Platelet Extracts Research Articles

Mechanism of action of novel NO‐releasing furoxan derivatives of aspirin in human platelets

Incorporation of a nitric oxide (NO)-releasing moiety in aspirin can overcome its gastric side effects. We investigated the NO-release patterns and antiplatelet effects of novel furoxan derivatives of aspirin (B8 and B7) in comparison to existing antiplatelet agents. Cyclooxygenase (COX) activity was investigated in purified enzyme using an electron paramagnetic resonance-based technique. Concentration-response curves for antiplatelet agents +/- the soluble guanylate cyclase inhibitor, ODQ (50 microM) were generated in platelet-rich plasma (PRP) and washed platelets (WP) activated with collagen using turbidometric aggregometry. NO was detected using an isolated NO electrode. The furoxan derivatives of aspirin (B8, B7) and their NO-free furazan equivalents (B16, B15; all 100 microM) significantly inhibited COX activity (P < 0.01; n = 6) in vitro and caused aspirin-independent, cGMP-dependent inhibition of collagen-induced platelet aggregation in WP. B8 was more potent than B7 (PRP IC(50) = 0.62 +/- 0.1 microM for B8; 400 +/- 89 microM for B7; P < 0.0001. WP IC(50)s = 0.6 +/- 0.1 and 62 +/- 10 microM, respectively). The NO-free furazan counterparts were less potent antiplatelet agents (WP IC(50)s = 54 +/- 3 microM and 62 +/- 10 microM, respectively; P < 0.0001, B8 vs B16). Of the hybrids investigated, only B8 retained antiplatelet activity in PRP.NO release from furoxan-aspirin hybrids was undetectable in buffer alone, but was accelerated in the presence of either plasma or plasma components, albumin (4%), glutathione (GSH; 3 microM) and ascorbate (50 microM), the effects of which were additive for B7 but not B8. NO generation from furoxans was greatly enhanced by platelet extract, an effect that could largely be explained by the synergistic effect of intracellular concentrations of GSH (3 mM) and ascorbate (1 mM). We conclude that the decomposition of furoxan-aspirin hybrids to generate biologically active NO is catalysed by endogenous agents which may instil a potential for primarily intracellular delivery of NO. The blunting of the aspirin effects of furoxan hybrids is likely to be due to loss of the acetyl moiety in plasma; the observed antiplatelet effects are thereby primarily mediated via NO release. Compounds of this class might represent a novel means of inhibiting platelet aggregation by a combination of NO generation and COX inhibition.

Spliced in the Cytoplasm

STKE In nucleated cells, noncoding introns are removed from pre-messenger RNA (pre-mRNA) transcripts by the spliceosome (a nuclear complex of proteins and RNAs) before export of the mRNA from the nucleus. Thus, one would not expect that platelets—anucleate blood cells that bud from megakaryocytes—would contain spliceosome components or pre-mRNAs. Nevertheless, Denis et al. show that components of the spliceosome are present in the cytoplasm of human megakaryocytes and also in circulating platelets. Interleukin-1β (IL-1β) pre-mRNA is present in the cytoplasm of quiescent platelets, whereas platelets that had been activated by adhesion to fibrinogen in the presence of thrombin contain mature IL-1β mRNA and protein. Moreover, IL-1β pre-mRNA could be converted into mature mRNA by a platelet extract. Thus, pre-mRNA splicing is a key regulatory point for cytokine production during platelet activation. — EMA Cell 122 , 379 (2005).

