Olfactory discrimination learning is blocked by Leupeptin, a thiol protease inhibitor

Rats were trained on successive two-odor discriminations with the cues randomly located in an 8-arm radial maze. After several days of training using different odor pairs, the thiol protease inhibitor leupeptin was infused into the ventricles and testing continued. Leupeptin caused a pronounced, dose-dependent and reversible deficit in performance in this task. Previous studies have shown that these drug concentrations do not influence spontaneous activity, feeding and drinking, or the acquisition and retention of avoidance conditioning. The results are interpreted as supporting the hypothesis that a calcium-sensitive proteinase is involved in certain forms of memory that require modification of telencephalic circuitries.     

Influence of hormones on platelet intracellular calcium

The pathophysiology of thromboembolic disease associated with estrogen therapy is poorly understood. There are innumerable calciumdependent activities involved in platelet function. To determine whether platelet calcium levels are affected by exogenous hormones, intracellular calcium and release were studied in platelets in various hormonal environments and findings were correlated with platelet adhesion and aggregation. Platelet intracellular calcium concentration and release was significantly decreased in women ingesting tamoxifen compared to controls and significantly increased, as was platelet adhesion, in oral contraceptive users. Platelets incubated ex vivo with estradiol had increased intracellular calcium and release but there was decreased adhesion to fibronectin. Intracellular calcium concentration and release were not affected when platelets were incubated with tamoxifen. Adhesion to collagen III was increased in tamoxifen-incubated platelets. Only oral contraceptive users had increased sensitivity to aggregating agents. This data suggests that 17β estradiol, progesterone, and tamoxifen likely have a nongenomic effect on platelet intracellular calcium and calcium release and that platelet calcium levels are closely related to the degree of platelet adhesion and aggregation in vivo.     

Cleavage of fibrinogen by human platelet calcium-activated protease

In lysates of washed human platelets produced by sonication or by addition of nonionic detergent, fibrinogen (Mr 340,000) was rapidly degraded, under conditions favorable to activation of the endogenous calcium-activated protease (CAP), to a core derivative (Mr 280-290,000) composed of partially degraded A alpha chains (Mr 47,000, 46,000, and 34,000) and B beta chains (Mr 56,000), and apparently intact gamma chains (Mr 53-54,000). Extensive degradation occurred within one minute at 4 degrees C, ambient temperature or at 37 degrees C, and was inhibited by leupeptin, EDTA, EGTA, or N-Ethylmaleimide, but not by soybean trypsin inhibitor, hirudin, aprotonin, benzamidine, phenylmethylsulfonyl fluoride or epsilon-aminocaproic acid. Purified plasma fibrinogen exposed to lysates containing active protease was cleaved in an identical fashion. The cleavage pattern of A alpha chains produced by this platelet protease activity is different from that produced by plasmin in vitro or that found in fibrinogen catabolites in vivo, and is unlike that produced by any cellular fibrinolytic enzyme yet described. In view of this finding, as well as the striking differential inhibitory effect of the agents cited above, we conclude that the degradation of platelet fibrinogen observed in these studies is due to direct proteolysis by platelet CAP.     

血小板激活在氧化应激诱发小鼠短暂性脑缺血发作中的作用和替罗非班的干预作用

目的:探讨氧化应激在小鼠短暂性脑缺血发作中的作用及其病理生理学机制。方法:采用尾静脉注射过氧化物加缺氧诱发的小鼠TIA模型,通过测定血清可溶性P-选择素水平的变化和小鼠症状的出现时间、模型评分,观察血小板激活和替罗非班的干预作用。结果:模型组血清可溶性P-选择素显著高于对照组,分别为(4.19±0.17)ng/ml和(0.82±0.07)ng/ml,替罗非班组与模型组比较无显著差异(P>0.05);替罗非班组小鼠症状的出现时间明显晚于模型组,分别为(4.95±1.19)d和(3.75±1.12)d;症状评分也低于模型组(P<0.05)。结论:氧化应激通过激活血小板,进而诱发微血栓的形成导致小鼠TIA发作;血小板活化后伴随的炎症反应可能也起了一定的作用。     

Thiolprotease inhibitor, EST, can inhibit thrombin-induced platelet activation

Participation of thiolprotease in platelet activation was investigated. When platelets were treated with EST (L- -epoxysuccinyl-leucylamide (3-methyl)butane-ethyl ester, a membrane-permeable thiolprotease inhibitor) for 30 min, thrombin-induced aggregation and secretion were inhibited, and remained so even when the platelets were washed and resuspended in EST-free medium after the pretreatment. The inhibitory action of EST on thrombin-induced platelet aggregation and secretion was both dose-dependent and incubation-time-dependent. The inhibitory action of EST on platelet aggregation and secretion was shown not only in response to thrombin but also to platelet activating factor (PAF). Pretreatment of platelets with 1 mM EST for 30 min inhibited the 65 % of calpain (thiolprotease) activity in platelets. The phosphorylation of 40 kDa and 20 kDa proteins of platelets caused by stimulation with thrombin was blocked by the pretreatment with 1 mM EST. Phosphatidylinositol hydrolysis and inositol-1-phosphate production, which appear after stimulation of platelets with thrombin, were also inhibited by the pretreatment with 1 mM EST. The results suggest that EST was incorporated to inside of platelets, and inhibited activation of platelet through inhibition of thiolprotease.     

