bluuebunny/crossref_metadata_2025 huggingface dataset:
Appearance of Active FVIII Subunits Derived from High M.W. FVIII in High Gravity Field
Authors: Vukovich Th. , Doleschel W. , Koller E. , Auerswald W.
Published: May 2019

To elucidate the type of binding of active subunits in the high M.W.FVIII, a commertial preparation, in isotonic saline, was put on Sepharose 4B and eluted with 0.15M Tris-NaCitrate-eACA at pH 7.0 (Fig.1). A sample of fraction A was centrifuged for 32min.at 2x105g in a UZ-cell with a filter paper pad. Globular Proteins of MW>106D sedimented into the pad while those of lower MW remained in both pad and supernatant. The latter was applied to S4B (Fig.2) and developed with buffer. Pad proteins were eluted with buffer and the eluate chromatographed (Fig.3). Uncen-trifuged sample fraction A was rechromatographed for control;FVIII activity was in void volume only. In contrast, several activity peaks were given by the treated test fractions. The appearance of lower MW factor VIII-active fractions resulting from application of a high gravity field to the >106D MW starting material is evi-\ dence against covalent binding of the former to give the latter.
Fibrin and Fibrinogen Proteolysis Products: Comparison Between Gel Filtration and SDS Polyacrylamide Electrophoresis Analysis
Authors: Alkjaersig N. , Davies A. , Fletcher A.
Published: May 2019

The proteolysis of purified human fibrinogen, stabilized and non-stabilized fibrin by plasmin were investigated by gel filtration analysis and SDS Polyacrylamide electrophoresis of the reaction products. Plasmin proteolysis of fibrinogen followed the sequential steps previously reported and the two analytical methods yielded concordant results. Large molecular weight proteolysis products, of substantially greater molecular weight than native fibrinogen, were identified by gel filtration analysis following dissolution of stabilized and non-stabilized fibrin clots; with further incubation with plasmin, these proteolysis products gradually diminished in size. On the other hand, SDS Polyacrylamide electrophoresis of these fibrin digests demonstrated that while non-stabilized fibrin yielded breakdown products similar in size to those obtained after proteolysis of fibrinogen, stabilized fibrin digests showed moieties of greater molecular size estimated to be of molecular weight 400, 000 to 800, 000. The final breakdown products of stabilized fibrin differed from those of fibrinogen and non-stabilized fibrin in that fragment D was present in the “double D” cross-linked form.
Classifying Potential Carriers of Hemophilia: Report of the Joint WFH-WHO Expert Committee
Authors: Graham John B.
Published: May 2019

The World Federation of Hemophilia and the World Health Organization convened an expert committee in Geneva, in Nov. 1976 to write a technical report on “carrier detection” in hemophilia. The committee consisted of:L.M. Aledort (USA), A.G. Bulanov (WHO), R.C. Elston (USA), E.K. Ginter (USSR), J.B. Graham (USA), J. Hermans (Neth), M.J. Larrieu (France), A.D. McLaren (UK), P.M. Mannucci (Italy), C.R.M. Prentice (UK), J.J. Veltkamp (Neth). It attempted to prepare a technical report which would cover all aspects of the matter, yet be useful to non-experts. Laboratory methods were described in moderate detail, but major emphasis was placed on methods of genetic and statistical analysis, since these are less accessible to hematologists. It was suggested that help from a biostatistician is usually necessary at the start. Once statistical methods are well in hand, however, the personnel of a coagulation lab can manage the routine calculations. Methods were suggested for choosing and testing the validity of reference populations of carriers and controls, for estimating the number of “possible carriers” in a population. The logical imperative of incorporating the “likelihood ratio” obtained from the laboratory with the “prior probability” obtained from the pedigree was stressed; methods for calculating prior probabilities and incorporating linkage information were also described. Finally, it was emphasized that genetic counseling consists of more than merely offering a consultand the probabilities favoring carriership; it involves giving counsel to a family about all aspects of hemophilia.
Prophylaxis for Hemophilic Arthropathy
Authors: Mackay S. Ross
Published: May 2019