Spliced in the Cytoplasm

Denis et al. made the unexpected discovery that precursor mRNAs (pre-mRNAs) and functional spliceosomes are found in the cytoplasm of platelets and, further, that splicing of this cytoplasmic pre-RNA is regulated by signals deriving from platelet activation. In nucleated cells, noncoding introns are removed from newly transcribed pre-mRNA by the spliceosome (a nuclear complex containing small nuclear ribonucleoproteins and auxiliary proteins), creating translatable mRNA, which may then be exported from the nucleus. Thus, one would not expect that platelets, anucleate blood cells that bud from megakaryocytes, would contain spliceosome components or pre-mRNAs. Denis et al. found, however, that components of the spliceosome (both proteins and small nuclear splicing RNAs) were present not only in the cytoplasm of human megakaryocytes but also in circulating platelets. Interleukin-1β (IL-1β) pre-mRNA was present in the cytoplasm of quiescent platelets (and in the cytoplasm of megakaryocytes extending the proplatelets that give rise to platelets), whereas platelets activated by adhesion to fibrinogen in the presence of thrombin contained mature IL-1β mRNA and protein. Indeed, reverse transcription polymerase chain reaction (RT-PCR) revealed a time-dependent decrease in IL-1β pre-mRNA and an increase in IL-1β mRNA after platelet activation. Moreover, IL-1β pre-mRNA that was transcribed in vitro was spliced into mRNA by a fraction of platelet extract. Thus, pre-mRNA splicing not only takes place in the cytoplasm of platelets but also provides a mechanism for regulating cytokine production after platelet activation. Meshorer and Misteli provide context in a Preview and speculate on the possibility that pre-mRNA splicing could occur in the cytoplasm of other cell types. M. M. Denis, N. D. Tolley, M. Bunting, H. Schwertz, H. Jiang, S. Lindemann, C. C. Yost, F. J. Rubner, K. H. Albertine, K. J. Swoboda, C. M. Fratto, E. Tolley, L. W. Kraiss, T. M. McIntyre, G. A. Zimmerman, A. S. Weyrich, Escaping the nuclear confines: Signal-dependent pre-mRNA splicing in anucleate platelets. Cell 122 , 379-391 (2005). [PubMed] E. Meshorer, T. Misteli, Splicing misplaced. Cell 122 , 317-318 (2005). [PubMed]

Quantitative validation of different protein precipitation methods in proteome analysis of blood platelets

For the preparation of proteins for proteome analysis, precipitation is frequently used to concentrate proteins and to remove interfering compounds. Various methods for protein precipitation are applied, which rely on different chemical principles. This study compares the changes in the protein composition of human blood platelet extracts after precipitation with ethanol (EtOH) or trichloroacetic acid (TCA). Both methods yielded the same amount of proteins from the platelet preparations. However, the EtOH-precipitated samples had to be dialyzed because of the considerable salt content. To characterize single platelet proteins, samples were analyzed by two-dimensional fluorescence differential gel electrophoresis. More than 90% of all the spots were equally present in the EtOH- and TCA-precipitated samples. However, both precipitation methods showed a smaller correlation with nonprecipitated samples (EtOH 74.9%, TCA 79.2%). Several proteins were either reduced or relatively enriched in the precipitated samples. The proteins varied randomly in molecular weight and isoelectric point. This study shows that protein precipitation leads to specific changes in the protein composition of proteomics samples. This depends more on the specific structure of the protein than on the precipitating agent used in the experiment.

The development of a quantitative enzyme‐linked immunosorbent assay to detect human platelet factor 4

Platelet factor 4 (PF4) is a marker for in vitro and in vivo tests of platelet (PLT) activation and alpha-granule secretion. PF4 is also a major CXC cytokine released during storage. Cytokines released during PLT storage are a potential cause of nonhemolytic transfusion reactions. Quantitative measurement of PF4 requires an assay that is both reliable and sensitive. To achieve this goal, a sensitive, cost-effective, sandwich enzyme-linked immunosorbent assay (ELISA) was developed with commercially available antibodies to human PF4. An ELISA was developed for measuring PF4 from whole human PLTs or secreted from activated PLTs. Optimal concentrations of capture antibody, detection antibody, and enzyme-conjugate were determined with serial twofold dilutions of recombinant PF4. This assay was used to determine the ideal sample dilutions needed for reliable quantitation of PF4 in releasates or from whole PLT extracts. Serial dilutions of recombinant PF4 resulted in a sigmoid titration curve with a maximal sensitivity of 10 pg and a dynamic quantitative range from 100 to 2500 pg. This ELISA was used to measure secretion from permeabilized PLTs stimulated with free calcium. In a secretion experiment with 2.5 x 10|*bsup*|8|*esup*| PLTs per mL, samples required a 1:10-fold dilution to reliably evaluate alpha-granule release. The parameters described yield an ELISA method with low background and high sensitivity over a range of PF4 concentrations. Using the commercial reagents described makes this assay cost-effective and therefore suitable for analyzing multiple samples in the research setting.

