Activity In Human Plasma Research Articles

In vitro nerve-growth-promoting activity of human plasma alpha 1-acid glycoprotein.

Human plasma alpha 1-acid glycoprotein or orosomucoid (OR), and its derivatives, prepared by sequential enzymatic cleavage of the carbohydrate units, were tested for their nerve-growth-promoting activities with explants of whole dorsal root ganglia from chick embryos. The results showed that the OR derivatives with terminal galactose, N-acetylglucosamine, or mannose have marked neurite-promoting activities. These preparations at a concentration of 100 micrograms/ml are equivalent to 5% fetal bovine serum (protein concentration 3,000 micrograms/ml) in their ability to elicit extensive neurite outgrowth and collateral branching. The asialo-OR, or ASOR, is the most potent form: its activity is estimated to be 20 times higher than that of transferrin and 100 times over that of fibronectin; it is approximately 1/1,500 that of NGF. The neurite-promoting activity of OR is independent of the non-neuronal cells and their products and can be blocked by a specific antiserum against OR. The mode of action of OR on the in vitro nerve growth is discussed and the pathophysiological significance of this plasma glycoprotein is considered in light of data from recent clinical and pathological studies.

Monoclonal antibodies to the Mr 74,000 cholesteryl ester transfer protein neutralize all of the cholesteryl ester and triglyceride transfer activities in human plasma.

A cholesteryl ester transfer protein (CETP) of apparent Mr 74,000 has recently been purified from human plasma. Three monoclonal neutralizing antibodies to the CETP were obtained by immunizing mice with purified CETP. The antibodies, each recognizing a similar epitope on CETP, caused parallel and complete immunotitration of plasma cholesteryl ester and triglyceride transfer activities but only partial inhibition of phospholipid transfer activity. Monoclonal immunoaffinity chromatography of plasma or its fractions showed complete removal of cholesteryl ester and triglyceride transfer activities but incomplete removal of phospholipid transfer activity. Sodium dodecyl sulfate gel electrophoresis and immunoblotting of the immunoaffinity-retained fractions showed that only the Mr 74,000 protein was immunoreactive. The results suggest that the previously characterized CETP accounts for all of the cholesteryl ester and triglyceride transfer activity in human plasma but only part of the phospholipid transfer activity.

339 Comparison of methods for measuring plasminogen activator inhibitor (PAI-1) activity in human plasma

339 Comparison of methods for measuring plasminogen activator inhibitor (PAI-1) activity in human plasma

Induction of Fibrinolysis by Polyanions in Human Plasma

The fibrinolytic potency of several polyanions was comparatively investigated. Fibrinolytic activity was measured in a whole plasma assay using H-D-Val-Leu-Lys-pNA (S-2251) as chromogenic substrate and by a fibrin plate assay using plasminogen rich fibrin plates. In the chromogenic substrate assay potent fibrinolytic polyanions comprised dextran sulfate, GAGPS, pentosan polysulfate, polyanethol sulfate, l-carrageenan and i-carrageenan. Chondroitin sulfates A, B, C, keratan sulfate, ribonucleic acid, k-carrageenan and heparin were weakly fibrinolytic. Hyaluronic acid and lipopolysaccharide from E. coli were inactive. Similar results were obtained when fibrinolytic activity was measured by a fibrin plate assay. All polyanions except lipopolysaccharide produced lysis zones. Induction of fibrinolytic activity in human plasma was shown to be at least partially dependent on Hageman factor. In factor XII deficient plasma no fibrinolysis was induced by any of the polyanions when measured in the fibrin plate assay. In the chromogenic substrate assay corn Hageman factor inhibitor (CHFI) inhibited the activation of S-2251 cleaving enzyme by GAGPS, pentosan polysulfate, polyanethol sulfate, heparin, and ribonucleic acid near completely. The activation by dextran sulfate was inhibited by 45%. Heparin, pentosan polysulfate and GAGPS, three polyanions of therapeutic interest were separately compared. In both assays GAGPS proved the most potent activator, while pentosan polysulfate exhibited 83% and 44% and heparin 32% and 14% of GAGPS fibrinolytic activity in the chromogenic substrate test and the fibrin plate assay, respectively.

