Flow cytometric analysis of mouse sperm using monoclonal anti-sperm antibody A-1

We have previously prepared an anti-mouse sperm monoclonal antibody (A-1) which inhibited sperm penetration into the egg zona pellucida. By indirect immunofluorescence (IIF), the A-1 antibody was shown to recognize an antigen localized in the acrosomal area of sperm. This antibody bound negligibly to fresh sperm, while binding to methanol-fixed sperm was almost complete. After methanol fixation, no sperm that penetrated into the zona were immunoreactive for this antibody. In the present study we examined the localization and fate of A-1 antigen during the acrosome reaction by IIF and flow cytometry (FCM). Cauda epididymal sperm were treated with either calcium ionophore A23187 or zona solution, immunostained indirectly, and subjected to FCM. Treatment with A23187 reduced the percentage of immunoreactive sperm to 59% from the 80% obtained in the untreated sperm. The treatment also reduced the average fluorescence intensity per fluorescence-positive spermatocyte to 65 channels, while this intensity was 89 channels in the untreated sperm. A similar result was obtained from treatment with zona solution. The proportion of sperm that was immunoreactive with A-1 antibody was reduced to 55% by incubation in zona-containing media from the 80% obtained in zona-free media. On the other hand, neither A23187 nor the zona solution affected the immunoreactivity or the fluorescence intensity of caput epididymal sperm, while the A-1 antigen was present in both the immature sperm from the caput epididymis of adult mice and in the mature sperm from the cauda epididymis of the same mice. These findings suggest that the intramembrane antigen recognized by the A-1 monoclonal antibody is released from sperm as a result of the acrosome reaction. © 1994 Wiley-Liss, Inc.     

A novel rat monoclonal antibody directed against a population of activated mouse peritoneal macrophages

A hybridoma clone secreting rat monoclonal antibody (MAB) designated as 3F3.5F and which reacted with a population of activated tumoricidal mouse peritoneal macrophage (M phi) was produced by the fusion of mouse myeloma cells with rat spleen cells immunized against adherent BCG-activated mouse peritoneal exudate cells (adherent BCG-PEC). The antibody was cytotoxic and of the rat IgM class. The specific reactivity of the antibody with mouse primary cells and cell lines was examined by complement-dependent cytotoxicity and indirect immunofluorescence flow cytometry analysis. The antibody was found to bind to about 40% of the adherent BCG-PEC activated in vivo and elicited peritoneal macrophages activated in vitro by lymphokine and lipopolysaccharide (LPS), to about 35% of polymorphonuclear neutrophils (PMN) 15 hr after intraperitoneal injection of BCG, to about 30% of bone marrow cells from BCG-infected mice, to about 10% of P815 mastocytoma cells and to thioglycollate-induced PEC to some degree. It did not bind to other cells tested including BCG-induced peritoneal lymphocytes, non-tumoricidal PEC, thymocytes, spleen cells, resting bone marrow cells from normal mice, lymphomas, myelomas, fibroblasts, or macrophage-cell lines. Pretreatment of adherent BCG-PEC with MAB 3F3.5F and rabbit complement caused a considerable decrease in tumor cytotoxicity toward P815 cells, but the same pretreatment of non-adherent BCG-PEC had no inhibitory effect on natural killer activity for YAC-1 cells.     

ICln, a novel integrin alphaIIbbeta3-associated protein, functionally regulates platelet activation