Hemophilia patients at the Rochester, New York, Hemophilia Center Clinic on three different modes of transfusion therapy were reviewed. It was found that those patients receiving prophylactic transfusion on a home infusion basis required less material over twelve months than those on home infusion or out-patient therapy. It was concluded that many hemorrhages may have a psychomotor origin and that prophylactic transfusion at home suppressed hemorrhage on a psychomotor basis. The mode of therapy may thus be a factor in slowing the rate of hemophilic arthritis.
Recent Reduction in Thrombogenic Enzyme Content of Prothrombin Complex Concentrates
Authors: Kingdon H.S.
Published: May 2019

In previous studies of prothrombin complex concentrates, it was demonstrated that there was a high content of potentially thrombogenic enzymes in the products from certain manufacturers, and that the enzyme conteat correlated closely with in vivo assays for thrombogenicity and with the observed incidence of thrombotic episodes following infusion (Thromb. Diath. Haemorrh. 33, 617, 1 975\Subsequently, it was shown that the thrombogenic enzymes could be generated by prolonged contact with DEAE-cellulose or calcium ions during preparation or later handling (Blood, 49, 159, 1977). In view of. these observations, efforts have been made to reduce the contact time of plasma fractions with either DEAE-cellulose or calcium ion during purification of these fractions for therapeutic use. In vitro assays for thrombogenic enzymes using the nonactivated partial thromboplastin time (NAPTT) were recently repeated on some of the currently available therapeutic materials. Assays were standardized and compared with previous results by using a provisional standard provided by Dr. David Aronson, Bureau of Biologies, USFDA. In the 1975 study, one manufacturer was identified as making a concentrate virtually free of thrombogenic enzyme. The concentrate currently being made by this manufacturer still does not significantly shorten the NAPTT. In the 1975 study, 2 manufacturers were shown to be making concentrates with high titers of thrombogenic enzyme. The current products of these two manufacturers contain detectable but significantly lower levels of thrombogenic enzymes. Thus it appears that for these two manufacturers, minor changes in production procedures have led to a product containing less potentially thrombogenic material.
Factor VIII and Factor VIII Related Antigen in Normal Pregnancy
Authors: Melo G. O. S. de
Published: May 2019

Factor VIII and factor VIII related antigen were found to change proportionately during normal pregnancy. There was a clear tendency for levels of activity and antigen to increase as the duration of the pregnancy advanced. In the pregnant women studied a significant difference was observed in factor VIII levels between Blood Group A and Blood Group O. A similar difference was found in factor VIII related antigen levels. Age, weight and previous use of contraceptives seems to have no influence on factor VIII and factor VIII related antigen values in pregnancy.
Factor VIII Deficient Plasma for Laboratory Tests Prepared from Normal Plasma and a Human Antibody
Authors: Rickard K.A. , Exner T. , Kronenberg H.
Published: May 2019

Plasma from a patient with a potent factor VIII inhibitor was mixed at several low concentrations with fresh normal plasma. The factor VIII deficient plasmas obtained after a short incubation period were used as substrate plasmas in the normal one stage factor VIII assay method. Results obtained using 0.5% to 1% of the inhibitor plasma in normal plasma compared favourably with those obtained using normal haemophilia A factor VIII-deficient plasma provided that tests were carried out without delay. A single plasma exchange of this patient provided enough antibody to prepare more than 500 litres of factor VIII-deficient plasma by simple mixing with fresh normal plasma.
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.
Dose Requirement for Substitution Therapy in Hemophilia A.
Authors: Allain J.P.
Published: May 2019

