Potent platelet aggregation inhibitor from Trimeresurus gramineus snake venom

Using DEAE-Sephadex A-50 column chromatography and gel filtration, a potent platelet aggregation inhibitor from Trimeresurus gramineus venom was purified. It was an acidic phospholipase a, rich in aspartic acid, glutamic acid and half-cystine, with an isoelectric point of 3.6. At a concentration of 10 μg/ml, the purified inhibitor showed a marked inhibitory effect on platelet aggregations induced by adenosine diphosphate, collagen, sodium arachidonate and ionophore A-23187 in rabbit platelet-rich plasma, washed platelet suspension, as well as in thrombin-degranulated platelet suspension. The ID₅₀ of this venom inhibitor was about 2.5–5 μg/ml in platelet aggregations induced by all these aggregation inducers. The action of this inhibitor could be partially antagonized by phosphatidylethanolamine. High concentration of Ca²⁺ (5 mM) did not reverse the inhibitory action even in the presence of ionophore A-238187. The [¹⁴C]serotonin release induced by sodium arachidonate and thrombin was unaffected. Malonic dialdehyde formation induced by these aggregation inducers remained unchanged. Basal and prostaglandin E₁-stimulated cAMP levels were not altered by this inhibitor. No lactate dehydrogenase was released even at a concentration of 62.5 μg/ml. Polylysine-induced platelet agglutination was not affected. β-Mercaptoethanol inactivated both its phospholiase A enzymatic and platelet inhibitory activities, while p-bromophenacyl bromide only inactivated the former activity. The possibility of acting on a common final step of platelet aggregation, i.e. the intercellular adhesion between the activated platelets, was proposed.     

Characterization of a potent platelet aggregation inhibitor from Agkistrodon rhodostoma snake venom

By means of CM-Sephadex C-50 column chromatography and gel filtration on Sephadex G-75 and G-50 columns, a potent platelet aggregation inhibitor was purified and characterized. It was a glycoprotein with a molecular weight of 31,000. It was devoid of phospholipase A, ADPase, esterase and fibrino(geno)lytic activities. It inhibited dose-dependently the aggregation of washed platelets induced by collagen, thrombin, sodium arachidonate, platelet activating factor and ionophore A23187 with a similar IC50 (5-10 micrograms/ml). It was also active in platelet-rich plasma, with an IC50 of 10-15 micrograms/ml. The venom inhibitor reduced the elasticity of whole blood clot and inhibited the thrombin-induced clot retraction of platelet-rich plasma. These activities were related to its inhibitory activity on platelet aggregation rather than blood coagulation. The venom inhibitor had various effects on [14C]serotonin release stimulated by aggregation agonists. It had no effect on thromboxane B2 formation of platelets stimulated by sodium arachidonate, collagen and ionophore A23187. The presence of this venom inhibitor prior to the initiation of aggregation was a prerequisite for the maintenance of its maximal activity. It showed a similar inhibitory effect on collagen or thrombin-induced aggregation even when it was added after the platelets had undergone the shape change. High fibrinogen levels partially antagonized its activity. The venom inhibitor completely inhibited the fibrinogen-induced aggregation of alpha-chymotrypsin-treated platelets. It is concluded that this venom inhibitor interferes with the interaction of fibrinogen with fibrinogen receptors, leading to inhibition of aggregation.     

Role of platelet endothelial form of nitric oxide synthase in collagen-platelet interaction: regulation by phosphorylation

Different pathways have been reported to be involved in platelet-collagen interaction. We have reported that the platelet endothelial form of nitric oxide synthase (eNOS) and the platelet receptor for type I collagen, p65, are closely associated. But the controlling mechanism underlying the generation of nitric oxide (NO) by the eNOS has not been fully explored. In this investigation, Western blot analyses of time course samples with anti-phosphorylated tyrosine, and anti-serine/threonine showed a marked increase in serine/threonine phosphorylation of eNOS during type I collagen-induced platelet aggregation. Meanwhile, the eNOS activity measured by the conversion of [3H]-arginine to [3H]-citrulline is significantly decreased. Correlation of type I collagen-induced platelet aggregation and the activity of eNOS in the presence of the serine/threonine phosphatase inhibitor, okadiac acid and the tyrosine phosphatase inhibitor, vanadate were performed with PRP. Results show the decrease in eNOS activity by adding okadiac acid correlated with the inhibitory effect on platelet aggregation in a dose-dependent manner. On the other hand, vanadate significantly inhibits platelet aggregation and also inhibits eNOS activity when the concentration of vanadate is greater than 2 mM. These results suggest that phosphorylation of serine/threonine and tyrosine residues control the activity of eNOS through different mechanisms to affect collagen-induced platelet aggregation.     

