[Cytomegalovirus detoxification and associated factors among preschool children in Yi'nan County].

Objective: To investigate cytomegalovirus detoxification and associated factors among preschool children in Yinan County, Shandong Province. Methods: Two kindergartens were selected from the county and township of Yinan respectively. A total of 250 children were investigated in October 2018. Case information was obtained through the child's guardian. Saliva samples of children and their mothers were collected for cytomegalovirus realtime-PCR detection.The status of CMV detoxification of children was explored and the associated factors were analyzed. Results: A total of 242 preschool children were investigated, and the detoxification rate of cytomegalovirus among them was 22.31% (54/242, 95%CI: 17.0%-27.6%). Logistic regression analysis showed that the rate of detoxification was higher in children whose mothers were cytomegalovirus detoxified (OR=12.39, 95%CI:1.73-88.65)and whose school was located in the county (OR=3.58, 95%CI:1.34-9.55). Conclusions: The detoxification rate of cytomegalovirus in preschool children is high, and there is mutual transmission between children and mothers. Women of childbearing age should pay attention to prevent congenital cytomegalovirus infection when they come into contact with children.

[Expression of nucleolar protein 14 and CD31 in pancreatic cancer mouse model and its correlation with tumor progression].

Objective: To investigate expression of nucleolar protein 14(NOP14) and CD31 in pancreatic cancer mouse model and its correlation with tumor progression. Methods: Clinicopathological data of 5 patients with pathologically confirmed pancreatic ductal adenocarcinoma(PDAC) and hepatic metastasis between January 2013 and December 2015 was collected in Department of General Surgery, Peking Union Medical College Hospital. Immunohistochemistry staining was employed to detect the expression of NOP14 in matched primary PDAC and relevant metastasis.Pancreatic cancer cells with NOP14 stably knocked down were established by transfecting lentivirus with NOP14 targeted silencing RNA.The inhibition efficacy was detected by quantitative real time PCR and western blot.Microvascular density(MVD) in pancreatic cancer transplantation mouse model was determined by CD31 immunohistochemistry staining analysis and correlated with NOP14 expression and tumor progression. Results: NOP14 had a significant higher expression in liver metastasis than primary pancreatic adenocarcinoma (2.09±0.45 vs. 1.31±0.27, P=0.028). NOP14 was knocked down 86 percent on mRNA level determined by qPCR and 78 percents on protein level detected by western blot. MVD was significantly decreased in NOP14-inhibited tumor from both pancreatic cancer cells subcutaneously and orthotopically grafted tumor mouse model with the value of 61.40±13.85 vs. 85.53±14.59 (P=0.041) and 38.33±10.91 vs. 59.33±15.37(P =0.037), respectively. Besides, MVD was positively associated with tumor volume(r=0.842, P<0.01) and metastasis (r=0.726, P=0.008). Conclusion: NOP14 presents higher expression in hepatic metastasis of pancreatic adenocarcinoma and might promote tumor progression by increasing microvascular density.

[The effect and factors associated with outcome of extracorporeal membrane oxygenation in refractory cardiogenic shock patients].

Objective: To observe the clinical efficacy and factors associated with outcome of extracorporeal membrane oxygenation (ECMO) in refractory cardiogenic shock patients. Methods: Patients with refractory cardiogenic shock received ECMO treatment in our hospital from May 2013 to November 2015 were retrospectively analyzed. The clinical status before ECMO support, ECMO timing, complications and outcome were observed and analyzed.The hemodynamic data and the amount of vasoactive drugs at 2 hours before ECMO support and at 2, 6, 24 and 48 hours after ECMO support were collected and compared. Results: Ten refractory cardiogenic shock patients were included in this study (5 acute fulminant myocarditis patients, 4 acute myocardial infarction patients, 1 myocardial rupture patient (6 males, 4 females, age ranged 12 to 56 years). Before ECMO, the mean left ventricular ejection fraction (LVEF) was (31.4±10.2)%, the mean score of APACHE Ⅱ was 26.6±10.8. Eight patients developed cardiac arrests and the duration of CPR ranged from 10 to 300 minutes and three patients received IABP. CVP decreased, BP increased, HR decreased, ScVO2 increased, dose of dobutamine decreased at 2 hours after ECMO support. After ECMO support for 6 hours, lactate decreased, dose of norepinephrine decreased. After ECMO support for 24 and 48 hours, hemodynamics became stable and shock was significantly improved. Complication including infection of limb and catheterization site occurred in 3 patients, femoral arterial thrombosis occurred in 2 patients, critical limb ischemia occurred in 2 patients, hemorrhage at the catheterization site occurred in 2 patients. The duration of ECMO ranged from 2 to 220 hours. Nine patients could be weaned off ECMO support and 6 patients survived to hospital discharge. Two patients died due to too late ECMO support, the other two patients died due to severe complication of limb. Conclusions: ECMO can rapidly improve hemodynamic stability of patients with cardiogenic shock. Accurate assessing the timing of ECMO support and decreasing complication of limb play a critical role on improving outcome in refractory cardiogenic shock patients.