Inhibition of ethanol-induced platelet activation by agents that elevate cAMP

Ethanol activates phosphoinositide-specific phospholipase C in human platelets resulting in the mobilization of intracellular calcium and shape change (Arch. Biochem. Biophys. 260, 480–492, 1988). Preincubation of platelets with agents that increase levels of cAMP (i.e., forskolin,prostaeyclin) inhibited these responses to ethanol in a concentration- and time-dependent manner. The inhibitory effect was potentiated by the phoshpodiesterase inhibitor, isomethybutylxanthine. When added after ethanol, these agents also reversed platelet shape change and lowered cytosolic free calcium to basal levels. The results demonstrate a direct inhibitory effect of cAMP on the ethanol-induced activation of phospholipase C.     

Effect of calcium dobesilate on platelet function

Platelet-active drugs are of potential benefit in prevention of diabetic microangiopathy as well as other thromboembolic complications. The present investigation examines the effect of an angio-protective agent, calcium dobesilate (Doxium®) on platelets and suggests its mechanism of action. Calcium dobesilate reduces aggregation and the release reaction induced by thrombin and collagen in rabbit platelets. Calcium dobesilate also increases platelet cAMP levels and . The experimental results indicate that the inhibitory effect of calcium dobesilate on platelet function is mediated through the cyclic AMP pathway, probably through activation of adenyl cyclase.     

Phospholipid-induced human platelet activation: Effects of calcium channel blockers and calcium chelators

Human platelet activation (aggregation, [¹⁴C]-5HT release and TxB₂ production) induced by the phospholipids, PAF and lysophosphatidic acid (LPA) was inhibited by EGTA, TMB-8 (an intracellular calcium antagonist) and by phenylalkylamine (Class II) but not 1,4-dihydropyridine (Class I) calcium channel blockers. Primary aggregation induced by PAF was selectively inhibited by phenylalkylamine (verapamil, methoxyverapamil) calcium channel blockers. Phospholipid-induced human platelet activation depends predominantly on the influx of extracellular calcium, possibly via specific receptor-operated calcium channels.     

管状材料对血小板激活作用的体外评价

背景：ISO10993-4及GB/T 16886.4中将血液相容性的评价分为5个方面：血栓形成、凝血、血小板、补体、血液学。目前国内较为确定和成熟的体外血液相容体外评价方法有溶血试验、凝血试验及血小板黏附试验,而对血小板激活及补体系统激活方面的研究很少。目的：评价聚乙烯、聚氯乙烯及聚甲基乙烯基硅氧烷3种基础材料管在体外对血小板的激活作用,初步建立一种体外评价管状材料对血小板激活作用的方法。方法：制备聚氯乙烯管、聚乙烯管、硅橡胶管的内径3.7 mm,外径5 mm,长35 cm。每管1 mL血液注入聚乙烯、聚氯乙烯、硅橡胶管,管的两端用两通连接,置于恒温培养振荡器中,30°倾斜,接口向上,37 ℃,140 r/min,振荡3.5 h。放射免疫法检测材料与血液接触后贫血小板血浆中血小板α颗粒蛋白水平,流式细胞仪检测材料与血液接触后血液中血小板α颗粒蛋白阳性血小板百分率、活化的gpⅡb/Ⅲa复合物阳性血小板百分率。结果与结论：放射免疫法检测结果显示聚乙烯、聚氯乙烯管与血液接触后贫血小板血浆中血小板α颗粒蛋白水平大于硅橡胶管(P < 0.05)。聚乙烯和聚氯乙烯管与血液接触后贫血小板血浆中血小板α颗粒蛋白水平差异无显著性意义(P > 0.05)。流式细胞术检测结果显示聚乙烯、聚氯乙烯管与血液接触后血液中血小板α颗粒蛋白阳性血小板百分率大于硅橡胶管(P < 0.05),聚乙烯管与血液接触后血液中血小板α颗粒蛋白阳性血小板百分率大于聚氯乙烯管(P < 0.05)。3种材料与血液接触后血液中活化的gpⅡb/Ⅲa复合物阳性血小板百分率差异无显著性意义(P > 0.05)。实验初步建立一种管材料与血液较为合理的接触方式,并可以考虑血浆血小板α颗粒蛋白是较好的反映血小板激活程度的评价指标。用流式细胞术检测血浆血小板α颗粒蛋白阳性血小板百分率更为敏感。关键词：管状材料;血小板激活;血浆血小板α颗粒蛋白;血液相容性;生物材料doi:10.3969/j.issn.1673-8225.2010.08.006     