In order to determine the relationship between different doses of factor VIII and their clinical effect,70 children with severe hemophilia A treated with single doses of cryoprecipitate were studied. Plasma factor VIII levels were tested by a one stage assay on samples taken before, and 30 and 60 min. post-infusion. Clinical failure was defined as an absence of clinical improvement or the occurrence of bleeding in another site within 24 hours post-treatment. The relationship between plasma factor VIII levels or doses injected in u/kg and clinical result followed an exponential curve. Calculated from 153 factor VIII recoveries, plasma factor VIII levels of 0.35 and 0.52 u/ml corresponded to 95 and 99% satisfactory treatment, respectively. The same clinical result was obtained with 20 and 31 u of factor VIII/kg when studied in 1043 infusions of lyophilized cryoprecipitate. The in vivo recovery being 0.015 u factor VIII activity/ml for 1 u/kg injected, these dosages provided plasma factor VIII levels of 0.30 and 0.47 u/ml respectively. Since home treatment is largely based on single infusions of factor VIII, it is suggested that most current bleedings can be adequately treated with a dose of factor VIII providing a plasma factor VIII activity of 0.30-0.35 u/ml.
Factor 8 Activity in a Hemophiliac Patient with Acute Lymphocytic Leukemia
Authors: Meyer S.C. , Troner M.B. , Lian E.C.Y. , Silverman M.A.
Published: May 2019

We have treated a 21 year old white male with classic hemophilia A for acute lymphoblastic leukemia. At presentation the patient had a white blood count of 30,000 with 90 per cent lymphocytes. Bone marrow asperate revealed 80 per cent blasts which were periodic acid-schiff positive and peroxidose negative. Remission was induced with a course of methotrexate, vincristine and prednisone. Factor 8 activity was measured by the method of kaolin activated partial thromboplastin time using congenitally deficient plasma at presentation and while in remission. Factor 8 activity was less than one per cent at presentation and the same while in complete remission. At no time during the course of the disease did the measured factor 8 level spontaneously rise above one per cent concentration. Bleeds felt to be typical of hemophilia occurred at occasional intervals during the patients two year course of treatment. These did not appear to occur with any less frequency than prior to the onset of the acute leukemia. These findings are in contrast to previously published reports of increased factor 8 levels during the uncontrolled phase of acute lymphocytic leukemia.
Non-Surgical Methods for Correction of Knee Flexion Contracture.
Authors: Boone D.C.
Published: May 2019

Knee flexion contractures have presented problems for the physical rehabilitation of hemophilic patients. These deformities reduce the functional mobility of the patient and their presence increases the amount of energy which is expended in walking. Hinges which extend the knee and tend to desublux the tibia are incorporated in plaster cylinders. Daily adjustments are made in the extension and subluxation screws. Serial castings may be required when correcting a flexion deformity greater than 30-35 degrees. Maximum correction can be expected in two to three weeks. Maintenance of the corrected position is vital and is obtained through a sequence of cylinder and open-front castings. The extension desubluxation hinge and the open-front cylinder cast will be demonstrated and discussed.
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.
Current Organization of General Dental Programs for Hemophiliacs
Authors: Lewis B.
Published: May 2019

Dentistry should be as accessible to the hemophiliac as to any other individual. The normal high standards of dental treatment should not he compromised because of hemophilia. A proper understanding of this problem and cooperation between the patient, physician, and dentist should underscore the precautions necessary for treatment. Preventive dentistry is very important. Proper brushing, flossing, and diet should keep the soft tissues healthy and firm. Bleeding problems can be adequately handled when they occur. Dental checkups at determined intervals will allow the practioner to discover any problems at a beginning stage where it can be easily restored with minimal trauma.
Nerve Lesions in Haemophilia
Authors: Fernandez-Palazzi F. , Bosch N.B.
Published: May 2019

Nerve lesions in haemophilia are due to external compression or traction upon the nerve due to sudden muscular haematoma, producing a neuroapraxia or an axonotmesis, and thus interrupting nerve function. After treating the muscular haematoma and so the interference with the nerve function, this recovers almost totally, except for the reflexes that remain absent. Since our unit opened in July 1973, we have seen a total of 12 nerve lesions (femoral nerve 9, ulnar nerve 2 and tibialis posterior nerve 1). The clinic of these lesions as well as orthopaedic and haematological treatment is discussed. Total recovery of function took from three to six months after hemorrhage.
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.
Platelet Membrane Actin and Myosin
Authors: Horák H. , Barton P.G. , Gibbs C.M.
Published: May 2019

