bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Chromogenic Semi-Micro Method for the Determination of Factor Xa, Factor Xa Inhibitor and Mini-Dose Heparin Levels
Authors: Scully M.F. , Kakkar V.V. 
Published: May 2019

Conditions influencing the measurement of heparin levels in the range 0 to 0.3 units/ml of plasma using the chromogenic substrate, B2-Ile-Glu-Gly-Arg-pNA HCl (S2222, KabitVitrum Ltd) have been analysed to find the most suitable incubation mixture.Factor Xa chromogenic activity was observed to be stable between pH 5.5 and 9.0 at 37°C. An optimal pH of 7.9 was found for the action of Factor Xa upon the substrate and also for the action of plasma inhibitor upon Factor Xa. A Michaelis constant (Km) of 0.6mM was determined for the hydrolytic cleavage of the substrate by Factor Xa. At a substrate concentration of 3 times Km, Factor Xa was measureable in the range of 0.02 to 0.12 units/incubation mixture with a change in optical density at 405 mμ of 0.06/second/unit corresponding to 5.7 n Kat/unit.Using 100 μl pooled plasma/unit of factor Xa, 55% of the Xa activity was inhibited at 3 minutes. Taking 3 minutes as a preincubation period, Xa inhibitor was measureable in the range 5 to 25 μl normal plasma per 0.25 units of Xa with a net change in optical density at 405 mμ of 0.8. The greatest difference between the percentage inhibition of Factor Xa by heparinised (0.3 units/ml) and non heparinised plasma, however, was observed to occur after 1 minute preincubation. Using these observations a heparin calibration curve was constructed using 10 μl aliquots of heparinised plasma. Heparin was measured in the range 0.06 to 0.3 units/ml plasma with a net change in optical density at 400 mμ of 0.8.
Further Physicochemical and Immunological Data Concerning the D-Dimer Complex
Authors: Gaffney P.J. , Brasher M. , Joe F. 
Published: May 2019

It has been suggested that a sensitive assay of D-dimer, a fragment found in plasmin-mediated crosslinked fibrin digests, might contribute to our understanding of natural and induced fibrinolysis in man (P.J. Gaffney, Lancet (1972) ii, 1422). Towards this end, further characterisation of the heterogeneity of the D-dimer complex was attempted using gel electrophoretic techniques, while some attempts to raise rabbit antisera to D-dimer were also assessed.It was confirmed that D-dimer, following plasmin-mediated release from crosslinked fibrin, was non-covalently linked to fragment E. The E fragment was released from the D-dimer/E complex during progressive digestion. The earliest digests examined showed that more than 50% of the total E fragment (immunologically measured) was complexed with D-dimer while some E remained in the complexed form even after intensive digestion. During examination of various timed digests it was observed that the electrophoretic properties of the various D-dimer complexes changed while the detergent gel patterns remained consistent. Antisera raised in rabbits to the D-dimer/E complex, following absorption with fragments D and E, reacted with purified fibrinogen and suggests that these antibodies may be recognising the dimeric D structure in fibrinogen.It was concluded that studies of the immunological properties of the D-dimer/E complex, this being the earliest form of soluble crosslinked fibrin fragments, might be the most useful in eventually establishing an assay which would further our understanding of natural and induced fibrinolysis.
Platelet Plasma Membrane Glycoproteins in Normal and Genetically Abnormal Platelets
Authors: Phillips David R. 
Published: May 2019

One approach to determine what specialized structures on the platelet membrane surface perform platelet specific functions is to compare the surface of normal to genetically abnormal platelets. We have used three techniques; lactoperoxidase-catalyzed iodination, neuraminidase/galactose oxidase/(3H)-NaBH4 labeling, and periodate/(3H)-NaBH4 labeling, to investigate the molecular organization of the proteins in these membranes. Several generalizations can be made about the normal membrane: (i) all of the major proteins exposed on the surface (12 have been identified thus far), are glycosylated, (ii) some of these glycoproteins are trans-membrane (i.e., they span the thickness of the membrane), (iii) the glycosylated segments are exposed to the outside of the cell, (iv) many disulfides (both inter- and intramolecular) are present in the platelet membrane glycoproteins, and (v) most of these glycoproteins contain sialic acid.Analysis of the glycoprotein composition of the platelets from 16 individuals with Glanzmann’s thrombasthenia (characterized by a lack of aggregation) showed a decreased concentration of glycoproteins IIb and III. In contrast, platelets from an individual with Bernard-Soulier syndrome (characterized by absent adhesion) had primarily a decrease in glycoprotein Ib. The data demonstrate that both genetic abnormalities are caused by different defects in platelet membrane glycoproteins and suggest molecular entities which may be involved in specific platelet functions. Supported by NIH Career Development Award HL-0080 and Grant HL-15616.
Blood Coagulation, Fibrinolytic Activity, Platelet Function and Immunoglobulins in Frostbite
Authors: Chohan I.S. , Singh I. 
Published: May 2019

