A modification of a method of evaluating the coagulation activity of factor VIII

The authors suggest that factor VIII activity be calculated relative to the value of activated partial thromboplastin time of substrate hemophilic plasma. This value is determined by replacing the tested plasma in the reaction mixture with veronal-acetate buffer. This modification has been developed to eliminate the shortcomings of the single-step technique for measuring this factor activity, that are explained by the use of nonstandard donor plasma and by the necessity to have such plasma during the investigation. A formula has been derived for the calculation of factor VIII coagulation activity in hemophilic patients and the advantages of the suggested modification are demonstrated.     

Characteristics of beta 2-adrenergic regulation of gastric secretion, hemostasis and cardiac rhythm in patients with duodenal ulcer and a history of hemorrhagic complications (effect of alupent)

The effect of intravenous infusion of alupent (0.1 microgram/kg/min for 1 h) on the main parameters of gastric secretion, thromboelastogram, electrocoagulogram, ADP-induced platelet aggregation, the kinetics of fibrinolysis and vegetative regulation of the cardiac rhythm was studied in 59 subjects (10 healthy males, 23 patients suffering from peptic ulcer of the duodenum with a history of hemorrhages, 26 patients with uncomplicated duodenal ulcer). In healthy subjects and in patients with uncomplicated ulcer, the beta 2-adrenoagonist caused an increase in the volume of basal secretion, production of acid and pepsin, acceleration of blood coagulation, and fibrinolysis activation. At the same time in 2/3 of patients with a history of hemorrhages due to duodenal ulcer alupent inhibited gastric secretion of acid and pepsin and, along with stimulation of plasmic factors of coagulation, inhibited activated fibrinolysis. Vegetative dysfunction in patients with uncomplicated ulcer was marked by the predominance of the parasympathetic regulation of the cardiac rhythm, whereas in patients with a history of hemorrhages by sympatheticotonia. These features of beta 2-adrenergic regulation can be used for predicting hemorrhages and development of pathogenetically validated treatment methods.     

Plasma lecithin: cholesterol acyltransferase activity in liver disease

Abstract. In liver disease the proportion of plasma cholesterol present in the form of ester is lower than that found in normal subjects. Recent work has suggested that a plasma enzyme, lecithin: cholesterol acyltransferase (LCAT), may be a major f actorin the physiological regulation of plasma cholesterol ester levels. In patients with a variety of hepatobiliary disorders LCAT activity was found to be reduced and a study of the effects of interaction between normal and jaundiced plasmas supported the hypothesis that the low LCAT activity was due mainly to a reduction in the plasma concentration of the enzyme. When bile salts were added to an in vitro system clear evidence of inhibition of LCAT was produced only with concentrations higher than those normally found in the plasma of patients with liver disease. This casts doubt on the suggested role of bile salts as in vivo inhibitors of the enzyme. The cholesterol ester concentration of plasma showed good correlation with its LCAT activity when this was measured in a standard substrate. Our results suggest that reduction in LCAT activity may be an important factor in the production of the low ester: free ratio found in almost all hepatic disorders.     

Effect of anticoagulants on the plasma hyaluronidase activities

Mammalian hyaluronidases (HAases) are an endo‐β‐N‐acetyl‐hexosaminidases that degrade hyaluronan (HA) and have been implicated in diverse pathophysiological functions. Several pathological conditions, such as diabetes, monoclonal gammapathy, and bladder and prostate tumors, report the distorted plasma HAase activity. However, the plasma HAase (hHyal‐1) activity has been presumed to change with the circulating HA level and serves as an early marker for several diseases. It has been generally practised to use the anticoagulants such as tri‐sodium citrate/di‐sodium EDTA/heparin for the preparation of plasma for both biochemical and clinical analyses. In the present investigation, theeffect of anticoagulants on plasma HAaseactivity was evaluated and compared with the serum HAase activity that is devoid of anticoagulants as no study provides information in this regard. The results suggested that the plasma HAase activity in the presence of the recommended concentration of EDTA was highly comparable/similar to that of the serum HAase activity. In contrast, citrated or heparinized plasma recorded a significantly reduced level of activity than that of the serum HAase activity. In conclusion, our results suggested that the EDTA‐treated plasma samples are a better choice compared with heparin and citrated samples to assess the HAase activity. J. Clin. Lab. Anal. 23:29–33, 2009. © 2009 Wiley‐Liss, Inc.     