Boophilus microplus: its saliva contains microphilin, a small thrombin inhibitor

Saliva of the cattle tick Boophilus microplus contains two thrombin inhibitors, BmAP and microphilin. This work presents the purification and characterization of microphilin. It was purified from the saliva by gel filtration, ultrafiltration through a 3 kDa cut-off membrane and affinity chromatography in a thrombin-Sepharose column. Analysis by mass spectrometry showed a molecular mass of 1770 Da. Microphilin is the smallest salivary thrombin inhibitor peptide known to date. It inhibits fibrinocoagulation and thrombin-induced platelet aggregation with an IC(50) of 5.5 microM, is temperature resistant and its inhibitory activity was abolished by protease K treatment. Microphilin did not inhibit the amidolytic activity of the enzyme upon a small chromogenic substrate, but inhibited the hydrolysis of a substrate that binds both catalytic site and exosite I. Therefore, we propose that microphilin blocks thrombin at exosite I.     

Aggretin, a C-type lectin protein, induces platelet aggregation via integrin alpha(2)beta(1) and GPIb in a phosphatidylinositol 3-kinase independent pathway.

Aggretin purified from Calloselasma rhodostoma venom was previously identified as alpha(2)beta(1) agonist in triggering platelet aggregation, and exists as a heterodimer sharing a great homologous sequence to GPIb binding proteins. We show here that binding to GPIb is also required in aggregation-inducing activity of aggretin. A2-IIE10, an anti-integrin alpha(2) monoclonal antibody, delayed platelet aggregation while agkistin, a GPIb antagonist, only slightly inhibited platelet aggregation caused by aggretin. However, the aggretin-induced platelet aggregation was completely abolished by a combination of A2-IIE10 and agkistin. Either A2-IIE10 or agkistin significantly inhibited the binding of FITC-aggretin toward fixed platelets. Aggretin and collagen induced a similar signal transduction in platelets involving a time-dependent tyrosine phosphorylation of p125(FAK) and PLCgamma2, but aggretin caused a much-delayed tyrosine-phosphorylation of PI 3-kinase compared with collagen. LY294002, a PI 3-kinase inhibitor, showed a significant inhibitory effect on collagen, but not aggretin-stimulated platelet aggregation. These findings indicate aggretin induces platelet aggregation via binding of alpha(2)beta(1) and GPIb, causing phosphorylation of p125(FAK) and PLCgamma2 leading to platelet activation without the involvement of PI 3-kinase activation.     

A potent platelet aggregation inducer from Trimeresurus gramineus snake venom

A potent platelet aggregation inducer (platelet aggregoserpentin) was purified from Trimeresurus gramineus snake venom by DEAE-Sephadex A-50 and Sephacryl S-300 column chromatography. It was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It elicited dose-dependently platelet aggregation and serotonin release reaction in rabbit platelet-rich plasma and platelet suspension. Exogenous calcium was required for its activity. Creatine phosphate/creatine phosphokinase and apyrase showed no significant inhibitory effect on aggregoserpentin-induced platelet aggregation in platelet suspension. Aggregoserpentin induced aggregation in ADP-refractory platelet-rich plasma. It caused no detectable malonic dialdehyde formation in the process of platelet aggregation. Indomethacin did not inhibit aggregoserpentin-induced platelet aggregation. Mepacrine abolished preferentially its aggregating activity, while prostaglandin E1 completely blocked both aggregoserpentin-induced aggregation and release reaction. Furthermore, platelet aggregoserpentin lowered basal and prostaglandin E1-stimulated cAMP levels in platelet suspension. Nitroprusside inhibited both its aggregating and releasing activity, while verapamil preferentially blocked its aggregating activity. It is concluded that aggregoserpentin activated platelets through lowering cAMP levels or the activation of endogenous phospholipase A2, resulting in the formation of platelet activating factor, but not of prostaglandins.     

