Effect of RGD-containing peptides on platelet aggregation

The influence of new synthetic peptides ARGDS-NH2 and RGD-dFK (synthesized by the fermentative method) and VPNLRGDLQVLA (a fragment of the foot-and-mouth virus's surface peptide) on the ADP-induced human platelet aggregation in vitro was studied. All peptides were found to inhibit the human platelet aggregation, but the synthetic peptides (ARGDS-NH2 and RGD-dFK) showed the most pronounced effect. Significant decrease in the platelet aggregation was observed at their concentrations within 0.1-10 mM. ARGDS-NH2 and RGD-dFK inhibited the platelet aggregation stronger than the reference drug pentoxifylline at equivalent concentrations.     

荭草苷的抗血栓作用研究

目的:研究荭草苷的抗血栓作用。方法:观察预防给予荭草苷后对小鼠体外凝血时间、小鼠全血凝块溶解作用、家兔血浆凝血酶原时间(PT)、家兔血浆白陶土部分凝血活酶时间(KPTT)、家兔血浆凝血酶时间(TT)、二磷酸腺苷二钠(ADP)诱导的家兔血小板聚集作用、家兔优球蛋白溶解时间(ELT)的影响。结果:与生理盐水组比较,荭草苷(1、2、4mg/kg)组能显著延长小鼠体外凝血时间,对小鼠全血凝块溶解作用无明显影响;荭草苷(0·625、1·25、2·5mg/kg)组能明显延长家兔PT、KPTT及TT,并能显著抑制ADP引起的血小板聚集,对家兔ELT无明显影响。结论:荭草苷具有明显的抗血栓形成作用。     

Synthesis and Antithrombotic Effect of Xanthone Derivatives

A series of xanthone derivatives was synthesized and tested in-vitro for their ability to inhibit aggregation of rabbit washed platelets and human platelet-rich plasma (PRP) induced by various inducers. 2-Prenyloxyxanthone showed the most potent inhibition of rabbit washed platelet aggregation induced by arachidonic acid (IC50 = 10.2 μM). Of the compounds tested in human PRP, 2-[3 (propylamino)-2-hydroxypropoxy]xanthone (4) hydrochloride salt exhibited the most potent inhibition of platelet aggregation induced by adrenaline (IC50 = 4.4 μM), whereas in evaluation of mouse antithrombotic activity, compound 4 exhibited the most potent protection of mice from thrombotic challenge. Compound 4, 2-[3-(isopropylamino)-2-hydroxypropoxylxanthone hydrochloride salt and 2,5 dihydroxyxanthone suppressed the secondary aggregation induced by adrenaline in human PRP. We conclude that the antiplatelet effects of these compounds are mainly due to an inhibitory effect on thromboxane formation.     

低分子肝素的抗血栓作用

研究了低分子肝素 (lowmoleculerweightheparin ,LMWH)的抗血栓作用 .研究表明 ,静脉注射LMWH (40 0、2 0 0IU/kg)能明显降低家兔高、中、低切比粘度 (P <0 .0 1) ,并显著降低家兔体内实验性血栓湿重及干重 (P <0 .0 0 1) ;静脉注射LMWH 40 0、2 0 0IU/kg及 30 0、15 0IU/kg能分别抑制家兔及小鼠ADP诱导的血小板聚集作用 (P <0 .0 0 1) .实验证明 ,相对分子质量为 6 40 0的低分子肝素有抗血栓作用 .     

Suppressive Effect of Recombinant TNF-gelatin Conjugate on Murine Tumour Growth In-vivo

Abstract— A recombinant human tumour necrosis factor α (TNF) was conjugated to gelatin by means of carbodiimide to improve the in-vivo stability of TNF. About 55% of TNF activity was retained after gelatin conjugation as judged by the cytotoxicity assay using L-M cells in-vitro. Intraperitoneal injection of the TNF-gelatin conjugate significantly suppressed the in-vivo growth of murine Meth A fibrosarcoma cells (SS2 cells) in mouse peritoneum, in comparison with that of free TNF at the same dosage (P < 0·05). After intraperitoneal injection, TNF activity of the conjugate was detected in both the serum and the peritoneal cavity of mice for a longer period than was free TNF, irrespective of the presence of SS2 cells. Chromatographic studies of the conjugate demonstrated that the increase in the apparent molecular weight of TNF was consistent with gelatin conjugation. It is likely that this leads to a prolonged retention of TNF activity in-vivo. In addition, the TNF-gelatin conjugate suppressed the in-vitro growth of SS2 cells to the same extent as free TNF. Thus, it is possible that the longer retention period of the conjugate brought about an increase in the chance of contact between TNF and SS2 cells, resulting in the enhanced suppressive effect of TNF on in-vivo tumour cell growth. Gelatin conjugation is an effective method for increasing the in-vivo antitumour activity of TNF.     

