Biochemical and pharmacological characterization of human embryo-derived platelet activating factor

The soluble platelet activating factor (PAF) produced by mouseembryos was shown to have properties similar to 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphocholine(PAF-acether). In this study PAF was extracted from the mediumIn which human embryos were cultured for 18 h prior to transfer.The extracted embryo-derived PAF moved on silica thin layerchromatograms with the same R_F of 0.26 ± 0.03 (n = 26)as PAF-acether. Embryo-derived PAF or PAF-acether activity wasassayed by monitoring the decrease in the proportion of singleplatelets in rabbit whole blood due to aggregation on incubationat 37°C. The two agonists were said to be of the same activity,if they induced the same degree of platelet aggregation after15 min incubation. PAF-acether (93 nM) and embryo-derived PAFof similar activity induced an Identical time response of plateletaggregation, the response being maximal by 6 min. PAF-acether,over the range 5.6–200 nM, induced a decrease that waslinear when plotted on a log-log scale. Embryo-derived PAF andPAF-acether (184 nM) gave identical dose responses when seriallydiluted to 16 nM. Pharmacologically, the action of embryo-derivedPAF and PAF-acether (46 nM) on platelet aggregation was significantlyinhibited by 3.75 µM of the PAF-speeific receptor inhibitor,SRI 63-441, and completely inhibited at 15 µM SRI 63-441.Embryo-derived PAF and PAF-acether (184 nM) were inactivatedto the same degree by incubation with 5–13 IU/ml phospholipaseA₂ (pA₂) Thus, the embryo-derived PAF from the human was shownon the basis of biochemical and pharmacological characterizationto be homologous to PAF-acether.     

A Novel Bioassay for Detection of Preimplantation Factor (PIF)

PROBLEM: To identify the presence of vital preimplantation embryos in vivo in humans, a newly observed phenomenon based on autorosette formation between lymphocytes and platelets, when treated with pregnant sera, was used as a marker. METHOD: Serum samples were obtained from 65 patients on the fourth day after embryo transfer (ET). Sera from 10 healthy males and 47 nonpregnant women were used as controls. The preimplantation factor (PIF) was detected by using blood group 0+ donor lymphocytes and platelets incubated with blinded serum in the presence of anti-CD2 antibody and rabbit complement. Human chorion gonadotropin (hCG) concentrations were determined 7 days later and compared with results of the lymphocyte-platelets assay. Implantation was confirmed by ultrasonographic evidence of presence of an intrauterine gestational sac. The role of platelet activating factor (PAF) in the observed phenomena was studied experimentally. RESULTS: Significantly more lymphocyte-platelet rosette formations were observed when sera from women who successfully implanted were compared to sera from women who failed to implant. This assay yielded a specificity of 95%, sensitivity of 88%, positive predictive value of 94%, and negative predictability of 90%. PAF added directly to the cell suspension and tested sera controls did not influence the percentage of lymphocyte/platelets rosettes. CONCLUSION: The application of PIF assay will enable the identification and study of early pregnancy events before the implantation occur. PAF by itself is not responsible for the rosette formation.     

Development and Validation of an Assay for Measuring Preimplantation Factor (PIF) of Embryonal Origin

PROBLEM: Tests to determine presence of embryos prior to implantation are needed. METHODS: Sera from women after embryo transfer were tested for preimplantation factor (PIF) using the lymphocyte/platelet binding assay. Autorosettes were counted using blood type O+ donor lymphocytes and platelets incubated with blinded serum in the presence of antiCD2 antibody and rabbit complement. Human chorion gonadotropin (hCG) concentrations were determined 7 days later and compared with results of the lymphocyte/platelet assay. Implantation was confirmed by ultrasonographic evidence of presence of an intrauterine gestational sac. The roles of platelet activating factor (PAF) and chaperonin 10 in the observed phenomena were studied experimentally. RESULTS: Significantly more lymphocyte/platelet rosette formations were observed when sera from women who successfully implanted were compared to sera from women who failed to implant. Neither PAF nor chaparonin added to the tested sera controls influenced the percentage of lymphocyte/platelets rosettes. CONCLUSIONS: PIF is a likely candidate to be the next frontier of diagnosing the presence of viable preimplantation embryos in vivo.     

