bluuebunny/crossref_metadata_2025 huggingface dataset:
The “LUPUS” Anticoagulant: Manifestations and Possible Mechanisms of Action
Authors: Hougie C.
Published: May 2019

With the more general use of the partial thromboplastin time for pre-operative screening, an increasing number of patients with an inhibitor of the type associated with lupus erythematosus are being detected. This is now the most frequent type of inhibitor seen by us. Although the inhibitor is referred to as the “lupus” anticoagulant, in at least one-third of cases this disease is not present. The patients have a prolonged partial thromboplastin time (PTT), while their plasma prolongs the PTT of normal plasma, and this lengthening is not accentuated by prior incubation of the mixture. Thromboplastin generation is normal except when the patient’s plasma is used as substrate. It has been suggested that the inhibitor interferes with the reaction of prothrombinase with prothrombin, more specifically by an action on the phospholipid moiety. In a small proportion of cases, the prothrombin time is prolonged due to a true deficiency of prothrombin. Those factors involved in the intrinsic system are usually decreased when measured by conventional one-stage techniques. As the level of the factor is often higher when tested at a 1:10 or 1:20 dilution rather than the conventional 1:5 dilution, the decrease is generally attributed to non-specific inhibition rather than a true deficiency. New evidence will be presented indicating that in some cases a true deficiency of one or more of these factors may exist, probably due to specific inhibition. The lupus anticoagulants studied by us differed in certain respects from each other in patient to patient. Their mode of action and clinical significance will be discussed.
Two-Dimensional Electrophoretic “Mapping” of Human Platelet Membrane Proteins
Authors: Nichols W. L. , Mann K. G.
Published: May 2019

Two-dimensional electrophoresis of solubilized human platelet membrane proteins utilizing Polyacrylamide gel isoelectric focusing followed by sodium dodecyl sulfate (SDS) slab gel electrophoresis with discontinuous buffer systems yields a high resolution “fingerprint” of platelet membrane proteins. Membranes were prepared from citrate-phosphate-dextrose (CPD) platelet concentrates by differential centrifugation, repeated washes and lysis by means of sonication or glycerol hypotonic lysis. Membrane vesicles were isolated on step gradients of 27%(w/v) or 30% (w/w) sucrose. Following further washing of the vesicles, membrane pellets were extracted by heating in SDS and β-mercaptoethanol. After pelleting the insoluble material, the resulting supernatant was found to contain most of the total protein content of the membrane, and to yield SDS gel patterns similar to platelet membranes totally solubilized with larger amounts of SDS. Extracts were dialyzed against 9M urea, 2% Triton X-100 at 37° for 12 hrs., then electrofocused in large-pore gels containing 9M urea, 1% Triton X-100 and 1.6% to 2.0% ampholyte (pH 3–10). Electrofocused samples were equilibrated with 1% SDS in half-strength stacking gel buffer (D. M. Neville, J. Biol. Chem. 246: 6328, 1971)and electrophoresed in the second dimension into slab gels.

Coomassie Blue R-250 staining revealed more than 40 components, the majority of which electro-focused between pH 4.5 – 7. The major glycoproteins, as assessed by periodic acid-Schiff staining, electrofocus at acidic pH. The greater resolution obtained by this analytical technique will be useful in the further characterization of the membrane proteins of normal and abnormal platelets.
Soluble Fibrin Complexes During Hormonal Contraception
Authors: Asbeck F. , Bebber J. , van de Loo J.
Published: May 2019

In a prospective study, 2o healthy women (16–35 yrs.) were treated with an oral contraceptive (o,25 mg D-norgestrel, o,o5 mg ethinyl-estradiol). Derivatives of fibrinogen and fibrin were investigated before the beginning of medication, after the 1st cycle, and after the 3rd cycle. The investigations included: determination of thrombin-clottable protein, cold precipitable protein (4°C) , ethanol precipitable protein (11,5%), ethanol gelation test, protamin sulfate test, fibrin-fibrinogen degradation products in serum, and quantitation of high-and low molecular weight derivatives of fibrinogen and fibrin by agarose gel filtration of the fresh patient plasmas.

There was a significant increase of thrombin clottable protein (212 vs. 236 mg/dl, p<0,001) and an increase of ethanol precipitable protein (117 vs. 168 mg/dl, p<0,003). There was no change in the cold precipitable protein nor in the paracoagulation phenomena. Fibrin-fibrinogen degradation products were not elevated. By agarose gel filtration, a significant increase in soluble fibrin complexes (p<0,02) could be demonstrated; the highest concentration of the SFC elu-ted in the position of the dimers.

The study confirms a low grade activation of the coagulation system even in healthy women taking a low dose estrogen containing oral contraceptive.
Behaviour of Soluble DES-AB Fibrin and Fibrinogen in Man
Authors: Mahn I. , Müller-Berghaus G.
Published: May 2019

Human des-AB fibrin was generated from I-131-fibrinogen by clotting with thrombin in the presence of EDTA and aprotinin, and solubilized in buffered urea. About 1 to 2 mg of fibrin was injected simultaneously with I-125-fibrinogen dissolved in buffered urea into 14 male healthy volunteers.

The mean distribution volume of the soluble fibrin was 48.5 ml/kg in comparison to 46.1 ml/kg of the injected fibrinogen demonstrating a homogeneous distribution of both the labelled proteins in the circulating blood. The elimination characteristics of the soluble fibrin did not represent a monophasic exponential decay. 24 hours after injection a mean of 24.3 % of the initial value of the fibrin was traced in the blood in comparison to a mean of 53.3 % of the fibrinogen at that time. The terminal slope of the fibrin elimination curve between 2 and 8 days after injection was a monophasic exponential curve with a mean T 1/2 of 2.3 days. The fibrinogen elimination curve showed a T 1/2 of 4.2 days during that period of time.

