bluuebunny/crossref_metadata_2025 huggingface dataset:
Thrombinaemia and Hematological Changes Induced in Dogs by the Artificial Kidney
Authors: Veenhof C. H. N. , Prakke E. M. , Vreeken J. , Bosch E. , Meer J. van der
Published: June 2019

The aim of the investigation was 1) to trace the chronological sequence of coagulation events and haematological changes induced by the artificial kidney and the reaction of the living organism; and 2) to study the influence of various drugs.

Group A, non-treated: in the venous blood factor V activity and fibrinopeptide A, generated in vitro, reached maximal values at 15 min. At 10-15 min fibrinomonomers were detected. From 15 min onwards FDP appeared. Granulocyte and platelet counts reached very low values at 10-15 min. The concentration of serotonin (5HT) was maximal at 10—15 min.

Group B : heparin and coumarin prevented thrombin activity. However, they did not inhibit the initial fall of leucocytes and platelets. Release of 5HT did not occur when no thrombin activity was detected.

Group C, treated with Acetylic Salicylic Acid (ASA), sulfinpyrazon, dipyridamol in combination with ASA, polyphloretinphosphate : thrombin generation was postponed to a small degree. However, the initial loss of leucocytes and platelets was not diminished. The release of 5HT was reduced.

In all three groups the manifestations of thrombin activity were much less pronounced in the arterial line indicating a clearing mechanism in the body. The FDP values in the arterial blood were not different from those on the venous side. The initial decrease of leucocyte and platelet counts, especially in the arterial side suggests trapping in the body. Moreover, the arterial-venous difference in platelet counts indicates retention of platelets in the extracorporeal circuit. The drugs used prevented or diminished generation of thrombin. To influence the initial decrease in leucocyte and platelet counts attention should be directed more to the properties of the different elements of an artificial kidney than to the blood components.
The Photometric Aggregation Test (PAT III). A Screening Test for Enhanced Platelet Aggregation
Authors: Krzywanek H. J. , Breddin K.
Published: June 2019

PAT III has been developed for the photometric registration of “spontaneous” platelet aggregation. Citrated PRP is rotated at 20 rpm and 37° C using a disc shaped polystyrol cuvette. Aggregation inducing substances are not used. Changes in optical density which occur when platelets aggregate, are registered on a chart recorder.

The following parameters are of special interest : Tr = time from start of rotation to onset of aggregation; angle alpha2 as a measure of max. aggregation speed; and α2/Tr. These parameters allow a more precise differentiation of the spontaneous platelet aggregation.

Our previous findings with PAT I were confirmed by our recent studies: In 124 normal subjects we found a gradual decrease of missing spontaneous aggregation (α 0-40°) with rising age, whereas the number of persons with enhanced aggregation (α 41-90°) reached 70-80% beyond the age of 50. The percentage of 183 patients with diabetes mellitus and enhanced platelet aggregation was greater than that of age matched controls.
Automated Monitoring of Platelet Aggregation in Multiple Samples of whole Blood
Authors: Clarke N. , McCabe V.
Published: June 2019

Whole blood may be first agitated in a circular transparent rotor chamber, and then rotated continuously to produce plasma/red cell separation for monitoring. The rotor is driven by a low inertia, printed circuit d.c. motor (108 Watt) which is automatically controlled. The pattern of rotation is determined by a signal generator which provides various waveforms at adjustable frequencies and also at fixed speeds.

By segmenting the rotor into multiple cuvettes (Vol = 0.4 ml) it is possible to monitor the platelet concentration, after agitation, as each cuvette spins through the stationary optical system. This consists of a light-emitting diode (peak: 900 nm), pulsed at 10 kHz in order to eliminate ambient light effects, and a photodetector with a built-in amplifier, fed to an amplifying circuit which eliminates other signals. An electronic circuit permits selection of the signal from each cuvette on a direct reading meter. Cuvette No. 1 is identified by a photocell-detector trigger.

Platelet-free plasma is about zero O. D. in the infra red with respect to a water blank, and with attention to specific criteria such as optical geometry and light path, platelet concentration may be accurately determined.
Two Cases of von Willebrand’s Disease with Specific Inhibitors of Ristocetin Induced Aggregation of Normal Platelets
Authors: Blombäch M. , Egberg N.
Published: June 2019

Ten patients (8 females and 2 males) with severe form of von Willebrand’s disease were investigated for the occurrence of inhibitors blocking Ristocetin induced aggregation of normal platelets. All these patients had been multiply transfused and had the classical signs of von Willebrand’s disease, prolonged bleeding time, less than 10% of normal factor VIII activity and correspondingly low factor VIII antigen levels, normal ADP induced platelet aggregation but no Ristocetin induced aggregation. In none of these patients circulating anticoagulants neutralizing factor VIII activity had been demonstrated. Plasma samples from 2 of these patients were found to block Ristocetin induced platelet aggregation of normal plasma. In one of these patients the inhibitor was found to be of IgG type. This patient otherwise had a normal serum electrophoretic pattern. The inhibitor of the other patient is being investigated.
Determination of Fibrinmonomer (FM) from Plasma by Affinity Chromatography on Insolubilized Fibrinogen (FG-ag)
Authors: Matthias F. R. , Reinicke R. , Heene D. L. , Lasch H. G.
Published: June 2019

Quantitative estimation of soluble fibrinmonomer in plasma may be achieved by affinity chromatography on insolubilized fibrinogen (FG-ag), as described by us (Heene, Matthias: Thrombosis Res. 2, 137, 1973). The procedure is based on the reversible adsorption of fibrinmonomer to FG-ag. Column: 0.9 × 28 cm FG-ag; sample volume: 2 ml plasma; adsorption buffer A: 0.05 M tris-H3PO4, 0.1 M NaCl, 0.001 M TAME, 0.005 M EDTA, 100 KIU Trasylol/ml, pH 7.8 ; desorption buffer B : 0.05 M tris-H3P04, 2 M NaBr, 0.001 M TAME, 0.005 M EDTA, 100 KIU Trasylol/ml, pH 5.3. Determination of fibrinmonomer from peak B was performed by the staphylococcal clumping test. The experimental conditions were standardized for a quantitative evaluation by use of radioactive labelled fibrinogen and fibrinmonomer.

