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PubMed1991

S. pombe pac1+, whose overexpression inhibits sexual development, encodes a ribonuclease III-like RNase.

The Schizosaccharomyces pombe pac1 gene is a multicopy suppressor of the pat1 temperature-sensitive mutation, which directs uncontrolled meiosis at the restrictive temperature. Overexpression of the pac1 gene had no apparent effect on vegetative growth but inhibited mating and sporulation in wild type S. pombe cells. In such cells, expression of certain genes required for mating or meiosis was inhibited. The pac1 gene is essential for vegetative cell growth. The deduced pac1 gene product has 363 amino acids. Its C-terminal 230 residues revealed 25% amino acid identity with ribonuclease III, an enzyme that digests double-stranded RNA and is involved in processing ribosomal RNA precursors and certain mRNAs in Escherichia coli. The pac1 gene product could degrade double-stranded RNA in vitro. These observations establish the presence of a RNase III homolog in eukaryotic cells. The pac1 gene product probably inhibits mating and meiosis by degrading a specific mRNA(s) required for sexual development. It is likely that mRNA processing is involved in the regulation of sexual development in fission yeast.

Amino Acid Sequence↗
PubMed1977

The isolation and characterization of lipopolysaccharide-defective mutants of Pseudomonas aeruginosa PAC1.

Mutants with defective lipopolysaccharides (LPSs) were isolated from Pseudomonas aeruginosa PACIR (Habs serogroup 3) by selection for resistance to aeruginocin from P. aeruginosa PI6 Carbenicillin-sensitive mutants were isolated from P. aeruginosa PACI but not all had defective LPSs. Rough colonial morphology and resistance to bacteriophage II9X appeared to be independent of LPS composition. The LPSs from five mutants were analysed and compared with that of the parent strain. Separation of partially-degraded polysaccharides from LPS from PACI on Sephadex G75 yielded two different high molecular weight fractions and a phosphorylated low molecular weight fraction (L). The mutant LPSs lacked most or all of the high molecular weight fractions but retained some low molecular weight material. That from PACI and two of the mutants was separated by elution from Biogel P6 into two fractions. One, L2, was the core polysaccharide while the other, LI, contained short antigenic side-chains attached to the core like the semi-rough (SR) LPSs of the Enterobacteriaceae. The two mutants which gave the LI fraction with Habs 3 and PACI antisera as did the parent strain. The other three mutants were unreactive and their LPSs contained core components only. One appeared to have a complete core while the other two lacked rhamnose and rhammose plus glucose respectively. Thus there may be four types of LPS in PACI: one contains unsubstituted core polysaccharide and yields L2 on acid hydrolysis, another has short antigenic side-chains of the SR type and yields the LI fraction, while the two high molecular weight fractions are derived from core polysaccharides with different side-chains.

Carbenicillin↗
PubMed1992

Anti-idiotypic antibodies against an antibody to the platelet glycoprotein (GP) IIb-IIIa complex mimic GP IIb-IIIa by recognizing fibrinogen.

Binding of the adhesive ligand fibrinogen and the monoclonal antibody PAC1 to platelet glycoprotein (GP) IIb-IIIa is dependent on cell activation and inhibited by Arg-Gly-Asp (RGD)-containing peptides. Previously, we identified a sequence in a hypervariable region of PAC1 (mu-CDR3) that mimics the activity of the antibody. Here we examine whether monoclonal antibodies to this idiotypic determinant in PAC1 can mimic GP IIb-IIIa by binding to fibrinogen. Mice were immunized with a peptide derived from the mu-CDR3 of PAC1. Four antibodies were obtained that recognized fibrinogen as well as a recombinant form of the variable region of PAC1. However, they did not bind to other RGD-containing proteins, including von Willebrand factor, fibronectin, and vitronectin. Several studies suggested that these anti-PAC1 peptide antibodies were specific for GP IIb-IIIa recognition sites in fibrinogen. Three such sites have been proposed: two RGD-containing regions in the A alpha chain, and the COOH terminus of the gamma chain (gamma 400-411). Two of the antibodies inhibited fibrinogen binding to activated platelets, and all four antibodies bound to the fibrinogen A alpha chain on immunoblots. Antibody binding to immobilized fibrinogen was partially inhibited by monoclonal antibodies specific for the two A alpha chain RGD regions. However, the anti-PAC1 peptide antibodies also bound to plasmin-derived fibrinogen fragments X and D100, which contain gamma 400-411 but lack one or both A alpha RGD regions. This binding was inhibited by an antibody specific for gamma 400-411. When fragment D100 was converted to D80, which lacks gamma 400-411, antibody binding was reduced significantly (p less than 0.01). Electron microscopy of fibrinogen-antibody complexes confirmed that each antibody could bind to sites on the A alpha and gamma chains. These studies demonstrate that certain anti-PAC1 peptide antibodies mimic GP IIb-IIIa by binding to platelet recognition sites in fibrinogen. Furthermore, they suggest that the gamma 400-411 region of fibrinogen may exist in a conformation similar to that of an A alpha RGD region of the molecule.