Platelet activating factor enhances the acrosome reaction, fertilization in vitro by subzonal sperm injection and resulting embryonic development in the rabbit

This study was conducted to investigate the effect of plateletactivating factor (PAF) on the acrosome reaction and fertilizingcapacity of spermatozoa, and development of the resulting embryosin the rabbit. Rabbit spermatozoa were exposed to PAF, Iyso-PAF,or high ionic strength medium (HIS) prior to subzonal sperminjection (SUZI) into 326 mature oocytes, or morphological assessmentof the acrosome reaction. The rates of fertilization and blastocystformation were compared among the three treatment groups. Acrosomereaction was assessed by fluorescein isothiocyanate-conjugatedPisum sativum agglutinin (FITC–PSA) staining and electronmicroscopy. PAF-treated spermatozoa fertilized the oocytes ata significantly higher rate (56.1%) than did lyso-PAF-(36.8%,P< 0.01) or HIS- (38.2%, P < 0.05) treated spermatozoa.The embryos produced by PAF-treated spermatozoa showed significantlyhigher blastocyst formation rates (34.0%) than lyso-PAF- (8.6%,P < 0.050) or HIS-(8.8%, P< 0.05) treated spermatozoa.FITC-PSA staining demonstrated a significantly higher incidenceof acrosome reaction in PAF-treated spermatozoa (45.8%) thanin Iyso-PAF- (28.0%, P < 0.01) or HIS- (34.9%, P < 0.01)treated spermatozoa. Acrosome reaction of PAF-treated spermatozoawas also confirmed by electron microscopy. PAF treatment ofspermatozoa enhances fertilizing capacity for SUZI possiblyby augmenting the acrosome reaction. Enhanced embryonic developmentwas also found in the oocytes fertilized by SUZI of PAF-treatedspermatozoa.     

Hypothesis: hemolytic transfusion reactions represent an alternative type of anaphylaxis

Classical anaphylaxis is the most severe, and potentially fatal, type of allergic reaction, manifested by hypotension, bronchoconstriction, and vascular permeability. Similarly, a hemolytic transfusion reaction (HTR) is the most feared consequence of blood transfusion. Evidence for the existence of an alternative, IgG-mediated pathway of anaphylaxis may be relevant for explaining the pathophysiology of IgG-mediated-HTRs. The purpose of this review is to summarize the evidence for this alternative pathway of anaphylaxis and to present the hypothesis that an IgG-mediated HTR is one example of this type of anaphylaxis.     

Influence of prostaglandins and platelet activating factor on implantation

Several studies indicate that prostaglandins and platelet activating factor (PAF) affect implantation of the embryo. Most studies relate to animal data on prostaglandin synthesis at the site of implantation and on prostaglandin production by blastocysts. The few experimental data available on human implantation suggest that prostaglandins and PAF behave similarly in man. A review of the literature on this subject suggests possible mechanisms for prostaglandin and PAF participation in the process of human implantation.     

血小板激活因子在小肠缺血再灌注损伤中的意义及山莨菪碱的保护作用

在大鼠小肠原位灌注模型上,发现小肠缺血—再灌注(I-R)损伤时,肠道产生的PAF显著增加;山莨菪碱灌流明显抑制其PAF的产生,并有与PAF受体拮抗剂Kadsurenone相似的抗I-R损伤效应。但山莨菪碱对外源PAF灌流引起的小肠组织损伤无显著防治效果。提示PAF在小肠I-R损伤中具有发病学意义,山莨菪碱可能是通过抑制肠道PAF的产生,而主要不是通过拮抗PAF,以改善小肠的I-R损伤。     

Involvement of platelet activation in experimental osteonecrosis in rabbits

Osteonecrosis (ON) was produced experimentally in rabbits by intravenous injection of platelet activating factor (PAF) in combination with lipopolysaccharides (LPS) on two occasions separated by a week-long interval. Eleven of 15 rabbits (73%), with administration of both LPS (50 microg/kg) and PAF (10 microg/kg), exhibited microcirculatory injuries including extravasation of erythrocytes into sinusoidal spaces and formation of microthrombi in arterioles near regions of erythrocyte extravasation. Seven of 15 rabbits (47%), which received both LPS (50 microg/kg) and PAF (10 microg/kg), exhibited necrosis of trabeculae and 8 of 15 (53%) exhibited bone marrow necrosis. In addition, PAF receptor antagonist (0.3 and 3.0 mg/kg) significantly reduced the incidence of trabecular necrosis (0%) in this model. The findings of the present study suggest that platelet activation may play an important role in inducible ON, and that suppression of platelet activation may contribute prevention of ON.     

Enhanced production of platelet activating factor by peripheral granulocytes from children with asthma

Schauer U, Koch B, Michl U, Jäger R, Rieger CHL. Enhanced production of platelet activating factor by peripheral granulocytes from children with asthma.
Granulocytes from 23 asthmatic children aged 4–15 years and 32 age-matched healthy children were studied. Cells were purified by Dextran sedimentation and Percoll gradient centrifugation from heparinized blood. After in vitro stimulation by ionophore A23187 the amount of newly synthesized PAF and LTC₄ was assessed by radio receptor assay or radioimmunoassay respectively. Eight patients had symptoms of asthma within the last 3 weeks before examination. Granulocytes from the symptomatic patients showed a significantly higher PAF generation (median 125 ng/10⁶ cells, range 7–189 ng/10⁶ cells) when compared to asymptomatic patients ( p < 0.001. median 14 ng/10⁶cells, range 6–33 ng/10⁶ cells) or controls ( p < 0.001, median 11 ng/10⁶ cells, range 3–26 ng/10⁶ cells). In contrast, LTC₄ generation was increased in both patient groups. The results suggest a regulatory role of PAF in the exacerbation of asthma.     