Western blot analysis of human and rat serotonin transporter in platelets and brain using site-specific antibodies: Evidence that transporter undergoes endoproteolytic cleavage

Background Serotonin transporter (SERT) is important target molecule for many antidepressive drugs and substances of abuse and is implicated in psychiatric disorders. We performed immunoblotting analysis of human and rat SERT in platelets and brain using the panel of eight site-specific SERT monoclonal and polyclonal antibodies (mAbs and pAbs). Methods SDS-PAGE/Western blotting was conducted using peroxidase-labeled DEAE and affinity purified SERT antibodies under conditions preventing SERT post-extraction degradation. Results Immunoreactive polypeptides of 14, 22, 32, 35, 37, 56, 68, and ∼150–200 kDa were revealed in human platelet extracts using N-terminal and C-terminal SERT antibodies. In rat brain, C-terminal mAbs detected 68, 56, and 37 kDa proteins, in postmortem human brain predominated 35–37 kDa proteins. The immunoreactivity was abolished after antibody preadsorption with antigens. N-terminal pAbs recognized the 68 kDa protein, affinity purified on C-terminal mAbs, confirming its identity as full-size human SERT (the predicted size ∼70.5 kDa). Conclusions The explanation of the results of immunoblotting most likely is a site-specific SERT endoproteolytic cleavage and a marked difference in glycosylation rather than nonspecific protein degradation, cross-reactivity with other epitopes or SERT alternative splicing.

Early Tumor Detection Using Platelet Uptake of Angiogenesis Regulators.

We report a new function for platelets: selective sequestration of tumor-derived angiogenesis regulatory proteins above the concentration of these molecules in plasma.Iodinated VEGF in a Matrigel pellet (from 100 to 600 ng/100 microl), implanted subcutaneously in mice, accumulates almost exclusively in platelets in a dose-dependent manner over a period as long as 2–3 weeks, without raising plasma levels of VEGF. Similarly, platelet VEGF increases in the presence of a single microscopic VEGF-secreting human tumor of up to only 1 mm3 in SCID mice without any increase of VEGF in plasma. In addition to VEGF, other factors such as bFGF, PDGF, BDNF, endostatin and other regulators of angiogenesis are taken up by platelets in a selective and quantifiable manner which is dependent on tumor generation of these molecules. Our data show that these proteins are not simply associated with the platelet surface, but are internalized. Furthermore, they are protected from degradation within the platelet, and are not released by classical degranulating agents, such as thrombin, ADP or epinephrine. Incubation of human platelets with endostatin at above physiological levels results in decrease of the majority of platelet-associated VEGF and bFGF in a concentration-dependent manner.Using SELDI-ToF mass spectroscopy of platelet extracts, we have found that this novel property of platelets enables the detection of microscopic tumors that undetectable by any presently available diagnostic method. The platelet angiogenic profile is more inclusive than a single biomarker because it can detect a wide range of tumor types and tumor sizes. Relative changes in the platelet angiogenic profile permit the tracking of a tumor throughout its development, beginning from an early in situ cancer.Conclusions: (i) While the half-life of mouse platelets is approximately 3 days, the platelet angiogenic profile persists for as long as the tumor (or Matrigel pellet) is present. This indicates that platelets may continuously scavenge proteins which regulate angiogenesis. (ii) The fact that the presence of a human tumor can now be detected at microscopic size, suggests that it may not be necessary to know the type and location of a tumor before initiating treatment, especially since it is feasible to use anti-cancer therapies of little or no toxicity. [Display omitted]

Human Platelet Dense Granules Contain Polyphosphate and Are Similar to Acidocalcisomes of Bacteria and Unicellular Eukaryotes