Evidence for an alternative mechanism of human platelet secretion involving peripheralization of secretory granules and formation of membrane-associated multivesicular structures

Stimulated platelets release the content of their granules to the environment by a process known as platelet secretion. The precise mechanism of platelet secretion is poorly understood. The most widely-held theory suggests that, during platelet activation, secretory granules centralize and their content is released into the surface-connected canalicular system. Using fixation techniques directed at preserving membrane structures, morphological evidence is provided that human platelet activation is associated with secretory granules migrating to the periphery of platelets, where they undergo transition to participate in the formation of membrane-associated multivesicular structures. These multivesicular structures then undergo dissolution resulting in granule content secretion to the environment. The formation of the membrane-associated multivesicular structures occurs in platelets in which some of the secretory granules have also become centralized. This suggests that during the activation of human platelets, membrane-associated multivesicular structure release may occur concurrently with other mechanisms of secretory granule content release. The present observations are consistent, however, with the hypothesis that during human platelet activation the formation of membrane-associated multivesicular structures by platelet secretory granules contributes importantly to the mechanism of platelet secretion.     

Two forms of Ca++-activated neutral protease in platelets

Bovine thrombin is more sensitive than human thrombin in detecting plasma heparin levels, and this is due to the more rapid neutralization of the bovine thrombin by antithrombin III heparin complex. The peptidase activities are qualitatively similar to the coagulant activities, when the thrombins are compared using the synthetic substrate S 2238.     

Nicotinic depolarization activates calcium dependent gK in myenteric neurons

Intracellular recordings were made from S type neurons in the myenteric plexus of the guinea-pig ileum. Acetylcholine (ACh) was applied directly onto the soma membrane by iontophoresis with visual placement of the iontophoretic pipette. Fast nicotinic depolarizations were evoked which were followed by slow muscarinic depolarizations. After application of hyoscine to block the slow muscarinic depolarization, the nicotinic depolarization was found to be followed by a hyperpolarization. This hyperpolarization was several mV in amplitude and 1-5 s in total duration. It disappeared when the preceding nicotinic depolarization was blocked by hexamethonium. The ACh induced by hyperpolarization was due to calcium entry and subsequent opening of potassium channels.     

An endogenous inhibitor of calcium-activated neutral protease in UMX 7.1 hamster dystrophy

An endogenous inhibitor for calcium-activated neutral protease (CANP) from skeletal and cardiac muscles of muscular dystrophic hamsters (UMX 7.1) was compared with that from normal control animals at 4 and 10 weeks of age by Western blotting using antibody raised against CANP inhibitor. Fragmented CANP inhibitor was found in dystrophic skeletal muscles in all cases at both ages, while only intact inhibitor was detected in the skeletal muscle of the normal hamsters. A total absence of intact inhibitor was shown in one 10-week-old dystrophic hamster. In contrast, there was little difference in CANP inhibitor from heart between dystrophic and control hamsters at 4 weeks. However, fragmentation similar to that in skeletal muscle was seen in the heart inhibitor in a few of the 10-week-old dystrophic hamsters.     

Influence of lipids metabolism on platelet activation in vivo

Platelet aggregability and plasma factor VIII-related antigen (F. VIIIR:AG) level in 16 ischemic heart disease (IHD) patients were increased by isometric exercise and these changes were prevented by administration of a lipid lowering agent, simfibrate, a derivative of clofibrate. Serum total cholesterol (TC) level decreased and the high density lipoprotein-cholesterol (HDL-C)/TC ratio increased with the treatment. Another 7 hyperlipidemics were administered with simfibrate. Platelet malondialdehyde (MDA) production decreased with improvement in lipid profile. In an in vitro study, platelet aggregability and the plasma level of von Willebrand factor (vWF) and F.VIIIR:AG of normal citrated blood were increased by passing it through a glass bead column. Combining above results of the three separate studies, it would be suggested that hyperlipidemia might enhance platelet activation in vivo, which occurred through contact of platelets to atherosclerotic rough vessel surface. The anti-platelet effect of simfibrate might be mediated through its effect on arachidonic pathway in platelets.     

