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Abstract Objective to determine the presence of marginal bacterial microfiltration in the IAI in different implant/abutment systems, in vitro. Material and methods Fifty‐six implants from seven different brand names, 4 with cone and 3 with straight connections were used, implant and abutment were connected using the Ncm tightening as indicated by each of the manufacturers and then were sealed. The samples were subjected occlusal load and thermal cycling, a first sample of each group was observed by micro CT and in a second sample (both samples randomly selected) length of connection was measured, while the rest of the samples were mounted on devices according to the bacterial microfiltration model with Porphyromonas gingivalis. Results Two of the conical connection system groups did not present bacterial microfiltration, one of the three straight connection groups only microfiltered in one sample, while the other two conical as well as the two straight connection samples showed different and important levels of bacterial microfiltration, all groups presented a direct relationship between the implant‐abutment adjustment determined by micro‐CT and bacterial microfiltration levels, not related to the connection length. Conclusion: Only two conical connection systems presented no bacterial microfiltration.

Podocalyxin (PODXL), a cell surface sialomucin expressed in diverse types of normal and malignant cells, mediates cellular adhesion to extracellular matrix and cell-to-cell interaction. A previous study reported the expression of PODXL protein on monocytes undergoing macrophage differentiation, yet the expression of this molecule in other antigen presenting cells (APCs) and its function in the immune system still remain undetermined. In this study, we report that PODXL is expressed in human monocyte-derived immature dendritic cells at both the mRNA and protein levels. Following dendritric cells maturation using pro-inflammatory stimuli, PODXL expression level decreased substantially. Furthermore, we found that PODXL expression is positively regulated by IL-4 through MEK/ERK and JAK3/STAT6 signaling pathways. Our results revealed a polarized distribution of PODXL during the interaction of APCs with CD4 + T cells, partially colocalizing with F-actin. Notably, PODXL overexpression in APCs promoted their interaction with CD4 + T cells and CD8 + T cells and decreased the expression of MHC-I, MHC-II, and the costimulatory molecule CD86. In addition, PODXL reduced the translocation of CD4 + T-cell centrosome toward the APC-contact site. These findings suggest a regulatory role for PODXL expressed by APCs in immune responses, thus representing a potential target for therapeutic blockade in infection and cancer., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Amo, Díez-García, Tamayo-Orbegozo, Maruri and Larrucea.)

Mature B-cell non-Hodgkin lymphoma (B-NHL) constitutes a group of heterogeneous malignant lymphoproliferative diseases ranging from indolent to highly aggressive forms. Although the survival after chemo-immunotherapy treatment of mature B-NHL has increased over the last years, many patients relapse or remain refractory due to drug resistance, presenting an unfavorable prognosis. Hence, there is an urgent need to identify new prognostic markers and therapeutic targets. Podocalyxin (PODXL), a sialomucin overexpressed in a variety of tumor cell types and associated with their aggressiveness, has been implicated in multiple aspects of cancer progression, although its participation in hematological malignancies remains unexplored. New evidence points to a role for PODXL in mature B-NHL cell proliferation, survival, migration, drug resistance, and metabolic reprogramming, as well as enhanced levels of PODXL in mature B-NHL. Here, we review the current knowledge on the contribution of PODXL to tumorigenesis, highlighting and discussing its role in mature B-NHL progression., Competing Interests: Funding: This study has been funded by Instituto de Salud Carlos III through the project PI18/00629 (Co-funded by European Regional Development Fund; “A way to make Europe”).