Association between HLA-A and -B polymorphisms and susceptibility to Henoch-Schönlein purpura in Han and Mongolian children from Inner Mongolia.

We examined a possible association between HLA-A and -B polymorphisms and susceptibility to Henoch-Schönlein purpura (HSP) in Han and Mongolian children in Inner Mongolia, through a case-control study. Two hundred and sixty-eight unrelated children were enrolled, including 56 Mongolian and 50 Han children with HSP, 66 healthy Mongolian and 96 healthy Han children as a control group. HLA-A and -B alleles were indentified by PCR-sequence-specific oligonucleotide analysis and were further analyzed by PCR-sequencing-based typing (SBT). Frequencies of HLA-A*11, HLA-B*15 in Mongolian patients and HLA-A*26, HLA-B*35, HLA-B*52 in Han patients were higher than those in the corresponding control group (P < 0.05), while frequencies of HLA-B*07 and -B*40 in Mongolian HSP patients were lower than those in the control group (P < 0.05). Further analysis using PCR-SBT showed that all HLA-A*11 were HLA-A*1101, and most HLA-B*15 were HLA-B*1501 in Mongolian HSP patients. All HLA-A*26 were HLA-A*2601 and HLA-B*35 were mostly HLA-B*3503 in Han patients. There were more Han patients with severe manifestations than Mongolian patients (P < 0.05). Frequencies of HLA-A*26, HLA-B*35 and HLA-B*52 in Han patients were higher than in Mongolian patients (P < 0.05). We conclude that HLA-A*11(*1101) and -B*15(*1501) are associated with susceptibility to HSP in Mongolian children and HLA-A*26(*2601), HLA-B*35(*3503) and HLA-B*52 are associated with susceptibility to HSP in Han children. HLA-B*07 and -B*40 may be protective genes in Mongolian children. The different frequencies of HLA-A and -B in Mongolian and Han children may be responsible for the different manifestations in these two ethnic groups.

Long noncoding RNA UCA1 promotes proliferation and metastasis of thyroid cancer cells by sponging miR-497-3p.

OBJECTIVE: Recently, long noncoding RNAs (lncRNAs) have attracted much attention for their roles in tumor progression. The aim of this study was to investigate the exact role of lncRNA UCA1 in the development of thyroid cancer (TC) and to explore the possible underlying mechanism. PATIENTS AND METHODS: UCA1 expression in both TC cells and tissues was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Colony formation assay, cell proliferation, and transwell assay were conducted in vitro. Subsequent luciferase reporter gene assay was applied to investigate the underlying mechanism. Furthermore, the function of UCA1 in vivo was monitored as well. RESULTS: UCA1 expression level in TC samples was significantly higher than that of corresponding normal tissues. After UCA1 was knocked down in vitro and in vivo, the proliferation, migration, and invasion of TC cells were significantly inhibited. Moreover, the expression of miR-497-3p was repressed after the knockdown of UCA1. Furthermore, miR-497-3p was directly targeted by UCA1. CONCLUSIONS: Knockdown of UCA1 could inhibit TC cell proliferation and metastasis via sponging miR-497-3p. Our findings might offer a new therapeutic intervention for TC patients.

Long noncoding RNA UCA1 promotes proliferation and metastasis of thyroid cancer cells by sponging miR-497-3p.

