bluuebunny/crossref_metadata_2025 huggingface dataset:
Chemical Synovectomy in Induced Haemophiliac Synovitis
Authors: Darracott J. , Cole F.M. , Belbeck L.
Published: May 2019

The response to chemical synovectomy with Yttrium 90 Colloid Citrate in induced Haemophiliac synovitis was studied.

The synovitis was induced in five Haemophilia ‘B’ (Factor IX deficient) dogs by injection of 2.5 ml. of each dogs blood into their own knee joints at least 26 times over a period of one year. 100 uC of Y90 Colloid Citrate was injected into one knee of each of the three surviving dogs. One year later the synovium and articular cartilage of both knees of the surviving dogs were compared.

The joints injected with Y90 macroscopically appeared normal; microscopically there was minimal evidence of inflammation and iron accumulation; there was no discernable change in the articular cartilage on routine and metachromatic stains.

The joints not injected with Y90 showed heavy pigmentation and thickening macroscopically; microscopically there was marked inflammatory infiltrate and hemosiderin deposition.

Chemical synovectomy is relatively atraumatic and it is suggested that consideration be given to its use in man, in the treatment of haemophiliac synovitis.
Arterio-Venous Fistula to Facilitate Self Transfusion for the Hemophiliac
Authors: Hiloartner M.W. , Stubenbord W.T.
Published: May 2019

The veins of many multiply transfused older hemophiliacs have been destroyed through misuse making self transfusion extremely impossible and transfusion by trained personnel difficult. To improve the superficial veins in the forearm 4 arterio-venous fistulae were performed in 3 of 125 patients in the Hemophilia Clinic of the New York Hospi tal-Cornel 1 Medical Center. In 2/3 a modified Brescia procedure was done in the forearm and anti-cubital fossa. The fistulae matured in 3 weeks to 2 months producing sufficient venous hypertrophy which allowed the patient to be trained for and successfully carry out self transfusion. The only complication has been occlusion of the fistula through pressure,, This procedure should be considered before veins are destroyed and frequent hemorrhage in the forearm and elbow produce thickening of the skin, superficial tissues with limited movement of the wrist and elbow and complicate the surgery. The outcome of the procedure with freedom for the patient from medical personnel outweighs the additional surgical procedure in patients with hemophilia.
Nitro Bleu Tetrazolium Reduction test in Hemophilia
Authors: Birardi F. , Morfini M. , Salti F. , Seri A. , Rossi Ferrini P.
Published: May 2019

Nitro Bleu Tetrazolium (NBT) reduction by neutrophils has been reported in many patients with bacterial infections or other inflammatory states. Occasionally it has been observed that hemophiliacs frequently displayed high NBT-reduction values beyond infectious diseases. NBT histochemical reduction by leukocytes was investigated in 47 hemophiliacs and 20 apparently health volunteers. Leukocytes were isolated by sedimentation from heparinized blood (10 U./ml). The buffy coat is incubated with 0,2% NBT solution v/v for 30 min. at 37°C and therefore for 15 min. at room temperature. Thin smears were stained with Pappenheimer’s stain. A low score (NBT-positive neutrophils<10%) in 17 normal subjects whereas in 34 hemophiliacs a significant (X2=7,21; p<0,05) high score (NBT-positive neutrophils >10%) was observed. Correlation with factor VIII or IX levels, frequency of hemarthrosis or transfusions is not significant (p>0,5). Nevertheless in 3 young hemophiliacs, unaffected from hemarthrosis, the NBT-test is costantly negative. The findings suggest that in hemophiliacs the phagocytic function by neutrophils is frequently enhanced in course of the chronic synovitis.
Elbow Arthroplasty in Hemophilic Arthritis
Authors: Martinson A.M.
Published: May 2019

The elbow is the second most commonly affected joint in hemophiliacs. In the adult patient with pain and frequently recurring bleeding episodes in an elbow, unresponsive to splintage, exercise and steroids, we have used resectional arthroplasty of the radial head and subtotal synovectomy for symptom relief. At one hospital 13 patients (15 elbows) ranging in age from 17 to 35 have had this procedure. All patients have had significant relief of pain and bleeding and this relief has been lasting; our longest followup is 8 years. No patient has made a significant gain in overall range of motion, however many have gained a small amount of rotation which has resulted in considerable functional improvement. One total elbow was inserted for recurrent pain and decreasing motion in a previously-operated elbow which sustained significant trauma. Two years later the patient has excellent function without bleeding but x-rays show the components to be loose. Revision at a later date will most likely be required.
Alternatives to oral Surgery for Hemophiliacs
Authors: Sachs S.A.
Published: May 2019

Modern hemophilia therapy allows comprehensive dental care to be performed safely. Essential to this is a mutual understanding between patient, hemotologist and dentist. Although much attention has been focused on the management of hemophiliacs who require the removal of teeth, care should be directed towards means of conserving the dentition and supporting structures. The relationships between pulpal and periodontal disease, the procedures utilized to treat them and hemophilia will be discussed.
Evaluation and Approach to Indicated Oral Surgery
Authors: Mulkey T.F.
Published: May 2019

The proper hematologic, psychological and clinical evaluations, including factor assay, inhibitor level, if any, previous bleeding history, response to previous dental treatment and status of dental health are all essential prior to any oral surgical treatment. In depth explanations of procedure, infusion of missing factor to the 20%-50% level, modified surgical technique and rigid post extraction instruction, have proven to be very effective in the management of oral surgery on hemophiliac patients.
Component Therapy Fractionation and the Economics of a Regionalized Blood Delivery System
Authors: Aledort L.
Published: May 2019

