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Glycosylation is one of the most complex post-translational modifications and may have significant influence on the proper function of the corresponding proteins. Bacteria and yeast are, because of easy handling and cost reasons, the most frequently used systems for recombinant protein expression. Bacteria generally do not glycosylate proteins and yeast might tend to hyperglycosylate. Insect cell- and mammalian cell-based expression systems are able to produce complex N-glycosylation structures but are more complex to handle and more expensive. The nonpathogenic protozoa Leishmania tarentolae is an easy-to-handle alternative expression system for production of proteins requiring the eukaryotic protein folding machinery and post-translational modifications. We used and evaluated the system for the secretory expression of extracellular domains from human glycoprotein VI and the receptor for advanced glycation end products from rat. Both proteins were well expressed and homogeneously glycosylated. Analysis of the glycosylation pattern identified the structure as the conserved core pentasaccharide Man 3 GlcNac 2 .

Phytochemical investigation on the bark of Goniothalamus tapis Miq. and G. uvaroides King has resulted in the isolation of eight styryl-lactones, (-)-cryptomeridiol, liriodenine, 3-methyl-1H-benz[f]indole-4,9-dione, (-)-stigmasterol and dimethyl terephthalate. The structures of the compounds were elucidated by spectroscopic techniques. The compounds were evaluated for their effect on platelet-activating factor (PAF) receptor binding on rabbit platelets using (3) H-PAF as a ligand. Among the compounds tested, (-)-cryptomeridiol, (+)-goniothalamin and (+)-isoaltholactone exhibited a significant and concentration-dependent inhibitory effect on PAF receptor binding, with inhibitory concentration (IC)(50) values of 17.5, 19.7 and 46.5 µm, respectively. The inhibitory effects of the first two compounds were comparable to that obtained from the positive control, cedrol. The results indicated that these compounds were strong PAF receptor binding inhibitors., (Copyright © 2011 John Wiley & Sons, Ltd.)

Objective: To seek the key platelet functional proteins (PFPs) for the occurrence of blood-stasis pattern (BP) in patients with coronary heart disease (CHD)., Methods: Peripheral blood platelet protein of 22 patients and 24 healthy person (for control) were extracted respectively in 4 batches for carrying 4 times of the test out. Differential PFPs in samples were screened out by two-dimensional fluorescence difference gel electrophoresis, and identified with matrix-assisted laser desorption/ionization-time of flight mass spectrometry; then the identified proteins were further authenticated by Western-blotting., Results: Thirteen differential PFPs were screened out, and among them the 7 identified by spectrometry were: isoform 1 of integrin alpha- II b, isoform 2 of integrin alpha- II b, actin-cytoplasmic 1, actin-cytoplasmic 2, cDNA FLJ52842, cDNA FLJ55253, and cDNA FLJ43573 fis. Among them isoform 2 of integrin alpha- II b (CD41) and actin-cytoplasmic 2 (Acting) were authenticated successfully., Conclusion: CD41 and acting are the possible marker proteins, and the other PFPs might play crucial roles in the occurrence and development of BSS in CHD.

Being central players in thrombosis and hemostasis, platelets react in manifold and complex ways to extracellular stimuli. Cell-matrix and cell-cell interactions are mandatory for initial adhesion as well as for final development of stable plugs. Primary interfaces for interactions are plasma membrane proteins, of which many have been identified over the past decades in individual studies. However, due to their enucleate structure, platelets are not accessible to large-scale genomic screens and thus a comprehensive inventory of membrane proteins is still missing. For this reason, we here present an advanced proteomic setup for the detailed analysis of enriched platelet plasma membranes and the so far most complete collection of platelet membrane proteins. In summary, 1282 proteins were identified, of which more than half are termed to be of membrane origin. This study provides a brief overview of gene ontology subcellular and functional classification, as well as interaction network analysis. In addition, the mass spectrometric data were used to assemble a first tentative relative quantification of large-scale data on the protein level. We therefore estimate the presented data to be of major interest to the platelet research field and to support rational design of functional studies.

A starting point for many glycosylation analysis pathways is marked by the determination of the respectivecarbohydrate attachment sites to the polypeptide backbone of proteins. Several methods have been reported for this purpose in the past, commonly divided into a three-step approach (1) affinity purification of glycoproteins/-peptides, (2) processing/trimming of the glycopeptides and (3) elucidation of the glycan attachment site by mass spectrometry. For N-glycosylation site analysis the last two steps are usually similar, while methods differ in the affinity purification step. Here, we describe the oxidative derivatisation of carbohydrate moieties and covalent trapping of glycopeptides on hydrazide functionalized beads. This method is suitable for large scale analysis of glycoproteins as well as isolated glycoproteins and can be applied readily to a number of different samples. In the described protocol, the elucidation of N-glycosylation sites of human platelet proteins is demonstrated as an example.

A range of natural and unnatural phomactins, recently synthesised in our laboratory, were found to exhibit PAF antagonism with pIC(50) values in the range 5.6-6.2. The variation in structural and stereochemical features between the phomactins was found to have only a modest effect on the inhibition of binding of PAF to its human platelet receptors.

Platelet-collagen interactions play a fundamental role in the process of arterial thrombosis. The major platelet collagen receptor is the glycoprotein VI (GPVI). Here, we determined the effects of a soluble dimeric form of GPVI on platelet adhesion in vitro and in vivo. We fused the extracellular domain of GPVI with the human immunoglobulin Fc domain. The soluble dimeric form of GPVI (GPVI-Fc) specifically bound to immobilized collagen. Binding of GPVI-Fc to collagen was inhibited competitively by soluble GPVI-Fc, but not control Fc lacking the external GPVI domain. GPVI-Fc inhibited the adhesion of CHO cells that stably express human GPVI and of platelets on collagen and attenuated thrombus formation under shear conditions in vitro. To test the effects of GPVI-Fc in vivo, arterial thrombosis was induced in the mouse carotid artery, and platelet-vessel wall interactions were visualized by intravital fluorescence microscopy. Infusion of GPVI-Fc but not of control Fc virtually abolished stable arrest and aggregation of platelets following vascular injury. Importantly, GPVI-Fc but not control Fc, was detected at areas of vascular injury. These findings further substantiate the critical role of the collagen receptor GPVI in the initiation of thrombus formation at sites of vascular injury and identify soluble GPVI as a promising antithrombotic strategy.

CEACAM1 is a cell adhesion molecule that has been implicated in a number of physiological processes (eg, tumor suppressor in epithelial tissues, potent angiogenic factor in microvessel formation, microbial receptor in human granulocytes and epithelial cells). The mechanism of CEACAM1 action is still largely unresolved but recent findings demonstrated that the cytoplasmic CEACAM1 domain is linked indirectly to the actin-based cytoskeleton. We have isolated integrin beta(3) as an associated protein using CEACAM1 tail affinity purification. This association depends on phosphorylation of Tyr-488 in the CEACAM1 cytoplasmic domain. Confocal laser scanning microscopy confirmed in vivo colocalization of both molecules in human granulocytes and epithelial cells. Furthermore, the concentrated colocalization at the tumor-stroma interface of invading melanoma masses suggests a functional role of CEACAM1-integrin beta(3) interaction in melanoma invasion. Moreover, colocalization of the two adhesion molecules is also found at the apical surface of glandular cells of pregnancy endometrium. Colocalization of CEACAM1 and integrin beta(3) at the transitional zone from proliferative to invasive extravillous trophoblast of the maternal-fetal interface supports a role for CEACAM1/integrin beta(3) complexes in cell invasion.

Platelet glycoprotein VI (GP VI) was purified from platelet membranes and its internal amino acid sequences were determined. The cloned cDNA of GP VI indicates an open reading frame coding for 20 amino acid signal sequences and a mature protein of 319 amino acids. Its extracellular region has two Ig-like domains and a mucin-like, Ser/Thr-rich region, suggesting that GP VI is a member of the paired Ig-like receptor family. GP VI-transfected cells contained convulxin-(reactive) and antibody against recombinant GP VI-reactive protein bands that migrated at the same position as platelet GP VI in SDS/PAGE-electroblotting. These data indicate that the protein deduced from the cloned cDNA corresponds to platelet GP VI.

