bluuebunny/crossref_metadata_2025 huggingface dataset:
Peptidase Activity of Amidinated Thrombin
Authors: Kang E.P.
Published: May 2019

Human thrombin, free of plasminogen and plasmin, was treated with ethyl acetimidate hydrochloride in order to modify the lysyl residues of the protein. By monitoring the enzymic activity in the modification mixture, it was found that the reaction was completed in about one hour and the loss of activity of thrombin was proportional to the amount of modification. After the removal of the excess ethyl acetimidate, approximately 25% of the clotting activity and of the hydrolysis activity for small peptides remained. Amino acid analysis of this modified thrombin indicated about 80% of the lysyl residues had been modified with no apparent change of other amino acid residues. By studying the thrombolytic hydrolysis of Bz-phe-val-arg-pNA, the kcat of the amidinated thrombin was about 8% of the control while the KM Secreased to 0.056 μM from 0.098 μM. The modification of the lysyl residues of thrombin, therefore, has lowered the catalytic efficiency of the enzyme with a rather small change in binding affinity. This suggests that modification of lysyl residues in the neighborhood of the active site hinders the catalytic hydrolysis of the small peptides.
The Influence of Pivka II on Thrombin Generation in Liver Disease
Authors: Malia R.G. , Preton F.E.
Published: May 2019

Thrombin generation and factor Xa activation have been studied in plasma samples from normal controls and from patients with various types of hepatic dysfunction. The patients were subdivided into groups according to the ratio procoagulant II/PIVKA II. PIVKA II was measured by Immunoelectrophoresis using a rabbit antibody raised against prothrombin. For those patients with a ratio greater than 0.70 the thrombin generation and Xa activation correlates well with the procoagulant factor II. Conversely patients with a ratio less than 0.5 exhibit inhibition within these same test systems. The administration of vitamin K abolishes this effect.

It is concluded that PIVKA II plays an important role in the coagulation mechanism of patients with hepatic dysfunction associated with vitamin K deficiency.
Platelet Function in Pre-Eclampsia
Authors: Whigham K.A.E. , Howie P.W. , Drummond A.H. , Prentice C.R.M.
Published: May 2019

As pre-eclampsia is associated with thrombocytopenia, it is possible that platelets contribute to the intravascular coagulation in this disease. Platelet functions were studied in non-pregnant and normal pregnant women, and in women with mild or severe preeclampsia. Platelet aggregation was measured in response to six aggregating agents; maximum aggregation rates and the concentration of each agent required to induce half-maximal aggregation (E. C. 50 value) were determined. Platelets from women with severe pre-eclampsia were less responsive than normal, with significantly lower maximum aggregation rates in response to collagen, vasopressin and arachidonic acid, and a higher than normal E. C. 50 value for collagen. Women with severe pre-eclampsia also had a significantly raised plasma adenine nucleotide concentration (2. 99 ± 2. 35 vs 0. 74 ± 0. 85 nmol/108 cells) and a reduced platelet 5-hydroxytryptamine concentration (0. 190 ± 0. 293 vs 0. 508 ± 0. 340 μg/109 cells). In mild pre-eclampsia, only the maximum rate of collagen-induced aggregation differed from normal. Platelets in patients with severe preeclampsia may have undergone the release reaction in vivo, in keeping with the close association between pre-eclampsia and intravascular coagulation.
ß-Thromboglobulin Clearance
Authors: Dawes Joan , Hunter WH , Smith RC , Duncanson Jeanette , Ruckley CV , Allan NC , Pepper PS , Cash JD
Published: May 2019

The clearance of β-Thromboglobulin (BTG)from the plasma has been studied by different techniques in normal subjects. Infusion of homologous purified iodinated BTG showed complex kinetics of clearance. The initial plasma half life was approximately 20 minutes but this increased to approximately 100 minutes during the latter part of the clearance curve, which was followed for 5 hours. Three per cent of the 125i excreted was immunoprecipitable. In a second study BTG clearance from the plasma and the patterns of urine excretion have been studied by autologous serum infusion 48 hours after withdrawal of 500 mis of blood. The plasma BTG returned to normal levels within 1-2 hours and elevated urinary levels were observed for 3-4 hours post-infusion.
On the Activation of Factor X-Activator of the Russell’s Viper-Venom by Calcium
Authors: Nath B.B. , Chattopadhyay Swapan
Published: May 2019

The relationship between Ca2+ ion and factor X-activator of Russell’s-viper-venom (RVV) seems to point to some sort of stoichiometry as evidenced from the composition and stability studies using an approximate concept of the Job’s method of continuous variation. The concept of the method, used in co-ordination chemistry to ascertain the metal-ligand interacting ratio has been used, in order to interpret the results. The clotting time was measured by varying Ca2+ ion and venom concentrations respectively and ‘log of clotting time’ was plotted against concentration in each case, and the minimum in the curve was taken to represent the maximum formation of the complex of Ca2+ ion with factor X-activator of RW. The plot of clotting time against the ratios of venom and Ca2+ ion concentrations also pointed to an interacting ratio. At higher concentrations either of Caion or of venom, longer clotting time points respectively to the salt-effect and the denaturising effect of the venom on plasma-proteins. Accordingly it is concluded that Ca2+ ion activates the factor X-activator in definite ratio before the activated metal-enzyme complex (M-E), acts on its substrate to produce E-M-S or E<SM for the formation of the products.
Multiple Molecular forms of Factor VIII Antigen in Normal Individuals and von Willebrand’s Disease Patients
Authors: Zimmerman T. , Kimball J. , Edgington T. , Abildgaard C.
Published: May 2019

Factor VIII antigen is present in normal individuals in multiple molecular forms which can be separated according to size. The smaller forms have little or no ristocetin co-factor activity. In order to evaluate the possibility that Factor VIII antigen forms of large size may be an artifact of in vitro aggregation, we have ultracentrifuged plasma on a 20% sucrose cushion at 37 C for 10 min at 254,000 xg (peak). The rate of clearing of Factor VIII antigen was compared to that of other plasma proteins. The results indicate that Factor VIII-related antigen forms of high S exist even when plasma is maintained at physiological temperature and analyzed with minimal delay, suggesting that these larger molecular forms also exist as such in vivo.