These experiments indicate that solubilized fibrin injected into man can be traced in the circulating blood for a long period of time. The catabolic behaviour of fibrin differs significantly from that of fibrinogen.
Degradation of Soluble Fibrin Complexes, Fibrinogen, and Fibrin by Macrophage Enzymes
Authors: Chang M. L. , Bang N. U. , Truex L. , Boxer L. , Mattier L. E. , Marks C. A.
Published: May 2019

Stimulated macrophages are known to hydrolyze fibrinogen (F) and fibrin (f) through the elaboration of plasminogen (P) activator. We report here on the degradation of soluble fibrin complexes (SFC), F, and noncrosslinked f by rabbit alveolar macrophages (RAM) via a nonplasmin-mediated pathway. 125I SFC in P-containing and P-free systems adhering to intact RAM were continuously degraded at equal rates into less-than 30,000 Dal tons, 60–80% trichloroacetic acid-soluble peptides, as evidenced by Bio Gel® A-5m gel chromatography of cell-free supernatants. SFC degradation was blocked by 10-3 M iodoacetamide. P-free F, incubated with enzymes from lysed RAM, rapidly became incoagulable and potently inhibited normal F-to-f conversion. SDS gel electrophoresis (SDSGE) (nonreducing conditions) demonstrated only a modest reduction of the MW of P-free F and f during initial incubation; peptides released were too small to be visualized on 6% SDS gels; after prolonged incubation, SDSGE demonstrated no discreet peptides. SDSGE (reducing conditions) revealed the initial proteolytic attack to involve F-f(A)α chains, followed by proteolytic cleavage of (B)β and γ chains. RAM enzymes hydrolyzed f more rapidly than F. Thus, RAM enzymes, in contrast to plasmin, degraded SFC, F, and f extensively, leaving no demonstrable “core fragments” resistant to further proteolytic attack. Since SFC have been shown to be rapidly removed from the circulation by the reticuloendothelial system, the catabolic pathways delineated here may have pathophysiological significance.
Binding of Calcium to Fibrinogen: Some Related Properties
Authors: Marguerie G.
Published: May 2019

The calcium binding properties of bovin fibrinogen have been studied using equilibrium dialysis method. At pH 7.5 fibrinogen has 3 specific calcium binding sites of high affinity and several non specific binding sites of low affinity. Direct titration of the calcium induced proton release indicates that the binding center is a chelate. Thermal an acid denaturation is found to be markedly influenced by the presence of Ca++, suggesting that structural features are related to the binding. However the circular dichroism spectra show that no generalized conformational change is induced when Ca++ is bound to the protein.

The plasminic digestion of fibrinogen is also found to be specificaly influenced by Ca++. The velocity of the initial cleavages is slightly reduced in the presence of calcium. It is therefore suggested that the C-terminal part of the Aα chain is involved in the binding.

Considering the dimeric structure of the fibrinogen molecule, the presence of only 3 calcium binding sites of high affinity suggests the existence of “salt bridges” between the constitutive polypeptide chains.
Development of Monospecific Antibodies to Human Factor IX
Authors: Tiarks Cheryl Y. , Chang Chin-Hai , Pechet Liberto
Published: May 2019

The purpose of this research was to develop neutralizing and precipitating antibodies to factor IX. Human factor IX, purified by the method of Rosenberg et.al. (J. Biol. Chem. 250:8883, 1975), was electrophoresed on acrylamide gel. Two major bands migrating adjacently were eluted. They contained factor IX activity only. The eluates and their homogenized gel segments 7 and 8 were injected separately into two rabbits, Rl and R2, respectively. On immunodiffusion the antiserum Rl showed one precipitating line with normal plasma. It neutralized human factor IX (20 Bethesda units) and also slightly neutralized factor X. It had no effect on factors II and VII. Following absorption of this antiserum with purified factor X it neutralized factor IX only. With continuous immunization, however, this antiserum revealed two new precipitating contaminants. The antiserum R2 neutralized only factor IX; it reached 220 Bethesda inhibitory units. On immunodiffusion it showed two precipitating lines, one of which disappeared after absorption with human albumin. On immunodiffusion and Laurell immunoelectrophoresis, the albumin-absorbed R2 antiserum showed one precipitin line of identity, or one rocket, with normal plasma, a Red Cross factor IX preparation (rich in factors IX, II and X), the original eluates 7 and 8, and a Hemophilia-B antigen-positive plasma. No line or rocket developed with normal plasma absorbed with aluminum hydroxide or with antigen-negative Hemophilia-B plasma. We conclude that the antisera Rl and R2 contain factor IX neutralizing antibodies and that albumin-absorbed R2 has monospecific precipitating antibodies to human non-activated factor IX.
Disorders of Fibrinogen and Factor XIII - Introduction
Authors: Nossel H.L.
Published: May 2019

Transformations of the fibrinogen molecule are amongst the best biochemically defined reactions of the hemostatic mechanism. The exact peptide bonds cleaved by thrombin are known and the initial sites of plasmin attack are defined. The gamma chain cross-links produced by Factor XIIIa are characterized, and work is proceeding on the identification of the alpha-chain cross-links.

