Surface Of Human Platelets Research Articles

Thrombocyte HLA molecules retain nonrenewable endogenous peptides of megakaryocyte lineage and do not stimulate direct allocytotoxicity in vitro

AbstractThe origin and the function of HLA class I molecules present on the surface of human platelets are still unclear. In particular, it is controversial which fraction of these class I molecules represents integral membrane components derived from the megakaryocyte-platelet lineage versus soluble plasma HLA molecules acquired by adsorption. Results of the present study show that HLA-A2 ligands isolated from platelets possess the same peptide motif as described for HLA-A2-associated peptides obtained from nucleated cells. Sequencing of these platelet-derived peptides reveals that they originate mainly from ubiquitously expressed proteins also present in the megakaryocyte-platelet lineage. Moreover, one of these peptides derives from the GPIX protein, which is specifically expressed by platelets and their precursors. Platelet HLA molecules are unstable in vitro at 37°C, but can be partially stabilized by addition of exogenous β2-microglobulin and HLA class I binding peptide, suggesting that platelets cannot load HLA molecules with endogenous peptides. In in vitro experiments platelets were used to stimulate peripheral blood mononuclear cells. No allospecific cytotoxicity was observed after primary stimulation, or secondary restimulation, with allogenic resting or activated platelets, even in the presence of additional third-party helper activity. These data indicate that HLA class I molecules from platelets cannot directly induce allogenic CD8+ cytotoxic T-cell response in vitro.

Thrombocyte HLA molecules retain nonrenewable endogenous peptides of megakaryocyte lineage and do not stimulate direct allocytotoxicity in vitro

The origin and the function of HLA class I molecules present on the surface of human platelets are still unclear. In particular, it is controversial which fraction of these class I molecules represents integral membrane components derived from the megakaryocyte-platelet lineage versus soluble plasma HLA molecules acquired by adsorption. Results of the present study show that HLA-A2 ligands isolated from platelets possess the same peptide motif as described for HLA-A2-associated peptides obtained from nucleated cells. Sequencing of these platelet-derived peptides reveals that they originate mainly from ubiquitously expressed proteins also present in the megakaryocyte-platelet lineage. Moreover, one of these peptides derives from the GPIX protein, which is specifically expressed by platelets and their precursors. Platelet HLA molecules are unstable in vitro at 37°C, but can be partially stabilized by addition of exogenous β2-microglobulin and HLA class I binding peptide, suggesting that platelets cannot load HLA molecules with endogenous peptides. In in vitro experiments platelets were used to stimulate peripheral blood mononuclear cells. No allospecific cytotoxicity was observed after primary stimulation, or secondary restimulation, with allogenic resting or activated platelets, even in the presence of additional third-party helper activity. These data indicate that HLA class I molecules from platelets cannot directly induce allogenic CD8+ cytotoxic T-cell response in vitro.

Biphasic kinetics of activation and signaling for PAR1 and PAR4 thrombin receptors in platelets.

Thrombin activates platelets in an ordered sequence of events that includes shape change, increase in cytoplasmic Ca(2+), activation of the alphaIIbbeta3 integrin, granule secretion, aggregation, and formation of a stable hemostatic plug. Activation of this process has also been implicated in the pathogenesis of atherosclerosis, stroke, and thrombosis. There are two identified thrombin-activated receptors on the surface of human platelets. PAR1 is a high-affinity thrombin receptor, and PAR4 is a low apparent affinity thrombin receptor of uncertain function. The goal of these studies is to determine the kinetics of thrombin activation of PAR1 and PAR4 and to relate the individual inputs from each receptor to platelet Ca(2+) signaling, secondary autocrine stimulation, and aggregation. Using a combination of PAR-specific peptide ligands and anti-PAR1 reagents, we separated the biphasic thrombin Ca(2+) response of platelets into two discrete components-a rapid spike response caused by PAR1, followed by a slower prolonged response from PAR4. Despite having a 20-70-fold slower rate of activation, PAR4 produces the majority of the integrated Ca(2+) signal that is sustained by the continuous presence of catalytically active thrombin. Surprisingly, PAR4 activation is much more effective than PAR1 activation in mounting secondary autocrine Ca(2+) signals from secreted ADP. The strong ADP response due to activated PAR4, however, requires prior activation of PAR1 as would normally occur during treatment of platelets with thrombin. Thus, the late signal generated by activated PAR4 is not redundant with the early signal from PAR1 and instead serves to greatly extend the high intracellular Ca(2+) levels that support the late phase of the platelet aggregation process.