In von Willebrand’s disease (vWd) all forms of Factor VIII antigen may be present but decreased in quantity (Type I) or large forms may be missing with smaller forms present in normal or increased quantities (Type II). Factor VIII antigen was isolated from plasma of three patients with Type I and three patients with Type II vWd by counter immunoelectrophoresis. The Factor VIII antigen was then reduced and electrophoresed on SDS-containing Polyacrylamide gels. The presence of carbohydrate was evaluated by staining with perchloric acid-Schiff’s reagent (PAS). The 210,000 MW Factor VIII antigen subunit from each patient was PAS-positive. Though subtle changes in carbohydrate content or composition could not be evaluated by this technique, a total defect of glycosylation is unlikely in this sample of vWd patients.
Femoral Fractures in Severe Hemophilia
Authors: Allain J.P. , Ageron M.H. , Laurian Y. , De La Caffinière J.Y.
Published: May 2019

Increasing physical activity of hemophilic children has resulted in more frequent fractures. Within the last ten years, fourteen severe hemophiliacs (8-19 years)sustained femoral fractures. Four of these hemophiliacs had an inhibitor. Eight were the result of minimal trauma. There was no obvious correlation in these eight cases with prolonged immobilisation and/or treatment with steroids. Co-existing knee hemarthrosis or thigh hematoma often led to a delay in the diagnosis of the fracture. Metaphyseal fractures(8 cases) were treated by plaster cast (4) or, skin or pin traction (4) with good anatomical result and normal union. Replacement therapy was given in 3 cases for 3 days and in 1 case for 2 weeks. No complication occurred in any of the non transfused patients(4). Epiphyseal and femoral shaft fractures were treated by skin or pin traction (3) or internal plate fixation (2)associated with replacement therapy for at least 2 weeks. Unsatisfactory anatomical result was obtained in three cases. One impacted fractured neck of femur was discovered on X-ray examination 3 weeks after trauma. Guide lines for orthopedic and medical treatment of femoral fractures in hemophiliacs are suggested according to the anatomical feature.
Case Histories
Authors: Evans Bruce E. , Cavalcanti Wellington
Published: May 2019

Case 1. 71 year old Factor VIII 2.8%

Diagnosis: five dental extractions, peripheral giant cell granuloma

Treatment: local hemostatic agents

Case 2. 48 year old Factor VIII<1% (with inhibitor 100 B.U.)

Diagnosis: four dental extractions

Treatment: local hemostatic agents; antifibrinolytic agents

Case 3. 36 year old Factor VIII 5.5%

Diagnosis: 3 day old tongue laceration

Treatment: cryoprecipitate infusions; antifibrinolytic agents

Case 4. 26 year old Factor VIII<1%

Diagnosis: 3 orthodontic extractions; 4 third molar extractions

Treatment: factor VIII concentrate infusions
History of Care for the Hemophiliac in France: Movement from Special Hospital to Home Care
Authors: Allain J.P.
Published: May 2019

In the modern context of hemophilia care the boarding school for hemophiliacs has become a comprehensive center capable of recognizing and adapting to the needs of hemophiliacs through a diversified structure and a multidisciplinary approach. In and out patient care are provided. The duration of stay varies from days to years according to the common project of the child, his parents, and the institution. An educational program for both hemophiliacs and parents has been developed. Since 1972,90 children ranging in age from 10 to 17 participated in the training for self-treatment. Eighty per cent succeeded and guidelines for optimal training were drawn considering age, psychological status and parents involvment. Since 1974 105 families participated in the program. Among 64 families whose children were not involved in self-treatment, 32 are treating their child, 11 were unsuccessful (5 with children under the age of 4, 6 for major psychological problems) and 21 had insufficient attendance. Twice a month, groups of 3-4 families meet successively for an hour with a nurse, a physiotherapist, a physician and an educator. The last two staff members moderate free discussions rather than teach. This training carried out over several months induced profound beneficial changes in the family’s approach to hemophilia.
A Summer Camp for Hemophilic Boys: A Film.
Authors: Seligsohn U. , Horoszowski H.
Published: May 2019

The great advantages of organizing a summer camp for hemophilic boys in a Kibutz are presented in a 10 minute film. They include: a) Psycho-social advantages: Holding guided group discussions, removal of parental restrictions, development of new interests, interaction with normal tolerant children living a communal life, and temporary relief for parents from the constant burden of raising hemophilic boys, b) Medical advantages - teaching and practising: self infusion, oral hygiene, preventive physiotherapy and providing general education about the disease.
Home Treatment for Children with Hemophilia
Authors: McWilliams N.B. , Ownby R. , Anderson L.C.
Published: May 2019

Twenty-four hemophilic boys between the ages of 2 and 21, 4 with factor IX and 20 with factor VIII deficiency, have been on a home transfusion program for 2 to 22 months. Criteria for participation include: moderate or severe disease, family cooperation, accessible veins and attendance at Comprehensive Hemophilia Clinic. Strict guidelines are given to families for treating hemorrhages without physician consultation at home, versus those that must be seen by a physician.