Horse platelet membranes isolated by the glycerol lysis technique and subjected to SDS-PAGE showed large amounts of actin and variable amounts of myosin relative to other membrane proteins and glycoproteins. [14C]-2-dinitrothioadenosine diphosphate, when briefly incubated with whole cells, rapidly labeled the membrane actin component. Retention of myosin by the membranes during their isolation was optimized by lysing the cells and resuspending the membranes in Tris-HC1, pH 7.35, with 0.13 M KCl, 0.01 M NaCl, 2 mM MgCl2 and 0.01 mM CaCl2. Subsequently, significant amounts of actin and myosin could be eluted from the membranes with 10-3 M ADP but not with CDP, GDP or UDP. Actin was also eluted effectively from membranes prepared in Tris-NaCl, pH 7.35, by washing with 0.1 mM EDTA (in presence or absence of ADP). Despite repeated washings with either elution system, more than 50% of the actin remained associated with the membranes. When membrane vesicles with right side out (RO) and inside out (IO) orientation, separated by chromatography on Con A-Sepharose, were similarly washed identical results were obtained. Two dimensional electrophoresis of the membrane protein of IO vesicles separated two major actin components, one of which was differentially removed by prior treatment of the vesicles with 10-3 M ADP. It is concluded that (1) platelet myosin and two forms of platelet actin are associated with the cell membrane and that myosin and one form of actin can be displaced by ADP or EDTA while the second form of actin is more firmly attached, and (2) some actin is present on both membrane surfaces.
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.
Platelet Aggregation and Secretion: Effects of Anticoagulants and Divalent Cation Chelators
Authors: Lages B.A. , Weiss H.J.
Published: May 2019

The aggregation and secretion responses of human platelets to ADP and epinephrine have been studied in citrated platelet-rich plasma (cPRP) and in heparinized platelet-rich plasma (hPRP) containing varying concentrations of divalent cation chelating agents. ADP induced a single wave of aggregation in hPRP while E induced biphasic aggregation in both hPRP and cPRP. Secretion of 14C-serotonin (5HT*) occurred in both hPRP and cPRP in response to ADP and E, but generally to a lesser extent in hPRP. The extent of 5HT* secretion in hPRP, relative to that in cPRP, decreased with increasing ADP concentration but remained essentially constant oyer the same range of E concentration. Addition of citrate or EDTA to hPRP produced a clear maximum in ADP-induced 5HT* secretion at levels of 5-20mM citrate or 2mM EDTA and, in some cases, was associated with the appearance of a biphasic aggregation response. With citrate, but not with EDTA, the chelator concentration corresponding to maximum secretion varied directly with the concentration of ADP. These results indicate that ADP-induced secretion is a function of divalent cation concentration but does not occur only at the low levels of divalent cation present in cPRP and that divalent cation may be more directly associated with ADP-platelet interactions than in interactions of platelets and E.
Characterization of Multiple forms of Dicoumarol-Induced Prothrombins and Their Fragments, F1
Authors: Malhotra O.P.
Published: May 2019

To confirm that multiple forms of prothrombin are induced by dicoumarol, we isolated prothrombins from the plasma of dicoumarol-treated steers and divided them into three groups: 1) barium (Ba) citrate variant adsorbable by Ba citrate; 2) Ba oxalate material adsorbable by Ba oxalate but not by Ba citrate; and 3) the alumina atypical proteins(s) adsorbable by alumina Cγ-gel but not by the Ba salts. The purified atypical varieties showed a single component by several physicochemical criteria and contained as much antigenic material as did normal prothrombins, but bound different amounts of Ca2+. The Ca2+-binding ability of these proteins depends on the carboxylation of the glutamyl residues on the amino portion of prothrombin. The extra car-boxyl groups appeared to have little influence on the isoelectric points of prothrombins because the Ba citrate variant binding Ca2+, equal to 38% of the normal, focused at the same pH (pH 4.55 or 4.6) as normal prothrombin. Ba oxalate proteins bound Ca2+ equal to 25% of normal and focused at pH 4.75; the alumina variant(s) bound little, if any Ca2+ and focused at pH 4.82. To prove unequivocally whether or not multiple forms of dicoumarol-induced prothrombins are produced, normal and atypical prothrombins were digested separately with thrombin to yield Fragment 1 (F1) and Intermediate 1 (II). Both F1 and I1 were obtained in highly purified forms by employing DEAE-cellulose chromatography, gel-filtration, and affinity chromatography on heparin-agarose. Since the Ca2+ binding sites reside in Fl, they were characterized (Ca2+-binding abilities, electrophoretic mobilities, antigenic properties and gel-isoelectrofocusing). In general, these experiments confirmed our studies with intact proteins: i.e., that different forms of atypical prothrombins are induced by dicoumarol and they differ from each other primarily in the extent of carboxylation of the glutamyl residues present in the amino portion of prothrombin.
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.
Human Plasmin and Protease Inhibitors: Interaction in a Whole Plasma System
Authors: Highsmith R.F. , Burnett C.J. , Weirich C.J.
Published: May 2019