The delivery of blood and its components to a large urban area is a major challenge. Donor recruitment, processing, delivery and financial support are significant problems. The hemophilia demands in an area with over 1,000 patients heightens the problem. The evaluation and operation of such a program will be demonstrated. Emphasis on the technology of component therapy will be stressed. Blood bankers, Food and Drug Administrators, third party payers, clinicians, as well as representatives from different national blood delivery systems will speak.
Legal Ways to Contest job Discrimination Among Hemophiliacs
Authors: Amitrani Paul , Goldman Donald
Published: May 2019

Despite many major advances made in the treatment of hemophilia in the past decade, many employers remain extremely reluctant to hire a hemophiliac. This is the case even when the individual is not even mildly disabled by the ravages of the affliction, and when his bleeding episodes are under control. The employment problem among many hemophiliacs is chronic, arising from a vicious cycle in the past of missed educational opportunities, inability to work steadily, severe orthopedic problems and lack of positive reinforcement.

We describe the methods hemophiliacs have used to obtain and maintain gainful employment. We also describe legal ways in which the hemophiliac can prevent discrimination in hiring and which they can use to contest dismissals. The need for public education of employers will be emphasized and the resources available to victims of disability or chronic unemployment will be discussed.
“Santal” a Hemophiliac Data Bank in the Network Cyclades
Authors: Bosser C. , Duport J.P. , Frommel D. , Noël B. , Saint Paul E.
Published: May 2019

For the past two years a group of sixty hemophiliacs regularly treated by the same medical team has produced the data of “Santal”. This data base is running in a real context on the network Cyclades (which is the precursary system of Transpac) and which enables clinicians and health economists of countries connected to the network to collaborate. The initial aim of this data base was to gain efficiency in the qualitative value and optimality of the therapeutics of hemophilia.

Santal seems to be an illustration on a large scale of a pool of knowledge and ability for the healing of chronic diseases requiring continuous or discontinuous treatments. By using informatics and some branches of mathematics in the medical field, this work has benefitted from R. Thom’s theories in an interdisciplinary and qualitative approach to discontinuous phenomena.
Effects of Psychiatric Intervention on Concentrate Usage
Authors: Ackerman L.L. , Eyster M.E. , Charney D. , Haverstick J.
Published: May 2019

To investigate possible psychosocial causes for increased Factor VIII requirements, concentrate usage was assessed from records submitted monthly by a 16 year old boy with a Factor VIII level of < 1%. This patient was using almost 5 times more concentrate per year than our average home therapy patient.

Group, family and individual psychotherapy, as well as relaxation techniques were utilized with the psychiatrist unaware of the amount of concentrate used. Concentrate usage during the 15 month period immediately preceding therapy was 23,051 ± 3,441.44 Units/month for a total of 344,900 units. For the 15 month period following initiation of therapy, usage was 14,942 ± 3,204.81 units/month for a total of 221,595 units. This represented a 36% decrease in concentrate usage and a saving of approximately $12,000 while the total cost of psychiatric therapy was $1,115.

This case study illustrates that psychological factors may affect concentrate usage. The results also suggest that psychological dependencies and stress induced needs that develop in some patients on home therapy can be effectively treated by psychosocial intervention.
A Comparison of Current Prophylactic Therapy in Canada and the United Kingdom
Authors: Aronstam A. , Inwood M.J. , Arblaster P.G.
Published: May 2019

A survey of Canadian and British physicians actively treating hemophiliacs was undertaken to assess whether the difference in the two countries’ health care delivery systems influenced their attitudes to prophylactic therapy. Questionnaires defining prophylactic therapy and relating its use to a number of activities were sent to directors of hemophilia treatment centres representing adequate geographic coverage in both countries. 47 replies were received (10/13 Canadian and 37/40 British). 50% of the United Kingdom (UK) physicians used prophylactic therapy on occasion as compared to 92% of Canadian physicians. However, only 20% UK physicians versus 80% Canadian used it on a routine basis for selected patients. 43% British physicians compared to 50% Canadian gave prophylaxis prior to certain athletic or social events. The mean dose of Factor VIII or IX given by Canadian physicians was higher and individual doses were given more frequently. The increased acceptability of this form of therapy in Canada may be related to easier availability of Factor concentrates and a more flexible health care system.
Further Experience on the Effect of High Pre-Donation Factor VIII Levels in Blood Donors on the Factor VIII Content of Small-Pool Fractions
Authors: Beeser H. , Eqli H.
Published: May 2019

Because of the well known wide normal range of the factor VIII activity between 60 to 170% I man, selecting of donors with high activity levels would be of advantaae for the preparation of factor VIII concentrates. This is especially true for preparing small-pool fractions, as for technical reasons the final product cannot be controlled for its factor VIII content. In preliminary investigations, we reported on elsewhere, high factor VIII activity in donors estimated before a donation had been rarely reproducible before a second donation after 8-12 weeks. So as a preliminary result of finding a donor’s factor VIII level varying from donation to donation selecting of plasmas with high factor VIII content for concentrate preparation could only be establishedby re-estimating the activity before each donation. Proceeding in this way would be much too troublesome. To get more reliable information whether a healthy subject’s high factor VIII plasma level is distinctly varying or rather constant we assayed the plasma of 200 donors with factor VIII activity > 120% two times more before donation. The results confirmed our preliminary findings, especially the fact that a high plasma factor VIII activity in experienced donors was rarely reproducible when re-estimated before a second and third donation. As a consequence selecting of donors with high factor VIII procoaqulant activity for preparing small-pool factor VIII concentrates is impracticable.
Biomechanical Alterations Caused by Musculo’s Keletal Bleedings.
Authors: Hofmann P. , Schumpe G. , Brackmann H.H.
Published: May 2019