Only 1 child has required hospitalization for bleeding. One child has had clinical hepatitis on 2 occasions. No patient has developed an inhibitor. No complications related to nonprofessional I.V. administration have occurred. Of 18 boys tested for hepatitis B antibodies, 17 were positive. None has been positive for hepatitis B antigen. Of 12 boys with established severe arthropathy, 8 have had symptomatic improvement and 4 are stable. Of 12 boys with minimal or no arthropathy, all are stable except 1 who has developed chronic synovitis.

Psycho-social benefits include a new found independence, improved living routine for family units and opportunities to participate in more activities. The mean cost/boy/month was $397.

We conclude that home treatment for hemophilic children is both safe and effective and should be available to all children meeting the above criteria.
Recurrent Acute Hepatitis Following the Use of Antihemophilic Factor Concentrates
Authors: Tembrevilla-Zubiri C.L. , Rickles F.R. , Klatsky A.U.
Published: May 2019

A patient with Hemophilia A experienced 5 well-documented episodes of acute icteric hepatitis within five to ten days following 5 separate infusions of concentrates of antihemophilic factor over a 3 year period. We were unable to demonstrate either consistent in vitro lymphocyte transformation or in vivo antibody formation in response to a variety of concentrates of antihemophilic factor. Nevertheless, although the mechanism remains unclear, it is likely that these episodes represented an immune response of the patient’s hepatocytes to protein (s) contained in the antihemophilic concentrates. Further studies to define the exact mechanism of hepatocellular injury in this case are in progress. Meanwhile, this communication is, to our knowledge, the first reported case of probable “immune complex” hepatitis as a regular complication of replacement therapy in hemophilia. This unusual complication must be considered in the differential diagnosis of post-transfusion liver dysfunction.
HBsAg in Donors, Patients, Laboratory Personnel and Housestaff of a Military Hospital in IRAQ: A Seroepidemiologic Study
Authors: Kassira E. , Karim S. , Hossaini A.
Published: May 2019

This study is an evaluation of the magnitude of the problem of hepatitis in the Iraqi army using a seroepi-demiological investigation conducted in Al-Rashid Military Hospital. Four hundred and fourteen blood samples collected from patients with acute viral hepatitis, blood donors, hospital, laboratory staff and of family contacts of carriers were tested for HBsAg by the CIEP and HA technics. In addition, a questionaire (relating to the epidemiological behavior of the disease) was filled in for each individual following a direct interview. The HBsAg incidence was 3.5% among volunteer blood donors, 4% among military unit contacts of carriers, 20% among wives of carriers, 8% of other members of the family, 8% of laboratory staff, 7% of the operating theater staff, 6.5% of surgical ward staff, 21. 5% of patients in the dialysis unit and 8% of its staff. No statistically significant difference in incidence was found in relation to age, blood groups and urban or rural living. Most carriers showed normal transaminase and serum protein levels. Of patients with acute hepatitis, 50% showed detectable antigen when samples were collected during the first week of jaundice and 30% when sera were collected and examined 4-6 wks. following onset of jaundice. The antigen disappeared in 95% of these cases within 3 months. In conclusion, this study provides statistical evidence for the importance of the degree of contact with carriers or with blood products in the transmission and prevalence of viral heoatitis.
Human Factor VIII Concentrates in Hemophiliac Dogs
Authors: Lewis Jessica H. , Hasiba Ute , Spero Joel A.
Published: May 2019

Human factor VIII corrects the clotting defect in dog hemophilic plasma in vitro. The present studies were undertaken to see if this happened in vivo and to look for and document the development of an inhibitor. Four hemophiliac dogs were infused with factor VIII concentrates, the first two on five occasions, the others three times. Factor VIII:C, VIIIR:Ag (defined with antibody to human VIII) and VIIIR:vW were followed at pre, 10 minutes, 2 and 24 hours post infusion. The pre-infusion VIII:C (assayed with human substrate) averaged 0.23 U/ml compared to 6.93 U/ml for normal dogs; VIIIR:Ag was absent in both. VIIIR:vW was low but variable. Following the first injection, all four dogs responded in VIII:C about as calculated. The amounts of VIIIR:Ag and vW were much greater than VIII:C in the concentrates and in the post-first infusion samples from the dogs. On subsequent infusions rises in VIIIR:Ag were not detected and increases in VIII:C and VIIIR:vW were minimal. Precipitating anti-human VIII was found on the third infusion and thereafter. After the first infusion reactions were marked. Vomiting and diarrhea occurred in all, and one dog died in anaphylactic shock about one hour after the third infusion. Lack of response in VIIIR:Ag occurred before anti-VIII could be demonstrated in vitro. This rapid development of an inhibitor suggests that prolonged cross-species VIII therapy will not be successful. The ability of the precipitating anti-VIII elicited in the dogs to destroy VIII:C, VIIIR:Ag and VIIIR:vW is analagous to the in vitro effects of heterologous anti-VIIIs (rabbit and goat).
Application of the Partial Thromboplastin time for Diagnosis and Treatment of Hemophilia
Authors: Sutor A.H. , Schorn P.
Published: May 2019