As a central event in the human coagulation and fibrinolytic pathways, intravascular proteolysis is normally limited by the presence of numerous circulating protease inhibitors. Plasmin, the fibrinolytic enzyme, is inhibited in vitro by purified preparations of antithrombin, Cl-esterase inhibitor, α1-antitrypsin, α2-niacroglobulin and inter-α-trypsin inhibitor. Using trace amounts of 125I-plasminogen and conventional gel filtration techniques, the elution profile of the labeled zymogen was studied in whole plasma before and after activation with urokinase (UK) or streptokinase (SK). Following activation, the major 125I-plasmin peak shifted to a higher molecular weight fraction of plasma than before activation. Lysine-sepharose affinity chromatography (LSAC), SDS-gel electrophoresis and immunodiffusion studies on the peak radioactive fractions revealed that a small percentage of the 125I-plasmin formed was bound to α2-macroglob-ulin while a majority was complexed to a component in plasma (mol. wt. ~ 60,000) immunologically distinct from the known human antiplasmins. Similar results were obtained when UK or SK activated plasmawas directly subjected to LSAC without prior fractionation. Minor differences were seen when the rate of activation was varied. However, in the presence of heparin, a significant amount of 125I-plasmin was complexed to antithrombin. These results, obtained in a whole plasma system, lend support for the presence of a new antiplasmin which is believed to be the same inhibitor recently described by others.Also, these data suggest that the other antiplasmins may play a minor, yet important role in the regulation of plasmin activity under different physiological conditions.
On the Reaction of Human Plasmin with the Primary Inhibitor of Plasmin from Human Plasma
Authors: Christensen Ulla
Published: May 2019

The interaction of human plasmin with the recently discovered primary inhibitor of plasmin from human plasma has been investigated. The inhibitor is an α2-glycoprotein with a molecular weight of 60000. Pure preparations of plasmin and the inhibitor were incubated for a specific time, after which residual plasmin was determined from measurements of the rate of consumption of α-N-bensoyl-L-arginine ethyl ester. Experiments were made over ranges of both plasmin and inhibitor concentrations and for a variety of incubation times. The results show that the reaction consists of at least two steps: rapid, reversible formation of an enzyme-inhibitor complex with the dissociation constant, K = 3nM, followed by a slow, irreversible transition with a rate constant, k = 6.5 10-3s-1. Amino acids with anti-fibrinolytic effect inhibit the formation of the first complex, but not its further transition.
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.
The Procoagulant Activity of Human Monocytes
Authors: Hiller E. , Ostendorf P. , Saal J.G.
Published: May 2019

Human leukocytes were separated into granulocytes, lymphocytes and monocytes. The pro-coagulant activity of the cell suspensions was assayed before and after endotoxin stimulation using one-stage methods and a two-stage method specific for tissue factor. The results indicate that the procoagulant activity in mixed leukocytes is almost exclusively derived from monocytes. Also mechanisms other than endotoxin stimulation, e.g., adherence to plastic surfaces, can cause monocytes to release tissue factor. Electron microscopic studies revealed the lysosomes of endotoxin-stimulated monocytes to be of giant size. Activated monocytes may play an important role in mediating the generalized Shwartzman reaction.
Influence of Deep Hypothermia on Coagulation Factors in Cardiac Surgery
Authors: Doutremepuich Ch. , Chauve A. , Oca C. , Boisseau M. , Fontan F.
Published: May 2019