Lack of thrombopoietin potentiation of platelet collagen activation in the first trimester is associated with preeclampsia

To determine whether a difference exists in platelet reactivity to collagen and potentiation by thrombopoietin (TPO) between preeclamptic and non-preeclamptic patients, 187 first trimester pregnant patients were prospectively followed through pregnancy. Citrated blood, drawn at first (<14 weeks estimated gestational age) and third trimesters (>28 weeks), when patients were admitted for delivery, and 4-6 weeks postpartum, was assayed by a Whole Blood Impedance Aggregometer measuring platelet activation by 0.4 mug/ml collagen, +/-10 ng/ml TPO. There was no significant difference in 1st trimester platelet collagen activation by unpaired t-test between groups. Significant TPO potentiation of collagen activation (P<0.05, paired t-test) was observed in non-preeclamptic patients at the first and third trimesters. In contrast, preeclamptic patients' platelets show no significant (P>0.8, paired t-test) TPO potentiation at any time. While the mechanism for this difference in thrombopoietin potentiation of platelet activation by collagen as early as the first trimester is unknown, it may be one of the initiating events in this syndrome.     

Actinoidin: a new inhibitor of ristocetin- and ristomycin-induced platelet agglutination

Actinoidin, like vancomycin, inhibits ristocetin-and ristomycin-induced VIIIR:WF-dependent agglutination of untreated or formaldehyde-fixed human platelets but does not interfere with bovine factor VIII-induced agglutination. The socalled “direct effect” of ristocetin and ristomycin on fixed platelets, which is represented by an immediate increase in light absorbancy in the absence of cofactor, was not blocked by actinoidin at concentrations which totally inhibited agglutination in the presence of VIIIR:WF. Actinoidin does not inhibit thrombin-induced platelet aggregation. This represents a further argument against a common platelet membrane receptor site for thrombin and ristocetin or a ristocetin-VIIIR:WF complex on the platelet membrane.     

急性脑梗死患者血小板钙含量的变化

目的 研究急性脑梗死患者血小板钙含量的改变,探讨前、后循环区脑梗死与血小板钙含量的关系.方法 采用流式细胞仪检测87例急性脑梗死患者(前循环区脑梗死40例、后循环区脑梗死47例)及20名健康体检者(正常对照组)外周血血小板钙含量,比较前循环区与后循环区脑梗死患者及与正常对照组的血小板钙含量.结果 急性脑梗死组血小板钙含量显著高于正常对照组(P＜0.01);前循环梗死组血小板钙含量显著高于后循环梗死组(P＜0.05).结论 急性脑梗死患者血小板钙含量显著增高,尤以前循环区脑梗死更明显.     

Effect of cetiedil on platelet aggregation and thromboxane synthesis

Cetiedil was found to inhibit platelet aggregation and thromboxane synthesis induced by thrombin and arachidonic acid. When platelets were activated by thrombin, half maximal inhibition (ED₅₀ effective dose of cetiedil necessary for 50% inhibition) for platelet aggregation was 100 μM while that for thromboxane B₂ (TXB₂) production was 50 pM. When arachidonic acid was used, the ED₅₀ for platelet aggregation was 100 μM while that for TXB₂ production was 150 μM. The presence of calcium ions did not affect on the inhibitory effects of cetiedil. The cAMP level in platelets did not increase after incubation with cetiedil. Cetiedil appears to inhibit the activation of platelets related to thromboxane synthesis.     

Factors influencing shear-induced platelet alterations: platelet lysis is independent of platelet aggregation and release

To gain further insight into the mechanisms involved in fluid shear-induced platelet alterations, we examined conditions and factors that might affect shear-induced platelet reactions in human citrated platelet-rich plasma (C-PRP) under well-defined laminar flow conditions at shear stresses between 0 and 160 dyn/cm² using a Couette rotational viscometer. Prevention of excessive alkalinization of C-PRP during shear due to CO₂ loss did not appreciably affect shear-induced platelet aggregation (PAG), adenine nucleotide (AN) release or platelet lysis. Shear-induced PAG and AN release were significantly greater in C-PRP stored and sheared at 24°C as compared to C-PRP stored at 24°C or 37°C and sheared at 37°C whereas platelet lysis was not affected by temperature. When C-PRP was sheared in the presence of EDTA or EGTA, shear-induced PAG up to shear stresses of 80 dyn/cm² was almost completely suppressed whereas AN release and lysis were unaffected. Exposure of C-PRP to PGE₁ and theophylline before shear virtually abolished shear-induced PAG and AN release at shear stresses up to 80 dyn/cm² but had no demonstrable effect on shear-induced platelet lysis. These findings seem to indicate that ADP released from platelets by shear and extracellular Ca⁺⁺ or in the presence of PGE₁ and theophylline. These findings seem to indicate that the structural and biochemical changes associated with shear-induced PAG and release in our system do not predispose platelets to shear-induced lysis.