Diagnosis of deep vein thrombosis using autologous indium-III-labelled platelets.

Forty-eight patients who had undergone surgical reduction of a fractured neck of femur or in whom deep vein thrombosis was suspected clinically were studied by ascending phlebography and imaging after injection of autologous indium-111-labelled platelets to assess the accuracy and value of the radioisotopic technique in diagnosing deep vein thrombosis. Imaging was performed with a wide-field gammacamera linked with data display facilities. Phlebography showed thrombi in 26 out of 54 limbs examined and a thrombus in the inferior vena cava of one patient; imaging the labelled platelets showed the thrombi in 24 of the 26 limbs and the thrombus in the inferior vena cava. The accumulation of indium-111 at sites corresponding to those at which venous thrombi have been shown phlebographically indicates that this radioisotopic technique is a useful addition to methods already available for the detection of deep vein thrombosis.     

Platelets and leukocytes in the lungs after acute hypobaric hypoxia

To determine if decompression from sea level causes aggregation and embolization of platelets or leukocytes to the lungs, we have measured the accumulation of 51Cr-labeled platelets or 111In-labeled leukocytes in the lungs of rabbits decompressed to 440 or 350 Torr for 18 or 40 h. To be certain that any increased accumulation of labeled platelets (or leukocytes) in the lungs was not just caused by an increased pulmonary blood volume we also labeled the rabbits red blood cells with 59Fe. There was no detectable accumulation of labeled platelets in the lungs on decompression. In control animals there were 22 times as many labeled leukocytes in the lungs as could be accounted for by the volume of blood in the lungs. In experimental animals at 326 Torr for 18 h this figure was reduced to 13.6. Hypobaric hypoxia caused an increase in circulating granulocytes from a mean of 3.3 +/- 1.6 X 10(9)/l to 5.3 +/- 2.1 X 10(9)/l. (P less than 0.005). Our results suggest that decompressions to 6,100 m for 18 h does not cause platelet sequestration in the lungs but does cause a significant reduction in leukocytes in the lungs and a peripheral granulocytosis.     

Immunocytochemical localization of platelets in baboon hepatic sinusoids using monoclonal mouse anti-human platelet glycoprotein IIIa following induction of thrombocytopenia.

A commercially available mouse monoclonal antibody to human platelet glycoprotein IIIa was used to demonstrate sequestration of platelets in hepatic biopsies obtained from baboons following intravenous infusion of echistatin, a novel fibrinogen receptor antagonist derived from the venom of the snake Echis carinatus. Biopsies of liver and spleen were taken prior to administration of echistatin. The hepatic biopsies were either snap-frozen in Freon-22/liquid nitrogen or fixed in 10% neutral buffered formalin. Biopsies of spleen were snap-frozen. During infusion of echistatin (2.3 micrograms/kg/min), circulating platelet counts decreased from 331,000/mm3 to 167,000/mm3. Selective sequestration within the liver was confirmed using whole body gamma camera imaging to demonstrate 111Indium-oxine labeled platelet accumulation within the liver during the thrombocytopenic episode. Hepatic biopsies were again taken and either snap-frozen in Freon-22/liquid nitrogen or fixed in 10% neutral buffered formalin. Biopsies of spleen and inguinal lymph node were also snap-frozen. Platelet rich plasma smears, included as positive controls, dewaxed paraffin sections, and cryosections of liver, spleen, and lymph node were stained with monoclonal mouse anti-human platelet glycoprotein IIIa using an avidin biotinylated peroxidase complex (ABC) technique. Prior to infusion of echistatin, platelet staining within the liver was minimal. After echistatin infusion, hepatic cryosections showed prominent platelet staining within hepatic sinusoids. No localization was shown in lymph node, however, the spleen showed prominent platelet staining both before and after echistatin infusion. Platelet rich plasma smears were intensely positive. No prominent platelet staining was observed in formalin-fixed, paraffin-embedded material. Thus, this immunocytochemical technique may help localize platelets in cryosections of tissues from baboons and other primate species.     

Cigarette Smoking and Platelet Aggregation

The influence of cigarette smoking on platelet aggregation was studied in habitual and non-habitual smokers. The results indicate that habitual smokers have a greater tendency to platelet aggregation than do non-habitual smokers. Acute effects of cigarette smoking were, however, not significant. The nucleotide content and the serotonin content of the platelets were analyzed. The adenosine nucleotide and serotonin contents were similar in smokers and non-smokers in the control state and neither showed significant changes on cigarette smoking. There were significant correlations between the control concentrations of the various nucleotides in both groups and there were even higher correlations after smoking. Platelet aggregation bore no demonstrable relationship to the nucleotide or serotonin contents of the platelet. We conclude that the long-term effect of smoking is probably more important than the acute effect.     

