bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification of α2-Antiplasmin by a Single-Step Affinity Chromatographic Procedure
Authors: Wiman B
Published: May 2019

A new and efficient single-step purification method for human α2-antiplasmin has been elaborated. The method is based on the interaction between α2-antiplasmin and a fragment (LBSI) constituting the three NH2-terminal triple-loop structures in plasminogen produced by elastase digestion. This fragment has been purified and coupled to Sepharose and used for affinity chromatographic purification of α2-antiplasmin using plasminogen depleted plasma as starting material. After adsorption and washing at high ionic strength the α2-antiplasmin is specifically eluted with 6-aminohexanoic acid. The inhibitor preparation obtained in this way is over 90% pure as judged from SDS polyacrylamide gel electrophoresis and activity measurements. About 40-45 mg pure α2-antiplasmin per liter plasma is obtained representing a yield of about 60%. LBS-I Sepharose has much higher capacity for α2-antiplasmin and is also much more specific than plasminogen-Sepharose. Repetitive treatment of plasma with LBS I-Sepharose failed to adsorb the last 20% of α2-antiplasmin as judged by Laurell electrophoresis. This supports the recent finding ot Clemmensen (1979) on partially purified α2-antiplasmin that a form of the inhibitor with less affinity for the lysine-binding sites in plasminogen may exist, even in unfractionated plasma. The major part of this type of α2-antiplasmin is also a functional antiplasmin since it can form a complex with plasmin.
Biochemical and Immunochemical Characterization of the in Vitro Thromboplastin Generation by Human Monocytes
Authors: van Ginkel C , van Mourik J , Vreeken J , van Aken W
Published: May 2019

During short-term culture monocytes (M) only, not granulocytes or lymphocytes, generate thromboplastin (TP) activity which is absent immediately after isolation. To clarify the mechanism whereby M generate TP,3 approaches were applied; I.To investigate the presence of hidden TP,freshly isolated M were subjected either to mechanical disruption, to enzymatic stripping or to delipidation, followed by reconstitution of the lipoproteins with exogenous phospholipids. No TP activity could be detected after any of these treatments. II. Using an antiserum against human brain TP apoprotein which cross-reacted with monocyte TP, it was shown that intact and disrupted freshly isolated M lack TP related antigen whereas cultured M display binding of anti-TP antibodies. However, M cultured in the presence of protein synthesis inhibitors and cultured lymphocytes, lacked TP antigen.III. Using several agents known to affect cellular processes,TP generation was shown to be dependent on:protein and RNA synthesis,energy metabolism,cAMP metabolism,assemblage of microtubules and divalent cations. Inhibition of prostaglandin synthesis by indomethacin did not affect TP generation. M from 3 patients with CGD displayed normal TP generation. These two observations suggest that prostaglandin synthesis and oxygen radicals are not involved in TP generation. In conclusion,our data indicates that de novo synthesis of TP apoprotein is responsible for the in vitro generation of TP activity by monocytes.
Ultramicroscopic and Coagulation-Fibrinolytic Findings Induced by Tissue Thromboplastin
Authors: Nagata H , Seya T , Oguma Y , Yamauchi M , Murakoshi T , Hasegawa H
Published: May 2019

We have studied the ultrastructures of tissue thromboplastin (T.Tbp) to demonstrate how It changes during coagulation.

[Materials and Methods] T.Tbp from lungs of rabbits was used for these studies. It was injected into ear veins of rabbits. Lungs were resected at several seconds, 10sec, 1 min, 5 min, 24 hrs or 48 hrs after the injection. They were examined by transmission electron microscope.

[Results] Concentrically arranged membrane structures of the injected T.Tbp disappeared in extremely short time after the injection. 1 min after the injection, fibrin fibers were seen between single sheet of membrane and endothelial cells of capillaries. In the rabbit which had died suddenly after the injection of T.Tbp, multiple pulmonary thrombi made of fibrin and platelets were seen in capillaries. The endothelial cells of capillaries were destroyed and interstitial tissues were edematous.

The hypercoagulable state was seen 10~30sec after the start of the injection, indicating the shortening of r of TEG. Then, it gradually returned the level before injection. Moreover, changes of the measurements of fibrinogen, antiplasmin and prekallikrein were also seen after the injection.
Association of Factor VIII Procoagulant Activity with Factor VIII Related Antigen
Authors: Armitage H
Published: May 2019

Using the technique of coagulation in an agarose gel following rapid two dimensional Immunoelectrophoresis, the relationship of factor VIII procoagulant activity (VIIIC) with factor VIII related antigen (VIIIRA) was studied in plasma from patients with von Willebrand's disease and carriers of haemophilia. In each case, VIIIC was closely associated with both the fast and slow moving forms of factor VIIIRA as demonstrated by the formation of fibrin in the clotting gel in a similar position to the precipitin arc in the antibody containing gel.

Mixtures of small molecular weight VIIIC with no detectable antigen and factor VIIIRA devoid of clotting activity, from either (a) haemophilic plasma, (b) Dithiothreitol treated haemophilic plasma, (c) normal serum or (d) von Willebrand serum were also studied using this technique. The samples of VIIIC and antigen were either electrophoresed in tandem or mixed in a test tube immediately prior to electrophoresis and an aliquot placed in a single well. Again fibrin formation occurred in the clotting gel in a similar position to the immune precipate. There was no peak of fibrin formation when the VIIIC was electrophoresed alone. All types of factor VIIIRA tested appeared to have an affinity for factor VIIIC and appeared to be capable of acting as a carrier protein.
Electrophoretic Distribution of FVIII:C Measured By Chromogenic and Clotting Methods and Comparison with Other FVIII-Associated Activities
Authors: Seghatchian M. J.
Published: May 2019