Platelet aggregating activity in human embryo culture media free of PAF-acether

A factor activating human platelets and liberated by the embryowas sought in the culture media of human embryos using two bioassays.The first bioassay demonstrated the existence of a thrombocytopaenicfactor after the i.p. injection of culture media into splenectomizedmice. This factor was found more frequently in media which containedan embryo compared to those which contained a non-fertilizedoocyte. PAF-acether (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine)was searched for with a specific bioassay, using washed rabbitplatelets. This remained negative for all the media studied,including those which had contained an embryo giving rise toa pregnancy. In these experiments it was not possible to relateembryo-derived platelet activating factor (EDPAF) to PAF-acether.Neither were we able to use the detection of EDPAF to test embryonicviability, or attempt to identify those embryos which were susceptibleto lead to a pregnancy after intrauterine transfer from amongall the embryos transferred.     

牛脑微血管平滑肌细胞的体外培养及其产生血小板激活因子的观察

本文采用匀浆及149μm尼龙网过滤法分离脑微血管,并用胶原酶处理后接种于培养瓶中,静置一周后.可见有散在的细胞生长,再经一周后可长成致密单层。细胞经光镜、电镜检查均符合平滑肌细胞的特征。光镜下可见细胞呈梭形,火焰状排列,电镜下可见为平滑肌细胞特征的肌丝。VIII因子相关抗原鉴定呈阴性,培养的脑徽血管平滑肌细胞,在A-23187刺激下可产生少量血小板激活因子(platelet activating faetor,PAF),其产量较内皮细胞要少.这也是平滑肌细胞与内皮细胞的重要区别之一。     

Modulating action of adenosine on human platelet activation

Research Center for Molecular Diagnosis and Treatment, Ministry of Health of Russia, Moscow. (Presented by Academician of the Russian Academy of Medical Sciences R. V. Petrov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 113, No. 6, pp. 606–608, June, 1992.     

Andrology: Effects of platelet activating factor on human sperm function in vitro

The direct effects of platelet activating factor (PAF) and thespecific PAF receptor antagonist, CV-3988, on the fertilizingability of human spermatozoa were investigated. PAF (10^–7–10^–11M) increased the human sperm penetration rates in a sperm penetrationassay at all doses >10^–11 M. In contrast, treatmentof the spermatozoa with 10^–5 CV-3988 caused a significantdecrease in human sperm penetration of zona-free hamster oocytesand adversely affected sperm motility after 24 h of incubation.This suppression was reversed by the addition of PAF. The acrosomereaction was also enhanced by PAF treatment of spermatozoa butthis effect was not observed in calcium-free medium. While 10^–5M CV-3988 decreased the acrosome reaction, the inhibition wasalso reversed by the addition of PAF. These results suggestthat PAF may have a direct role in the fertilizing capacityof human spermatozoa. These findings also suggest that PAF mayhave a clinical application in an in-vitro fertilization programme.     

A 12-week placebo-controlled study of rupatadine 10 mg once daily compared with cetirizine 10 mg once daily, in the treatment of persistent allergic rhinitis

Background:  With the current increasing incidence of allergies worldwide, new treatments showing efficacy and long term safety are needed for chronic conditions such as persistent allergic rhinitis (PER). New generation H1-antihistamines have demonstrated anti-allergic properties, which could possibly enhance their effectiveness in long-term periods of treatment.
Objective:  To investigate the efficacy of rupatadine, in controlling symptoms of PER over a 12-week period.
Methods:  A randomized, double blind, parallel-group, placebo-controlled study was carried out in patients aged older than 12 years with PER. Main inclusion criteria were: instantaneous total symptom score (i6TSS) ≥45, nasal obstruction score ≤12, and overall assessment of PER ≥2 as moderate during the first visit. The primary efficacy endpoint was the 12-week average change from baseline of the patients' i6TSS.
Results:  In all, 736 patients were selected. Of them, 543 (73.8%) were randomized in three different groups: placebo ( n  = 185), cetirizine ( n  = 175) and rupatadine ( n  = 183). Rupatadine ( P  = 0.008) but not cetirizine ( P  = 0.07) statistically reduced the baseline i6TSS vs placebo (47.8%, 44.7% and 38.8%, respectively), after 12 weeks. Onset of action was observed at the first 24 h for both treatments (rupatadine vs placebo, P  = 0.013; cetirizine vs placebo, P  = 0.015). Furthermore, instantaneous total nasal symptoms score (iTNSS) (including nasal blockage) mean change from baseline showed a significant reduction with rupatadine 10 mg in comparison with placebo, along all treatment duration of 12 weeks. Study treatments were well tolerated.
Conclusion:  Rupatadine significantly relieves symptoms of PER, providing a rapid onset of action and maintains its effects over a long period of 12-weeks.     