Since this article has been suspected of research misconduct and the corresponding authors did not respond to our request to prove originality of data and figures, "Long noncoding RNA UCA1 promotes proliferation and metastasis of thyroid cancer cells by sponging miR-497-3p, by H. Gao, J.-Y. Yang, L.-X. Tong, H. Jin, C.-Z. Liu, published in Eur Rev Med Pharmacol Sci 2020; 24 (2): 728-734-DOI: 10.26355/eurrev_202001_20052-PMID: 32016975" has been withdrawn. The Publisher apologizes for any inconvenience this may cause. https://www.europeanreview.org/article/20052.

Gene polymorphism of adiponectin in restenosis after coronary stenting.

OBJECTIVE: The objective of the present study was to investigate the relationship between adiponectin (APN)+45T/G and +276G/T polymorphisms and in-stent restenosis (ISR). PATIENTS AND METHODS: A total of 150 patients treated with percutaneous coronary intervention (PCI) were divided into the ISR group and non-ISR group. The levels of blood biochemical indicators were measured, and APN+45T/G and +276G/T polymorphisms were detected by TaqMan probes. RESULTS: Cholesterol levels in the IRS group were significantly higher than those in the non-ISR group (p<0.05). The frequency of the GG genotype and G allele of the APN+45T/G locus in the ISR group were significantly higher than those in the non-ISR group (p<0.05). The frequency of the GG genotype and G allele of the APN+276G/T locus in the ISR group were significantly higher than those in the non-ISR group (p<0.05). CONCLUSIONS: APN+45T/G and +276G/T polymorphisms were associated with susceptibility to ISR, and carrying the G allele of the APN+45T/G and +276G/T loci can significantly increase the risk of ISR.

On the process capability of the solid free-form fabrication: a case study of scaffold moulds for tissue engineering.

This study applies the methodology and procedure of process capability to investigate a solid free-form fabrication technique as a manufacturing method to produce scaffold moulds for tissue engineering. The process capability Cpk and process performance Ppk of scaffold mould manufacture using a solid free-form fabrication technique has been analysed with respect to the dimension deviations. A solid free-form fabrication machine T66 was used to fabricate scaffold moulds in this study and is able to create features that ranged from 200 microm to 1000 microm. The analysis showed that the printing process under the normal cooling conditions of the printing chamber was in statistical control but gave low process capability indices, indicating that the process was 'inadequate' for production of 'dimension-consistent' scaffold moulds. The study demonstrates that, by lowering the temperature of the cooling conditions, the capability Cpk of the printing process can be improved (about threefold) sufficiently to ensure the consistent production of scaffold moulds with dimension characteristics within their specification limits.

Rasagiline, an inhibitor of MAO-B, decreases colonic motility through elevating colonic dopamine content.

BACKGROUND: Dopamine (DA) is a negative modulator of gut motility. Monoamine oxidase-B (MAO-B) is an important metabolic enzyme degrading DA. Rasagiline, an irreversible MAO-B inhibitor, is used to treat Parkinson's disease because of its neuroprotective effect and increasing central DA. However, it is unclear whether MAO-B exists in the colon and rasagiline increases colonic DA, thereby affecting colonic motility. METHODS: Immunohistochemistry, western blotting, enzyme activity assay, colonic motility recording, gut transit test, and high-performance liquid chromatography-electrochemical detection were employed in this study. KEY RESULTS: Monoamine oxidase-B was distributed in the colonic muscular layers including neurons and glias of rat and human. When oral treatment of rats with rasagiline for 4 weeks, in vitro colonic motility was significantly reduced, but it was greatly reversed by SCH-23390, an antagonist of DA D1 receptor. The rasagiline-treated rats also manifested decreased MAO-B activity and increased DA content in the colonic muscular layer, but no alterations were detected in the protein expressions of D1 and D2 receptors, and MAO-A and MAO-B, as well as in the content of 5-hydroxytryptamine and noradrenaline. Moreover, acute administration of rasagiline did not affect the colonic motility in vitro and the colonic DA level in rats, although MAO-B activity was significantly inhibited. CONCLUSIONS & INFERENCES: Monoamine oxidase-B is abundant in the colonic muscular layer including myenteric plexus of rat and human. Long-term administration of rasagiline can increase colonic DA thereby inhibiting colonic motility, suggesting that colonic MAO-B could be a potential drug target for colonic dysmotility.

