bluuebunny/crossref_metadata_2025 huggingface dataset:
Thrombolytic Therapy with Streptokinase in Acute Deep Venous Thrombosis
Authors: Schmutzler R.
Published: May 2019

The difficulty is known to determin the real date of the beginning of venous thrombosis. Acute deep thrombosis of the femoral and pelvic veins can be followed by two severe complications: pulmonary embolism and chronic venous insufficiency. Primarily causal treatment has to be aspired: thrombectomy or thrombolysis. Confrontation of two groups: 34 successfully thrombolysed occlusions with streptokinase (SK) contrary to 34 heparin treated occlusions without success. Control 3 years later: clear-cut advantage of thrombolytic therapy. Evaluation of 93 patients in a study over ten years with acute, deep venous thrombosis of the extremities after SK treatment: success rate 68%. Comment on the situation of acute postoperative thrombosis with the possibility of thrombolytic treatment based on our experimental examinations. In summary, it can be stated that thrombolytic treatment cannot be started before the seventh postoperative day without risk of hemorrhage. Side effects respectively complications of SK treatment have to be mentioned like febrile reactions and hemorrhages. Fever appeared in 57% of the patients in a large study, hemorrhaging in 29% of the cases, 22% of which could be considered mild. Pulmonary embolism during thrombolysis occured in 4-8%, 1-2% lethal. An over-all lethality rate for SK therapy in the mentioned study is between 1, 2 to 1, 4%.
Plasma Fibrinogen in Heparinized Patients
Authors: Saleem A. , Kreig A. F. , Fretz K.
Published: May 2019

We present a simple and reproducible micro-procedure (using 20 ul) for plasma fibrinogen based on Ellis Stransky principle of clot density measurement. In this procedure we use polybrene buffer as a plasma diluent which neutralizes up to 16 units of heparin per ml of plasma. The measurement of absorbance at 340 nm provides improved sensitivity at low fibrinogen levels. The procedure compares favorably (r=0.99) with a reference procedure on total clottable protein.
Heparin Cofactor During Early Stage of Coagulation in Vitro and During Bleeding in Vivo
Authors: Birk-Jensen A. Hurlet , Moriau M. , Beys C. Col-De , Gillet C. , Masure R.
Published: May 2019

Heparin cofactor activity is measured with the amidolytic method according to 0degard (1975) during in vitro coagulation and during bleeding.

Heparin cofactor activity decreases differently during coagulation process of PRP or PFP. It is suggested this is due to heparin neutralization by PF4 and/or to thrombin formation. Antithrombin III level is not modified.

During bleeding, the same phenomenon is observed when the amount of heparin in the buffer is low enough. Decrease in heparin cofactor activity is already observed on a sample collected less than 90 seconds after the puncture, before any detectable thrombin formation.

Decrease in heparin cofactor activity during bleeding is studied in pathological conditions. It is more marked in some hypercoagulable states.
Method for the Control of Fibrinolytic Inhibitors
Authors: Badimón L. , Villaverde C.A. , Borruel M.T. , Valdecasas F.G.
Published: May 2019

To determine easily and with precision the inhibitors of fibrinolysis, we have developed a method studying the critical Urokinase amount that can produce the fibrin plate lysis in 20 hours of incubation (37° C), by its addition to the fibrinogen solution when the fibrin plate is prepared. Placed on the coagulated fibrin layer samples (20 μl) of blood, plasma, serum or isolated inhibitors from euglobulins supernatant, we obtained circular areas of unlysed fibrin; plotting sample dilution, in logarithmic scale, against unlysed fibrin diameter, in decimal scale, a linear response is obtained. To give a standarization pattern we assayed a Trasylol curve that showed a linear response between 1 and 40 I.U./ml., so the results can be expressed in diameters of unlysed fibrin or in Trasylol units. As standard can also be used Iniprol in a range of 0.5 to 8 I.U./ml. or a pool of normal plasma, or whatever we have to evaluate in progressive dilutions. We have been contrasted this method with thromboelastographic and Blix ones, obtaining correlative results.