Inorganic polyphosphate (polyP) has been identified and measured in human platelets. Millimolar levels (in terms of Pi residues) of short chain polyP were found. The presence of polyP of approximately 70-75 phosphate units was identified by 31P NMR and by urea-polyacrylamide gel electrophoresis of platelet extracts. An analysis of human platelet dense granules, purified using metrizamide gradient centrifugation, indicated that polyP was preferentially located in these organelles. This was confirmed by visualization of polyP in the dense granules using 4',6-diamidino-2-phenylindole and by its release together with pyrophosphate and serotonin upon thrombin stimulation of intact platelets. Dense granules were also shown to contain large amounts of calcium and potassium and both bafilomycin A1-sensitive ATPase and pyrophosphatase activities. In agreement with these results, when human platelets were loaded with the fluorescent calcium indicator Fura-2 acetoxymethyl ester to measure their intracellular Ca2+ concentration ([Ca2+]i), they were shown to possess a significant amount of Ca2+ stored in an acidic compartment. This was indicated by the following: 1) the increase in [Ca2+]i induced by nigericin, monensin, or the weak base, NH4Cl, in the nominal absence of extracellular Ca2 and 2) the effect of ionomycin, which could not take Ca2+ out of acidic organelles and was more effective after alkalinization of this compartment by the previous addition of nigericin, monensin, or NH4Cl. All of these characteristics of the platelet dense granules, together with their known acidity and high density (both by weight and by electron microscopy), are similar to those of acidocalcisomes (volutin granules, polyP bodies) of bacteria and unicellular eukaryotes. The results suggest that acidocalcisomes have been conserved during evolution from bacteria to humans.

Analysis of serotonin transporter in human platelets by immunoblotting using site-specific antibodies.

We have produced a panel of site-specific antibodies recognizing different regions of the human serotonin transporter (SERT). This panel included: 1) monoclonal antibodies 23C5 (mAbs 23C5) to the C-terminal region (amino acid residues 597-630); 2) polyclonal antibodies (pAbs) to the N-terminal region (amino acid residues 69-83); 3) pAbs to the region (amino acid residues 86-100) in the beginning of the first transmembrane domain (TMD). The antibodies were produced using recombinant proteins and synthetic peptides (containing certain sequences of SERT) as antigens. These antibodies were purified by affinity chromatography, conjugated to horseradish peroxidase (HRP), and used for immunoblotting analysis of SERT in extracts of human platelets. Sodium dodecyl sulfate extracts were prepared under conditions preventing non-specific proteolytic degradation of the proteins. In platelet extracts, all antibodies were able to detect the 67 kD protein, apparently corresponding to full-length SERT molecule (its theoretical mass is about 70 kD). These antibodies also detected several polypeptides of smaller size (56, 37, 35, 32, 22, and 14 kD), apparently corresponding to N-terminal, C-terminal, and non-terminal SERT fragments. Specificity of immunostaining was confirmed by preincubation of HRP-labeled anti-SERT antibodies with excess of corresponding antigen, which resulted in disappearance of protein band staining. It is suggested that SERT undergoes a programmed proteolytic cleavage (processing) resulting in formation of several SERT-derived polypeptides of smaller size. It is possible that one of the cleaved SERT species is required for serotonin transport activity. Possible sites for specific proteolysis may be located in the region near TMD1 and in the intracellular loop between TMD4 and TMD5.