This study was conducted on a group of 55 children with ages ranging from 15 days to 35 months. Surgery was performed in deep hypothermia for congenital cyanotic and non cyanotic heart disease. In 9 of the cases, an additionnai study was made of the patient’s haemostasis at normal body temperature and later in deep hypothermia (average 23°C) before the heparin injection required for the extra-corporeal circulation. Test results showed, in deep hypothermia, a decrease of platelet count(

= 126000 ± 62650/mm3), prothrombin value(

= 69.9 ± 9.02%), fibrinogen (

= 1.84 ± 0.4lg/l), a lenghthening of thrombin time (

= 39.33 ± 8.49 sec)and A.P.T.T.(

= 55-12 ± 8.70 sec), an increase of fibrinolytic activity. Heparin level in the blood for the total group of patients(55) measured after rewarming and the neutralization of heparin by protamin, was found to be within acceptable bounds(0.10 - 0.20 UI/ml) in 41.81% of the cases. In comparison, out of a group of 850 patients who underwent surgery and under moderate hypothermia (average 28°C) 68.88of the cases showed a satisfactory residual blood heparin level (0.10 - 0.20 Ul/ml).
Isolation of the Antithrombin-Thrombin Complex and Platelet Factor 4 by Solid Phase Heparin
Authors: Bleyland H. , Róka L.
Published: May 2019

Solid-phase-heparin as heparin-agarose [Hep-Ag] was used for the isolation of the complex formed by antithrombin and thrombin. This complex has a lower affinity to heparin than antithrombin III. The complex has no antithrombin activity. The increase of the complex during blood coagulation can be seen by the two-dimensional Immunoelectrophoresis in agaroee, which contains heparin (Sas et al., Thromb.Res. 6, 87-91, 1975).

Platelet factor 4 (PF 4) was isolated by a three step procedure: 1. precipitation by divalent cations according to Burstein, 2. adsorption to Hep-Ag and desorption with high salt concentrations, 3. desalting over Sephadex G 25 fine. PF 4 was prepared from isolated platelets, platelet rich plasma and serum.
Role of Sialic Acid in the Dysfibrinogenemia Associated with Liver Disease
Authors: Martinez J. , Palascak J. , Kwasniak D. , Shapiro S.S.
Published: May 2019

We have described an abnormal fibrinogen in 6 patients with liver disease who had prolonged plasma thrombin times due to impaired fibrin monomer aggregation. To investigate the role of sialic acid in this functional abnormality, fibrinogen was purified from normal and patient plasmas by the glycine precipitation method. Sialic acid content of the fibrinogens was measured by the thriobarbituric acid assay after acid hydrolysis. Normal fibrinogen had 6.1 ± 0.5 residues per molecule of fibrinogen, whereas patient fibrinogen sialic acid content ranged between 7.5 and 10 residues per molecule. The reduced fibrinogen demonstrated normal mobility of Aα, B3 and γ chains on SDS Polyacrylamide gel electrophoresis when stained for protein and, similar to normal fibrinogen, only the Bβ and γ chains stained with PAS. The degree of prolongation of the thrombin times of the purified patient fibrinogens appeared to correlate with the increase in the fibrinogen sialic acid. The effect on fibrin monomer aggregation of decreasing patient fibrinogen sialic acid content was studied. Partially desialated patient fibrinogen was prepared by treating the protein with Vibrio cholerae neuraminidase for varying periods of time. Partial removal of sialic acid from patient fibrinogen resulted in normalization of the thrombin time and improvement in fibrin monomer aggregation. Thrombin times ranged from 31.5 to 49.5 seconds prior to removal of excess sialic acid compared to 20.5 to 25.5 seconds post removal. These findings indicate that the dysfibrinogenemia associated with liver disease is biochemically characterized by increased sialic acid content and removal of this sialic acid results in a functional normalization of the protein.
Platelet Coagulant Activities in Patients with Thrombotic Disorders
Authors: Walsh P.N.
Published: May 2019