The cellular distribution of osteoclast integrin subunits alpha(v) and beta(3), the tissue distribution, and level of the apparent ligand osteopontin (OPN) as well as of the putative regulatory enzyme tartrate-resistant acid phosphatase (TRAP) were studied along with the intracellular distribution of the activation marker c-src in osteopetrotic ia/ia (incisors-absent) mutant rats and their normal littermates. In ia/ia rats, the osteoclasts are incapable of bone matrix resorption. Ultrastructurally the cells exhibit extended clear zones at the expense of ordinary ruffled borders. A secretory dysfunction in the mutant is strongly suggested by the absence of detectable extracellular TRAP, concomitant with an accumulation of the enzyme in abundant small cytoplasmic vesicles. Moreover, TRAP mRNA, protein content, as well as enzymatic activity were elevated. Furthermore, increased levels of integrin subunits alpha(v) and beta(3) were detected at the clear zone of mutant osteoclasts. OPN mRNA levels were elevated in long bones from mutants. In ia/ia rats, immunolabeling for OPN was homogeneously distributed at the surface facing osteoclasts, while in normal littermates it was concentrated at the clear zone area and barely detectable at ruffled borders. The absence of OPN labeling in the abundant, putative intracellular secretory vesicles in mutant osteoclasts suggests that these cells do not produce OPN. The osteoclasts of ia/ia rats appeared to produce and translocate the c-src protein to the cell membrane. In ia/ia a defect ruffled border-formation is observed along with extensive clear zone formation and decreased secretory function. The lesion may be due to a signaling defect, but in that case the defect seems to be located downstream to or not involving the c-src pathway. Our results illustrate the close relationship between secretory function and ruffled border formation in osteoclasts, a relationship that appears to be necessary for proper resorptive function., (Copyright 1999 Academic Press.)

Although platelet-activating factor (PAF) receptors have been found on B lymphoblastoid cell lines, the action of PAF on freshly isolated human B cells has not been clearly demonstrated. Using a sensitive semiquantitative reverse-transcriptase PCR, we have found PAF receptor mRNA expressed by tonsillar B lymphocytes, but little in T lymphocytes. Examination of Percoll-fractionated tonsillar B cells indicated that the low density (primarily germinal center cells) and medium density fractions had approximately twofold more PAF receptor mRNA relative to the high density fraction. PAF (10-7 M) stimulated increases in intracellular Ca2+ that were consistently higher in the low and medium density B lymphocytes compared with high density cells. The PAF receptor antagonist Web 2170 inhibited this. Addition of PAF, but not lyso- or enantio-PAF, induced four- to sixfold greater synthesis of IgM and IgG in low and medium density cells compared with unstimulated controls, but had little effect on Ig production by high density cells. To investigate how PAF may influence Ig synthesis, PAF-stimulated B cells were examined for production of the Th2-type cytokines IL-4 and IL-13. PAF induced IL-4 and IL-13 mRNA expression in 17% of CD20+ cells, and IL-4 was detected in cell supernatants after 48-72 h of culture. Together, these data strongly suggest that functional PAF receptors are expressed on B cells in tonsils.

Signaling across integrins is regulated by interaction of these receptors with cytoskeletal proteins and signaling molecules. To identify molecules interacting with the cytoplasmic domain of the beta3-integrin subunit (glycoprotein IIIa), a placental cDNA library was screened in the yeast two-hybrid system. Two identical clones coding for a 96-amino acid sequence were identified. This sequence was 100% identical to a sequence in skelemin, a protein identified previously in skeletal muscle. Skelemin is a member of a superfamily of cytoskeletal proteins that contain fibronectin-type III-like motifs and immunoglobulin C2-like motifs and that regulate the organization of myosin filaments in muscle. The amino acid residues in the isolated clones encompassed C2 motifs 4 and 5 of skelemin. A recombinant skelemin protein consisting of C2 motifs 3-7 interacted with beta1- and beta3-integrin cytoplasmic domains expressed as glutathione S-transferase (GST) fusion proteins, but not with GST-beta2-integrin cytoplasmic tail or GST alone. The skelemin-binding region was in the membrane proximal cytoplasmic domains of the integrins. Full-length skelemin interacted with integrin in intact cells as demonstrated by the colocalization of hemagglutinin-tagged skelemin in Chinese hamster ovary (CHO) cells containing alphaIIbbeta3-integrin and by the finding that microinjection of C2 motif 4 of skelemin into C2C12 mouse myoblast cells caused spread cells to round up. A skelemin-like protein was detected in CHO cells, endothelial cells, and platelets, and this protein colocalized with beta1- and beta3-integrins in CHO cells. This study suggests the presence of a skelemin-like protein in non-muscle cells and provides evidence that it may be involved in linking integrins to the cytoskeleton.

A monoclonal antibody (MAb) designated FC-5.01 (IgG2a) was generated that binds to several human carcinomas and malignant melanoma. It has revealed no or very low reactivity with most human normal tissues, except for the fact that FC-5.01 binds to some cells from the neuroendocrine system, macrophages, and some renal proximal convolute tubules with an intracellular pattern. Biochemical studies indicate that FC-5.01 recognizes a heterogeneous glycoprotein (broadband between 30-60 kDa) in melanoma tumors. The epitopes reside in the protein core and are presumably conformational, with disulphide bonds implicated in MAb recognition. The current study presents evidence that MAb FC-5.01 reacts with CD63 antigen (Ag), which has been initially described as a melanoma associated Ag, and is a member of the tetraspan family. Reactivity of MAb FC-5.01 with CD63 was demonstrated by Western blot, immunodepletion assay, and FACS analysis of the CD63-negative melanoma cells (KM3) after transfection with the genomic copy of CD63. The epitope recognized by MAb FC-5.01 was shown to be different from the epitope recognized by another anti-CD63 MAb, ME491, by an inhibition radioimmunoassay. Opposite to what has been stated for MAb ME491, no significant differences were found in CD63 expression between primary and metastatic melanoma using MAb FC-5.01.

Murine monoclonal antibodies were raised against porcine platelets in order to provide tools for investigating interactions of human blood cells and natural antibodies with porcine tissues. Hybridomas were screened by cellular ELISA on porcine platelets and endothelial cells. Positive clones were tested by flow cytometry for reactivity with isolated endothelial cells. One clone, NaM160-1A3, produced an antibody that stained porcine but not human endothelial cells and lymphocytes. The antibody bound to a 116 kDa glycoprotein on Western blot of both platelets and endothelial cells. The antigen was purified from a platelet lysate by affinity chromatography, first on a ConA column and then on a column presenting the immobilized NaM160-1A3 antibody. Two glycoproteins were obtained: one (116 kDa) was recognized by the antibody and one (150 kDa) was not. The 116 kDa protein had an internal decapeptide identical with human beta 1 integrin, and the 150 kDa protein had an internal amino acid sequence belonging to porcine alpha 2 integrin. Therefore, the NaM160-1A3 antibody was directed against porcine beta 1 integrin and allowed the purification of the complex alpha 2 beta 1, also termed Very Late Antigen 2 (VLA-2). It did not recognize human beta 1 integrin.