Podocalyxin (PCLP1) is a CD34-related sialomucin expressed by some normal cells and a variety of malignant tumors, including leukemia, and associated with the most aggressive cancers and poor clinical outcome. PCLP1 increases breast tumor growth, migration and invasion; however, its role in hematologic malignancies still remains undetermined. The purpose of this study was to investigate the expression and function of PCLP1 in mature B-cell lymphoma cells. We found that overexpression of PCLP1 significantly increases proliferation, cell-to-cell interaction, clonogenicity, and migration of B-cell lymphoma cells. Furthermore, PCLP1 overexpression results in higher resistance to death induced by dexamethasone, reactive oxygen species and type II anti-CD20 monoclonal antibody obinutuzumab. Strikingly, enforced expression of PCLP1 enhances lipid droplet formation as well as pentose phosphate pathway and glutamine dependence, indicative of metabolic reprogramming necessary to support the abnormal proliferation rate of tumor cells. Flow cytometry analysis revealed augmented levels of PCLP1 in malignant cells from some patients with mature B-cell lymphoma compared to their normal B-cell counterparts. In summary, our results demonstrate that PCLP1 contributes to proliferation and survival of mature B-cell lymphoma cells, suggesting that PCLP1 may promote lymphomagenesis and represents a therapeutic target for the treatment of B-cell lymphomas., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Podocalyxin-like protein 1 (PCLP1), a CD34-related sialomucin involved in the regulation of cellular morphology and adhesion, is expressed by a number of normal cells and various tumor cells. In breast malignancies PCLP1 overexpression has been associated with the most aggressive, metastatic cancers and poor prognosis. These observations suggest that PCLP1 expression could provide a mechanism to evade the immune response, thereby promoting metastatic progression of cancer. In the present work, we aimed to determine the effect of PCLP1 overexpressed in MCF7 breast cancer cells on natural killer (NK) cell cytotoxicity, dendritic cell maturation, and agonist-induced T cell proliferation. The results showed that PCLP1 expressed in MCF7 breast cancer cells confers resistance to NK cell-mediated cytolysis and impairs T cell proliferation. Furthermore, PCLP1 decreased the levels of NK cell activating receptors NKG2D, NKp30, NKp44, NKp46, DNAM-1, and CD16 on cell surface in a contact-dependent manner. Moreover, NK cells acquired PCLP1 from MCF7 cells by a process known as trogocytosis. These data reveal a new function of PCLP1 expressed on tumor cells as an immunomodulatory molecule, which may represent a mechanism to evade the immune response., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Natural killer (NK) cells play an essential role in the fight against tumor development. Over the last years, the progress made in the NK-cell biology field and in deciphering how NK-cell function is regulated, is driving efforts to utilize NK-cell-based immunotherapy as a promising approach for the treatment of malignant diseases. Therapies involving NK cells may be accomplished by activating and expanding endogenous NK cells by means of cytokine treatment or by transferring exogenous cells by adoptive cell therapy and/or by hematopoietic stem cell transplantation. NK cells that are suitable for adoptive cell therapy can be derived from different sources, including ex vivo expansion of autologous NK cells, unstimulated or expanded allogeneic NK cells from peripheral blood, derived from CD34+ hematopoietic progenitors from peripheral blood and umbilical cord blood, and NK-cell lines. Besides, genetically modified NK cells expressing chimeric antigen receptors or cytokines genes may also have a relevant future as therapeutic tools. Recently, it has been described the derivation of large numbers of functional and mature NK cells from pluripotent stem cells, both embryonic stem cells and induced pluripotent stem cells, which adds another tool to the expanding NK-cell-based cancer immunotherapy arsenal.

Besides their essential role in hemostasis and thrombosis, platelets are involved in the onset of cancer metastasis by interacting with tumor cells. Platelets release secretory factors that promote tumor growth, angiogenesis, and metastasis. Furthermore, the formation of platelet-tumor cell aggregates in the bloodstream provides cancer cells with an immune escape mechanism by protecting circulating malignant cells from immune-mediated lysis by natural killer (NK) cells. Platelet-tumor cell interaction is accomplished by specific adhesion molecules, including integrins, selectins, and their ligands. Podocalyxin-like protein 1 (PCLP1) is a selectin-ligand protein in which overexpression has been associated with several aggressive cancers. PCLP1 expression enhances cell adherence to platelets in an integrin-dependent process and through the interaction with P-selectin expressed on activated platelets. However, the involvement of PCLP1-induced tumor-platelet interaction in tumor immune evasion still remains unexplored. The identification of selectin ligands involved in the interaction of platelets with tumor cells may provide help for the development of effective therapies to restrain cancer cell dissemination. This article summarizes the current knowledge on molecules that participate in platelet-tumor cell interaction as well as discusses the potential role of PCLP1 as a molecule implicated in tumor immune evasion.