Amino Acid Sequence↗
PubMed1987

Induction of the fibrinogen receptor on human platelets by intracellular mediators.

We have used platelets permeabilized with saponin to examine the mechanism by which platelet activation causes the exposure of surface receptors for fibrinogen. Receptor exposure was detected using 125I-fibrinogen and 125I-PAC1, a monoclonal antibody specific for the activated form of the fibrinogen receptor. The potential mediators that were studied included guanyl-5'-yl imidodiphosphate (Gpp(NH)p) and guanosine 5'O-(thiotriphosphate) (GTP gamma S), which cause G protein-dependent phospholipase C activation in platelets; inositol 1,4,5-triphosphate (IP3), which causes Ca2+ release from the platelet dense tubular system; and diacylglycerol and phorbol ester, which activate protein kinase C. Each of these molecules caused fibrinogen and PAC1 binding. The effect of IP3 was mimicked by raising the cytosolic free Ca2+ concentration in the permeabilized platelets. However, IP3 and Ca2+-induced PAC1 binding were abolished by indomethacin or aspirin, which had no effect on PAC1 binding caused by Gpp(NH)p, phorbol ester, or diacylglycerol. This suggests that the response to IP3 and Ca2+ is due to the formation of metabolites of arachidonic acid. One such metabolite, TxA2, is believed to activate platelets by stimulating G protein-dependent phosphoinositide hydrolysis. Indeed, we found that the G protein inhibitor guanyl-5'-yl thiophosphate (GDP beta S) inhibited PAC1 binding caused by a thromboxane A2 analog (U46619), IP3, and Ca2+, but had no effect on diacylglycerol or phorbol ester-induced PAC1 binding. Thrombin-induced PAC1 binding and phosphoinositide hydrolysis were also inhibited by GDP beta S and by pertussis toxin. Increasing the thrombin concentration overcame the inhibition of PAC1 binding caused by GDP beta S but did not overcome the inhibition of phosphoinositide hydrolysis. These observations demonstrate that fibrinogen receptor exposure occurs by at least two routes. One of these, in response to agonists such as thrombin and U46619, is initiated by G protein-dependent phosphoinositide hydrolysis and involves the formation of IP3 and diacylglycerol. IP3 appears to act by stimulating Ca2+-dependent arachidonic acid metabolism which, in turn, triggers further phosphoinositide hydrolysis. Diacylglycerol acts by stimulating protein kinase C. A second route is activated by high concentrations of thrombin and is independent of phosphoinositide hydrolysis.

Antibodies, Monoclonal↗
PubMed1989

A monoclonal antibody against the platelet fibrinogen receptor contains a sequence that mimics a receptor recognition domain in fibrinogen.