The new technicme of measuring FVIII-associated activities in agrose gel after electrophoresis has made it possible to investigate the molecular nature of FVIII components and their electrophoretic distribution. The method has been successfully used for the characterization of the neterogeneous nature of FVIIIR:Ag in commercially available FVIII concentrate and cryoprecipitate obtained from normal, haemophilia and VWD patients and their response to transfusion of FVIII concentrates. The mobility of FVIIIR:Ag measured by X-immunoelectrophoresis in these samples was compared with the VIII:C distribution by chromogenic, one stage and two stage clotting, and FVIIIR:RCF measured by both the slope of ristocetin induced platelet aggregation (RIPA) and by the measurement of non aggregated platelets. Different methods measured various forms of FVIII to differing degrees. FVIII:C methods had the highest sensitivity for the identification of FVIII, particularly the smaller forms, while RIPA and antigen methods preferentially measured the larger aggregates. The presence of a FVIII subunit with FXa generation activity and a faster mobility than the main FVIIIR:Ag became apparent in all the preparation analysed. The high sensitivity, and the staining properties of chromogenic methods, particularly after the colour intensifying reaction made this method also a good visual marker of FVIII distribution in agrose: Thus in certain types of immunochemical investigation the FVIIIR:Ag assay may be replaced by quantitative chromogenic FVIII:C. The parallel measurement of FVIII related activities provided a new approach to the diagnosis and the control of management of FVIII functional abnormalities.
An Experimental Study of The Pathogenesis of Venous Thrombosis Versus DIC: The Role of Intrinsic and Extrinsic Coagulation Pathways
Authors: Gurewich V. , Nunn T. , Lipinski B.
Published: May 2019

Clinically, DIC is rarely accompanied by venous thromboembolism. These two entities were studied in rabbits in order to explain this paradox. Ellagic acid (0.1M, lOml/kg), aged serum (1.3-10ml/kg), Factor IXa concentrate (0.5-3.Oml/kg), Xa concentrate (1.0-5. Oml/kg), tissue (rabbit lung) extracts or si-mplastin . (l-4ml/kg) and purified thrombin(10-200u/kg) were infused. Venous segments were ligated 15 seconds after infusion, examined after 10 minutes stasis and thrombosis was graded (0-4+). Fibrinogen concentration, PTT, TT, PT and fibrin monomer (FM) were measured. Ellagic acid, serum and IXa each caused 4+ thrombi at low doses whereas even at the highest doses, they caused no change in fibrinogen concentration and only moderate FM (2+) elaboration. The PTT shortened after all these infusions. By contrast, Xa, thrombin and the tissue extracts caused fibrinogen consumption (approx. 25-50%), substantial FM (4+) elaboration but no venous thrombosis. The PTT, but not the TT or PT, was prolonged ≥1-1/2 baseline) for 2-3h. after the infusions. Mixing post-infusion and baseline plasmas (1:1), prolonged the PTT of the latter. These findings suggest that activation of the intrinsic pathway readily induces thrombosis in areas of stasis but not DIC. The latter is more readily induced via the extrinsic pathway and is associated with the elaboration of a potent, labile inhibitor of intrinsic coagulation which may be responsible for the absence of stasis thrombi.
Fibronectin: Its Blood Clearance and Interaction with Fibrin in Vivo
Authors: Sherman Laurence , Lee Joan
Published: May 2019

Fibronectin (cold insoluble globulin, CIG) binds to fibrin in vitro by both H+ bonds and F.XIII crosslinking. Potential in vivo significance was studied in a rabbit model. 131 I-ciG purified by usual methods was ≥ 40% denatured as evinced by rapid blood clearance and complex t½. in contrast, the remaining nondenatured 131 I-CIG in animals given 3 H-amino acids had the same blood t½ (70-75 hours) and Cl(0.8) as did the 3 H-CIG. The CIG t½ was greater than that found for fibrinogen (55-65 hours). A second series received 131-fibrinogen and nondenatured 125I-CIG i.v. Subsequently, thromboplastin or thrombin (50 u/Kg/lhr) was injected and both 125 I-CIG and 131I-fibrinogen levels dropped during injection to 50-70% of preinjection levels. When ancrod was injected instead, 131I-fibrinogen fell by >90%, but 125I-CIG levels did not change. When animals were initially defibrinated with ancrod CIG levels remained unchanged during subsequent thrombin injection. The results indicate that CIG has a longer normal blood t½ than fibrinogen, but the body distributions were approximately the same. When fibrin is formed, CIG and fibrin complex in vivo, and are rapidly cleared from the blood. The abnormal fibrin formed by ancrod apparently does not bind to CIG in vivo, or else does so reversibly prior to clearance from the blood.
A Hitherto Undescribed Naturally Occurring Plasma Antagonist of Activated Factor X Inhibitor (Antithrombin III)
Authors: Yin E. T. , Salsgiver W. J. , Tangen O.
Published: May 2019

Circumstantial evidence suggested that normal human plasma contained a substance regulating the neutralization of F.Xa by F.Xa inhibitor(XaI), (Yin et.al.,Adv.Exper. Med. & Biol., 52 : 239, 1975, Plenum Press, N.Y.).

This plasma component has now been isolated and partially purified in our laboratory, and tentatively designated as “Anti-XaI”.

In experiments employing purified components, when Anti-XaI was incubated at 37°C with F.Xa, Xal and heparin for two minutes at pH7.5, the amount of F.Xa inhibited was inversely proportional to the Anti-XaI concentration. But, when the F.Xa was replaced by thrombin in the incubation mixture, the neutralization of thrombin clotting activity was undisturbed.Anti-XaI was found to be neither PF3 nor PF4.