10,000 bleeding events into the musculo-skeletal system of 500 hemophiliacs were brought into statistical relation with the clinical and radiological findings. A significant interaction between alteration of distinct biomechanical parameters and the frequency of musculo-skeletal - especially intraarticular - hemorrhages was seen. The initial change due to intraarticular hemorrhage was found to be muscular imbalance leading to a) malpositions of the articular axes, b)excentric restriction of movement, c) reduced weight bearing capacity. Unphysiological gait results and induces an increased frequency of local bleedings as well as biomechanically predetermined overstresses within ipsilateral joints and muscles. The latter induces joint or muscle bleedings in the presence of hemophilic coagulation defect. The alterations mentioned above are amplified by skeletal growth.
Local use of Epsilon-Aminocaproic Acid in Dental Therapy
Authors: Berry P.R. , Coster A.B. , Berry E.W.
Published: May 2019

Oral bleeding following trauma or dental extraction has been treated with local application of Epsilon-Aminocaproic Acid (S-ACA) in six patients including one with factor VIII antibodies. This treatment has produced rapid cessation of oozing with the formation of firm clots. Following the initial application E-ACA usage has been continued as a mouthwash. Clinically this appears to be a rapid and safe adjuvant therapy for oral bleeding.
HBS and “e” Antigen-Antibody Systems in a Population of Hemophiliacs
Authors: Mariani G. , Bottini F.R. , Mazzucconi M.G. , DeBac C. , Taliani G.
Published: May 2019

In a population of 70 patients with congenital coagulation disorders, most of them hemophiliacs (61), HBS antigen (Ag), HBg antibody (Ab) as well as “e” Ag and Ab have been studied. These patients have been repeatedly exposed in the past to replacement therapy especially with commercial freeze-dried concentrates. Fourteen patients (20%) experienced juandice in the past four years. HBsAg was detected in 3 patients (4%) and HBsAb in 47 (67%). “e”Ag was detected in 8 patients (11%) and “e”Ab in 7(10%) . All the ‘e”Ag-positive patients but one were HBsAb-positive, although at a low titre. None of the “e”Ag-positive patients had raised serum-transaminases levels.

The clinical and prognostic significance of these hepatitis B-related humoral findings remains obscure.
Surgical Problems on Patients with Coagulation Defects
Authors: Mandalaki T. , Golematis B. , Loizou C. , Delikaris P. , Papalabros E. , Tsigris Ch.
Published: May 2019

Surgical experience over a period of three years referring to 40 cases of hemophiliacs, with special reference to rare surgical complications, is presented. Critical evaluation of the postoperative results suggests that two main points should be equally taken into consideration for a successful surgical treatment, a) Hematology-substitution treatment, b) Surgery-local hemostasis. Substitution therapy is adjusted according to the nature and severity of the defect. Full preoperative correction of missing factor to normal levels. A level over 30% should be maintained post-operatively for at least 15 days, particularly in major abdominal surgery. The intensity of substitution treatment must be related to the kind of operation. Regarding Surgery meticulous local hemostasis using fine silk ligatures and atraumatic needle chrome cat-gut, for the gastrointestinal system, preferably with interrupted sutures. Electrocoagulation is avoided. Removal of skin sutures should be delayed, as well as peroral alimentation in gastrointestinal tract Surgery.
The Natural History of “Inhibitors” to Factors VIII and IX in Hemophiliacs
Authors: Harris C.E.C.
Published: May 2019

Since 1954 an increasing number of adult hemophiliacs have been followed and “inhibitors” have been found in a minority of patients. A retrospective study was made of 9 factor-VIII and of 9 factor-IX cases who have attended with sufficient regularity for adequate documentation. The presence of this complication was, until recently, suspected because of failure clinically to respond to replacement therapy and confirmed by the original Biggs’ technique in the laboratory. In the past k years, this has been replaced by her method using serial dilutions at room temperature and at 37°C, incubated for 1 hour. In addition, studies have been made of factor recovery and survival following infusions.

In no case were immuno-suppressive agents used. There were no deaths. Life-threatening crises were met by conservative measures, including hypothermia, and by the use of porcine factor-VIII. Instances when in vivo survival of infused concentrate was shortened were usually managed successfully by more frequent administration of equal or lesser dosage quantities; it was exceptional in such cases to witness enhancement of “inhibitor” potency as a consequence.

In no less than 6 of the 9 patients would it seem that the “inhibitor” has disappeared and full clinical response is now being obtained with standard dosage schedules. The nature of “inhibitors” would not seem to be well understood.
Oral Prophylactic Therapy in Hemophilia A and B (Factor VIII and IX Deficiencies)
Authors: Stapp W.F. , Boudreaux H.B.
Published: May 2019

The purpose of this ongoing nutritional study with massive doses of choline ultimately is to try and establish an alternative means of prophylactic therapy to recently proposed use of plasma fractions, plasma factor concentrates and earlier so-called “bleeding and clotting diets”. Contrary to other prophylactic therapy studies, our premise is that if all other hemostatic and homeostatic factors are near normal through optimum nutrition, the over-all importance of Factor VIII and Factor IX deficiencies are less significant.