This is an easy performance method that can be of clinical interest to study a great number of samples and to control the fibrinolytic agent treatment.
Increased Procoagulant Activity of Phospholipid in Thrombo-Embolic Diseases
Authors: Boffa M.C. , Gozin D.
Published: May 2019

Procoagulant activity of phospholipid has been measured in recalcified plasma as previously described (l), by means of a viperidae anticoagulant phospholi pase A2, specific inhibitor of phospholipid in clotting (2).

With reference to a control normal group and to a group of atherosclerotic patients, phospholipid activity was found significantly increased in thrombo-embolic diseases, with maximum frequency after recent embolism,, In the oral contraceptives group, this assay appeared as useful for selecting women predisposed to thrombosis : in fact, about 5% of the treated women showed a high phospholipid activity contrarly to the control group. One month after interruption of the oestrogen treatment, the phospholipid activity became normal. The few cases examined during thrombosis presented a very high activity.

In patients with high plasma phospholipid content (hyperlipoproteinemia) phospholipid activity was practically normaL This shows that procoagulant activity of phospholipid is related only to a very small fraction of phospholipid, indépendant of the lipoprotein phospholipid and very likely from platelet origin.
Haemostasis and High Altitude
Authors: Singh I. , Chohan I.S.
Published: May 2019

On arrival at high altitude there is a tendency towards hypercoaguiation associated with an increase in platelet count, factors X and XII, and thrombo-test activity (TA) which is reflected by decrease in prothrombin time (PT), bleeding time (BT), clotting time in glass (CT-gl) and in silicone (CT-sl), and stypven time (ST). Clot retraction is impaired. This hypercoaguiation state is countered by a compensatory rise in fibrinolytic activity reflected by reduction of clot lysis time (CLT), plasma fibrinogen, and factor VIII.

This hypercoaguiation state persists throughout the early fortnight after arrival at high altitude and then starts regressing. On day 3, factor V decreases, BT, and ST are further reduced, and factor VIII shows a rise. On day 7, a progressive rise occurs in factors V, VIII, X and XII, TA, platelet counts and platelet factor 3 (PF-3).CT-gl, CT-sl, PT and ST are further shortened. On day 14, haematocrit rises and of all the parameters, factors V and X and clot retraction return to normal. Throughout the fortnight, factor XII remains high, CLT is short, and platelet adhesiveness remains within normal range.

After 2 years’ stay at high altitude, a regression in hypercoagulability occurs and is indicated by persistent short CLT, prolonged BT, CT-sl, PT, ST and reduced TA. Platelet adhesiveness, PF-3, factors V, VIII and XII and clot retraction are restored to normal.
Purification of Membrane-Bound Glycoprotein
Authors: Hasitz I.M. , Okumura T. , Jamieson G.A.
Published: May 2019

Glycocalicin (Mr 150,000) is a component of the platelet glycocalyx and is released in soluble form on platelet homogenization. Glycocalicin appears to play a central role in platelet aggregation induced by thrombin and ristocetin. In addition, the membrane fraction can be resolved into four glycoprotein bands by one-dimensional gel SDS electrophoresis (Mr 150,000, 120,000, 100,000, and 85,000). Since glycoprotein I and glycocalicin are similar electrophoretically and in the nature of their macroglycopeptides, we have endavored to solubilize and characterize membrane-bound glycoprotein I. Extraction of isolated platelet plasma membranes with 1 M sodium chloride or other salts (KBr, KI, KCNS) consistently solubilized 40-60% of glycoprotein I together with thrombosthenin and other components. Homogeneous glycoprotein I was obtained following chromatography on hydroxylapatite. Glycoprotein I purified in this way and glycocalicin, itself, had identical thrombin-binding ability as measured by the Majerus technique. These results suggest that the two types of molecule are funtionally identical but that the molecule can present itself in the platelet in-three different forms — glycocalicin, saline-soluble glycoprotein I and detergent soluble glycoprotein I.
Isolation and Properties of Human Vascular Plasminogen Activator
Authors: Allen R. , Pepper D.S. , Cash J.D.
Published: May 2019