These and other data strongly suggest that the “Antithrombin III pathway” is more complex than currently believed to be. In circulating blood an equilibrium state must exist between Anti-XaI and XaI.Under certain conditions when the Anti-XaI activity is predominant the rate of F.Xa neutralization bv XaI then becomes slower than the activation of prothrombin to thrombin by F.Xa.
Mechanism of Heparin Rebound
Authors: Fabian I. , Aronson M.
Published: May 2019

Heparin rebound is occasionally encountered following protamin sulfate administration for the neutralization of excess of heparin. in these situations the anticoagulatory properties of heparin are initially abolished, but within several hours the blood display again an increased clotting time. The purpose of this work was to try to reproduce the phenomenon under in vitro conditions, and to provide a working hypothesis for its explanation. Under the condition used the following parameters were obtained (according to the APTT method): clotting time of untreated plasma 50-55 seconds; with the addition of 4 units heparin/ml plasma>3 minutes; and with the addition of 50-100 μg of protamin to the heparinized plasma the clotting time reverts to 50-55 seconds. It was, however, found that incubation of heparin-protamin complex with the plasma at 37° for several hours, reduced the effectivity of the protamin, in other words, a longer coagulation time was observed. Subsequently, we found by electrophoretical methods that (heparin bound) protamin is proteolitically degraded upon incubation in plasma, the anticoagulatory properties of the heparin remaining intact. in summary, our results are compatible with the hypothesis that heparin rebound can be explained by the degradation of protamin by plasma enzymes and the release of this newly available heparin Into the circulation. The importance of this phenomenon in conjunction with other observations previously described by us are discussed.
Enhancement of Platelet Activity by Inhibition of Adenylate Cyclase
Authors: Salzman E. W. , Macintyre D. E. , Gordon J. L. , Steer M.
Published: May 2019

Induction of the platelet response to ADP and many other agonists is accompanied by reduction from basal level of cyclic AMP (cAMP) . The Squibb compound 22536 (9-(tetrahydro-2-furyl)-adenine), an inhibitor of lung adenylate cyclase (Harris et al. Fed. Proc. 34:617, 1975), reverses the inhibition of platelet function and prevents elevation of platelet cAMP by drugs that stimulate adenylate cyclase, including PGE1, PGD2 and adenosine. It reduces basal and PGE1-or NaF-stimulated adenylate cyclase activity and enhances the inhibition of basal activity induced by epinephrine. SQ22536 reduces basal cAMP levels but does not initiate the release reaction or platelet aggregation. It does, however, increase the effect on cAMP of platelet stimulants such as ADP and epinephrine and enhances serotonin release and aggregation induced by ADP, epinephrine, collagen, thrombin, arachidonic acid, endoperoxide PGG2, centrifugation, and rewarming after chilling. Enhancement of platelet activity by SQ22536 in citrated PRP is demonstrable only over a narrow range of agonist concentrations where primary aggregation without release is transformed to complete aggregation with release. SQ22536 does not enhance platelet stimulation by vasopressin, serotonin, or the calcium ionophore Lilly A23187; these substances activate platelets without lowering cyclic AMP, unlike the stimuli listed above. In heparinized PRP, ADP-induced platelet aggregation is accompanied by serotonin release in the presence (but not in the absence) of SQ22536. These results suggest that reduction of cAMP may be involved in the initiation of release in platelets conditioned by a variety of phenomena, including primary aggregation.
In Vitro Studies of Synthesis and Release of Factor VIII Related Protein by Endothelial Cells
Authors: Tuddenham E. G. D. , Hoyer L. W.
Published: May 2019

In vivo studies have shown that many stimuli such as epinephrine, exercise and pregnancy lead to a rise in factor VIII levels. However, the physiologic mechanisms controlling factor VIII levels are poorly, if at all, understood. Since endothelial cells synthesize factor VIII related protein (FVIII:RP) and can be grown in tissue culture, they provide a suitable in vitro model to study synthesis and release of FVIII:RP. Endothelial cells were harvested by collagenase digestion from human umbilical cords and grown in medium 199 supplemented with 20 to 30% pooled human serum. Confluent cultures were washed and then maintained in medium 199 supplemented with 20% fetal calf serum. Release of FVIII:RP into the medium was measured by immunoradiometric assay. Labeled amino acids were added to the medium for studies of FVIII:RP synthesis. Incorporation of radioactive label into FVIII:RP was measured in binding studies using a specific immunoadsorbent. Epinephrine in concentrations from 1 ng to 10 ug per ml had no effect on rate of release of FVIII:RP from cultured endothelial cells, suggesting that the in vivo effect of epinephrine is not due to a direct action on endothelial cells. In a separate experiment,exogenous FVIII:RP was added to the culture medium at a high concentration (2 units FVIII:RP per ml) along with 3H Leucine. A control without exogenous VIII:RP incorporated as much radioactivity into VIII:RP as did the culture with added FVIII:RP. This result suggests that there is no end product inhibition of FVIII:RP synthesis which operates on the endothelial cell.
Different Forms of Factor VIII Related Antigens in Endothelial Cell Cultures, Cryoprecipitate Supernatant and Normal Plasma
Authors: Peake I. R. , Bloom A. L. , Shearn S. A. M.
Published: May 2019

The nature of factor VIII related antigen (FVIIIRAG) was studied in endothelial cell cultures (EC), cryoprecipitate supernatant (cryo-S) and gel-filtered plasma fractions (GPF). Antigenic reactivity (AR) was assessed using the Laurell electroimmunoassay (LA) and a two-site immunoradiometric assay (IRMA) using a non-coagulation inhibitory rabbit antiserum raised against a factor VIII concentrate prepared by cryoprecipitation and gel filtration. Electrophoretic mobility (EM) of FVIIIRAG was assessed by two dimensional crossed immunoelectrophoresis (2DCIE), ristocetin co-factor activity (RiCoF) was estimated using fixed platelets. In normal plasma the results of LA, RiCoF assay and IRMA were in agreement. FVIIIRAG synthesised by EC showed increased EM and, in the IRMA decreased AR when compared to LA often with non-parallel dose response curves (DRC). This effect was not prevented by the presence of protease inhibitors in the culture medium. RiCoF activity was reduced compared with. LA and was related to the parallelism of the DRC in the IRMA. In cryo-S and the late eluting GPFs increased EM of FVIIIRAG was seen, and in these preparations also the DRC of the IRMA were not parallel to the normal plasma DRC. AR and RiCoF activity were disproportionately reduced when compared to the LA. It is concluded that the IRMA is sensitive to the presence of different forms of FVIIIRAG, particularly those which exhibit low levels of RiCoF activity and increased EM. some of these forms are present in normal plasma.
Detection and Partial Purification of a Natural Heparin Inhibitor
Authors: Chien Mingjien , Yue Robert H. , Gertler Menard M.
Published: May 2019