A simple fluorimetric microassay for adenine compounds in platelets and plasma and its application to studies on the platelet release reaction

1. The formation of a stable fluorescent product between chloroacetaldehyde and adenine or its derivatives provides the basis of a rapid simple assay for total adenine compounds in blood platelets and in plasma. The assay will measure down to 200pmol of adenine nucleotides. An evaluation of the method established the optimum conditions for the production of maximum fluorescence. 2. Values obtained for total adenine compounds in platelets were 12.9nmol/10(8) cells in man and 7.8nmol/10(8) cells in rat. These closely agree with previous values for total platelet adenine nucleotides found by using a firefly luciferase assay, or a recycled NAD-linked photometric assay. This supports the concept that the chloroacetaldehyde reaction measures total adenine nucleotides in platelets. 3. Platelet aggregation induced by collagen was studied photometrically in 0.1ml volumes of citrated platelet-rich plasma, and total adenine nucleotides were assayed in platelets and plasma before and after aggregation. During aggregation 58% of adenine nucleotides were released from human platelets, and 36% from rat platelets. 4. The chloroacetaldehyde assay is no substitute for more sophisticated procedures, but is a simple sensitive means of monitoring the release of adenine nucleotides from blood platelets and is particularly valuable when small plasma samples must be used.     

Sidenafil Pre-Treatment Promotes Decompression Sickness in Rats

Vascular bubble formation after decompression contributes to endothelial injuries which form the basis for the development of decompression sickness (DCS). Nitric oxide (NO) is a powerful vasodilator that contributes to vessel homeostasis. It has been shown that NO-releasing agent may reduce bubble formation and prevent serious decompression sickness. The use of sildenafil, a well-known, phosphodiesterase-5 blocker, which act by potentiating the vasodilatory effect on smooth muscle relaxation, has never been studied in DCS. The purpose of the present study was to evaluate the clinical effects of sildenafil pre-treatment on DCS in a rat model. 67 rats were subjected to a simulated dive at 90 msw for 45 min before staged decompression. The experimental group received 10 mg/kg of sildenafil one hour before exposure (n = 35) while controls were not treated (n = 32). Clinical assessment took place over a period of 30 min after surfacing. At the end, blood samples were collected for blood cells counts and the level of circulating bubbles in the right cavities was quantified. There were significantly more manifestations of DCS in the sildenafil group than in the controls (34.3% vs 6.25%, respectively, p = 0.012). Platelet count was more reduced in treated rats than in controls (−21.7% vs −7%, respectively, p = 0.029), whereas bubble grades did not differ between groups. We concluded that pre-treatment with sildenafil promotes the onset and severity of neurological DCS. When considering the use of phosphodiesterase-5 blockers in the context of diving, careful discussion with physician should be recommended.     

Energy transduction and adenine nucleotides in mitochondria from rat liver after hypoxic perfusion.

The characteristics of mitochondria isolated from perfused livers of rats under hypoxic or oxic conditions were studied. The electron transfer activity was about 60% of normal after hypoxic perfusion for 3 h, but respiratory control was abolished almost completely. These parameters recovered considerably on subsequent oxic perfusion. The adenine nucleotide contents and their net uptake decreased in hypoxia, closely correlated with the energy transduction. Energy-dependent nicotinamide nucleotide transhydrogenase activity and NAD reduction by succinate in submitochondrial particles were most severely inhibited after hypoxic perfusion and were also correlated with adenine nucleotide contents in the particles. These results are discussed in terms of the involvement of adenine nucleotides in energy-transducing systems in mitochondrial membranes.     

