLC increasing to about 2.1 times than hHC1690+hLC. ELISA results also showed that there was no significant difference in FVIII antigen expression when hHC745 and hHC1690 combined with rLC and hLC. The specific activity of hHC745+rLC increased to about 4.2 times compared with hHC745+hLC, while that of hHC1690+rLC increased to about 1.8 times compared with hHC1690+hLC. In addition, the activity of hHC1690 combined with rLC was significantly higher than that of hHC745 combined with rLC. CONCLUSION: rLC can significantly enhance the coagulation activity of FVIII when co-expressed with hHC of different length including hHC745 and hHC1690.

Lymphadenopathy in POEMS syndrome: a correlation between clinical features and imaging findings.

Lymphadenopathy is an important characteristic of POEMS syndrome, and a Castleman disease (CD)-like pathologic change in the lymph nodes is one of the major diagnostic criteria. However, the characteristics of lymphadenopathy in POEMS still have not been completely elucidated. The lymph node biopsies are available only for a small proportion of patients. A simple and safe way is needed to rule CD in or out. This study aimed to analyse the features of lymphadenopathy and estimate the role of imaging methods, including computed tomography (CT) and positron emission tomography-CT (PET/CT), in the diagnosis of lymphadenopathy in patients with POEMS syndrome. We conducted a retrospective analysis of 23 patients with confirmed POEMS syndrome. All of the patients received chest and abdominal CT scan and/or superficial ultrasound examinations. Four patients underwent PET/CT examinations, and 6 patients received lymph node biopsies. Enlarged lymph nodes (short diameter ≥ 1 cm) were found in 48% (11/23) of patients, but only 1 patient had an enlarged lymph node with a diameter ≥ 2 cm. Lymph nodes with CD-like pathologic changes from 2 patients showed increased maximum standard uptake values (SUVmax) of 18F-deoxyglucose (18FDG) on PET/CT, while lymph nodes with reactive pathologic changes from 2 other patients showed a normal metabolic PET/CT profile. The extent of lymph node enlargement in patients with POEMS was less than that in patients with CD per se. We draw the conclusion that most of the enlarged lymph nodes had diameters ≤ 2 cm, which is less than that in cases of CD per se and PET/CT may be helpful in determining whether enlarged lymph nodes are characterized by CD-like changes or not.

4,5,6-Tri-O-acetyl-2,3-di-S-ethyl-2,3-dithio-d-allose diethyl dithio-acetal.

The title compound, C(20)H(36)O(6)S(4), was obtained by ethanethiol-ysis of 3,5,6-tri-O-acetyl-1,2-O-isopropyl-idene-α-d-gluco-furan--ose. One of the ethyl groups is disordered over two sites with refined occupancies of 0.869 (6) and 0.131 (6). Compared with the precursor, the absolute configuration of the stereocenters at positions C-3 and C-2 are inverted and maintained, respectively.

[Effect of Amino Acid Motifs in Integrin ß3 Cytoplasmic Tail on αâ ¡bß3-Mediated Cell function in 293T cell models].

OBJECTIVE: To establish 293T cell lines stably expressing Calpain-cleavage related α3 cytoplasmic tail mutants, and to explore the effect of amino acid motifs in integrin ß3 cytoplasmic tail on αⅡbß3-mediated cell function. METHODS: 293T cell lines stably co-expressing human wild type integrin αⅡb and full length ß3 or mutant ß3, including ß3-ΔNITY (ß3 cytoplasmic tail NITY motif deleted), ß3-Δ754 (ß3 cytoplasmic tail TNITYRGT motif deleted) and ß3-Δ759 (ß3 cytoplasmic tail RGT motif deleted) were established. Spreading and adhesion of these stable cell lines on immobilized fibrinogen were tested. RESULTS: 293T-αⅡbß3ΔNITY, 293T-αⅡbß3Δ754, 293T-αⅡbß3Δ759 and 293T-αⅡbß3 cell lines were successfully established. Compared with the 293T cells, 293T-αⅡbß3 cells which expressed full ß3, possessed well adhesion and spread ability on immobilized fibrinogen, suggesting it can be as a surrogate for platelet. Compared with 293T-αⅡbß3 cells, the 293T-αⅡbß3ΔNITY cells showed a partial impairment of adhesion and spreadability on immobilized fibrinogen. while the 293T-αⅡbß3Δ754 cells and 293T-αⅡbß3Δ759 cells failed to adhere or spread on immobilized fibrinogen. CONCLUSION: To the cell spreading function mediated by integrin ß3, RGT motif is vital, while NITY can be dispensable. These established 293T cell lines stably expressing different ß3 mutants provide a solid basis for a further analysis of mass spectrometry.

Trans-repressive effect of NUP98-PMX1 on PMX1-regulated c-FOS gene through recruitment of histone deacetylase 1 by FG repeats.

