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 FcγRIIA. The importance of this association was demonstrated by the ability of the FcγRIIA 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.

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.

Factor J (FJ) is a complement inhibitor that acts on the classical and the alternative pathways. We demonstrated FJ-cell interactions in fluid phase by flow cytometry experiments using the cell lines Jurkat, K562, JY, and peripheral blood lymphocytes. FJ bound to plastic plates was able to induce in vitro adhesion of these cells with potency equivalent to fibronectin. As evidence for the specificity of this reaction, the adhesion was blocked by MAJ2, an anti-FJ monoclonal antibody, and by soluble FJ. Attachment of the cells required active metabolism and cytoskeletal integrity. The glycosaminoglycans heparin, heparan sulfate, or chondroitin sulfates A, B, and C inhibited to varying degrees the binding of FJ to cells, as did treatment with chondroitinase ABC. In the search for a putative receptor, a protein of 110 kDa was isolated by affinity chromatography, and microsequence analysis identified this protein as nucleolin. Confocal microscopy evidenced the presence of nucleolin in cell membrane by immunofluorescence with monoclonal (D3) and polyclonal anti-nucleolin antibodies in Jurkat cells. The interaction FJ-nucleolin was evidenced by Western blot and enzyme-linked immunosorbent assay. Furthermore, purified nucleolin and D3 inhibited adhesion of Jurkat cells to immobilized FJ, suggesting that the interaction was specific and that nucleolin mediated the binding. Factor J (FJ)1 is a complement inhibitor that is able to regulate both the classical and the alternative pathways of complement (1, 2). FJ was initially found as a soluble molecule in urine and serum (1, 3). FJ is a peculiar protein having a high sugar content and a pI Ն 9.6 (4). In addition, the existence of FJ-related molecules has been described on the surface of human circulating cells and lymphoid cell lines (5). Flow cytometry analysis with an anti-FJ monoclonal antibody (mAb) has revealed staining in a small but consistent population of lymphocytes (mean: 11%) but not in monocytes, granulocytes, erythrocytes, or platelets. Moreover, we have also found that anti-FJ stained several cell lines (Jurkat, U937, K562, and Ramos) at varying percentages.Relationships between complement and adhesion processes have been described previously; moreover, complement regulators such as vitronectin (VN) (6), clusterin (7), and more recently, factor H-like protein 1 (8) and factor H (9) have been implicated in adhesion processes. Affinity purification identified Mac1 (CD11b/CD18) as a factor H binding receptor (9). Moreover, CD11b/CD18 (Mac1) binds several soluble ligands including the complement fragment iC3b (10). Some characteristics of FJ resemble VN. Among them are the ability to regulate complement, the tendency to form aggregates, and the potential to bind heparin (11). VN is a ligand for ␤ 1 and ␤ 3 integrins. Clusterin, a different complement regulator, is capable of promoting the aggregation and adhesion of renal epithelial cells.FJ also strongly binds heparin (12). Heparin-binding proteins constitute a diverse group that includes extracellular matrix mo...

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 ␣ IIb ␤ 3 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 ␣ IIb ␤ 3 , is a noncovalent heterodimer of glycoproteins IIb and IIIa. This work was aimed at elucidating the role played by the carboxy-terminal extracellular, transmembrane, and cytoplasmic regions of the glycoprotein ␤ 3 in the formation of functional complexes with ␣ subunits. Progressive carboxy-terminal deletions of ␤ 3 revealed that surface exposure of ␣ IIb ␤ 3 or ␣ v ␤ 3 could not occur in the absence of the transmembrane domain of ␤ 3 . In con- IntroductionThe glycoprotein (GP) IIb-IIIa complex, integrin ␣ IIb ␤ 3 , is a calcium-dependent, noncovalent heterodimer formed by GPIIb and GPIIIa. This complex is found in the plasma membrane of megakaryocytes, platelets, and some tumor tissues 1-3 and functions as a receptor for fibrinogen and other adhesive proteins like the von Willebrand factor, fibronectin, or vitronectin. 4 The ␤ 3 subunit may also complex the GP ␣ v to form the vitronectin receptor (integrin ␣ v ␤ 3 ) that shares with ␣ IIb ␤ 3 the binding of fibrinogen although with different affinity. 5 The platelet ␣ IIb ␤ 3 complex is essential to maintain a normal hemostasis. Unlike other platelet receptors that are constitutively active, the ␣ IIb ␤ 3 is maintained in a low-affinity state for its ligands. Disruption of the vascular endothelium and exposure of platelets to the action of agonists and adhesive proteins from the subendothelial matrix induces a cellular activation. The activated cells interact with adhesive proteins from the extracellular matrix, 6,7 and the ␣ IIb ␤ 3 receptors are able to bind fibrinogen with high affinity (insideout signaling), resulting in platelet aggregation. 8 Conversely, ligand-bound ␣ IIb ␤ 3 propagates signals to the interior of the cell (outside-in signaling) leading to enhanced interaction with the cytoskeleton, clustering of receptors (increased ligand avidity), and formation of focal contacts rich in signaling complexes. 9,10 The agonist-induced increase in ligand affinity of ␣ IIb ␤ 3 is thought to be the result of conformational changes of the heterodimer [11][12][13] initiated by the interaction of the cytoplasmic tails of ␣ and ␤ 3 subunits with cytosolic proteins. Despite the pathophysiologic importance of the platelet ␣ IIb ␤ 3 receptor, the knowledge of the mechanisms controlling its state of activation is rather limited.Unlike previous reports, 14 recent work from our laboratory 15 showed that a truncated form of ␤ 3 lacking the transmembrane and cytosolic domains failed to associate with ␣ IIb . The present work was aimed at further investigating the role played by the carboxyterminal domain of ␤ 3 in the surface expression and function of ␤ 3 heterodimers. The results obtained in this study indicate that surface expression of ␣ IIb ␤ 3 could not occur in the absence of the transmembrane domain of ␤ 3 . The present study has also revealed that either deletion of the carboxy-terminal region of the ␤ 3 ectodomain or disruption of the 663-687 disulfide bridge confers constitutive activity to the ␤ 3 integr...