Structure and Function of Murine αIIbβ3 (GPIIb/IIIa): Studies Using Monoclonal Antibodies and β3-null Mice

The alphaIIbeta3 receptor (GPIIb/IIIa) is the only platelet-specific integrin receptor and the most abundant adhesion/aggregation receptor on the surface of human platelets. Since mice are increasingly being used as models of human disease, we analyzed the structure and function of murine platelet alphaIIbeta3, utilizing both beta3 integrin-deficient mice, who have a phenotype that resembles Glanzmann thrombasthenia, and our hamster monoclonal antibody (mAb) 1B5 to murine alphaIIbbeta3. By immunoblot analysis, flow cytometry, and mAb binding studies, mouse platelets express abundant amounts of alphaIIbbeta3 (60-80,000 copies/platelet). Like their human counterparts, murine alphaIIb and beta3 exhibit different electrophoretic motilities under nonreducing (aIIb 135k Da; beta3 92k Da) and reducing (aIIb 120k Da; beta3 108k Da) conditions, and the alphaIIbbeta3 complex is dissociated by EDTA at pH 8 and 37 degrees C. Murine beta3 is less susceptible to proteolysis by plasmin than is human beta3. In addition to defective platelet aggregation, mouse platelets lacking alphaIIbbeta3 and alphaVbeta3 are unable to adhere to fibrinogen and prothrombin, but retain the ability to adhere to fibronectin and collagen. Following platelet activation, beta3-null platelets express slightly less P-selectin than do wild-type mouse platelets. Moreover, beta3-null platelets have altered tyrosine phosphorylation patterns following thrombin- and collagen-induced aggregation. These results suggest fundamental similarities between human and mouse platelet activation and aggregation, but delineate subtle differences that need to be considered when comparing studies from mice and humans.

A CD9, alphaIIbbeta3, integrin-associated protein, and GPIb/V/IX complex on the surface of human platelets is influenced by alphaIIbbeta3 conformational states.

A noncovalently associated complex comprising of CD9, the fibrinogen (Fg) receptor alphaIIbbeta3, integrin-associated protein (IAP), and glycoprotein (GP) Ib/V/IX complex was isolated from Chaps-solubilized human platelets. The CD9 complex was immunoprecipitated by mAbs specific for CD9 (mAb7), IAP (BRIC126), GPIb (SZ1), GPIX (GR-P), beta3 (AP3) and alphaIIb (C3). Additionally, the association between CD9 and alphaIIbbeta3 was demonstrated by ELISA. In this system, CD9 did not bind to vitronectin receptor (alphavbeta3) suggesting that CD9/alphaIIbbeta3 association was alphaIIb-subunit or alphaIIbbeta3-complex dependent. D3, an alphaIIbbeta3-activating mAb that is also an anti-LIBS (ligand-induced binding site), immunoprecipitated primarily alphaIIbbeta3 with GPIb and IAP. CD9 was not detected in D3 immunoprecipitates. D3 binding induced platelet aggregation via direct alphaIIbbeta3 activation and was upregulated by the alphaIIbbeta3 antagonist eptifibatide. In contrast, AP3 and C3 exhibited neither effect. In addition, D3 also inhibited whole blood clot retraction, in contrast to AP3 and C3, suggesting that conformational constraints on alphaIIbbeta3 by D3 binding not only influenced the CD9 complex but also affected alphaIIbbeta3 post receptor occupancy events. The CD9 complex was immunoprecipitated in the presence of eptifibatide, demonstrating that alphaIIbbeta3 receptor occupancy was not sufficient to cause complex dissociation. CD9 complex isolation was also independent of platelet activation, although a twofold increase in the quantity of CD9 complex was seen after platelet activation by alpha-thrombin in the presence of CaCl2 compared with that present in EDTA. Stirred platelets showed fibrinogen-mediated aggregation by alpha-thrombin in the presence of CaCl2 but not with EDTA, suggesting that fibrinogen crosslinking of CD9 complexes via alphaIIbbeta3 could be partially responsible for this increase. These findings imply that the platelet CD9 complex is independent of platelet activation although it is dependent upon the conformation state of alphaIIbbeta3.