All 27 hemophiliacs (hemophilia A or B) with severe, moderately severe, or mild form of hemophilia, had a pathologically prolonged partial thromboplastin time (PTT) when compared to age adjusted normal values (<40 sec.). However, the degree of the hemophilia could not be determined by the PTT. PTT-determinations during therapy with antihemophilic globulin (AHG) showed pathological values when the F VIII-activity was below 32%. In addition a drop of F VIII-activity below 13% was associated with a PTT of more than 48 seconds. The clinical importance of PTT-control during AHG-substitution is demonstrated by the case of a hemophiliac who underwent surgery.
Are There any Differences in Thrombosis Prophylaxis and Side Effects Between Sodium and Calcium Heparin?
Authors: Bergqvist D. , Hallböök T.
Published: May 2019

In clinical studies on low dose heparin both sodium and calcium heparin have been used but no comparative investigation has been made. The aim of this study therefore was to perform a double blind randomized comparison between sodium and calcium heparin on prophylaxis of postoperative thrombosis and possible side effects. 75 patients were included in the study, 39 receiving sodium heparin and 36 calcium heparin (Heparin Vitrum from the same heparin batch was used and the following dosis schedule was followed: 5000 IU s.c. 2 hours preoperatively and then every 12th hour for 5 days). The injection was given on the lateral aspect of the thigh. Deep vein thrombosis was diagnosed with 125I-fibrinogen test. The activity over the site for heparin injection was also measured. The patients were interviewed by a nurse for local reaction and she also judged haematoma formation from photographs. Bleeding tendency was measured as blood loss during operation and total transfusion need. The groups were comparable concerning age, sex and type of operation. The frequency of thrombosis was 40% in the sodium heparin group and 43% in the calcium heparin group, and the blood loss 318 and 454 ml respectively (p>0.05). There were no differences in the patients’ subjective local reaction to heparin injection or haematoma formation judged from the photographs. Nor did the 125I-fibrinogen activity over heparin injection sites differ between the two types of heparin.

This study has thus shown that sodium and calcium heparin are identical from the point of view of prophylaxis of postoperative deep vein thrombosis and side effects, provided that the heparin batch is the same in both.
Pathways of Thrombin-Induced Aggregation and Release with Human and Rabbit Platelets
Authors: Kinlough-Rathbone R.L. , Perry D.W. , Packham M.A. , Mustard J.F.
Published: May 2019

There are at least 3 mechanisms involved in thrombin-induced aggregation and release: (1) released ADP, (2) formation of thromboxane A2 and (3) a third mechanism(s). We have examined whether the third pathway is due to formation or release of a substance from platelets which affects other platelets. Washed human platelets were exposed to thrombin (2.5 u/ml) for 15 min at 37°C in the presence of indomethacin to block thromboxane A2 formation. Platelets were removed by centrifugation and the thrombin neutralized with hirudin or DFP. Addition of the superna te to washed human platelets prelabeled with 14C-serotonin caused platelet aggregation but release did not occur. Treatment of the supernate with apyrase, CP/CPK or dialysis abolished aggregation, indicating that the material was ADP. Thus, the mechanism by which thrombin induces aggregation and release with human platelets in the presence of agents which destroy ADP and block the formation of thromboxane A2 is a direct effect of thrombin on platelets and does not involve a substance freed from platelets. In contrast, when washed rabbit platelets were treated with thrombin in the presence of indomethacin and the released ADP was removed, material remained in the supernate which caused aggregation and release from washed rabbit platelets but was without effect on washed human platelets. The activity of this material (MW > 10,000) was not abolished by dialysis or boiling. Therefore rabbit platelets differ from human platelets because they have a mechanism in addition to released ADP, thromboxane A2 and the direct effect of thrombin on platelets that can cause aggregation and release.
Platelet Alterations Caused by Antiplatelet Antibodies in the Circulation
Authors: Nagasawa T. , Kim B.K. , Baldini M.G.
Published: May 2019

It is known that antiplatelet antibodies cause loss of platelet cytoplasmic and granular contents in vitro. It is, however, unknown whether similar platelet changes occur in vivo, in the circulation, leading to destruction and phagocytosis of platelets in the R.E. system. To study this possibility a rabbit model was devised. Severe and stable thrombocytopenia was first produced in rabbits by one intravenous injection of Adriamycin. Large numbers of allogenic platelets labeled in vitro with 51Cr and 14C-serotonin were then infused to raise the circulating platelet count to 180-250 × 103/mm3. A dilute heteroimmune antiplatelet serum prepared in the guinea pig was infused intravenously and platelet samples were collected four times during the subsequent 30 minutes to 24 hours. Platelet hexokinase and β-glucuronidase, 14C-serotonin and 51Cr were measured. Within the first 60 min the specific activity of 51Cr in platelets decreased by 21%, 14C-serotonin declined by 30%, hexokinase by 5% and β-glucuronidase by 29%. During the subsequent 24 hours only 51Cr and hexokinase registered a mild decrease but 51C-serotonin and β-glucuronidase remained essentially unchanged. In a second series of experiments the effect of platelet alloantibodies was studied in rabbits previously immunized with allogenic platelets. The decline in the specific activities of the enzymes and 14C-serotonin was similar to that observed in animals treated with heteroimmune sera but loss of 51Cr was more severe. These results demonstrate that the platelets remaining in the circulation after the disappearance of the immediate effect of hetero- or alloantibodies were qualitatively altered with a severe reduction of their granular and cytoplasmic contents.
Separation of the Binding Step from the Collagen Platelet Reaction
Authors: Kronick P.L. , Jimenez S.A.
Published: May 2019