A critical role for the conserved alpha-integrin cytoplasmic motif, KVGFFKR, is recognized in the regulation of activation of the platelet integrin alpha(IIb)beta(3). To understand the molecular mechanisms of this regulation, we sought to determine the nature of the protein interactions with this cytoplasmic motif. We used a tagged synthetic peptide, biotin-KVGFFKR, to probe a high density protein expression array (37,200 recombinant human proteins) for high affinity interactions. A number of potential integrin-binding proteins were identified. One such protein, a chloride channel regulatory protein, ICln, was characterized further because its affinity for the integrin peptide was highest as was its expression in platelets. We verified the presence of ICln in human platelets by PCR, Western blots, immunohistochemistry, and its co-association with alpha(IIb)beta(3) by surface plasmon resonance. The affinity of this interaction was 82.2 +/- 24.4 nm in a cell free assay. ICln co-immunoprecipitates with alpha(IIb)beta(3) in platelet lysates demonstrating that this interaction is physiologically relevant. Furthermore, immobilized KVGFFKR peptides, but not control KAAAAAR peptides, specifically extract ICln from platelet lysates. Acyclovir (100 microm to 5 mm), a pharmacological inhibitor of the ICln chloride channel, specifically inhibits integrin activation (PAC-1 expression) and platelet aggregation without affecting CD62 P expression confirming a specific role for ICln in integrin activation. In parallel, a cell-permeable peptide corresponding to the potential integrin-recognition domain on ICln (AKFEEE, 10-100 microm) also inhibits platelet function. Thus, we have identified, verified, and characterized a novel functional interaction between the platelet integrin and ICln, in the platelet membrane.     

Flow cytometric estimation of cell cycle parameters using a monoclonal antibody to bromodeoxyuridine

An estimation of cell kinetic parameters was made by simultaneous flow cytometric measurements of DNA and bromodeoxyuridine (BrdUrd) contents of cells. The procedure described in this paper involves the incorporation of BrdUrd by S phase cells, labeling the BrdUrd with an indirect immunofluorescent technique using a monoclonal anti-BrdUrd antibody, and staining DNA with propidium iodide (PI). The amount of incorporated BrdUrd in HeLa cells was proportional to that of synthesized DNA through S phase. For all cell lines examined, the pattern of BrdUrd incorporation was essentially the same and the rate of DNA synthesis during S phase was not constant. The bivariate BrdUrd/DNA distributions showed a horse-shoe pattern, maximum in the mid S phase and minimum in the early and late S phases. Furthermore, the durations of cell cycle (Tc) and S phase (Ts) were estimated from a FLSm (fraction of labeled cells in mid S phase) curve that was generated by plotting the percentage of BrdUrd pulse-labeled cells in a narrow window defined in the mid S phase of the DNA histogram. The values of these parameters in NIH 3T3, HeLa S3, and HL-60 cells were in good accordance with the reported data. This FCM method using the monoclonal anti-BrdUrd antibody allows rapid determination of both cell cycle compartments and also Ts and Tc without the use of radioactive DNA precursors.     

Bidirectional transmembrane modulation of integrin alphaIIbbeta3 conformations

Activation of blood platelets by physiological stimuli (e.g. thrombin, ADP) at sites of vascular injury induces inside-out signaling, resulting in a conformational change of the prototype integrin alphaIIbbeta3 from an inactive to an active state competent to bind soluble fibrinogen. Furthermore, ligand occupancy of alphaIIbbeta3 initiates outside-in signaling and additional conformational changes of the receptor, leading to the exposure of extracellular neoepitopes termed ligand-induced binding sites (LIBS), which are recognized by anti-LIBS monoclonal antibodies. To date, the mechanism of bidirectional transmembrane signaling of alphaIIbbeta3 has not been established. In this study, using our newly developed anti-LIBScyt1 monoclonal antibody, we showed that extracellular ligand binding to alphaIIbbeta3 on blood platelets induces a transmembrane conformational change in alphaIIbbeta3, thereby exposing the LIBScyt1 epitope in the alphaIIb cytoplasmic sequence between Lys994 and Asp1003. In addition, a point mutation at this site (P998A/P999A) renders alphaIIbbeta3 constitutively active to bind extracellular ligands, resulting in fibrinogen-dependent cell-cell aggregation. Taken collectively, these results demonstrated that the extracellular ligand-binding site and a cytoplasmic LIBS epitope in integrin alphaIIbbeta3 are conformationally and functionally coupled. Such bidirectional modulation of alphaIIbbeta3 conformation across the cell membrane may play a key role in inside-out and outside-in signaling via this integrin.     