Platelets can initiate intrinsic coagulation by two alternative mechanism, one involving the activation of factor XII by ADP-stimulated platelets (contact product forming activity), and the other involving activation of platelet-associated factor XI by collagen-stimulated platelets (collagen-induced coagulant activity).Subsequently, platelet membrane phospholipo-proteins are made available by which platelets can first promote factor-X activation (intrinsic factor -Xa forming activity) and finally prothrombin activation. Specific assay techniques for each of these platelet coagulant activities have been applied to the study of patients with thrombotic and prethrombotic disorders. We have demonstrated an association between platelet coagulant hyperactivity and the development of post-operative deep vein thrombosis in patients undergoing reconstructive surgery of the hip. In patients with acute primary retinal vein occlusion two- to three-fold elevations of platelet coagulant activities concerned with the initiation and early stages of intrinsic coagulation have been observed whereas plasma coagulation and platelet aggregation studies have been normal. In patients with transient cerebral ischemic attacks and normal serum lipids we have found two- to three-fold elevations of platelet coagulant activities concerned with the initiation and early phases of intrinsic coagulation but normal platelet factor 3 activity and normal plasma coagulation and platelet aggregation studies. These studies indicate an association between platelet coagulant hyperactivity and various venous and arterial thrombotic disorders. This association may have important pathogenetic implications.
The Role of γ-COOH Glutamic Acids in the Enzymatic Functions of Factor Xa.
Authors: Lindhout M.J. , Kop-Klaassen B.H.M. , Hemker H.C.
Published: May 2019

In order to study the function of γ-carboxyglutamic acid residues (gla) in factor X(a) we compared the properties of factor X(a) and abnormal factor X(a) from anticoagulated cowl (PIVKA-X(a)), We studied Ca++ and phospholipid binding, spectral changes upon Ca++-binding and the activation by a specific fraction from+ Russells Viper venom (RW). In factor X but+ not in PIVKA-X a cooperative binding of about 5 Caions as well as a cooperative effect of Caions in spectral shift was observed.PIVKA-X can be converted into an active esterase by RVV. This reaction is Ca++ dependent. The Hill coefficient of this dependence is 2.8 for factor X and 1.0 for PIVKA-X. Factor Xa binds to micelles of an equimolar mixture of (dioleoyl) phosphatidyl-serine and dioleoyl phosphatidylcholine in the presence of Ca++ ions. Consistent with the inability of PIVKA-X to bind Ca++ ions cooperatively, PIVKA-X does not bind to phospholipid vesicles. Kinetic experiments on the activity of factor Xand PIVKA-X , in the absence of Ca+ions and phospholipids towards amides shows that both enzymes have identical Km values for each of the substrates but different kcat values. The results indicate a positive effect of gla residues on the rate of hydrolysis of amides. These studies suggest that the gla residues not only function to anchor factor X(a), on phospholipid via Ca++ ions, but that they also assist in obtaining conformational changes in the protein molecule as distant as to influence the active site. Indeed even the “anchor function” may depend upon such changes.
Species Differences in Platelet Glycoproteins
Authors: Nurden A.T.
Published: May 2019

Reports of considerable species differences in the mammalian erythrocyte membrane glycoprotein composition led us to conduct a preliminary investigation of the platelet membrane glycoprotein composition in a range of mammals (15 species). Electrophoretic studies of detergent solubilized membranes and protease released acidic glycopeptides suggest considerable species differences in the extent of glycosylation of surface orientated proteins, with differences observed even between closely related species of primates. The failure to locate the large PAS-staining GP I band following SDS-polyacrylamide gel electrophoresis of cat platelet membranes, even though cat platelets have a comparable sialic acid content (21.3±2.3 ug per 109 platelets) to human platelets, may have implications concerning the proposed roles for this glycoprotein in platelet function. It is suggested that account should be taken of possible species differences in platelet surface structure during the interpretation of platelet function tests using animal models.
Powerful Stimulation by Formaldehyde of Platelet Functions
Authors: Holmsen H. , Dangelmaier C.A.
Published: May 2019