Besides normal samples plasma of patients with consumption-coagulopathy, acute myocardial infarction and with carcinomas were investigated. The elevation of fibrinmonomer in plasma was dependent on the severity of the disease. In many cases an elevation of fibrinmonomer was found even before the analysis of clotting factors or the ethanol test indicated hypercoagulability or consumption-coagulopathy. Under conditions of an increased turn-over of clotting factors it is possible to follow up quantitatively changes in the concentration of fibrinmonomer in plasma during the course of the disease.
Improvement of the Preparation of Factor VIII Concentrates
Authors: Vermeer C. , Soute B.A.M. , Brummelhuis H. G. J.
Published: June 2019

The effect of some variables on the yield of factor VIII procoagulant activity during the preparation of factor VIII concentrates was investigated. The results may be summarized as follows:

- The pH of the anticoagulant solution, which results in an optimal recovery of factor VIII in plasma varies with the time, during which the blood or plasma has to be stored before the plasma is frozen. The temperature during the storage of blood and plasma should be higher than 10° C.

- A high freezing and thawing rate of the plasma results in a high recovery of factor VIII and less contaminating proteins and lipids in the cryoprecipitate.

- After centrifugation and dissolving of the precipitate, the preparation may be lyophilized. An appreciable loss of activity is found when the temperature during this process exceeds 20° C.

- From the cryoprecipitate factor VIII can be further purified by precipitation with PEG 4000. The fraction between 2½% at 20° C and 6% at 0° C was used. Within certain limits this precipitation is independent on the protein concentration, pH, time, and ionic strength. Mandelate (0.3% w/v) prevents irreversible precipitation of fibrinogen during all operations.

- Sterilization of the dissolved precipitate was achieved by filtration through membrane filters (0.3 μ) on which the filter aids Celite 535 and Hyflo supercel were layered.

- PEG precipitation and sterilization induce a 20% loss.

- The clinical use of the product was assayed in series of infusions in hemophiliacs.
Human and Bovine Prothrombin: Sequence Homology
Authors: Walz D. A. , Hewett-Emmett D. , Reuterby J. , McCoy L. E. , Seegers W. H.
Published: June 2019

Bovine and human prothrombins can be selectively activated to release PR fragment and 0 fragment. In addition, the generation of human thrombin is accompanied by the loss of a 13 residue peptide, designated Al. We have determined the complete amino acid sequence of the non-thrombin portion of bovine prothrombin and have established regions of significant internal homology. A region of bovine PR fragment (66-128) is homologous to a region of the bovine 0 fragment (15-77). In addition, residues 1-14 of the 0 fragment and residues 17-30 of the A chain of thrombin are homologous. A theoretical primordial prothrombin gene can be proposed: chromosomal misalignment and an unequal crossover of this gene would account for the internal homology and for the molecular size of prothrombin which is approximately 16,000 daltons greater than its related molecules, autoprothrombin III (Factor X) and Factor IX. Preliminary structure work on human prothrombin supports this gene duplication hypothesis. The initial sequence of human PR fragment is nearly identical to that of bovine PR fragment, presumably due to the functionally important modified glutamic acid residues. The 0 fragment and A chain of human prothrombin are also homologous.
Effects of Bovine Factor VIII-Related Protein on Human Platelets and Isolated Human Platelet Membranes
Authors: Solum N. O.
Published: June 2019

The first phase of aggregation of human platelets induced by bovine factor VIII-related protein does not require an intact energy metabolism and probably represents a passive agglutination phenomenon due to receptors for the protein on the outside of the platelet cytoplasmic membrane. However, washed human platelets lost their ability to be aggregated by bovine factor VIH-related protein after freezing and thawing of the platelets. Furthermore, isolated human platelet membranes were not flocculated by the bovine protein in the absence of added calcium ions. Additions of calcium chloride (2.1 mM) alone flocculated the isolated membranes. The membranes used represented the material sedimenting between 10,000 and 100,00 g from a homogenate obtained by homogenizing washed platelets suspended in 0.27 M sucrose in an Aminco-French pressure cell (1.361 atm. 2 × 1 min). Subsequent sucrose density gradient centrifugation of the preparations did not reveal bands of particulate matter at a higher sucrose density than 1.12. In contrast, freezing and thawing of formalin-treated human platelets (4 mg/ml formaldehyde) did not destroy their ability to be aggregated by bovine factor Vlll-related protein. Application of the method described above to the formalin-treated platelets resulted in a low yield of 10,000-100,000 g sedimenting material and to more particulate bands on subsequent sucrose density gradient centrifugation. The stabilizing effect of the formalin treatment (through protein polymerization ?) may also be seen from the fact that such platelets are aggregated by bovine factor VHI-related protein even after their ability to aggregate with thrombin has been lost.
Platelet Phospholipid Synthesis During” Thrombin-Induced Platelet Aggregation
Authors: Kaulen H. D. , Gross R.
Published: June 2019