Case reports of patients with Hemophilia A and B are presented, which indicate a comparative decrease in the plasma fraction requirements for the treatment of the bleeding episodes in these patients during long term therapy with megavitamin doses of choline (a refinement of Boudreaux’s “peanut factor”). One case (WFS) of severe Hemophilia A with less than 1% Factor VIII, under therapy for over 10 yeara has had periods of remission not requiring plasma factor infusions for almost 2 years.

From our combined studies, we conclude that a long term nutritional prophylactic therapy program with emphasis on choline in Hemophilia A and B can result in a decrease in plasma needs. antibody formation and the occurrence of hepatitis.
Interactions of Human Fibrinogen in Solution
Authors: Serrallach E. , Känzig W. , Hofmann V. , Straub P.W. , Zulauf M.
Published: May 2019

The intriguing diversity of published translational diffusion constants for the fibrinogen molecule can hardly be explained, unless interactions between the molecules are postulated. In the present study we have investigated the possible effect of molecular association and electrostatic intermolecular interactions on the Brownian motion. The translational diffusion coefficient DT, the rotational diffusion coefficient around the minor axis DR and the sedimentation coefficient have been measured. The methods used were dynamic light scattering and analytical ultracentrifugation. The samples were solutions of purified human fibrinogen. The correlation-function corresponding to DT deviates from a single exponential. The initial slope is found to depend on concentration, being DT = (1.7 ± 0.3) 10-7 cm2/s at 10mg/ml, pH 7.4 and 0.15 molar Tris-NaCl, and increases at fibrinogen concentrations below 2mg/ml. These results are compatible with a polydispers solution, in which single molecules are in equilibrium with pair and higher aggregates. The nature of the aggregates is end-to-end as indicated from the difference between the two rotational diffusion constants DR = 40000 ± 20% and DR = 10000 ±30% s-1. On the basis of the Hall-Slayter model and assumption of end-to-end association we calculated the ratio of the sedimentation coefficient of single, pair and triplet associates, being 1:1.14:1.20. Therefore, it is difficult to separate them in a sedimentation run. For ionic strength below 0.05 molar and low fibrinogen concentration (0.lmg/ml) a fast decay appears in the correlation, indicating that the Brownian motion is strongly influenced by electrostatic interactions.
Platelet Factor 4 (PF4) and Protamine Neutralisation of Heparin in Plasma
Authors: Michalski R. , Lane D.A. , Pepper D. , Kakkar V.V.
Published: May 2019

The ability of PF4 and protamine sulphate to neutralise heparin in plasma has been studied using a specific anti-Factor Xa assay and a KCCT assay to measure residual heparin. When heparin is added to plasma in vitro PF4 and protamine neutralise almost equivalent amounts of heparin on a weight basis, 1.0 unit of heparin being neutralised by approximately 20 μg of PF4 and 15 μg of protamine. Similar results are obtained using either of the heparin assays. However, following intravenous injection of heparin only about one half of the circulating heparin could be neutralised in vitro by PF4 or protamine when it was measured by anti-Factor Xa assay. Total neutralisation was obtained with both neutralising agents in the KCCT assay system. These results demonstrate that the choice of assay is important when a protamine titration is used to measure plasma heparin levels, and that PF4 and protamine are unable to totally neutralise circulating antithrombotic heparin activity.
Comparison of Sodium and Calcium Heparin in Prophylaxis of Venous Thromboembolism
Authors: Cade J.F.
Published: May 2019

Sodium and calcium heparin in minidose have both been shown to be highly effective in reducing the incidence of venous thromboembolism in hospital patients but there is little comparative data on their relative efficacy. In the present study, sodium heparin, calcium heparin or placebo (isotonic saline) was given to 148 high-risk patients. Treatment was allocated randomly, was double blind and was given by subcutaneous injection 12 hourly for 10 days or until the patient was fully ambulant. The heparin was prepared by the same manufacturer from the same batch and both active preparations (each 5000 U) and placebo were made into coded ampoules of 1 ml. Leg scanning was performed daily, the sites of injection were checked for pain or bruising, the partial thromboplastin time (PTT) was measured 3 and 6 hours after injection on at least two occasions and a lung scan was obtained when possible on the tenth day. Positive leg scans were found in 31% of control patients, 8% of patients given sodium heparin and 4% of patients given calcium heparin. These differences are statistically significant. There were no significant differences in PTT’s between groups nor in lung scans. It is concluded that both sodium and calcium heparin are effective in reducing the incidence of calf vein thrombi but that in equivalent dose calcium heparin may be somewhat more effective.
Primary Structure of Bovine Factor IX
Authors: Titani K. , Enfield D.L. , Katayama K. , Ericsson L.H. , Fujikawa K. , Walsh K.A. , Neurath H.
Published: May 2019

Factor IX is the zymogen of a serine protease which participates in the middle phase of intrinsic blood coagulation. Bovine factor IX is a single chain glycoprotein with a molecular weight of 55,000. Upon activation by factor XIa (act ivated factor XI), cleavage of two specific peptide bonds (Arg-Ala and Arg-Val) gives rise to factor IXa (act ivated factor IX) having a two chain structure(mol wt 46,000) and an activation peptide(mol wt 9,000)(K. Fujikawa, M.E. Legaz, H. Kato and E. W. Davie, Biochemistry, 13 4508 (1974). A protease from Russell’s viper venom (RVV) also activates factor IX by cleaving only one specific peptide bond (Arg-Val) (P.A. Lind-quist, K. Fujikawa and E. W. Davie, Fed. Proc., 35, 1353 (1976)). Bovine factor IXa(RVV) thus activated has a molecular weight of 55,000 identical to that of factor IX but is composed of two peptide chains held together by one or more disulfide bonds. After reduction and alkylation of disulfide bonds, two peptide chains were separated on a column of SP-Sephadex C-25 by a salt and pH gradient in the presence of 7 M urea. The sequence analysis of factor IX is in progress on these two peptide chains using their subpeptides produced by cleavage at methionine, lysine, arginine and tryptophan. Preliminary results indicate that the sequence of factor IX is homologous to that of factor X and related also to prothrombin and other vitamin K-dependent blood coagulation factors. A high degree of sequence homology is also observed between the heavy chain (the carboxy1-terminal half of the molecule) of factor IXa and pancreatic serine protease such as trypsin, chymotrypsin and elastase.
Molecular Mechanisms of Contact Activation
Authors: Griffin John H.
Published: May 2019