Effects of ovariectomy on aggregation,secretion, and metalloproteinases in porcine platelets

Differences in the aggregation and release of growth factors including matrix metalloproteinases (MMPs) after loss of ovarian hormones could contribute to an exaggerated response to injury in arteries of ovariectomized animals. Therefore, experiments were designed to compare aggregation, dense granular ATP release, expression of MMPs (MMP-2, MMP-9, and MMP-14) and tissue inhibitors of metalloproteinase (TIMP-1 and TIMP-2) in circulating platelets from sexually mature (7 mo old) gonadally intact and ovariectomized (4 wk) female pigs. Numbers of circulating platelets did not change after ovariectomy, but the percentage of reticulated platelets increased significantly. Platelet aggregation and dense granular ATP secretion also increased significantly with ovariectomy. In platelet lysates, active MMP-2 increased, whereas MMP-14 significantly decreased, after ovariectomy; the expression of TIMP-1, TIMP-2, and P-selectin did not change. These results suggest that platelet turnover, aggregation, and ATP secretion increase with ovariectomy. Also, ovarian hormones selectively regulate the expression and activity of MMPs in porcine platelets. Increased platelet aggregation and activity of MMP-2 would alter platelet-platelet and platelet-vessel wall interactions, contributing to an exaggerated response to injury with loss of ovarian hormones.     

The adenine nucleotide translocator in foetal, suckling and adult rat liver mitochondria

The adenine nucleotide content of rat liver mitochondria was shown to increase significantly after birth. On the other hand, it was found that the ligand-binding properties of the adenine nucleotide translocator were essentially the same in foetal, suckling and adult rat liver mitochondria. These results are compatible with the proposal that the accumulation of adenine nucleotides which occurs during mitochondrial biogenesis and maturation is effected by a pathway different from the adenine nucleotide translocator.     

Leukotriene B4 increases pulmonary transvascular filtration by a neutrophil-independent mechanism

We examined the role of circulating granulocytes in the pulmonary microvascular response to leukotriene B4 (LTB4) by prior depletion of circulating granulocytes using hydroxyurea. LTB4 (2 micrograms/kg injection followed by infusion of 2 micrograms/kg over 15 min) produced transient increases in pulmonary arterial pressure and pulmonary vascular resistance, indicating that neutrophils were not required for the pulmonary hemodynamic effects of LTB4. Infusion of LTB4 in granulocyte-depleted sheep also resulted in transient increases in pulmonary lymph flow (QL) with no significant change in the lymph-to-plasma protein concentration ratio (L/P), findings similar to those in control animals. In vitro studies indicated that LTB4 (10(-7) or 10(-9) M) produced a transient adherence of neutrophils to cultured pulmonary artery endothelial monolayers. Maximal responses occurred at 10 min after the addition of LTB4 to the endothelial cell-neutrophil coculture system, and the adherence decreased to base line within 60 min. LTB4 infusion in sheep also produced a transient uptake of autologous 111In-oxine-labeled neutrophils. The results indicate that LTB4-mediated increase in pulmonary transvascular protein clearance (QL x L/P) is independent of circulating granulocytes.     

高压氧预处理对急性减压所致的大鼠肺组织细胞凋亡及Bcl-2/Bax表达的影响

目的:探讨高压氧预处理对减压病大鼠肺组织细胞凋亡的影响相关蛋白表达的影响。方法:雄性SD大鼠24只,随机分为3组,正常对照组(NC group)、HBO预处理组(HBOP group)、减压组(DCS group),每组8只。连续进行HBO预处理5天后进行减压病模型制备,取左侧肺组织进行湿干重比值测定,右侧肺组织用于病理实验;HE染色观察肺组织病理学改变,免疫组织化学法标记Bcl-2、Bax、Caspase-3与MMP-9阳性细胞表达,并对bcl-2/bax值进行分析。结果:减压组肺组织Bax、Caspase-3与MMP-9阳性细胞数明显增加(P0.05),而Bcl-2阳性细胞表达减少(P0.05);高压氧预处理组与减压组相比,Bax、Caspase-3与MMP-9阳性细胞数明显减少(P0.05),而Bcl-2阳性细胞表达增加(P0.05);大鼠肺组织减压组与高压氧预处理组Bcl-2/Bax值较对照组明显降低(P0.05);与减压组相比,高压氧预处理组明显升高(P0.05)。结论:HBO预处理可以减轻减压对肺组织的病理损伤,减轻肺泡和支气管上皮细胞的变性坏死,抑制细胞凋亡,从而起到对减压病的保护作用。     

Gray platelet syndrome: selective alpha-granule deficiency and thrombocytopenia due to increased platelet turnover

Clinical and laboratory studies of two siblings, both suffering from gray platelet syndrome (GPS) are described. The patients had a mild bleeding disorder, their platelets were blue-gray in panoptic stains, and alpha-granules were markedly reduced, as shown by electron microscopy. The platelet content of platelet factor 4 and that of beta-thromboglobulin were significantly reduced (3%-7% of normal). Platelet count was decreased (33-150 X 10(9)/1) and small platelets were increased in platelet volume distribution. Bleeding time was prolonged on most occasions. Bone marrow aspiration was performed in one patient and revealed increased reticulin fibers, however, megakaryocyte count was normal. The mean platelet survival was 4.8 days using 111indium-labelled platelets. In this patient, platelet-associated IgG was within the normal range. Prednisone therapy failed to increase platelet count. Dental surgery was performed under cover of desmopressin and no bleeding complication occurred; however, no improvement of bleeding time was observed. The patient delivered a healthy male infant without hemorrhaging while under concurrent platelet transfusion therapy.     