The binding of fibrinogen to its platelet receptor, the glycoprotein IIb-IIIa complex, is mediated, in part, by an Arg-Gly-Asp (RGD) sequence within the fibrinogen A alpha chain. PAC1 is an IgM-kappa murine monoclonal antibody that binds to the platelet fibrinogen receptor, and its binding is inhibited by both fibrinogen and RGD-containing peptides. To identify the regions of PAC1 that interact with the fibrinogen receptor, we determined the mRNA sequences of PAC1 immunoglobulin heavy and light chain variable regions. Five out of the six complementarity-determining regions (CDRs) of PAC1 had entirely germline sequences with no regions of similarity to fibrinogen. However, CDR3 of the PAC1 heavy chain (H-CDR3) was very large and unique due to the insertion of a novel D region segment. H-CDR3 contained a sequence, Arg-Tyr-Asp (RYD), that, if present in the proper conformation, might behave like the RGD sequence in fibrinogen. A 21-residue synthetic peptide encompassing the H-CDR3 region inhibited fibrinogen-dependent platelet aggregation as well as the binding of PAC1 (Ki = 10 microM) and fibrinogen (Ki = 5 microM) to activated platelets. The RYD region of H-CDR3 appeared to be central to its function, because substitution of the tyrosine with glycine increased the inhibitory potency of the peptide by 10-fold, while replacing the tyrosine with D-alanine or inverting the RYD sequence sharply reduced the inhibitory potency. Thus, the linear sequence, RYD, within H-CDR3 of PAC1 appears to mimic the RGD receptor recognition sequence in fibrinogen. This type of immunologic approach could be useful in studying the structural basis of other receptor-ligand interactions.

Amino Acid Sequence↗
PubMed1987

Detection of activated platelets in whole blood using activation-dependent monoclonal antibodies and flow cytometry.

Platelets may become activated in a number of clinical disorders and participate in thrombus formation. We developed a direct test for activated platelets in whole blood using flow cytometry. Whole blood was incubated with either biotin-PAC1, a monoclonal antibody specific for the fibrinogen receptor on activated platelets, or biotin-S12, an antibody specific for an alpha-granule membrane protein that associates with the platelet surface during secretion. Platelet-bound antibodies were detected with streptavidin conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE). Platelets were differentiated from the larger erythrocytes and WBCs by their light-scatter profile. Alternatively, platelets could be identified with FITC-AP1, an antibody specific for platelet membrane glycoprotein Ib, and analyzed further for PAC1 or S12 binding with PE-streptavidin. No centrifugation or washing steps were required. With gel-filtered platelets, there was a direct correlation between ADP-induced biotin-PAC1 binding and binding determined in a conventional 125I-PAC1 binding assay (r = .99; P less than .001). Furthermore, as few as 0.8% activated platelets could be detected by flow cytometry when activated platelets were mixed with unstimulated platelets. In whole blood, unstimulated platelets demonstrated no PAC1- or S12-specific fluorescence, indicating that they did not bind these antibodies. On stimulation with agonists, however, the platelets demonstrated a dose-dependent increase in fluorescence similar to that observed for platelets in plasma or buffer. Low concentrations of ADP and epinephrine, which induce fibrinogen receptors but little secretion, stimulated near-maximal PAC1 binding but little S12 binding. On the other hand, a concentration of phorbol myristate acetate (TPA) that evokes full platelet aggregation and secretion induced maximal PAC1 and S12 binding. Activated platelets could also be analyzed in whole blood samples that had been fixed with paraformaldehyde. These studies demonstrate that activated platelets can be reliably detected in whole blood using activation-dependent monoclonal antibodies and flow cytometry. This technique may be useful to assess the degree of platelet activation and the efficacy of antiplatelet therapy in clinical disorders.

Adenosine Diphosphate↗
PubMed1991

Dynamic measurements of the platelet membrane glycoprotein IIb-IIIa receptor for fibrinogen by flow cytometry. I. Methodology, theory and results for two distinct activators.