To study the effect of aggregation and release on platelet membrane metabolism human platelet phospholipids were labeled with either glycerol-C 14 or P 32. After washing off the residual label in order to avoid interference of aggregation with uptake, the changes in the distribution of the label in the various phospholipid classes were followed during the first four minutes of thrombin-induced aggregation. Glycerol-C 14 was incorporated into all glycerophospholipids except the polyphosphoinositides, indicating the various rates of de-novo phospholipid synthesis, and this incorporation was not changed by aggregation. P 32, labeling preferentially the phosphoinositides, was rapidly incorporated over non-aggregated controls in the course of aggregation from water-soluble precursors into phosphatidic acid, di- and triphosphoinositide ositide. As no new de-novo synthesis is taking place it is concluded that phospholipid phosphokinase reactions at the platelet membrane are activated with thrombin aggregation. Accordingly, phosphoinositide kinases have been demonstrated in isolated platelet membranes.

(Supported by the Deutsche Forschungsgemeinschaft.)
The Effect of a Platelet Inhibiting Drug (Sulfinpyrazone) in the Therapy of Patients with Transient Ischemic Attacks (Tia’s) and Minor Strokes
Authors: Robertson J. T. , Dugdale M. , Salky N. , Robinson H.
Published: June 2019

A prospective double blind randomized trial of sulfinpyrazone and placebo is described in patients with TIA’S and minor strokes. The criteria for entry into the Study were patients with at least two transient ischemic attacks within a two-week period or patients with a minor fixed neurological deficit resulting from cerebrovascular occlusive disease. All patients had routine laboratory, special platelet aggregation studies, and cerebral arteriography. Several had initial and subsequent platelet survival studies. To insure equivalence of the two groups, patients were randomly assigned to categories reflecting their risk factors and age. Patients were followed every three months with appropriate laboratory and platelet studies. Interval follow-up occured when side effects or additional symptoms appeared.

When patients failed on sulfinpyrazone, they were placed on sulfinpyrazone and Coumadin. Patients who failed on placebo were placed on sulfinpyrazone followed by sulfinpyrazone and Coumadin if subsequent failure occured. Failure was defined as two transient ischemic attacks within a two-week period or an additional stroke syndrome.

New patients will be enrolled for three years, and all patients will be followed for five years. All data is incorporated into computer files.

Preliminary results relating to the first sixty patients will be presented.
The Role of Heparin on the Inhibition of Platelet Adhesion to Damaged Arterial Endothelial Surface
Authors: Essien E. M. , Kinlough-Rathbone R. , Moore S. , Mustard J. Fraser
Published: June 2019

A technique for the isolation of rabbit thoracic aorta in a form in which it could withstand normal pressure in an in vitro system was developed. Selected segments of the aortae from different animals were damaged mechanically. These were then flushed through rapidly with a suitable dilution of heparin from hog mucosa and the excess heparin was washed off. When such treated aortae were exposed to rabbit platelets, adhesion of platelets to such surfaces compared to non-heparinised ones was significantly depressed (P < 0.001). A similar effect was demonstrable on undamaged aortic surfaces (P < 0.006).

It is suggested that heparin may interact directly and rapidly with damaged vascular surfaces and inhibit platelet adhesion to them. The significance of these results with particular reference to therapeutic concepts is discussed.
Rabbit Subendothelium - Human Platelet Interaction, a New Tool for Pathogenesis of Long Bleeding Time
Authors: Michel H. , Bodevin E. , Caen J. P.
Published: June 2019

Interaction of human platelets with previously denuded rabbit aorta provides a new tool for the estimation of platelet function in inherited and acquired platelet disorders. The interaction with the sub endothelium was estimated morphometrically by the Baumgartner technique.

It is already known that in the Bernard-Soulier syndrome, adhesion-aggregation to purified collagen and platelet aggregation in the presence of all the inducers acting through ADP are normal. In contrast bovine factor VIII and ristocetin do not aggregate the Bernard-Soulier platelets which adhere to subendothelium at only approximately 10 percent of the normal range.

In thrombasthenia, adhesion to collagen is normal but primary or secondary ADP-induced aggregation is absent. Adhesion to rabbit subendothelium is reduced by 50 percent but always a monolayer of cells was seen.

In a 65 year old patient who has an acquired haemorrhagic disorder with a long bleeding time and a normal platelet count her isolated platelets do not respond to ristocetin while they aggregate normally with collagen and ADP. Adhesion of her platelets to subendothelium is reduced by 50 per cent.