Micro- and nano-injectable composite biomaterials containing calcium phosphate coated with poly(DL-lactide-co-glycolide).

Calcium phosphate/poly(dl-lactide-co-glycolide) (CP/DLPLG) composite biomaterial, in which each CP particle was coated with DLPLG, was synthesized. Two kinds of composites were prepared: microcomposite, with particles 150-200mum in size, and nanocomposite, with the particles 40+/-5nm in size. Using nanoparticles, a new class of injectible composite biomaterials was produced. Based on scanning electron microscopy, atomic force microscopy, differential thermal analysis, thermogravimetric analysis, differential scanning calorimetry and Fourier transform infrared analyses, the structure and phase organization in both biomaterials was identified and in both studied cases CP particles were coated with DLPLG polymer. An injectable composite biomaterial, the characteristics of which depend on the ratio of the phases, was prepared by mixing physiological solution with the nano-CP/DLPLG composite. Rheological studies indicated a possible agglomeration of particles of the injectable nano-CP/DLPLG composite biomaterial with a CP content of 65%.

Effect of tobacco smoke on hydrogen sulfide-induced rat thoracic aorta relaxation.

Levels of hydrogen sulfide (H2S), a gaseous signaling molecule, are reduced in the serum of individuals who smoke. We hypothesized that tobacco smoke influenced smooth muscle relaxation by decreasing H2S levels and this effect could also influence expression of cystathionine γ-lyase (CSE) and sulfonylurea receptor-2 (SUR-2). The aim of this study was to explore the effect of tobacco smoke on H2S-mediated rat thoracic aorta relaxation and its possible mechanism. Thirty-two Sprague-Dawley rats were divided into four groups: control (C) group, short-term smoker (SS) group, mid-term smoker (MS) group, and long-term smoker (LS) group. H2S concentrations in serum, action of H2S on rat aortic vascular relaxation, and expression of CSE and SUR-2 in thoracic aortic smooth muscle were measured. Although there was no significant difference in H2S between the C and the SS groups, concentration of H2S was significantly reduced in both the LS and MS groups compared to control (P<0.01). Furthermore, H2S was significantly lower in the LS than in the MS group (P<0.05). Rat aortic vascular relaxation was lower in all three treatment groups compared to the control, with the most significant decrease observed in the LS group (P<0.05 compared to the MS group). Expression of CSE and SUR-2 was reduced in the LS and MS groups compared to control (P<0.05), with the lowest levels observed in the LS group (P<0.05). Therefore, tobacco smoke reduced expression of CSE and SUR-2 in rat thoracic aorta, which may inhibit H2S production and vascular dilation.

Increased expression of type 2 3alpha-hydroxysteroid dehydrogenase/type 5 17beta-hydroxysteroid dehydrogenase (AKR1C3) and its relationship with androgen receptor in prostate carcinoma.

Type 2 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) is a multi-functional enzyme that possesses 3alpha-, 17beta- and 20alpha-HSD, as well as prostaglandin (PG) F synthase activities and catalyzes androgen, estrogen, progestin and PG metabolism. Type 2 3alpha-HSD was cloned from human prostate, is a member of the aldo-keto reductase (AKR) superfamily and was named AKR1C3. In androgen target tissues such as the prostate, AKR1C3 catalyzes the conversion of Delta(4)-androstene-3,17-dione to testosterone, 5alpha-dihydrotestosterone to 5alpha-androstane-3alpha,17beta-diol (3alpha-diol), and 3alpha-diol to androsterone. Thus AKR1C3 may regulate the balance of androgens and hence trans-activation of the androgen receptor in these tissues. Tissue distribution studies indicate that AKR1C3 transcripts are highly expressed in human prostate. To measure AKR1C3 protein expression and its distribution in the prostate, we raised a monoclonal antibody specifically recognizing AKR1C3. This antibody allowed us to distinguish AKR1C3 from other AKR1C family members in human tissues. Immunoblot analysis showed that this monoclonal antibody binds to one species of protein in primary cultures of prostate epithelial cells and in LNCaP prostate cancer cells. Immunohistochemistry with this antibody on human prostate detected strong nuclear immunoreactivity in normal stromal and smooth muscle cells, perineurial cells, urothelial (transitional) cells, and endothelial cells. Normal prostate epithelial cells were only faintly immunoreactive or negative. Positive immunoreactivity was demonstrated in primary prostatic adenocarcinoma in 9 of 11 cases. Variable increases in immunoreactivity for AKR1C3 was also demonstrated in non-neoplastic changes in the prostate including chronic inflammation, atrophy and urothelial (transitional) cell metaplasia. We conclude that elevated expression of AKR1C3 is highly associated with prostate carcinoma. Although the biological significance of elevated AKR1C3 in prostatic carcinoma is uncertain, AKR1C3 may be responsible for the trophic effects of androgens and/or PGs on prostatic epithelial cells.