Platelet aggregation, which occurs within seconds of activation, is generally considered to be mediated by fibrinogen binding to glycoprotein IIb-IIIa which becomes expressed as a fibrinogen receptor (FbR) on the activated platelet surface. This receptor expression has, however, only been measured to date at relatively long activation times (greater than 15 min). We have therefore developed a theoretical and experimental approach for determining FbR expression within seconds of platelet activation using flow cytometry. The fluorescently labeled IgM monoclonal antibody FITC-PAC1, was used to report on the GPIIb-IIIa receptor for Fb (FbR). Human citrated platelet-rich plasma (PRP; diluted 1:10) was incubated with adenosine diphosphate (ADP) or phorbol myristate acetate (PMA) for varying times (tau = 0-10 s, out to 60 min), followed by incubation with fluorescein isothiocyanate (FITC)-PAC1 antibody at saturating concentrations. The time course of FITC-PAC1 binding was then measured for these variously preactivated samples (different tau) from the mean platelet-bound fluorescence (Fl), determined for greater than or equal to 5 s of PAC1 addition by dilution quenching and determination of fluorescence intensity histograms with the FACSTAR or FACSCAN (Becton-Dickinson Canada, Mississauga, Ontario) flow cytometers. Both rapid, initial rate of increase in Fl (nu) (related to PAC1 on-rates) and maximal extent of increase (Flmax) were thus determined for different tau values. These measurements yield the rate of formation of FbR (k1), and both the rate (k2) and efficiency (alpha) of binding of PAC1 to FbR as a function of activator type and time of action. We have found that ADP appears to cause rapid, maximal expression of FbR within 1-3 s (k1 greater than 20 min-1), whereas PMA expresses FbR in a slow, biphasic manner (k1 - 0.01 and 0.2 min-1). However, k2 and alpha for maximal PMA activation are about two and three times greater, respectively, than for maximal ADP-activation. Moreover, k2 decreases with post ADP activation time. These differences are discussed in terms of altered FbR organization and accessibility. This kinetic approach can be widely used to analyze the dynamics and organization of molecules on cell surfaces by flow cytometry, including studies of size-dependent subpopulations (see Part II, Frojmovic, M., and T. Wong. 1991. Biophys. J. 59:828-837).

Adenosine Diphosphate↗
PubMed1990

Direct detection of activated platelets and platelet-derived microparticles in humans.

Flow cytometry was used to determine whether activated platelets and platelet-derived microparticles can be detected directly in whole blood after a hemostatic insult. Two different in vivo models of platelet activation were examined: (1) a standardized bleeding time, and (2) cardiopulmonary bypass. Platelets and microplatelets were identified with a biotinylated anti-glycoprotein (GP)lb antibody and a fluorophore, phycoerythrin-streptavidin. Microparticles were distinguished from platelets by light scatter. Activated platelets were detected with three fluorescein-labeled monoclonal antibodies (MoAbs): (1) PAC1, which binds to the activated form of GPIIb-IIIa; (2) 9F9, a newly developed antibody that is specific for fibrinogen bound to the surface of activated platelets; and (3) S12, which binds to an alpha-granule membrane protein expressed on the platelet surface after granule secretion. In nine normal subjects, bleeding times ranged from 4.5 to 7.5 minutes. Over this time, there was a progressive increase in the amount of PAC1, 9F9, and S12 bound to platelets in blood emerging from the bleeding time wound. With all three antibodies, platelet activation was apparent as early as 30 seconds after the incision (P less than .03). Activation was accompanied by a progressive decrease in the concentration of platelets in blood from the wound, while the concentration of microparticles increased slightly. In nine patients undergoing open heart surgery, 1 hour of cardiopulmonary bypass caused a 2.2-fold increase in the relative proportion of microparticles in circulating blood (P less than .001). Moreover, bypass caused platelet activation as evidenced by a mean two- to threefold increase in PAC1 binding to platelets. Although this increase was significant (P less than .02), PAC1 binding exceeded the normal range for unstimulated control platelets in only 5 of 9 patients, and 9F9 and S12 binding exceeded the normal range in only two patients. Taken together, these studies demonstrate that it is now feasible using flow cytometry to evaluate the extent of platelet activation and the presence of platelet-derived microparticles in the circulation of humans.

Adenosine Diphosphate↗
PubMed1989

Epinephrine induces platelet fibrinogen receptor expression, fibrinogen binding, and aggregation in whole blood in the absence of other excitatory agonists.