Mechanistic insights from a refined three-dimensional model of integrin alphaIIbbeta3

The integrin alpha(IIb)beta(3) plays an important role in platelet function, and abnormalities of this protein result in a serious bleeding disorder, known as Glanzmann thrombasthenia. Although crystallographic data exist for the related integrin alpha(V)beta(3), to date, there are no high resolution structures of integrin alpha(IIb)beta(3) available in the literature. Therefore, it is still unclear how specific elements of the alpha(IIb) subunit contribute to integrin alpha(IIb)beta(3) function. Here we describe a refined model of the alpha(IIb) N-terminal portion of integrin alpha(IIb)beta(3) obtained by using the alpha(V)beta(3) template combined with a new method for predicting the conformations of the unique alpha(IIb) loop regions comprising residues 71-85, 114-125, and 148-164. The refined model was probed based on a structural prediction that differentiates it from standard homology models: specifically, that Lys-118 of alpha(IIb) contacts Glu-171 of beta(3). To test this hypothesis experimentally, the mutant integrin chains alpha(IIb) K118C and beta(3) E171C were cotransfected into HEK 293 cells. We show that the cells expressed the mutants alpha(IIb)beta(3) on their surface as a disulfide-linked dimer, supporting the close proximity between alpha(IIb) Lys-118 and beta(3) Glu-171 predicted from the refined model. This validated model provides a specific structural context for the analysis and interpretation of structure-function relations of integrin alpha(IIb)beta(3). In addition, it suggests mechanistic hypotheses pertaining to both naturally occurring mutations responsible for Glanzmann thrombasthenia and to point mutations that affect ligand binding.     

Reconstructing and deconstructing agonist-induced activation of integrin alphaIIbbeta3

BACKGROUND: Integrin receptors, composed of transmembrane alpha and beta subunits, are essential for the development and functioning of multicellular animals. Agonist stimulation leads cells to regulate integrin affinity ("activation"), thus controlling cell adhesion and migration, controlling extracellular-matrix assembly, and contributing to angiogenesis, tumor cell metastasis, inflammation, the immune response, and hemostasis. A final step in integrin activation is the binding of talin, a cytoskeletal protein, to integrin beta cytoplasmic domains. Many different signaling molecules that regulate integrin affinity have been described, but a pathway that connects agonist stimulation to talin binding and activation has not been mapped. RESULTS: We used forward, reverse, and synthetic genetics to engineer and order an integrin activation pathway in cells expressing a prototype activatable integrin, platelet alphaIIbbeta3. Phorbol myristate acetate (PMA) activated alphaIIbbeta3 only after the increased expression of both recombinant protein kinase Calpha (PKCalpha) and talin to levels approximating those in platelets. Inhibition of Rap1 GTPase reduced alphaIIbbeta3 activation, whereas activated Rap1A(G12V) bypassed the requirement for PKC, establishing that Rap1 is downstream of PKC. Talin binding to integrins mediates Rap1-induced activation because Rap1A(G12V) failed to activate alphaIIbbeta3 in cells expressing integrin binding-defective talin (W359A). Rap1 activated integrins by forming an integrin-associated complex containing talin in combination with the Rap effector, RIAM. Furthermore, siRNA-mediated knockdown of RIAM blocked integrin activation. CONCLUSIONS: We have, for the first time, ordered a pathway from agonist stimulation to integrin activation and established the Rap1-induced formation of an "integrin activation complex," containing RIAM and talin, that binds to and activates the integrin.     

Binding of a fibrinogen mimetic stabilizes integrin alphaIIbbeta3's open conformation

The platelet integrin alphaIIbbeta3 is representative of a class of heterodimeric receptors that upon activation bind extracellular macromolecular ligands and form signaling clusters. This study examined how occupancy of alphaIIbbeta3's fibrinogen binding site affected the receptor's solution structure and stability. Eptifibatide, an integrin antagonist developed to treat cardiovascular disease, served as a high-affinity, monovalent model ligand with fibrinogen-like selectivity for alphaIIbbeta3. Eptifibatide binding promptly and reversibly perturbed the conformation of the alphaIIbbeta3 complex. Ligand-specific decreases in its diffusion and sedimentation coefficient were observed at near-stoichiometric eptifibatide concentrations, in contrast to the receptor-perturbing effects of RGD ligands that we previously observed only at a 70-fold molar excess. Eptifibatide promoted alphaIIbbeta3 dimerization 10-fold more effectively than less selective RGD ligands, as determined by sedimentation equilibrium. Eptifibatide-bound integrin receptors displayed an ectodomain separation and enhanced assembly of dimers and larger oligomers linked through their stalk regions, as seen by transmission electron microscopy. Ligation with eptifibatide protected alphaIIbbeta3 from SDS-induced subunit dissociation, an effect on electrophoretic mobility not seen with RGD ligands. Despite its distinct cleft, the open conformer resisted guanidine unfolding as effectively as the ligand-free integrin. Thus, we provide the first demonstration that binding a monovalent ligand to alphaIIbbeta3's extracellular fibrinogen-recognition site stabilizes the receptor's open conformation and enhances self-association through its distant transmembrane and/or cytoplasmic domains. By showing how eptifibatide and RGD peptides, ligands with distinct binding sites, each affects alphaIIbbeta3's conformation, our findings provide new mechanistic insights into ligand-linked integrin activation, clustering and signaling.     