Design and testing of novel cytotoxic polyamine analogues.

We designed three polyamine analogues, 1,14-diamino-N5-methyl-5,10- diazatetradecane (5me-4-4-4), 1,14-diamino-N5,N5-dimethyl-5,10-diazatetradecane (Q-Amm-4-4-4), and 1,14-bis-(ethylamino)-N5,N5-dimethyl-5,10-diazatetradecane (BE-Q-Amm-4-4-4), on the basis of computer modeling and physical-chemical studies of polyamine-DNA interactions. These analogues differ from natural polyamines and from one another in the charge distribution on their aliphatic backbone. We found that 10 microM 5me-4-4-4 did not inhibit growth and was not cytotoxic to the human brain tumor cell lines SF-767 and SF-126. The same concentrations of Q-Amm-4-4-4 and BE-Q-Amm-4-4-4 inhibited cell growth and killed more than 90% of each cell type on day 7 of the experiment. BE-Q-Amm-4-4-4 was slightly more toxic than Q-Amm-4-4-4 in both cell lines. All three agents either decreased or completely depleted intracellular putrescine and spermidine. Q-Amm-4-4-4 and BE-Q-Amm-4-4-4 each also lowered spermine. The fact that 5me-4-4-4 was nontoxic but that Q-Amm-4-4-4 was cytotoxic and inhibited growth suggests that the charge distribution along the surface of the aliphatic backbone of polyamines is important in determining growth inhibition and cytotoxicity.

Characterization of the promoter region and genomic organization of GLI, a member of the Sonic hedgehog-Patched signaling pathway.

GLI is the prototype for the Gli-Kruppel gene family characterized by a consensus C2-H2 zinc finger domain and is believed to function as a transcription activator in the vertebrate Sonic hedgehog-Patched signal transduction pathway. Understanding GLI gene regulation may be of importance to understanding causes of human birth defects and cancer. To begin to understand the regulation of this developmentally important gene we have cloned the human GLI gene and functionally characterized its 5' flanking region. The GLI gene is composed of 12 exons and 11 introns and in the zinc finger coding region shares a highly conserved splicing pattern with several other Gli family members in both vertebrates and C. elegans. A major transcription initiation site was identified upstream of the GLI translation start site along with three minor transcription initiation sites. The region surrounding the transcription initiation sites lacks TATA and CCAAT consensus sequences, has a high GC content, includes a CpG island, and contains several GC boxes. A 487bp segment surrounding the transcription initiation sites increased expression of a luciferase reporter gene 15-fold in Tera-1 cells and was defined as the core promoter region of human GLI. In transgenic mice this region directed beta-galactosidase expression to the central nervous system on embryonic days 10.5-12.5 and to sites of endochondral ossification on embryonic days 12.5 and 13.5 in a pattern comparable to the endogenous expression pattern of mouse gli within these tissues. The previously identified gastrointestinal expression of gli was not driven by this region and may require elements outside of the core promoter. Sequence analysis of the 5' flanking region of the mouse gli gene and the full-length mouse gli cDNA demonstrated high homology with human GLI, suggesting conservation of GLI regulation and function.

The desferrithiocin pharmacophore.