Antioxidant systems of erythrocytes in rheumatism

Patients with inactive and active rheumatic fever were examined for the intensity of lipid peroxidation of red blood cell membranes, lipid antioxidant system of tocopherol, non-lipid system of glutathione and related total activity of the pentosophosphate pathway of carbohydrate metabolism in red blood cells. It was established that activation of rheumatic process is accompanied by a increase in the content of diene conjugates in lipids of red blood cell membranes, a reduction of the tocopherol content in blood plasma, activation of glutathione reductase and glucose-6-phosphate dehydrogenase with the lowering of the total activity of the pentose cycle in red blood cells. The characteristics under study depended to a lesser degree on circulation insufficiency. The interrelationship between the shifts revealed is discussed, the mechanism by which they develop is suggested.     

Plasma somatomedin activity in vitamin A deficient children.

A role for vitamin A in the sulphation of mucopolysaccharides has been suggested. Somatomedin, a growth hormone dependent serum factor, has also been shown to stimulate the uptake of sulphate by cartilage. Therefore studies were undertaken on vitamin A deficient children to examine the possible interrelationship between vitamin A and somatomedin activity. Plasma somatomedin activity was markedly lowered in vitamin A deficient children and plasma HGH levels were in the normal range. The data suggest that vitamin A and somatomedin may be interrelated and also that plasma somatomedin activity may not always be determined by plasma growth hormone levels.     

A method of determining heparin in blood plasma based on its antithrombin activity]

The suggested method for measuring blood plasma heparin is based on heparin ability to enhance antithrombin activity of antithrombin III (AT-III), the major Xa and thrombin inhibitor. The method consists in measurement of blood plasma AT-III activity in the presence and absence of protamine sulfate that destroys the heparin--AT-III complex. Heparin content in U/ml is determined from the difference in the activities of heparin--AT-III complex and AT-III proper activity represented on the calibration curve. The method is sufficiently sensitive, it permits registration of heparin concentrations in a wide band (from 0.01 U/ml to 0.75 U/ml of plasma).     

乳腺癌患者血浆多药耐药基因谷胱甘肽巯基转移酶活性分析及其临床应用

目的：通过对乳腺癌患者血浆样本中多药耐药基因谷胱甘肽巯基转移酶（GST）活性检测,掌握治疗过程中耐药信息的变化,为临床治疗提供参考依据。方法：采用生化反应方法,将血浆样本与化学试剂加在一起混匀,根据反应前后透光率的不同,计算每个样本GST活性。结果：治疗前GST活性平均为（842±606）nmol·min^-1·mL^-1,化疗3个周期后GST活性平均为（796±587）nmol·min^-1·mL^-1。有淋巴结转移与无淋巴结转移者相比较,治疗前GST活性差异有统计学意义（P〈0．05）。结论：乳腺癌患者血浆GST活性测定,对乳腺癌化疗用药选择及预后具有参考价值。     

The effect of heparin in vitro on human plasm post-heparin lipolytic activity.

Human plasma post-heparin lipolytic activity was reduced when collected in tubes containing lithium heparin as compared with samples collected in lithium sequestrene. The addition of heparin in vitro to samples abolished the inhibitory effect of protamine sulphate on plasma post-heparin lipolytic activity. It is suggested that heparin should not be used as an anticoagulant for the collection of samples for assay of post-heparin lipolytic activity.     