氨基酸和霉菌水提物对深层发酵桦褐孔菌菌丝体黄酮积累及其抗氧化活性的影响

黄酮类化合物是桦褐孔菌菌丝体中多酚类化合物的重要组成部分,也是该菌治疗众多疾病的有效成分之一。然而人工培养桦褐孔菌黄酮等酚类化合物积累甚少,导致药理活性的明显下降。为此,我们研究了3种氨基酸和4种霉菌水提物对深层发酵桦褐孔菌黄酮的积累及其抗氧化能力的影响。在所试验的3种氨基酸和4种霉菌水提物中,L-酪氨酸,黄曲霉和毛霉水提物能有效地增加该菌黄酮的积累。人工培养菌体中的黄酮至少由4种黄酮苷组成,苷元分别是槲皮素、柚皮素、山奈酚和异鼠李素。深层发酵菌丝体具有一定的抗氧化能力,并与总黄酮的含量呈正相关。由L-酪氨酸,黄曲霉和毛霉水提物调控生长的桦褐孔菌菌丝体,能有效地清除超氧阴离子、羟基自由基和DPPH自由基。     

An inhibitor selective for collagen-stimulated platelet aggregation from the salivary glands of hard tick Haemaphysalis longicornis and its mechanism of action

Soluble materials of salivary glands from Haemaphysalis longicornis were found to inhibit collagen, ADP, and thrombin-stimulated platelet aggregation. One inhibitory component was purified to salivary gland homogeneity by a combination of gel filtration, ion-exchange, and C_8 reverse phase HPLC. The purified activity, named longieornin, is a protein of moleeular weight 16 000 on SDS-PAGE under both reduced and nonredueed conditions. Collagen-mediated aggregation of platelets in plasma and of washed platelets (IC_(50) was approximately 60 nmol/L) was inhibited with the same efficacy. No inhibition of aggregation stimulated by other effeetors, including ADP, arachidonic acid, thrombin, ristocetin, calcium ionophore A23187, thromboxane A2 mimetic U46619 and 12-O-phorbol-13-myristate acetate, was observed. Longieonin had no effect on platelet adhension to collagen. Not only platelet aggregation but also release reaction, and increase of intraeellar Ca~(2 ) level of platelets in response to collagen were com     

Purification and functional characterization of AAV1, a novel P-III metalloproteinase, from Formosan Agkistrodon acutus venom

AAV1, an alkaline glycoprotein (GP), was purified from Agkistrodon acutus venom by two chromatographic steps on successive DEAE-Sephadex A-50 and Superdex 75 FPLC columns. AAV1 on SDS-PAGE under non-reducing conditions migrated as a monomeric and a polymeric forms with apparent molecular mass of 57 and 180 kDa, respectively. Upon reduction, it appeared as a single broad band with a mass of 50.3 kDa corresponding to the size of a typical P-III metalloproteinase acurhagin. The N-terminal sequence of an autoproteolytical 30 kDa-fragment of AAV1 showed a high homology to that of venom proteins with Metalloproteinase, Disintegrin-like, and Cysteine-rich (MDC) domains. Although it was devoid of cleaving activity toward gelatin, fibronectin and prothrombin, AAV1 preferentially digested the Aalpha chain of fibrinogen and followed by the Bbeta chain, leading to the inhibition of fibrinogen-induced platelet aggregation in elastase-treated human platelets. However, the proteolytic activity of AAV1 was completely inactivated by the chelating agent but not serine proteinase inhibitor. Furthermore, AAV1 could concentration-dependently inhibit platelet aggregation and suppress tyrosine phosphorylation of intracellular proteins in collagen- and convulxin-stimulated platelets, respectively. The interaction of MDC domains in AAV1 molecule with platelet GPVI was responsible for the inhibitory effect of AAV1 on collagen- and convulxin-induced platelet aggregation. Taken together, these pieces of evidence suggest that AAV1 from Formosan viper venom belongs to a new member of high-molecular mass metalloproteinase family and functions as a GPVI antagonist.     