The formation of fusion genes between NUP98 and members of the HOX family represents a critical factor for the genesis of acute leukemia or acute transformation of chronic myeloid leukemia (CML). To gain insights into the molecular mechanisms underlying the leukemogenesis of NUP98-HOX fusion products, we cloned NUP98-PMX1 from a CML-blast crisis patient with t(1;11) as a secondary chromosomal translocation, and functionally studied the fusion products in detail through various molecular and protein biochemical assays. In addition to many interesting features, we have found that the NUP98-PMX1 fusion protein exerts a repressive effect on PMX1 or serum response factor-mediated c-FOS activation, probably through the recruitment of a common corepressor histone deacetylase 1 by FG domains of the NUP98-PMX1 fusion protein. Moreover, we have provided evidence that the FG domains of NUP98-PMX1 and two other NUP98-containing fusion proteins, i.e., NUP98-HOXA9 and NUP98-HOXC11, all exhibit dual binding ability to both CREB binding protein, a coactivator, and histone deacetylase 1, a corepressor. Accordingly, we have hypothesized that this dual binding activity is shared by most, if not all, NUP98-HOX-involved fusion proteins, enabling these fusion proteins to act as both trans-activators and trans-repressors, and contributing to the genesis of acute leukemia or acute transformation of CML.

[Role and Mechanism of the Interaction of BCR-ABL with E3 Ligase c-CBL in Targeting Therapy of Chronic Myelogenous Leukemia].

OBJECTIVE: To explore the interaction domains between BCR-ABL and E3 liagase c-CBL, so as to reveal the structure-basis for the arsenic to treat chronic myelogenous leukemia(CML). METHODS: The interactional interface of BCR-ABL and c-CBL was simulated and analyzed according to the available structure model. Based on the structural information, the WT and mutant Migr1-BCR-ABL-GFP (ΔSH2,ΔTyrKC,ΔSH2/TyrKC (S/H) and pFlag-c-CBL (ΔRF) were constructed and co-transfected into the 293T and HeLa cells. The co-immunoprecipitation (Co-IP) was performed by using M2 beads (anti-Flag), anti-GFP antibody and protein A beads, and the interaction was identified by using GFP and M2 antibody, respectively. Moreover, the colocalization of BCR-ABL and c-CBL was further evaluated by using immunofluorescent(IF) assay in transfected HeLa cells. RESULTS: Co-IP demonstrated that the TyrKC domain of BCR-ABL was primarily involved in the interaction with c-CBL, while both the SH2 domain of BCR-ABL and the RF domain of c-CBL also participated in the interaction to a certain degree, which were consistent with the structure-based simulation. IF elucidated that the colocalization of BCR-ABL and c-CBL was almost entirely vanished when the deleted TyrKC domain of BCR-ABL was co-transfected with c-CBL, which were elegantly coincident with the results from Co-IP. CONCLUSION: The TyrKC domain of BCR-ABL is sufficient and necessary to mediate the interaction between BCR-ABL and c-CBL, the SH2 domain of BCR-ABL and the RF domain of c-CBL are also involved in the association between the two proteins. It suggests that the association of BCR-ABL and c-CBL can modulate the stability and degradation of BCR-ABL, thus illustrating the molecular mechanisms of the targeting therapy for CML by arsenic.

[Myr-RKEFAK Peptide Selectively Regulates Outside-in Signaling Transduction-related Functions in Human Platelets].

OBJECTIVE: To study the effect of interaction of the talin rod domain integrin binding site 2 with integrin ß3 on platelet signal transduction. METHODS: A peptide that mimics the membrane proximal α helix 6 residues R724 KEFAK729 of the integrin ß3 cytoplasmic tails was designed and synthesized, to which the myristoylation was covalently linked to the N-terminal of the peptide enabling membrane penetration. The effects of myr-RKEFAK peptide on the typical platelet outside-in signaling ovent (stable adhesion and spreading on immobilized fibrinogen, aggregation, fibrin clot retraction) and inside-out signaling events (soluble fibrinogen binding) were tested. RESULTS: myr-RKEFAK peptide dose-dependently inhibited platelet stable adhesion and spreading on immobilized fibrinogen, irreversible aggregation, as well as fibrin clot retraction, but not soluble fibrinogen binding and reversible phase of platelet aggregation. CONCLUSION: The cell-penetrating peptide myr-RKEFAK causes an inhibitory effect on integrin ß3 outside-in signaling-regulated platelets functions, but did not affect inside-out signaling-regulated platelets functions.

[Effect of the Integrin ß3 Cytoplasmic NITY Motif on α II bß3-Mediated Cell Functions in CHO Cell Model].

UNLABELLED: OBJLECTIVE: To investigate the effect of integrin ß3 cytoplasmic NITY motif on αIIbß3-mediated cell functions. METHODS: Stable Chinese hamster ovary (CHO) cell lines that co-express human wild type integrin αIIb and wild type ß3 or mutant ß3ΔNITY (ß3 deleting cytoplasmic NITY motif) were established. Expression of αIIb and ß3 were tested by Western blot and flow cytometry in CHO cell lines. Spreading and adhesion of stable cell lines on immobilized fibrinogen were examined. The co-immunoprecipitation was used to detect protein interactions. RESULTS: CHO-αIIbß3, CHO-αIIbß3ΔNITY cells were successfully established. The CHO cells transfected with wild type αIIbß3 had the ability of adhesion and spreading. Compared with CHO-αIIbß3 cells, CHO-αIIbß3ΔNITY cells showed an impaired capacity of adhesion but no significant difference was observed in spreading of adhered cells. The co-immunoprecipitation showed that kindlin-2 associated with wild type integrin αIIbß3. The ß3ΔNITY mutation substantially reduced kindlin-2 association. CONCLUSION: Deletion of NITY motif causes an impaired ability of adhesion. The deletion mutation can suppress kindlin-2 binding to integrin ß3, thereby partially inhibit the integrin ß3 signaling.