Convulxin Binds to Native, Human Glycoprotein Ibα

Convulxin (CVX), a C-type snake protein from Crotalus durissus terrificus venom, is the quintessential agonist for studies of the collagen receptor, glycoprotein VI (GPVI) and its role in platelet adhesion to collagens. In this study, CVX, purified from venom, behaves as expected, i.e. it binds to platelet GPVI and recombinant human GPVI, induces platelet aggregation and platelet prothrombinase activity, and binds uniquely to GPVI in ligand blots of SDS-denatured proteins. Nonetheless, we find that CVX has a dual specificity for both GPVI and native but not denatured human GPIb alpha. First, CVX binds to human GPIb alpha expressed on the surface of CHO cells. Second, CVX binds weakly to murine platelet GPIb alpha but more strongly to human platelet GPIb alpha, as evidenced by comparative binding to wild-type, GPVI(-/-), FcR gamma (-/-), and human GPIb transgenic mice. Third, the binding of CVX to human GPIb alpha is inhibited by soluble, recombinant human GPVI. Fourth, CVX binding to GPIb alpha is disrupted by phenylalanine substitutions at GPIb alpha tyrosine-276, tyrosine-278, and tyrosine-279, which also disrupts von Willebrand factor and alpha-thrombin binding to GPIb alpha. Fifth, CVX binding to GPIb alpha on Chinese hamster ovary cell transfectants is inhibited by function-blocking murine monoclonal anti-GPIb alpha antibodies. Lastly, CVX fails to bind to denatured GPIb alpha in detergent extracts of platelets. Three separate preparations of CVX (two purified by the authors; one obtained commercially) produced equivalent results. These results indicate that CVX exhibits dual specificity for both native GPIb alpha and GPVI. Furthermore, the binding site on GPIb alpha for CVX may be close to that for von Willebrand factor. Therefore, a contribution of GPIb alpha to CVX-induced platelet responses needs to be carefully re-evaluated.

Antimicrobial peptides from human platelets.

Platelets share structural and functional similarities with granulocytes known to participate in antimicrobial host defense. To evaluate the potential antimicrobial activities of platelet proteins, normal human platelets were stimulated with human thrombin in vitro. Components of the stimulated-platelet supernatants were purified to homogeneity by reversed-phase high-performance liquid chromatography. Purified peptides with inhibitory activity against Escherichia coli ML35 in an agar diffusion antimicrobial assay were characterized by mass spectrometry, amino acid analysis, and sequence determination. These analyses enabled the identification of seven thrombin-releasable antimicrobial peptides from human platelets: platelet factor 4 (PF-4), RANTES, connective tissue activating peptide 3 (CTAP-3), platelet basic protein, thymosin beta-4 (Tbeta-4), fibrinopeptide B (FP-B), and fibrinopeptide A (FP-A). With the exception of FP-A and FP-B, all peptides were also purified from acid extracts of nonstimulated platelets. The in vitro antimicrobial activities of the seven released peptides were further tested against bacteria (E. coli and Staphylococcus aureus) and fungi (Candida albicans and Cryptococcus neoformans). Each peptide exerted activity against at least two organisms. Generally, the peptides were more potent against bacteria than fungi, activity was greater at acidic pHs, and antimicrobial activities were dose dependent. Exceptions to these observations were observed with PF-4, which displayed a bimodal dose-response relationship in microbicidal assays, and Tbeta-4, which had greater activity at alkaline pHs. At concentrations at which they were individually sublethal, PF-4 and CTAP-3 exerted synergistic microbicidal activity against E. coli. Collectively, these findings suggest a direct antimicrobial role for platelets as they are activated to release peptides in response to trauma or mediators of inflammation.

High throughput peptide mass fingerprinting and protein macroarray analysis using chemical printing strategies.

We describe a chemical printer that uses piezoelectric pulsing for rapid, accurate, and non-contact microdispensing of fluid for proteomic analysis of immobilized protein macroarrays. We demonstrate protein digestion and peptide mass fingerprinting analysis of human plasma and platelet proteins direct from a membrane surface subsequent to defined microdispensing of trypsin and matrix solutions, hence bypassing multiple liquid-handling steps. Detection of low abundance, alkaline proteins from whole human platelet extracts has been highlighted. Membrane immobilization of protein permits archiving of samples pre-/post-analysis and provides a means for subanalysis using multiple chemistries. This study highlights the ability to increase sequence coverage for protein identification using multiple enzymes and to characterize N-glycosylation modifications using a combination of PNGase F and trypsin. We also demonstrate microdispensing of multiple serum samples in a quantitative microenzyme-linked immunosorbent assay format to rapidly screen protein macroarrays for pathogen-derived antigens. We anticipate the chemical printer will be a major component of proteomic platforms for high throughput protein identification and characterization with widespread applications in biomedical and diagnostic discovery.