Functional Expression of the High Affinity Receptor for IgE (FcɛRI) in Human Platelets and Its' Intracellular Expression in Human Megakaryocytes

AbstractThe high affinity IgE receptor (FcɛRI) expressed on the cell surface of mast cells and basophils is the key molecule in triggering the IgE-mediated allergic reaction. Recently, it was elucidated that the FcɛRI is expressed on a variety of other cells like Langerhans cells, monocytes, and eosinophils, and the functional importance of the FcɛRI expression in Langerhans cells was also shown. Some studies suggest that human platelets may play important roles in allergic inflammation through the cell-surface expression of the FcɛRII and FcγRII. Here, we report that human platelets and megakaryocytes constitutively express the messenger RNA and protein for the FcɛRI. Although the FcɛRI is expressed on the cell surface of human platelets, it is only detected in the cytoplasm of human megakaryocytes. We also confirmed that human platelets express the genes for the α, β, and γ chains of the FcɛRI without any defined mutations. Furthermore, stimulation of human platelets via the FcɛRI induced the release of serotonin and RANTES (Regulated on Activation, Normal T Expressed, and presumably Secreted). Taken together, these results suggest a novel and important role for human platelets in perpetuating allergic inflammation through the expression of and activation via the FcɛRI.

Functional Expression of the High Affinity Receptor for IgE (FcɛRI) in Human Platelets and Its’ Intracellular Expression in Human Megakaryocytes

The high affinity IgE receptor (FcepsilonRI) expressed on the cell surface of mast cells and basophils is the key molecule in triggering the IgE-mediated allergic reaction. Recently, it was elucidated that the FcepsilonRI is expressed on a variety of other cells like Langerhans cells, monocytes, and eosinophils, and the functional importance of the FcepsilonRI expression in Langerhans cells was also shown. Some studies suggest that human platelets may play important roles in allergic inflammation through the cell-surface expression of the FcepsilonRII and FcgammaRII. Here, we report that human platelets and megakaryocytes constitutively express the messenger RNA and protein for the FcepsilonRI. Although the FcepsilonRI is expressed on the cell surface of human platelets, it is only detected in the cytoplasm of human megakaryocytes. We also confirmed that human platelets express the genes for the alpha, beta, and gamma chains of the FcepsilonRI without any defined mutations. Furthermore, stimulation of human platelets via the FcepsilonRI induced the release of serotonin and RANTES (Regulated on Activation, Normal T Expressed, and presumably Secreted). Taken together, these results suggest a novel and important role for human platelets in perpetuating allergic inflammation through the expression of and activation via the FcepsilonRI.

The Platelet High Affinity Binding Site for Thrombin Mimics Hirudin, Modulates Thrombin-induced Platelet Activation, and Is Distinct from the Glycoprotein Ib-IX-V Complex