Design and Characterization of an Acid-Activated Antimicrobial Peptide

Dental caries is a microbial biofilm infection in which the metabolic activities of plaque bacteria result in a dramatic pH decrease and shift the demineralization/remineralization equilibrium on the tooth surface towards demineralization. In addition to causing a net loss in tooth minerals, creation of an acidic environment favors growth of acid-enduring and acid-generating species, which causes further reduction in the plaque pH. In this study, we developed a prototype antimicrobial peptide capable of achieving high activity exclusively at low environmental pH to target bacterial species like Streptococcus mutans that produce acid and thrive under the low pH conditions detrimental for tooth integrity. The features of clavanin A, a naturally occurring peptide rich in histidine and phenylalanine residues with pH-dependent antimicrobial activity, served as a design basis for these prototype ‘acid-activated peptides’ (AAPs). Employing the major cariogenic species S. mutans as a model system, the two AAPs characterized in this study exhibited a striking pH-dependent antimicrobial activity, which correlated well with the calculated charge distribution. This type of peptide represents a potential new way to combat dental caries.     

乌苏里藜芦生物碱单体新计巴丁的抗血栓作用

目的:研究乌苏里藜芦生物碱单体新计巴丁(neogermbudine,NG)的抗动、静脉血栓形成作用及初步作用机制探讨.方法:应用电刺激诱发大鼠颈总动脉血栓形成模型和淤血诱发下腔静脉血栓形成模型评价NG的抗动、静脉血栓形成作用;采用Born法测定NG在体、内外对血小板聚集的抑制活性;采用凝血酶时间法测定NG对血凝时间的影响.结果:与生理氯化钠溶液(NS)组相比,NG 4种剂量(5～40 μg·kg-1)组血管阻塞时间(OT)均显著延长,下腔静脉淤血形成血栓的干重明显降低;体内、外血小板聚集率均显著下降,凝血酶时间显著延长.上述指标均呈良好的量效关系.结论:NG具有较强的抗动、静脉血栓形成作用,该作用与NG的抗血小板作用和抗凝作用有关.     

In-vivo anti-reflux and raft properties of alginates

The comparative efficacy of two alginate-containing anti-reflux preparations (Gaviscon, Algicon) was assessed in a single blind crossover study of 20 patients with gastro-oesophageal reflux disease. The clinical efficacy study was preceded by two studies in healthy volunteers to assess the intragastric effects of Algicon and Gaviscon by pH measurement, endoscopic visualization and gamma scintigraphy. Algicon and Gaviscon were shown to form a raft in the fasting and fed human stomach, with Algicon alone having a potent antacid effect below and within the raft. Both Algicon and Gaviscon liquids significantly reduced the frequency and severity of reflux symptoms from baseline when given at their recommended doses (10 ml and 20 ml four times daily, respectively). There were no significant differences between Algicon and Gaviscon, although 12 patients preferred Algicon (vs 5 for Gaviscon) for control of reflux symptoms. It was concluded that both Algicon and Gaviscon were effective for the symptomatic control of gastro-oesophageal reflux disease.     

Ex-vivo uses and applications of cytokines for adoptive immunotherapy in cancer

Harnessing the power of the immune system to eliminate infection and cancer is a long-standing goal in clinical immunology. The development of a robust immune response to foreign antigen relies, in part, on communication between cellular components of the immune system. The proteins involved in governing the magnitude and longevity of an immune response are collectively called cytokines. Cytokines act directly on immune cells to induce proliferation, differentiation and tolerance, and signaling errors can lead to autoimmune disease or immune deficiency. Identification of the molecules involved in these signaling processes has allowed investigators to manipulate immune cells for therapeutic effect, both in vivo and ex vivo. While in vivo immune modulation using cytokines has produced some exciting results, the toxicity involved with systemic administration has limited their broad use in the clinic. Therefore, research has been focused on the ex vivo manipulation of immune cells for adoptive transfer to treat cancer and infection. This review will focus on the ex vivo manipulation of immune cells with particular emphasis on stimulating cytotoxic T cell responses. Adoptive transfer of ex vivo generated cell types may then be used to treat malignant and viral disease.     