Flow cytometric detection of erythropoietic cytotoxicity in mouse bone marrow.

Erythroblast proliferation and maturation in bone marrow are the processes leading to the formation of polychromatic erythrocytes (PE) and normochromatic erythrocytes (NE), respectively. PE contain RNA but no DNA, and can therefore be distinguished both from NE (which lack both RNA and DNA) and from nucleated cells (which contain both DNA and RNA). Cytotoxic agents that induce impairment of the maturation process change the PE:NE ratio. We have developed a simple and rapid method of determining the PE:NE ratio, based on flow cytometric analysis of formaldehyde-fixed, acridine orange (AO)-stained cells. The effects of cyclophosphamide (CP), mitomycin C (MMC), and vincristine (VC) were tested and the PE:NE ratio was evaluated over 7 days of treatment. In this study we monitored the kinetics of these compounds and were able to demonstrate both a time- and a dose-dependent effect. We detected a difference between the effects of the alkylating agents tested and those induced by the spindle inhibitor tested. Flow cytometry of fixed bone marrow samples stained with AO provides more information, better and more rapid statistical analysis, than conventional microscopic methods for counting the PE:NE ratio.     

Flow cytometric and immunoblot assays for cell surface ADP-ribosylation using a monoclonal antibody specific for ethenoadenosine

NAD-dependent ADP-ribosylation is one of the posttranslational protein modifications. On mammalian cells, glycosylphosphatidylinositol-anchored cell surface ADP-ribosyltransferases (ARTs) ADP-ribosylate other cell surface proteins and thereby affect important cellular functions. Here we describe convenient flow-cytometric and immunoblot assays for monitoring ADP-ribosylation of cell surface proteins on living cells by exploiting the capacity of ARTs to utilize etheno-NAD as substrate. Etheno-ADP-ribosylation of cell surface proteins can be detected by flow cytometry with 1G4, a monoclonal antibody specific for ethenoadenosine. Labeling of cells with 1G4 is dependent on the expression of cell surface ARTs and occurs only after incubation of ART-expressing cells with etheno-NAD and not with etheno-ADP-ribose. Dose-response analyses show efficient 1G4 staining of ART-expressing cells at micromolar etheno-NAD concentrations. Half-maximal staining is obtained with 1-2 micro M etheno-NAD, saturation is reached at 5-20 micro M etheno-NAD. Immunoblot analyses confirm that ART-expressing cells incorporate ethenoadenosine covalently (i.e., SDS resistant) into several cell surface proteins. The flow-cytometric 1G4 staining assay can be used to identify subpopulations of cells expressing cell surface ART activity and to select ART(hi) cell variants. The immunoblot 1G4 staining assay can also be used to identify etheno-ADP-ribosylated target proteins. These new assays hold promise for many interesting applications in biochemistry and cell biology.     