The platelet high affinity binding site for thrombin appears to be described by a classical receptor-ligand interaction that is distinct from the platelet thrombin receptor/substrate, PAR-1. However, the identification and function of the high affinity binding site with respect to its physiological importance have continued to elude investigators. Prior studies using two mutant thrombins suggested that thrombin interaction with the platelet high affinity binding site is mediated through an extensive portion of the thrombin molecule involving residues within the substrate binding pocket and the anion binding exosite (Leong, L., Henriksen, R. A., Kermode, J. C., Rittenhouse, S. E., and Tracy, P. B. (1992) Biochemistry 31, 2567-2575) and may mimic a thrombin-hirudin interaction. To test this hypothesis, an anti-hirudin peptide antibody (anti-hirpeptide Ab) was raised against a peptide mimicking the COOH terminus of hirudin. The Ab recognized adherent platelets and those in suspension as determined by enzyme-linked immunosorbent assay and immunofluorescence microscopy, respectively. 125I-Thrombin binding to platelets was inhibited in the presence of the anti-hirpeptide Ab in a dose-dependent manner with maximal inhibition >90%. Analyses of data from binding studies of 125I-thrombin to platelets at a fixed Ab concentration indicated that the anti-hirpeptide Ab inhibited the high affinity binding interaction exclusively. In addition, thrombin-induced increases in platelet [Ca2+]i were enhanced by blocking the high affinity binding site with the Ab due to redistribution of the agonist to PAR-1. Thrombin Quick I-induced platelet calcium mobilization was unaffected by the presence of the Ab, consistent with the inability of thrombin Quick I to bind to the high affinity site. Even though glycoprotein (GP) Ib contains a hirudin-like region within the alpha subunit, the postulated high affinity binding site, direct binding of 125I-thrombin could not be demonstrated to transfected Chinese hamster ovary and L cells expressing the GP Ib-IX-V complex. Furthermore, an anti-GP Ib Ab, raised to the peptide region proposed as the thrombin high affinity site, did not enhance thrombin-induced platelet calcium mobilization. The anti-hirpeptide Ab recognized a population of platelet membrane proteins distinct from PAR-1 and GP Ib by three-color immunofluorescence using confocal microscopy. These combined studies demonstrate that the high affinity binding site for thrombin is a unique platelet protein distinct from GP Ib which modulates the effective thrombin concentration localized at the human platelet surface.

Crossreactivity of Human versus Swine Platelet Surface Antigens Is Similar for Glycoproteins Ib and IIIa, but Not for the Glycoprotein IIb/IIIa Complex.

Swine platalets are very similar to those of humans and are therefore relevant to cardiovascular research. The swine coronary circulation mimics the human circulation and is large enough to obtain multiple blood samples in survival experiments. In swine regional ischemia similar to the human condition is easily obtainable, which makes the porcine model an ideal choice to study coronary artery disease. However, little is known about the similarity between swine and human platelet surface antigens. We tested the hypothesis that certain swine platelet antigens could crossreact with antihuman antibodies. Using FITC-conjugated monoclonal murine antihuman platelet antibodies, surface antigen expression was determined for human and Yorkshire swine platelets. Expression of CD9 (p24), CD42B (Ib), CD41b, (Ilb), CD61 (IIIa), CD41a (Ilb/IlIa), CD49b (VLA-2), CD62p, (P selectin), CD31 (PECAM-1D, and CD51/CD61 (vitronectin) was measured by flow cytometry. Significant crossreactivity with human platelets was observed consistently for swine platelet GP 1b and GP IIIa. Crossreactivity of the swine GPIb, and GP IIIa with the human receptors is evidence of receptor similarity between human and swine platelets. The implications of significant crossreactivity of these antigens and the lack of recognition of IIb/IIIa needs to be understood in cardiovascular research. Determining commercially available antihuman GP Ib and GP IIIa, rather than GP IIb/IIIa, would contribute to better elucidation of the effect of von Willebrand factor and the booming family of platelet inhibitors in the swine model of ischemia-reperfusion.

Immunoaffinity Method to Identify Aggregin, a Putative ADP–Receptor in Human Blood Platelets

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.

High-resolution backscatter electron detection of cell surface molecules on human platelets using the double-layer coating method and cryo field emission scanning electron microscopy.