130 Activated protein C-induced increase of tissue plasminogen activator (tPA) activity in human plasma: Evidence for the quenching of tPA-inhibitor (PAI-1) and for the interaction with tPA-PAI complexes

130 Activated protein C-induced increase of tissue plasminogen activator (tPA) activity in human plasma: Evidence for the quenching of tPA-inhibitor (PAI-1) and for the interaction with tPA-PAI complexes

Megakaryocyte colony growth-supporting activities in human plasma: modification by platelets and platelet membranes.

Human megakaryocyte colonies are grown in methylcellulose with platelet-poor plasma and medium conditioned by phytohemagglutinin-stimulated leukocytes (PHA-LCM) as a source of megakaryocyte colony stimulating factor (MEG-CSF). The megakaryocyte colony growth-supporting activity in human plasma can be absorbed by intact platelets or degranulated platelet membranes. It was possible to recover the activity by solubilizing platelet membranes with cholic acid. Filtration of the solubilized platelet membrane preparations through a Sephadex G-100 column yielded at least two activity peaks. The molecular weight of these two activities differs from that of the growth-promoting activity in PHA-LCM.

Characterization of a lectin in human plasma analogous to bovine conglutinin.

The structural characteristics of a human plasma protein analogous to bovine conglutinin were studied. The protein was previously found to bind to complement-reacted IgG in a calcium-dependent and N-acetyl-D-glucosamine-inhibitable manner and it further shows cross-reactivity with anti-bovine conglutinin antibody. By gel permeation chromatography the conglutinin activity in human plasma was localized to fractions containing proteins of Mr at around 700,000. The conglutinin was localized by one ELISA for antigen determinants and by another for biological activity. When analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions these fractions were shown to contain proteins of about 300,000. When human conglutinin-like protein, partially purified by affinity chromatography, was analysed unreduced by SDS-PAGE followed by western blotting, the cross-reacting anti-bovine conglutinin antibody bound to a protein with an Mr of 330,000. When the sample was reduced and alkylated before electrophoresis a band of 66,000 was immunostained. The 330,000 and 66,000 proteins were shown to be collagenase sensitive. 125I-iC3b was seen to bind to the 330,000 band when incubated with western blots of partially purified human conglutinin.

Initiation and Regulation of Fibrinolysis in Human Plasma at the Plasminogen Activator Level

The initiation and regulation of fibrinolysis has been studied by reconstitution of fibrinolytic activity in human plasma in vitro. Depletion of tissue plasminogen activator (tPA) antigen by immunoadsorption of human plasma with anti-tPA Ig Sepharose 4B leads to total loss of spontaneous fibrinolytic activity determined by lysis of a thrombin-induced clot. Addition of physiological concentrations of purified tPA to tPA-depleted plasma restores fibrinolytic activity as a function of the length of time between tPA addition and clotting. Addition of free tPA to tPA-depleted plasma followed by immediate clotting results in a high rate of fibrinolysis. In contrast, when free tPA is allowed to incubate in plasma for 10 to 60 minutes prior to clot formation, the fibrinolytic activity of tPA is gradually lost. The loss of tPA-induced fibrinolytic activity in unclotted plasma is accompanied by decreased partitioning of tPA antigen into fibrin after clotting and is kinetically correlated with the formation of a 100 kilodalton (kDa) tPA complex as demonstrated by SDS-gel electrophoresis and fibrin-agar zymography. These results suggest that free tPA is susceptible to complexation by the plasma inhibitor in the absence of a clot. Fibrin formation renders tPA relatively inaccessible to inhibition. The tPA antigen isolated from stored plasma consists mainly of 100 kDa activity in SDS-gel electrophoresis and zymography, indicating that the tPA complex is resistant to dissociation by SDS. Upon rezymography of the sliced gel, only a 60 kDa tPA activity is found, suggesting that the activity at 100 kDa is at least partly due to free tPA dissociated from the complex during the first zymography. Conversion of tPA complex to enzymatically active free tPA also occurs with brief SDS exposure followed by incubation in the presence of excess Triton X-100 or by hydroxylamine treatment. These results reconcile the apparent discrepancy of the 100 kDA inhibitor-tPA complex manifesting plasminogen activation activity during zymography. The plasma tPA-inhibitor complex is precipitated strongly by antisera against plasminogen activator inhibitors (PAIs) of human Hep G2 hepatoma and HT-1080 fibrosarcoma cells and weakly by antiserum against bovine aortic endothelial cell PAI but not by antiserum against a placental PAI (PAI-2) suggesting that the plasma inhibitor is immunologically related to Hep G2, HT-1080 and possibly endothedial cell PAIs. Based on the above findings, a simple model for the initiation and regulation of plasma fibrinolysis at the PA level has been formulated.