Low concentrations of formaldehyde (HCHO) increased both the rate and the extent of thrombin-induced secretion of ATP, ADP, preabsorbed 14C-serotonin, β-N-acetyl glucosaminidase and β-galactosidase from gel-filtered human platelets (GFP). The effect was optimal (7-fold stimulation) with 0.03-0.07 u/ml of thrombin; above 0.1 u/ml there was little effect. With 0.05 u/ml of thrombin maximal stimulation was seen with 0.03-0.5 mM HCHO while the aldehyde was inhibitory above 1 mM. The degree of stimulation increased with the time of exposure of the platelets to HCHO and was maximal after 15-20 min. (37°C). Incubation of control GFP (without HCHO) at 37°C caused a progressive increase in the secretory response 2-3 fold above preincubation values. The degree of stimulation was not altered by indomethacin or aspirin suggesting that prostaglandin/thromboxane formation was not involved.HCHO (0.3 mM) caused a progressive reduction in the steady-state level of metabolic ATP which leveled off after 15-20 min. of incubation. There was no change in the adenylate energy charge, but HCHO caused a transient increase in lactate production in the presence of antimycin A. Similar results were obtained with ADP and epinephrine as inducers of serotonin and adenine nucleotide secretion while the response to collagen was abolished by these low HCHO concentrations. The primary and secondary aggregation responses to ADP and epinephrine, which had been markedly depressed by GFP maintained at 37°C for longer periods of time, was restored by exposure to HCHO. The mechanism of activation, preservation and restoration of platelet activities by HCHO is obscure.
A Tentative Cause of Discrepancy Between FVIII:C Estimation
Authors: Seghatchian M.J.
Published: May 2019

Biochemical analysis based on the molecular size and change was performed on normal, patient plasmas and clinical concentrate to differentiate various molecular forms of FVIII:C. Assay of the effluent fractions by one stage, two stage and chromogenic methods revealed two major peaks of activities, measured to differing extents by various methods. Thromboplastin generation methods (two stage clotting and chromogenic) were particularly sensitive to smaller forms, which had a faster electrophoresis mobility and eluted in the gamma globulin region. The one stage method measured mainly the aggregated forms (MW> 300.000), with the slower electrophoretic mobility.

A discrepancy of about 30% between the two methods could account for the detection of the smaller form, which predominated in plasma rather than concentrate. This had a preferential adsorption on Al(OH)3 and some FVIII.C neutralising activity. A similar peak of activity was identified in haemophilia and V.Wd. plasma isolated on sepharose suggesting that the whole plasma contained an inhibitory factor acting on the clotting activity. Excess factor X and traces or thrombin-like enzymes could be identified in this region. Thus the role of these factors in accelerating the rate of FXa generation by activating FVIII can not be excluded.

Regardless of the molecular nature of this form of FVIII, this activity may play an important role in controlling haemstasis via VIII activation and may explain the discrepancy between assay of plasma versus concentrate.
Photometric Assay of Factor VIIIC With a Chromogenic Substrate
Authors: Vinazzer H.
Published: May 2019

By the aid of chromogenic substrates, highly specific assays of some serine proteases can be carried out. The substrate used for factor VIIIC-assays was Bz-Ile-Glu-Gly-Arg-pNA [S-2222 KABI) which measures factor Xa. When all components necessary for factor Xa activation except factor VIIIC are kept at constant levels, the resulting Xa-activity is in direct relation to the concentration of factor VIIIC. The substrate plasma was a mixture of hemophilia A plasma with factor VIII inhibitor plasma with a remaining inhibitor activity of between 0.1 and 0.5 units per ml. This substrate was defibrinated by ancrod. For assays of factor VIIIC, this plasma was mixed with the diluted test plasma, cepheloplastin, and calcium chloride at 37°C After a constant activation time, the chromogenic substrate was added and the difference in OD/min was measured at 405 nm. The calibration curve was linear between 1% and over 200% factor VIIIC activity, and the average CV was 7.9%. This method was compared to a standard one-stage method for factor VIIIC. Identical results were obtained in plasma samples of normal individuals, in samples of high factor VIIIC, activity, in plasma from hemophilia A patients, and in factor VIII concentrates. The advantages of this method over the clotting method are: independence of the results from variations of factors V,II, and I in the reaction mixture, stability of the reagents, a better discrimination of factor VIIIC levels in the range between 30% and over 100%, and the possibility of automatization of the method.