A naturally occurring heparin inhibitor has been detected in the mucosa of the fresh hog small intestine and has been partially purified. After the homogenized mucosa was extracted with Tris buffer overnight (3°) and the resulting supernatant was fractionated with ammonium sulfate, a large quantity of antiheparin activity was detected in the ammonium sulfate precipitate. This precipitate contains antiheparin activity with a specific activity of 0. 68 unit/mg of protein. Therefore, each hog small intestine contains an amount of this inhibitor enough to inhibit approximately 20, 000 units of heparin. Further purification of this heparin inhibitor was carried out by the technique of heparin affinity chromatography (covalently linked the heparin by the cyanogen bromide procedure). Eluted by a controlled NaCl and buffered gradient at 3°, the chromatogram contains a major peak and a minor peak. Antiheparin activity was located in the minor peak and has a specific activity of 9·7 units/mg of protein. Thus, we have achieved a 14-fold purification of this heparin inhibitor. This partially purified protein inhibits heparin stoichiometrically. Further experiments to purify this heparin inhibitor are in progress. This naturally occurring heparin inhibitor probably has an important biological function in balancing the action of heparin which is an important factor in maintaining Mood fluidity.
Lecithin Analog as a New Platelet Aggregating Agent
Authors: Hawiqer J. , Hooper H. W.
Published: May 2019

Lecithin comprises 32% of the human platelet phospholipids which are the source of arachidonic acid required for biosynthesis of prostaglandin endoperoxides, potent inducers of platelet aggregation. A synthetic analog of lecithin, dimethly-DL-2,3-distearoyloxypropyl-2’-hydroxy-ethylammonium acetate known as a phospholipase A inhibitor, showed a profound in vitro effect upon human platelets. Blood platelets obtained from normal, healthy, fasting volunteers and suspended in autologous plasma aggregated in response to lecithin analog. Platelet aggregation was dose-dependent within the range of 10-5 to 10-4M of lecithin analog and accompanied by release of [3H] serotonin. In contrast, addition of equimolar or higher amounts of lecithin to human platelets in vitro remained without a measurable effect on their function. Sensitivity of human platelets to the lecithin analog used was increased at least 10-fold by separation of platelets from the bulk of plasma proteins by gel filtration on Sepharose 2B. Release of [3H] serotonin from gel-filtered platelets induced by the lecithin analog studied was dose-dependent and partly reversible due to reuptake of serotonin. Thus, the lecithin analog used in our experiments is a new platelet activating agent which is a “false phospholipid” and phospholipase A inhibitor acting through a previously unrecognized mechanism triggering membrane-mediated functions of human platelets.
Amplification of Color Yield Obtained Using a Chromogenic Substrate for Determining Plasminogen Activator
Authors: Friedmaa Robert B. , Kwaan Hau C. , Szczecinski Marija
Published: May 2019

A synthetic chromogenic substrate has recently been reported for use in the specific measurement of urokinase. This material (Chromozym UK, marketed by Pentapharm, Ltd., Basle) is a synthetic tripeptide linked through an amide bond to the chromogen para-nitroaniline (PNA). The release of free PNA can be monitored spectrophotometrically by measuring the increase of absorbance of light in the range of 380 to 410 nm. The present communication describes our efforts at enhancing the color yield of the assay as well as changing the absorption maximum to a wavelength which can more readily and accurately be measured on inexpensive laboratory spectrophotometers .

Bratton and Marshall have developed a method which can be used for the determination of aromatic amines in biological systems. We have modified this method for use in semimicro applications and have adapted it to the measurement of free PNA in solution. After acidification of an aliquot of the enzyme digest with trichloroacetic acid (TCA), the liberated PNA is diazotized with sodium nitrite. Excess nitrous acid is removed with ammonium sulfamate, and the diazotized PNA is reacted with 1-naphthylethylenediamine reagent. The product has a magenta-red color with an absorption maximum of 545 nm. The substrate is not affected by exposure to TCA, and when fresh reagents are used, a low background is obtained. The color yield during actual enzyme assay is amplified twenty fold. Other coupling agents are also currently under investigation.
Photometric Measurement of Urokinase and Similar Activities with a New Artificial Chromogenic Substrate
Authors: Wenzel E. , Svendsen L. , Nienhaus K. H. , Pfordt L.
Published: May 2019