Physiologically fibrinogen participates in a number of platelet functions including adhesion to collagen and foreign surfaces and ADP-induced aggregation. Polymerizing fibrin aggregates platelets. Fibrinogen not only acts as a substrate for thrombin but is a powerful reversible inhibitor of thrombin activity.

Congenitally abnormal fibrinogen may be associated with a thrombotic tendency, a hemorrhagic tendency or be asymptomatic. Acquired molecular changes occur in thromboembolic disease, intravascular coagulation and possibly in liver disease. Assays for molecular alteration find increasing clinical application. In the present session the following topics will be dicussed:

1. Relationship between fibrinogen and platelets

2. Fibrin polymerization - biochemistry

- physiological and clinical significance

3. Cross-linking - biochemistry

- physiological and clinical significance
Clotting Studies Performed on Blood Stored in Half-Strength ACD-A
Authors: Cederholm-Williams S. , Mishler J.M. , Darley J.H.
Published: May 2019

Six hundred ml of whole blood from each of five healthy male donors was equally divided and stored at 4-6°C in either standard ACD-A (2.2g trisodium citrate, 0.8g citric acid, 2.45g dextrose/dl anticoagulant solution, pH 4.9) or half-strength ACD-A (l.lg trisodium citrate, 0.8g citric acid, 2.45g dextrose, pH 4.3) to determine if low citrate concentrations adversely effected the following: prothrombin time (PT), thrombin time (TT), Kaolin-cephalin clotting time (KCCT), ethanol gel (EG) and fibrinogen levels. Low citrate concentrations had no significant effect (Student’s t test for paired scores) on any clotting indice tested (see table below).
Immune Thromboses of the Pulmonary Vessels
Authors: Andreev S.V. , Kubatiev A.A.
Published: May 2019

According to current concepts, pathogenesis of intravascular trombus formation is underlaid by three crucial factors: lesion of the vascular wall, impairment of hemodynamics and hemostatic properties of the blood. While admitting the important role of each of these factors, one should acknowledge that the true nature of thrombosis is much more complicated and does not always fit the framework of this triad. In our experiments on rabbits, it was demonstrated that the thrombotic process in the basin of the pulmonary vessels could be successfully reproduced even in intact animals under conditions of disturbed immune homeostasis. A distinctive feature of immune thrombosis of the pulmonary vessels was a generalized lesion of the microcirculatory bed, gradual increase in the thrombotic masses and involvement of the major branches of the pulmonary vessels (PV). Morphologically, a picture of lymphoid-cellular infiltration and localization of the antigenic complex in the affected PV was revealed. As a result of progressive decrease in the pulmonary arterial blood circulation and increasing resistance in the system of the lesser circulation there were noted, already during the first hours after the onset of the capillary thrombosis, a compensatory hyperfunction of the right ventricle of the heart which was manifested in its highly increased contractility, higher levels of cyclic AMP and phosphorylation potential. At a later period, however, the compensatory possibilities of the right ventricle of the heart failed to overcome this resistance with resulting incompetence of the organ starting to develop within 2-3 days.
Relation Between Platelet Volumes and Platelet Kinetics in Idiopathic Thrombocytopenic Purpura(ITP)
Authors: Tsukada T.
Published: May 2019

Platelet volume distribution and appearance rate of megathrombocytes were measured with Coulter counter Model-B in 24 patients with ITP during platelet kinetic studies. Normal range of platelet volumes in EDTA-blood is as follows: mode; 6.8 ±0.8 μm3 (mean ± 1 SD), median; 7.6 ±0.8 μm3 and mean volume; 9.4 ±1.1 μm3. In 15 patients platelets were increased in mean volumes, 8 patients showed normal size and one patient decreased in mean volumes. Increase of megathrombocytes (larger than 14 μm3) was noted in 14 cases. Remaining 10 cases showed normal appearance rate of megathrombocytes (15 ±4.7%). In 5 patients, highest peak was deviated to the smaller elements although the mean volumes were remained in normal range or increased. These findings suggest that at least two population of platelets are present simultaneously in these cases. No close correlation was noted between platelet volumes and platelet counts, mean platelet survival time or platelet turnover. The appearance rate of megathrombocytes also had no correlation with the results of platelet kinetics. Three out of 11 cases whose platelet counts increased to the normal after splenectomy- showed still larger platelet volumes than the normal.

It could be concluded that young platelet could be of variable size in ITP and that it might be impossible to suspect the amounts of effective platelet production from the platelet volumes or the appearance rate of megathrombocyte s.
Detection of Hemophilia a Carriers by Repeated Factor VIII Activity/Antigen Determinations
Authors: Seligsohn U. , Zivelin A. , Peretz H. , Modan M.
Published: May 2019

Detection of hemophilia A carriers (C), using factor VIII (fVIII) activity (Ac) and antigenicity (Ag) measurements, has been hampered by an overlap of results obtained in normal females (NF) and obligatory carriers (OC). In an attempt to improve the specificity and sensitivity of C detection, 29 OC and 33 NF were examined 3 times. FVIII Ac and fVIII Ag were determined in fresh plasma samples, employing a one stage method and the Laurell technic with rabbit anti fVIII antiserum, respectively. Using the mean of the 3 pairs of measurements, a discriminant function, based on Bayes’ theorem, was obtained for calculating the probability that a woman is a C. Three ranges of measurements were defined: Definite C, possible C and definite NF. The results were as follows:

If only the first pair of measurements was used, the results were much less satisfactory:

Thus, for each suspected C, the relative odds of being a C can be assigned by using this function, but the results should be based on the mean of 3 measurements.
Thromboregulation by Platelet Phospholipid Fatty Acids: Effect of Dietary Linoleate
Authors: Hornstra G.
Published: May 2019

It is thought that fatty acids, released from certain platelet phospholipids regulate the platelet release reaction and, thereby, thrombus formation. Arachidonic acid (AA) has been shown to be of major importance in this respect, since it is converted by the cyclo-oxygenase (CO-) system to release-inducing substances. However, the AA-CO interaction is inhibited competitively by other poly-unsaturated fatty acids such as linoleic acid (LA). This implies that the ultimate thrombotic response will depend on the ratio between AA and competing fatty acids, released upon platelet activation and, consequently, on the fatty acid composition of certain platelet phospholipids.