A potent platelet aggregation inducer from Trimeresurus gramineus snake venom

A potent platelet aggregation inducer (platelet aggregoserpentin) was purified from Trimeresurus gramineus snake venom by DEAE-Sephadex A-50 and Sepharyl S-300 column chromatography. It was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It elicited dose-dependently platelet aggregation and serotonin release action in rabbit platelet suspension. Exogenous calcium was required for its activity. Creatine phosphate/creatine phosphokinase and apyrase showed no significant inhibitory effect on aggregoserpentin-induced platelet aggregation in platelet suspension. Aggregoserpentin induced aggregation in ADP-refractory platelet-rich plasma. It caused no detectable molonic dialdehyde formation in the process of platelet aggregation. Indomethacin did not inhibit aggregoserpentin-induced platelet aggregation. Mepacrine abolished preferentially its aggregating activity, while prostaglandin E₁ completely blocked both aggregoserpentine-induced aggregation and release reaction. Furthermore, platelet aggregoserpentine lowered basal and prostaglandin E₁-stimulated cAMP levels in platelet suspension. Nitroprusside inhibited both its aggregating and releasing activity, while verapamil preferentially blocked its aggregating activity. It is concluded that aggregoserpentin activated platelets through lowering cAMP levels or the activation of endogenous phospholipase A₂, resulting in the formation of platelet activating factor, but not of prostaglandins.     

Platelet-collagen interaction. Inhibition by a monoclonal antibody raised against collagen receptor

Monoclonal antibodies to the purified platelet type I collagen receptor were produced to study platelet receptor function. The antibody specifically reacted with the platelet receptor in immunoblot experiments. The IgG purified from the monoclonal antibodies and isolated Fab' fragments inhibited the binding of radiolabeled alpha 1(I) chain to washed platelets competitively. Soluble and fibrillar type I collagen-induced platelet aggregations were inhibited by purified IgG suggesting that soluble and fibrillar collagens shared a common receptor. The adhesion of platelets to an artificial collagen matrix was also inhibited by the monoclonal antibody. However, adenosine diphosphate-induced platelet aggregation was not inhibited by the same amount of IgG that inhibited collagen-induced platelet aggregation. The results suggest that collagen-induced platelet aggregation is mediated through the interaction of collagen with the platelet receptor.     

Inhibitory effect of sulfur-containing compounds in Scorodocarpus borneensis Becc. on the aggregation of rabbit platelets

The inhibitory effects of three pure compounds isolated from wood garlic, 2,4,5-trithiahexane (I), 2,4,5,7-tetrathiaoctane (II), and 2,4,5,7-tetrathiaoctane 2,2-dioxide (III), on rabbit platelet aggregation induced by collagen, arachidonic acid, U46619, ADP (adenosine 5'-diphosphate), PAF (platelet aggregating factor), and thrombin were studied in vitro. The anti-aggregating activity of 2,4,5,7-tetrathiaoctane 4,4-dioxide (IV) was also measured with collagen and arachidonic acid. I, II, III, and IV inhibited the platelet aggregation induced by all tested agonists. I, II, and III exhibited a stronger inhibitory effect against the thrombin-induced aggregation of GFP (gel-filtered platelets) than against the aggregation induced by the other agonists. Notably, the IC50 value for III was 4 microM, which is approximately 2.5 times stronger than MATS (methyl allyl trisulfide), a major anti-platelet compound isolated from garlic. In inhibiting collagen-induced aggregation, II was as potent as MATS and aspirin, with a marked disaggregation effect on the secondary aggregation by arachidonic acid, at the rate of 47.05%/min at a concentration of 10(-4) M. I, II, and III also suppressed U46619-induced aggregation. These results suggest that sulfur-containing compounds in wood garlic not only inhibit arachidonic acid metabolism but also suppress aggregation in association with the function of the platelet plasma membrane.     

桦褐孔菌三萜类物质的提取与含量测定

以桦褐孔菌发酵菌丝体为材料,通过对溶剂乙醇(95%)、甲醇、乙酸乙酯、丙酮、异丙醇、正己烷和氯仿的提取效果比较表明,提取三萜类化合物的最佳溶剂为异丙醇,提取时间为24h,每个样品所需溶剂量(6mL)和菌丝体样品量(100mg)较少,并可同时对大量样品进行有效提取。以白桦脂醇为标准品,对香草醛-冰醋酸-高氯酸分光光度法进行评价,证明该方法简单快速、准确度高、试验误差小、重复性好。利用此方法对桦褐孔菌的野生菌核和发酵菌丝体内三萜类化合物含量进行测定,结果表明野生菌核(59.86mg/g)和发酵菌丝体(94.92mg/g)中都含有很高的三萜类化合物,且发酵菌丝体中三萜类化合物含量高于野生菌核。因此在桦褐孔菌产品开发中,发酵菌丝体可代替野生菌核进行大工业化生产。     