Plasma Renin Activity in Diabetic Autonomic Neuropathy

Postural changes in plasma renin activity were studied in three groups of age and duration-matched male diabetics (potent, impotent and with postural hypotension) and in non-diabetic control subjects. Those diabetic subjects with postural hypotension due to automatic neuropathy had no increase in plasma renin activity to the erect posture whereas both the potent and impotent groups had similar plasma renin activity responses to the control subjects. There was a significant inverse correlation between the rise in plasma renin activity on standing and the postural drop in blood pressure (r = 0. 476, P < 0.01) but no correlation with other tests of autonomic reflex function such as the Valsalva manoeuvre and blood pressure response to sustained handgrip. The results suggested that the lesion responsible for the postural hypotension is in the efferent sympathetic pathway. However, neuropathy per se did not wholly explain the decreased postural plasma renin activity response. Diabetic nephropathy, with involvement of cells of juxtaglomerular apparatus, may also be implicated.     

A kinetic micromethod of determining the activity of antithrombin III

A method for measuring the blood plasma antithrombin III (AT III) activity is suggested, based on measurement of the rate of inactivation of added standard volume (0.05 ml) of plasma thrombin AT III. The thrombin residual activity in the mixture is detectable over the course of fibrinogen tests. Curves are plotted, reflecting the rate of inactivation of thrombin AT III in the tested and reference plasma samples; by modeling and comparing these curves the values of the examined sample AT III activity are obtained, with the effects of fibrinogen and fast antithrombins various levels eliminated.     

异丙酚对肝缺血/再灌注损伤时黄嘌呤氧化酶活性的影响

目的　探讨异丙酚对肝缺血 /再灌注损伤 (HIRI)时黄嘌呤氧化酶 (XO)活性的影响。方法　选择HIRI实验兔及肝癌手术患者 ,动态观察血浆XO活性的变化及异丙酚对它的影响。结果　HIRI期间 ,血浆及肝组织内XO活性明显增强(P<0 0 5和 P<0 0 1) ;使用异丙酚后 ,XO活性的异常变化显著减轻 ,其差异有显著性意义 (P <0 0 5和 P<0 0 1)。结论　异丙酚可有效地抑制HIRI时XO活性而减少氧自由基的形成。     

Plasma and peripheral leukocyte beta-N-acetylhexosaminidase isoenzymes and disease activity in rheumatoid arthritis

OBJECTIVES: Recently it has been suggested that serum beta-N-acetylhexosaminidase (Hex) could be a joint destruction marker in rheumatoid arthritis (RA) patients. However, a large amount of serum Hex activity has its source from platelets, and the blood platelet-count is often increased in RA, which may have masked the significance of the results. The purpose of this study was to investigate the relationship between plasma activity of Hex and disease activity or severity. DESIGN AND METHODS: In 51 patients with RA, with an evolution period for the illness of 10.9 +/- 1.2 yr (range 1-40 yr), we determined the total Hex activity together with its Hex A and B isoenzymes in plasma and in mononuclear (MN) and polymorphonuclear (PMN) leukocytes. RESULTS: The plasma activity of total Hex and Hex B isoenzyme was slightly higher in the group of patients studied (p < 0.01), together with the specific activity of total Hex, Hex A and B in PMN leukocytes (p < 0.001) than in the control group. No significant correlation was found between plasma or leukocyte Hex and the radiologic evaluation of the disease (Sharp's modified method), or the patient's functional capacity (modified Health Assessment Questionnaire). Likewise, a significant correlation between Hex activity and laboratory inflammation markers (C reactive protein, sialic acid, erythrocyte sedimentation rate) or the evolution time of the disease was not found. CONCLUSIONS: The plasma activity of total Hex, or even of its isoenzymes Hex A and Hex B, does not appear to be a reliable marker of erosion and cartilage degradation in RA patients. Liver function appears to be the major determinant for the plasma Hex activity in these patients.     

A novel anticoagulant activity assay of tissue factor pathway inhibitor I (TFPI).