Adenylate energy charge of rat and human cultured hepatocytes

Summary A simple and rapid method for the assay of adenine nucleotides (ATP, ADP, and AMP) was established to evaluate the adenylate energy charge (ATP+ADP/2)/(ATP+ADP+AMP) of cultured hepatocytes. The effects of inhibitors of glycolysis, fatty acid oxidation, or oxidative phosphorylation on the energy charge were examined. The energy charges of cultured hepatocytes in rats and human were almost identical and were maintained at a high level between 6 and 24 h after changing the media (rat: 0.908±0.008n=9, human: 0.918±0.014n=6, mean ± SD). Inhibition of glycolysis with sodium fluoride or oxidative phosphorylation with antimycin A irreversibly reduced both the adenine nucleotide contents and the energy charge. However, the inhibition of fatty acid oxidation with 2-tetradecylglycidic acid did not affect the nucleotide contents, and the energy charge only decreased transiently to recover within 8 h. When the inhibitor of oxidative phosphorylation was removed, the recovery in the energy charge preceded the recovery in the adenine nucleotide contents. These findings suggest that the adenylate energy charge is a more sensitive measure of the changes in energy metabolism than the adenine nucleotide contents. Furthermore, energy charge regulates adenine nucleotide contents in cultured hepatocytes. It is important to confirm that the high energy charge of the cultured hepatocytes is maintained when these cells are used for metabolic studies.     

Immunocytochemical localization of platelets in baboon hepatic sinusoids using monoclonal mouse anti-human platelet glycoprotein IIIa following induction of thrombocytopenia

Summary A commercially available mouse monoclonal antibody to human platelet glycoprotein IIIa was used to demonstrate sequestration of platelets in hepatic biopsies obtained from baboons following intravenous infusion of echistatin, a novel fibrinogen receptor antagonist derived from the venom of the snake Echis carinatus. Biopsies of liver and spleen were taken prior to administration of echistatin. The hepatic biopsies were either snap-frozen in Freon-22/liquid nitrogen or fixed in 10% neutral buffered formalin. Biopsies of spleen were snapfrozen. During infusion of echistatin (2.3 µg/kg/min), circulating platelet counts decreased from 331000/mm³ to 167000/mm³. Selective sequestration within the liver was confirmed using whole body gamma camera imaging to demonstrate ¹¹¹Indium-oxine labeled platelet accumulation within the liver during the thrombocytopenic episode. Hepatic biopsies were again taken and either snap-frozen in Freon-22/liquid nitrogen or fixed in 10% neutral buffered formalin. Biopsies of spleen and inguinal lymph node were also snap-frozen. Platelet rich plasma smears, included as positive controls, dewaxed paraffin sections, and cryosections of liver, spleen, and lymph node were stained with monoclonal mouse anti-human platelet glycoprotein IIIa using an avidin biotinylated peroxidase complex (ABC) technique. Prior to infusion of echistatin, platelet staining within the liver was minimal. After echistatin infusion, hepatic cryosections showed prominent platelet staining within hepatic sinusoids. No localization was shown in lymph node, however, the spleen showed prominent platelet staining both before and after echistatin infusion. Platelet rich plasma smears were intensely positive. No prominent platelet staining was observed in formalin-fixed, paraffin-embedded material. Thus, this immunocytochemical technique may help localize platelets in cryosections of tissues from baboons and other primate species.     

Adenine nucleotide metabolism of blood platelets. IX. Time course of secretion and changes in energy metabolism in thrombin-treated platelets.