Initiation and regulation of fibrinolysis in human plasma at the plasminogen activator level

The initiation and regulation of fibrinolysis has been studied by reconstitution of fibrinolytic activity in human plasma in vitro. Depletion of tissue plasminogen activator (tPA) antigen by immunoadsorption of human plasma with anti-tPA Ig Sepharose 4B leads to total loss of spontaneous fibrinolytic activity determined by lysis of a thrombin-induced clot. Addition of physiological concentrations of purified tPA to tPA-depleted plasma restores fibrinolytic activity as a function of the length of time between tPA addition and clotting. Addition of free tPA to tPA-depleted plasma followed by immediate clotting results in a high rate of fibrinolysis. In contrast, when free tPA is allowed to incubate in plasma for 10 to 60 minutes prior to clot formation, the fibrinolytic activity of tPA is gradually lost. The loss of tPA-induced fibrinolytic activity in unclotted plasma is accompanied by decreased partitioning of tPA antigen into fibrin after clotting and is kinetically correlated with the formation of a 100 kilodalton (kDa) tPA complex as demonstrated by SDS-gel electrophoresis and fibrin-agar zymography. These results suggest that free tPA is susceptible to complexation by the plasma inhibitor in the absence of a clot. Fibrin formation renders tPA relatively inaccessible to inhibition. The tPA antigen isolated from stored plasma consists mainly of 100 kDa activity in SDS-gel electrophoresis and zymography, indicating that the tPA complex is resistant to dissociation by SDS. Upon rezymography of the sliced gel, only a 60 kDa tPA activity is found, suggesting that the activity at 100 kDa is at least partly due to free tPA dissociated from the complex during the first zymography. Conversion of tPA complex to enzymatically active free tPA also occurs with brief SDS exposure followed by incubation in the presence of excess Triton X-100 or by hydroxylamine treatment. These results reconcile the apparent discrepancy of the 100 kDA inhibitor-tPA complex manifesting plasminogen activation activity during zymography. The plasma tPA- inhibitor complex is precipitated strongly by antisera against plasminogen activator inhibitors (PAIs) of human Hep G2 hepatoma and HT- 1080 fibrosarcoma cells and weakly by antiserum against bovine aortic endothelial cell PAI but not by antiserum against a placental PAI (PAI- 2) suggesting that the plasma inhibitor is immunologically related to Hep G2, HT-1080 and possibly endothedial cell PAIs. Based on the above findings, a simple model for the initiation and regulation of plasma fibrinolysis at the PA level has been formulated.