The ADP-receptor on the surface of human platelets and cells of megakaryocytic lineage has been classified as P2T purinergic receptor for which ADP is an agonist and ATP is an antagonist. Although it is one of the earliest identified of the important cellular receptors, it has neither been purified nor cloned. We have developed an immunoaffinity method for rapidly identifying the platelet ADP-receptor and this method can be extended to the purification of the receptor. A polyclonal antibody to glutamate dehydrogenase (GDH) covalently modified by 5'-p-fluorosulfonylbenzoyladenosine (FSBA) recognized neither FSBA nor glutamate dehydrogenase. Immunoblot of the gel obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized FSBA-labeled platelets showed the presence of a protein band at 100 kDa and this band was absent in the immunoblots of platelets that were preincubated with ADP and ATP or covalently modified by the chemically reactive ADP-affinity analogs, 2- and 8-(4-bromo-2,3-dioxobutylthio)adenosine-5'-diphosphate (2- and 8BDB-TADP) and 2-(3-bromo-2-oxopropylthio)adenosine-5'-diphosphate (2-BOP-TADP), prior to treatment with FSBA. FSBA as well as 2- and 8-BDB-TADP and 2-BOP-TADP have been previously shown to inhibit ADP-induced platelet responses by selectively and covalently modifying aggregin (100 kDa), an ADP-receptor in intact human blood platelets. The results show that polyclonal antibody to FSBA-labeled GDH is capable of recognizing FSBA-labeled aggregin on platelets and, thus, could be used to purify aggregin by immunoaffinity column chromatography. The immunoaffinity method was found to be far more sensitive than the radiochemical methods to identify aggregin previously developed in our laboratory. Since FSBA is also capable of reacting with enzymes that require ATP for their catalytic function, the polyclonal antibody may be used to identify and purify other P2-type purinergic receptors that require binding of ATP before eliciting cellular responses., (Copyright 1997 Academic Press.)

The method for isolation of glycoprotein IIbIIIa by chromatography on Concanavalin A-Sepharose and DEAE-Sephacel columns have been proposed. The 66 kDa, 27 kDa and 6 kDa fragments were produced by chymotrypsin digestion of GpIIbIIIa and purified with FPLC system on Superose-12 column under non denaturating conditions. N-terminal sequence analysis of fragments revealed that 66 kDa fragment contained NH2-terminal region, disulphide rich node, transmembrane and cytoplasmatic regions of IIIa subunit. 27 kDa fragment was a part of Ca(2+)-binding site of IIb subunit. 6 kDa fragment was shown to be from transmembrane and cytoplasmatic regions of IIb subunit. GpIIIa was isolated by gel-filtration on Sephacryl S-300 column of the GpIIbIIIa dissociated.

Megakaryocytic cell lines, established from the blood of patients with leukaemia, provide us with a unique opportunity to study the proliferation, differentiation and maturation of megakaryocytes. Eighteen human and three animal cell lines that express some megakaryocytic features have been described in the literature. Many of these cell lines have primitive multiphenotypic properties of erythroid, myeloid and megakaryocytic cells, while some show more restricted megakaryocyte-specific markers. The most consistent cell marker of megakaryocytic cell lines is the presence of platelet membrane glycoprotein (GPIIb-IIIa) in human cell lines and that of acetylcholinesterase in mouse or rat cell lines. The expressions of GPIb, von Willebrand factor and platelet peroxidase are variable among different cell lines, perhaps reflecting different stages of differentiation or a neoplastic nature of immortal cell lines. Treatment of many of these cell lines with phorbol esters leads to enhanced expression of the megakaryocytic programme.

Enzymatic and immunochemical assays show a phosphatidylinositol 4-kinase in novel and specific complexes with proteins (CD63 and CD81) of the transmembrane 4 superfamily (TM4SF) and an integrin (alpha3beta1). The size (55 kDa) and other properties of the phosphatidylinositol 4-kinase (PI 4-K) (stimulated by nonionic detergent, inhibited by adenosine, inhibited by monoclonal antibody 4CG5) are consistent with PI 4-K type II. Not only was PI 4-K associated with alpha3beta1-CD63 complexes in alpha3-transfected K562 cells, but also it could be co-purified from CD63 in untransfected K562 cells lacking alpha3beta1. Thus, TM4SF proteins may link PI 4-K activity to the alpha3beta1 integrin. The alpha5beta1 integrin, which does not associate with TM4SF proteins, was not associated with PI 4-K. Notably, alpha3beta1-CD63-CD81-PI 4-K complexes are located in focal complexes at the cell periphery rather than in focal adhesions. The novel linkage between integrins, transmembrane 4 proteins, and phosphoinositide signaling at the cell periphery may play a key role in cell motility and provides a signaling pathway distinct from conventional integrin signaling through focal adhesion kinase.

Anti-alpha 4 integrin mAb coprecipitated CD81 (TAPA-1), a 25-kDa cell surface protein, from various alpha 4 beta1 -positive hemopoietic cell lines, including Molt4, Jurkat, Ramos, and alpha 4-transfected K562 (KX4C4) cells. In reciprocal experiments, the integrin alpha 4 beta 1 (VLA4, CD49d/CD29) could be reprecipitated from CD81 immunoprecipitates. Anti-alpha 4 integrin mAb also coprecipitated CD81 from the alpha 4 beta 7-positive B cell line RPMI 8866. In contrast, no CD81 was identified in alpha 2 beta 1, alpha 5 beta 1, or alpha L beta 2 immunoprecipitates. Abs to other members of the transmembrane-4 superfamily, including CD53, CD63, and CD82, also coprecipitated alpha 4 beta 1. As shown by confocal microscopy, CD81 and CD82 colocalized with alpha 4 beta 1 in cell surface clusters. The cytoplasmic domain of the alpha 4 integrin was not necessary for alpha 4 beta 1/CD81 association, nor was the association influenced by divalent cations, EDTA, integrin-activating mAb, or alpha 4 subunit cleavage. Notably, two independent alpha 4 adhesion-deficient mutants (D346E and D408E) were deficient in their ability to associate with CD81. Thus, CD81 and other transmembrane-4 superfamily members may participate in functionally relevant interactions with alpha 4 beta 1 and other integrins.

Human natural "anti-Gal" antibodies are specifically directed to Gal alpha 1-3Gal beta 1-4GlcNAc residues expressed on non-primate mammal and new world monkey cells. We investigated the relative involvement of purified IgG and IgM anti-Gal as xenoreactive natural antibodies (XNA). IgG and IgM were isolated from human plasma, and anti-Gal antibodies were purified by affinity chromatography on a Synsorb-14 column (Chembiomed, Edmonton, Alberta, Canada). Anti-Gal of both IgM and IgG classes represent the bulk of human XNA that bind to porcine platelets in enzyme-linked immunosorbent assay (ELISA). On immunoblots, normal human sera, as well as purified IgM and IgG fractions, reacted with 115-, 125-, 135-, 150-, 180-, 210-, and 240-kd) pig platelet proteins, whereas purified anti-Gal antibodies of both IgM and IgG classes mainly bound to 135-, 150-, 180-, and 210-kD glycoproteins. A low reactivity was observed in ELISA with anti-Gal free IgM and IgG, indicating that xenoantibodies are not solely directed to galactosyl epitopes. These antibodies revealed bands of 115, 125, and 240 kD, alpha-Galactosidase treatment of porcine platelet glycoproteins (gps) enriched by affinity chromatography abrogated the reactivity of 135- and 210-kD proteins. N- and O-glycosidase treatments demonstrated that alpha-galactosyl residues are located on the O-glycans of the 135-kD component. Finally, glycoproteins of 90 and 135 kD were identified by amino acid sequencing as the pig analogs of the human glycoproteins IIIa and IIb, respectively, whereas the 240-kD) component was identified as the porcine fibrinogen, using a new murine monoclonal antibody (naM147-7B6; IgG1) specific for its beta-chain.