Determination of activity of most agents in stimulating platelets to aggregate or release adenine nucleotides is conveniently done by titrating the platelet reaction with the agent. Platelets have previously been titrated with different types of collagen (types I, II, and III) in this way to compare the activities of the collagens. It has been concluded that the order of activity is type III>I>II. Whether this order is due to differences in binding was not obvious from these experiments because the binding was not determined directly. We have developed a method of comparing activities by measuring the targeted dose for each point in the titration - the amount of collagen which actually binds to platelets. The collagens used in these experiments were prepared in vitro from embryonic chick tissue to give labelled products of extremely high specific activity without structural alteration. We find that type I collagen is at least 20 times as active as previously reported, and that the activity of Type III collagen is not significantly higher when the amounts bound are taken into account. The fraction of the labelled tendon collagen which was bound to platelets was identified as type I by its hydroxyproline/proline ratio. Direct measurement of the bound fraction in dose-response studies is required for valid comparison of collagen activities.
Leukocyte Generated - Renal Dialysis Membrane Induced - Procoagulant (Tissue Factor) Activity
Authors: Muhlfelder T. , Niemetz J.
Published: May 2019

Leukocytes are capable of generating procoagulant-tissue factor activity (TFa) if appropriately stimulated. One such well established stimulus is bacterial lipopolysaccharide. Other stimuli of Tfa by leukocytes have been described. We have recently reported that leukocytes harvested from renal hemodialysis membranes have a significant TFa. Experiments were done to elucidate the mechanism whereby this stimulation occurred.Sterile, pyrogen free, cuprophane membrane segments from a renal hemodialysis apparatus were incubated for 18 hours with human leukocytes, platelet rich or platelet poor heparinized plasma, tissue culture media etc., and tested for activity after removal of plasma and sonic disruption of the leukocytes. Maximum generation of TFa required leukocytes, platelet rich plasma and membranes. When membranes were omitted significantly less TFa was generated (110 vs 40 units P<0.02). No TFa was generated in the absence of leukocytes or plasma. The effect of membranes on plasma was also tested. Heparinized plasma shaken with membranes (or Zymosan) had the ability to stimulate leukocytes to generate TFa, while EDTA or citrated plasma had no such stimulatory effect. These date suggest that a factor (s) in plasma is activated which induces leukocytes to generate TFa.
The Effect of Vitamin K on Pivka and its Associated Procoagulant Proteins in Liver Disease
Authors: Malia R.G. , Preston F.E. , Blackburn E.K.
Published: May 2019

Vitamin K deficiency is associated with low procoagulant activity of the vitamin K-dependent clotting factors together with normal concentrations of an immunologically cross-reacting protein associated with the appropriate clotting factor.

In this study we have examined the response of the clotting factors II, VII and X together with their appropriate related antigens to the intravenous administration of vitamin K1 in 14 patients with various forms of liver disease. We have also related the observed changes to alterations of the inhibitor activity of PIVKA as detected by the modified thrombotest.

A relationship has been established between the rate of appearance of procoagulant activity and the rate of disappearance of PIVKA after the intravenous administration of vitamin K1 in those patients with thrombotest inhibitor activity. In this same group of patients it can also be shown that PIVKA is physico-chemically dissimilar to the related protein of normal individuals.

Patients without thrombotest inhibitor activity appear to synthesise decreased amounts of a structurally normal protein which is not influenced by vitamin K.
Metabolism of I-131 Antithrombin III in Dogs.
Authors: Wentland S.H. , Damus P.S. , Leonard B.D. , Zamzam A.A. , Reeve E.B.
Published: May 2019

The turnover of canine antithrombin III (AT3) was studied in dogs as a probe of the generation and removal of proteases (e.g. thrombin). AT3 was prepared from dog plasma by affinity chromatography using heparin Sepharose. It was homogeneous by Polyacrylamide gel electrophoresis, and showed one major and one minor band by isoelectric focussing. The specific activity (relative units/0.D.) was 1400-fold that of the starting defibrinated plasma. Under suitable conditions labelling efficiency was 80%, ca. 1.2 gram atoms iodine per mole AT3 were incorporated, and specific activity of the labelled AT3 averaged 1200 relative units/0.D. Turnover studies were made by measuring both plasma and whole body counts over 15 days. Later decay of whole body counts was exponential and paralleled that of plasma counts indicating the presence of a single labelled protein degraded at a constant rate. Less active preparations labelled with 1-131 were found to have longer half-lives than this preparation and slopes that became flatter with time, indicating contaminating slower metabolizing components. 131-I-AT3 turnover studies in dogs that were sedated (to facilitate whole body counting) did not differ significantly from those in untreated dogs. Analysis of fractions obtained from plasma some days after 131-I-AT3 injection suggests formation of higher molecular weight metabolites. Some of these are comparable to complexes of AT3 and bovine thrombin formed in vitro. Thus, we have prepared a highly active, labelled AT3, demonstrated its suitability for use in turnover studies and have obtained preliminary observations concerning thrombin turnover. (Supported in part by USPHS NIH HL02262; Colorado Heart Assoc.)
The Comparative Evaluation of γ-Linolenic Acid and Dihomo-γ-Linolenic Acid, their Esters and Possible Precursors as Potential Antithrombotic/Thrombolytic Agents
Authors: Sim A.K. , McCraw A.P.
Published: May 2019