In rats, the ratio between AA and LA from platelet phosphotidylcholine is found to be negatively related to the dietary LA content. Since phosphotidylcholine has been shown to be the main source of AA, this finding may explain the well-established anti-thrombotic effect of dietary linoleate.
Changes Brought about in Blood Coagulation in the Newborn by Adminstration of Vitamin K2
Authors: Suzuki S. , Ichinoe K.
Published: May 2019

Deficiency of the blood coagulation factor, especially of vitamin K-dependent factors, is a major disturbance of extrauterine life in the neonatal period. It is well known that vitamin K1 is converted to vitamin K2 in the human body. In order to compare the effectiveness of vitamin K1 and that of vitamin K2 in the newborn period, changes in coagulability were studied not only at the end of the day after birth but also along the time course. Six hrs after birth, 2 mg of vitamin K1 or vitamin K2 was injected intramuscularly into the thigh of the newborn, and the effects were observed by thrombotest and normotest 3, 15, and 24 hrs after injection. In this experiment, the infants treated with vitamin K2 showed higher values in the thrombotest than those treated with vitamin K1 after 3 and 15 hrs. After 24hrs however, there was no longer any significant difference between the K1 and K2 groups.

As a clinical application of vitamin K2, gastrointestinal bleeding was studied polarographically with the emulsion of meconium or stool. Since the origin of vitamin K2 is the gastrointestinal bacterial flora, changes in the occult blood were studied in one group given 2 mg of vitamin K2 and mixed milk (18 cases), a second group given 2 mg of vitamin K1 and mixed milk (21 cases), and a control group (24 cases). Haemoglobin percentage of newbornfaces are greatly diminished by a oral adminstration of vitamin K2. Both results may indicate rapid action of vitamin K2.
ReAct Test: Recalcification of Activated Whole Blood Using the Sonoclot
Authors: Ens G.E. , Hamstra R.D.
Published: May 2019

A rapid, simple, reliable laboratory method for detecting thrombotic tendency, estimating heparin dosage, and monitoring heparin effect is available using a new thrombokinetic instrument (Sonoclot) and recalcified activated clotting (ReACT).. The same method can be used to follow bleeding disorders such as hemophilia. 0.2 ml of carefully mixed, citrated whole blood (1 part citrate plus 9 parts blood) was measured into a disposable unsiliconized glass cuvette containing a disposable magnetic stir bar. 0.1 ml of ReACT mixture (siliceous earth plus 0.025 M CaCl2) was added to the cuvette, mixed for 5 seconds, and allowed to clot while monitoring with the Sonoclot. The ReACT procedure was modified for hemophiliac studies, platelet poor plasma was clotted with siliceous earth plus 0.05 M CaCl2. A continuous line chart was analyzed for Clot Onset (C0), an arbitrary time end point, ReACT Time (RT), and Clot Formation Rate (CFR). Studies from normal adults (50) show a C0 of 100-150 sec, and RT of 140-190 sec, and CFR of 10-20 chart units/minute. Patients with clinically evident clotting problems exhibit a slightly shorter Co, shortened RT and increased CFR. Heparin added in vitro at doses of 0.05 u/ml to 0.5 u/ml lengthens the C0 and RT while the CFR changes little. ReACT results from heparinized patients are similar. Estimation of needs based on in vitro data have been made with smooth heparization. Platelet poor plasma from untreated classical hemophiliacs shows a markedly prolonged C0 and RT with a low CFR. Serial dilution with normal plasma causes normalization of the Sonoclot curve between 0% and 10% factor VIII levels.
Purification and Metabolism of Rat Antithrombin III
Authors: Okuda S. , Nakagawa M. , Okada T. , Sawada K. , Ijichi H.
Published: May 2019

The previously reported heparin sepharose affinity column has been proved to be reduced in its recovery on the repeated uses because of loose binding of ligand to sepharose. We tried to obtain the better recovery of antithrombin III (AT-III) using four different affinity columns of CNBr activated sepharose 4B, AH-sepharose 4B, CH-sepharose 4B, and Epoxy activated sepharose 6B. 3H-labeled heparin was used to check its binding capacity to sepharose gels. Binding of heparin to gel surface was found to be tight in AH-sepharose 4B, CH-sepharose 4B, and Epoxy activated sepharose 6B because of the difference of the binding sites of heparin free radicals but their recovery and purity of AT-III were poor.

According to this experimental results, CNBr activated sepharose 4B was used for the purification of rat tissue AT-III and the metabolism of AT-III was investigated from the stand point of 14C-glycine. incorporation into AT-III fractions. 14C radioactivity in the tissue AT-III fractions were observed immediately after injection and reached to the maximum at 6 hours and appeared to be released into circulating pool in 2 hours after injection.
The Influence of Anticoagulant Treatment on Blood Coagulation System Findings in Patients with Acute Myocardial Infarction
Authors: Fletcher A. , Alkjaersig N. , Ghani M. , Tulevski V.
Published: May 2019

Two hundred and twenty patients admitted to an acute coronary care unit were studied by serial plasma fibrinogen chromatography; 110 of these patients had documented acute myocardial infarction. Statistically significant increases in high molecular weight fibrin(ogen) complexes (HMWFC) were noted within 24 hours of disease onset and a further increase (p < 0.001) occurred within 48 hours in 43 patients who did not receive anticoagulant therapy. Plasma HMWFC usually returned to near normal values 12-20 days after disease onset.