Exposure of human plasma to various negatively charged surfaces (glass, kaolin, connective tissue components, urate crystals, endotoxin, etc.) leads to ‘contact activation”of plasma and thereby initiates the activation of the intrinsic coagulation, the kinin-forming, and the plasma fibrinolytic systems. Studies of plasmas which are functionally defective in contact activation reactions implicate the following proteins in contact activation reactions: (1) Hageman Factor (HF) (Factor XII); (2) prekallikrein (Fletcher Factor); (3) high MW kininogen (Fitzgerald, Flau¡eac, or Williams Factor) (Contact Activation Cofactor); and (4) Factor XI. Recent studies suggest that a previously postulated HF-dependent plasminogen proactivator is identical to prekallikrein.

A mixture of purified HF, high MW kininogen, prekallikrein, and kaolin gives the same rapid rate of activation of purified Factor XI as does an equivalent aliquot of plasma plus kaolin, thus suggesting that contact activation of Factor XI is fully explicable in terms of the interactions between these proteins. Using these purified proteins, the molecular mechanisms of contact activation have been studied extensively, and the results which will be presented support the following new hypothesis. Surface-binding of HF does not convert a detectable fraction (<1%) of HF molecules to active enzymes as previously supposed, but instead it renders HF much more susceptible (100 to 400-fold) to proteolytic activation by plasmin or kallikrein in the presence of high MW kininogen. High MW kininogen is a surface-bound HF cofactor for each step in the reciprocal activation of prekallikrein and HF, and for the limited cleavage of Factor XI by activated HF.
Antithrombin III: Critical Review of Assay Methods. Significance of Variations in Health and Disease
Authors: Ødegård O.R. , Abildgaard U.
Published: May 2019

Thrombin and factor Xa which are added to plasma are inactivated by At-III. The reactions are accelerated by heparin, permitting assay systems which rapidly measure At-III content of diluted plasma. Without heparin, the (slow) inactivation rate may be measured. The review of existing activity assays(fibrinogen or chro-mogenic substrates), and semiquantitative assays, will focus upon their usefulness and limitations, and to the correlation to results of immunoassay.

In health, a narrow range of At-III concentration (and activity) is found. The level is low in infancy. Fertile women have on the average lower levels than men. In old age the level tends to drop. In the normal material that will be reported, one man was the propositus of a “new” family with At-III deficiency and thrombophilia. In a clinical material (n=2000), studied with amidolytic assay, subnormal At-III levels were found in hereditary deficiency, liver cirrhosis, disseminated intravascular coagulation and in some cases with acute thrombosis. Piasma samples containing soluble fibrin (positive ethanol gelation test) were classified as to the degree of hypercoagulation according to the values of other coagulation tests (grade 1-4, grade 4=DIC). The mean At-III plasma level decreased with increasing degree of hypercoagulation. Due to its high accuracy and simplicity, we have chosen the amidolytic activity assay with heparin and thrombin (Thromb. Res. 6, 287, 1975) for routine work.
Hereditary Deficiency of Antithrombin III.
Authors: Marciniak E.
Published: May 2019

The recognition of biological importance of antithrombin III (AT III) stems from the description of a familial thrombotic tendency associated with reduced levels of this protein in blood. A limited number of such families has been reported in the literature. All of the affected members are heterozygous for the abnormal gene. In many, the quantity of AT III as evaluated by antigenic determinants correlates with the capacity of plasma to inactivate coagulation enzymes. Both the existence of genetic polymorphism and the prevalence of the trait are still open to speculation. Although low level of AT III is an important risk factor for the development of venous thromboembolism, it does not preclude longevity. Liver is the major source of AT III synthesis but little is otherwise known of its metabolism. Subjects with hereditary deficiency treated withCoumadin often respond with a moderate increase in both AT III antigen and binding capacity. One can assume that this reflects a diminished rate of AT III utilization in vivo since the requirement for inhibitor apparently decreases with the reduction in procoagulants. More puzzling, however, is the fact that continuous intravenous treatment with heparin markedly diminishes the quantity of circulating AT III in man. This decrease is without proportion to the starting AT III level and a patient with congenital deficiency is at risk of attaining alarmingly low inhibitor level during therapy with heparin. Although the mechamism remains conjectural, the fact that heparin markedly lowers AT III in blood gives some new perspective to AT III metabolism.
Platelet Antithrombin Deficiency: A New Clinical Entity
Authors: Tullis J.L. , Watanabe K.
Published: May 2019