Characterization of the platelet aggregation inducer and inhibitor isolated from Crocus sativus

Bulbs of Crocus sativus variety Cartwrightianus were found to contain both a platelet aggregation inducer and inhibitor. The aggregating factor has a Mr of 42 kDa estimated by a Sephadex G75 column and SDS-polyacrylamide slab gel electrophoresis. It was found to lack enzymatic activity such as proteinase, esterase and acid or alkaline phosphatase. The inhibitory factor was also purified to homogeneity by different chromatographic techniques and shows a Mr of 27 kDa as it was estimated by Biogel P30 column and SDS-polyacrylamide slab gel electrophoresis. It was found to possess strong proteinase activity.     

Anti-cancer effect and structural characterization of endo-polysaccharide from cultivated mycelia of Inonotus obliquus

The endo-polysaccharide extracted from mycelia of Inonotus obliquus (Pers.:Fr.) Pil. (Hymenochaetaceae) is a specific activator of B cells and macrophages. However, the in vivo anti-cancer effects and the chemical structure of the endo-polysaccharide are unknown. We purified the endo-polysaccharide, investigated its anti-cancer effects via in vitro and in vivo assays, and performed a structural characterization. The endo-polysaccharide was extracted from I. obliquus mycelia cultivated in a 300-l pilot fermenter, followed by hot water extraction and ethanol precipitation. Purification was achieved by DEAE-cellulose ion-exchange chromatography and gel-permeation chromatography. Chemical analysis revealed that the purified endo-polysaccharide is an alpha-linked fucoglucomannan with a molecular weight of approximately 1,000 kDa. The anti-cancer activities of the endo-polysaccharide against various types of tumor cells were determined. No direct toxicity against either cancer or normal cells was observed. Intraperitoneal administration of the endo-polysaccharide significantly prolonged the survival rate of B16F10-implanted mice, resulting in a 4.07-fold increase in the survival rate at a dose of 30 mg/kg/day. After 60 days of feeding, approximately 67% of the initial number of mice survived with no tumor incidence based on macroscopic examination. These results indicate that the anti-cancer effect of endo-polysaccharide is not directly tumorcidal but rather is immuno-stimulating.     

Folin-Ciocalteu比色法测定桦褐孔菌多酚的条件优化

以没食子酸为标准品,通过正交试验和单因素实验研究了Folin-Ciocalteu 比色法测定桦褐孔菌中多酚含量的适宜条件。结果表明,优化后的显色条件为Folin-Ciocalteu试剂用量0.3mL、10% Na2CO3溶液0.6mL,25℃时避光反应30min,于750nm处测定其吸光值。多酚质量浓度在0.0032－0.0256mg/mL范围内与吸光值有良好的线性关系。根据拟合的线性回归方程对桦褐孔菌多酚进行定量测定,野生桦褐孔菌和人工培养桦褐孔菌中多酚含量最高分别为（72.05±0.08）mg/g、（52.76±0.06）mg/g（n=6）。不同加标水平的加标回收率测定实验获得的平均回收率为98.95%,RSD为1.27%。该法测定桦褐孔菌多酚具有快速、准确,稳定性强,重现性好,精密度高的特点,可应用于实际样品的测定。     

Prostacyclin (PGI2) inhibits the development in human platelets of ADP and arachidonic acid-induced shape change and procoagulant activity

Platelets which change shape from discs to spheres concomitantly develop platelet procoagulant activity which is independent of and precedes aggregation or the release reaction. Since prostacyclin (PGI₂) is known to be potent inhibitor of platelet aggregation and releae, the effect of PGI₂ on platelet shape change and the development of platelet procoagulant activity was measured. Platelet shape change (percent discs and spheres) was assayed by a light transmission technique. Platelet procoagulant activity was assayed using recalcified clotting times measured concurrently (by aggregometry) with platelet shape assays. PGI₂ inhibited the development of platelet shape change and procoagulant activity induced by the addition of ADP (0.7 μM); the 50% inhibitory dose of PGI₂ was 2 nM. PGI₂ also inhibited arachidonic acid (0.3–1.2 mM) induced platelet shape change and procoagulant activity; the 50% inhibitory dose of PGI₂ was 2.3 nM. Thus, physiologic concentrations of PGI₂ inhibit platelet shape change and prevent the development of sphering associated procoagulant activity.     