Murine tumor-induced platelet aggregation and coagulation: mechanisms, inhibitors, and species differences

This study examined the platelet-aggregating and procoagulant activities of two hematogenously disseminating tumors, a mouse lymphoblastic leukemia (L5178Y) and a mouse renal adenocarcinoma (RAG). Tumor-induced human platelet aggregation was inhibited by addition of the following agents to platelet-rich plasma (PRP): a calcium channel blocker (verapamil), a chelator of divalent cations (EDTA), stimulators of adenylate cyclase (2-fluoroadenosine and forskolin), and inhibitors of cAMP phosphodiesterase (oxagrelate and papaverine). The platelet aggregating activities of both cell lines were completely blocked by treatment of the cells with heat, sonication, phospholipase A 2, and Triton X-100. These data suggest that L5178Y and RAG cell-induced human platelet aggregation are dependent on a heat-labile phospholipid component of the tumor cell membrane. L5178Y cells had greater platelet-aggregating activity in human plasma than in rat or mouse plasma, whereas RAG cells had greater procoagulant activity in rat or mouse plasma than in human plasma. The procoagulant activity of RAG cells in rat and mouse plasma was demonstrated by three lines of evidence: (a) RAG cells induced heparinized PRP to clot; (b) the thrombin inhibitor DAPA lengthened of the clotting time and the lag time before aggregation; and (c) RAG cells shortened of the recalcification time of the plasma. The above data indicate that RAG cell-induced murine platelet aggregation and coagulation is dependent on thrombin generation.

Acute oral captopril inhibits angiotensin converting enzyme activity in human cerebrospinal fluid.

Angiotensin converting enzyme (ACE) activity in human plasma and cerebrospinal fluid (CSF), was measured by a fluorimetric method, following an acute oral dose (75 mg) of captopril. A decrease of approximately 60% in enzyme activity was observed in CSF, suggesting that the drug penetrates the blood/brain barrier (BBB) in sufficient amounts to inhibit the ACE in CSF. The activity of CSF enkephalinase, an enzyme present in human brain and inhibited in vitro by an elevated concentration of captopril, was, however, unaffected by the acute drug administration. It is proposed that the antihypertensive effect of captopril in humans may be due, at least in part, to the inhibition of ACE contained within brain structures.

Assay for dopamine β-hydroxylase in human plasma and rat serum by high-performance liquid chromatography with fluorimetric detection

A highly sensitive assay for dopamine β-hydroxylase activity in human plasma and rat serum by high-performance liquid chromatography with fluorimetric detection is described. Norepinephrine, formed enzymatically from the substrate dopamine, and epinephrine (internal standard), after clean-up with a cation-exchange cartridge (Toyopak IC-SP M), are converted into the corresponding fluorescent compounds by reaction with 1,2-diphenylethylenediamine. The derivatives are separated by reversed-phase chromatography on TSK gel OSD-80TM. The detection limit for norepinephrine formed enzymatically is 5 pmol per assay tube.

Activation by puff adder venom of inactive renin in normal and hypertensive rat plasma.

Inactive renin has been studied extensively in human plasma, but in animal plasma its accurate quantification has proved more difficult, due partly to higher activity of plasma protease inhibitors. Such activity in human plasma can be conveniently destroyed by a metalloprotease in Bitis arietans venom, with concommitant release of endogenous enzyme activities, such as plasma kallikrein, that then activate inactive renin. It was therefore of interest to look for inactive renin in rat and rabbit plasma using this approach, so providing, in addition, a comparison for the disparate data of other groups who have used trypsin or acid for activation. In both rat and rabbit plasma the proportion of inactive renin was 62% of total renin, whereas human plasma contained more inactive renin and a higher proportion, 82%. A higher concentration of venom was required for rat (33 ug venom/ml plasma) and rabbit (4 micrograms/ml) than needed for activation, at a similar rate, in human plasma (1 microgram/ml). When applied to studies of rats made hypertensive and hyper-reninaemic by aortic ligation for 5 days, higher total (active + inactive) renin was observed. The proportion of inactive renin, as a percentage of total renin in plasma collected at this time, was, however, found to diminish significantly. In conclusion, puff adder venom activates inactive renin in rat and rabbit plasma and can be used to study physiological changes in inactive renin in such animal plasma.