To define the molecular basis of human chemoattractant receptor regulation, rat basophilic leukemia RBL-2H3 cells, which are thrombin-responsive, were transfected to stably express epitope-tagged receptors for C5a, interleukin-8 (IL-8), formylpeptides (e.g. N-formyl-methionyl-leucyl-phenylalanine (fMLP)), and platelet-activating factor (PAF). Here we demonstrate that both thrombin and a synthetic peptide ligand for the thrombin receptor (sequence SFLLRN) caused phosphorylation and heterologous desensitization of the receptors for C5a, IL-8, and PAF but not that for formylpeptides as measured by agonist-stimulated [35S]guanosine 5'-3-O-(thio)triphosphate binding to membranes. Consistent with the PAF receptor phosphorylation, both thrombin and thrombin receptor peptide inhibited phosphoinositide hydrolysis, Ca2+ mobilization, and degranulation stimulated by PAF. Unexpectedly, despite heterologous desensitization at the level of receptor/G protein activation, there was enhancement ("priming") by thrombin of subsequent activities stimulated by C5a and IL-8 as well as fMLP. The priming effect of thrombin was blocked by its inhibitor, hirudin. However, two other activators of the thrombin receptor, the peptide SFLLRN and trypsin, stimulated Ca2+ mobilization in RBL-2H3 cells but did not cause priming. In addition, SFLLRN and the thrombin receptor antagonist peptide FLLRN both inhibited thrombin-induced Ca2+ mobilization but not priming. Furthermore, the proteolytically active gamma-thrombin, which does not stimulate the tethered ligand thrombin receptor and caused little or no Ca2+ mobilization in RBL-2H3 cells, effectively primed the response to fMLP. These data demonstrate that heterologous receptor phosphorylation and attenuation of G protein activation are not, by themselves, sufficient for the inhibition of biological responses mediated by C5a and IL-8. Moreover, thrombin appears to utilize mechanism(s) independent of its tethered ligand receptor to selectively prime phospholipase C-mediated biological responses of the C5a, IL-8, and formylpeptide receptors but not PAF. Because C5a, IL-8, and formylpeptide activate phospholipase Cbeta2, whereas PAF stimulates a different phospholipase C, the striking selectivity of thrombin's priming may be mediated via its ability to enhance receptor-mediated activation of phospholipase Cbeta2.

ADP is an important platelet agonist which initiates platelet shape change, aggregation, exposure of fibrinogen receptors, and calcium mobilization. Because of the limitations of previously used affinity analogs and photo-labeling studies as well as controversies surrounding the identity of an ADP receptor on platelets, we have used an affinity label capable of alkylating a putative exofacial receptor on platelets. We now report that 8-(4-bromo-2,3-dioxobutylthio)adenosine-5'-diphosphate (8-BDB-TADP), which is an analog of the natural ligand ADP, blocked ADP-induced platelet shape change, aggregation, exposure of fibrinogen-binding sites, secretion, and calcium mobilization. Following modification by 8-BDB-TADP, the rates of aggregation of platelets induced by thrombin, a calcium ionophore (A23187) or a stimulator of protein kinase C (phorbol myristate acetate) were minimally affected. However, the 8-BDB-TADP-modified platelets exhibited decreased rates of aggregation in response to ADP, as well as collagen and a thromboxane mimetic (U46619), both of which partially require ADP. Autoradiograms of the gels obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized platelets modified by either [beta-32P]8-BDB-TADP, or 8-BDB-TADP and NaB[3H]4 showed the presence of a single radiolabeled protein band at 100 kDa. The intensity of this band was reduced when platelets were preincubated with ADP, ATP, and 8-bromo-ADP prior to labeling by the radioactive 8-BDB-TADP. The results show that 8-BDB-TADP selectively and covalently labeled aggregin (100 kDa), a putative ADP receptor, resulting in a loss of ADP-induced platelet responses.

We previously suggested that platelet 62 kDa protein (p62, GP VI) is a putative collagen receptor by the studies on platelets and antiplatelet antibody in a patient (YA) with idiopathic thrombocytopenic purpura (ITP) who showed defective collagen-induced platelet function. Herein, we confirm that p62/p57 (reduced/unreduced conditions) is a specific membrane protein recognized by YA antibody using an improved immunoprecipitation technique. To further study the functional role of this protein, a crude p57 fraction ('p57') was obtained from human platelets by serial chromatography including DEAE-Sepharose CL-6B and WGA-agarose followed by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This 'p57' diminished the platelet-aggregating activity of collagen and of YA immunoglobulin G (IgG). N-terminal amino acid sequence analysis of 'p57' showed that it contained intercellular adhesion molecule 2 (ICAM-2). Immunoprecipitation experiments using platelet lysates and anti-ICAM-2 monoclonal antibody demonstrated that ICAM-2 molecules had a molecular weight similar to p62 on SDS-PAGE. However, ICAM-2 was also expressed on YA's p62-deficient platelets both on immunoprecipitation and flow cytometric analysis. Thus, ICAM-2 seems to be different from p62.

Transient phosphorylation of histidine characterizes the two-component systems in prokaryotes that control important physiological functions, but analogous events have not been implicated in signal transduction in mammalian cells. To explore histidine phosphorylation during activation of human cells, stimulated platelets were analyzed for the formation of protein phosphohistidine in a model system employing P-selectin. P-selectin, a leukocyte adhesion molecule, undergoes rapid phosphorylation and selective dephosphorylation of tyrosine, serine, and threonine. We now establish that phosphorylation following platelet activation with thrombin or collagen generates phosphohistidine at histidines on the cytoplasmic tail of P-selectin. With thrombin stimulation, the kinetics of phosphohistidine appearance and disappearance of P-selectin are very rapid. Platelets exhibit a novel ligand-induced signaling pathway to generate phosphohistidine. These results provide direct biochemical evidence for the induction of rapid and reversible histidine phosphorylation in mammalian cells upon cell activation and represent a novel paradigm for mammalian cell signaling.

The specificity of tetrafibricin was examined by comparing its activities on GPIIb/IIIa and on the vitronectin receptor (alpha v beta 3) with those of Arg-Gly-Asp-Ser (RGDS) on the same receptors. Tetrafibricin, which inhibited fibrinogen-GPIIb/IIIa binding 10 times more potently than RGDS, was three orders of magnitude less potent compared to RGDS on the inhibition of fibrinogen binding to alpha v beta 3. Furthermore, tetrafibricin potently inhibited platelet adhesion to both fibrinogen and von Willebrand factor. Whereas, there was no significant inhibition observed in the GPIIb/IIIa-independent cellular adhesions. These results suggest that tetrafibricin is highly selective for GPIIb/IIIa.

Leukocyte rolling in post-capillary venules is mediated by adhesion molecules of the selectin family expressed on both leukocytes (L-selectin) and endothelial cells (E- and P-selectin). With the use of intravital fluorescence microscopy, the effects of antibodies against these selectins were analyzed in the skinfold chamber model of BALB/c mice and the ear model of homozygous hairless mice (hr/hr) that permit chronic observation of striated muscle and skin microcirculation in awake animals, respectively. Mice were injected intravenously with monoclonal antibodies (MAb) to murine L-selectin and E-selectin and affinity-purified polyclonal antibodies to P-selectin. The antibodies, which are known to block cell adhesion, were tested by immunoprecipitation to selectively bind to L-, E-, or P-selectin. Leukocyte rolling was a constant finding in both microcirculation models in the absence of inflammatory stimuli. In both models, injection of anti-P-selectin antibodies completely prevented baseline leukocyte rolling over an observation period of 2 h (P < 0.01 vs. baseline), while no effects were seen after administration of either anti-L-selectin or anti-E-selectin MAb. Treatment with the isotype-matched control antibodies did not affect leukocyte rolling in either model. We conclude that leukocyte rolling in postcapillary venules of murine striated muscle and skin is a physiological process mediated via P-selectin, whereas L- and E-selectin appear not to play a significant role under these circumstances.