The (S)-desferrithiocin (DFT) skeleton is shown to be a useful pharmacophore on which to design orally effective iron chelators. While the study clearly indicates that formal reduction of the desazadesmethyldesferrithiocin thiazoline to a thiazolidine (6), expansion of the desmethyldesferrithiocin thiazoline to a thiazine (7), or substitution of the thiazoline sulfur of of desazedes-methyldesferrithiocin by an oxygen (8 and 9) lead to a substantial loss of activity, conversion of (S)-desmethyldesferrithiocin (1) to an N-methylhydroxamate (4) or to the hexacoordinate dihydroxamate ligand (5) results in active compounds. This investigation thus demonstrates which structural components of the siderophore are required for iron clearance after oral administration and suggests the use of the desferrithiocin platform as a vector for other chelators.

Antiproliferative properties of polyamine analogues: a structure-activity study.

A basis set of polyamine analogues was designed and synthesized. These compounds were used to initiate a systematic investigation of the role of chain length, terminal nitrogen alkyl group size, and symmetry of the methylene backbone in the antineoplastic properties of polyamine analogues. New synthetic methods predicated on our earlier polyamine fragment synthesis are described for accessing the tetraamines of interest. An unsymmetrically substituted diamine reagent, N-(tert-butoxycarbonyl)-N,N'-bis(mesitylenesulfonyl)-1,4-diaminobu tane, was developed for entry into unsymmetrical tetraamines. All of the tetraamines synthesized were first evaluated in a murine leukemia L1210 cell IC50 assay at 48 and 96 h. In an attempt to correlate this behavior with some aspect of polyamine metabolism, each compound was tested for its ability to compete with spermidine for the polyamine uptake apparatus, its impact on the polyamine biosynthetic enzymes ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), and its effect on the polyamine-catabolizing enzyme spermidine/spermine N1-acetyltransferase (SSAT) and on polyamine pools. While there was no obvious correlation between the 48 and 96 h IC50's and the impact of the analogues on polyamine metabolism, there were other structure-activity relationships. Correlations were observed to exist between chain length and IC50's and between terminal alkyl substituents and impact on Ki, ODC, and AdoMetDC. Also, preliminary studies suggest a relationship may exist between the 48 and 96 h IC50 activities and the analogue's chronic toxicity in vivo. Finally, when the overall length of the polyamine backbone was held constant, the symmetry of the methylene chains of the polyamine fragments was shown to be unimportant to the compound's activity.

Measurement of glycoprotein IIb/IIIa blockade by flow cytometry with fluorescein isothiocyanate-conjugated crotavirin, a member of disintegrins.

The blockade of platelet membrane glycoprotein IIb/IIIa by a monoclonal antibody, 7E3, was measured by flow cytometry using a fluorescein isothiocyanate-conjugated disintegrin, FITC-crotavirin, as the probe. After treatment of platelets with 7E3 or 7E3 F(ab')2, there is a good correlation between the inhibition of platelet aggregation and the blockade of FITC-crotavirin's binding to platelets. The content of glycoprotein IIb/IIIa for the subsequent binding of FITC-crotavirin to the 7E3-pretreated platelets highly correlated to the extent of glycoprotein IIb/IIIa, remaining available. It was evidenced by the observation that the sum of glycoprotein IIb/IIIa occupation by 7E3 and that of FITC-crotavirin approached the total amount of glycoprotein IIb/IIIa expressed on the platelet membrane. This indicates that the percentage inhibition of FITC-crotavirin's binding at the saturation dose reflects the extent of glycoprotein IIb/IIIa blockade by 7E3. At the saturation binding concentration (5 micrograms/ml), FITC-crotavirin did not displace platelet bound 7E3. Gating the light-scattering profile for platelets, the binding of FITC-crotavirin to platelet glycoprotein IIb/IIIa could be easily determined in diluted whole blood by direct stain method. The available unoccupied glycoprotein IIb/IIIa of platelets in the 7E3 or 7E3 F(ab')2-pretreated whole blood were measured by flow cytometry at the saturation binding dose of FITC-crotavirin (4 micrograms/ml) and the data showed that the higher deconcentration of antibody added into whole blood, the lower debinding of FITC-crotavirin to platelets. This technique may provide an alternative rapid method for measuring the blockade of glycoprotein IIb/IIIa by 7E3, a promising anti-thrombotic agent, thus providing a monitoring method for adjusting the therapeutic dose of 7E3 or its related derivatives.

Analysis of human platelet glycoprotein IIb-IIIa by fluorescein isothiocyanate-conjugated disintegrins with flow cytometry.