The exposure of fibrinogen receptors is an early event in agonist-induced platelet activation. Previous measurements of fibrinogen binding or aggregation in platelet-rich plasma or washed platelets have failed to define whether the initial response to epinephrine results solely from a direct effect of this agonist. To address this problem, we have measured fibrinogen receptor exposure on platelets in whole blood by using flow cytometry and a fluorescein isothiocyanate-labeled monoclonal antibody specific for the activated fibrinogen receptor (FITC-PAC1). We also measured platelet-bound fibrinogen with an antifibrinogen monoclonal antibody (FITC-9F9) as well as platelet aggregation in whole blood. In blood anticoagulated with citrate and in the presence of a cyclooxygenase inhibitor, epinephrine (0.1 to 100 mumol/L) caused significant FITC-PAC1 binding (P less than .001) that was maximal at 10 mumol/L epinephrine. The maximal epinephrine response was one third of that observed with 10 mumol/L adenosine diphosphate (ADP) and was eliminated by yohimbine, an alpha 2-adrenergic antagonist. Incubation of the blood with apyrase or phosphoenolpyruvate plus pyruvate kinase to remove extracellular ADP resulted in a 40% to 50% reduction in the epinephrine response. Despite this, FITC-PAC1 binding was still significant at epinephrine greater than or equal to 1 mumol/L (P less than .05). No reduction in epinephrine-induced FITC-PAC1 binding was observed in the presence of ATP alpha S, an ADP receptor antagonist; cinanserin, a serotonin antagonist; or WEB-2086, a platelet activating factor antagonist. Furthermore, addition of the thrombin inhibitors hirudin or leupeptin to citrated blood had no effect on the extent of the epinephrine response. Blood anticoagulated with hirudin also demonstrated an epinephrine response, even in the presence of apyrase. Similar results were obtained when FITC-9F9 was used to detect fibrinogen binding or when aggregation was assessed by a decrease in the number of single platelets. We conclude that epinephrine itself can induce fibrinogen receptor exposure, fibrinogen binding, and aggregation. This primary response is independent of synergistic interaction of epinephrine with traces of ADP, serotonin, platelet activating factor, or thrombin. However, such synergistic interaction with ADP present in whole blood may enhance the responses induced by epinephrine.

Adenosine Diphosphate↗
PubMed1989

Functional expression of human glucose-6-phosphate dehydrogenase in Escherichia coli.

The complete coding sequence for human glucose-6-phosphate-dehydrogenase (G6PD) was inserted downstream from the tac promoter of a plasmid, pJF118EH, which also carries the lacIq repressor gene. When Escherichia coli strains (that are unable to grow on glucose due to the absence of functional zwf (G6PD-) and pgi genes) were transformed with this plasmid (pAC1), they were able to grow on glucose as sole carbon source. The rate of growth on glucose was faster in the presence of the inducer of the tac promoter, isopropyl-beta-D-thiogalactopyranoside (IPTG). Extracts of the transformed cells contained a G6PD activity that was not detectable in the parental strains and that was inducible by IPTG. The G6PD activities from normal E. coli and from pAC1-transformed cells comigrated with human G6PD when subjected to electrophoresis on agarose gels. However, when denatured, the G6PD produced by pAC1 was, like the human enzyme, distinguishable from the E. coli-encoded enzyme on the basis of its immunoreactivity with antibody specific for human G6PD. Therefore, human G6PD can be expressed in E. coli and can function to complement the bacterial enzyme deficiency.

Base Sequence↗
PubMed1991

Dynamic measurements of the platelet membrane glycoprotein IIb-IIIa receptor for fibrinogen by flow cytometry. II. Platelet size-dependent subpopulations.