Can total prorenin in human plasma be determined by current activation methods?

A proper evaluation of the physiological significance of plasma prorenin depends on its accurate determination. However, current activation methods do not necessarily measure total prorenin, or a known proportion of it, even when carried out to apparent completion. Thus, extending cold activation of human plasma at -4 degrees C generally revealed progressive increments of prorenin, mainly during the first 15 days, but the total and the time required to achieve it varied considerably among individuals. Similarly, the titration curves of individual plasmas varied with increments of added trypsin and achieved totals that were not necessarily greater than those obtained by cold activation. This indicates the inappropriateness of attributing greater effectiveness to one method over the other. When the two methods were paired in sequence, a synergism was apparent in that prorenin estimates increased consistently; in one case more than 10-fold. Thus, total prorenin by any single method generally fell short of the total achieved by double methods. However, this too may still not represent the unknown true total prorenin. The sequence of activation steps was important, providing clues as to the mechanism of the observed synergism. Trypsin-before-cold activation proved to be more effective than trypsin-after-cold activation, with no further advantage being gained from triple treatments involving cold before and after trypsin, or trypsin before and after cold. The inferiority of trypsin-after-cold activation was apparently due to sensitization of the plasma to the destructive effects of trypsin, shifting its titration curve to the left.(ABSTRACT TRUNCATED AT 250 WORDS)

Improved estimation of cholesteryl ester transfer/exchange activity in serum or plasma.

This simple, routine assay for measuring cholesteryl ester transfer/exchange activity in human plasma is based on the removal of interfering lipoproteins--very-low-density (VLDL) and low-density lipoproteins (LDL)--by precipitation with polyethylene glycol. High-density lipoproteins (HDL) in the samples do not affect the results. The supernate after precipitation is mixed with [14C]cholesteryl ester-labeled LDL as donor and with HDL as the acceptor for the cholesteryl ester. After incubation for 16 h at 37 degrees C, LDL is separated from HDL by precipitation with dextran sulfate and the radioactivity measured in the supernate, which contains the HDL. The assay is applicable to samples containing as much as 10 mmol of triglycerides per liter. The within-assay CV was 2.7%, the day-to-day CV 6.8%. Results compared well with those by conventional procedures.

Inhibition by FOY of kininase II in human plasma.

In this investigation we studied the inhibitory effect of FOY S 983 (gabexate mesilate) and FOY S 980 (camostate mesilate) on the kininase II activity in human plasma in vitro. Both compounds were able to inhibit kinase II, however, compared to captopril or EDTA only very weakly. The inhibitory effect of FOY S 983 or FOY S 980 could be diminished neither by NaOH-induced hydrolysis of the inhibitor nor by dialysis or ultrafiltration, but could be clearly reduced by dialysis against ZnCl2 solution. The inhibition of kininase II activity in human plasma by FOY S 983 was due to its 6-guanidinocaproate component probably acting as Zn2+-complexing agent. The ethyl 4-hydroxybenzoate component of FOY S 983 had no inhibitory effect.

Characterization of a peptide alpha-amidation activity in human plasma and tissues