A model system utilizing cryo scanning electron microscopy (SEM) for the detection of putative cell adhesion molecule(s) on the surface of human platelets is described. Plunge freezing was used for cryoimmobilization of unactivated and activated platelets after prefixation. Extracellular ice was removed by sublimation to expose the surface of the platelet membrane. Cryosamples were coated by the double-layer method, in which undirectional shadowing is performed at an angle of 45 degrees with 2 nm of platinum by thermal evaporation, followed by evaporation of 5 nm of carbon at an angle of 90 degrees for stabilization of the platinum film. The topography of the extracellular surface of the unstimulated platelet membrane was dominated by small spherical protrusions, while that of the activated platelet had not only similar spherical projections, but also possessed numerous rod-like protrusions, presumably representing the upregulation of the cell adhesion molecule, P-selectin, from intracellular a granules. These results clearly demonstrate that cryo field-emission SEM can detect molecular topography on the extracellular surface of cells consistent with the dimensions and shape of membrane cell adhesion molecules.

Co-localization of CD9 and GPIIb-IIIa (alpha IIb beta 3 integrin) on activated platelet pseudopods and alpha-granule membranes.

CD9 is a 24-kDa membrane glycoprotein expressed on the surface of human platelets and potentially involved in cellular activation and adhesion functions. This protein belongs to a recently delineated family of cell-surface antigens that span the membrane four times, called tetraspans, and found mainly in leucocytes and tumour cells. As a first approach to clarify the function of CD9, we used immunoelectron microscopy to determine the localization of this antigen in human platelets, and compared its distribution with that of the GPIIb-IIIa integrin, the platelet receptor for fibrinogen. Monoclonal antibodies against CD9 (MAb7) and GPIIb-IIIa (HP1-1D) coupled to colloidal gold of different sizes (5 and 15 nm) were incubated with intact platelets in suspension or on ultrathin sections of platelets embedded in LR white. CD9 was found in association with GPIIb-IIIa on the inner face of alpha-granule membranes. These two antigens also colocalized on pseudopods of activated platelets and in contact regions between adjacent platelets. CD63, another member of the tetraspan family, was absent from alpha-granules but was associated with lysosomal structures. Flow cytometric analysis of platelet CD9 with a series of monoclonal antibodies revealed an increased expression upon thrombin stimulation, confirming the presence of an intracellular granular pool. The observation that CD9 and GPIIb-IIIa are stored in the same intracellular structures and migrate to the same activation zones after platelet stimulation lends support to previous suggestions of a close association between CD9 and GPIIb-IIIa in human platelets and of a possible involvement of CD9 in adhesive functions of platelets.

Release of the Thrombin Receptor (PAR-1) N-terminus from the Surface of Human Platelets Activated by Thrombin

Platelet activation by thrombin is at least partially mediated by a G-protein-coupled receptor whose extended N-terminus is cleaved by thrombin. Theoretically, this should release a small fragment containing the original receptor N-terminus. However, the fate of this fragment is unknown, as is its biological role, if any. To begin to examine these issues, we have prepared monoclonal anti-receptor antibodies whose epitopes lie entirely N-terminal to the thrombin cleavage site. By flow cytometry and fluorescence microscopy of human platelets and megakaryoblastic CHRF-288 cells, these antibodies were found to recognize intact receptors and receptors activated by the agonist peptide, SFLLRN, but not receptors whose N-terminus had been cleaved by thrombin or cathepsin G. Incubating CHRF-288 cells with thrombin released pre-bound antibody from the cell surface. An assay based upon the antibodies was able to detect a fragment containing the original receptor N-terminus in the supernate of thrombin-treated human platelets. The concentration of the fragment obtained with platelets from 15 normal donors was 4.8 +/- 0.9 pmol per 10(9) platelets (mean +/- sem), which is similar to the value expected if all of the thrombin receptors present on human platelets have been cleaved. Taken together, these results demonstrate that 1) following receptor cleavage a fragment containing the original N-terminus of the receptor is released from the platelet surface, 2) based upon epitope mapping, this fragment is at least 15-20 residues long, 3) it is possible to quantitate the receptor fragment in the supernates of cells exposed to thrombin, and 4) the results of the quantitation suggest that on platelets all of the receptors have been cleaved and 100% of the fragment is present in the cell supernate. Depending on it survival time, measurements of the receptor fragment in blood or urine samples may eventually prove to be a useful marker for thrombin receptor activation in vivo.