Sixty-seven additional patients received conventionally administered anticoagulant therapy, 10 initial heparin only, 33 heparin plus warfarin and 24 warfarin alone. plasma fibrinogen chromatographic findings in the anticoagulant treated patients did not differ significantly (p > 0.1) from those of the control untreated group over the first five days of illness. Minor differences were evident in the comparison of the 5-10 day findings in those receiving long-term warfarin therapy.

Our findings demonstrate that patients with acute myocardial infarction develop a coagulopathy secondary to increased fibrin deposistion, presumably occurring at the infarct site. Since conventional anticoagulant therapy fails to control this phenomenon, the influence of fibrin deposition at the infarct site on disease outcome remains uncertain.
Aggregability of Mammalian and Avian Thrombocytes with Special Respect to the Effect of Various Antithymocyte and Antilymphocyte Globulins
Authors: Suba E.A. , Csákó G.
Published: May 2019

Although interest in platelets is rapidly growing, systematic comparative studies on platelet aggregation are still limited. Here, the aggregation in citrated platelet (thrombocyte)-rich plasmas (PRP) from 18 mammalian (man; chimpanzee; baboon; cynomologus, rhesus, pigtailed, stumptailed, and squirrel monkeys; dog; cat; cow; goat; sheep; horse; rabbit; rat; hamster; guinea pig) and one avian species (chicken) was measured by Bom’s turbidimetric method with agents at final concentrations as given below. Despite the quantitative differences, ADP (5 μM), acid-soluble calf skin collagen (120 μg/ml) or bovine thrombin (0.5 U/ml) aggregated in all mammalian PRPs. In contrast, epinephrine (25 μg/ml) caused aggregation in canine, feline and some primate PRPs, whereas ristocentin (1.5 mg/ml) did in PRPs of certain primates. Chicken thrombocytes were slightly reactive only to collagen and a 4-fold dose of thrombin. Because platelets share antigens with lymphocytes, it was not surprising that all unabsorbed antithy-mocyte (horse anti-human; rabbit anti-rat, -mouse, -chicken ATGs) and antilymphocyte globulins (rabbit anti-rhesus, -rat, -mouse, -hamster, -guinea pig ALGs) tested exhibited platelet aggregating activity. The cross-reactivity of ATGs or ALGs always followed the phylogenetic order qualitatively, but the quantitative relations (extent of aggregation) frequently differed from that pattern. As an explanation, variations in susceptibility to aggregation among various species can be suggested. In parallel studies, goat antirhesus and rabbit antibaboon platelet sera revealed wide cross-reactions between mammalian species, however, like anti-mammal i an ATGs or ALGs, not for the chicken.
Factor VIII Inhibitors: Therapeutic Response to Prothrombin Complex Concentrates
Authors: Permer J. , Kelly P.
Published: May 2019

Prothrombin complex concentrates, two commercial (Prople.c, Konyne), and two “activated” products (spontaneous - Auto IX, and controlled - CAP) were employed in the management of bleeding episodes in hemophilic and non-hemophilic patients with Factor VIII inhibitors. Auto IX’s response rate has been high - approximately 80% - providing a sufficient quantity is administered, while CAP’s, presently under investigation, appears similar. Commercial products have been less effective, as indicated by clinical and laboratory observations. Recent changes in manufacturing procedures have reduced the response rate of these non-activated concentrates.

The duration of effect achieved with these products appears short, perhaps 4 to 6 hours at most, and repeated administration at 6 or 8 hour intervals is recommended until bleeding is controlled. Doses capable of reducing partial thromboplastin time values to less than twice normal (less than 60 seconds)should be employed. A Factor VIII correction unit based on the product’s capacity to correct Factor VIII deficient plasma has been useful in predicting a clinical response, and provides values similar to that obtained by mixing the products with inhibitor plasma. Thrombogenic-ity has not proved to be a problem even when the concentrates have been employed in large amounts.
Bovine Factor VIII. Antigens and Biological Activities
Authors: Casillas G. , Simonetti C. , Pavlovsky A.
Published: May 2019

The procoagulant (PcgF) and the platelet aggregating (PAF) activities of bovine F. VIII were studied in order to establish their relation to the antigens A1 and A2, antigens similar to those synthetized by von Willebrand and hemophilia A patients respectively. Studies of stability to different agents (temperature. EDTA, thrombin, etc.) allowed us to establish that VIII (PcgF) and VIII (PAF) are not mutually dependenteAn homologous antibody of low species specificity against human F. VIII binds specifically with VIII (A,) antigenic moiety of bovine F. VIII. The complex so formed is purified by gel filtration and has VIII (PAF) activity but not VIII (PcgF) activity indicating that VIII(A1) and VIM (PcgF) activities associate,, Another complex, formed by bovine factor VIII and a rabbit antibody against the VIII (A2) moiety, was prepared,, This complex has VIII (PcgF) activity but not VIII (PAF) activity, indicating that VIII (A2) and VIII (PAF) associate,, The complex and its procoagulant activity sediment after centrifugation and may by re-covered by suspension of the precipitate. The following scheme showing the relationship between the antigenic moieties and the activities of bovine factor VIII is proposed: VIII(A1-PcgF) (A2-PAF).
A Laser Scalpel for Operations on Hemophiliacs
Authors: Horoszowski H. , Seligsohn U. , Heim M. , Farin I.
Published: May 2019