Multiplex ligation-dependent probe amplification (MLPA) and array- comparative genomic hybridization analysis have been proven to be useful in the identification of submicroscopic copy-number imbalances in families with nonsyndromic X-linked intellectual disability (NS-XLID). Here we report the first description of a child with mild intellectual disability and a submicroscopic duplication at Xp22.12 identified by MLPA with a P106 MRX kit (MRC-Holland, Amsterdam, Netherlands) and further confirmed and characterized with a custom 244-k oligo-array, fluorescence in situ hybridization, quantitative polymerase chain reaction (qPCR), and immunoblotting. This 1.05-megabase duplication encompasses 7 genes, RPS6KA3 being the only of these genes known to be related to ID. The proband was an 8-year-old boy referred to the genetics unit for psychomotor retardation and learning disabilities. Both maternal brothers also showed learning difficulties and delayed language during childhood in a similar way to the proband. These boys also carried the duplication, as did the healthy mother and grandmother of the proband. The same duplication was also observed in the 5-year-old younger brother who presented with features of developmental delay and learning disabilities during the previous year. Increased RPS6KA3/RSK2 levels were demonstrated in the proband by qPCR and immunoblotting. To our knowledge, this is the first family identified with a submicroscopic duplication including the entire RPS6KA3/RSK2 gene, and our findings suggest that an increased dose of this gene is responsible for a mild form of NS-XLID.

Podocalyxin (PODXL) is a type I membrane mucoprotein abundantly presented in the epithelial cells (podocytes) of kidney glomeruli where it plays an important role in maintaining the plasma filtration. PODXL is also expressed in other types of cells but its function is ignored. A recombinant soluble fragment of the PODXL ectodomain modifies the signaling of the membrane bound PODXL. Based on this antecedent, we aimed at investigating whether PODXL could be cleaved and released into the extracellular space as a soluble peptide. In this study, we used a fusion protein of human PODXL and green fluorescent protein expressed in CHO cells (CHO-PODXL-GFP) and a human tumor cell (Tera-1) inherently expressing PODXL. PODXL was detected by wide-field microscopy in the Golgi, the plasma membrane and in a vesicular form preferentially located at the leading edges of the cell and also progressing along the filopodium. We detected PODXL in the insoluble and soluble fractions of the extracellular medium of CHO-PODXL-GFP cells. Stimulation of protein kinase C (PKC) by Phorbol-12-myristate-13-acetate (PMA) enhanced the release of PODXL to the extracellular space whereas this effect was prevented either by inhibitors of PKC or specific inhibitors of matrix metalloproteinases. It is concluded that intact PODXL is released to the extracellular space as a cargo of microvesicles and also as a soluble cleaved fragment of ectodomain., (2011 Elsevier B.V. All rights reserved.)

Podocalyxin (Podxl) is a type I membrane sialoprotein of the CD34 family, originally described in the epithelial glomerular cells of the kidney (podocytes) in which it plays an important function. Podxl can also be found in megakaryocytes and platelets among other extrarenal places. The surface exposure of Podxl upon platelet activation suggested it could play some physiological role. To elucidate the function of Podxl in platelets, we generated mice with restricted ablation of the podxl gene in megakaryocytes using the Cre-LoxP gene targeting methodology. Mice with Podxl-null megakaryocytes did not show any apparent phenotypical change and their rates of growth, life span and fertility did not differ from the floxed controls. However, Podxl-null mice showed prolonged bleeding time and decreased platelet aggregation in response to physiological agonists. The number, size-distribution and polyploidy of Podxl-null megakaryocytes were similar to the floxed controls. Podxl-null platelets showed normal content of surface receptors and normal activation by agonists. However, the mice bearing Podxl-null platelets showed a significant retardation in the ferric chloride-induced occlusion of the carotid artery. Moreover, acute thrombosis induced by the i.v. injection of sublethal doses of collagen and phenylephrine produced a smaller fall in the number of circulating platelets in Podxl-null mice than in control mice. In addition, perfusion of uncoagulated blood from Podxl-null mice in parallel flow chamber showed reduced adhesion of platelets and formation of aggregates under high shear stress. It is concluded that platelet Podxl is involved in the control of hemostasis acting as a platelet co-stimulator, likely due to its pro-adhesive properties.