Antithrombotic effects of KBT-3022, a novel antiplatelet agent,in an arterial thrombosis model in the guinea-pig

We examined the antithrombotic effect of KBT-3022, a novel antiplatelet agent, on photochemically induced arterial thrombosis in the guinea-pig saphenous artery, and compared its effect with that of aspirin and that of ticlopidine. Pretreatment with KBT-3022 [0.1 mg (0.2 μmole), 0.3 mg (0.7 μmole), 1 mg (2.2 μmole) and 3 mg (6.7 μmole)/kg, p.o.] 3 h prior to thrombosis induction prolonged the time required to achieve the thrombotic occlusion and inhibited the collagen (low concentration, 0.2 μg/ml; high concentration, 1 μg/ml)-induced platelet aggregation in whole blood ex vivo, each effect being concentration-dependent. The effects were tested of ticlopidine [30 mg (99.9 μmole), 100 mg (333.1 μmole) and 300 mg (999.2 μmole)/kg, p.o.] and aspirin [10 mg (55.5 μmole), 30 mg (166.5 μmole) and 100 mg (555.1 μmole)/kg, p.o.]. Both drugs prolonged the time to occlusion (but only at their highest concentration), and also inhibited the collagen (low concentration)- induced platelet aggregation in whole blood ex vivo. Aspirin [100 mg (555.1 μmole)/kg, p.o.], but not ticlopidine (at any concentration), inhibited the collagen (high concentration)-induced platelet aggregation. A good correlation was found between the antithrombotic and antiplatelet-aggregating effects of both KBT-3022 and aspirin, but not of ticlopidine. The antithrombotic potency of KBT-3022 was 300 times that of aspirin and 1000 times that of ticlopidine. These observations suggest that KBT-3022 may be clinically effective against platelet-rich thrombosis. Drug Dev. Res. 40:217–222, 1997. © 1997 Wiley-Liss, Inc.     

In-vitro and Ex-vivo Inhibition of Blood Platelet Aggregation by Naftazone

Because of the considerable interest in the role of platelets and antiplatelet therapy in cardiovascular disease, including the aggregation of platelets to each other during arterial thrombosis and atherogenesis, we have studied the effect of naftazone (Etioven), an original vasculotropic drug on platelet aggregation. Rat and human platelets were prepared and incubated in-vitro with different concentrations of naftazone. We found that naftazone inhibited both platelet secretion and aggregation in platelet-rich plasma (PRP) and washed platelets after stimulation with thrombin or ADP. Rats were also treated intraperitoneally for five days with various naftazone doses (0.125-10 mg kg^?1) and ex-vivo platelet aggregation compared, at various times after the last injection, with that of control animals. Inhibition by naftazone was dose-dependent in both PRP and isolated platelets. The inhibition was transient, a maximum value (～ 50%) being obtained about 3–6 h after the last injection, with a return to near-control values after 24 h. Naftazone also facilitated platelet deaggregation after in-vitro stimulation with thrombin or ADP. In another series of experiments, rats were treated intraperitoneally for five days with 10 mg kg^?1 of aspirin, ticlopidine, dipyridamole or naftazone. Platelets were prepared and tested for aggregation 90 min after the last injection. Thrombin-induced aggregation in PRP and washed platelets was significantly reduced after in-vivo treatment with ticlopidine and naftazone. Except for dipyridamole, all the drugs inhibited ex-vivo ADP-induced aggregation in PRP. In isolated platelet preparation, only naftazone induced a significant inhibition of ADP-or thrombin-stimulated aggregation. We conclude that naftazone inhibits platelet aggregation in-vitro and ex-vivo.     

BM—13505抗栓作用及机理研究

BM 13505是新合成的血栓烷受体拮抗剂，BM 13505 0.45和0.23mg/kg iv延长大鼠颈动脉血管阻塞时间（P＜0.001和P＜0.01）。BM 13505 2mg/kg iv有效地防止AA 4mg/kg所致的大鼠脑血栓形成（P＜0.01）。BM 13505 10mg/kg ip可防止AA诱导的小鼠肺部栓塞（P＜0.01）。该药可延长小鼠尾出血时间。BM 13505显著抑制AA诱导的兔血小板聚集，半数抑制浓度IC50为0.17μmol/L，对ADP和胶原诱导的血小板聚集无影响。BM13505降低兔血小板及小鼠血浆中TXB2水平，对血小板cAMP水平无影响，在不远的将来，BM13505可能会发展成一种理想的抗血小板药及抗血栓药。     