A kindred with a history of multiple thromboses was studied for coagulant abnormalities. A deficiency of plasma antithrombin III was found in approximately half of the 13 family members by either coagulant or immunologic assay. No clear relationship between plasma antithrombin III deficiency and a history of thrombosis was present. Platelet antithrombin assays were studied in the same family. Ten of the 13 members were deficient. None of the three who was normal had a history of thrombosis. The plasma antithrombin III assays could be influenced by heparin or coumarin treatment. The same medications were without consistent effect on platelet antithrombin. On the basis of these findings, it is proposed that familial hypercoagulability in some cases may be due to a platelet antithrombin deficiency and that the plasma antithrombin III deficiency is a secondary effect.
Collagen-Induced Platelet Aggregation: Involvement of Endogenous Phosphorylation
Authors: Chiang T.M. , King L.E. , Kang A.H. , Phillips D.R.
Published: May 2019

It has previously been shown that the aggregating agents, collagen, the ccl chain and the al-CB5 glycopeptide cause a decrease in the activity of adenylate cyclase and of the cAMP content, and an increase in the activity of guanylate cyclase and of the cGMP content in platelets. The present study was performed to investigate the effect of exogenous cyclic nucleotides, DBcGMP and DBcAMP, and the above aggregating agents on protein kinase(s) and phosphorylation of platelets.

Platelets were loaded with radio-labelled inorganic pyrophosphate, washed, resuspended in autologous platelet-poor plasma or 0.02M Tris/0.15M NaCl/0.001M EDTA, pH 7.4, and reacted with various test agents. The phosphorylated components were separated by SDS-slab gel electrophoresis and analyzed by radioautography and densitometry.

The results showed that DBcGMP and the aggregating agents stimulated endogenous phosphorylation in platelets of two components separable by SDS-electrophoresis. The phosphorylated components were protein in nature with apparent molecular weights of 42,000 and 20,000. The activity of cGMP-dependent protein kinase(s) was substantially higher than that of cAMP-dependent enzyme. Analyses of the time-course following exposure to the aggregating agents revealed that the phosphorylation of the two protein components preceded visible aggregation detectable by aggregometry. These data suggest the possibility that the aggregating effects of collagen and the derived peptides may be mediated by protein kinase(s).
The Binding of Bovine Factor VIII of Human Platelets
Authors: Kirby Edward P. , Tang Sha May
Published: May 2019

Highly purified bovine Factor VIII (FVIII) was iodinated by the lactoperoxidase method. Subsequent chromatography on agarose and extensive dialysis against sodium iodide solutions was required to remove non-covalently bound 1-125 which adhered tightly to FVIII. Iodination destroyed the procoagulant activity (Antihemophilic Factor-AHF) of FVIII, but did not affect its Platelet Aggregating Factor (PAF) activity. Binding to human platelets was determined by incubating radio!abelled FVIII with formalin-treated platelets, centrifuging, and measuring both bound and free radioactivity. Results obtained by this method were much more precise than those obtained by measuring disappearance of unlabelled AHF, PAF, or FVIII-related antigen from the supernatant, although the estimates of total binding obtained were comparable. Binding of FVIII to formal in-treated platelets was approximately the same as to unfixed platelets, and the binding could be saturated by adding an excess of unlabelled FVIII. Maximal binding occurred within 1-2 minutes at 37° and binding could be blocked by dextran sulfate, Evans Blue or other inhibitors of FVIII-induced platelet agglutination. Treatment of platelets with trypsin inhibited binding of labelled F-VIII. Binding was not affected by the presence of plasma, or high levels of purified human fibrinogen or FVIII.
Morphologic Platelet Changes During Thrombus Formation in the Rat
Authors: Wiedemann R. , Weichert W. , Breddin K.
Published: May 2019

The film presents observations in small mesenteric vessels (diameter 10-20 μm) of the rat using high power Nomarski optics. Under stasis conditions platelets appear as flat discs. Leucocytes are often seen creeping slowly along the intact vessel wall. Vascular lesions are produced with a focused laser beam (Hadron 513 biolaser). Immediately after the lesion platelets stick to the site of the microburn either in their native disc like shape without apparent morphologic changes or with protrusions. Within seconds these platelets swell and form protrusions. After 3-10 min, depending on the size of the lesion the vessel is occluded by a platelet thrombus. Platelets undergo further swelling. Later the thrombus is partially or completely swept away and the vessel is recanal i zed. Irreversible fusion of platelets is rarely observed. . New, usually smaller thrombi form at the damaged vessel wall. The morphologic platelet changes observed differ markedly from the changes observed during aggregation in vitro. After injection of a new antithrombotic substance (Bay G 7565) the adhesion of platelets to the damaged area is remarkably diminished. The few platelets which adhere to the site of injury show the same swelling and transformation like in untreated animals. The film demonstrates that it is possible to investigate morphologic changes of single platelets during thrombus formation. It seems possible to adapt this model for the in vivo study of antithrombotic drugs.
Regulation of Fibrinolysis Through Activation and Inhibition of Activation of a Plasminogen Proactivator (Preurokinase).
Authors: Bernik M.B. , Oiler E.P.
Published: May 2019