Podocalyxin (PODXL) is a 145KDa sialoprotein abundantly expressed in the glycocalix of the intraglomerular kidney epithelial cells, essential in maintaining a normal renal function. PODXL is also found in vascular endothelial cells, megakaryocytes and platelets. The function of PODXL in platelets is ignored; however, its surface exposure upon platelet activation suggests its participation in controlling the hemostasis. We have generated mice (pralphaIIb-PODXL) overexpressing PODXL specifically in megakaryocytes , either alone or as a fusion protein with green fluorescent protein. The transgenic mice showed a phenotype characterized by decreased bleeding time, mild rebleeding and enhanced platelets aggregation upon agonist stimulation. The cytohematological exams as well as the prothrombin time (PT) and (APTT) tests did not differ from the control group. The biochemical analysis showed only a discrete hyperlipemia and a rise in plasma uric acid levels in the transgenic mice. The present data seem to indicate that PODXL may act as a costimulator of agonists in the activation of platelets and formation of a stable thrombus., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Podocalyxin (Podxl) is a type I membrane mucin-protein of the CD34 family abundantly expressed in kidney epithelial cells (podocytes) where it plays a crucial functional role. Podxl is also expressed in tissues other than kidney, like in brain, but its function is ignored. To investigate the functional role of podocalyxin (Podxl) in brain we produced the specific brain-ablation of the Podxl gen in mice by crossing Podxl(floxed/floxed) mice, generated in our laboratory, to mice with pan-neural expression of recombinase Cre (Cre3). Podxl(-/-) mice show no apparent behavioral phenotype but their brains showed enlargement of ventricular volumes detected in vivo by MR imaging. The pattern of brain vasculature was of normal appearance but the thickness of the main carotid artery was significantly increased. Moreover, the histological analysis showed increased number of choroidal capillaries lining the ventricular spaces. These findings are analyzed in the light of the role likely played by podocalyxin in cell migration and cell-cell recognition during brain development and also on the consistent findings of increased ventricular spaces in human pathological disorders like schizophrenia., (Copyright 2009 Elsevier Inc. All rights reserved.)

Podocalyxin (PODXL) is an anti-adhesive glycoprotein expressed abundantly in the epithelial cells of kidney glomeruli. In contrast, we report herein that expression of podocalyxin(GFP) (PODXL(GFP)) in CHO cells increased the adherence to immobilized fibronectin, spreading, and migration. The transient knockdown of PODXL or the expression of PODXL lacking the cytosolic carboxyterminal domain (PODXL-Delta(451)) inhibited cell adherence. Moreover, the effect of PODXL was prevented by the ectodomain of podocalyxin (PODXL-Delta(429)), by RGD peptides, or by inhibitors of the vitronectin receptor (alphavbeta3). CHO-PODXL(GFP) also showed adherence to human vascular endothelial cells (HUVEC), exhibiting polarization of granular PODXL and emission of long and thin, spike-like, protrusions with PODXL granules progressing along. We found PODXL colocalized with beta1 integrins at membrane ruffle regions on the leading edge of the cell and a blocking beta1 mAb prevented the spreading of cells. PODXL was also associated with submembrane actin in lamellipodia ruffles, or with vinculin at cell protrusions. The proadhesive effects of PODXL were absent in sialic acid deficient O-glycomutant CHO cells. To conclude, we present evidence indicating that human PODXL enhances the adherence of cells to immobilized ligands and to vascular endothelial cells through a mechanism(s) dependent on the activity of integrins.