The platelet antigens, PlA1 and PlA2, are responsible for most cases of posttransfusion purpura (PTP) and neonatal alloimmune thrombocytopenia (NAIT) in the caucasian population and are determined by two allelic forms of the platelet glycoprotein GPIIIa gene. To study the interaction between these antigens and their respective antibodies, we inserted the sequence that encodes the signal peptide and the N-terminal 66 amino acids of the PlA1 form of GPIIIa into the expression vector pGEX1. To express the PlA2 antigen, nucleotide 196 of the PlA1 coding sequence was mutated to the PlA2 allelic form. When transformed and induced in Escherichia coli, the two constructs produce glutathione S-transferase (GST)/N-terminal GPIIIa fusion proteins, one containing leucine at position 33 (PlA1), the other proline (PlA2). These proteins are easily purified in milligram quantities using glutathione-Sepharose and react specifically with their respective antibodies by immunoblot and enzyme-linked immunosorbent assay. Antigenicity of the PlA1 fusion protein in reduced glutathione increases with time; moreover, the addition of oxidized glutathione accelerates this process, presumably because of formation of the native disulfide bonds. Neutralization assays indicate that the PlA1 fusion protein competes for all of the anti-PlA1 antibody in the serum of patients with PTP and NAIT that is capable of interacting with the surface of intact platelets. This study shows that the GST/N-terminal GPIIIa fusion proteins contain conformational epitopes that mimic those involved in alloimmunization, and that regions other than the amino terminal 66 amino acids of GPIIIa are not likely to contain or be required for the development of functional PlA1 epitopes. Furthermore, these recombinant proteins can be used for the affinity-purification of clinical anti-PlA1 antibodies and specific antibody identification by western blotting, making them useful in the diagnosis of patients alloimmunized to PlA1 alloantigens.

Platelet adhesion to subendothelial von Willebrand factor involves receptor recognition by the platelet glycoprotein (GP) Ib-IX and initiates activation signals that contribute to primary hemostasis. We show here that GPIb-IX is specifically associated with an intracellular 29-kDa protein. The physicochemical characteristics and amino acid sequence of this protein indicate that it is identical to the human zeta-isoform 14-3-3 protein, previously characterized as a platelet phospholipase A2 (PLA2). As activation of PLA2 is an early event in GPIb-IX-mediated signaling, this result suggests that ligand occupancy of GPIb-IX may directly activate PLA2, leading to platelet activation.

We have analyzed the reactivity of a new mouse monoclonal antibody (mAb), 11C3, which identifies a cell marker detected on the surface of chicken thrombocytes. Tissue distribution studies have shown that only cells of the thrombocytic lineage in blood, spleen, and bone marrow are stained by 11C3. However, it does not react with other species such as quail, mouse, and man. The 11C3 mAb immunoprecipitates an heterodimeric molecule made of two bands with an apparent molecular weight of 112 and 90 kDa under nonreducing conditions and 112 and 26 kDa following reduction. This pattern of migration is similar to the one observed for members of the integrin family of cell adhesion molecules. We have used the previously described mAb AP-2, which is specific for the human platelet integrin GPIIb-IIIa and cross-reacts with chicken thrombocytes. We have shown that it immunoprecipitates two bands with an identical electrophoretic mobility. Cross-inhibition and immunodepletion studies reveal that the two antibodies recognize two different isoforms or two conformational variants of the same molecule. Moreover, our data demonstrate that in contrast with AP-2, 11C3 is a potent inducer of thrombocyte activation measured by cell aggregation, chemiluminescence, or release of [3H]serotonin. It also inhibits the adhesion of thrombin-activated thrombocytes to fibrinogen and, to a lesser degree, to fibronectin, in a dose-dependent manner. Altogether, these results indicate that this antibody identifies the avian homolog of the mammalian platelet integrin and fibrinogen receptor GPIIb-IIIa.

The stimulation of human platelets with physiological agonists results in the incorporation of several proteins into the cytoskeleton, fibrinogen binding, and platelet aggregation. We recently demonstrated that the Ras-related low molecular weight GTP-binding protein Rap2B associates with the cytoskeleton in activated platelets and that this interaction requires platelet aggregation. In the present study we demonstrate that agonist-induced actin polymerization is necessary for the translocation of Rap2B to the cytoskeleton, suggesting that Rap2B interacts with the newly formed actin filaments. Moreover, the association of Rap2B with Triton X-100-insoluble material from platelets was totally blocked by treatment of intact platelets with monoclonal antibodies against the fibrinogen receptor glycoprotein IIb-IIIa. Platelets from patients affected by Glanzmann thrombastenia, a genetic disorder in which platelet plasma membranes lack glycoprotein IIb-IIIa but possess normal levels of Ras-related proteins, failed to incorporate Rap2B into the cytoskeleton upon activation by thrombin. Comparative immunoblotting revealed that the translocation of Rap2B to the cytoskeleton during platelet aggregation was accompanied by the simultaneous translocation of glycoprotein IIb-IIIa. Moreover, the cytoskeleton from aggregated platelets contained Rap2B and glycoprotein IIb-IIIa in comparable amounts. These results demonstrate the association of Rap2B and glycoprotein IIb-IIIa and their translocation to the cytoskeleton in aggregated human platelets.

Glycoprotein (GP) Ib alpha is required for expression of the highest affinity alpha-thrombin-binding site on platelets, possibly contributing to platelet activation through a pathway involving cleavage of a specific receptor. This function may be important for the initiation of hemostasis and may also play a role in the development of pathological vascular occlusion. We have now identified a discrete sequence in the extracytoplasmic domain of GP Ib alpha, including residues 271-284 of the mature protein, which appears to be part of the high affinity alpha-thrombin-binding site. Synthetic peptidyl mimetics of this sequence inhibit alpha-thrombin binding to GP Ib as well as platelet activation and aggregation induced by subnanomolar concentrations of the agonist; they also inhibit alpha-thrombin binding to purified glycocalicin, the isolated extracytoplasmic portion of GP Ib alpha. The inhibitory peptides interfere with the clotting of fibrinogen by alpha-thrombin but not with the amidolytic activity of the enzyme on a small synthetic substrate, a finding compatible with the concept that the identified GP Ib alpha sequence interacts with the anion-binding exosite of alpha-thrombin but not with its active proteolytic site. The crucial structural elements of this sequence necessary for thrombin binding appear to be a cluster of negatively charged residues as well as three tyrosine residues that, in the native protein, may be sulfated. GP Ib alpha has no significant overall sequence homology with the thrombin inhibitor, hirudin, nor with the specific thrombin receptor on platelets; all three molecules, however, possess a distinct region rich in negatively charged residues that appear to be involved in thrombin binding. This may represent a case of convergent evolution of unrelated proteins for high affinity interaction with the same ligand.

Stabilization of a clot is dependent on fibrin cross-linking mediated by the transglutaminase, factor XIIIa (FXIIIa). In addition to fibrin stabilization, FXIIIa acts on a number of platelet-reactive proteins, including fibronectin and vitronectin, as well as the platelet proteins, glycoprotein (GP) IIb-IIIa, myosin, and actin. However, conditions inducing the platelet-activation dependent binding of FXIIIa have not been characterized nor have the sites mediating FXIIIa binding been identified. The generation of FXIIIa and consequent detection of FXIIIa on the platelet surface were compared with other thrombin-induced activation events; the rate at which FXIIIa bound to activated platelets was much slower than platelet degranulation or fibrin(ogen) binding. Whereas platelets could be rapidly induced to express a functional receptor for FXIIIa, the rate of FXIIIa binding to platelets is limited by the rate of conversion of FXIII to FXIIIa. Immunoprecipitation of radiolabeled platelets using polyclonal anti-FXIII A-chain antibody identified two proteins corresponding to GPIIb and GPIIIa. Preincubation of intact platelets with 7E3, a monoclonal antibody that blocks the fibrinogen binding site, or GRGDSP peptide inhibited FXIIIa binding by about 95% when measured by flow cytometry; FXIIIa binding to purified GPIIb-IIIa was also inhibited by 7E3. The binding of FXIIIa to purified GPIIb-IIIa was enhanced by the addition of fibrinogen, but not by that of fibronectin or thrombospondin, suggesting that FXIIIa also binds to fibrinogen associated with the complex. These observations suggest that activated platelets bearing FXIIIa may enhance stabilization of platelet-rich thrombi through surface-localized cross-linking events.