The vascular tree of cadaver legs was washed out with a detergent - glucose mixture and the soluble activator precipitated with 8%PEG 6,000. The redissolved precipitate was chromatogranhed on 4% agarose where it exhibited an apparent molecular weight of 8 × 105 and was well resolved from lipids, factor VIII antigen, albumin and haemoglobin. The active material eluted in the void volume of G-200 Sephadex when using 0.15 M NaCl eluant, but was dissociated and retarded by re-chromatography in 1 M NaCl or lysine. The dissociated, purified enzyme had an apparent molecular weight of 56,000 and was a serine protease. The enzymic activity of the carrier complex was stable for several days at 4°C, but the dissociated enzyme was rapidly inactivated at 4°C.
Fibrinogen Heterogeneity, Fibrinolysis and Blood Coagulation in Burned Patients
Authors: Lipinski B. , Szyfelbein S.K. , Lipinska I. , Gurewich V. , Burke J.F.
Published: May 2019

The aim of this study was to investigate with recently developed methods the blood coagulation and fibrinolytic abnormalities frequently seen in major thermal injury. Total fibrinogen (F), its high molecular weight (HMW) and lower molecular weight (LMW) fractions evaluated by means of 3.5% SDS-polyacrylamide gel electrophoresis, fibrin monomer (FM) in plasma, serum fibrin degradation products (FDP), fibrinolytic activity (FA) in euglobulins, thrombin time (TT), PT and PTT were determined on 24 occasions 3-35 days after burn and immediately pre- and post-primary incisions of burn eschar. It was found that the content of HMW-F, its proportion to LMW-F and to the total plasma proteins was significantly increased in all patients, suggesting accelerated F synthesis. The high concentration of HMW-F may contribute to the increased blood viscosity observed in burned patients. The content of plasma HMW-F in these patients did not correlate with decreased FA nor elevated serum FDP. FM was detected in only 2 instances. A significant prolongation of TT was observed in 22 out of 24 occasions (mean 25"/15") and was found not to correlate with FDP levels. The TT was corrected by higher concentrations of thrombin, the addition of 0.015 M CaCl2 but not MgCl2 and mixing (1:1) with normal plasma.PT and PTT were within the normal range in all patients. These patients showed clinical evidence of defective hemostasis which appeared to correlate with the TT. It is concluded that significant intravascular coagulation did not occur in the burned patients and that the TT prolongation is probably due to the presence of an as yet unidentified abnormality in plasma interfering with fibrin polymerization.
Antibody-Induced von Willebrand’s Disease. Dissociation of Factor VIII Molecular Complex Activities by the Autoantibody
Authors: Gazengel C. , Prieur A.M. , Jacques C. , Girot R. , Josso F.
Published: May 2019

Acquired Von Willebrand’s disease is reported in a 16 year old girl with systemic lupus erythematosus. Routine coagulation studies showed a normal platelet count, prolonged bleeding time and abnormal glass bead retention. Factor VIII molecular complex respective activities were 8% for VIIIAHF and undetectable for VIIIVWF (Ristocetin aggregation of washed platelets) and VIIIAGN (electro-immunodiffusion).

In vitro, the patient’s plasma exhibited an inhibitory activity against exogenous VIIIAGN and VIIIywF but did not neutralize VIIIAHF activity of control plasma, even after a 2 hour incubation at 37°C. This inhibitory activity was supported by the purified plasma IgG fraction. In vivo, following cryoprecipitate administration (20U. VIIIAHF/kg), only 50% of the infused VIIIAHF activity was recovered after 15 mn and the original level was reached 4 hours later. It was only observed a transient peak of VIIIVWF activity and VIIIAGN level did not increase at all after the infusion.

After the start of immuno depressive therapy the three factor VIII related activities returned to normal level in the following order : VIIIAHF and VIIIVWF (9-12 days); VIIIAGN (3 weeks).