Peptidyl glycine α-amidation activity has been detected in human plasma and in several human tissues known to synthesize biologically active α-amidated peptides. Activity was monitored by measuring conversion of mono-[ 125I]-D-Tyr-Val-Gly into mono-[ 125I]-D-Tyr-Val-NH 2. The plasma α-amidation activity is dependent on molecular oxygen, copper, and ascorbic acid and appears to recognize a variety of peptide substrates which contain carboxyl terminal glycine residues. Kinetic analyses demonstrated Michaelis-Menten kinetics with a Km of 14 μmol/L for D-Tyr-Val-Gly. Based on gel filtration, the apparent molecular weight of the peptidyl glycine α-amidation activity in human serum is 60,000. The level of peptidyl glycine α-amidation activity in adult plasma (N = 17) was 106 ± 3 pmol/mL/h (Mean ± SEM) with no difference between male and female subjects (range 84 to 126 pmol/mL/h). In subjects under 15 years old (N = 10), mean plasma activity was 128 ± 10 pmol/mL/h, higher than values for adult control plasma ( P < .05). In serum from hypothyroid adults (N = 13), mean serum activity was 141 ± 11 pmol/mL/hr, higher than euthyroid controls ( P < .025). The most striking elevations in α-amidation activity occurred in plasma from patients with peptide-secreting tumors. Patients with medullary thyroid carcinoma (N = 19) had a mean plasma peptidyl glycine α-amidation activity of 142 ± 52 pmol/mL/h (range 84 to 435 pmol/mL/h). The level of plasma α-amidation activity in one patient with metastatic carcinoid tumor was 560 pmol/mL/h. Peptidyl glycine α-amidation activity was detected in extracts of pituitary and central nervous system tissue, but was not detected in significant amount in extracts of various peripheral tissues. Extracts of peptide-secreting tumors also contained peptidyl glycine α-amidation activity. Human peptidyl glycine α-amidation activity may be co-secreted from tissue along with amidated peptide hormones and serve as a useful marker for certain endocrine tumors.

Determination of prolinase activity in plasma. Application to liver disease and its relation with prolidase activity

We describe prolinase (EC 3.4.13.8) activity in human plasma for the first time. Optimum activity was obtained with prolylvaline as substrate and 0.02 mmol/L manganese concentration at pH 9.0. Moreover, preincubation with manganese was not required, contrary to prolidase (EC 3.4.13.9) activity. The mean value observed in 106 subjects without liver and renal disorders was 16 U/L +/- 14 (2 SD). We determined this plasma enzyme activity in patients with acute hepatitis and chronic liver disease. Plasma prolinase activity was strongly dependent upon cytolysis because of the high activity in liver and the low activity in plasma. Of 24 patients with chronic liver disease (4 chronic hepatitis and 20 cirrhosis) and without cytolysis, prolinase activity was slightly increased in only three patients, whereas prolidase activity was increased in 13. This could be due to a difference in the activation of these two enzymes in liver during the fibrotic process.

Determination of Biotinidase Activity in Human Plasma Using [14C]Biocytin as Substrate

Determination of Biotinidase Activity in Human Plasma Using [¹⁴C]Biocytin as Substrate

Effects of Antihypertensive Agents Propranolol, Metoprolol, Nadolol, Prazosin, and Chlorthalidone on ACAT Activity in Rabbit and Rat Aortas and on LCAT Activity in Human Plasma In Vitro

Various antihypertensive agents were studied in vitro to determine their effects on cholesterol esterification by arterial ACAT (acylCoA:cholesterol acyltransferase; E.C. 2.3.1.26) and on the activity of plasma LCAT (lecithin:cholesterol acyltransferase; E.C. 2.3.1.43). Propranolol inhibited ACAT in normal rat aorta, atheromatous rabbit aorta, and in isolated microsomes from atheromatous rabbit aorta, and in isolated microsomes from atheromatous rabbit aorta. Inhibition reached 50% in microsomes at approximately 0.8 mM. Metoprolol, prazosin, and chlorthalidone also inhibited microsomal ACAT, but to a lesser extent than propranolol; nadolol had no effect on the enzyme. Propranolol, metoprolol, prazosin, and chlorthalidone also inhibited LCAT in human plasma, whereas nadolol showed no inhibitory effect. Fifty percent inhibition occurred at 2 mM with prazosin and chlorthalidone and at 4-5 mM with propranolol. Metoprolol showed a weak dose-dependent inhibition that ranged from 2 to 10% over the concentration range 0.5-5 mM. The data suggest a mechanistic basis for altered lipoprotein profiles observed clinically with certain antihypertensive therapies and suggest that a direct effect of beta-blockers on arterial wall metabolism may account for their recognized ability to reduce the development of experimental atherosclerosis and to improve survival in post-myocardial infarction patients.