The human integrin glycoprotein (GP)IIb/IIIa plays a central role in haemostasis as an inducible receptor for fibrinogen and other RGD-containing adhesive proteins at the platelet plasma membrane. Expression of the fibrinogen receptor on platelet activation involves conformational changes in the quaternary structure of GPIIb/IIIa. Little is known, however, about the nature of this conformational transition. Given that isolated GPIIb/IIIa contains a mixture of RGD-binding and non-RGD-binding heterodimers, we used limited proteolysis as a tool for investigating the structural differences between the two conformers. Comparison of their fragmentation patterns shows that, whereas in the non-RGD-binding form of GPIIb/IIIa the N-terminal half of the heavy chain of GPIIb (GPIIbH) and the central region of GPIIIa are cleaved by endoproteinase Arg-C, these domains associate tightly with one another in the RGD-binding GPIIb/IIIa and are thus protected from proteolysis. In addition, the C-terminal half of GPIIb becomes more susceptible to degradation in the non-RGD-binding GPIIb/IIIa conformer. Our interpretation, in the context of available structural and functional data, is that a major relative reorientation of the GPIIbH and GPIIIa extracellular domains takes place along the subunit interface during the conformational transition of the platelet integrin.

A well-characterized in vitro model system composed of thrombin-stimulated gel-filtered human platelets, fibrin-(ogen), plasminogen, and recombinant tissue plasminogen activator (rt-PA) was used to examine the relationship between platelet-fibrin adhesive interactions and the lytic resistance of a platelet-rich thrombus. Laser light scattering kinetic experiments demonstrated that the ligand-mimetic peptide D-RGDW and an anti-alpha IIb beta 3 monoclonal antibody both inhibited clot retraction, but neither integrin-targeted reagent affected the overall delay in lysis of "bulk" fibrin caused by thrombin-stimulated platelets. However, lysis of the model platelet-rich thrombus did proceed some 30% more quickly when treated with a plasminogen activator inhibitor (PAI)-resistant t-PA variant. Taken together, these results confirm that platelet-released PAI-1 is a major determinant of global lytic resistance. Next events occurring during fibrinolysis in the unique microenvironment near the platelet surface were monitored by scanning electron microscopy and quantitative fluorescence microscopy. Scanning electron micrographs of the partially lysed model thrombus in the presence of 200 mumol/L of D-RGDW showed no platelet aggregates, and fibrin was attached by fewer strands to the platelets. Quantitative fluorescence microscopy, using fluorescein-labeled fibrin, showed that fibrin adherent to the surface of thrombin-stimulated platelets lysed 20% to 50% more slowly than bulk fibrin (monitored in parallel by laser light scattering). Furthermore, this microspectroscopic technique showed that D-RGDW reduced the quantity of platelet-bound fibrin, and accelerated lysis near the platelet surface with both native rt-PA and the PAI-resistant variant. These observations suggest that the dense network of fibrin bound to the platelet surface is protected from fibrinolysis by tissue-type plasminogen activators. Further, uncoupling fibrin from its platelet receptors uniquely hastens fibrinolysis at the cell/fibrin interface.

Cellular receptors for blood proteases regulate chemotaxis, extracellular proteolysis, and growth behavior of normal and malignant cells. Binding of the coagulation protease factor Xa to leukocytes is contributed by a recently identified molecule, denominated Effector cell Protease Receptor-1 (EPR-1). Monoclonal antibodies were generated against EPR-1+ MOLT13 lymphocytes and selected for reactivity with lymphocyte surface proteins by flow cytometry and with affinity-purified EPR-1 in Western blots. Antibody-based functional cloning of the cDNA for EPR-1 reveals the sequence of a novel molecule encoded by a prominent 1.9-kilobase mRNA. The cDNA predicts a glycoprotein of 337 amino acids, characterized by a unique cysteine-rich extracellular module, a single membrane-spanning domain, and a serine-rich (26%) cytoplasmic tail featuring at least 15 potential phosphorylation sites. Genetically engineered EPR-1 transfectants were recognized by monoclonal antibodies to EPR-1 and bound 125I-factor Xa in a specific and saturable manner (Kd approximately 10-15 nM). In the absence of factor V/Va, EPR-1 transfectants promoted prothrombin activation in a factor Xa concentration-dependent reaction, inhibited by a monoclonal antibody to EPR-1. These findings define EPR-1 as a novel cell surface receptor for factor Xa potentially implicated in protease-dependent cellular effector functions.

We have applied flow cytometry to the detection of activated platelets in patients with coronary heart disease. Paraformaldehyde-fixed platelets were incubated with one of the following monoclonal antibodies (MAbs): Bx-1 (anti-GP Ib), AP-2 (anti-GP IIb-IIIa complex), VH10 (anti-GMP-140, a glycoprotein of the alpha-granule membrane), or PAC-1 (directed against an activation-dependent determinant on GP IIb-IIIa complexes). Bound antibody was quantitated after the addition of FITC-conjugated anti-immunoglobulin. This report highlights studies on 16 unstable angina patients undergoing transluminal angioplasty. Blood samples were taken at different periods before and after the angioplasty. Levels of activated platelets were variable, remaining in the 2-4% range of control donors for some, but increasing to 10-30% post-angioplasty for others (despite all patients receiving heparin and aspirin). Maximum numbers of activated platelets were detected at 24 or 48 h. Nonetheless, the amount of antibody bound to individual platelets rarely reached the levels seen when control platelets were stimulated with thrombin in vitro. Results with VH10 and PAC-1 often, but not always, correlated suggesting different pathways of platelet activation.

The platelet plasma membrane is lined with a membrane skeleton composed of short actin filaments, actin-binding protein, spectrin, vinculin, and other unidentified proteins. It is connected to the outside of the cell through association with the cytoplasmic domains of transmembrane receptors. In detergent-lysed platelets, cytoplasmic actin filaments are sedimented by centrifugation at 15,600 x g, but the sedimentation of membrane skeleton fragments requires higher g-forces (100,000 x g). In the present study, we show that the major platelet integrin, glycoprotein (GP) IIb-IIIa, sediments from detergent-lysed platelets at 100,000 x g together with fragments of the membrane skeleton that contain the cytoskeletal proteins spectrin, vinculin, and talin. In addition, this cell fraction contained the tyrosine kinases pp60c-src and pp62c-yes and the p21ras GTPase-activating protein (GAP). After thrombin-induced platelet aggregation mediated by fibrinogen binding to GP IIb-IIIa on adjacent platelets, we detected a redistribution of spectrin, talin, vinculin, pp60c-src, and pp62c-yes to the fraction that sediments at 15,600 x g. The redistribution of these proteins from the high-speed detergent-insoluble fraction to the low-speed fraction correlated with the extent of aggregation and was not detected in aggregation-defective thrombasthenic platelets (which lack the GP IIb-IIIa complex). In addition, many of the proteins phosphorylated on tyrosine in activated platelets were present in detergent-insoluble fractions. These results are consistent with the possibilities that 1) GP IIb-IIIa, pp60c-src, pp62c-yes, and GAP associate with a membrane skeleton fraction that contains spectrin, vinculin, and talin, 2) the association of GP IIb-IIIa with adhesive ligand in a platelet aggregate causes components of the membrane skeleton to undergo altered association with cytoplasmic actin filaments, and 3) many of the proteins phosphorylated on tyrosine residues in activated platelets are components of the cytoskeleton. The results imply that the membrane skeleton may play an important role in binding signaling molecules at sites of integrin-cytoskeleton interactions and in mediating signal transduction events in platelets. Further, GP IIb-IIIa-induced redistribution of components of the membrane skeleton and associated signaling molecules may represent an important step in regulating integrin-induced motile events in platelets.