Introduction: Hyperactivity of platelets has been associated with thrombotic episodes by molecular mechanisms not yet elucidated. The present work aimed at identifying whether the platelet protein content from patients who had suffered an arterial thrombosis episode differed from that of platelets obtained from normal healthy donors., Methods: Differential platelet protein profiles were determined by 2-dimensional (2-D) gel electrophoresis and Western blot analysis of total platelet lysates. Identification of differentially expressed proteins was carried out by mass spectrometry (MALDI-TOF)., Results: We found a decreased platelet content of three protein spots in patients of arterial thrombosis: integrin linked kinase (ILK), fructose bisphosphate aldolase (aldolase) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) whereas the content of four other protein spots was increased: actin binding protein, coronine like (p57), non-muscle myosin heavy chain (NMMHC-A), pyruvate kinase M2 isoenzyme (PK) and phosphoglycerate kinase (PGK). The variations in ILK, GAPDH and PK were validated by Western blot analysis. The proteins showing a decreased platelet content in arterial thrombosis patients are associated with the cytoskeletal insoluble fraction and the detected increase in some proteins seems to be due to the generation of peptides caused by a limited proteolysis. Differences in the protein profiles of circulating platelets from arterial thrombosis were maintained months after the acute thrombotic event and disappear in the long term., Conclusions: The observed variations in some platelet proteins suggest the existence of a perturbation in the cytoskeletal organization and increased proteolysis, both indicative of a platelet pro-active state, persistent after the thrombotic event.

Background: Podocalyxin (podxl) is a heavily glycosylated transmembrane protein mainly found on the apical membrane of rat podocytes and also in endothelial, hematopoietic, and tumor cells. Despite of its interest no much is known about the transcriptional regulation of podxl in different cells. Thus, we aimed at studying the functional features of the 5'-regulatory region of the human Podxl gene., Results: The promoter region of the human Podxl gene has been cloned and its structure and function were analyzed. The primary DNA sequence is rich in G+C and is devoid of TATA or CAAT boxes. The sequence contains recognition sites for several putative transcription factors; however, the basic promoter activity seems to rely entirely on Sp1 transcription factor since supershift analysis was positive only for this factor. The region encompassed by 66 to -111 nts conferred the minimal transcriptional activity that increases as the number of Sp1 sites augmented with the length of the promoter fragment. In Sp1-lacking insect cells the Podxl promoter constructs showed activity only if cotransfected with an Sp1 expression plasmid. Finally, mutation of the Sp1 sites reduced the promoter activity. We analyzed whether methylation of the CpG dinucleotides present in the first approximately 600 nts of the promoter region of Podxl could explain the variable rates of expression in different types of cells. Inactivation of methyltransferases by 5'-aza-2'deoxicitidine showed a dose-dependent increase in the podxl content. Moreover, in vitro methylation of the promoter constructs -111,-181 and -210 led to an almost complete reduction of the promoter activity. A correlation was found between the degree of methylation of the CpG promoter dinucleotides and the rate of podxl expression in different cell lines., Conclusion: Our results indicate that transcriptional regulation of Podxl is supported primarily by Sp1 site(s) and that DNA-methylation of the CpG promoter islands contributes to control the tissue specific expression of podxl.

This work reports the functional studies of CHO cells coexpressing alpha-adrenergic (alphaAR) and human fibrinogen (Fg) receptors (integrin alphaIIbbeta3). Stimulation of these cells with alpha-agonists produced a transient rise in the free cytosolic calcium (Ca(++)) accompanied by enhanced binding to soluble Fg, and these effects were prevented by specific alphaAR antagonists. The alpha-adrenergic-induced activation of alphaIIbbeta3 in CHO-alphaIIbbeta3-alphaAR increased the rate of adhesion and extension of cells onto Fg coated plates, and also induced a soluble Fg- and alphaIIbbeta3-dependent formation of cell aggregates, whereas no effects were observed by the stimulation of CHO-alphaIIbbeta3 cells. alpha-Adrenergic antagonists, the ligand mimetic peptide RGDS, pertussis toxin (PTX), or EDTA, they all prevented the alpha-adrenergic stimulation of adhesion and aggregation. However, inhibition of PKC prevented the alpha-adrenergic stimulation of cell adherence, whereas blocking the intracellular Ca(++) mobilization impeded the stimulation of cell aggregation. The alpha-adrenergic activation was associated with phosphorylation of a protein of approximately 100 kDa and proteins of the MAPK family. The former was selectively phosphorylated by alpha-adrenergic stimulation whereas the latter were phosphorylated by the binding of cells to Fg and markedly intensified by alpha-adrenergic stimulation.