It is therefore postulated that rabbit subendothelium-human platelet interaction provides a method for a better understanding of pathogenesis of long bleeding time.
Delayed Adhesion to Subendothelium and Defective Aggregation of Platelets from Rats with Storage Pool Disease (SPD)
Authors: Tschopp Th. B. , Baumgartner H. R.
Published: June 2019

The inherited hemorrhagic diathesis in the Fawn-hooded rat has been shown to closely resemble the SPD in human patients. To study platelet interaction with surfaces, pooled citrated rat blood was circulated for periods of 2.5, 5 or 10 min through a perfusion chamber containing 2 everted segments of rabbit aorta previously denuded of endothelium. One segment was untreated, the second digested with a-chymotrypsin which removes amorphous material and microfibrils, and thus exposes elastin and islands of collagen fibrils. After perfusion, platelet-surface interaction was evaluated by a morphometric technique. On subendothelium exposed to blood of control rats, platelet adhesion amounted to 61%, 85% and 82% after 2.5, 5 or 10 min respectively. Significantly diminished adhesion (31% and 47%) with blood of SPD rats after 2.5 and 5 min became unsignificant (60%) after a 10 min perfusion period. This delayed adhesion was paralleled by an inhibition of SPD rat platelets to spread out on subendothelium as indicated by an increased ratio of contact/spread platelets of 1.4 compared with 0.2 found in controls after perfusion for 2.5 min. Platelet adhesion was unaltered on digested segments. Platelet thrombus formation (aggregation) observed with control blood was small (< 5%) and transient on sub-endothelium. On digested subendothelium however, platelet thrombi covered 50% of the surface after 10 min exposure. In contrast, virtually no platelet thrombi were observed with blood of SPD rats on either surface.

Inhibition of spreading by an unknown, probably membrane associated defect explains the delayed platelet adhesion reaction observed with blood of SPD rats. The strong inhibition of platelet accumulation is probably due to diminished storage pool adenine-nucleotides and thus to a defective release reaction during spreading.
Anticoagulant Prophylaxis in Chronic Haemodialysis
Authors: Remde W. , Funke K.
Published: June 2019

In 15 patients with chronic uraemic syndrome anticoagulant prophylaxis was carried out with chloride phenindione during three to twelve months. Indications were: repeated clotting of the Scribner shunt, unfavourable localisation of the shunt or difficulties during the implantation which let expect a shunt clotting.

In spite of the heparinisation during the haemodialysis we succeeded in maintaining the Quick level between 15 and 35 percent. In all cases the incidence of shunt clotting was considerably reduced. Severe bleeding complications did not occur.
Heparin Therapy Monitoring Tests – Effects of Method and Patient Variables upon Accuracy
Authors: Mink I. B. , Fitzpatrick J. E.
Published: June 2019

Tests utilizing the recalcification time of whole blood or plasma (e.g. A. P. T. T.) to monitor heparin therapy of a patient with malignancy and an accompanying thrombotic disorder, are subject to unpredictable in-vitro changes which occur during the time interval between withdrawal of blood from the patient and eventual testing in the laboratory. Significant reduction or prolongation of clotting time occurs in as little as five minutes, necessitating the performance of the test (whole blood recalcification or alternately centrifrigation and plasma recalcification) as rapidly as possible after patient contact.

Correlations are presented between the direction and magnitude of these time-related changes and methodology variables (venipuncture stress, tube surface, decalcifying agent, storage temperature) as well as patient variables (heparin dosage, PF-4 activity, clotting factor lability, heparin tolerance). Repeated testing of the same blood samples may provide a, pattern of change which reflects in-vivo heparin catabolism.
The Significance of Prothrombin Assay Method Using Echis Carinatus Venom in Disseminated Intravascular Coagulation Syndrome
Authors: Sakuragawa N. , Takahashi K. , Matsuoka M.
Published: June 2019

Prothrombin is decreased in disseminated intravascular coagulation syndrome (DIC). Prothrombin is estimated by the assay method using echis carinatus venom: 0.1 ml of 0.1% fibrinogen solution, 0.1 ml of one to ten diluted specimen and 0.1 ml of echis carinatus venom solution (0.01 mg/ml) are mixed in the test tube at 37° 0, and checked clotting time. From the standard calibration curve, prothrombin is shown by percentage in a normal person.

Prothrombin activity by the regular assay method using tissue thromboplastin is lower than the assay method using echis carinatus venom in DIC.

Thrombin which is produced in the blood of DIC splits prothrombin molecule to prethrombin. Prothrombin cannot be assayed by using tissue thromboplastin, but assayed rather by echis carinatus venom. This fact is proven from the results of SDS-dise electropboresis”pattern of purified prothrombin activated by thrombin or echis carinatus venom, by animal studies of DIC rabbit infused with thrombin, and by examination of DIC blood.

‘ The assay method using echis carinatus venom is concluded to be useful to diagnose DIC.
Disturbance of Haemostasis in Rats Caused by Partial Liver-Necrosis
Authors: Nemesânszky E. , Riesz T. , Jákó J. M.
Published: June 2019

A partial liver-necrosis was induced in albino rats by ligation of the left hepatic lobe and the changes of the haemostatic system were investigated in the course of sterile autolytic process caused. In the test animals, changes characteristic to diffuse intravascular coagulation (DIC) developed after surgery. This process proved to be reversible and undervent spontaneous remission within 48 h. The alterations observed in the blood clotting screening tests rapidly normalized and the fibrin precipitate, demonstrable in the kidney by histological method were only detectable until the second day following surgery. Even after the normalisation of haemostasis, fibrinogen products (FDP) were detectable by immunological methods for several days.
The Elimination of Labelled Fibrinogen in Galactosamine-Induced Experimental Hepatitis in Rabbits
Authors: Mahn I. , Reuter C. , Merkel H. , Müller-Berghaus G.
Published: June 2019

The intravenous injection of D-galactosamine-HCl into animals induces a liver disease resembling virus hepatitis in man in its histological and clinico-phatological features. In a previous study disseminated intravascular coagulation was demonstrated by tracing fibrin-rich microdots in the renal glomerular capillaries, especially if the fibrinolytic system was inhibited by EACA (Thromb. Res. 1, 473, 1972). In order to differentiate between disturbance of synthesis and disseminated intravascular coagulation, investigations with 125I-fibrinogen were performed in rabbits treated with D-galactosamine (1 g/kg) and EACA (0.5 g/kg xhr). In rabbits infused with galactosamine and EACA the elimination of 125I-fibrinogen was increased in comparison to the control animals treated with EACA or isotonic saline only. If heparin (750 u/kg × hr) was infused additionally to galactosamine and EACA, the accelerated decay of labelled fibrinogen was prevented. The occurrence of 125I-activity in organs was pronounced in animals exhibiting microdot formation. These experiments indicate that due to a diminished synthesis of coagulation factors in this model of hepatitis disseminated intravascular coagulation may contribute to the coagulation defect.