Platelet aggregation has previously been shown to occur within 1 s of activation with 100 microM adenosine diphosphate (ADP) for both large (L) and small (S) platelet subpopulations, but L platelets were about twofold more sensitive and more rapidly recruited into microaggregates than were S platelets after correcting for differences in platelet surface area. Because platelet aggregation normally requires fibrinogen binding to glycoprotein IIb-IIIa receptors (FbR) expressed on the activated platelet surface, we wished to compare the kinetics and nature of FbR expression induced by ADP for L versus S platelets, and to measure size-dependent differences in FbR expression for platelets maximally activated with phorbol myristate acetate (PMA). We presented the theory and methodology in Part I (Frojmovic, M., T. Wong, and T. van de Ven. 1991. Biophys. J. 59:815-827) for measuring the rate of FbR expression (k1) and both the rate (k2) and efficiency (alpha) of binding of PAC1 to FbR as a function of activation conditions from the initial on-rate of FITC-PAC1 to FbR (V) and the maximal number of FbR expressed: these are measured, respectively, from the initial rate of increase in platelet-bound fluorescence (v) and the maximal increase in mean fluorescence (Flmax). We extended these analyses to L and S platelets, selected by electronic gating of forward scatter profiles (FSC), with corresponding fluorescence (Fl) histograms retrieved analytically. Platelet size (V) and surface area (SA), determined directly for cells separated with a cell sorter, were highly correlated with FSC, allowing v and Flmax values to be expressed per unit area of membrane for L:S comparisons. Surprisingly, ADP activation appeared to express all FbR within 1-3 s of ADP activation for both L and S platelets, whereas k1 was similar for PMA activation. In addition, L platelets maximally expressed two and three times more FbR per unit area than did S platelets when maximally stimulated, respectively, with ADP or PMA. Whereas k2 was independent of platelet size for a given activator, the efficiency of PAC1 binding (alpha), per unit area of membrane, was two times greater for L than for S platelets, for either ADP or PMA activation. Our data suggest that the FbR structure, its microenvironment, or its surface organization may vary with platelet size or activator type. Major reorganization of FbR and/or its environment appears to occur after approximately 5 min of ADP activation equally for both L and S platelets. A model is presented to account for size-dependent differences in FbR expression with implications for regulation of platelet aggregation.

Adenosine Diphosphate↗
PubMed1990

Affinity modulation of the alpha IIb beta 3 integrin (platelet GPIIb-IIIa) is an intrinsic property of the receptor.

To analyze the basis of affinity modulation of integrin function, we studied cloned stable Chinese hamster ovary cell lines expressing recombinant integrins of the beta 3 family (alpha IIb beta 3 and alpha v beta 3). Antigenic and peptide recognition specificities of the recombinant receptors resembled those of the native receptors found in platelets or endothelial cells. The alpha IIb beta 3-expressing cell line (A5) bound RGD peptides and immobilized fibrinogen (Fg) but not soluble fibrinogen or the activation-specific monoclonal anti-alpha IIb beta 3 (PAC1), indicating that it was in the affinity state found on resting platelets. Several platelet agonists failed to alter the affinity state of ("activate") recombinant alpha IIb beta 3. The binding of soluble Fg and PAC1, however, was stimulated in both platelets and A5 cells by addition of IgG papain-digestion products (Fab) fragments of certain beta 3-specific monoclonal antibodies. These antibodies stimulated PAC1 binding to platelets fixed under conditions rendering them unresponsive to other agonists. Addition of these antibodies to detergent-solubilized alpha IIb beta 3 also stimulated specific Fg binding. These data demonstrate that certain anti-beta 3 antibodies activate alpha IIb beta 3 by acting directly on the receptor, possibly by altering its conformation. Furthermore, they indicate that the activation state of alpha IIb beta 3 is a property of the receptor itself rather than of the surrounding cell membrane microenvironment.

Animals↗
PubMed1992

Activation of platelets in blood perfusing angioplasty-damaged coronary arteries. Flow cytometric detection.

Fluorescence-activated flow cytometry has been used to investigate platelet activation in blood flowing through atherosclerotic coronary arteries after sustaining mechanical damage induced by percutaneous transluminal angioplasty (PTCA). For flow cytometry, platelets and platelet-derived microparticles were identified by biotinylated anti-glycoprotein (GP) Ib monoclonal antibody (mAb) and a fluorophore, phycoerythrin-streptavidin. Activated platelets were detected by using a panel of fluoresceinated mAbs specific for activation-dependent platelet epitopes, including 1) activated GPIIb-IIIa complex (PAC1); 2) fibrinogen bound to platelet GPIIb-IIIa (9F9); 3) ligand-induced binding sites on GPIIIa (anti-LIBS1); and 4) P-selectin, an alpha-granule membrane protein expressed on the platelet surface after secretion (S12). The binding of antibodies to platelets was determined in blood that was sampled continuously via heparin-coated catheters from the coronary sinus in 1) patients before, during, and for 30 minutes after PTCA and 2) control patients undergoing coronary angiography without PTCA. Platelets in coronary sinus blood showed significant binding of mAbs that specifically detect activation epitopes associated with the GPIIb-IIIa complex (PAC1, anti-LIBS1, and 9F9) during and for 30 minutes after angioplasty in four of the five patients. The relative proportion of platelets positive for PAC1 and anti-LIBS1 increased from baseline values of 2.0 +/- 0.3% (mean +/- SD) and 2.0 +/- 0.5% to 18 +/- 14% and 28 +/- 14%, respectively, during PTCA or 30 minutes after PTCA (p < 0.01 in both cases). Binding with 9F9 was less prominent. The expression of P-selectin was detected in one of the five patients. By contrast, activation-specific mAbs failed to bind detectably with platelets obtained from 1) the peripheral blood during coronary angiography in eight patients or 2) coronary sinus blood obtained via catheter throughout control catheterization procedures in three patients or before PTCA in five. We conclude that circulating platelets become activated while flowing through PTCA-damaged stenotic coronary arteries and that this process of platelet activation is readily demonstrated by measuring the expression of activation-specific membrane GP epitopes by flow cytometric analysis.