Purified preparations of a plasminogen proactivator, preurokinase (preUK), the enzyme plasmin and thrombin and native inhibitors of these enzymes (C’l-esterase inhibitor and antithrombin III) were used to study factors and events which may participate in the regulation of fibrinolysis by facilitating or hindering conversion of preUK to UK.PreUK, expressed in CTA units of generated UK, was purified from supernates of kidney cultures by chromatography on Sephadex G-100 columns to specific activity of 20,000 CTA U/mg protein and was studied by immunodiffusion and immunoelectrophoresis with antisera to highly purified urinary and tissue culture UK. In assays on fibrin plates, generation of UK activity (3-4 CTA U/ml) from pre UK occurred with traces of plasmin (0.01 CTA< U/ml) or trypsin (10 BAEE U/ml) but not with thrombin (0.01-100 NIH U/ml). Inhibition of UK generation occurred in two ways: 1) by inhibition of the activating enzymes, and 2) by inactivation of pre UK itself. The former was demonstrated with C’l-esterase inhibitor which inhibited the fibrinolytic as well as preUK-activating properties of plasmin and trypsin, and the latter with thrombin which inactivated preUK progressively in a dose-response fashion without being itself consumed.Thus, 3 CTA U/nl preUK were inactivated in 20 min or less at 37°C by 2.5 NIH U/ml thrombin and in more than 3 hr by 0.01 NIH U/ml. Thrombin-induced preUK inactivation was inhibited by antithrombin III indicating that while thrombin acted as an inhibitor of fibrinolysis by inactivating preUK, antithrombin III facilitated fibrinolysis indirectly by protecting preUK from thrombin.
An Immunoenzyme Histochemical Technique for Detecting Platelet Antibodies from the Serum of Children with Idiopathic Thrombocytopenic Purpura (ITP)
Authors: Tate D. , Sorenson R. , Gerrard J.M. , Krivit W. , White J.G.
Published: May 2019

A simple reproducible, semiquantitative immunohistochemical assay for the detection of antiplatelet antibodies has been developed and is adaptable for clinical use. Blood smears from normal donors are incubated with test sera and platelet bound immunoglobulins are detected using goat anti-human immunoglobulin G, A, M, conjugated to horseradish peroxidase. The blood smears are observed under phase contrast microscopy and the labelling of peroxidase on individual human platelets is quantitated, by comparison with standards graded 0-4. For each test sera a hundred platelets are examined. Control platelets showed indices which ranged from 0.34 to 1.06 (mean 0.67 ± 0.06 SE, n = 12). Sera samples from ten patients with active ITP showed significantly increased antiplatelet antibody levels with indices ranging from 1.47 to 3.04 ( mean 1.99 ± 0.18 SE). Seven children with thrombocytopenia due to other causes were in the same range as normal children (mean 0.62 ± 0.07 SE). Five patients who had had multiple platelet transfusions did have significantly elevated levels of antiplatelet antibodies (mean 1.16 ± 0.12 SE).However, when these individuals were evaluated using blood smears from HLA matched donors levels of antiplatelet antibody decreased to control levels (mean 0.46 ± 0.08 SE). The results suggest that this test may be useful for the evaluation of children with ITP and can differentiate between autoantibodies due to ITP and isoantibodies resulting from multiple platelet transfusions.
Two Human Immunochemically Distinct low Molecular Fibrinolytic Inhibitors
Authors: Hedner U. , Gallimore M.
Published: May 2019

Recently a fast reacting α2-antiplasmin (Müllertz 1972, Collen et al 1975) not identical with the inhibitor of the plasminogen activation (Hedner and Collen 1976) was described. Using the antiserum of Dr Aoki his inhibitor (Moroi and Aoki 19 76) was found to be identical with the α2-antiplasmin but distinct from the inhibitor of the plasminogen activation. Furthermore the inter-α-anti-plasmin described by Gallimore (1975) was found to contain both material reacting with the antiserum of Collen and with the antiserum against the inhibitor of the plasminogen activation. By means of specific immunoadsorption of the fraction eluted close to the plasminogen activation inhibitor by the method described 1973 (Hedner 1973) these two inhibitors have been separated into two fractions, which do not cross react immunochemically. In a caseinolytic system plasma from which the α2-antiplasmin was removed still has got an inhibitory activity against plasminogen activation. Also the α2-antiplasmin, plasminogen, activation inhibitors as well as α2-macroglobulin did not show any covariation in patients with different diagnoses. The inhibitor of the plasminogen activation did not interact with purified urokinase or plasmin but the α2-antiplasmin forms complexes both with human and porcine plasmin.

Thus there seem to exist at least two different low molecular fibrinolytic inhibitors migrating as α2-globulins in human blood.
Human α-Thrombin Preparations
Authors: Fenton J.W. , Finlayson J.S. , Fasco M.J. , Landis B.H. , Zabinski M.P. , Aronson D.L.
Published: May 2019

Thrombin production from human Cohn fraction III paste (>20 g; 340 ± 110 mg/kg paste, N = 65) has yielded predominantly α-thrombin (97 ± 3%, N = 29), with high clotting and esterase activities (2.8 + 0.4 US “NIH” units/μg protein; 88 ± 8% esterase active, N = 65), free of detectable plasmin(ogen) and Factors II, VII, IX(a) or X(a). Preparations in 0.75 M NaCl (pH/v6) at 2-7 mg/ml (A250nm = 1,83 m-1 cm-1 cm 0.10 M NaOH) retain activities frozen at <-50°C for >1 year. The enzyme (My 36,600 + 800, N = 9) consists of two polypeptides (A chain Mr 4,600 ± 200, N = l6; B chain Mp 32,000 ± 1,000, N = 22), is an isoionically heterogeneous glycoprotein (pI 7.0, 7.3, 7.6; carbohydrate-staining B chain), displays sparing solubility (e.g., in 0.15 M NaCl, pH~7; precipitates in H2O), is easily denatured (low I; pH <5 or >10; T1/2 53°C), and autolyzes to non-clotting derivative forms (β- followed by 7-thrombin; both <1% clotting of α-thrombin). Chloride salts (I 3, pH 8.3) of Group I (except Li) enhanced a-thrombin (1 mg/ml) thermal stability (T1/2 60-61 vs 49°C for Li+), whereas Group II salts reduced its stability (T1/2 40, 44, 48°C for Ca2+, Ba2+, Sr2+, respectively). Autolytic loss of clotting activity (22°C, 48 h) followed the series: Na+ (>90% loss) > K+ (90$) > Rb+ (70%) >Cs+ (30%) > Li+ (10%)~Ca2+~Sr2+~Mg2+ > Ba2+ (<1%). Clotting activity was correlated with enzymic homogeneity of α-thrombin (3.09 ± 0.57 US units/μg extrapolated pure-active enzyme, N = 29). (Supported in part by USPHS Grant HL I3160)
Dansyl and Anthraniloyl Human α-Thrombins. Highly Sensitive Active Site Probes of Thrombin Conformation.
Authors: Shen Y.Y. , Berliner L.J.
Published: May 2019