The CO2 laser scalpel has an excellent hemostatic effect producing a minimal parainci-sional necrotic zone. Four severe hemophilia A patients were operated with the laser scalpel. Three underwent knee synovectomy 2 of whom without placing a tourniquet. The fourth patient underwent an operation for hip contracture. Factor VIII levels at the beginning of the operations were gradually reduced from the first operation to the fourth as follows: 100%, 80%, 14.5% and 1.4% respectively. No significant bleeding was observed during any operation. The 3 patients who underwent synovectomy were ambulated as early as 3 days after operation. They were discharged after 20 - 26 days, which is considerably shorter than the mean stay of 39 days in the hospital in 8 patients who had been synovectomized previously by the conventional method. An attempt to stop replacement therapy in the fourth patient on the 6th postoperative day resulted in bleeding. Thus, the advantages of the laser scalpel are: Excellent local hemostasis, tourniquet unecessary, low levels of factor VIII sufficient at operation, early ambulation and discharge. It is not as yet clear whether the amount of postoperative replacement therapy can be reduced. The disadvantages are: Longer operation time (by about a third); smoke produced at theatre; the price of the instrument.
Social Support Systems in the Treatment Programme of Haemophilia in new South Wales, Australia
Authors: Bellamy A.
Published: May 2019

A system of social supports for the person with haemophilia and his family is vital if he is to realize his personal capacities as a fully functioning member of his family group and in the community. Negative attitudes within the family, in health care personnel, in educational and occupational systems constitute barriers to the acceptance of the haemophiliac person as capable of becoming self-sufficient and economically self-supporting. The importance of education and development of positive attitudes in parents, health professionals, teachers and employers is paramounto. Therapeutic groups for parents, particularly mothers, for the adolescent boys, informative interviews with educational personnel directed towards attitude change are a vital part in the overall frame of treatment and rehabilitation. Techniques in family and community education have been an integral part of the social work programme of the Haemophilia Treatment Centre, Royal Prince Alfred Hospital, Sydney, Australia.
Fluctuations in Factor VIII Procoagulant, Antigen and Von Willebrand Factor in Normal Individuals
Authors: Yang H.C. , Vaudreuil C.
Published: May 2019

Fluctuations in the relationship among factor VIII procoagulant (VIIIC), factor VIII antigen (VIIIAG) and von Willebrand factor (vWf, ristocetin cofactor) were studied in two normal females (A&B) and one normal male (C). Biweekly fasting morning plasma samples were obtained over a three week period and assayed on 2 different days. The mean VIIIC/VIIIAG, VIIIC/vWf and VIIIAG/vWf ratios were not significantly different from each other (p>.05) in each of the subjects. However, there were marked day-to-day changes in the three ratios as indicated by the ranges.

The results demonstrate that although the ratios among factor VIII procoagulant, factor VIII antigen and von Willebrand factor are relatively fixed, there are significant day-to-day fluctuations.
The Role of Factor IX (Christmas Factor) in Blood Coagulation
Authors: Davie Earl W. , Fujikawa Kazuo , Lindquist Patricia , Scipio Richard Di , Kurachi Kotoku , Heimark Ronald
Published: May 2019

Factor IX participates in the middle phase of the intrinsic pathway of blood coagulation. The reactions leading to the activation of factor IX involve prekallikrein, high molecular weight kininogen, and factor XII. These proteins interact in the presence of a surface such as kaolin and give rise to the activation of factor XI. Factor XIa then converts factor IX to factor IXa in the presence of calcium ions. In this reaction, factor IX (a single-chain glycoprotein of mol. wt.-~55,000) is converted to factor IXa in a two-step reaction. In the first step, an internal peptide bond is cleaved leading to the formation of an intermediate lacking enzymatic activity. This intermediate contains two polypeptide chains held together by a disulfide bond(s). In the second step, an activation peptide is split from the heavy chain of the intermediate giving rise to factor IXa (mol. wt. ~45,000). Factor IXa is composed of a heavy chain (mol. wt.~27,000) and a light chain (mol. wt. ~16,000) held together by a disulfide bond(s). The activation mechanism is essentially identical for human and bovine factor IX. Factor IXa is a serine protease with esterase activity and is sensitive to protease inhibitors such as antithrombin III. Factor IX is also activated by the protease from Russell’s viper venom, but this reaction involves only a single cleavage in the precursor molecule. The critical step in the activation of factor IX by factor XIa or the protease from Russell’s viper venom is the cleavage of the same internal Arg-Val peptide bond and the formation of a new amino-terminal sequence of Val-Val-Gly-Gly- in the heavy chain of the enzyme.
Bone Marrow Plasminogen Activator in Malignant Disease
Authors: Hampton J.W. , Weidenbach A.S. , Rao S.
Published: May 2019