Aged↗
PubMed1992

Regulation of glycoprotein IIb-IIIa receptor function studied with platelets permeabilized by the pore-forming complement proteins C5b-9.

Recent evidence suggests that the cytoplasmic domains of platelet glycoprotein (GP) IIb-IIIa are involved in the agonist-initiated transformation of this integrin into a receptor for fibrinogen. To identify intracellular reactions that regulate the receptor function of GP IIb-IIIa, membrane-impermeable agonists and antagonists were introduced into the platelet by permeabilizing the plasma membrane with the pore-forming complement proteins C5b-9. Platelet responses were then analyzed by flow cytometry. Non-lytic concentrations of C5b-9 caused permeabilization of the platelet plasma membrane, as determined by uptake of a water-soluble fluorescent tracer dye. The complement pores were large enough to permit the entry of fluorescein isothiocyanate (FITC)-labeled oligopeptides in a size-dependent manner. Under conditions of low external Ca2+, C5b-9 treatment per se did not activate GP IIb-IIIa, as measured by binding of the activation-dependent antibody FITC-PAC1. However, FITC-PAC1 binding to C5b-9-permeabilized platelets was stimulated by a thrombin receptor agonist acting at the cell surface and by guanosine 5'-O-(thiotriphosphate), a membrane-impermeable activator of G proteins. Permeabilization also permitted the entry of cyclic AMP and the peptide, RFARKGALRQKNV, a pseudo-substrate inhibitor of protein kinase C. Each of these inhibited agonist-induced FITC-PAC1 binding to permeabilized platelets but not to intact platelets. Agonist-induced GP IIb-IIIa activation in permeabilized platelets was also inhibited by tyrphostin-23, a protein tyrosine kinase inhibitor. Thus, C5b-9 can be used to permeabilize the plasma membrane to permit the selective entry of small peptides and other bioactive compounds into permeabilized platelets. Results obtained with these platelets indicate that GP IIb-IIIa receptor function is regulated by a network of signaling reactions involving G proteins, serine/threonine kinases, and tyrosine kinases.

Amino Acid Sequence↗
PubMed1990

Analysis of platelet aggregation disorders based on flow cytometric analysis of membrane glycoprotein IIb-IIIa with conformation-specific monoclonal antibodies.

Normal primary platelet aggregation requires agonist-mediated activation of membrane GPIIb-IIIa, binding of fibrinogen to GPIIb-IIIa, and cellular events after ligand binding. PAC1 monoclonal antibody distinguishes between resting and activated states of GPIIb-IIIa, and other antibodies preferentially recognize GPIIb (PMI-1) or IIIa (anti-LIBS1) after the binding of fibrinogen or fibrinogen-mimetic peptides, such as GRGDSP. Using these antibodies and platelet flow cytometry, we studied two distinct persistent platelet aggregation abnormalities. Platelets from a thrombasthenic variant, which contained near-normal amounts of GPIIb-IIIa, failed to aggregate or bind PAC1 in response to agonists. In addition, GRGDSP, which binds to normal GPIIb-IIIa without prior cell activation, failed to increase the binding of PMI-1 or anti-LIBS1 to the thrombasthenic platelets, suggesting a primary defect in ligand binding. Chromatography of detergent-solubilized platelets on a KYGRGDS affinity column confirmed that the patient's GPIIb-IIIa lacked the fibrinogen binding site. In another patient with myelofibrosis and defective aggregation, PAC1 failed to bind to adenosine diphosphate-stimulated platelets, but did bind when protein kinase C was directly activated with phorbol myristate acetate. Furthermore, the binding of PMI-1 and anti-LIBS1 increased in response to GRGDSP, confirming a defect in agonist-mediated fibrinogen receptor activation rather than in fibrinogen binding or events distal to binding. These studies indicate that this immunochemical approach is useful in classification of clinical abnormalities of platelet aggregation as defects in either (a) fibrinogen receptor activation, (b) fibrinogen binding, or (c) postoccupancy events.