These findings could be explained by the formation of a short-living cirulating immune complex between the antibody and the factor VIII molecular complex. In such an hypothesis the auto-antibody would respect the site of VIIIAHF activity and would mask the site reacting with anti VIIIAGN hetero antibodies.
The Clinical Application of Feiba Immuno in Factor VIII Inhibitor Patients
Authors: Anderle K.
Published: May 2019

The aim of this report is to summarize the clinical and laboratory effect and possible side-effects arising in the use of FEIBA IMMUNO for treating Factor VIII and IX inhibitor patients.

In the time between 22nd November 1974 and today FEIBA IMMUNO was supplied for 104 treatment periods. More than 70 patients in 22 hemophilia centers all over Europe were treated, 59 case reports from 19 of the centers were sent to IMMUNO AG for further evaluation.

In 8 cases minor surgery was carried out, of which only one case was complicated by post-operative bleeding. In all other cases different bleeding complications were treated. The clinical effect was difficult to evaluate, but the impression was favourable.

FEIBA’s effect on WBCT, the r-value of TEG, and on aPTT are statistically evaluated for two different mean dosages.

Acute side-effects were allergic reactions in 5 cases and laboratory signs of DIC in 4 cases, two of which had received extremely high dosages. Calculation of the mean pre- and post-treatment values for platelets and fibrinogen did not show any differences.
Factor IX Related Antigen in Hemophllia B
Authors: Yang H.C. , Levine P.H.
Published: May 2019

The presence of factor IX related antigen (FIXRA) was studied in 15 hemophilia B patients, 20 normals, 2 obligate carriers of hemophilia B, 10 hemophilia A patients and 2 patients on Coumadin therapy. A monospecific rabbit antiserum for factor IX was used in a counterimmuno-electrophoresis (CIEP) system. Only 3 out of the 15 hemophilia B patients, representing 3 of 10 pedigrees, had a factor IX related antigen as demonstrated by a precipitin line in CIEP; the other 34 subjects all had demonstrable FIXRA. The presence of FIXRA in hemophilia B did not correlate with the factor IX procoagulant level. The hemophilia B group with FIXRA could partially neutralize a human inhibitor to factor IX. Hemophilia B is a heterogeneous disease in which a minority have a factor IX related antigen.
The use of Ultrasonography in the Management of Haemophilia
Authors: McVerry B.A. , Voke Jennifer , Vicary F.R. , Dormandy Katharine
Published: May 2019

Four patients, three with factor VIII antibodies and one with von Willebrand’s disease, are described, in whom ultrasonic scanning was useful in the diagnosis as well as in locating and assessing the size of bleeding into a hidden area. In this technique, the recorded pattern of reflected waves indicates an unusual space occupying lesion by the presence of a transonic area. In haemophiliacs who do not have antibodies, this technique is only of limited value since they respond readily to the infusion of factor VIII or IX and therefore do not require further investigation. In less straightforward cases such as the four reported, ultrasound proved a useful diagnostic tool. In each of these, a space occupying lesion was clearly demonstrated. Three of the patients had retroperitoneal haemorrhages and one an intramuscular haematoma. In the second patient, the results of the scan actually led to the haematological investigations on which the diagnosis of spontaneous factor VIII antibody was based.
Oral Surgery and Dental Treatment for Hemophiliacs
Authors: Evans Bruce E.
Published: May 2019

The dental needs of hemophilic patients should be managed routinely by conventional dental treatment. Unfortunately many patients do not receive optimal dental care. Consequently such patients develop complications requiring oral surgical procedures. Management of oral surgery for the hemophiliac is based on:

1. Thorough preoperative evaluation in conjunction with the hematology service.

2. Surgical treatment including antifibrinolytic therapy and local hemostatic measures.

3. Postoperative management of complications and followup.
Michigan’s Summer camp for Hemophiliacs
Authors: Boutaugh M. , Penner J.A. , Kelly P. , Lusher J.M.
Published: May 2019