(Supported by the Deutsche Forschungsgemeinschaft, Bad Godesberg, Germany.)
Hemorrhagic Diathesis in Amanita Phalloides Poisoning
Authors: Zimmermann R. , Fauser U. , Andrassy K. , Bleiler H.
Published: June 2019

In human amanita phalloides poisoning the severity of the disease as well as the different hospitalisation time after toxin uptake allow no clear interpretation of blood clotting analysis. Therefore the coagulation changes were examined in a standardized animal model in relation to the given dose. Due to the main responsibility of amanitin for the clinical picture Gamma-Methyl-Amanitin (0,05–0,08 mg/kg) was administered intravenously in 20 beagle dogs. Depending on the given dose three different groups could be observed: Group I (amanitin > 0,07 mg/kg): Death in hypoglycemic shock 20-40 hours after poisoning. No manifest hemorrhagic diathesis. Histologically no fibrin clots. Coagulation analysis: DIC (presence of fibrinmonomers) with predominant fibrinolysis, increased level of FDP (> 120 ug/ml). Within 24 hours decrease of coagulation factors, Fibrinogen, F. II, V, VIII, XIII (< 10%) and platelets (< 30,000). Group II (amanitin 0,05–0,07 mg/kg): A. 70% of the animals survived with signs of hemorrhagic diathesis. Histologically no fibrin clots. Coagulation analysis: Decrease of clotting factors and of platelets to about 50% with nadir after 48 h. Maximal fibrinolysis after 36 h. Normalisation after 120 h. B. Protracted decrease of the clotting factors and platelets to 7000 after 60 h. Marked fibrinolysis with death in hemorrhagic shock. Histologically fibrin clots. Comment: In relation to the dose a different reaction of the clotting system can be stated: I. In cases of massive poisoning an endotoxinshock-like picture could be shown. Predominant fibrinolysis. II. With lower dose of amanitin a moderate consumption coagulopathy with moderate, secondary fibrinolysis (A), or (B) predominant consumption coagulopathy with marked fibrinolysis and death in hemorrhagic shock.
Thromboplastic Effects ot Human Plasma Lipoproteins
Authors: Andersson L.-O. , Sandberg H.
Published: June 2019

Lipoprotein fractions from human plasma was prepared by ultracentrifugal flotation. Additions of those fractions to plasma containing various amounts of platelets showed that in platelet-poor and platelet-free plasma there was a clear clot-promoting effect of the additions. In platelet-rich plasma, this effect was negligible. Measurements on the thrombo-plastine and Stypven clotting times showed that the high density lipoprotein fraction affected both the prothrombin and the Factor X activation steps whereas the low density lipoproteins only influenced the prothrombin activation step. Addition of antibodies against high density lipoproteins to platelet-free plasma caused a prolongation of the thromboplastin time.

The relation between lipoprotein structure, phospholipid content and thromboplastic effects is dicussed.
Isolation, Characterization and Radioimmunoassay of a Peptide Resulting from Limited Proteolysis of Human Fibrinogen by Plasmin.
Authors: Harfenist E. J. , Lauer R. C. , Canfield R. E. , Nossel H. L.
Published: June 2019

A polypeptide with a molecular weight of approximately 20,000 daltons, produced at an early stage in the degradation of human fibrinogen by plasmin, has been isolated. Its amino acid composition is unique in that most of the residues are hydrophilie, with serine, glycine and proline making up more than 40% of the total. The polypeptide, which has tentatively been designated fragment H, has also been isolated from the Aα chain of fibrinogen and from fibrin. A radioimmunoassay for H, capable of measuring concentrations in vitro of less than 1 pmol/ml, has been developed for the purpose of obtaining a sensitive and quantitative measure of plasmin action on fibrinogen in clinical blood samples. This assay has been used to study the pattern of release of H from fibrinogen and fibrin and to confirm that it is cleaved from the Aα chain. Since fibrinogen cross reacts in the assay, it is necessary to remove it quantitatively from clinical samples. Preliminary studies show that normal serum levels are in the range of 50-200 pmols/inl, while elevated levels have been detected in patients with coagulation disorders associated with excessive proteolysis.
Different Types of von Wiilebrand’s Disease (vWd): A Study of 101 Cases
Authors: Barbui T. , Baudo F. , Capitanio A. , Ciavarella N. , Mannucci P. M. , Mariani G. , Pareti F. I. , Ruggeri Z. M.
Published: June 2019

The following tests have been carried out in 101 patients with vWd: bleeding time (BT), ristocetin aggregation in PRP (RA), platelet retention to glass bead columns (PR), antihemophilic factor (VIIIAHF). factor-VIII antigen (VIIAGN) and Willebrand factor (VIIIVWF. measured with a washed platelet system), 86 patients had a clinical form of the disease of moderate severity and autosomal dominant pattern of inheritance. In 59 of them VIIIAHF. VIIIAGN and VIIIVWF were concomitantly reduced, PR was usually low and BT moderately prolonged (type I vWd). These patients could be further divided into three subtypes according to the behaviour of RA, which was normal in type la, decreased in type lb and increased in type lc. Typo I vWd is thought to represent an example of decreased synthesis of factor VIII, as suggested by the concomitant decreased of the three factor-VIII related properties. Since RA was found to be either decreased, normal or increased in presence of reduced plasma levels of VTIIVWF. the latter cannot bes solely responsible of the extent of RA in PRP, which may be also related to the variations of a platelet component.