Platelet activating factor (PAF) increases plasma protein extravasation and induces lowering of interstitial fluid pressure (Pif) in rat skin

Aim: To investigate the ability of the microdialysis technique to measure capillary selectivity of different sized plasma proteins induced by local administration of platelet activating factor (PAF). Methods: We used hollow plasmapheresis fibres with 3 cm membrane (cut off 3000 kDa) placed on the back of anaesthetized rats. Results: Platelet activating factor (50 μg mL^?1) administered locally via the fibre, increased extravasation of radiolabelled ¹²⁵I‐HSA from plasma to the microdialysis fibre by approximately 900% compared both to baseline and the control fibre within 70 min (n = 6, P < 0.05). The extravasation in the control fibre did not change over time. HPLC measurement of plasma proteins in the microdialysis perfusate also demonstrated decreased capillary selectivity for proteins in the diameter range of 73 Å, 56 Å and 39 Å after local administration of PAF (n = 6, P < 0.05). PAF also significantly lowered interstitial fluid (P_if) pressure after subcutaneous administration (50 μg mL^?1). Mean arterial pressure (MAP) after intravenous injection of PAF (0.4 μg kg^?1) fell instantly by about 50 mmHg, and stabilized at 50 mmHg after 15 min (n = 6). MAP was unaltered when PAF was given through the microdialysis fibre (n = 4). Both total tissue water (TTW) and extravasation of albumin, measured as the plasma‐to‐tissue clearance (E‐alb) showed a significant increase after PAF (n = 7, P < 0.05). Conclusions: The present study demonstrates that PAF induces plasma protein extravasation and decrease capillary selectivity of different sized plasma proteins. It also increases transcapillary fluid flux, and lowers P_if, indicating a role for PAF in the interstitium for generation of transcapillary transport of water and large molecules followed by formation of oedema.     

Cardiodepressor action of platelet activating factor

Biophysics Research Laboratory, A. V. Vishnevskii Institute of Surgery, Academy of Medical Sciences of the USSR, Moscow. Laboratory of Lipid Biochemistry, Research Institute of Biomedical Technology, Moscow. (Presented by Academecian of the Academy of Medical Sciences of the USSR D. S. Sarkisov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 107, No. 1, pp. 27–30, January, 1989.     

An atypical protein kinase C, PKC zeta, regulates human eosinophil effector functions

Protein kinase (PK) C comprises a family of isoenzymes that play key roles in downstream signalling and cell functions. We studied PKC zeta participation in the effector functions of human eosinophils stimulated with platelet-activating factor (PAF) or complement (C) 5a. After pretreating eosinophils with a myristoylated specific PKC zeta inhibitor; bisindlolylmaleimide I (BisI), an inhibitor of conventional and novel PKCs; or rottlerin, a PKC delta inhibitor, we examined PAF- and C5a-evoked functions. Induced PKC translocation was characterized by confocal laser scanning microscopy. The PKC zeta inhibitor blocked PAF- or C5a-induced eosinophil superoxide anion generation as effectively as BisI or rottlerin. The PKC zeta inhibitor also attenuated PAF- or C5a-induced eosinophil degranulation and adhesion. In contrast, the PKC zeta inhibitor did not affect PAF- or C5a-induced CD11b expression. Finally, both eosinophil shape changes and the translocation of PKC zeta and p47phox, a component of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, to the plasma membrane induced by PAF or C5a were completely inhibited by the PKC inhibitor. Thus, the atypical PKC zeta regulates human eosinophil adhesion and effector functions.     

Platelet activating factor in culture media as an indicator of human embryonic development after in-vitro fertilization.

Although human chorionic gonadotrophin can detect trophoblast after implantation of the conceptus, there is a need to detect the conceptus before implantation. We have investigated whether human embryo-derived platelet activating factor is formed during embryonic development after in-vitro fertilization. A total of 99 ova from 12 patients were cultured and the 54 media were analysed. Platelet activating factor was also measured by radioimmunoassay after extraction. Fertilization increased the amount of platelet activating factor 4-fold over non-fertilized ova to a level of 4 ng/ml. This increase was also dependent on the degree of embryonic development with a maximum level of platelet activating factor of 7 ng/ml at the 2-cell stage. The follicular inducing agent used to treat the patient also had an effect on platelet activating factor; buserelin treatment gave embryos with a higher level than did clomiphene citrate treatment. These results indicate that platelet activating factor may have a role in embryonic development before implantation and may serve as a useful marker for fertilization and the developmental stage of the embryo.