Enhanced blood fibrinolytic activity has been reported with metastatic malignancy and with primary hepatic disease. The demonstration of a plasminogen activator in a case of acute myelomonocytic leukemia prompted us to test bone marrow concentrates from healthy individuals (7), individuals with cirrhosis (3), carcinoma of the prostate (1), and with leukemia (17). Three mis of bone marrow was obtained by sternal marrow aspiration and mixed with 1 ml sodium heparin (10,000 u/ml). Using progressively smaller bore needles the particles were fractured and layered with a Ficoll-Hypaque solution. The cells are resuspended in a final concentration of 105 cells/ml. Using affinity-chromatographed plasminogen, 10μ1 of the cell suspension was added to a bovine fibrin plate. After 17 hours the diameter of the plate was measured and converted to Ploug units using a standard urokinase curve. Healthy individuals showed generally no or very low activity. The patients with cirrhosis showed 2-6.5 u/105 cells. The single patient with metastatic carcinoma of the prostate showed 24 u/105 cells. Patients with acute leukemia showed from 1-40 u/10b cells. There was a rough correlation between the height of activity and the percent blast cells in the marrow.Triton-X added to the marrow cells caused the release of the membrane activity in experiments with 3-22 u/105 cells. Immunodiffusion of bone marrow activator from a patient with leukemia showed identity with the activator from a patient with cirrhosis and with primary fibrinolysis. We conclude that the bone marrow plasminogen activator may reflect the altered stem cell associated with leukemia.
The Parasinusoidal Location of Megakaryocytes in Marrow: A Determinant of Platelet Release and a Physiologic Version of Vascular Invasion and Metastasis
Authors: Lichtman M.A. , Chamberlain J.K.
Published: May 2019

Megakaryocytopoiesis occurs in the hematopoietic (extravascular) compartment of marrow. Thus, platelets must traverse the wall of the vascular sinuses of marrow to enter the circulation. We have examined mouse and rat marrow, fixed by rapid immersion so as to maintain anatomical relationships as close to the natural state as possible. Quantitative transmission electron microscopy (TEM) of random transections of femurs established that megakaryocytes reside less than lym from a marrow sinus wall with a probability unlikely to be the result of chance (P<.001). An intimate relationship exists between the megakaryocyte periphery and the abluminal surface of the endothelial lining cell. At the time of platelet release megakaryocyte cytoplasm invaginates (invades) and penetrates the endothelial lining cell. The penetrating cytoplasm is detached and enters the marrow circulation. From their dimensions in comparison to circulating platelets, the released cytoplasm represents a packet of platelets that undergoes further fragmentation in the circulation. The parasinusoidal location of megakaryocytes and the process of sinus wall penetration and platelet delivery was observed both by TEM and scanning electron microscopy.

These studies provide quantitative support for a specific anatomical arrangement of megakaryocytes in marrow. Moreover, the process of platelet release appears to be a physiological form of metastasis with invasion of vascular walls and vascular spread of cells, that are in this case amitotic.
Membrane Microenvironmental Changes During Platelet Aggregation: A Study with Fluorescent Probes
Authors: Nathan T. , Dvilansky A. , Fleisher G. , Parola A.H.
Published: May 2019

Studies were carried out with fluorescent probes which covalently bound with varied selectivity to plasma membrane proteins. Comparison with non-covalently bound fluorescent probes, Aniline naphthalene sulfonic acid and 1,6-diphenyl hexatriene, should reveal the role of various membranal constituents in the aggregation process. Combined phase contrast and fluorescent microscopy studies revealed essentially no alterations of platelet morphology, caused by the probes. Washed or sepharose 2-B separated labeled platelets exhibited normal aggregation curves. Fluorescent emission intensity decreased 3 to 4 fold during aggregation. The observed decay of fluorescence was not merely a result of the gross macroenvironmental changes in turbidity, as evident by the different initial rates of the processes. Rotational dynamics of the labeled plasma protein were followed by the fluorescence polarization method. An increase in fluorescence polarization values during initial aggregation stages was observed. These results may indicate the motion of the labeled proteins from the internal hydrophobic membrane region to the exposed hydrophilic environment resulting in the reduction of the rotational freedom of those labeled proteins.
Effects of Sodium Nitroprusside on Platelet Adhesion, Subsequent Aggregation and Vasoconstriction
Authors: Baumgartner H.R.
Published: May 2019

Sodium nitroprusside (SNP), a potent vasodilator, has shown beneficial effects in acute myocardial infarction. Since platelets may play an important role in the pathogenesis of myocardial infarction, the effect of SNP on their interaction with rabbit aorta subendothelium was investigated in vivo and under controlled blood flow conditions ex vivo and in vitro.

One iliac artery and the abdominal aorta were denuded of endothelium by balloon catheter injury during infusion of glucose, SNP at 6 or 12 μg/kg/min in groups of 12, 6 and 7 rabbits respectively. The aorta and their branches were perfuse-fixed under controlled pressure 10 min after denudation. Morphometric evaluation showed dose-dependent and significant (2p < 0.01 or 0.001) inhibition of platelet spreading, adhesion and aggregation. The latter was abolished at the higher dose of SNP. Denudation and subsequent platelet adhesion caused strong vasoconstriction (2p < 0.001) which was inhibited by SNP (2p < 0.01).

By exposure of subendothelium to either citrated blood or native blood in a flow chamber (2000 sec-1 shear rate) strong inhibition of spreading and adhesion-induced aggregation was again demonstrated at 6 and 12 μg/kg/min SNP. In vitro, adhesion-induced aggregation was completely abolished after the addition of SNP to rabbit (at 20 μg/ml) or human blood (2 μg/ml). 1 μg/ml PGE1 was needed to induce a similar inhibitory effect.