Fifteen males, 19-45 years old, stationed between altitudes 3690 and 5540 m in the Western Himalayas who were frostbitten were studied within 24 hours of the injury and then 4 weeks and 1 year after for blood coagulation defects. The following disturbances were found: fibrinogen degradation products and factor VIII-related antigen were increased; fibrinogen, platelet counts and haematocrit were decreased; platelet adhesiveness was increased; euglobulin lysis time was prolonged; antithrombin III,α-1 antitrypsin and α-2 macroglobulin were markedly decreased; IgG and IgA immunoglobulins and cryoglobulins were increased; serum albumin was decreased and IgM immunoglobulin consumption was increased.These abnormalities increase platelet adhesiveness and diminish fibrinolytic activity and promote intravascular thrombosis.Furosemide increases fibrinolytic activity and suppresses platelet adhesiveness in vivo (I. Singh and I.S. Chohan, Int. J. Biometeor. 17, 73, 1973). Its use in the prevention of frostbite is under investigation.
Platelet Function, Ultrastructure and Management in Glanzmann’s Thrombasthenia
Authors: Lusher J.M. , Barnhart M.I. , Pullen J. , Warrier A.I. 
Published: May 2019

This exhibit will demonstrate laboratory abnormalities found in longitudinal studies on six patients with Glanzmann’s thrombasthenia. All have had mucous membrane bleeding (predominantly epistaxis) since infancy. In addition to prolonged bleeding times, absent clot retraction and little or no platelet retention in a glass bead column, all six have defective platelet factor 3 release with Kaolin and their platelets do not aggregate with ADP, epinephrine or collagen. The platelets of all six do aggregate with ristocetin, however. Transmission electron microscopy (TEM) of thin sectioned platelets revealed normal ultrastructure. However, in the TEM platelet function test (Thromb. Diath. Haemorrh., Suppl. 42:321-344, 1970) there was difficulty in capturing the thrombasthenic platelets even on the formvar surface. Those platelets which did adhere to the formvar exhibited normal percentages of dendritic and spread forms although pseudopod detail was unusual. Inspection by scanning electron microscopy (SEM) of surfaces of platelets responding to contact activation and captured on companion formvar coverslips revealed surface membrane features consistent with inhibition of membrane activation. Platelets were swollen with convoluted membranes while pseudopod outgrowth from discrete regions was not prominent. Suppression of membrane activation was also noted by SEM of platelets obtained by cytocentrifugation of buffy coat preparations. Upon exposure to collagen platelet adhesion and morphologic features of aggregation were abnormal under our test conditions.In addition to local measures, recommended management of severe bleeding episodes is infusion of single donor platelet concentrates, obtained from an HLA matched donor by plateletpheresis.
Heparin Concentrations in Plasma During Surgery in Heparin Low-Dose
Authors: Kruse-Blinkenberg H. O. , Gormsen J. 
Published: May 2019

The concentration of heparin in plasma was measured by the method of Denson & Bonnar and by a method using a chromogenic substrate for activated factor X (S-2222, Bofors, Sweden), pre-, per- and postoperatively in 126 patients undergoing major abdominal-thoracic, gynaecologic or orthopedic surgery. The patients received heparin (Heparin (R), Novo) 5,000 units 2 hours preoperatively and every 8 hours postoperatively for 1 week. No significant correlation was found between concentrations of heparin, sex, age, presence of malignant diseases, the type of operation or frequency or extent of per- and postoperative bleeding. Zero values were found immediately postoperatively in 41 of 126 patients, most frequently after abdominal surgery.The correlation between the values found by the 2 methods was satisfactory.
On the Nature of Fibrinolytic Activity of Tissues
Authors: Donner L. , Klener P. , Vodáková L. 
Published: May 2019

