Fresh Human Plasma Research Articles

Inhibition of urokinase by complex formation with human [formula omitted

Human α 1- antitrypsin was prepared from fresh human plasma by (NH 4) 2SO 4-precipitation, gel filtration, affinity chromatography on concanavalin A, ion exchange chromatography and isotachophoresis. Human urokinase (EC 3.4.99.26) (plasminogen activator from urine) with M r 46 000 and 36 000 was further purified from Urokinase Leo reagent preparation by gel filtration on Sephadex G-100 Superfine. The hydrolytic activity of urokinase on acetyl-glycyl- l-lysine methyl ester acetate (Ac-Gly-Lys-OMeAc) was inhibited in a strong timedependent manner by α 1- antitrypsin . Complex formation between enzyme and inhibitor could be demonstrated in crossed immunoelectrophoresis against antiα 1- antitrypsin and anti-urokinase serum as well as by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The latter method revealed the formation of 1:1 and 2:1 molar enzyme-inhibitor complexes.

Purification of human plasma kallikrein

Attempts to purify human plasma kallikrein activated by contact with a glass surface are described. Fresh human plasma was shaken with glass beads and the adsorbed protein was eluted from the glass surface with glycinate-NaCl buffer, pH 10. The kallikrein obtained was further purified by exclusion chromatography on DEAE-cellulose followed by chromatography on P-cellulose or SP-Sephadex. The most active preparation obtained had a specific activity of 460 (μg-equivalents bradykinin/mg protein) and an esterase activity of 27·4 (μM/mg protein per min), using TAME as substrate.

Release of serotonin from human platelets by acoustic microstreaming.

Fresh human platelet-rich plasma was prepared from adult blood anticoagulated with EDTA, and the platelets allowed to incorporate tritiated serotonin. Samples were sheared for 5 min at 23°C by the acoustic microstreaming field generated around submerged portions of a transversely oscillating steel wire (115-μm radius driven at 20 kHz). Immediately following irradiation, each sample was centrifuged for 5 min to sediment intact cells and debris, and the quantity of serotonin released into the supernatant was measured using a liquid scintillation counter. There was no measurable threshold below which serotonin was not liberated, after allowances had been made for the spontaneous release of serotonin. In addition, it was found that the amount of serotonin released by irradiation alone varied with the square of the wire-displacement amplitude, indicating that hydrodynamic shear stress and velocity gradient are probably the agents responsible for the release of serotonin (together with the simultaneous release of clot promoting and/or accelerating factors).

Contributions to the optimal use of human blood. 3. Large-scale preparation of human c1 esterase inhibitor concentrate for clinical use.

Abstract. A C1 esterase inhibitor concentrate was prepared in large quantities from fresh human plasma. After removal of the cryoprecipitate and prothrombin complex, the inhibitor was adsorbed batchwise onto an anion‐exchanger.2.5 g (dry weight) of DEAE‐Sephadex A‐50 was used per liter of ‘plasma’. After extensive washing with 0.15 M NaCl, the inhibitor was eluted from the anion‐exchanger with 2 m NaCl. The eluate was fractionated with ammonium sulphate. The fraction precipitating between 50 and 65% saturation with ammonium sulphate was collected, desalted, sterilized by filtration, and freeze‐dried.The yield of C1 esterase inhibitor was 35%. The method gave a 100‐ to 150‐fold purification on a protein basis. The final preparation contained 2 mg/ml albumin, 6 mg/ml ceruloplasmin and traces of other plasma proteins. The final product passed the test for general safety and was not pyrogenic. To test the effect of the concentrate, 20 ml portions equal in activity to 2 1 of fresh plasma were infused into 5 adults with hereditary angioedema during remission. The activity of C1 esterase inhibitor in the serum of a patient increased from zero to about 75% of normal serum and decreased to about 25% after 24 h.Preliminary observations concerning the risk of transmitting serum hepatitis by the C1 esterase inhibitor concentrate are described.

Study of Efficiency of some Modifications of Medium B-2 for the Primary Cultivation of Tubercle Bacteria

It was found that the addition of 0.5% of glucose to the medium B-2 decreases the latter’s efficiency. The same was true for the addition of ribonucleic acid. Penicillin was a little more effective as decontaminant than malachite green. The medium prepared with fresh human plasma gave the same results as the medium prepared with human serum.

Studies on soluble fibrin in plasma. I a. N-terminal analysis of a modified fraction I (Cohn) from plasma containing fibrinolytic inhibitors, and incubated with plasminogen-free human thrombin.