BM－13505抗栓作用及机理研究

ＢＭ１３５０５是新合成的血栓烷受体拮抗剂，ＢＭ１３５０５０．４５和０．２３ｍｇ／ｋｇｉｖ延长大鼠颈动脉血管阻塞时间（ｐ＜０．００１和ｐ＜０．０１）．ＢＭ１３５０５２ｍｇ／ｋｇｉｖ有效地防止ＡＡ４ｍｇ／ｋｇ所致的大鼠脑血栓形成（ｐ＜０．０１）。ＢＭ１３５０５１０ｍｇ／ｋｇｉｐ可防止ＡＡ诱导的小鼠肺部栓塞（ｐ＜０．０１）。该药可延长小鼠尾出血时间。ＢＭ１３５０５显著抑制ＡＡ诱导的兔血小板聚集，半数抑制浓度ＩＣ＿（５０）为０．１７Ｖｍｏｌ／Ｌ，对ＡＤＰ和胶原诱导的血小板聚集无影响。ＢＭ１３５０５降低兔血小板及小鼠血浆中ＴＸＢ＿２水平，对血小板ｃＡＭＰ水平无影响。在不远的将来，ＢＭ１３５０５可能会发展成一种理想的抗血小板药及抗血栓药。     

Antithrombotic and profibrinolytic activities of phloroglucinol

Phloroglucinol is the monomeric units of phlorotannins abundant in brown algae. Several biological effects of phloroglucinol have been reported, however, antithrombotic and profibrinolytic activities of phloroglucinol have not been studied yet. In this study, the anticoagulant properties of phloroglucinol were determined by assays of activated partial thromboplastin time (aPTT), prothrombin time (PT) and cell based thrombin and activated factor X (FXa) generation activities. And the effects of phloroglucinol on the expression of plasminogen activator inhibitor type 1 (PAI-1) and tissue-type plasminogen activator (t-PA) were tested in tumor necrosis factor-α (TNF-α) activated human endothelial cells (HUVECs). I found that phloroglucinol prolonged aPTT and PT significantly and inhibited thrombin and FXa generation in HUVECs. Furthermore, phloroglucinol inhibited TNF-α induced PAI-1 production. I then used pathway inhibitors to investigate which step of the TNF-α induced signaling pathway was targeted by phloroglucinol. I observed that the c-Jun N-terminal kinase (JNK) inhibitor increased the inhibitory effects of phloroglucinol, whereas the nuclear factor factor-κB (NF-κB) and the extracellular signal-regulated kinase (ERK) inhibitor did not. Therefore these results suggest that phloroglucinol possess antithrombotic and profibrinolytic activities and that NF-κB and ERK pathways are possible targets of phloroglucinol in the regulation of TNF-α stimulated PAI-1 production in HUVECs.     

On the Cellular Uptake and Membrane Effect of the Multifunctional Peptide,TatLK15

Recently, the multifunctional peptide TatLK15 resulting from the fusion of the cell penetrating peptide Tat and the amphipathic peptide LK15 was shown to be efficient at mediating siRNA and shRNA delivery in leukemia cells to silence the bcr-abl oncoprotein. The present study focused on TatLK15 peptide cellular uptake and defining conditions for its use within a range of doses exhibiting minimal toxicity. The initial part of the study carried out in solution confirmed that the insertion of a glycine bridge allowed retention of the LK15 α-helicity, and fluorescence correlation spectroscopy did not reveal preferential conformations at the studied concentrations. In the second part, TatLK15 uptake mechanisms appeared peptide dose- and cell line- dependent as well as requiring membrane potential. Below a critical dose, TatLK15 toxicity appeared limited for approximately three hours as demonstrated by the combined use of lactate dehydrogenase release, MTT assays, and time-dependent observation of membrane-impermeant dye uptake using high content screening apparatus. Furthermore, toxicity was observed to occur rapidly at higher peptide doses. Finally, a comparison between TatLK15 and another Tat amphipathic peptide construct suggested that α-helix content should be viewed as a key element in the development of similar peptides. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:293–304, 2014     

RGD肽对TPTX大鼠给予外源PTH_（1-34）后血钙水平的影响

目的探讨精氨酸-甘氨酸-天门冬氨酸（RGD）序列肽对TPTX低钙模型大鼠给予外源PTH后血钙水平的影响。方法雄性SD大鼠于麻醉下行TPTX术,建立低钙模型,16只造模成功按体重随机分为NS组和RGD组,分别予PTH1-34联合生理盐水和PTH1-34联合人工合成RGD序列肽（RGDY）持续输注6 h,监测血钙水平的动态变化。结果两组大鼠TPTX术后24 h血钙水平均降低至基线水平的70%左右,提示TPTX大鼠低钙血症模型建立。给予外源PTH后RGD组血钙水平恢复且显著低于NS组（F=16.465,P〈0.05）。结论 RGDY阻抑了TPTX大鼠给予甲状旁腺激素后血钙的上升,RGD序列肽可能具有抑制破骨细胞骨吸收的功能,作为防治代谢性骨病的新型药物开发值得关注。