γ-linolenic acid, dihomo-γ-linolenic acid and Naudicelle, a naturally-occurring rich source of essential unsaturated fatty acids have been evaluated as potential antithrombotic agents. A sequential series of tests was used to compare each substance in vitro (human plasma), in vivo using an artificially induced thrombus in the microcirculation of small laboratory animals and ex vivo in non-human primates with a spontaneous pathological thrombotic tendency. Finally the test substances were compared in man after oral administration. Particular reference was made to the effects of the compounds on platelet function, the fibrinolytic system, thrombus formation and on circulating blood lipids. The compounds which are possible prostaglandin precursors, have been shown to have a positive (antithrombotic) effect in the test systems used. Platelet aggregation was inhibited over 24 hours in non-human primates and in man at oral dose levels of 1.5 mg/kg. In addition plasma cholesterol and triglycerides were decreased by up to 50% on the same dose regime.
In Vitro Spontaneous Platelet Aggregation in Cerebrovascular Disease
Authors: Cate J.W.ten
Published: May 2019

Fifty patients from a group of 130 patients with transient ischemic attacks or cerebral infarction were found to demonstrate in vitro spontaneous platelet aggregation (SPA). This phenomenon occurred within 15 minutes when platelet-rich plasma samples (PRP) of these patients were stirred at 37°C in an aggregometer.

In addition all patients showing SPA also demonstrated a lowthreshold concentration for the onset of ADP-induced second wave aggregation (ADP f.c. < 0.25 uM; normal range 0.75 + 0.2 uM). Of the remaining 80 patients 25 patients were found to be sensitive tolow concentrations of ADP.

SPA remained present in samples of 8 patients studied when stored at room temperature for two hours. SPA was found to be dependant upon the presence of divalent cations and could be prevented by adenosine, phentolamine and aspirin. The following additional findings point towards a possible platelet defect:

1. Platelets from 10 patients with SPA when isolated and resuspended in normal plasma still demonstrated SPA while isolated normal platelets in patients did not.

2. Platelets demonstrating SPA showed an increased aggregation tendency upon incubation with ADP while normal platelets developed the expected refractory state for ADP.
Platelet-Dependent proteolytic activation of factor XI
Authors: Walsh P.N. , Griffin J.H.
Published: May 2019

To investigate the hypothesis that human platelets can promote the activation of Factor XI, human platelets were washed by albumin density gradient centrifugation, gel filtered on Sepharose 2B and incubated with purified Factor XI in the presence and absence of purified Factor XII, kallikrein and high MW kininogen (HMWK). The activation of XI was tested in clotting assays using plasmas deficient in XI, IX or VII and the limited proteolytic cleavage of 125I-labelled XI was assessed using SDS gel electrophoresis. The proteolytic activation of XI in the presence of either XIIa and HMWK or XII, HMWK and kallikrein was enhanced 5-l5-fold by ADP-treated or collagen-treated platelets. Significant platelet-dependent proteolytic activation of XI occurred in the absence of added XII and was further enhanced 3-fold by added XII. The results demonstrate that ADP-treated or collagen-treated platelets promote the proteolytic activation of Factor XI in the presence of XII, HMWK and kallikrein and that appreciable proteolytic activation of XI can occur in the presence of collagen-treated platelets and kallikrein without added HMWK or XII.
Chronic Idiopathic Thrombocytopenic Purpura (ITP) 51-Cr-Platelet Kinetics and Splenectomy in 197 Patients
Authors: Gugliotta L. , Isacchi G. , Motta M. R. , Guarini A. , Bachetti G. , Mandelli F. , Tura S.
Published: May 2019

51cr-Platelet Kinetics was studied in 197 patients with Chronic ITP of which 111 were submitted to splenectomy after the study.

Platelet half life-span was always decreased (< 24 hours).

Platelet, sequestration was splenic, spleno-hepatic, hepato-splenic, hepatic and diffuse in 56%, 18%, 1%, 6% and 19% of patients respectively. Platelet life-span and sequestration were not correlated with sex, age, platelet number, duration and severity of disease.

101 patients with splenic and spleno-hepatic platelet sequestration underwent splenectomy. Their follow-up is 1-5 years. Complete (steady platelet level > 150,000μl) or partial (platelet level in between 80,000 and 15,000/μ1) remission was obtained in 83% and 6% of them respectively. In 10 patients with non-splenic platelet segue-stration complete and partial remission’after splenectomy were observed in 5 and 1 gases respectively.

51Cr-Platelet Kinetics is a reliable and useful predictor of the therapeutic value of splenectomy. However splenectomy should be considered also in selected case with non splenic platelet sequestration.
International, Quality Control in Blood Coagulation: Measurement of Fibrinogen
Authors: Thomson Jean M. , Poller L. , Yee K.F.
Published: May 2019

Fibrinogen estimations were included in four of the collaborative exercises from the above Centre between 1976-1078. More than thirty different techniques were employed by participants in over four hundred laboratories. The thrombin time, fibrinogen titre and turbidity techniques showed poor discrimination between normal and low fibrinogen levels compared with other routine methods e.g. Clauss technique, immunological and clot weight methods.
An Evaluation of Currently Available Methods for Plasma Fibrinogen
Authors: Rickard K.A. , Burridge J. , Exner T. , Power P.
Published: May 2019