Fresh human plasma containing fibrinolytic inhibitors was incubated with traces of plasminogen-free human thrombin. Subsequently, the clottable proteins were precipitated with either 16 or 8% ethanol. N-terminal analysis demonstrated that these proteins contained more N-terminal glycine (indicating soluble fibrin) when precipitated with 16 than with 8% ethanol. This probably indicates that the presence of more than one type of soluble fibrin in thrombin-incubated plasma is not due to plasmin activity in vitro.

Action of lecithin-cholesterol acyltransferase on sonicated dispersions of lecithin and cholesterol and on lecithin-cholesterol-protein complexes

The plasma enzyme lecithin-cholesterol acyltransferase was shown to be able to utilize lecithin-cholesterol mixed dispersions as substrate for esterification. However, dispersion with a lecithin/cholesterol molar ratio of at least 3 are required for effective esterification of dispersed cholesterol. Furthermore, dispersed cholesterol and lecithin appear to serve as substrate only after their incorporation into residual fraction ( d > 1.210) proteins to yield newly formed lipid-protein complexes, the majority of which have an apparent hydrated density between 1.063 and 1.210. This appears to reflect the ability of the esterifying enzyme to carry out effective transfer of the fatty acyl group from lecithin to cholesterol only when these components are associated with certain lipid-binding proteins in a specific structural arrangement. Both the molar ratio of lecithin/cholesterol and the ratio of these lipids to the combining proteins seem to influence the structural arrangement of the formed lipidprotein complexes. The proteins capable of associating with dispersed cholesterol and lecithin are almost absent in residual fractions isolated from fresh human plasma. Incubation of the plasma at 37° appears to promote the formation of these proteins and with it the utilization of lecithin-cholesterol dispersions as substrate for cholesterol esterification.

A controlled trial on therapy for newborns weighing 750-1 250 g. I. Clinical findings and mortality in the newborn period.

SummaryA controlled therapeutic trial was performed in newborns with birthweight of 750‐1 250 g. 40 infants (the “treatment” group) received an i.v. infusion with glucose and NaHCO3 from the 1st to the 4th‐7th day of life, and increasing amounts of human milk from the 2nd day of life. They also received, during the newborn period, 3 antibiotics (sodium penicillin G 500 000 U + methicillin 100 mg + colistin sulf. 8 mg per kg/day), a gamma globulin preparation (0.5 ml/day for 3 days) and fresh human plasma (3 ml every 2nd day in the first 5 days of birth). 40 infants (the “control” group) received only oral feeding with 10% glucose and human milk, starting in the 2nd day of life, and i.m. kanamycin (20 mg/kg/day for 5 days).No difference between treated and control babies was observed with respect to the following findings: neonatal mortality rate and survival curve on the whole series; neonatal mortality rate in babies with birthweight above or below 1.04 kg, with gestational age below 28 weeks, with above or below median respiratory rate or arterial pH on admission, with or without marked abnormality on chest film, and in males or females (but, in subjects with gestational age above 27 weeks, a significantly lower mortality rate was found in controls); postmortem findings; incidence of apnoeic spells, tremors and cloni, and pattern of muscular tonus. Bradycardia associated with 2: 1 atrioventricular block was observed in 6 controls in the 2nd day of birth. No adverse effects presumably due to the therapeutic procedures so contrasted were demonstrated.It was concluded that in the present series of newborns with very low weight: (a) the massive anti‐infectious therapy was not superior to the administration of a single broadspectrum antibiotic; (b) the administration of fresh human plasma was not effective in preventing hemorrhage (but further evaluation is needed); (c) the routine i.v. infusion with glucose and NaHCO3 did not improve neonatal survival as compared with oral feeding.However, previous studies, as well as observations on the present series reported elsewhere (11), have shown that several biochemical abnormalities commonly seen in orally fed newborns with very low weight can be prevented or corrected, at least in part, by the early infusion with glucose and NaHCO3. Since some of these abnormalities are potentially dangerous to the central nervous system, the final evaluation of this therapy must await long‐term follow‐up studies in survivors. It is therefore suggested that, before such studies are available, the early i.v. infusion with glucose and NaHCO3 should be given routinely to newborns with very low weight.