Plasminogen activator of the vessel wall of thoracic, abdominal aortas, coronary, renal and carotic arteries was examined by means of the Todd’s histochemical method.Activator was removed from sections by treatment for one hour with kalium thiocyanate. After saline wash possible proactivator was converted with streptokinase or urokinase solution and tested again with the histochemical method. Sections treated with thiocyanate showed no or very low fibrinolytic activity. After streptokinase or urokinase treatment a local fibrinolysis appeared again. It is suggested that thiocyanate despite its power to remove activator is unable to extract proactivator from tissue. No differences were found in different types of arteries examined neither in atheroclerotic or normal arteries.
Determination of Antithrombin, Heparin Co-Factor and Heparin in Plasma Using the Chromogenic Substrate S2238
Authors: Scully M.F. , Kakkar V.V. 
Published: May 2019

Antithrombin, the thrombin inhibiting capacity of human plasma, was measured in normal plasma and also as a partially purified protein with the synthetic substrate, H-D-Phe-Pip-Arg-p-nitroanilide-2HC1. (S2238, KabiVitrum, London). A method was developed which was sensitive in the range 0.05 to 0.25 NIH units of thrombin in a total volume of 0.5 ml. Under these conditions a change in optical density of 0.36 per minute per 0.1 units of thrombin was observed. The substrate is, therefore, 5 to 6 times more sensitive than the previously used B2-L-Phe-Val-Arg-p-nitroanilide (S2160, KabiVitrum, London). Using this procedure antithrombin levels in up to 10 pis of plasma were measured without preliminary defibrination.The method has been adapted for the automated determination of thrombin (0.025 to 0.4 units/ incubate and also antithrombin levels in 5-50μls of plasma using an LKB 2086 reaction rate ana-lzer. Triplicate measurements of 25 μl aliquots of 15 random samples of normal human plasma agreed within each sample set to ± 3% (1 standard deviation). A correlation was shown with antithrombin III levels measured by a clotting method. A modification of the method produced a more sensitive assay measuring in the range 80-l007o plasma antithrombin. Heparin cofactor was measured in 10 μl aliquots of heparinized plasma (NIH/ml). An adaption allowed automated measurement of heparin levels in the range 0.2 to 1.0 units/ml of plasma.Preliminary experience indicates that with the use of this substrate various clotting tests may be substituted with a method which is automated and readily reproducible.
Feiba Immuno: A Preparation with Factor Eight Inhibitor Bypassing Activity
Authors: Elsinger F. 
Published: May 2019

FEIBA IMMUNO is a preparation in which a new activity is generated capable of bypassing factor VIII. The preparation which is used to treat patients with inhibitors (especially inhibitors to factor VIII) is standardized in FEIBA units, i.e. in terms of its in vitro capacity to shorten the activated PTT of a factor VIII inhibitor plasma.It could be concluded from different in vitro experiments that none of the classic’ activated coagulation factors is responsible for the factor VIII bypassing reaction; FEIB-activity seems to be correlated to a new complex of coagulation factors.To get an answer to the question which coagulation factors are essential for FEIB-activity, we tried to generate this activity from different deficient plasmas; from these experiments the following conclusions could be drawn:, the presence of at least factors VII, IX, and X is essential for the generation of the molecular species responsible for factor VIII as well as factor X bypassing activity, but factor V is not bypassed. This activity is not factor Xa itself. Factors VIII and V are not necessary for the generation of this active principle, but factor V is finally needed for its bypassing action.
Large Scale Purification and Characterisation of Porcine Factor VIII for Clinical Use
Authors: Lee Helen , Chavin S.I. , Blewitt Rita E. , Austen D. E. G. 
Published: May 2019

Animal factor VIII can be useful in the treatment of haemophilic patients with high titres of human factor VIII antibody when the antibody shows low cross-reactivity towards animal factor VIII. The use of currently available preparations of animal factor VIII is associated with rigor, anaphylaxis and transient thrombocytopaenia, which limit their therapeutic value. Analysis of some of these preparations has shown low IgM and high IgG concentrations and haemagglutinating activity against a panel of human red blood cells. It has been demonstrated that purified porcine IgG and IgN agglutinate human red blood cells, while IgM is also complement fixing.A large scale purification procedure has been developed for producing porcine factor VIII from platelet-poor plasma. It involves various precipitations followed by gel filtration through Sepharose 4B-CL on a KS sectional column. The pooled void-volume fractions are concentrated by using the Pellicon cassette system, giving a final yield of approximately 20% of the starting plasma. Porcine factor VIII prepared by this method is stable. Its greatly reduced fibrinogen content facilitates final filtration and prevents the accumulation of fibrinogen in patients treated with high doses. Non-specific haemagglutinins which may have contributed to some of the post-transfusion reactions have been removed. Specific activities of at least 50 U/mg of protein are obtained, and doses greater than 10,000 units can be given in 10 ml.
Surgical Management and Complcations.
Authors: Gilbert M.S. 
Published: May 2019