Human α-thrombin was labeled at the active site with the two serine esterase fluorescent probes, dansyl fluoride (Dans) and paranitrophenylanthraniloate (Anth). Both derivatives show enhanced quantum yields and blue shifts in their emission maxima. Of most significance is a strong energy coupling to tryptophan (or tyrosine) residues near the catalytic center. Changes in the conformation of the active site region or other changes which alter the environment of the probe will effect the extent of this fluorescence energy transfer between the probe and the protein. Both derivatives, particularly the Anth derivative, show marked changes in their energy coupled spectra upon binding with a small basic ligand such as valerylamidine, while Anth shows even greater effects upon association with the prothrombin activation Fragment II. These fluorescent thrombin derivatives are quite powerful in examining thrombin structure alone (α-, β-, and γ-forms) as well as their interactions with other coagulation factors at concentrations more closely approaching serum levels than are attainable with other physical techniques.
Exo-Site Affinity Labeling of Human α(CLOTTING) and β/γ(NONCLOTTING) m[o-2-CHLORO-5-Sulfonylfluorophenylureido) Phenoxybutoxy] Benzamidine (mCP(PBA)-F)
Authors: Bing D.H. , Fenton J.W. , Cory M.
Published: May 2019

Human α-thrombin was selectively and irreversibly inactivated with mCP(PBA)-F (pseudo first order rate constant k1=3.7x10-3 sec-1 23°). The initial velocities of inactivation followed simple enzyme-substrate kinetics (Km
(app)=72.7 μM, k2=3x10-3 sec-1). mCP(PBA)-F was determined to be an affinity labeling reagent for both α and β/γ-thrombins (7% α, 20% β and 78% γ) as (1)[3H]mCP(PBA)-F was stochiometrically incorporated (1.07 mole/mole for α thrombin and 1.19 mole/mole for β/γ-thrombin), (2) [14C]iPr2P-F inactivated α and β/γ-thrombin did not react with mCP(PBA)-F, and (3) the analog o-(2-chloro-5-sulfonylf luorophenylureido)phenoxy benzene lacking the benzamidine, only caused 14% inactivation of α-thrombin. [14C]iPr2P-F was only found on the B chain (Mr 32,000) but [3H]m(PBA)-F label was distributed 20% on the A chain and 80% on the B chain in reduced and alkylated α-thrombin electrophoresed in 10% PAGE-SDS-6 M urea. The labeling pattern of β/γ-thrombin with [14C]iPr2-F and [3H]mCP(PBA)-F revealed that 14C label was on the B chain active site fragments of β-thrombin (Mr ≈ 23,000) and γ-thrombin (Mr ≈ 12,000), but the 3H label was on 20% the A chain and 80% on fragments of Mr 6000-10,000. The results suggest that mCP(PBA)-F reacts with residues exclusive of the active site serine within ~ 10 Å radius of the anionic binding site.(Supported by NIH Grants AI 11231, AM 17351, CA 17376, HL 13160 and HL 18825)
Platelet Adherence of Collagen: A Simple Reproducible Method
Authors: Mant M.J.
Published: May 2019

An optimal method for measuring platelet adherence to collagen should be simple, quantitative, reproducible, adaptable to varying experimental conditions and have no interfering platelet aggregation nor use reagents potentially influencing the results. An adaptation of previously described techniques has been developed which satisfies most of these requirements. Platelets were stirred with a stable collagen suspension in 0.2% EDTA at 37°C. Platelet counts were performed with a Coulter counter before and after stirring and %. adherent platelets calculated. Platelet aggregation was excluded by phase contrast microscopy and by unchanged adherence after addition of aggregating agents (ADP and adrenalin) and an inhibitor of aggregation (prostaglandin El). The counting technique was validated by absence of particle counts with collagen alone and by simultaneous “manual” post-adherence platelet counts. Adherence % was found to plateau at a maximum value with time, increasing collagen concentration and increasing stirring speed. It fell exponentially with increasing platelet count and linearly when pH was reduced. Utilizing platelet rich plasma (PRP) at a platelet count of 300,000/μl, a final collagen concentration approximating 0.05 mg/ml, a stir speed of 900 rpm and a pH of 7 ± 0.5 adherence % on 30 normal donors varied from 43.0 to 59.3%. A normal range of 51.6 ± 8.2% (± 2 SD) was established. Replicate single sample testing gave a mean ± 2 SD of 51.9 ± 2.8%. Serial adhérences in 1 individual showed only minimal weekly variation. This simple, reproducible, quantitative method can be adapted to a wide variety of experimental conditions. It should therefore be useful for investigating many aspects of platelet adherence to collagen.