The sensitivity and the specificity of the photometric method for measuring urokinase and similar activities (thrombin, plasmin, trypsin, kallicrein) with an artificial chromogenic substrate (BZ – VAL – Gly – Arg – PNA = UK – Chromozym) were evaluated. For reference, a urokinase from Abbott Laboratories (Lot No. 143/291 Abbott, North Chicago, Illinois) was used. For a final concentration of UK – Chromozym of 0.1 x 10-3, 1 mU UK – Chromozym was found to be adequate to 17.2 U (Abbott). For 0.6 x 10-3 M, 1 mU UK – Chromozym was found to be in accordance to 10.4 U (Urokinase Abbott). In a preparation of 1 NIH thrombin (Boehringer Mannheim Diagnostica), an activity of 0.82 mU UK – Chromozym was measured. In plasmin (human, Kabi München, 1 Casein U), 84.8 mU UK – Chromozym were detected (1 Casein U per ml = 0.848 Δ OD/min.). For 1 NF trypsin, 19.7 mU Urokinase Chromozym were measured (1 Δ OD/min. = 0.197). For comparing the different activities in commercially available urokinase preparations (Sereno, Heidelberg, Urokinase Green Cross, Osaka), a Tris-Imidazol buffer was used (ionic strength 0.15; pH 8.4; containing 100 mg albumin, bovine per 1 and 10 U Aprotinin per 1). Thus, unspecific activities similar to urokinase activity were blocked (plasmin, trypsin, kallicrein, etc.). The same system was used for measuring the anti-urokinase-activity in the plasma of 25 healthy human volunteers and in 21 patients suffering from arterial occlusive diseases or from venous thrombosis before, during and after fibrinolysis therapy and before and after treatment with anticoagulants. These first results of clinical investigations will be discussed.
Methods for the Determination of Plasmin, Antiplasmin and Plasminogen by Means of the Substrate S-2251
Authors: Friberger P. , Knös M. , Gustavsson S. , Aurell L. , Claeson G.
Published: May 2019

The chromogenic peptide substrate H-D-Val-L-Leu-L-Lys-pNA (S-2251) has been chosen from a series of potent plasmin substrates. It is a highly specific and sensitive substrate towards plasmin (Km 2.5·10-4 mol/1, V 0.5·10-6 mol/min CTA-U) and its solubility in tris buffer (1·10-2 mol/1)is sufficient to obtain substrate saturation. Tris buffer pH 7.4 and I 0.15 serves as an optimal medium for the determination of plasmin. In this buffer, antiplasmins in plasma have been determined by using 0.05 CU of plasmin and 20 ul of plasma. At least two inhibitors have been found. One fast (<15 sec. incubation time) and the other(s)slow (5 min. incubation time).

The substrate has also been used for plasminogen determinations after activation of 10 ul of plasma with UK (500 Ploug-U) or an excess (40 times) of SK. It was found that neither plasminogen free plasma (100% antiplasmin activity) nor an excess (>1000 times) of SBTI inhibited the substrate activity of the SK activated plasminogen. Optimal conditions for the SK-activated plasminogen was tris buffer pH 7.4, I 0.05. Km = 2.4.10-4 mol/1 for both purified plasminogen and plasma and V = 1.10-4 mol/min.CU or 3.10-6 mol/min-ml plasma.
Separation of Human Blood Platelets from Factor VIII (Von Willebrand’s Factor) by Combined Albumin Gradient-Gel Filtration Method
Authors: Timmons S. , Hawiger J.
Published: May 2019

Currently used gel filtration methods were investigated in order to obtain a system which would effectively separate platelets from plasma proteins, such as Factor VIII/von Willebrand’s Factor (FVIII/vWF), in relatively small samples of human blood which are not amenable to repeated centrifugation. BioGel A150, which has a higher exclusion limit than the presently used Sepharose 2B, was found to be equally effective for preparing gel-filtered platelets. However, it did not completely separate the FVIII/vWF as judged by ristocetin-induced platelet aggregation. Therefore, a stepwise albumin gradient was employed as a preceeding step to gel filtration on BioGel A150. Platelets obtained by this modification were not responsive to ristocetin unless human plasma, as a source of FVIII/vWF, was added. Tyrode buffer, which shows variation in pH due to changes in pCO2, was replaced by the more stable zwitterionic buffer containing N-2-hydroxyethylpiperazine-N’2-ethanesulfonic acid (HEPES) pH 7.4. The need for control of CO2 was thereby obviated, reactivity of platelets in vitro was maintained, and their functional life span was extended from 1 to 2 hours. This modification of existing methods allows efficient separation of human platelets from FVIII/vWF and from other plasma macromolecules in relatively small samples of blood with preservation of the functional integrity of obtained platelet preparations.
High Molecular Weight Factor VIII Coagulant Activity in Cryoprecipitate and Polyethylene Glycol Precipitates
Authors: Rickard K. A. , Exner T. , Kronenberg H.
Published: May 2019

Gel filtration of human plasma cryoprecipitate on Sepharose 2B indicated the molecular weight of factor VIII coagulant activity (VIIIc) to be significantly greater than that found in antihaemophilic concentrate. Polyethylene glycol at 3% concentration precipitated approximately half of the VIIIc from cryoprecipitate. This activity eluted as high molecular weight material on gel filtration. The addition of more polyethylene glycol to a concentration of 8% precipitated most of the remaining VIIIc from cryoprecipitate. This activity appeared to be of significantly lower molecular weight, approximately corresponding in elution volume to that observed for antihaemophilic concentrate. The possibility that an antibody to VIIIc generated in a patient treated with cryoprecipitate might be directed against the higher molecular weight form of factor VIII was investigated. However, no significant differences between the higher and lower molecular weight forms of factor VIII either in stability or in reactivity with human antibody to factor VIII were found.
Partial Chemical Characterization of S-Carboxymethylated Chains of Rabbit Fibrin(OGEN): Evidence for Separate Chain Biosynthesis
Authors: Alving B. , Murano G. , Walz D.
Published: May 2019