Disintegrins are a group of snake venom peptides which inhibit human platelet aggregation by acting as glycoprotein IIb-IIIa (GPIIb-IIIa) antagonists. They are cysteine-rich, Arg-Gly-Asp (RGD)-containing peptides, and bind to GPIIb-IIIa complex on platelet membrane with a very high affinity (Kd, 10(-7)-10(-8) M). In this study, we analyzed GPIIb-IIIa complex on platelet membrane by flow cytometry using fluorescein isothiocyanate (FITC)-conjugated disintegrins as probes. Of these FITC-conjugated disintegrins, FITC-Rhodostomin is the most sensitive probe because Rhodostomin was conjugated with more FITC molecules than Trigramin and Halysin were. The binding fluorescence intensity of FITC-Trigramin (FITC-Tg), FITC-Halysin (FITC-Hy) and FITC-Rhodostomin (FITC-Rn) was measured in both resting and ADP-activated platelets of diluted human platelet-rich plasma. The binding fluorescence of FITC-disintegrins was abolished by EDTA and 7E3, a monoclonal antibody against GPIIb-IIIa. ADP markedly increased the fluorescence intensity of FITC-Tg and FITC-Hy bound on platelets especially when lower doses of these probes were used, whereas it had little effect on that of FITC-Rn. Therefore, FITC-Tg and FITC-Hy can be used for the detection of the activated platelets as noted by a higher ratio of fluorescence intensity (approx. 2-4) between ADP-activated and resting platelets as compared with that (approx. 1-1.3) in the case of FITC-Rn as the probe. The platelets from three patients with Glanzmann's thrombasthenia were probed with FITC-disintegrins.(ABSTRACT TRUNCATED AT 250 WORDS)

Halysin, an antiplatelet Arg-Gly-Asp-containing snake venom peptide, as fibrinogen receptor antagonist.

By means of Sephadex G-75 and CM-Sephadex C-50 column chromatography and reverse-phase HPLC, a low molecular weight (Mr = 7500), cysteine-rich peptide, halysin, was purified from Agkistrodon halys (mamushi) snake venom. Halysin is a potent platelet aggregation inhibitor that concentration-dependently inhibited human platelet aggregation stimulated by ADP, thrombin and collagen (IC50 = 0.16 to 0.36 microM) without affecting platelet secretion. It was active in inhibiting platelet aggregation of platelet-rich plasma and whole blood. Halysin had no effect on thromboxane B2 formation of platelets or intracellular Ca2+ mobilization of Quin 2-AM loaded platelets stimulated by thrombin. It inhibited the fibrinogen-induced aggregation of elastase-treated platelets. Halysin concentration-dependently inhibited the 125I-fibrinogen binding to ADP-stimulated platelets in a competitive manner (IC50 = 0.16 microM). 125I-Halysin bound to resting platelets (Kd = 1.6 x 10(-7) M) and to ADP-stimulated platelets (Kd = 3.4 x 10(-8) M) in a saturable manner. EDTA, the Arg-Gly-Asp (RGD)-containing snake venom peptides trigamin and rhodostomin, Arg-Gly-Asp-Ser (RGDS), and Gly-Gln-Gln-His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val blocked both 125I-fibrinogen binding and 125I-halysin binding to ADP-stimulated platelets. The monoclonal antibody, 7E3, raised against glycoprotein IIb-IIIa complex blocked both 125I-fibrinogen and 125I-halysin binding, whereas 10E5 had no significant effect on halysin binding to ADP-stimulated platelets, indicating that 7E3 and halysin bind to an epitope which is different from that of 10E5. RGDS concentration-dependently inhibited 125I-halysin binding in a competitive manner. We determined the primary structure of halysin which is a single peptide chain of 71 amino acid residues. An RGD sequence appeared in the carboxy-terminal domain of halysin. Halysin showed about an 85% identical sequence with trigamin which is a specific antagonist of fibrinogen receptor associated with glycoprotein IIb-IIIa complex. In conclusion, halysin inhibited platelet aggregation by interfering with fibrinogen binding to the fibrinogen receptor of the activated platelets. The RGD sequence of halysin plays an important role in the expression of its biological activity.