Tissue factor (TF) pathway inhibitor I (TFPI) is the physiological inhibitor of TF-induced blood coagulation. Circulating blood contains full-length TFPI and TFPI truncated at the C-terminal end. Previous studies have shown that full-length TFPI exerts a stronger anticoagulant effect on diluted prothrombin time (DPT) than truncated TFPI, and it has been suggested that full-length TFPI is biologically more important in vivo. The objective of this study was to develop and validate an assay of TFPI anticoagulant activity. TFPI anticoagulant activity was assayed using a modified DPT assay. Plasmas were incubated in the absence and the presence of TFPI-blocking antibodies. Results were expressed as a ratio with the clotting time in the presence of anti-TFPI as the denominator. The ratio was normalized against a ratio obtained with a reference plasma. The assay was compared with assays of TFPI free antigen, total antigen, and bound TFPI, and TFPI chromogenic substrate activity. We performed all tests in 436 healthy individuals. The normalized TFPI anticoagulant ratio was strongly associated with TFPI free antigen (r = 0.73) but was weakly associated with TFPI chromogenic substrate activity (r = 0.46), TFPI total antigen (r = 0.48), and bound TFPI (r = 0.30). TFPI chromogenic substrate activity was strongly associated with TFPI total antigen (r = 0.73). We have developed a novel assay of TFPI anticoagulant activity in plasma, which may be considered a functional assay of full-length TFPI. Further studies are needed to establish the role of TFPI anticoagulant activity for thrombotic disorders.     

Low postheparin plasma hepatic lipase activity in familial type IIa hyperlipoproteinemia.

A subnormal activity of postheparin plasma hepatic lipase was demonstrated in nine of 16 patients with familial type II hypercholesterolemia. On the other hand, in patients with combined hyperlipidemia (type II b) the hepatic lipase activity was mostly in upper normal range. The postheparin plasma lipoprotein lipase activity was normal in both patient groups. It is suggested that the low hepatic lipase activity may have a role in the patholgenesis of one form of familial hypercholesterolemia.     

Oral melphalan pharmacokinetics: influence of interferon-induced fever

The influence of interferon-induced fever on oral melphalan pharmacokinetics has been studied in 10 myeloma patients in a randomized crossover design. The melphalan dose (0.25 mg/kg) was given alone and 5 hours after the administration of human interferon alpha (7 x 10(6) IU/m2), respectively. The plasma concentration of melphalan was determined by liquid chromatography with fluorometric detection after derivatization of melphalan with N-acetylcysteine. The area under the plasma concentration-time curve (AUC) was significantly lower (p = 0.02) when melphalan was given with interferon. There was a significant negative correlation (p = 0.008) between body temperature and dose normalized AUC, whereas no effect was noticed on the maximum plasma concentration (Cmax) and on the time to obtain Cmax. The rate of elimination showed a tendency (p = 0.06) to increase with increasing body temperature. It is suggested that the cytotoxicity of the drug is most probably enhanced because of the higher alkylating activity of the compound at elevated body temperatures.     

Activation of hageman factor by collagen

Purified acid-soluble and insoluble human collagen accelerated the clotting of plateletpoor plasma in silicone-treated tubes. The clot-promoting effect did not appear to be due to thromboplastic activity since the collagen preparations did not activate factor X in the presence of factor VII and calcium. Instead, collagen appeared to accelerate clotting by activating Hageman factor (factor XII) on the basis of the following findings: collagen increased the clot-promoting activity of partially purified Hageman factor but exerted no further effect in the presence of kaolin, a known activator of Hageman factor; clot-promoting eluates were obtained from collagen exposed to normal, hemophilic, or PTC-deficient plasma but not from collagen exposed to Hageman or PTA-deficient plasma. The collagen molecule itself appeared to be required for the clot-promoting activity since digestion with collagenase or thermal denaturation at pH 2.5 (about 35 degrees C) resulted in very marked reduction in clot-promoting activity. Since thermal denaturation is associated with transformation of collagen structure from triple helical to random coil form, it is suggested that the native form of collagen is essential for the ability to activate Hageman factor.Blockage of the free amino groups by treatment with nitrous acid or dinitrofluorobenzene only slightly reduced the clot-promoting activity of collagen. In contrast, since addition of cationic proteins to collagen markedly reduced pro-coagulant activity it is suggested that negatively charged sites on the collagen molecule are critical for Hageman factor activation. This suggestion is supported by the finding that pepsin treatment of collagen, which removes the predominantly negatively charged telopeptides, results in significant decrease in coagulant activity. Esterification of collagen, which neutralizes 80-90% of the free carboxyl groups, reduced coagulant activity by over 90% and it is suggested that the free carboxyl groups of glutamic and aspartic acids provide the negatively charged sites critical for Hageman factor activation.     