Phospholipase A(2) with platelet aggregation inhibitor activity from Austrelaps superbus venom: protein purification and cDNA cloning

Four phospholipase A(2) (PLA(2)) enzymes (Superbins a, b, c, and d) with varying platelet aggregation inhibitor activities have been purified from Austrelaps superbus by a combination of gel filtration, ion-exchange, and reversed-phase high-pressure liquid chromatography. Purity and homogeneity of the superbins have been confirmed by high-performance capillary zone electrophoresis and mass spectrometry. The electron spray ionization mass spectrometry data showed that their molecular masses range from 13,140 to 13,236 Da. Each of the proteins has been found to be basic and exhibit varying degrees of PLA(2) activity. They also displayed different platelet aggregation inhibitory activities. Superbin a was found to possess the most potent inhibitory activity with an IC(50) of 9.0 nM, whereas Superbin d was found to be least effective with an IC(50) of 3.0 microM. Superbins b and c were moderately effective with IC(50) values of 0.05 and 0.5 microM, respectively. The amino-terminal sequencing confirmed the identity of these superbins. cDNA cloning resulted in the identification of 17 more PLA(2) isoforms in A. superbus venom. It has also provided complete information on the precursor PLA(2). The precursor PLA(2) contained a 27-amino-acid signal peptide and 117- to 125-amino-acid PLA(2) (molecular mass ranging from 13,000 to 14,000 Da). Two of these PLA(2) enzymes resembled more closely (87%) Superbin a in structure. Two unique PLA(2) enzymes containing an extra pancreatic loop also have been identified among the isoforms.     

Anti-platelet activity of a three-finger toxin (3FTx) from Indian monocled cobra (Naja kaouthia) venom

A low molecular weight anti-platelet peptide (6.9 kDa) has been purified from Naja kaouthia venom and was named KT-6.9. MALDI-TOF/TOF mass spectrometry analysis revealed the homology of KT-6.9 peptide sequence with many three finger toxin family members. KT-6.9 inhibited human platelet aggregation process in a dose dependent manner. It has inhibited ADP, thrombin and arachidonic acid induced platelet aggregation process in dose dependent manner, but did not inhibit collagen and ristocetin induced platelet aggregation. Strong inhibition (70%) of the ADP induced platelet aggregation by KT-6.9 suggests competition with ADP for its receptors on platelet surface. Anti-platelet activity of KT-6.9 was found to be 25 times stronger than that of anti-platelet drug clopidogrel. Binding of KT-6.9 to platelet surface was confirmed by surface plasma resonance analysis using BIAcore X100. Binding was also observed by a modified sandwich ELISA method using anti-KT-6.9 antibodies. KT-6.9 is probably the first 3FTx from Indian monocled cobra venom reported as a platelet aggregation inhibitor.     

Inhibition of platelet aggregation by S-(1,2-dicarboxyethyl)glutathione, intrinsic tripeptide in liver, heart, and lens

S-(1,2-Dicarboxyethyl)glutathione (DCE-GS) found in animal tissues or baker's yeast showed strong inhibitory effects on blood coagulation and platelet aggregation. The inhibitory effect of blood coagulation was almost the same as those of EDTA, oxalate, and citrate. DCE-GS did not show chelating activity. As for ADP- or thrombin-induced platelet aggregations, DCE-GS exerted a potent effect on the secondary aggregation, while it was less active in the primary aggregation. DCE-GS gave a distinct lag period in the time course of the secondary aggregation induced by collagen and inhibited most strongly the aggregation induced by arachidonic acid compared with those elicited by ADP, thrombin, and collagen. The peptide, however, did not inhibit the platelet aggregation induced by 12-O-tetradecanoylphorbol-13-acetate. Although both DCE-GS and EDTA inhibited the platelet aggregation which was triggered by ADP, their inhibitory manners were entirely different.