The purpose of this study was twofold: 1) chemically characterize the isolated polypeptide chains of rabbit fibrin(ogen), and 2) explore their mode of biosynthesis. The three S-carboxy-methyl polypeptide chain derivatives of rabbit fibrin (α, β and γ) were isolated by cation exchange chromatography. Their amino acid composition was similar to the human with a methionine distribution (mole/mole) as follows: γ = 9; β = 14, α = 14. Their molecular size, (SDS electrophoresis) was estimated as follows: γ = 46,000; β = 54,000; α = 63,500. The N-terminal amino acid sequence (12 steps) of the β derivative was:Gly-His-Arg-Pro-Ile-Asp-Arg-Arg-Arg-Glu-Glu-Leu-. To determine whether the three chains are synthesized sequentially (one continuous chain, later split into three) or in parallel, turpentine-stimulated male New Zealand rabbits were given ~40 μCi of [75Se] selenomethionine (SeM) and its incorporation into fibrinogen (F) was followed. F was clotted from plasma samples, washed, reduced, and constituent chains separated by gel electrophoresis in the presence of SDS-urea. The radioactivity of each chain (expressed as percent of total F radioactivity) was determined, and the specific methionine radioactivity calculated for each chain isolated at 20, 25, and 30 min after SeM injection. During this interval the specific activity of the α and the γ chains was essentially the same (within 3%) while that of the β chain was 42 to 97% greater than that of the α chain. The similar activity of the α and γ chains during the early phase of SeM incorporation suggests that these two chains are not synthesized sequentially, rather they are synthesized in parallel.
Platelet and Fibrinogen Production: Relative Sensitivities to Endotoxin
Authors: Evatt B. L. , Alving B. M. , Bell W. R. , Levin J.
Published: May 2019

Both the threshold, and possible mechanisms, for increased platelet and fibrinogen production after endotoxin were determined in male New Zealand rabbits. Animals received E. coli endotoxin (Difco, 026:B6) in single IV doses of 0.1, 0.5, 5.0, 10.0, or 50.0 μg/kg body mass. Selenomethionine-75 Se (75seM) was injected 18 h after endotoxin, and the percent incorporation into platelets and fibrinogen was used to measure thrombopoiesis and fibrinogen synthesis. Endotoxin was detected with the limulus test only in the blood of rabbits that had received 10.0 or 50.0 μg/kg. However, platelet production was increased after 0.5 μg/kg, the smallest endotoxin dose which caused significant reduction of platelet counts. Fibrinogen synthesis was increased after 5.0 μg/kg. An endotoxin dose of 50.0 μg/kg caused the greatest increase in 75SeM-labeled platelets and fibrinogen (150% and 304%, respectively). Platelet counts were lowest at this dose; however, fibrinogen levels were not decreased nor were fibrin(ogen) degradation products detected. Therefore, stimulation of fibrinogen synthesis by endotoxin does not appear to be mediated through intial reduction of circulating fibrinogen levels. In contrast, a direct relationship was demonstrated among endotoxin dose, decrease in platelet count, and increase in production. The data suggest that endotoxin may alter platelet production by its effect on levels of circulating platelets.
Constitutional Thrombocytopathia with Abnormal Aggregation Different from Storage-Pool Disease and Aspirin-Like Syndrome
Authors: Conard J. , Samama M. , Vargaftig B. , Lecrubier C. , Breton-Gorius J. , Rendu F. , Bernadou A.
Published: May 2019

A thrombocytopathi a associated with a lifelong hemorragic diathesis has been observed in a 18 years old woman. The bleeding time is prolonged. Platelet count and size, and clot retraction are normal. Adrenalin-induced aggregation is abolished and response to ADP and arachidonic acid are impaired. A storage-pool disease is unlikely since platelet ultrastructural aspects appear normal and the number of dense bodies is overnormal, that is confirmed by examination of platelets charged with mepacrine. The uptake of 14(c) serotonin is not increased and it is in correlation with abnormal fluorescence of meracrine-stained dense bodies. Contrasting with aspirin-like syndrome, the first phase aggregation is decreased and in vitro aspirin tolerance test abnormal. Finally, although platelets do not aggregate normally to arachidonic acid, production of Tromboxane A2 and transferable platelet aggregating activity are present. Hence, the thrombocytorathia reported here could not be classified, but a congenital defect is suspected.
Infusion of Cryoprecipitate Partly Corrects the Prolonged Bleeding Time in Patients with Storage Pool Deficiency
Authors: Gerritsen S. M. , Akkerman J. W. N. , Sixma J. J.
Published: May 2019

Four patients with Hermansky-Pudlak Syndroora (HPS)— storage pool deficiency, albinism and ceroid containing bone marrow macrophages — from one family, one unrelated hps patient and one patient with storage pool deficiency (SPD) were alternately treated with cryoprecipitate from 16 donors or an equal amount of human albumin. Prior to infusion of cryoprecipitate template bleeding times were all longer than 20 min. Within 2 hrs after infusion bleeding times decreased 70% of initial values. This affect lasted for at least 6 hrs but had disappeared after 24 hrs. Infusion of albumin had no effect. Similar results were obtained in 2 additional cases of SPD that were treated with cryoprecipitate only. The abnormal platelet function tests and the biochemical abnormalities remained unchanged. Infusion of cryoprecipitate protected 4 SPD patients from bleeding during surgery. Infusion of cryoprecipitate may prevent bleeding in spd patients. Its mechanism is still obscure.
Contribution of Platelets to the Cardiovascular Effects of ADP in the Rat
Authors: Dejana E. , Castelli M. G. , Gaetano G. de , Bonaccorsi A.
Published: May 2019

The contribution of platelets to the cardiovascular effects of ADP was investigated in rats in different experimental conditions. Following rapid i. v. bolus injections of ADP(from 0.001 to 0.03 mg/kg b. w.) only a dose-related fall in blood pressure could be detected. Increasing the dose of ADP (up to 1 mg/kg b. w.), platelet fall and changes in cardiac rhythm (bradycardia, A. V. blocks and ectopic beats) became evident. All these phenomena were rapidly reversed. Inhibition of platelet aggregation by a pyrimido-pyrimidine compound (SH 869) or thrombocytopenia induced by Busulfan or antiplatelet antiserum did not significantly protect the animals from the cardiovascular effects of ADP. The fall in blood pressure, however, was reduced. Adenosine, at equimolar concentrations, caused ECG changes similar to those induced by ADP with no platelet aggregation and a less pronounced blood pressure fall. These results suggest that most of the cardiovascular modifications induced by rapid injection of ADP are largely independent of platelets. Platelets appeared to play a more important role when ADP was given for a longer period of time. A slow i. v. infusion of ADP (6 mg/kg b. w. for 10 min) was accompained by platelet fall, cardiovascular collapse and ECG alterations typical of myocardial ischaemia. All these effects persisted throughout the ADP infusion but disappeared soon after its termination. They were almost completely inhibited in rats given SH 869 or made thrombocytopenic .