Arp2/3 complex is required for actin polymerization during platelet shape change

Platelets undergo a series of actin-dependent morphologic changes when activated by thrombin receptor activating peptide (TRAP) or when spreading on glass. Polymerization of actin results in the sequential formation of filopodia, lamellipodia, and stress fibers, but the molecular mechanisms regulating this polymerization are unknown. The Arp2/3 complex nucleates actin polymerization in vitro and could perform this function inside cells as well. To test whether Arp2/3 regulated platelet actin polymerization, we used recombinant Arp2 protein (rArp2) to generate Arp2-specific antibodies (alpha Arp2). Intact and Fab fragments of alpha Arp2 inhibited TRAP-stimulated actin-polymerizing activity in platelet extracts as measured by the pyrene assay. Inhibition was reversed by the addition of rArp2 protein. To test the effect of Arp2/3 inhibition on the formation of specific actin structures, we designed a new method to permeabilize resting platelets while preserving their ability to adhere and to form filopodia and lamellipodia on exposure to glass. Inhibition of Arp2/3 froze platelets at the rounded, early stage of activation, before the formation of filopodia and lamellipodia. By morphometric analysis, the proportion of platelets in the rounded stage rose from 2.85% in untreated to 63% after treatment with alpha Arp2. This effect was also seen with Fab fragments and was reversed by the addition of rArp2 protein. By immunofluorescence of platelets at various stages of spreading, the Arp2/3 complex was found in filopodia and lamellipodia. These results suggest that activation of the Arp2/3 complex at the cortex by TRAP stimulation initiates an explosive polymerization of actin filaments that is required for all subsequent actin-dependent events.

Role of anti-Galα1,3Gal and anti–platelet antibodies in hyperacute rejection of pig lung by human blood

Background. Previous work has shown that antibodies against porcine antigens are an important trigger of hyperacute lung rejection (HALR). The relative importance of Galα1,3Gal epitopes and other antigens, such as those expressed on pig platelet membranes or lung itself, has not been defined. This study compares the efficiency of three anti–pig antibody depletion strategies, and their efficacy with regard to attenuation of HALR. Methods. Plasma pooled from three human donors was adsorbed against Galα1,3Gal disaccharide or porcine platelet extract (PPE), or passed through pig lung vasculature. Whole blood reconstituted using adsorbed plasma was then used to perfuse piglet lung, and results were compared with unmodified human blood. Results. Depletion of lung-reactive anti–Galα1-3Gal antibodies was most efficient with the αGal column (99% ± 0.5% vs 87% to 93% ± 11% for PPE and 92% to 95% ± 8% for lung, p < 0.01 vs αGal column). PPE column tended to be more efficient (77% to 84% ± 12%) in removing anti-PPE antibodies than pig lung (66% to 70% ± 14%) or the αGal column (56% to 63% ± 16%, p < 0.05). Lung survival and function with each antibody depletion strategy was improved relative to unmodified controls (mean survival ≥146 minutes vs 8 minutes for controls). Although αGal and lung adsorption yielded more consistent lung protection (survival beyond 2 hours) than did PPE, no approach proved significantly superior. Complement C3a elaboration at 10 minutes was attenuated >80% by each adsorption strategy, an effect that was most pronounced in the lung adsorption group (95%, p < 0.01). Histamine elaboration was blunted significantly by PPE adsorption but not in other groups ( p < 0.05). Platelet but not leukocyte sequestration was decreased with antibody depletion compared with the nondepleted group (44% to 50% vs 82%, p < 0.01). Conclusions. Each antibody depletion strategy tested significantly prolongs lung xenograft survival and function compared with unmodified human blood, but none was sufficient to reliably prevent HALR. Depletion of antibodies against both αGal and additional cell membrane antigens, or control of antibody-independent pathogenic pathways, may be necessary to consistently prevent HALR.

A novel form of platelet-type 12-lipoxygenase mRNA in human vascular smooth muscle cells.