EFFECT OF MONOCLONAL ANTIBODY ON THE ADHESION OF PLATELETS TO AORTIC SEGMENTS

A purified monoclonal antibody raised against a purified platelet 65 kDa protein that binds to type I collagen receptor was used to see if the antibody can block the adhesion of platelets to damaged aortic segments. The purified antibody binds to the surface of human platelets by flow cytometry. The adhesion of platelets to the damaged aortic segments was significantly reduced by the preincubation of the platelet-rich plasma with the antibody, suggesting that the antibody binds to platelets and blocks the adhesion of platelets to the aortic segments. Copyright © 1996 Elsevier Science Ltd

Evidence for cAMP-dependent Platelet Ectoprotein Kinase Activity That Phosphorylates Platelet Glycoprotein IV (CD36)

The dephosphorylating enzyme alkaline phosphatase, by removing phosphate groups from the external platelet membrane proteins, modulates platelet activation (Hatmi, M., Haye, B., Gavaret, J. M., Vargaftig, B. B., and Jacquemin, C. (1991) Br. J. Pharmacol. 104, 554-558). This observation, together with findings reported by others (Ehrlich, Y. H., Davis, T. B., Bock, E., Kornecki, E., and Lenox, R. H. (1986) Nature 320, 67-70; Dusenbery, K. E., Mendiola, J. R., and Skubitz, K. M. (1988) Biochem. Biophys. Res. Commun. 153, 7-13), indicate the existence of ectoprotein kinase activity on the blood platelet surface. In this study, we demonstrate that washed human platelets phosphorylate the synthetic heptapeptide kemptide in a cAMP-dependent mode. The intensity of the phosphorylation was concentration-dependent for kemptide. In addition, incubation of platelets with [gamma-32P]ATP resulted in a rapid incorporation of [32P] phosphate into proteins at the outer membrane surface that was sensitive to alkaline phosphatase treatment. When cAMP was added to the medium, major phosphorylation of an 88-kDa ectoprotein occurred. Its isoelectric point determined by isoelectric focusing SDS-polyacrylamide gel electrophoresis was around pH 6.2. Phosphorylations of this 88-kDa polypeptide and of the exogenous kemptide substrate were both prevented by the specific protein kinase A inhibitor peptide. When platelets were preincubated with [32P]inorganic phosphate to label intracellular proteins, the protein phosphorylation pattern was different from that obtained with [gamma-32P]ATP, indicating that the latter occurred at the outer surface of the cells. Prostacyclin, which induces the increase of intracellular cAMP levels and, consequently, its liberation into the extracellular medium, increased phosphorylation of both kemptide and platelet 88-kDa polypeptide. The major protein of 88-kDa, which was phosphorylated in the presence of cAMP and external [gamma-32P]ATP, was identified by immunoprecipitation to GPIV (CD36), one of thrombospondin and collagen binding sites on platelets. The phosphorylation of CD36 also occurred in platelet-rich plasma, suggesting a physiological role for this ectoenzyme. In the present study, we clearly demonstrate the presence of an ectoprotein kinase A activity at the surface of intact human platelets, and we revealed its principal endogenous substrate as being CD36.