A CONTROLLED TRIAL ON THERAPY FOR NEWBORNS WEIGHING 750‐1 250 g

SummaryA controlled therapeutic trial was performed in newborns with birthweight of 750‐1 250 g. 40 infants (the “treatment” group) received an i.v. infusion with glucose and NaHCO, from the 1st to the 4th‐7th day of life, and increasing amount of human milk from the 2nd day of life. They also received in the newborn period 3 antibiotics (penicillin, methicillin, and colistin), gamma globulin, and fresh human plasma. 40 infants (the “control” group) received only oral feeding with 10% glucose and human milk, starting in the 2nd day of life, and i.m. kanamycin.As compared to controls, in treated infants acidemia decreased more rapidly in the first two days of birth, but hypercarbia developed after the 3rd day.Blood electrolytes and ECG studies were performed in part of the infants in the 2nd day of life. Markedly increased serum K and and BUN levels were observed in controls, and significantly lower values in treated infants. Severe ECG abnormalities were associated with hyperkalemia in controls, and were never observed in treated infants. Serum Ca levels were low and similar in the two groups. Hyperbilirubinemia occurred more frequently controls, and exchange‐transfusion was performed only in this group of infants.Since many metabolic abnormalities commonly occurring in fasting newborns with very low weight are potentially dangerous to the central nervous system, the conclusive evaluation of the i.v. infusion with glucose and NaHC03 must await long‐term follow‐up studies in survivors. While waiting for this evidence, it is suggested that this therapy should be given routinely to newborns with very low weight. The present observations also suggest that, if a reasonably fast oral feeding schedule is provided, in most circumstances the infusion therapy should not be continued after the 3rd day of birth.

Studies on soluble fibrin in plasma. I. N-terminal analysis of a modified fraction I (Cohn) from normal and thrombin-incubated plasma.

Fresh human plasma was incubated with urokinase to the extent that the thrombin clotting time was doubled. The clottable proteins were examined by N-terminal amino acid analysis. Increased amounts of N-terminal aspartic acid, glutamic acid, and alanine were found, whereas N-terminal tyrosine and glycine remained almost constant. Urokinase-treated plasma, saturated with soluble fibrin (by subsequent incubation with traces of thrombin), contained 0.92 μmol of N-terminal glycine per μmol clottable protein, as compared with 0.24 μmol in normal, fibrin-saturated plasma. Thus, increased fibrin solubility probably contributes to the abnormal coagulation of plasmas containing fibrinogen degradation products.

Isolation and characterization of C1-Inactivator from human plasma

A simple procedure for the isolation of C‐Inactivator from fresh human ACD plasma is described consisting of the following fractionation steps:DEAE chromatography, ammonium sulfate precipitation, Sephadex G‐150 gel filtration and zone electrophoresis. A 250‐fold concentration was achieved in good agreement with an immunologically determined C‐Inactivator concentration of 25 mg/100 ml serum.C‐Inactivator is a glycoprotein containing 35% carbohydrates; it migrates with the α2‐globulins at pH 8.6 and in the PS‐1 zone at pH 4.4. The isoelectric point is between 2.7–2.8. It had s20,w of 3.67 and a molecular weight of 104000 according sedimentation equilibrium patterns. C‐Inactivator could not be dissoziated in subunits even not by reduction with 2‐mercaptoethanol and subsequent alkylation with iodoacetamide. 1 mg C‐Inactivator neutralizes 17 C‐Inactivator units respectively 5 Remmert and Cohen plasmin units. Only weak affinities for trypsin and chymotrypsin were found.

Characterization of human plasminogen: I. On the relationship between different molecular forms of plasminogen demonstrated in plasma and found in purified preparations

Using starch gel electrophoresis in combination with a zymographic technique, up to six main plasminogen components have been demonstrated in normal fresh human plasma or serum from single individuals. Plasminogen components with the same electrophoretic mobilities as these are usually found in purified preparations. In addition, plasminogen forms with markedly deviating mobilities are often detected in significant amounts. The latter components, which probably are degraded forms of plasminogen, may be present in the plasma fractions used as starting materials. However they are especially abundant if spontaneous proteolytic activity is present or allowed to develop during the purification. A procedure for purification of human plasminogen from plasma fraction III is described. Great care is taken to remove contaminating proteolytic activity and to prevent partial activation, which easily occurs in later stages of the procedure. Prior to the last step, chromatography on DEAE-Sephadex, all traces of contaminating plasmin (EC 3.4.4.14) have to be removed. This is done by treatment with an inhibitor (Trasylol). The purified plasminogen obtained contains up to ten electrophoretic components all having plasminogen activity. Those components, which migrate differently from the plasminogen forms of plasma, may be removed by proper pooling of the effluent. Thus, preparations have been obtained containing 4–6 components, electrophoretically identical to the plasminogen forms of plasma. Glutamic acid constituted the major detectable N-terminal amino acid of these preparations (approx. 1 mole per mole plasminogen). Edman degradation in six steps showed the following N-terminal sequence: Glu-Pro-Leu-Asp-Asp-Tyr-.