The carbohydrate structures of a mouse monoclonal IgG antibody OKT3

A mouse monoclonal antibody OKT3, of IgG2a isotype, was isolated from hybridoma culture fluid. Sugar analysis showed the presence of sialic acid, galactose, mannose, fucose, and N-acetylglucosamine, i.e. sugars typical for N-glycosidically linked carbohydrate chains. The absence of N-acetylgalactosamine revealed that O-glycosidically linked carbohydrates were not present. The purified antibody was reduced, alkylated, and separated into heavy and light chains, and all carbohydrates were shown to be associated with the heavy chains. The N-linked carbohydrate chains were isolated as alditols using strong alkaline-borohydride degradation and further fractionated on a concanavalin A-Sepharose column and high performance ion exchange chromatography with pulsed amperometric detection. Structural analysis was carried out on the isolated oligosaccharide alditols by chemical analyses, fast atom bombardment mass spectrometry, and 500-MHz 1H NMR spectroscopy. Triantennary and biantenary types of structures were found. The triantennary structures were present as trisialo and tetrasialo forms without fucose; the tetrasialo forms were shown to contain a sequence of Neu5Ac alpha 2-3Gal beta 1-3[Neu5Ac alpha 2-6]GlcNAc beta 1- on one of the branches. The biantennary structures were present as completely sialylated nonfucosylated species and as asialo-, agalacto-, and partially fucosylated structures.     

Flow cytometric analysis of circadian changes in platelet activation using anti-GMP-140 monoclonal antibody.

The hemostatic activity of blood shows a circadian variation with a higher frequency of acute coronary events in the morning. The thrombotic tendency of blood is influenced by many factors, including platelets. Diurnal changes of in vivo platelet activation were investigated by whole blood flow cytometry in 10 young healthy male volunteers using anti-GMP-140 (anti-alpha-granule membrane protein 140 kD) monoclonal antibody at 3h intervals from 06:00 to 24:00. We also studied circulating platelet aggregates to investigate whether there exists a similarity between the results of these methods. Results of flow cytometric analysis indicate that there is an increase in platelet activation during the period from 06:00 to 09:00. Platelet activation then decreases gradually during the period from noon to midnight. These changes are accompanied by a similar trend in circulating platelet aggregates. This suggests that GMP-140 expression on platelets is synchronized with or followed by platelet aggregate formation in vivo, and increased platelet activation may predispose individuals to thrombosis at this time.     

Platelet fragmentation requires a specific structural conformation of human monoclonal antibody against beta3 integrin

We have described an autoantibody against beta3 (GPIIIa49-66), a region of platelet integrin alphaIIbbeta3 that is unique. It induces platelet fragmentation in the absence of complement via antibody activation of platelet NADPH oxidase and 12-lipoxygenase to release reactive oxygen species, which destroy platelets. To study the mechanism of anti-GPIIIa antibody-induced platelet fragmentation, we screened a human single chain Fv antibody library with the GPIIIa49-66 peptide. Nine monoclonal antibodies were identified that were capable of binding to GPIIIa49-66. Surprisingly, binding avidity for GPIIIa49-66 did not correlate with activity of induction of platelet fragmentation. We therefore investigated the requirements for platelet fragmentation. Mutations were introduced into the heavy chain complementary-determining region-3 of clones 11, 43, and 54 by site-directed mutagenesis. The capability of these clones to induce platelet fragmentation or bind to GPIIIa49-66 subsequently changed. Molecular modeling of these clones with their mutants revealed that the ability to induce platelet fragmentation is affected by the side chain orientation of positively charged amino acids in the heavy chain of residues 99-102. Thus, a structural change in the conformation of anti-GPIIIa49-66 antibody contributes to its binding to the beta3 integrin and subsequent antibody-induced platelet fragmentation and aggregate dissolution.     

Flow cytometric analysis and sorting of human endometrial cells after immunocytochemical labeling for cytokeratin using a monoclonal antibody

Endometrial cells in suspension were stained with propidium iodide and a monoclonal antibody against a cytokeratin intermediate filament protein specific for glandular and columnar cells (RGE 53). In this way columnar epithelial cells of the normal endometrium and of adenocarcinomas can be distinguished and separated by flow cytometry from non-epithelial cells (fibroblasts and inflammatory cells) and squamous epithelial cells, all of which are negative for RGE 53. This makes it possible to analyse and also sort pure fractions of this particular tissue type for further studies. The use of propidium iodide allows simultaneous DNA flow cytometry of these columnar epithelial cells. Therefore, the use of antibodies to cytokeratin in combination with propidium iodide can be of help in analyzing and sorting pure fractions of both normal and malignant cells. This allows a more refined examination of complex cell mixtures using flow cytometry.     