Thus SNP is a strong inhibitor of platelet function and of injury + platelet induced vasoconstriction. These findings may explain its beneficial effect in acute myocardial infarction.
Studies on NAD(P)H Dehydrogenase and its Role in Vitamin K Dependent Carboxylation
Authors: Wallin R.
Published: May 2019

A simple two-step method has been established for the purification of NAD(P)H dehydrogenase (DT diaphorase) from rat liver by affinity chromatography. The same method has been used to remove selectively the enzyme from the detergent-solubilized, microsomal vitamin K-dependent carboxylating system. The removal of NAD(P)H dehydrogenase inactivated the system, but the activity is restored if purified enzyme is added back to the system.
The WFH/ICTH Study of the Sensitivity of Different Partial Thromboplastin Time Tests in Mild Haemophilia
Authors: O’Brien P.F. , North W.R.S. , Ingram G.I.C.
Published: May 2019

The second phase of the WFH/ICTH study has been conducted by distributing some 40 snap-frozen coded plasma samples from normal subjects and mild haemophiliacs to seven U. K. laboratories, where they were tested with the Manchester PTT reagent (Dr. L. Poller) and seven commercial reagents. Each laboratory also assayed each sample (under different code) for VIIIc by the local method. Each laboratory used the Manchester reagent and a given pair of the commercial reagents throughout the study, so that each commercial reagent was used by two different laboratories. The correlation between VIIIc and PTT was imperfect, with consistant discrepancies across reagents for given samples, making clear that the PTT was affected by other plasma characteristics (presumably within the normal range) besides the low VIIIc. The ability of reagents to discriminate between ca 30 iu/dl and 100 iu/dl VIIIc appeared to be correlated with the slope of PTT on VIIIc in the range 5-30 iu/dl. The results from mild haemophiliacs and normal subjects will be reported in detail for each PTT reagent.
Urinary β-Thromeoglobulin in Essential Hypertension
Authors: Anderton J.L. , Fananapazir L. , Dawes Joan
Published: May 2019

Urinary concentrations of the platelet-specific protein β-thromboglobulin (β-TG) were measured in patients with essential hypertension. Values obtained for patients with normal renal function fell within the same population as those for a control group of normal individuals, but nevertheless 21% of these patients had urinary β-TG values above, the upper limit of the normal range. Values for patients with renal insufficiency were significantly different from the other groups, and 44% of these individuals had abnormally elevated urinary β-TG concentrations. These elevated values may reflect an increased plasma concentration of β-TG in patients with renal impairment or they could be an indicator of renal damage.
Coagulation Factor XIII and Serum Triglycerides in Patients with Chronic Renal Disease on Conservative and Regular Dialysis Treatment
Authors: Carmassi F. , Mariani G. , Palla R. , Fusani L. , Dianchi R.
Published: May 2019

The behaviour of coagulation factor XIII (fibrin stabilizing factor, FSF) and of serum triglycerides was studied in patients with chronic renal disease, using a specific antiserum to the active subunit (FSFA) for the direct measurement of the active fraction according to the Laurell’s method. The plasma FSFA concentration was measured in the following patients: I) 31 patients with chronic renal disease and serum creatinine below 1.5 mg/dl; II) 41 patients with chronic renal failure on conservative treatment; III) 53 chronic uremic patients on regular hemodialysis. FSFA concentration was significantly higher than normal (P<0.005) in the patients with chronic renal disease without renal failure, and similarly increased in the 41 patients with chronic renal failure on conservative therapy (P<0.001), as well as in the patients on chronic hemodialysis (P<0.001). Plasma FSFA levels were significantly higher in those patients with serum triglycerides above the upper normal limit and a significant positive correlation was found between serum triglycerides and FSFA plasma levels. These findings suggest a possible interaction of FSF with blood VLDLs in the genesis of atherosclerosis, early and severe complication of uremia.
Free Zone Electrophoresis of Clotting Factors
Authors: Cartwright T. , Dickerson C.H. , Austen D.E.G.
Published: May 2019

A free-zone electrophoresis system is used for rapid production of substantially purified factor VIII from either citrated plasma or from a factor VIII Concentrate of intermediate purety. Using concentrate, a total yield of 43% of clotting activity is obtained with fibrinogen reduced to approximately 30% of its starting level. Protein level is reduced to 20% of the original value. Using plasma, the albumin, factor VIII and fibrinogen were all separated from factor VIII. In these experiments, factor-VIII-related antigen migrates with the coagulant factor VIII illustrating that both have similar mobilities equivalent to that of an alpha-globulin, or else that they are connected together. The particular advantage of this separation is that it is carried out in a continuous flow apparatus in liquid buffer with no solid matrix and that once the apparatus is set up, material can flow through the machine at a rate of 5 mls/min being continuously separated. The main problem and area for further study is that of concentrating the relatively dilute fractions without undue loss.
Prostacyclin and the Mechanism of Action of Antithrombotic Drugs
Authors: Moncada S.
Published: May 2019

The balance between the proaggregating thromboxane A2 and the antiaggregating prostacyclin forms a homeostatic mechanism for the control of platelet aggregability in vivo. Prostacyclin is a circulating substance released from the lungs which acts by stimulating adenylate cyclase leading to an increase of cyclic AMP in the platelets. The mechanism of antithrombotic action of phosphodiesterase inhibitors like dipyridamole depends on their ability to potentiate the effect of endogenous PGI2. Prostacyclin and TXA2 are inhibited by aspirin but the enzyme which synthesises TXA2 is more sensitive to the inhibitory effect of aspirin, therefore it is possible to administer doses of aspirin which by inhibiting selectively TXA2 will have an antithrombotic effect. Human studies demonstrate that cutaneous bleeding time is controlled by the balance between TXA2 and PGI2 and can be manipulated by using different doses of aspirin.