We report the analysis of a variant case of thrombasthenic phenotype that is a compound heterozygote for two mutations located within the metal ion dependent adhesion site (MIDAS) of the beta3 subunit. The patient inherited a maternal allele carrying the Met124Val substitution and a paternal allele that changes Asp119 to Tyr. Phenotyping of the human platelet antigen 1 (HPA-1) showed that the platelet alphaIIbbeta3 complex in the patient was mostly accounted for by the Asp 119Tyr allele that does not bind to fibrinogen (Fg). The patient showed agonistinduced binding of platelets to Fg but neither binding to PAC-1 nor cell aggregation could be detected, most likely due to the minute expression (< or = 5%) of alphaIIb(124Val)beta3 receptors. CHO cells expressing (124Val)beta3 showed a diminished surface expression of alphaIIbbeta3, enhanced adhesion to immobilized Fg, and spontaneous aggregation in the presence of soluble Fg, suggesting that (124Val)beta3 may confer constitutive activity to the alphaIIb(124Val)beta3 receptors. A distinct feature of these cells is the failure of DTT to enhance the binding to soluble Fg and the formation of cell aggregates. The substitution of (124Met)beta3 by either a polar or a positively charged amino acid restored the surface exposure and function of the alphaIIbbeta3 receptors whereas a negatively charged residue did not.

As mouse models have become commonplace for studying hemostasis and thrombosis, we considered whether the mouse system had utility for assessing genetic alterations in platelet receptors. Platelets from 5 mouse strains (C57BL/6 [C57], FVB/N [FVB], BALB/c, C3H/He, and 129Sv) showed only minor differences in the expression of integrin alpha(IIb), integrin beta(3), glycoprotein (GP) Ib alpha, or GPVI across strains. However, FVB platelets expressed approximately 50% the level of integrin alpha(2) as platelets from other strains (P <.0001). We bred FVB mice with C57 and assessed alpha(2) expression in FVB/C57xFVB/C57 (F2) offspring. Linkage analysis demonstrated the gene responsible for alpha(2) levels is tightly linked to the D13mit260 marker (log odds [lod] score 6.7) near the alpha(2) gene. FVB platelets showed reduced aggregation and a longer lag phase to collagen. FVB and C57 platelets aggregated similarly to collagen-related peptide, but FVB platelets showed a reduction in rhodocytin-induced Syk and PLC gamma 2 tyrosine phosphorylation. Thus, FVB platelets express half the level of alpha(2) as other mouse strains, a trait linked to the alpha(2) gene and seemingly responsible for reduced platelet aggregation to collagen. These strain differences serve as a useful model for the 2-fold difference in human platelet alpha(2)beta(1) expression and demonstrate that alpha(2)beta(1) participates in signaling during platelet activation.

Objective: It has been reported that women fare worse after ischemic coronary events, but the mechanisms remain unclear. Because platelets play a central role in the formation of occlusive thrombi at sites of ruptured atherosclerotic plaques, we studied male/female paired mouse littermates for sex differences in platelet function., Methods and Results: We compared platelet reactivity in male/female mouse littermates by monitoring agonist-induced fibrinogen (FGN) binding and platelet aggregation. Compared with the platelets from males, platelets from females bound more FGN in response to low concentrations of thrombin and collagen-related peptide. Female platelets also demonstrated greater aggregation in response to adenosine diphosphate and collagen-related peptide. Platelet protein tyrosine phosphorylation on activation also showed small differences between sexes. These differences are independent of platelet size and surface expression of alphaIIbbeta3 and GPIb-IX-V, and they were not blocked by apyrase or aspirin. The sex differences we observed were intrinsic to platelets, because they were observed in washed platelets, but not when platelets were in plasma., Conclusions: The platelets of female mice were more reactive than those of males in a manner independent of COX-1 and secreted ADP.