Kistomin, a metalloprotease purified from venom of Calloselasma rhodostoma, dose- and time-dependently prolonged the latent period of aggregation and inhibited ATP secretion of human washed platelets stimulated by thrombin. It inhibited aggregation induced by low concentrations of thrombin (< or = 0.2 U/ml) whereas it had only slight effect on aggregation induced by high concentrations of thrombin (> or = 0.5 U/ml). Meanwhile it also inhibited ristocetin-induced platelet aggregation in a dose- and time-dependent manner. It significantly inhibited cytosolic calcium rise of Quin 2--loaded platelets, completely blocked thromboxane B2 formation, and blocked [3H]inositol phosphates formation of [3H]myoinositol loaded platelets stimulated by 0.1 U/ml of thrombin. Kistomin inhibited significantly thromboxane but not [3H]inositol phosphates formation of platelets stimulated by a high concentration of thrombin (1 U/ml). Incubation of platelets with kistomin resulted in a selective cleavage of platelet membrane glycoprotein Ib as revealed by SDS/PAGE stained by periodic acid/Schiff reagent. These results suggested that thrombin activates platelets at least through two receptors/or effectors-mediated events. In addition to glycoprotein Ib, other surface membrane component(s) (e.g., the seven transmembrane domain thrombin receptor) may also be important in regulating the biochemical events of human platelets in response to thrombin. However, the extent and rate of platelet aggregation stimulated by low concentrations of thrombin ( < or = 0.2 U/ml) are closely related with the intactness of glycoprotein Ib.

The transforming properties of v-jun, the viral counterpart of the transcription factor AP1, were investigated in avian hematopoietic cells. Two retroviruses, called JB and JBN, expressing both v-jun and v-erbB, were constructed using an avian erythroblastosis-based vector. We show that the cooperative action of both oncogenes allowed the virus to efficiently transform bone marrow cells. No such transformation was obtained with either oncogene alone. JB-transformed bone marrow cells expressed GATA-1, TAL-1, and histone H5, suggesting that they belong to the erythrocytic/thrombocytic lineage. (Thrombocytes are the avian homologues of mammal megakaryocytes.) Moreover, after induction with phorbol 12-myristate 13-acetate JB-transformed bone marrow cells began to differentiate and synthesized high levels of platelet glycoproteins, indicating that they were of thrombocytic origin. These results were confirmed by c-ets1 analysis since this transcription factor, specifically found in cells with megakaryocytic but not erythrocytic features, was clearly detected in these cells.

P-selectin is an adhesion receptor for leukocytes that is redistributed from secretory granule membranes to the surfaces of activated platelets and endothelial cells. The cytoplasmic domain of P-selectin contains two serines, two threonines, and one tyrosine that could potentially be phosphorylated. We found that P-selectin was phosphorylated in both platelets and endothelial cells and that phosphorylation rapidly increased after cell activation. Approximately 0.02, 0.05, and 0.08 mol of phosphate/mol of P-selectin were incorporated, respectively, into resting, thrombin-activated, and phorbol ester-activated platelets. Phosphorylation was completely inhibited by the protein kinase C inhibitors, staurosporine, H-7, and chelerythrine, and was enhanced by the phosphatase inhibitors, okadaic acid and calyculin-A. Phosphoamino acid analysis of 32P-labeled P-selectin showed that phosphorylation occurred predominantly on serine with lesser amounts on threonine. When expressed in transfected Chinese hamster ovary cells, P-selectin was also phosphorylated. Mutagenesis studies showed that Ser788 was the principal site of phosphorylation, with minor sites on the other serine and threonine residues of the cytoplasmic domain. Phosphorylation may regulate membrane trafficking or other functions of P-selectin.

P-selectin is an adhesion receptor for leukocytes on thrombin-activated platelets and endothelial cells. It contains a NH2-terminal carbohydrate-recognition domain, an epidermal growth factor motif, nine consensus repeats, a transmembrane domain, and a cytoplasmic tail. We expressed two soluble forms of P-selectin, one truncated after the ninth repeat (tPS) and the other lacking the transmembrane domain due to alternative RNA splicing (asPS). When visualized by electron microscopy, each was a monomeric rod-like structure with a globular domain at one end, whereas membrane P-selectin (mPS) from platelets formed rosettes with the globular domains facing outward. Sedimentation velocity and equilibrium studies confirmed that tPS and asPS were asymmetric monomers, whereas mPS was oligomeric. HL-60 cells adhered to immobilized tPS and asPS, although less efficiently than to mPS. 125I-Labeled tPS and asPS bound to approximately 25,000 sites/neutrophil and approximately 36,000 sites/HL-60 cell with an apparent Kd of 70 nM. Treatment of HL-60 cells with O-sialoglycoprotease eliminated the binding sites for asPS. We conclude that 1) P-selectin is a rigid, asymmetric protein; 2) monomeric soluble P-selectin binds to high affinity ligands with sialylated O-linked oligosaccharides on leukocytes; and 3) oligomerization of mPS enhances its avidity for leukocytes.

Immunohistochemical studies have localized thrombospondin (TSP), a platelet adhesive protein, to the atherosclerotic plaque. To investigate how TSP may become incorporated in the plaque, we evaluated the interaction of TSP with human plasma lipoproteins and apolipoproteins. Chylomicrons, VLDL, LDL, HDL, and apolipoproteins AI, AII, C were immobilized on microtiter plates. Binding to TSP was measured directly with [125I]TSP. Labeled TSP bound saturably to all the plasma lipoproteins tested, showing the highest capacity for binding to VLDL. This binding to VLDL was maximal in the presence of 1 mM CaCl2 and MgCl2 and only partially inhibited with EDTA. The binding was inhibited totally by incubation with fluid-phase lipoproteins, apolipoproteins or anti-TSP monoclonal antibodies. The dissociation constants (Kd) for VLDL and apo C were 153 nM and 150 nM, respectively. Thus, TSP exhibits specific and saturable binding with high affinity to VLDL, perhaps mediated by its surface apo C. This binding may facilitate TSP incorporation into nascent atherosclerotic plaques and delivery of VLDL cholesterol into these lesions.

The major platelet integrin, glycoprotein IIb-IIIa, binds soluble fibrinogen only after platelet activation. To investigate the mechanism by which platelets convert glycoprotein IIb-IIIa into a functional fibrinogen receptor, we characterized the opening and closing of fibrinogen-binding sites in isolated platelet membranes and compared the regulatory properties of membrane-bound glycoprotein IIb-IIIa with those of the detergent-solubilized receptor. Basal fibrinogen binding to the membranes possessed many of the properties of fibrinogen binding to activated platelets; however, less than 10% of glycoprotein IIb-IIIa in the membranes was capable of binding fibrinogen. Preincubating the membranes with either an activating glycoprotein IIb-IIIa antibody or alpha-chymotrypsin increased fibrinogen binding. In contrast, agents that require intracellular mediators, such as platelet agonists, guanine-nucleotide-binding-protein activators and purified protein kinase C, did not stimulate fibrinogen binding to the membranes, suggesting that cytosolic factor(s) may be required for activation of the receptor in platelets. Occupancy of glycoprotein IIb-IIIa in the membranes with RGD (Arg-Gly-Asp)-containing peptides reversibly exposed neoantigenic epitopes and fibrinogen-binding sites in the receptor. These conformational changes required membrane fixation to be maintained following peptide removal. Similar results were obtained with purified glycoprotein IIb-IIIa incorporated into phospholipid vesicles, indicating that the resting state of the receptor is favoured in these environments. In contrast, when the conformation of detergent-solubilized glycoprotein IIb-IIIa was altered by exposure to RGD-containing peptides, the receptor remained active even after incorporation into phospholipid vesicles. These results demonstrate that platelet membranes are a useful model in which to study the regulation of glycoprotein IIb-IIIa and suggest that the environment surrounding the receptor may have a profound influence on this process.