Flow cytometric detection of progastrin interaction with gastrointestinal cells

The unprocessed gastrin precursor, progastrin (PG), is often overexpressed in colon cancer and other malignancies where it appears to stimulate colonic growth. Overexpression of progastrin also stimulates proliferation of normal colonic mucosa, but the receptors mediating these effects have not been identified. Here we report the development of a non-radioactive assay for assessment of PG binding to normal and transformed cells. Progastrin was labeled using biotinylation, and binding of biotinylated PG to cells was assessed using flow cytometry. Using this approach, we show strong and specific binding of PG to some cell lines (IEC-6, IEC-18, HT-29, COLO320) and minimal binding to others (HeLa, DC2.4, Jurkat). We also found PG binding to several non-gut epithelial lines, such as CHO-K1, COS-6 and HEK293 cells. The specificity of binding was confirmed by competition with cold, unlabeled PG but not with glycine-extended gastrin or amidated gastrin-17. Binding was not influenced by the presence of the classical CCK-2 receptor, but was partially dependent on the charged glycosaminoglycans (GAG). The analysis of primary colonic tissues isolated from wild type C57BL/6 mouse, revealed a small epithelial subpopulation of non-hematopoietic (CD45-negative) cells that strongly interacted with PG. Surprisingly, this population was greatly expanded in gastrin knockout mice. This non-radioactive, FACS-based assay should prove useful for further characterization of cells expressing the progastrin receptor.     

Flow cytometric analysis of mouse hepatocyte ploidy

Summary The development of liver ploidy in mice aged up to 24 months was investigated by flow cytometry in four mouse strains. A mathematical procedure was applied for correction of flow cytometry histograms. In two of the mouse strains, C3H and DBA, both cellular and nuclear ploidy proceed in the same way. The octoploid cell with two tetraploid nuclei is the most numerous cell type in adulthood. On the other hand, strain NZB and the out-bred strain NMRI show at the corresponding age a higher proportion of diploid cells with strikingly low proportions of 4c cells. In addition, high values of 16c cells and nuclei are present in NMRI. In all strains the proportion of binucleate hepatocytes is in the same range (60%). However, the strains differ in ploidy classes of binucleate cells. Development of liver polyploidization does not depend on life span of the specific strain.     

Flow cytometric analysis of mouse hepatocyte ploidy

Preparative and mathematical procedures are presented for the investigation of the ploidy pattern of liver cells. The DNA content of enzymatically-isolated liver cells and of nuclei was measured by flow cytometry. The true DNA content could not be measured directly due to super-position of statistical coincidences (demanding "first mode correction") and incomplete separation of the nuclei in binucleate hepatocytes (demanding "second mode correction"). The statistical coincidences (caused by simultaneous measurement of two or more particles or subsequent reaggregation of particles) were corrected by splitting the "unnatural" i.e., aneuploid DNA content, and classifying it with the normal ploidy classes. In addition, the higher normal ploidy classes were reduced by the proportion of the measured coincidences in favour of the lower ones. The second mode correction applied to nuclear distributions only. It is a probability calculation based on counting nuclear pairs on microscope slides, and resulted in a 10% increase of diploid nuclei and a larger standard deviation between the age groups. 8c and 16c values were reduced. The tetraploid values were unchanged.     

Flow cytometric analysis of European flat oyster, Ostrea edulis, haemocytes using a monoclonal antibody specific for granulocytes