Changes in the energy metabolism of washed human platelets were compared with the kinetics of secretion induced by thrombin (5 units/ml). A 50% decrease in the level of metabolic ATP (3H-labelled), which was essentially complete in 30s, was matched in rate by adenine nucleotide secretion from storage in dense granules. Incubation of platelets with antimycin before thrombin addition increased the rate of fall in metabolic ATP, but did not affect the rate of adenine nucleotide secretion. beta-N-Acetylglucosaminidase secretion, which was slower than adenine nucleotide secretion in control platelets, was noticeably inhibited by antimycin, confirming previous reports that different regulatory mechanisms exist for dense and alpha-granule secretion. The rates of rephosphorylation of metabolic ADP to ATP via glycolysis and oxidative phosphorylation were estimated by measuring lactate production and O2 consumption in resting and thrombin-stimulated platelets and compared to the level of metabolic ATP (9-10 nmol/mg of platelet protein in the resting state). The rate of ATP production was stimulated at least two fold from 12 nmol to 24 nmol/min/mg within seconds of thrombin addition. This increased rate was maintained over the observed period of 5 min although the level of metabolic ATP had decreased to 4-5 nmol/mg within 30 s; the turnover of the remaining metabolic ATP thus increased four fold over the resting state although the actual stimulation of energy production was only two fold.     

内耳减压病豚鼠听力和耳蜗组织的病理变化

研究探讨了内耳减压病豚鼠皮层听觉诱发电位阈值、耳蜗火棉胶切片、酶组织化学和透射电镜观察的变化。结果表明,豚鼠内耳减压病导致听力损失,耳蜗广泛的病理损害.毛细胞琥珀酸脱氢酶活性降低。提出了加压治疗内耳减压病时配合改善微循环、增加能量供应等见解。     

高压氧预处理对减压病大鼠肺组织的影响

目的:研究高压氧预处理对减压病大鼠肺组织的影响及其意义。方法:SD大鼠30只,随机分为正常对照(CN)组,高压氧预处理(HBO)组,减压(DCS)组,减压组采用20min匀速升压至7.0ATA,停留20min使大鼠充分换气,2min内快速减压常压方案。减压24h后观察肺组织中谷胱甘肽过氧化物酶(GPx)、丙二醛(MDA)、超氧化物歧化酶(SOD)的变化;并通过HE染色观察肺组织病理学变化。结果:减压组肺泡腔不够完整,肺泡破裂融合,肺泡壁增厚,有中度炎性细胞浸润,高压氧组与减压组相比,病理改变明显减轻;与对照组相比,单纯减压使大鼠肺组织GPx、MDA升高,SOD降低,高压氧预处理组GPx、MDA降低,SOD降低升高;高压氧组与减压组相比GPx、MDA下降,SOD升高(P<0.05)。结论:高压氧预处理对减压病大鼠肺组织具有一定的保护作用。     

快速减压对豚鼠外周微循环和大脑血流量的影响

目的：探讨动物处于减压病（ＤＣＳ）临界发病状态时微及其血流动力作用的改变。方法：采用小型化激光微综在数测量仪及ＬＤＦ－３微区血流量仪,以检测动物高压暴露前及快速减压后微循环和血流动力作用的改变。结果：快速减压后动物微血管明显收缩;毛细血管开放数量减少;微循环中可见气泡并有血栓形成;白细胞、血小板与血管内皮粘附;血流中有料多白色微小血栓;细动脉血流速度平均比正常状态减慢０．９ｍｍ／ｓ,细静脉流速减慢     

Determinants of platelet kinetics: effects of pulmonary microembolism

We examined the mechanisms of platelet uptake in the lungs after alpha-thrombin-induced pulmonary microembolism. Platelets labeled with 111In-oxine were reinfused into chronically prepared sheep. Pulmonary microembolism resulted in an increase in lung platelet radioactivity (95.5 +/- 15.3%; n = 4), which was followed by an exponential washout (half-life = 115 +/- 4 min). Platelet uptake in the lungs was more sustained after prior fibrinolytic inhibition with tranexamic acid (half-life = 178 +/- 11 min), although the initial increase was similar (90.7 +/- 9.6%; n = 7). Prior depletion of fibrinogen with ancrod (Arvin), blunted the initial increase in lung platelet uptake after alpha-thrombin challenge (31.7 +/- 11.3%, n = 5), indicating that the effect of thrombin was markedly dependent on fibrinogen. We examined the role of circulating granulocytes, since platelets may bind to subendothelial matrix exposed after granulocyte-mediated lung vascular injury. Maximal pulmonary platelet uptake after thrombin in granulocytopenic sheep was not different from control (71.7 +/- 14.4%; n = 4). The results indicate that pulmonary microembolism results in lung platelet sequestration. Platelet uptake is not dependent on granulocyte-mediated vascular injury but requires fibrin deposition and is sustained if fibrinolysis is inhibited.