Surgery in the hemophiliac requires close coordination of a team familiar with hemophilia care. Minimum requirements include a hematologist, a diagnostic laboratory capable of performing factor and inhibitor assays, complete blood banking facilities and an orthopedic surgeon and physiotherapist familiar with the orthopedic complications of hemophilia. Pre-surgical evaluation must include an inhibitor study and an infusion study to determine levels and half life. A single batch of 60-100,000 units of concentrate is set aside for the patient.Surgery is scheduled for early in the day and early in the week. Intramuscular anaesthetic pre-medications are avoided. The infusion is given just before induction of anaesthesia and a post infusion level determined. Surgery is started immediately and special care is taken to avoid contamination of the operating room team because of the risk of hepatitis. Surgical technique differs little from that used in the non-hemophiliac with close attention to hemostasis. Tourniquets, electrocoagulation and suction drains have been used without problems.Factor levels are not allowed to fall below 30% until sutures are removed and healing is complete. Physiotherapy may require maintenance of levels for longer periods.
The All-American Blood Buddy: Donor Motivation for Hemophilia and Other Chronic Disorders
Authors: VanHarlingen D. , Child D. , Dzuback J. , Thompson F. 
Published: May 2019

A novel method of enlisting donors for hemophilia from among high school and college students will be presented. This method has been successfully used in New Jersey and in less than two years of operation has recruited thousands of donors specifically for hemophilia. The program is built on a particular philosophy of responsibility for blood replacement which enhances the importance of the donor in the process of giving blood. The program is aimed at those who would not ordinarily donate blood under conventional recriutment programs.This system is applicable to other regions and other chronic blood disorders. It has also been proven successful in motivating donors at ages other than the originally intended group. The importance of this approach to both the hemophiliac and the community at large is discussed. Statistics supporting the success and necessity of the program will be presented. We shall also present arguments which refute common criticisms of our system.
Identification of a Thrombin Proteolytic Receptor on Human Platelets
Authors: Phillips David R. , Agin Patricia Poh 
Published: May 2019

Thrombin is one of the most potent physiological agents causing platelet stimulation. It would appear that proteolysis is intimately linked to stimulation because trypsin, but not thrombin inactivated with PMSF, also stimulates platelets. Our approach to identifying the proteolytic substrate was to radioactively label the membrane surface proteins and determine which of these were hydrolyzed by thrombin. A glycoprotein labeling method (neuraminidase/galactose oxidase/(3H)-NaBH4) was employed. Twelve membrane glycoproteins were labeled, including most of those labeled by 1actoperoxidase-catalyzed iodination. Secretion and aggregation experiments showed that platelets labeled by this procedure are equally responsive to thrombin, collagen, and ADP as unlabeled platelets.Of the glycoproteins labeled by this procedure, only glycoprotein V (Mr = 89,000) was decreased as a result of thrombin action. Although low thrombin concentrations (0.2 U/ml) were sufficient to obtain significant hydrolysis, the complete loss of glycoprotein V occurred at the ratio of 1 U thrombin per 109 platelets; no further changes were observed when the thrombin concentration was increased to 10 U/109 platelets. A soluble glycopeptide hydrolytic product (Mr = 70,000) was released into solution. The kinetics of glycoprotein V hydrolysis were comparable to those of secretion and aggregation. Glycoprotein V hydrolysis was not observed when platelets were aggregated by collagen, ADP, or the Ca++ ionophore A-23187. It is proposed that glycoprotein V is a proteolytic receptor of thrombin.
Intracellular Regulation of the Metabolism of Arachidonic Acid in Human Platelets
Authors: Bills Thomas K. , Bryan Smith J. , Silver Melvin J. 
Published: May 2019