The importance of haemocytes in mollusc defence mechanisms can be inferred from their functions. They participate in pathogen elimination by phagocytosis (Cheng, 1981; Fisher, 1986). Hydrolytic enzymes and cytotoxic molecules produced by haemocytes contribute to the destruction of pathogenic organisms (Cheng, 1983; Leippe & Renwrantz, 1988; Charlet et al., 1996; Hubert et al., 1996; Roch et al., 1996). Haemocytes may also be involved in immunity modulation by the production of cytokines and neuropeptides (Hughes et al., 1990; Stefano et al., 1991; Ottaviani et al., 1996). As a result, the literature dealing with bivalve haemocyte studies has increased during the last two decades. Most of these publications use microscopy for morphological analysis (Seiler & Morse, 1988; Auffret, 1989; Hine & Wesney, 1994; Giamberini et al., 1996; Carballal et al., 1997; Lopez et al., 1997; Nakayama et al., 1997), and functional analysis (e.g. phagocytosis) (Hinsch & Hunte, 1990; Tripp, 1992; Mourton et al., 1992; Fryer & Bayne, 1996; Mortensen & Glette, 1996). Flow cytometry represents a rapid technique applicable to both morphological and functional studies of cells in suspension. While the measurements based on autofluorescence provide information on cell morphology, the analyses with fluorescent markers including labelled antibodies, offer data on phenotyping and cell functions. As a result, its application has greatly contributed to the investigation of immunocyte functions and differentiation in vertebrates (Stewart et al., 1986; Rothe & Valet, 1988; Ashmore et al., 1989; Koumans-van Diepen et al., 1994; Rombout et al., 1996; Caruso et al., 1997). Some authors studied oyster haemocyte populations by flow cytometry based on cellular autofluorescence (Friedl et al., 1988; Fisher & Ford, 1988; Ford et al., 1994). However, no analysis using specific monoclonal antibodies has been reported to date. In this study, a protocol for studying European flat oyster, Ostrea edulis, haemocytes by flow cytometry using a monoclonal antibody specific for granulocytes and an indirect immunofluorescence technique have been developed. European flat oysters, Ostrea edulis, 7-9 cm in shell length were obtained from shellfish farms in Marenne Oléron bay (Charente Maritime, France) on the French Atlantic coast. All individuals were purchased just before each experiment and processed without any previous treatment.     

CIB1 is an endogenous inhibitor of agonist-induced integrin alphaIIbbeta3 activation

In response to agonist stimulation, the alphaIIbbeta3 integrin on platelets is converted to an active conformation that binds fibrinogen and mediates platelet aggregation. This process contributes to both normal hemostasis and thrombosis. Activation of alphaIIbbeta3 is believed to occur in part via engagement of the beta3 cytoplasmic tail with talin; however, the role of the alphaIIb tail and its potential binding partners in regulating alphaIIbbeta3 activation is less clear. We report that calcium and integrin binding protein 1 (CIB1), which interacts directly with the alphaIIb tail, is an endogenous inhibitor of alphaIIbbeta3 activation; overexpression of CIB1 in megakaryocytes blocks agonist-induced alphaIIbbeta3 activation, whereas reduction of endogenous CIB1 via RNA interference enhances activation. CIB1 appears to inhibit integrin activation by competing with talin for binding to alphaIIbbeta3, thus providing a model for tightly controlled regulation of alphaIIbbeta3 activation.     

Mechanisms and consequences of agonist-induced talin recruitment to platelet integrin alphaIIbbeta3

Platelet aggregation requires agonist-induced alphaIIbbeta3 activation, a process mediated by Rap1 and talin. To study mechanisms, we engineered alphaIIbbeta3 Chinese hamster ovary (CHO) cells to conditionally express talin and protease-activated receptor (PAR) thrombin receptors. Human PAR1 or murine PAR4 stimulation activates alphaIIbbeta3, which was measured with antibody PAC-1, indicating complete pathway reconstitution. Knockdown of Rap1-guanosine triphosphate-interacting adaptor molecule (RIAM), a Rap1 effector, blocks this response. In living cells, RIAM overexpression stimulates and RIAM knockdown blocks talin recruitment to alphaIIbbeta3, which is monitored by bimolecular fluorescence complementation. Mutations in talin or beta3 that disrupt their mutual interaction block both talin recruitment and alphaIIbbeta3 activation. However, one talin mutant (L325R) is recruited to alphaIIbbeta3 but cannot activate it. In platelets, RIAM localizes to filopodia and lamellipodia, and, in megakaryocytes, RIAM knockdown blocks PAR4-mediated alphaIIbbeta3 activation. The RIAM-related protein lamellipodin promotes talin recruitment and alphaIIbbeta3 activity in CHO cells but is not expressed in megakaryocytes or platelets. Thus, talin recruitment to alphaIIbbeta3 by RIAM mediates agonist-induced alphaIIbbeta3 activation, with implications for hemostasis and thrombosis.