In 1969 Hemophilia of Michigan, a state chapter of the National Hemophilia Foundation, began a summer camp for young hemophiliacs. Availability of effective medical therapy and the increased awareness that hemophiliac children need physical activity and social interaction just like other children enabled Hemophilia of Michigan to launch this special camp. From 23 campers the first year, the camp has grown steadily in popularity and attendance. The 1976 Hemophilia Camp was four weeks long and a total of 170 children from sixteen States attended. In addition to professional staff (camp director, physician, registered nurse, social worker), 21 young men with hemophilia served as staffers. Planning for summer camp begins months ahead with recruitment of volunteer physicians and nurses and the gathering of medical information concerning each child who will attend. Although a smoothly run medical operation makes camp possible, it is the activities which are important to the campers. The camp provides an environment in which the hemophiliac’s medical problems become low-key -- the campers are not considered hemophiliacs, but youngsters who share basic needs with all children. Available activities include swimming, boating, arts and crafts and nature study. When bleeding episodes occur, early treatment is the rule so that the camper can return to fun activities with minimum delay. Since the camp has been in operation for eight years a wealth of information and experience are available to further assist in planning each session.
The Action of Heparin and Other Polysaccharides on the Rigidity of Surface Layers of Fibrinogen
Authors: Copley A.L. , King R.G.
Published: May 2019

Our earlier findings(Thromb. Res. 1:5,1972), that surface layers (SL) of heparin plasma showed decreased rigidity or torque values (J) when compared to SL of oxalate plasma from the same blood withdrawal, led to studies of effect of different’ commercial preparations of heparin (H) and other polysaccharides on SL of 0.4% fibrinogen (Fg) solutions. Depolymerized hyaluronate, chondroitin sulfate and dextran sulfate markedly decreased (J) of FgSL. 1% H lowered J more than 0.1% H. These findings were not consistent with all H preparations, some of which did not lower J. The anticoagulant activity (AA) of H, thus, does not correspond to this J reducing action of certain Hs on FgSL. This will be discussed in connection with studies of H uptake on the endothelium by Hiebert and Jaques who concluded that H may act as antithrombotic agent independently from its AA. (Artery 2:26, 1976). Our findings with H suggest that in thrombosis therapy any commercial H may not necessarily be useful, if merely its AA is taken into consideration, particularly since the initiation of thrombosis according to Copley’s concept is due to the clotting of Fg without thrombin interaction. Since we view FgSL formation on the endothelium to progress to a surface gel state with consequent large increase in volume, the polysaccharides and H preparations, found to reduce J, may have both surface gelation inhibiting (SL antigeloplastic) and antithrombotic actions. (Aided in part by NIH grant HL19013-02.)
Cyclic Nucleotides in Platelet Function
Authors: Haslam R.J.
Published: May 2019

Five lines of evidence indicate that cyclic AMP mediates the effects on platelets of inhibitory agonists, such as PGE1 and adenosine. Thus, these compounds activate adenylate cyclase in platelet particulate fractions (1) and increase cyclic AMP levels in intact platelets (2), while their effects are potentiated by inhibitors of cyclic AMP phosphodiesterase (3), are blocked by inhibitors of adenylate cyclase (4) and are mimicked by analogues of cyclic AMP (5). Apparent discrepancies between platelet cyclic AMP levels and the inhibition of aggregation can be explained by the ability of some aggregating agents to prevent increases in cyclic AMP and by postulating a delay before the effect of a change in platelet cyclic AMP level is expressed. A mechanism of action for cyclic AMP is suggested by observations that it causes phosphorylation of platelet membrane proteins and increases the uptake of Ca2+ ions by platelet membrane fractions. Studies on the effects of aggregating agents on cyclic AMP levels in resting platelets have given variable results but recent work indicating that compounds that can inhibit adenylate cyclase in intact platelets neither cause nor reproducibly potentiate aggregation or release suggests that cyclic AMP plays no role in the responses of platelets to aggregating agents, unless the platelet cyclic AMP level is elevated above resting values. Studies on platelet cyclic GMP levels have shown that increases usually occur in association with both platelet aggregation and the release reaction. While these findings are consistent with some role for cyclic GMP in these processes, conditions have been found in which some aggregation and release can occur without increases in cyclic GMP. In general, the available evidence suggests that Ca2+ ions are likely to be more important than cyclic GMP in mediating platelet responses.
Clinical Use of Factor IX Concentrates
Authors: Ménaché D.
Published: May 2019