The lipoxygenase pathway has been implicated in the growth, migration, and contraction of vascular smooth muscle cells (VSMCs). However, the precise type of lipoxygenase present in the vascular wall has not been characterized. In this study, we used a specific reverse-transcriptase polymerase chain reaction method with 2 sets of specific primers on total RNA and polyA (+)RNA of normal human VSMCs prepared from umbilical artery. Two forms of platelet-type 12-lipoxygenase mRNA were present in human VSMCs: the already published form cloned from human erythroleukemia cells and a variant form of platelet-type 12-lipoxygenase, which includes 2 additional sequences consistent with the 2 introns (D and E). This novel form of 12-lipoxygenase poly A (+)RNA was downregulated by lipopolysaccharide (10 ug/ml) and upregulated by epidermal growth factor (100 ng/ml) but was not affected by angiotensin II (10(-7) mol/l). We developed a rabbit anti-human platelet-type 12-lipoxygenase polyclonal antibody directed against a 24-amino acid peptide encoded within exon 4. Western immunoblotting of protein extracted from VSMCs and umbilical artery and platelet extract with this antibody showed a coordinate 110-kDa protein and the already-described 70-kDa band detected in platelets and cord homogenate. Another 120-kDa protein was consistently detected in cord extracts but not in platelet or VSMC homogenates. The immunohistochemistry study performed with the same antibody showed extensive cytoplasmic staining of VSMCs. The specific role of these different forms of platelet-type 12-lipoxygenase is subject to further investigation.

A cGMP-Dependent Protein Kinase Assay for High Throughput Screening Based on Time-Resolved Fluorescence Resonance Energy Transfer

Activation of cyclic GMP-dependent protein kinase (cGK) is an important event in the regulation of blood pressure and platelet function. Upstream signals are the generation of nitric oxide (NO) by NO synthases and the subsequent rise in cyclic GMP levels mediated by NO-dependent guanylyl cyclases (GCs). The identification of new cGK activators by high throughput screening (HTS) may lead to the development of a novel class of therapeutics for the treatment of cardiovascular diseases. Therefore, a homogeneous, nonradioactive assay for cGK activity was developed using a biotinylated peptide derived from vasodilator-stimulated phosphoprotein (VASP), a well-characterized natural cGK substrate. The phosphorylated peptide could be detected by a VASP-specific monoclonal phosphoserine antibody and a fluorescent detection system consisting of a europium-labeled secondary antibody and allophycocyanin (APC)-labeled streptavidin. Fluorescence resonance energy transfer (FRET) from europium to APC was detected in a time-resolved fashion (TR-FRET). Activation and inhibition constants for known substances determined by this new fluorescence-based assay correlated well with published results obtained by conventional radioactive cGK activity assays. The assay proved to be sensitive, robust, highly specific for cGK, and suitable for HTS in 96- and 384-well formats. This assay is applicable to purified enzymes as well as to complex samples such as human platelet extracts.

A cGMP-Dependent Protein Kinase Assay for High Throughput Screening Based on Time-Resolved Fluorescence Resonance Energy Transfer

Activation of cyclic GMP-dependent protein kinase (cGK) is an important event in the regulation of blood pressure and platelet function. Upstream signals are the generation of nitric oxide (NO) by NO syntheses and the subsequent rise in cyclic GMP levels mediated by NO-dependent guanylyl cyclases (GCs). The identification of new cGK activators by high throughput sreening (HTS) may lead to the development of a novel class of therapeutics for the treatment of cardiovascular diseases. Therefore, a homogeneous, nonradioactive assay for cGK activity was developed using a biotinylated peptide derived from vasodilator-stimulated phosphoprotein (VASP), a well-characterized natural cGK substrate. The phosphorylated peptide could be detected by a VASP-specific monoclonal phosphoserine antibody and a fluorescent detection system consisting of a europium-labeled secondary antibody and allophycocyanin (APC)-labeled streptavidin. Fluorescence resonance energy transfer (FRET) from europium to APC was detected in a time-resolved fashion (TR-FRET). Activation and inhibition constants for known substances determined by this new fluorescence-based assay correlated well with published results obtained by conventional radioactive cGK activity assays. The assay proved to be sensitive, robust, highly specific for cGK, and suitable for HTS in 96- and 384-well formats. This assay is applicable to purified enzymes as well as to complex samples such as human platelet extracts.