A Novel Method for Chemical Modification of Functional Groups Other Than a Carboxyl Group in Proteins byN-Ethyl-5-phenylisooxazolium-3′-sulfonate (Woodward's Reagent-K): Inhibition of ADP-Induced Platelet Responses Involves Covalent Modification of Aggregin, an ADP Receptor

The chemical reaction ofN-ethyl-5-phenylisooxazolium-3′-sulfonate (Woodward's Reagent-K, WR-K) with a carboxyl group yields an enol ester that cannot be reduced by sodium borohydride in an aqueous solution, while other nucleophiles such as sulfhydryl, hydroxyl, amino, and imidazole groups, react with WR-K to yield unsaturated ketones that are capable of being reduced by sodium borohydride in an aqueous medium. Aggregin, a 100-kDa protein on the surface of human blood platelets has been identified as an ADP receptor. Autoradiography of the gels obtained by sodium dodecyl sulfate–polyacrylamide gel electrophoresis of the samples of solubilized human blood platelets modified by WR-K and then reduced by tritiated sodium borohydride (NaB[3H]4) showed the presence of a prominent band corresponding to a 100-kDa radiolabeled protein. Labeling of platelets by WR-K and NaB[3H]4was inhibited by ADP, ATP, and thiol group modifying reagents. WR-K blocked completely labeling of platelets by [β-32P]-8-(4-bromo-2,3-dioxo-butylthio)adenosine-5′-diphosphate, an ADP-affinity analog that selectively and covalently labels aggregin (Puri, R. N., Kumar, A., Chen, H., Colman, R. F., and Colman, R. W. (1995)J. Biol. Chem.256, 24482–24488). WR-K also inhibited ADP-induced platelet shape change, aggregation, and mobilization of intracellular Ca2+and blocked ADP-induced inhibition of stimulated adenylate cyclase activity. The results show conclusively that WR-K inhibited ADP-induced platelet responses by preventing binding of ADP to aggregin and suggest that ADP binding domain of aggregin contains an essential thiol group. The method of labeling proteins by WR-K and NaB[3H]4, hitherto not used to distinguish among functional groups modified by WR-K, offers a useful and convenient alternative to previously used ultraviolet spectral methods which cannot be used to investigate the modified proteins in intact cellular systems.

Molecular Cloning of cDNA Encoding a Novel Platelet-Endothelial Cell Tetra-Span Antigen, PETA-3

Platelet-endothelial cell tetra-span antigen (PETA-3) was originally identified as a novel human platelet surface glycoprotein, gp27, which was detected by a monoclonal antibody (MoAb), 14A2.H1. Although this glycoprotein is present in low abundance on the platelet surface, MoAb 14A2.H1 stimulates platelet aggregation and mediator release. We now report isolation of a cDNA clone encoding PETA-3 from a library derived from the megakaryoblastic leukemia cell line MO7e. The clone encodes an open reading frame of 253 amino acids that displays 25% to 30% amino acid sequence identity with several members of the newly defined Tetraspan, or Transmembrane 4 superfamily. These proteins consist of four conserved putative transmembrane domains with a large divergent extracellular loop between the third and fourth membrane-spanning regions. PETA-3 has a single consensus sequence for N-linked glycosylation located in this extracellular loop. A single PETA-3 RNA transcript (1.6 kb) was detected in RNA isolated from MO7e cells, bone marrow stromal cells, the C11 endothelial cell line, and several myeloid leukemia cell lines. No transcript was detected in the lymphoblastoid cell lines MOLT-4 and BALM-1. This pattern correlates well with previous protein expression data. Northern blot analysis of RNA from a range of human tissues indicated that the transcript was present in most tissues, the notable exception being brain.

Increased expression of procoagulant activity on the surface of human platelets exposed to heavy-metal compounds