The clinical efficacy of Factor IX concentrates in the treatment of patients with Factor IX deficiency is well recognized. The availability of such concentrates has brought a radical change in the management of these patients. The basis for treatment is to obtain an effective Factor IX hemostatic level in vivo. In these conditions, concentrates have been used to reduce the incidence of hemorrhagic episodes in patients particularly exposed and more often to control hemorrhagic episodes or to prevent hemorrhage in the post operative period. Although thromboembolic complications have occured in some instances the major indication of Factor IX concentrates still remains replacement therapy in patients with Factor IX deficiency.

More recently Factor IX concentrates have been used for the treatment of patients with antibody to Factor VIII. Although the therapeutic principle(s) responsible for the Factor VIII inhibitor bypassing activity has not yet been characterized several beneficial effect of both “activated” and non activated Factor IX concentrates have been observed in such patients experiencing minor or severe bleeding episodes. On the other hand we are aware of some cases without beneficial effect of Factor IX concentrates in patients with Factor VIII inhibitor. The major complication would seem to be an anamnestic response in some patients resulting in either a moderate or a considerable increase of the Factor VIII inhibitor titer when compared to the initial level before Factor IX concentrates therapy. If Factor IX concentrates prove to be efficacious in the treatment of patients with Factor VIII antibody special attention would be required in the manufacturing processing in order to avoid an anamnestic response.
A Potentiometric Method for Studying the Platelet Release Reaction
Authors: Robblee L.S. , Kornstein L.B. , Shepro D.
Published: May 2019

A Potentiometrie method which permits the continuous recording of small changes in extracellular calcium (Ca) concentration (1-10 nmoles/ml) is used to study the thrombin-induced release of Ca from human platelets. The instrumentation consists of a Radiometer Ca ion electrode and pH meter, and a chart recorder. Platelets are suspended in Tris-buffered saline, pH 7.4, containing 50 μM Ca. The electrode is placed in a 2 ml aliquot of the platelet suspension in a beaker, thrombin is added, and the change in extracellular Ca (in mV) accompanying the release of Ca is recorded. Recorder tracings of the release reaction show three phases: 1) an initial lag period inversely related to thrombin concentration; 2) an exponential phase during which most of the Ca release occurs; and 3) a final linear phase during which a slow release of Ca is observed. The amount of Ca released is a direct function of both platelet and thrombin concentration. Because an open system is used and release is recorded continuously, considerable flexibility in experimental design is possible. Platelet and thrombin concentrations can be varied over a wide range; thrombin can be added in single or multiple increments and at different time intervals; fresh platelets can be introduced to a completed release reaction; and inhibitors can be added at various times relative to thrombin. Results obtained by using some of these variations suggest that platelets undergo a partial release reaction and become refractory to repeated low level stimuli. The method is both sensitive and inexpensive, and provides a rapid, non-traumatic means of studying temporal and quantitative aspects of the release reaction, and the effects of pathology, drugs or storage on this aspect of platelet function.
β -Thromboglobulin and Platelet Survival in Patients with Arterial Thromboembolic Disease
Authors: Ludlam C.A. , Davies A.B. , Mackay A.D. , Bentley Nancy J.
Published: May 2019