The effect of indomethacin and other anti-inflammatory drugs on the renin-angiotensin system.

The administration of two different doses of indomethacin, 9 and 18 mg/kg, to two different groups of rabbits was followed 6 h later by a significant decrease in plasma renin activity, and these levels were not increased by hemorrhage. The administration of 2 mg/kg of indomethacin did not alter the basal levels of plasma renin activity, but it was effective in diminishing the peripheral increase of renin produced by hemorrhage. Similar effects were obtained in other groups of rabbits treated with 9 mg/kg of meclofenamate or 18 mg or aspirin. The lowering effect of indomethacin on plasma renin activity is not specifically related to hemorrhage because it also prevented the increase in plasma renin activity elicited by 5 mg/kg of furosemide. Further studies showed that indomethacin did not exert any significant effect in vivo on the plasma level of renin substrate or on the generation of angiotensin from normal plasma by exogenous renin. And indomethacin did not interfere with the binding capacity of anti-angiotensin I for angiotensin I in the radioimmunoassay reaction or with the in vitro formation of angiotensin from hog renin-nephrectomized rabbit plasma reaction. The results thus indicate that the lowering effect of indomethacin on plasma renin activity is due to the interference with renal renin release. That this effect may be related to the blockade of prostaglandin synthesis is suggested by the similar effect exhibited by other blockers of prostaglandin synthesis.     

High microparticle concentration in cord plasma

We investigated if differences in the microparticle concentration and activity between newborn cord plasma and adult plasma exist. Methods: To enumerate and characterize microparticles (MP) FACS and ELISA were used.The effect of microparticles derived tissue factor (TF) on thrombin generation was measured indirectly by CAT (calibrated automated thrombography). Results: The flow cytometric measurements revealed an increased microparticle concentration in newborn cord compared with adult plasma. By the use of ELISA a significantly increased procoagulant activity of microparticles was found in newborn cord plasma as compared to adult plasma. Initiation of thrombin generation by adding phospholipids alone resulted in a significant lower prolongation of the lag time, time to peak in cord plasma, while the decrease of endogenous thrombin potential (ETP) and peak was comparable between newborns and adults. Conclusion: Our results show a higher impact of microparticles on the haemostatic system of newborns than on that of adults. The three methods suggest a somewhat increased microparticle activity in newborn cord plasma, but argue against strong platelet activation during birth.     

Effects of platelets and platelet-derived material on the activated partial thromboplastin time (Cephotest) coagulation test

OBJECTIVE: The Cephotest is an activated partial thromboplastin time (APTT) test used to measure the activity of the intrinsic pathway of coagulation. To perform this test, blood is usually centrifuged to obtain plasma that is almost without erythrocytes and leucocytes and with only a minimal amount of platelets. MATERIAL AND METHODS: In the present experiments blood was centrifuged at different speeds to produce either platelet-poor plasma (PPP) or platelet-rich plasma (PRP). PPP and PRP obtained from the same whole blood samples from each of several persons were tested in pairs using the standard Cephotest reagent to observe the consequences of Cephotest being performed on plasma containing platelets. The same procedure was used with a Cephotest reagent with a reduced concentration of phosphatidylserine. In succeeding experiments the PRP was preincubated with SFLLRN or Ca-ionophore to activate the platelets, a procedure also known to produce platelet-derived microparticles. PPP and PRP were compared by thrombin time and reptilase time tests to find out at which stage platelets might influence the Cephotest. RESULTS: The results showed that the platelets did influence Cephotest when using both the regular reagent and the phospholipid-reduced agent. When using the regular reagent, PRP showed a tendency towards a longer APTT than PPP. It is suggested that this was caused by platelets consuming some of the first traces of thrombin generated. CONCLUSIONS: When using the phospholipid-reduced reagent, PRP showed a shorter APTT than PPP, probably because the platelets contributed phosphatidylserine to the system. When the platelets were activated before testing, their effects on the tests were increased. Microparticles that formed during platelet activation may have contributed to these effects.