The platelet fibrinogen receptor, integrin alphaIIbbeta3, is a noncovalent heterodimer of glycoproteins IIb and IIIa. This work was aimed at elucidating the role played by the carboxy-terminal extracellular, trans-membrane, and cytoplasmic regions of the glycoprotein beta3 in the formation of functional complexes with alpha subunits. Progressive carboxy-terminal deletions of beta3 revealed that surface exposure of alphaIIbbeta3 or alphavbeta3 could not occur in the absence of the transmembrane domain of beta3. In contrast, internal deletions 616 to 690 of the carboxy-terminal regions of the beta3 ectodomain led to surface exposure of constitu tive active receptors in CHO cells, as indicated by the enhanced rate of cell adhesion to immobilized ligands and spontaneous binding to soluble fibrinogen or activation-dependent antibody PAC-1. The functional analysis of cysteine mutations within the 616 to 690 region of beta3 or chimeric beta3-beta7 subunits revealed that disruption of the C663-C687 disulfide bridge endows constitutive activity to the alphaIIbbeta3 receptor. It is concluded that the carboxy-terminal tail of the beta3 ectodomain, so-called beta tail domain (betaTD), is not essential for cell surface expression of beta3 receptors. However, a basal, nonactivated, low ligand-affinity state of the beta3 integrins demands a normal conformation of this domain.

The platelet glycoprotein (GP) Ib-IX-V complex mediates the attachment of platelets to the blood vessel wall by binding von Willebrand factor (VWF), an interaction that also transmits signals for platelet activation and aggregation. Because the complex is extensively palmitoylated, a modification known to target proteins to lipid rafts, we investigated the role of raft localization in GP Ib-IX-V functions. In unstimulated platelets, a minor portion of the complex localized to Triton-insoluble raft fractions; this portion increased three to sixfold with platelet activation by VWF. Raft-associated GP Ib-IX-V was selectively palmitoylated, with GP Ib-IX-V-associated palmitate increasing in the raft fraction on VWF-mediated activation. The raft fraction was also the site of association between GP Ib-IX-V and the Fc receptor FcgammaRIIA. The importance of this association was demonstrated by the ability of the FcgammaRIIA antibody IV.3 to inhibit shear-induced platelet aggregation. Disruption of rafts by depleting membrane cholesterol impaired several GP Ib-IX-V-dependent platelet fractions: aggregation to VWF under static conditions and under shear stress, tyrosine phosphorylation, and adhesion to a VWF surface. Partial restoration of membrane cholesterol content partially restored shear-induced platelet aggregation and tyrosine phosphorylation. Thus, localization of the GP Ib-IX-V complex within rafts is crucial for both platelet adhesion and postadhesion signaling.

This work reports the establishment of a Chinese hamster ovary (CHO) cell line stably coexpressing the human alphaIIbbeta3 integrin and the platelet-activating factor receptor (PAFR). These cells aggregate in response to PAF in a Ca(++), alphaIIbbeta3, and soluble fibrinogen (Fg)-dependent manner that is prevented by PAF antagonists or alphaIIbbeta3 blockade. The aggregating response is accompanied by enhanced binding of fibrinogen and the activation-dependent IgM PAC1. This model has permitted us to identify, for the first time, intracellular signals distinctly associated with either alphaIIbbeta3-mediated adhesion or aggregation. Nonreceptor activation of protein kinase C (PKC) by phorbol ester produced cellular adhesion and spreading onto immobilized Fg, but it was not a sufficient signal to provoke cellular aggregation. Moreover, inhibition of PKC impeded the PAF stimulation of cellular adhesion, whereas the aggregation was not prevented. The PAF-induced cellular aggregation was distinctly associated with signaling events arising from the liganded Fg receptor and the agonist-induced stimulation of a calcium/calmodulin-dependent signaling pathway. Sustained tyrosine phosphorylation of both mitogen-activated protein kinase (MAPK) and an approximately 100-kd protein was associated with the PAF-induced aggregation, whereas phosphorylation of focal adhesion kinase (FAK) was preferably associated with cellular adherence and spreading onto immobilized Fg.