A previous study has reported raised plasma levels of a platelet specific protein β-thromboglobulin (β-tg) in some patients with arterial thromboembolism (AT), rheumatic heart disease (RHD) and prosthetic cardiac valves (PCV). Comparative studies of β-tg levels and platelet survival are now reported in similar patients. β·tg was measured by a radioimmunoassay and platelet survival using 51Cr labelled autologous platelets. In normal individuals a correlation (r=0.9l) has been demonstrated between the reciprocal of the plasma β-tg concentration and the platelet survival. In 5 patients with AT the mean platelet lifespan was 7.4 days (range 5.7-9.3) and this was similar to 7.9 days (5.5-10.3) in control subjects. The mean plasma β-tg concentration in the patients, however, was 108ng/ml (range 45-235) and this was significantly greater (p=0.05) than 30ng/ml (range 17-72) in normal individuals. To determine if a single measurement of β-tg is representative of subsequent estimations in patients with RHD and PCV, samples were collected at monthly intervals from normal subjects and patients. In 11 patients the mean level was 50ng/ml and the coefficient of variation of repeat samples was 28% (range 13-68), while in normals the mean plasma concentration was 19ng/ml with a coefficient of variation of 26% (range 13-48). Treatment with dipyridamole resulted in decreased levels of plasma β-tg in some patients. It is concluded that in normal subjects and in patients with RHD and PCV the plasma β-tg is remarkably constant. It is suggested that the plasma β-tg concentration may be a more sensitive index of platelet activation than shortening of the platelet mean lifespan.
Modules of Structure and Function in the Evolution of Prothrombin, Plasminogen and Factors X and IX
Authors: Magnusson S. , Sottrup-Jensen L. , Petersen T.E.
Published: May 2019

Comparison of the amino acid sequences of prothrombin (Magnusson et al.), factor X1 (Enfield et al.: light chain; Titani et al.: heavy chain), plasminogen (Wiman: PAP? Sottrup-Jensen et al.: heavy chain region, Wallen & Wiman, Sottrup-Jensen et al.: light chain region) and what is known about factor IX (Fujikawa et al.) indicates that these zymogens contain three major structure modules of separate evolutionary origin: 1) A C-terminal serine protease module is common to all four zymogens and to other serine proteases. 2) The “middle” module is either a kringle · structure (duplicated in prothrombin, quintiplicated in plasminogen; all seven kringle structures mutually homologous), or a “pseudo-kringle” structure (one in factor X, residues appr. 45-142 of the light chain; probably one in factor IX, both mutually homologous). 3) The “N-terminal” module is either the vitamin-K dependent region (residues 1-41/42) of prothrombin, factor X and factor IX (all mutually homologous), or the PAP-region of plasminogen. Apparently then different variants of type 2) and 3) modules and also different numbers of type 2) modules can be “fused” to a C-terminal serine protease module during evolution to produce large serine protease zymogens with strictly defined binding affinities and activation characteristics. The “three-module” evolutionary origin of these zymogens leads to the assumption that each module in the zymogen has its own tertiary structure, largely independent of the rest of the molecule. This view is strengthened by the fact that large (1 mm x 0.5 mm x 0.5 mm) high-resolution (2.8 Å) crystals of native A-fragment (residues 1-156) from bovine prothrombin have been obtained (Olsson, Lindkvist, Sottrup-Jensen, Petersen & Magnusson).
A Molecular Model for the Calcium Mediated Binding of the Vitamin K Dependent Coagulation Factors of Phospholipid Surfaces
Authors: Jackson C.M. , Dombrose F.A. , Peng C.W.
Published: May 2019