An assessment of fibrinogen concentration is basic to any investigation of clotting dysfunction and often an estimate of fibrinogen level is needed rapidly as an indication of consumptive coagulopathy or fibrinolysis. Fibrinogen levels in a variety of clinical plasma samples were assessed concurrently by several methods. Results were correlated against a reference method based on Ancrod-clottable fibrinogen and calibrated by U.V. absorbance with alkaline solutions of carefully dried fibrin standard. The best correlations with the reference method were achieved by an immunologic method using the Centrifichem principle and by heat precipitation with quantitation by packing in micro-haematocrit tubes. A modified clot opacity method also gave acceptable results. The turbidimetric ammonium sulphate and sodium sulphite precipitation methods correlated less well with the reference method, and in particular the sodium sulphite technique gave high apparent fibrinogen levels with jaundiced plasmas. Neither of the turbidimeteric methods were useful for fibrinogen levels below 50mg/dl. The thrombin time method showed excellent sensitivity to fibrinogen, even at very low fibrinogen levels, but did not correlate well with the reference method. This apparently conflicts with the findings of a recent CAP survey which strongly favoured the thrombin time method. We believe there is a danger that such surveys promote test methods on which there is good inter-labotatory agreement, but which may not be specific in function.
Chemical Characterization of Duck and Goose Fibrinogen
Authors: Krajewski T. , Nowak P. , Cierniewski C.
Published: May 2019

Chemical composition of duck /A. platyrynchos/ and goose /A. anser/ fibrinogens as well as their polypeptide subunit structure were analyzed. A-mino acid composition studies of intact fibrinogens, isolated in the presence of proteolytic inhibitors, revealed their significant similarity. However, small differences between both fibrinogens in amounts of Tyr, Ser and Hys were detectable. Some differences between duck and goose fibrinogens in carbohydrate quantity were also found. They contained sialic acid /0.71 % and 0.78 %/, hexose /0.75% and 1.1 96/ and H-acetylhexosamine /0.59 % and 0.75%/. Electrophoretic analysis in 9% Polyacrylamide gels of reduced duck and goose fibrinogen showed the presence of two main bands /48 000 and 54 000 daltons/. The third, thrombin-sensitive band /59 000 daltons/ was weaker stained with Coomassie blue and amido black. Electrophoresis of thrombin digests of isolated fibrinogen and plasma fibrinogen demonstrated the identity of the heaviest peptide with the intact Aa chain. The remaining, probably partially degraded Aα chain, also sensitive to thrombin action, migrated together with the Bβ chain /54 000 daltons/. These observations were confirmed by isoelectric focusing analysis. Sub-unit polypeptide chains of duck and goose fibrinogen were isolated by CM-cellulose chromatography. Their chemical composition was also determined.
Aspirin Dosage and Antithrombotic Effect
Authors: Clagett Patrick G. , Russe Marianne , Hufnagel Howard
Published: May 2019

It is generally accepted that there is no correlation between aspirin (ASA) dosage and antithrombotic effect. This hypothesis was tested by devising an animal model of thrombosis consisting of an A-V shunt with flow rate controlled at 200 ml/sec. The occlusion time (T-O) was found to be dependent upon formation of a platelet rich thrombus confirmed by electron microscopy. Three groups of dogs (4 in each group) were treated with 3 dosages of ASA (160, 325, and 650 mg BID for 4 days) and mean T-O under treatment was divided by mean T-O during a control period to obtain an Index of prolongation of T-O by ASA in each animal. The mean indexes of prolongation for the groups were 1.31±0.14 (160 mg), 1.41±0.12 (325 mg), and 2.67±0.32 (650 mg), with the last being significantly greater (p <.01) than the others. This paralleled the salicylate levels: 1.0±0.37 (160 mg), 1.75±0.31 (325 mg), and 4.98±0.53 mg/dl (650 mg). The index of prolongation T-O for each animal was correlated with ASA dosage in mg/kg (r=0.86, ऩ <.001) and salicylate levels (r=0.77, p <.01). To confirm a dosage effect, 3 animals were treated with low (160 mg BID) and high (650 mg BID) dose ASA. In each animal, T-O (sees.) was significantly longer (p <.001) with the higher dose. ASA in all doses produced equivalent suppression of collagen-induced aggregation, 14C-serotonin release, and platelet prostaglandin synthesis as measured by thrombin-stimulated malonyldialdehyde production. These results suggest an antithrombotic dose dependency of ASA not apparent from in vitro platelet function tests.
Prolonged Inhibition of PGI2 Production and Associatedincreased Thrombogenic Effect in Arteries After Aspirin Administration
Authors: Buchanan M.R. , Dejana E. , Mustard J.F. , Hirsh J.
Published: May 2019

In recent studies in rabbits, high dose aspirin (ASA) which inhibited prostacyclin(PG I2) synthesis by veins augmented thrombus formation in jugular veins. This thrombogenic effect was short-lived and lasted only 1-2 hrs. we have shown that the inhibitory effect of ASA on PG I2 synthesis in arteries lasts much longer than in veins. We therefore examined the effects of high dose ASA on arterial thrombosis in rabbits by measuring PG I2 production, 51cr platelet adhesion and 125I-fibrin accretion onto a mechanically-induced injury site in carotid arteries. PG I2 production, quantitated by a thrombin-induced 14C-5HT release assay, was 0.13 ± 0.01 ng/10 mm2 vessel. 51Cr platelet adhesion and 125 I -fibrin accretion onto these vessels 1 nr after injury was 1.00 ± 0.13 × 106 platelets and 57 ± 5 μg fibrin/10 mm2 respectively (mean = SE, n = 22). After ASA treatment (100 mg/kg, IV), PG I2 production was inhibited by at least 84% for 6 hrs and returned to 56% of control by 20 hrs. This PG I2 inhibition was associated with a marked increase in platelet adhesion (>100%) which lasted for at least 20 hrs. There was no significant increase in fibrin accretion. These results show that the more prolonged effect of ASA on PG I2 production in arteries than in veins is paralleled by a more prolonged thrombogenic effect on arterial vessel wall injury.
Properties of an Inhibitor of Platelet Aggregation and Thromboxane Synthesis Isolated from Onion and Garlic
Authors: Makheja A.N. , Vanderhoek J.Y. , Bailey J.M.
Published: May 2019