One of the essential roles for platelets in haemostasis is in the potentiation of blood clotting due to the contribution of anionic phospholipid from the surface of the cells, as an essential cofactor to the proteolytic reactions of coagulation (platelet procoagulant activity). Only a limited number of agonists are known to initiate platelet procoagulant activity. In this study the rate of thrombin formation on the platelet surface was observed to increase in a dose-dependent manner upon treatment of washed platelets with heavy-metal compounds. Unlike the immediate increase observed upon treatment of platelets with calcium ionophore, A23187, the change due to these agents was progressive, approaching a maximum after 10 min. The maximum-fold acceleration of the rate of thrombin formation compared with control platelets was calculated for HgCl2 (56-fold), AgNO3 (42-fold) phenylmercuriacetate (24-fold) and thimerosal (14-fold), compared with 70-fold observed for calcium ionophore. The increase in procoagulant activity due to HgCl2 coincided with a large increase in intracellular calcium and phosphorylation of 22 and 45 kDa proteins. It is considered that the mechanism responsible for the increase in procoagulant activity is exposure of anionic phospholipids. This was detected by a 2-fold increase in the binding of 125I-annexin V upon addition of HgCl2, compared with resting platelets (3-fold on treatment of platelets with calcium ionophore). In contrast to the generation of activity by A23187 and other known agonists of this reaction, heavy-metal compounds appeared to cause little or no release of microparticles from the platelet surface. Since HgCl2 did not cause aggregation of platelets or significant release of serotinin, these findings may give further support to the need for exposure and ligation of glycoprotein IIb:IIIa for vesiculization to occur. Treatment of platelets with heavy metals may constitute a new approach to investigating the early changes in the cell membrane which lead to increased expression of anionic phospholipid.

The role of PECAM-1 in vascular cell biology.

Platelet/Endothelial Cell Adhesion Molecule-1 (PECAM-1) is a 130-kDa integral membrane glycoprotein found on the surface of human platelets and leukocytes, and at the intercellular junctions of endothelial cells. PECAM-1 is a member of the immunoglobulin (Ig) gene superfamily, with six Ig-like loops in the extracytoplasmic domain, a 19-residue transmembrane domain, and a 118-amino acid cytoplasmic tail that contains potential sites for phosphorylation and lipid modification that could potentially modulate its adhesive properties and/or subcellular distribution. Antibodies to PECAM-1 interfere with the ability of endothelial cells to form normal cellular junctions, suggesting that PECAM-1 functions in forming or stabilizing the vascular bed. Anti-PECAM-1 antibodies also have been reported to block neutrophil and monocyte chemotaxis in response to lipopolysaccaride, suggesting that PECAM-1 may play a role in leukocyte motility. PECAM-1-transfected murine L cells, but not untransfected L cells, aggregate in a calcium- and PECAM-1-dependent manner, demonstrating directly that PECAM-1 functions as a cell-cell adhesion molecule. PECAM-1 becomes highly phosphorylated in response to cellular activation and, coincident with phosphorylation, associates with the cytoskeleton of activated, but not resting platelets. The engagement of PECAM-1 with the platelet cytoskeleton enables it to move large distances within the plane of the membrane and may similarly account for the ability of PECAM-1 to localize to the intercellular borders of endothelial cells once cell-cell contact has been achieved. Finally, engagement of PECAM-1 causes up-regulation of integrin function on leukocytes, implicating PECAM-1 as a trigger molecule that may regulate leukocyte trafficking through the vessel wall.

Appearance of Additional Ouabain Binding Sites on Platelet Surfaces Following Activation

Na,K-ATPase is an integral membrane protein complex involved in the regulation of intracellular Na+ and K+ ion levels. The number of accessible Na,K-ATPase complexes on the surface of human platelets before and after activation by adenosine 5′-diphosphate (ADP) was quantitated using ouabain, a highly specific inhibitor of this enzyme. Studies with [3H]ouabain revealed an increase in the number of accessible ouabain binding sites from approximately 55 per resting platelet to approximately 625 per ADP-activated platelet. Ultrastructural localization of this ion transport complex was also studied in resting human platelets or following activation by ADP. Localization was performed using a novel colloidal gold affinity probe which employed ouabain (ouabain-GAP) as a ligand specific for the Na,K-ATPase. Qualitatively, the number of surface-associated ouabain-GAP labels per platelet was observed to increase following activation by ADP. Fracture-labeling studies of frozen preparations of resting and freshly activated platelets suggested ouabain binding sites were associated with intracellular platelet structures during the early stages of activation. Together these results suggest an increase in the amount of ouabain binding sites at the surface of human platelets as an event of activation and that at least some of these additional sites may originate from intracellular sites.