27 patients showed very prolonged BT, low PR and absent or markedly rudeced RA: VIIIVWF was much lower than VIIIAHF and VIIIAGN. 15 patients had no family history, a severe clinical form of the disease, unmeasurable levels of VIIIAHF, VIIIAGN and VIIIVWF and markedly abnormal BT, PR and RA. In the majority of their unaffected parents, VIIIVWF and VIIIAGN were much lower than VIIIAHF These patients are similar to the case of recessive vWd described by Veltkamp and Tilbury (N. Engl. J. Med. 289.. 882, 1973).

Supported by a grant of the Fondazione Angelo Bianchi Bonomi.
The Detection of Deep Venous Thrombosis Using Labelled Urokinase
Authors: Millar W. T. , Smith J. F. B.
Published: June 2019

The use of 125I-labelled fibrinogen in the detection of venous thrombosis in the lower limbs is well established. However, the technique is limited in applicability since only actively forming thrombi may be detected efficiently.

Urokinase, a fibrinolytic enzyme, has been labelled with 99mTc and found to be suitable for the detection and localisation of venous thrombi. Iodinated urokinase has also been investigated. The use of 99mTc-labelled urokinase allows the lower limbs and the pelvis to be scanned using either a rectilinear scanner or a gamma camera, whereas with iodinated urokinase thohand monitor method used in the fibrinogen technique is used. The fibrinolytic activity of the labelled urokinase was evaluated using the fibrin plate technique in order to verify that the urokinase was not denatured in the labelling process. In the series of patients studied, venography was undertaken when possible for comparison with the urokinase scan.

The time between the onset of clinical symptoms and urokinase scanning ranged from two days to several months suggesting that both active and formed thrombi may be detected. In order to exclude non-specific uptake by the thrombi, several patients with unilateral calf thrombosis were scanned with 99mTc-labelled human serum albumen.

The labelled urokinase was injected intravenously and scanning initiated after 15 minutes in order to allow the circulating blood level to stabilise.
Fibrino-peptide a Liberation Compared with Simultaneous Changes in other Coagulation Parameters, Fibrinolytic and Complement Responses after Allergen Provoked Cutaneous Vasculitis Attacks in Man
Authors: Theorell H. , Kockum C.
Published: June 2019

In cutaneous allergic vasculitis several hours pass between allergen exposure and development of clinical symptoms. Two patients with etiological allergy against food and inhalation allergens were examined. Serum complement levels, FDP, fibrinopeptide A, Normotest, Trombotest, platelets and other coagulation parameters have been followed during the first 24 hours after provocation with allergen.

Case 1 is a 32 year old woman with Gougerot type of vasculitis, Erythema Nodosum and hematoma tendency, A probably IgG-mediated allergy against milk with evidence of immune complex in milk test site is found. Ingestion of milk causes rapid transient fibrinolysis and fibrinopeptide A liberation within 3 hours, arthralgia and purpura. Intradermal grass pollen tests cause hypersensitivity reactions of type I and II/III, scrum complement fluctuations, fibrinolysis and factor VII consumption.

Case 2, a 43 year old male, has Zeek’s necrotizing angiitis and significant 3–24 hours intradermal reactions to bacteria, foods, pollens, and danders. Repeated inhalation with sheep-wool and also sneezing of birch pollen reproduce vasculitis with proceeding scrum complement (C3) reduction and fibrinopeptide A liberation in absence of fibrinolysis.

Elimination of allergens improves the clinical condition in both cases. In the latent period before symptoms, fibrinopeptide A appears to be the most sensitive indicator of allergen induced intravascular coagulation in vasculitis.
Reactivity of Fibrinogen Acceptor Crosslinking Sites in the Absence of Thrombin
Authors: Seelich T. , Furlan M. , Beck E. A.
Published: June 2019

The interaction of thrombin-free, solid-phase preactivated factor XIII with fibrinogen was followed by SDS-polyacrylamide gel electrophoresis. Effects of increased ionic strength, protamine sulfate, or urea were evaluated both in the presence or absence of monodansyleadaverine.

Our results suggest that high ionic strength in the absence of thrombin alters fibrinogen conformation in such a way as to render its crosslinking acceptor sites accessible to the action of F XIIIa,
The Sulphite Precipitation Method for Fibrinogen Measurement in Small Samples in the Presence of FDP’s
Authors: Rampling M. W. , Gaffney P. J.
Published: June 2019

A technique based on precipitation by sodium sulphite (Na2SO3) has been developed for measuring fibrinogen concentrations of small (100 μl) samples. Results obtained with this technique on fibrinogen solutions and on plasma samples are accurate to 10% and are not significantly different from those produced by the commonlyused thrombin-clotting method. Furthermore, sulphite precipitation offers considerable technical advantages over the clotting method, especially for measurement of dilute fibrinogen solutions.