In conclusion, platelets seem to contribute to the cardiovascular effects of ADP only in certain experimental conditions. In others, the nucleotide’s direct effects seem more important.
The Separation of Willebrand Factor from Factor VIII Related Antigen
Authors: Barrow E. S. , Reisner H. M. , Graham J. B.
Published: May 2019

Factor VIII (F. VIII) in normal plasma: a) shortens the prolonged clotting time of hemophilic plasma (F. VIII coagulant, VIII:C), b) precipitates with heterologous antisera (F. VIII related antigen, VIIIR:AG), and c) together with the antibiotic Ristocetin, aggregates platelets (F. VIII related Willebrand factor, VIIIR:WF). VIII:C has been shown by others to be separable from VIIIR:AG-WF proteins by gel filtration with buffers of high ionic strength. No one has, to our knowledge, clearly separated VIIIR:WF from VIIIR:AG. We seem to have accomplished this by sequential use of two antibodies to F. VIII. The IgG fractions of a precipitating rabbit anti-human VIII and of a human, non-precipitating anti-VIII were separately bound covalently to CNBr-activated Sepharose. The rabbit antibody had a high affinity for VIIIR: AG, but a low affinity for VIII:C and VIIIR:WF. Passage of human plasma over the rabbit antibody column completely removed VIIIR:AG, but not the VIII:C or VIIIR:WF. The VIIIR:AG-free plasma was then sent over the human antibody column which removed only VIII:C. The effluent retained 60% of the original VIIIR:WF activity but had no measurable VIII:C or VIIIR:AG. Bio-Gel A-15 filtration of the VIIIR:WF resulted in elution of the activity in the Vo fractions. These data suggest that the VIII:C, VIIIR: AG and VIIIR:WF activities may exist in plasma as separate entities.
Some Characteristics of Fibrin Monomer Preparations Made from Dissolved Fibrin Clots or from Fibrinogen and Immobilized Thrombin in Urea
Authors: Brosstad F. , Godal H. C. , Kierulf P.
Published: May 2019

Fibrin monomer was prepared from purified human fibrinogen by the action of insolubilized thrombin in the presence of 3.3 M urea. As judged by N-terminal amino acid analyses, complete conversion of fibrinogen to fibrin was achieved. The preparation proved homogeneous by gel chromatography and SDS electrophoresis and had retained clotting abilities identical to those of fibrin monomers arising from fibrinogen exposed to large amounts of thrombin. No changes were observed after freezing in liquid nitrogen or after storage at +4°C for 4 weeks. As to homogeneity and ability to polymerize the preparation proved superior to those fibrin monomer preparations obtained by dissolution of polymerized fibrin in various solvents.
Significant Alteration in the Stoichiometry of Factor VIII Inhibitor Interactions in the Presence of 0.25 M CaCl2
Authors: Harbury C. B. , Perry P. L. , Janszen M. E.
Published: May 2019

The major aim of this study was to assess whether the hemophilic VIII antibody (Ab) interacted more readily with “monomeric” factor VIII than with undissociated VIII. Conditions were chosen so that multiplicity of neutralization would occur with low frequency, favoring one Ab molecule per VIII molecule. Remaining VIII (R8) was kept at 65% to 85% of original VIII activity. Ab units were calculated by units of VIII neutralized x 1/dilution of antisera. As R8 fell below 65%, the apparent potency of Ab units decreased. A 25-donor pool of lyophilized plasma was made up with H2O or 0.25 M CaCl2. Thus, within and between experiments, unknown but constant concentrations of VIII molecules and VIII antigens were present. Unknown but constant concentrations and types of Ab molecules from each of three inhibitor plasmas were used. Samples were incubated 2 hours @ 22° C. Controls remained stable for the duration. The two-stage factor VIII assay of Pool et al allowed sufficient dilution of CaCl2 for accurate assay.

For 15 independent experiments,

= 53.4 ± 12 Ab u/ml and

= 25.8 ± 10 Ab u/ml

For 31 independent experiments,

= 437 ± 78 Ab u/ml and

= 174 ± 50 Ab u/ml

For 12 independent experiments,

= 80 ± 9 Ab u/ml and

= 32 ± 9 Ab u/ml

Thus, there was 2.07, 2.51, and 2.50 times more VIII neutralized in the 0.25 M CaCl2 vs. H2O incubation. Experiments were also run at 37° C, slightly increasing Ab u/ml for H2O; but the H2O control often lost significant activity. Possible explanations include: 1) the antibody is bivalent when VIII is monomeric; 2) Previously-hidden Vlll-neutralizing Antigen sites are revealed in the VIII monomeric form; or 3) antigen/antibody affinity is altered.
Immunological Evidence that Human Factor VIII is Composed of Two Linked Moieties
Authors: Koutts J. , Lavergne J.-M. , Meyer D.
Published: May 2019