Analysis of preformed xenoreactive antibodies in the discordant guinea pig to rat model using a guinea pig fibroblast-like cell line

In the discordant guinea pig (gp) to rat model of xenotransplantation, circulating xenoreactive natural antibodies (XNA) recognizing gp antigens are usually determined by an ELISA using membrane extracts of gp platelets. We analysed the lung-derived, fibroblast-like cell line JH 4 to detect XNA by ELISA or immunoblot, which was compared to primary gp cells, i.e. platelets, liver- and spleen cells. All membrane extracts proved to be useful to detect rat XNA directed against gp antigens by ELISA. In general, IgM responses of Lewis or C6 deficient PVG rats (PVG/C6-) were higher as compared to IgG responses. However, we observed great inter-individual variabilities. The strongest IgM response of Lewis rat sera was observed when the JH 4 cell line or gp liver cells were used as antigen. JH 4 cells also showed the strongest xenoreactivity with sera from PVG/C6- rats. These data demonstrate that JH 4 cells prove useful as antigen source for XNA ELISA. In immunoblot, individual sera of the two different rat strains showed the same antigen patterns using a gp membrane extract of one particular cell type. However, the different gp cell types showed a distinct pattern of antigen expression. Whereas the JH 4 cells, platelets and spleen cells express xenoreactive proteins of the same size, a unique pattern of proteins was detected in liver cells.

Calcium Influx Factor Directly Activates Store-operated Cation Channels in Vascular Smooth Muscle Cells

Recently, we described a novel 3-pS Ca(2+)-conducting channel that is activated by BAPTA and thapsigargin-induced passive depletion of intracellular Ca(2+) stores and likely to be a native store-operated channel in vascular smooth muscle cells (SMC). Neither Ca(2+) nor inositol 1,4,5-trisphosphate or other second messengers tested activated this channel in membrane patches excised from resting SMC. Here we report that these 3-pS channels are activated in inside-out membrane patches from SMC immediately upon application of Ca(2+) influx factor (CIF) extracted from mutant yeast, which has been previously shown to activate Ca(2+) influx in Xenopus oocytes and Ca(2+) release-activated Ca(2+) current in Jurkat cells. In bioassay experiments depletion of Ca(2+) stores in permeabilized human platelets resulted in the release of endogenous factor, which activated 3-pS channels in isolated inside-out membrane patches excised from SMC and exposed to permeabilized platelets. The same 3-pS channels in excised membrane patches were also activated by acid extracts of CIF derived from human platelets with depleted Ca(2+) stores, which also stimulated Ca(2+) influx upon injection into Xenopus oocytes. Specific high pressure liquid chromatography fractions of platelet extracts were found to have CIF activity when injected into oocytes and activate 3-pS channels in excised membrane patches. These data show for the first time that CIF produced by mammalian cells and yeast with depleted Ca(2+) stores directly activates native 3-pS cation channels, which in intact SMC are activated by Ca(2+) store depletion.

Immunohistochemical localization of cGMP-binding cGMP-specific phosphodiesterase (PDE5) in rat tissues.

We raised a polyclonal antibody against maltose binding protein fusion human cGMP-binding, cGMP-specific phosphodiesterase (PDE5) produced in E. coli. This antibody immunoreacted specifically with recombinant human and rat PDE5 proteins expressed in transfected COS-7 cells and with a native form of PDE5 in extracts of rat platelets, lung, and cerebellum. Immunohistochemical analysis showed that the anti-PDE5 antibody detected immunoactive materials in Purkinje cell layers of the cerebellum, proximal renal tubules, collecting renal ducts, and epithelial cells of pancreatic ducts in rats. Reverse transcriptase-polymerase chain reaction analysis demonstrated that PDE5 transcripts are also present in rat cerebellum, kidney, and pancreas. Here we described a cell-specific localization of PDE5 in various rat tissues, suggesting the possibility of the presence of a cGMP/PDE5 pathway in these tissues.