We report the molecular genetic analysis of the Bernard-Soulier syndrome (BSS) phenotype in two related patients showing absence of glycoprotein (GP) Ibalpha and detectable amounts of GPIX on the platelet surface, and a truncated form of GPIbalpha in solubilized platelets and plasma. They both were compound heterozygotes for the GPIbalpha gene: a maternal allele with a T insertion at position 1418 causing a translational frameshift and premature polypeptide termination, and a paternal allele with a T715A substitution chan-ino Cys209 to Ser. Heterozygotes for either one of these mutations were asymptomatic. Transient transfection of cells coexpressing GPIbbeta and GPIX failed to detect surface expression of the GPIbalpha mutants. Cells transfected with [1418insT]GPIbalpha-cDNA showed a truncated protein of the predicted size in both cell lysate and conditioned medium, indicating the inability of the mutant protein to anchor the plasma membrane. In contrast. transfection of [T715A]GPIbalpha-cDNA yield a mutated protein barely detectable in the cell lysate and absent in the medium, indicating that the loss of Cys209 renders GPIbalpha more vulnerable to proteolysis and unable to undergo the normal secretory pathway. Our findings indicate that the additive effects of both mutations are responsible for the BSS phenotype of the patients.

This work aimed at investigating the function of the [C674R] mutation in GPIIb that disrupts the intramolecular 674 to 687 disulfide bridge. Individuals heterozygous for this mutation show a platelet GPIIb-IIIa content approximately 30% of normal controls, which is less than expected from one normal functioning allele. Coexpression of normal [674C]GPIIb and mutant [674R]GPIIb with normal GPIIIa produced a [674R]GPIIb concentration-dependent inhibition of surface exposure of GPIIb-IIIa complexes in Chinese hamster ovary (CHO) cells, suggesting that [674R]GPIIb interferes with the association and/or intracellular trafficking of normal subunits. Mutation of either 674C or 687C had similar effects in reducing the surface exposure of GPIIb-IIIa. However, substitution of 674C for A produced a much lesser inhibition than R, suggesting that a positive-charged residue at that position renders a less efficient subunit conformation. The mutant [674R]GPIIb but not normal GPIIb was found associated with the endoplasmic reticulum chaperone BiP in transiently transfected CHO cells. BiP was also found associated with [674R]GPIIb-IIIa heterodimers, but not with normal GPIIIa or normal heterodimers. Overexpression of BiP did not increase the surface exposure of [674R]GPIIb-IIIa complexes, indicating that its availability was not a limiting step. Platelets from the thrombasthenic patient expressing [674R]GPIIb-IIIa were found to bind soluble fibrinogen in response to physiologic agonists or dithiothreitol treatment. Thus, the [674R]GPIIb mutation leads to a retardation of the secretory pathway, most likely related to its binding to the molecular chaperone BiP, with the result of a defective number of functional GPIIb-IIIa receptors in the cell surface.

Factor J (FJ) is a cationic glycoprotein with inhibitory activity in vitro against both classical and alternative pathways of complement activation. Recently FJ has been implicated in adhesion to several cell lines, through a membrane receptor identified as nucleolin. In the present work we study the events that follow the binding of FJ to cells. After incubation of K562 with FJ, this protein was internalized actively and localized in the cytoplasm and nucleus. Adhesion to immobilized FJ induced tyrosine phosphorylation of several intracellular proteins in Jurkat cell line with a similar pattern to that induced by fibronectin (FN), an extracellular matrix protein. This effect was maximal at 5 min and decreased after 10 min, and inhibited by anti-FJ monoclonal antibody (mAb). These results suggest that the binding of FJ to cells may play an important role in transduction of biochemical signals across the plasma membrane to the cell interior., (Copyright 1999 Academic Press.)