Whether the three measurable parameters of factor VIII (procoagulant activity, VIII:C; ristocetin cofactor activity, VIIIR:WF; and factor VIII related antigen, VIIIR:AG) reside on a single protein remains disputed. A solid phase immunoadsorption system, in which homologous antibodies to VIII:C arising in haemophiliacs were insolubilized onto Sepharose, was used to examine the action of such antibodies and the inter-relationship between VIII:C, VIIIR:WF and VIIIR:AG. Homologous antibodies were shown to bind specifically VIII:C and to induce a spontaneous separation of VIII:C from VIIIR:WF/VIIIR:AG. The bond between VIII:C and the homologous antibodies bound to Sepharose appeared to be very stable and could not be broken with the usual antigen-antibody dissociating agents. Following prolonged incubation with antibody-sepharose, concentrated VIIIR:WF/VIIIR:AG (20 u/ml), completely devoid of VIII:C and inhibitor-neutralizing activity, was obtained. The loss of VIII:C had no detectable effect on the molecular size, antigenicity or electrophoretic mobility of the original molecule. The concentrated VIIIR:WF/VIIIR:AG was used to absorb heterologous antisera raised against factor VIII. Specific heterologous antisera to VIII:C, no longer neutralizing VIIIR:WF nor precipitating with VIIIR:AG, were obtained. Immunization of rabbits with VIIIR:WF/VIIIR:AG resulted in antisera which potently neutralized VIIIR:WF and precipitated with VIIIR:AG but also weakly neutralized VIII:C. These antibodies, like 4 other heterologous antibodies to Factor VIII studied, did not neutralize VIII:C which had been dissociated from VIIIR:WF/VIIIR:AG.

The results indicate that VIII:C and VIIIR:WF/VIIIR:AG are two different, but linked entities.
Fibrinolytic Activity of Hydatidiform Molar Tissue
Authors: Tsakok F. H. M. , Ratnam S. S.
Published: May 2019

Intravascular coagulation has been previously suspected in hydatidiform molar pregnancy (McKay 1965) and isolated cases have been described (Egley 1974). Recently a comprehensive coagulation profile in 18 intact hydatidiform molar pregnancies has been reported (Tsakok 1976). These studies showed evidence of abnormal proteolysis with activation of the coagulation system and the fibrinolytic system in varying degrees. In the present work the fibrinolytic activity of fresh molar tissue from 16 patients was studied by the fibrin plate method (Nilsson 1962) and by histochemistry (Pandolfi 1972). The fibrinolytic activity was compared with fresh normal placental tissue.

Marked fibrinolytic activity was found in the hydatidiform molar tissue as compared to none in the normal placenta. Increased fibrinolytic activity of hydatidiform mole may be the cause of early, prolonged and heavy uterine bleeding in such abnormal pregnancies. This may be due to local activation of the fibrinolytic system.
Defective Fibrinolytic Activity in Coronary Heart Disease
Authors: Walker Isobel D. , Davidson J. F. , Hutton I.
Published: May 2019

The possibility of an association existing between defective fibrinolysis and the development of coronary heart disease (C.H.D.) has been examined.

Using resuspended plasma euglobulin precipitates on fibrin plates, fibrinolytic activity (F.A.) was studied in 36 male patients with angiographic evidence of C.H.D. and in 16 male controls with normal coronary angiograms. Spontaneous plasma F. A. and “fibrinolytic potential”, as assessed by a 5 minute venous occlusion test, were measured in each subject at 0 hours (9 a.m.), at 5 hours (2 p.m.) and at 72 hours (9 a.m.).13 of the 36 patients had defective fibrinolysis compared with the controls. In only 7 of these 13 patients was a defect obvious in the 9 a.m. basal measurements of spontaneous F. A. but all 13 had a defect of diurnal rhythm as evidenced by an absent or significant reduction in the diurnal increase in F. A. at 2 p.m. All 13 had a defect of “fibrinolytic potential” as shown by an absent or impaired response to the challenge of the 5 minute venous occlusion tests. A marked increase in F. A. at 2 p.m. and a normal “fibrinolytic potential” were found in all 16 controls.

By using a series of carefully timed resting and venous occlusion tests several aspects of F. A. can be studied and defects in diurnal rhythm and “fibrinolytic potential” demonstrated in 36% of patients with C. H.D.
Biological Effects of the Administration of an Equimolar Streptokinase-Plasminogen Complex in Man
Authors: Verstraete M. , Vermylen J. , Holleman W. , Barlow G. H.
Published: May 2019

Two different lots of a 1:1 stoichiometric streptokinase-plasminogen (SK-PIg) complex were prepared (J . of Biol. Chem. 242, 1419–1425, 1967). In vitro experiments suggested that the SK-Plg complex did not react with antisera against human plasminogen and SK indicating that the antigenicity of the complex might be different from that of the precursor molecules. These changes might result in a modified specificity and immunogenicity in man and therapeutic consequences both as far as the dose and repeatability of treatment are concerned. Therefore the SK-human plasminogen complex was administered in man. Five patients received a lot SK-PIg complex prepared with heat treated human plasminogen, 4 patients another non-lyophilized lot SK-PIg complex prepared with unheated human Pig. The administration of SK-PIg complex was associated with signs of immediate antigenicity in 3 and of immunogenicity in all patients.
Treatment of the Hemolytic-Uremic-Syndrome with Streptokinase and Antiplatelet Drugs
Authors: Sutor A. H. , Schindera F. , Wehinger H. , Eisenmann G. , Künzer W.
Published: May 2019

In total we treated 11 patients with the hemolytic-uremic-syndrome (HUS) with streptokinase (SK) and platelet-aggregation-inhibitors (acetylsalicylic acid and/or dipyridamol). 9 of them survived. One patient died from bleeding complications which were probably due to erraneous underdosage of SK. Another patient had a relapse of HUS and died 18 months after treatment with aggregation inhibitors had been discontinued. All patients had very high levels of plasminogen-proactivator. We therefore recommend a high initial SK-dose of 6000 U/kg and a maintenance dose of 1500 U/kg/h for at least 24 h. We control SK-therapy with PP-determinations to avoid hyperplasminemia which may cause bleeding complications. Follow-up studies 2 years later showed normal renal function in 5 of the 9 survivors.