Onion (allium cepa) and garlic (allium sativum) inhibit platelet aggregation both in vitro and in vivo. An oily chloroform extract of onion was prepared and the anti-platelet activity was purified using standard chromatographic procedures. Inhibitory activity from onion is associated with a non-polar material not inactivated by mild acid or alkali and stable to heating. Similar inhibitory properties were observed with both onion oil and garlic oil (I50/ml PRP = 30-100 μg with different samples of human and rabbit platelets). Platelets incubated with onion inhibitor and 1-14C arachidonic acid showed striking changes in the pattern of radioactive metabolites formed. Most apparent was the almost complete suppression of thromboxane B2 synthesis and the appearance of a new metabolite identified as a product of the platelet lipoxygenase. Measurements of oxygen consumption of treated platelets indicate that these materials inhibit the platelet cyclooxygenase. Similar inhibition of sheep vesicular gland cyclooxygenase was observed with onion oil but not with garlic. Gas chromatographic and mass spectrometric analyses of active extracts of onion and garlic show differences in several major components which may relate to the observed differences in biological activity. The results indicate that two members of the allium family commonly used in the diet contain chemically similar compounds which inhibit platelet aggregation by blocking thromboxane synthesis.
Fibrinogen New Orleans: An Inherited Variant with Abnormal Peptide Release
Authors: Chavin S.I. , Andes W.A. , Beltran W.G. , Stuckey W.J.
Published: May 2019

An inherited fibrinogen abnormality is described in a 30-year old woman with a history of several episodes of excessive bleeding. The initial laboratory finding was a prolonged thrombin time, and a comparable prolongation was present in the plasmas of her mother and daughter, but not of her father. The abnormal fibrinogen gave a reaction of complete fusion with normal fibrinogen in an Ouchterlony plate, and by immunoelectrophoresis, it had a slightly faster than normal anodal migration. The patient’s plasma and purified fibrinogen were able to prolong the clotting time of normal plasma. Using SDS-polyacrylamide gel electrophoresis, we have detected a marked delay in release of a major proportion of the A peptide and a lesser delay in release of the B peptide, after thrombin treatment. B peptide release appeared to be completed before that of A peptide release, in contrast to the situation with normal fibrinogen. Cross-linking of the resulting fibrin was delayed but eventually complete. The rate and extent of monomer aggregation, measured by spectrophotometry, were within normal limits.
Hereditary Hypodysfibrinogenemia with Defective Release of Fibrinogenopeptide A (Fibrinogen Freiburg)
Authors: Böttcher D. , Hasler K. , Köttgen E. , Maurath J.
Published: May 2019

A new autoaomally inherited hypodysfibrinogenemia was recognized in four members in three different generations of a family. Only one patient had a major bleeding episode after trauma, the other affected members had no history of excessive bleeding or thromboembolic disease.

The thrombin time and Reptilase time of plasma were greatly prolonged and partially corrected by the addition of calcium. Patient plasma prolonged the thrombin time of normal plasma. Fibrinogen levels ranged between 10 to 20 mg/100ml when measured as thrombin-clottable protein, whereas immunologically the fibrinogen levels were only slightly reduced.

Functionally the major defect was impaired release of fibrinogenopeptide A upon incubation of the purified abnormal fibrinogen (94% clottable protein) with thrombin and Reptilase. The abnormal fibrinogen showed a delayed polymerisation of its purified fibrin monomers. The described abnormal fibrinogen was indistinguishable from normal fibrinogen by polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate.
Relative Reactivity of Platelets Towards Nonendothelialized Surfaces
Authors: Hanson S.R. , Harker L.A. , Ratner B.D. , Hoffman A.S.
Published: May 2019

Rates of platelet consumption (PC) by subendothelium, vascular grafts and artificial surfaces have been measured in nonhuman primates. Studies included 1) 18 monkeys (M. nemestrina)with balloon catheter deendothelialization of 10 cm or 20 cm segments of the aorta, 2) 24 baboons with Dacron velour (USCI Sauvage) aortic grafts, and 3) 113 baboons with femoral A-V cannulae. In all studies PC was directly proportional (r =11.935) to the area of non endothelialized surface. Platelet-surface reactivities (plats/cm2/day × 10-8, ± 1 SD) were: subendothelium, 176 ± 35; aortic grafts, 42 ± 9; cannulas 1 ± 1 to 27 ± 3. Whereas serial measurements of PC by cannulae were unchanged, PC by subendothelium and aortic grafts decreased progressively over 3-5 weeks as a result of reendo-thelialization. PC by eight acrylic polymer cannulae ranged from 4.4 ± 1.5 to 27.2 ± 2.7 (54 studies). PC by five commercial polyurcthanes ranged from 2.7 ± 1.0 to 24.3 ± 0.8 (25 studies). PC by Teflon, Tygon, Silastic and polyethylene was less than 2.5 (34 studies). Cannula PC was independent of variable blood flow rates, circulating platelet concentrations, or heparin anticoagulation but corrated directly with surface polarity (as determined from surface spectroscopy and water sorption measurements). 12I-fibrinogen kinetics were normal. Cannula PC was completely interrupted by dipyridamole (10 mg/kg/day), sulfinpyrazone (100 mg/kg/day) but not by ASA (100 mg/kg/day). We conclude that platelet kinetic measurements provide an objective in vivo measure of the reactivity of nonendothelializcd surfaces and the efficacy of antithrombotic therapy.