The effects of the presence of FDP’s on the two methods were investigated. Sulphite was found to precipitate fragments X and Y in conjunction with fibrinogen but not D or E; thrombin, by contrast, clotted fragment X incompletely but did not clot Y, D or E. Neither technique was very sensitive to the presence of FDP’s in plasma, only when levels of fragments X and Y reached 10 to 15% of the fibrinogen did they have a significant effect, such levels rarely being reached in clinical specimens. However, in these circumstances the difficulty of assessing the stoichiometric interactions of X, Y and fibrinogen in the thrombin measurement suggest the sulphite method as a useful alternative.
Pharmacological Control of Disseminated Intravascular Coagulation (DIC) by Naturally Occurring and Synthetic Thrombin Inhibitors
Authors: Markwardt F.
Published: June 2019

In experimental animals, endotoxin- and thrombin-induced changes in the clotting system and morphological alterations corresponding to that of the pathologic phenomenon of DIC were studied. Simultaneously, oxygen consumption, elasticity and water content of the lung were measured. The mentioned alterations and functional disturbances of the respiratory system could be prevented by inhibition of the clotting enzyme by injection of hirudin or 4-amidinophenyl pyruvic acid (APPA).

Based on the pharmacological investigations, clinical trials with the synthetic inhibitor were performed mainly in patients suffering from chronic DIC. Under’ the treatment with APPA normalization of the fibrinogen level and platelet count as well as the disappearance of fibrin monomer complexes and degradation products were observed.
Long Term Therapy of Patients Suffering from Occlusive Arterial Diseases with Combined Therapy with Phenformin and Stanozolol
Authors: Bielawiec M. , Perzanowshi A. , Myśliwiec M.
Published: June 2019

Phenformin (Dibotin-Winthrop) 100 mg daily and Stanozolol (Stromba-Winthrop) 7,5 mg daily were given for at least half a year and longer periods to 75 patients suffering from coronary heart disease and obliterative arteriosclerosis of the lower limbs. Clinical improvement was observed in above 80% of the patients. Statisticaly significant increase of fibrinolytic activity and decrease of platelet function, fibrinogen, cholesterol, lipids, and beta-lipoproteins levels as well as platelet — leucocyte aggregates number in blood during the therapy were found. Readministration of phenformin and stanozolol after some time of cessation of the herapy caused the similar effects.

The results of our investigations show that combined therapy with phenformin and stanozolol may be of value in prophylaxis and treatment of thrombotic conditions and occlusive arterial diseases.
Isolation and Characterisation of Platelet “Tubulin”, a Sub-Unit Protein of the Microtubules
Authors: Crawford N. , Castle A. G.
Published: June 2019

From studies with whole platelets using microtubule binding agents such as colchicine and vinblastine, it appears that the microtubule have a cytoskeletal function and may be involved in shape changes, intracellular transport and other forms of cell motile activity.

A microtubule subunit protein, “tubulin”, has been isolated from pig platelets and shown to have properties similar to tubulins from cilia, flagella and mammalian brains. The protein binds SH-colchicine, has a sedimentation constant as the dimer of about 6 S and the monomer (MW±55,000) coelectrophoreses with rat, rabbit and guineapig brain tubulins, running in acrylamide gels as a closely spaced doublet similar to the α and β components of brain tubulin. Some higher molecular weight presumptive dyrnein components are also present. The polymer formed during a temperature dependent “in vitri.)” assembly procedure has been studied by electron microscopy. Long linear aggregates with side to side association can be seen, resembling the microtubule subfilamentous structures seen in whole platelet preparations.
Studies on Platelet-Heparin Interactions Utilizing a Tritium-Labelled Heparin
Authors: Shanberge J. N. , Kambayashi J.
Published: June 2019

Intact platelets were separated from platelet-rich plasma by means of sepharose 2B columns according to the method of Tangen, Eerman and Marfey. 90% of the platelets were recovered, free of plasma proteins. When the platelet-rich plasma was treated with tritium-labelled heparin prior to chromatography the platelets obtained by gel filtration contained radioactivity as measured in a liquid scintillation counter. Radioactivity also was found in the plasma protein fractions which came out of the column before the platelet fractions. However, these latter fractions were eluted earlier than active fractions obtained when tritium-labelled heparin alone was chromatographed. Platelets obtained by gel filtration were treated with tritium-labelled heparin and then rechromatographed. Again platelets with radioactivity were obtained. Treating the platelet-rich plasma with sodium dodecyl sulfate, which alters surface charge, prevents the platelets from taking up heparin while heparin-plasma protein complexing does not seem to be inhibited. Attempts to release heparin from intact platelets has been unsuccessful. Whether platelets combine with heparin by surface complexing or by absorption has not been determined, but the ability of platelets to take up heparin does not seem dependent on prior heparin-plasma protein complexing.
The Interaction Mechanism between Human α2-Macroblobulin and Plasmin
Authors: Lambin P. , Fine J. M. , Audran R. , Steinbuch M.
Published: June 2019