Antibodies, Monoclonal↗
PubMed1989

Cloning and expression of a collagen-analog-encoding synthetic gene in Escherichia coli.

A family of totally synthetic genes coding for multiple tandem repeats of the amino acid sequence (Gly-Pro-Pro) has been prepared and inserted into the ClaI cloning site of the expression vector pJL6. A representative recombinant plasmid, pAC1, with an insert of about 340 bp was established in an Escherichia coli strain bearing a defective lambda prophage, to study expression of the CII-collagen analog fusion protein produced from pAC1 upon heat induction. Authentic fusion protein production was demonstrated by nucleotide sequencing, Northern-blot analysis, and in vivo synthesis. Conversion of a wild-type rpoH allele to the rpoH165 mutation was shown to suppress proteolysis of the unstable fusion protein.

Amino Acid Sequence↗
PubMed1992

A variant of Glanzmann's thrombasthenia which fails to express a GPIIb-IIIa related epitope that is recognized by a specific monoclonal antibody (C17).

Binding of different antibodies to the GPIIb-IIIa complex in resting (AP2, EDU3, C17) or activated platelets (PAC1) was studied by flow cytometry in a patient with a platelet defect involving GPIIb-IIIa related functions. The patient has a mild history of bleeding. Aggregation induced by ADP and collagen were absent but normal response was obtained with ristocetin. Platelets from the patient do not bind fibrinogen. Perfusion studies with flowing blood showed that patient's platelets have a marked impairment in the process of spreading and aggregate formation on vascular subendothelium. Electrophoretic studies in SDS-polyacrylamide gels demonstrated the presence of normal amounts and normal mobility of GPIIb-IIIa. Fibrinogen was present in the patient's platelets (68-74% of controls). The binding of AP2 and EDU3 to patient's resting platelets was normal as assessed by flow cytometry. In contrast, a decreased presence of the C17 antigen (10 fold lower than control platelets) was detected in resting platelets and a markedly reduced binding of PAC1 was found in thrombin activated platelets. These studies suggest that C17 recognizes an epitope of the GPIIb-IIIa in resting platelets that is implicated in the regulation of adhesive and cohesive properties of GPIIb-IIIa. Studies on this patient might be helpful for the understanding of GPIIb-IIIa functions.

Adenosine Diphosphate↗
PubMed1975

Heterogeneity of the lipopolysaccharide from Pseudomonas aeruginosa.

Lipopolysaccharide isolated from pseudomonas aeruginosa PAC1 and its phage-resistant mutant was degraded by mild acid hydrolysis into lipid A and three major polysaccharide-containing fractions which were separated on Sephadex G-75. The low-molecular-weight fraction contained glucose, rhamnose, heptose, galactosamine, alanine and phosphate. The higher-molecular-weight fractions consisted mainly of glucose, rhamnose and glucosamine together with amino compounds. Alkaline degradation of the lipopolysaccharide produced at least four different species each of which contained a low-molecular-weight polysaccharide similar if not identical to that produced by acid hydrolysis. Under certain growth conditions an abnormal lipopolysaccharide was produced which was defective in the low-molecular-weight polysaccharide and contained mainly high-molecular-weight material. Strains of different serotype yielded lipopolysaccharides which also exhibited heterogeneity but contained a low-molecular-weight polysaccharide similar to that obtained from strain PAC1 and PAC1R. It is suggested that each strain of P. aeruginosa may produce several lipopolysaccharides each containing a polysaccharide common to all. The relative proportions of the various lipopolysaccharides may be changed by growth conditions.

Alanine↗