The synthesis of prostaglandins and thromboxanes by human platelets is limited by the availability of the fatty acid precursor, arachidonic acid. Although large amounts of arachidonic acid are esterified to platelet phospholipids, only the free acid can be utilized by the oxygenation pathways of platelets. Since there are only trace amounts of free arachidonic acid in platelets, the enzymatic liberation of this fatty acid from platelet phospholipids can be considered the initial and rate limiting step of these oxygenation pathways. This process is catalyzed by a phospholipase A2 whose role as the rate limiting enzyme makes it a prime target for the intracellular regulation of prostaglandin and thromboxane synthesis.A second mechanism for the regulation of prostaglandin synthesis has been hypothesized. Several commonly occurring unsaturated fatty acids, e.g., oleic and linoleic acids, are capable of inhibiting prostaglandin cyclooxygenase. If these fatty acids are liberated from phospholipids along with arachidonic acid, they could limit the production of prostaglandins and thromboxanes. Thus, the types and amounts of fatty acids released from platelet phospholipids could regulate the amounts of prostaglandins and thromboxanes produced.We have investigated these two types of intracellular regulation and have found that 1) intracellular cyclic nucleotides and divalent cations are involved in the regulation of the platelet phospholipase A2 activity stimulated by thrombin and 2) this phospholipase A2 activity catalyzes the specific release of arachidonic acid from phospholipids, thereby obviating the regulatory role of liberated oleic and linoleic acids.
Human Prekallikrein (Plasminogen Proactivator): Purification, Characterization and Activation by Activated Factor XII
Authors: Bouma Bonno N. , Griffin John H. 
Published: May 2019

In order to resolve conflicting reports about the possible identity of prekallikrein and Factor XII-dependent plasminogen proactivator (FXII-PPA), the γ-globulin fractions of prekallikrein-deficient (Fletcher trait) and of normal plasma were assayed for FXII-PPA. Based on both fibrin plate and clot lysis tests, FXII-PPA in the γ-globulin fractions of prekallikrein-deficient plasmas from 2 unrelated patients was undetectable, i.e. <1% of the FXII-PPA in the normal γ-globulin fraction. However, PPA independent of FXII was detected in both the Fletcher and the normal γ-globulin fractions at 4% of the FXII-PPA present in the normal γ-globulin fraction.Human plasma prekallikrein was purified 2,000-fold (specific clotting activity 22 units/mg) and was greater than 95% homogeneous on SDS-gels. FXII-PPA was always copurified with prekallikrein and was totally separated from Factor XI. No Factor XII-dependent or Factor XII-independent plasminogen activator activity was detected in purified Factor XI preparations at 40 units/ml. Purified prekallikrein in its precursor form gave 2 protein bands on SDS-gels at 82,000 and 78,000 MW. Upon reduction, a single 85,000 MW band was observed. Kallikrein and plasminogen activator activity were generated upon incubation with purified human Factor XIIa (28,000 MW form). Analysis of this reaction mixture on SDS-gels without reduction showed 2 bands with apparently identical MW’s as the precursor protein bands, whereas reduction showed cleavage of both protein bands.These results suggest that prekallikrein is identical to FXII-PPA in normal human plasma and that activation of this zymogen by Factor XIIa involves limited proteolytic cleavage.
Characterization of Multiple forms of Human Thrombin
Authors: Gorman J.J. , Castaldi P.A. 
Published: May 2019

Human thrombin was obtained by activation of partially purified human prothrombin with venom of the Australian Taipan (oxyuranus scutellatus scutellatus).The crude thrombin was precipitated with ammonium sulphate and subsequently purified by chromatography on Sephadex G-75 CM-Sephadex C-50 and the affinity resin am inobenzamidine-CH-Sepharose. The final preparation had a specific activity of 1700 units per absorbance unit (A| cm
280n m Was herterogenous as shown by urea-acrylamide gel electrophoresis at acid pH and by isoelectric focusing. SDS-acrylamide electrophoresis revealed molecular weights of 39,000, 28,000, 25-23,000 and 15-12,000 for these proteins. The 39,000 dalton species predominated (greater than 90%) when the enzyme was inhibited with phenyImethanesuI phony I fluoride prior to dialysis against 0.02M sod i urn phosphate (pH 8.0) containing 0.1% SDS. Lack of such inhibition reduced the amount of the 39,000 dalton species to less than 60% with concomitant increase in the smaller species. Increase in the smaller species also occurred during incubation in 0.IM NaCI-0.I M Tris buffer (pH 8.0).Peptide mapping studies indicated that the smaller species were structurally related to the 39,000 dalton species. Amino acid compositions of tryptic peptides indicated a high degree of homology with bovine thrombin.It has been established that human thrombin can exist in at least two secondary structural forms of different molecular weights, probably due to autolytic degradation of the largest (39,000 dalton) protein species.