Lectin-carbohydrate recognition between the selectins and their ligands are among the earliest events in leukocyte recirculation, leukocyte recruitment into inflamed areas, and abnormal egress of leukocytes in diseases. Previously, we have described a dimeric sialoglycoprotein from myeloid cells with subunits of molecular mass = 120 kDa, which is selectively recognized by P-selectin (Moore, K.L., Stults, N.L., Diaz, S., Smith, D.F., Cummings, R.D., Varki, A., and McEver, R.P. (1992) J. Cell Biol. 188, 445-456). Here, we demonstrate that this P-selectin ligand carries alpha 2-3-linked sialic acids and the sialyl-Lewisx (SLex) tetrasaccharide motif. This glycoprotein contains < 1% of the total membrane-bound sialic acids and a very small fraction of the total SLex on neutrophil membranes. In spite of a relative resistance to sialidase digestion, the predominant form of sialic acid on the ligand is N-acetylneuraminic acid. Selective periodate oxidation of the side chain of sialic acids does not affect P-selectin binding and allows the introduction of tritium label into the truncated sialic acids. beta-Elimination with alkaline borohydride releases labeled O-linked oligosaccharides both from the labeled neutrophil ligand and from the ligand purified from HL-60 cells metabolically labeled with [3H]glucosamine. The ligand from both neutrophils and HL-60 cells is also susceptible to cleavage by the enzyme O-sialoglycoprotease from Pasteurella hemolytica. Analysis of the specificity of this enzyme suggests that the P-selectin ligand carries large numbers of closely spaced sialylated O-linked oligosaccharides. O-Sialoglycoprotease abolishes both direct binding of P-selectin to HL-60 cells and the adhesion of HL-60 cells to immobilized P-selectin, without significantly decreasing overall cell surface SLex expression. This indicates that the 120-kDa ligand may be the major determinant of P-selectin:myeloid cell interaction in vivo. Finally, based on the current and previous data, we hypothesize that the high affinity recognition site(s) of this P-selectin ligand may be derived from a "clustered saccharide patch" of sialylated fucosylated O-linked oligosaccharide sequences.

We have used a series of Escherichia coli-expressed deletion mutants of the glycoprotein (GP) Ib-binding domain of von Willebrand factor (vWF) to study the structural basis of its function. In addition to the prototypic molecule (rvWF441-733), we constructed 11 mutants; seven had deletions of sequence on the amino- and/or carboxyl-terminal side of the Cys509-Cys695 intrachain disulfide loop, and four had limited deletions inside the loop. Other cysteine residues in addition to 509 and 695, when present in the corresponding native sequence, were mutated to glycine; all molecules were purified in the oxidized as well as reduced and alkylated state. The smallest species retaining the ability to interact with GP Ib in the absence of modulators was the oxidized rvWF508-696; the latter, as well as rvWF441-696, became inactive after reduction and alkylation. In contrast, all the other fragments with deletions outside of the loop, but extending at least to residue 700, showed better binding to platelets after reduction and alkylation than when the Cys509-Cys695 disulfide bond was oxidized. Any limited deletion of sequence inside the loop caused complete loss of GP Ib-binding function both in the absence or in the presence of botrocetin, and this persisted even after reduction and alkylation. In contrast, all mutants with intact sequence between residues 509 and 695 bound to GP Ib in the presence of botrocetin, regardless of whether the 2 cysteine residues were oxidized or reduced and alkylated. Ristocetin, unlike botrocetin, appeared to have no effect in modulating the binding of any of the expressed fragments to platelets. Our findings suggest that the GP Ib-binding domain of vWF contains multiple interaction sites, but integrity of the sequence 509-695 is important for function.

The cardioprotective effects of an mAb to P-selectin designated mAb PB1.3 was examined in a feline model of myocardial ischemia (MI) and reperfusion. PB1.3 (1 mg/kg), administered after 80 min of ischemia (i.e., 10 min before reperfusion), significantly attenuated myocardial necrosis compared to a non-blocking mAb (NBP1.6) for P-selectin (15 +/- 3 vs 35 +/- 3% of area at risk, P < 0.01). Moreover, endothelial release of endothelium derived relaxing factor, as assessed by relaxation to acetylcholine, was also significantly preserved in ischemic-reperfused coronary arteries isolated from cats treated with mAb PB1.3 compared to mAb NBP1.6 (67 +/- 6 vs 11 +/- 3, P < 0.01). This endothelial preservation was directly related to reduced endothelial adherence of PMNs in ischemic-reperfused coronary arteries. Immunohistochemical localization of P-selectin was significantly upregulated in the cytoplasm of endothelial cells that lined coronary arteries and veins after 90 min of ischemia and 20 min of reperfusion. The principal site of intracytoplasmic expression was in venous vessels. mAb PB1.3 significantly decreased (P < 0.01) adherence of unstimulated PMNs to thrombin and histamine stimulated endothelial cells in a concentration-dependent manner in vitro. These results demonstrate that PMN adherence to endothelium by P-selectin is an important early consequence of reperfusion injury, and a specific monoclonal antibody to P-selectin exerts significant endothelial preservation and cardioprotection in myocardial ischemia and reperfusion.

We report that the adhesion receptor P-selectin can be metabolically labeled with [3H]palmitic acid in human platelets. Analysis of alkaline methanolysis products from labeled protein demonstrated that the radioactivity associated with P-selectin was covalently bound palmitic acid. [3H]Palmitic acid was cleaved by hydroxylamine treatment at neutral pH and by reducing agents, indicating that acylation occurred through a thioester linkage. Both stearic acid and palmitic acid were detected by gas chromatography-mass spectrometry analysis of alkaline hydrolysates of purified P-selectin. Deletion or mutation of Cys766 eliminated [3H] palmitic acid labeling of P-selectin in transfected COS-7 cells. We conclude that the cytoplasmic domain of P-selectin is acylated at Cys766 through a thioester bond. Fatty acid acylation may regulate intracellular trafficking or other functions of P-selectin.

In addition to thyroglobulin, primary thyrocytes secrete into the culture medium significant quantities of p500, a protein so named because of its M(r) > or = 500,000. Epithelial monolayers cultured on porous filters serve as a useful model system in which to study protein secretion. From these monolayers, thyroglobulin, the precursor in thyroid hormonogenesis, was released with apical predominance, while p500 was found mostly in the basolateral medium. Thyrocyte exposure to thyrotropin augmented selectively thyroglobulin but not p500 production. By contrast, exposure to cycloheximide actually augmented p500 production, a response observed for immediate-early proto-oncogenes. Using thyrocyte conditioned medium, the p500 protein was purified to homogeneity. Peptide sequencing of tryptic fragments of purified p500 showed identity to thrombospondin. Immunoprecipitation of thrombospondin from media bathing primary thyrocytes and the FRTL5 cell line quantitatively recovered p500, confirming its identity and indicating an epithelial origin. Gel filtration of secreted thrombospondin eluted at a high molecular weight, suggesting complexation with components of the extracellular matrix. Further, immunofluorescence showed cellular codistribution of thrombospondin and thyroglobulin, although thrombospondin exhibited predominantly an extracellular, basolateral deposition. It seems likely that thrombospondin production by thyrocytes plays a role in the growth or development of the thyroid epithelium.

The prototypical integrin receptor, alpha IIb beta 3, isolated from the membrane fraction of human blood platelets by solubilization in Triton X-100 (reduced) and affinity chromatography on lentil lectin-agarose, has been further purified by gel filtration chromatography in octyl glucoside to obtain the intact receptor complex in a form suitable for hydrodynamic measurements. The molecular weight [(6.0 +/- 0.2) x 10(3)] and Stokes radius (2.3 +/- 0.1 nm) of detergent micelles formed in 0.03 M octyl glucoside have been determined by classical light scattering intensity and dynamic light scattering measurements, respectively. An algorithm has been developed which explicitly considers the contribution of detergent micelles to the intensity autocorrelation function of particles suspended in detergent. This procedure has been validated with polystyrene particles of known radius, as well as with the soluble protein fibrinogen. Application of these procedures to dynamic light scattering data obtained with alpha IIb beta 3 resulted in a translational diffusion coefficient (Dto(20,w)) of (2.78 +/- 0.31) x 10(-7) cm2 s-1, corresponding to a Strokes radius (Rs) of 7.67 +/- 0.85 nm for the integrin/octyl glucoside complex. Light scattering intensity measurements gave a molecular weight of (2.26 +/- 0.22) x 10(5) for the polypeptide moiety of the complex, in excellent agreement with the 2.28 x 10(5) value calculated from primary structure data. As a spherical, hydrated alpha IIb beta 3 complex, with bound detergent, would exhibit a Stokes radius of approximately 5 nm, these data indicate considerable asymmetry in the solution conformation of alpha IIb beta 3.