The Preparation of Human Kininogen and the Elicitation of Antibody for Use in a Radial Immuno-Diffusion Assay

Abstract Kininogen was purified from fresh human plasma using mild conditions and an inhibitor of the kinin-activating sequence. The material recovered after chromatography, gel filtration and electrophoresis was only of the low molecular weight variety. It has been shown to release kinin upon treatment with purified human plasma kallikrein, urinary kallikrein or trypsin. Antibody elicited in rabbits with purified kininogen reacted with biologically active kininogen eluted from disc gel fractions. This antibody was employed in the preparation of a radial immunodiffusion assay for kininogen in human biologic fluids.

Thromboplastin as a Reagent

SummaryTissue thromboplastin is the key reagent in the one-stage prothrombin time test. To obtain reliable results, a potent thromboplastin with constant activity and stability is required. This need is met by acetone-dehydrated rabbit brain. This reagent, when protected against oxidation by sealing in an evacuated tube, retains its full activity indefinitely. The basic prothrombin time serves as a screening test for depletion of prothrombin, factors V, VII and X. Thromboplastin is slowly inactivated by oxidation and also by bacterial action. Phenol prevents the latter reaction and therefore a phenolized extract of either human brain or acetone-dehydrated rabbit brain serves as a satisfactory reagent for the prothrombin time when employed to control oral anticoagulant therapy.The constant 12 sec prothrombin time of fresh normal human plasma is fixed by the concentration of active prothrombin. By modifying the basic prothrombin time by adding excess of factors V, VII or X, the test is made a specific quantitative measure of each of these factors.

Free Fatty Acids in Human Plasma U.S.P.

Human plasma U.S.P., a preparation used as plasma volume expander, showed unusually high levels of free fatty acids. Results of incubation studies with fresh human plasma samples, kept at 32 C for various lengths of time suggest that the high fatty acid levels in human plasma U.S.P. developed during the long incubation periods used in the preparation of the product. Sodium chloride (0.6M) reduced the release of fatty acids in the incubated fresh plasma samples.

Autologous and homologous fresh human plasma as a volume expander in hypovolemic subjects

Autologous and homologous fresh human plasma as a volume expander in hypovolemic subjects

Concentrated antihemophilic factor (AHF) from outdated blood.

The antihemophilic factor (AHF) content of bank blood decreases during storage under standard blood bank conditions. In the present study, an average of 58 per cent of the original activity was present after the usual shelf life of 21 days expired. This material was concentrated by a modification of the Pool cryoprecipitation method. Almost 60 per cent of the residual AHF in the outdated plasma was found in the precipitate. The resulting precipitate was resuspended in about 25 ml of the residual plasma. On a volume basis, this had an average of about four times the activity of fresh human plasma. The cold insoluble material resuspended in residual plasma can be used for therapy without further processing. Following intravenous injection into hemophilic patients, high levels of circulating AHF, in excess of 60 per cent of normal, have been achieved without causing circulatory overload.

Stability of ACTH Preparations in Human Plasma Incubatedin Vitro

The stability of the steroidogenic activity of ACTH was studied in human plasma incubated in vitro using a series of native and synthetic ACTH preparations. Native ACTH was stable over a period of 4 hr when incubated in fresh human plasma, although it was significantly inactivated during the same period in plasma stored overnight in a freezer. Synthetic 39 amino acid ACTH (αp1–39-ACTH) was as stable as native ACTH in fresh plasma. However, synthetic preparations with 26, 19 and 18 amino acids (αb1–26-ACTH, α1–19-ACTH and α1–18-ACTH) were progressively less stable in that order, than was the whole molecule. Octadecapeptide amide (α1–18 NH2-ACTH) was significantly more stable than α1–18-ACTH. These results may explain at least partly the differences in steroidogenic potency of these ACTH preparations. It would appear, therefore, that the chain length of the C-terminal portion of the ACTH molecule is an important factor in protecting ACTH from inactivation. It suggests also that the presence of a carboxamide...

Autologous and homologous fresh human plasma as a volume expander in hypovolemic subjects.

Autologous and homologous fresh human plasma as a volume expander in hypovolemic subjects.

Clinical use of human prothrombin complexes.

IN previous communications1 , 2 we have reported preliminary observations on the use of a prothrombin complex, factors II-VII-IX-X, prepared as an eluate from barium sulfate by the method of Surgenor et al.3 This report concerns clinical studies with such prothrombin preparations, combined with additional results obtained with a new, improved concentrate of prothrombin complex factors ( II, VII, IX and X) obtained by Chromatographic isolation from fresh human plasma by the method of Melin.4 A deficiency of one or more components of the prothrombin complex may complicate many clinical states: cirrhosis; hepatitis; biliary obstruction; malabsorption; childbirth; and the preoperative and postoperative . . .