When purified homogenous α2M is submitted to SDS polyacrylamide electrophoresis splitting in half molecules of MW of 380.000 is observed. If increasing amounts of plasmin are added to a given amount of α2M, a new component of MW 450,000 increases whereas as the MW 380,000 component decreases. This new component consists of one half molecule of α2M and one plasmin molecule. In the presence of reducing agents and SDS the half molecules of α2M are further cleaved showing that the molecule is composed of 4 chains having a MW of 180,000. If α2M/plasmin complexes are submitted to the same treatment, a major component MW of 95,000 appears proportionally to the amount of added plasmin. As already shown by Harpel this component is a split product to the proteolytic action of plasmin. Other components of major interest correspond to the heavy chain of plasmin MW 50,000 and a component of MW 120,000 which represents the association of the MW 95,000 fragment of α2M and the light chain of plasmin (25,000). This assumption is materialized by the use of Unlabeled plasmin. Both the 450,000 component and the 120,000 component are revaled by auto-radiography. Thus a stable, eventually covalent linkage between α2M and plasmin can be assumed.
Comparative Studies on Factor XIII in Plasma of Various Animal Species
Authors: Lopaciuk S. , McDonagh B. P. , McDonagh Jan
Published: June 2019

Factor XIII was assayed by the monodansylcadaverine incorporation method in platelet/thrombocyte-poor plasma of 14 species. In all plasmas activity was generated by thrombin. In horse, cat, goat, sheep, pig, rat, chicken, goose, and turkey values ranged from 55% to 120% of that found in human plasma. For rabbit, dog, guinea pig, and ox the values were 178%, 225%, 326%, and 471% respectively. Factor XIII activity was not correlated with fibrinogen concentration. The latter ranged from 1.37 to 4.44 mg/ml (mean: 3.31 ±0.82 mg/ml). Factor XIII of all species was neutralized to varying degrees by two human antibodies to factor XIII and, except for rabbit and the avian plasmas, by commercial rabbit antiserum to subunit A. No neutralization occurred with rabbit antiserum to subunit S, although in immunodiffusion studies a precipitin line was formed between this antiserum and plasmas of all species except rabbit and avian. Factor XIII was further characterized by gel filtration. For each species the elution properties were similar to that of human plasma factor XIII. These results indicate that there are both similarities and differences in the immunological and structural characteristics of plasms factor XIII in these mammalian and avian species.

(Supported by Grant HL 14228 from the National Institutes of Health, U.S.A.)
The Uptake and Metabolism of Adenosine in Human Blood Platelets
Authors: Lips J. P. M. , Sixma J. J. , Trieschnigg A. M. C. , Holmsen H.
Published: June 2019

Adenosine is taken up tino intact washed human blood platelets by two independent carrier-mediated processes. The ‘low Km system’ has a Km of 9 uM, a V of 792 pMol/min/109, is energy dependent and has a Q10 of 1.71. This system is competitively inhibited by papaverine and dipyridamol. The high Km system has a Km of 9.4 mM, as V of 106 nMol/min/109 is also energy dependent and has a Q10 of 1.31. This system is competitively inhibited by adenine, that has a very high affinity (K1 = 5,8 uM).

Evidence is presented that the low Km system transports adenosine in such a way, that it is directly incorporated into nucleotides. Adenosine transported by way of the high Km system arrives unchanged inside the platelets. At low concentrations all this adenosine is deaminated. At higher concentrations (5 mM) part of it is converted into adenine nucleotides and part of it is present in an unchanged form. Only a relatively small part is deaminated at high adenosine concentrations.

The high Km system is inhibited by purines. The low Km system is only inhibited by purine ribosides. The presence of a double uptake mechanism and metabolic pathway has direct bearing on experiments that have been effected about the relation between adenosine transport and inhibition of ADP aggregation.
Mathematical Study of the Blood Coagulation Phenomenon
Authors: Pieptea R. , Pieptea D. , Pieptea M.
Published: June 2019

Mathematical analysis of the coagulation process by means of thrombodynamogram made it possible to establish some new and important graphical elements (2) and the analytical expressions (1) of this phenomenon (F[x]):

where a = quantic coagulation parameter (a ≃ ym); λ = coagulation rhythm parameter

= thrombodynamic center (inflection point of the curve);

= arbitrary point on the descending slope. Correct identification and interpretation of these elements raises the expressivity of the thrombodynamogram for evaluating coagulation phenomena.
Development of an in Vitro Vessel Wall Model for Studying Certain Aspects of Platelet-Endothelial Interactions
Authors: Booyse F. M. , Bell S. , Sedlak B. , Rafelson M. E.
Published: June 2019

Cultured bovine aortic endothelial cells were characterized by the presence of Weibel-Palade bodies, pinocytotic vesicles, Factor VIII antigen and thrombostenin-like contractile proteins. Longitudinal sections of cells showed the presence of an extensive matrix of 100–110 Å extracellular filaments. Treatment of monolayers of these cells with serotonin, thrombin, trypsin, endotoxin and heat caused contraction and exposure of the 100–110 Å filaments. Cultured cells were grown on plastic cover slips (9 × 35 mm, 3.5–4 × 105 cells). Post confluent cultures (2 weeks) were treated (activated) with one of the above agents and exposed to platelets, laleled with 125I (lactoperoxidase method) or 14C-amino acids, for various periods of time. Cover slips were washed and counted. Initial platelet-endothelial interactions were linear and could be expressed quantitatively by plotting radioactivity (CPM or calculated number of platelets) per 3.5–4×105 endothelial cells vs. exposure time. Non-activated control values were subtracted in each case. PGE1 eAMP, ATP and aspirin inhibit this interaction. Citrate and EDTA decreased the interaction. Interaction was increased by red blood cells and rotational flow rates and unaffected by plasma proteins. Preliminary electron microscopic observations suggest platelet attachment at the sites of 100–110 Å microfilament abundance. The feasibility of developing a quantitative in vitro model for studying platelet-endothelial interactions under defined experimental conditions will be considered further.