Bovine Prothrombin Fragment 1 binding to phospholipid vesicles has been quantitatively characterized to provide a basis for understanding the calcium mediated binding of the vitamin K related proteins to phospholipids. Using equilibrium binding techniques, column chromatography, ultraviolet difference spectroscopy and analytical ultracentrifugation calcium binding to Fragment 1, calcium induced self association of Fragment 1 and calcium mediated binding of Fragment 1 to lipid vesicles has been characterized. Calcium induced self association of Fragment 1 explains the divergent results from various laboratories on calcium binding to this protein and is important in understanding binding of Fragment 1 to phospholipid. The number of Fragment 1 molecules binding to each phospholipid vesicle has been shown to be proportional to the number of negatively charged lipid molecules in the vesicle. At low calcium concentrations the affinity of Fragment 1 for lipid is dependent on calcium concentration; at higher concentrations binding becomes independent of calcium concentration. Binding is dependent on ionic strength implicating electrostatic contributions to the association. A model for calcium mediated binding of Fragment 1 to phospholipid which is consistent with calcium binding to the protein, calcium induced self association and calcium dependence of binding to lipid has been proposed.
Activation of Bovine Blood Coagulation Factor V, A Prerequisite for it of Bind Both Prothrombin and Factor Xa.
Authors: Guillin M.C. , Bezeaud A. , Freeman J.P. , Jackson C.M.
Published: May 2019

It is known that prior to bind bovine prothrombin and to become fully functional, bovine Factor V must itself be “activated” by either thrombin or an enzyme isolable from Russell’s viper venom. The purpose of this work was to determine if Factor V activation is also required in order for it to bind bovine Factor Xa.

This has been investigated by measuring the binding of both “native” (unactivated) Factor V and Factor V activated by the Russell’s viper venom activating enzyme, to a column of agarose-bound Factor Xa. The experiments were also performed using diisopropylfluorophosphate (DFP) inhibited Factor Xa covalently bound to agarose. Both purified bovine Factor V (Va) and bovine plasma were used and gave the same results. In order to prevent initiation of clotting in bovine plasma, heparin wad added to the plasma to promote inactivation of Factor Xa by antithrombin III.

The results indicate that Factor V activation is a prerequisite for it to bind Factor Xa ; Factor Va binds both Factor Xa and DFP inhibited Factor Xa, unmodified Factor V does not.

These experiments suggest that Factor V may not participate in prothrombin activation at all, until after some thrombin has been formed. If this is so, an alternate pathway by which the first thrombin is generated must be considered and may be proposed to be simply that involving Factor Xa, phospholipid and Ca2+ alone.
A New Model for Antiplatelet Drug Discovery
Authors: Saunders R.N. , Burns T.S. , Waskawic E.R. , Stelzer M.R.
Published: May 2019

Circulating platelet aggregates (CPA) have been observed by Wu and Hoak (Lancet 2: 294, 1974) in the blood of patients with transient ischemic attacks, acute myocardial infarction and acute peripheral arterial insufficiency. We have discovered that retired breeder (RB) male rats have spontaneous CPA which respond to therapy with clinically proven antiplatelet drugs. CPA are described as the ratio of the platelet count from blood drawn into a citrate/formalin solution compared to the platelet count from blood drawn into a citrate solution. A platelet aggregate ratio (PAR) of 1.0 is indicative of the absence of CPA. The PAR of 24 virgin male rats (0.94 ± .02, S.E.M.) was significantly greater (P<0.01) than that of 34 male RB rats (0.77 ± .02)-indicating the presence of increased CPA in the RB rats. Acetylsalicylic acid (ASA) and sulfinpyrazone (S) have been successfully used clinically in patients with CPA and transient ischemic attacks (Stroke 6: 521, 1975).RB rats treated with ASA or S (100 mg/kg/ day, i.g. for 8 days) demonstrated significantly (P < 0.01) increased PAR of 0.88 ± 0.02 and 0.99 ± 0.02 respectively. In vitro responses of RB platelets in citrate-treated platelet-rich plasma to adenosine diphosphate (0.4 yM to 3 uM/109 platelets) and collagen (420 μg to 1470 μg/109 platelets) were similar in sensitivity to platelets from virgin rats. This suggests that the hyperactivity of RB rat platelets expressed as CPA is related to an increased aggregate stimulation in vivo. The results indicate that the male RB rat may be a useful model for the preliminary evaluation of therapeutically useful antiplatelet agents.
The Activation of Plasminogen by Hageman Factor and Hageman Factor Fragments
Authors: Goldsmith G. , Saito H. , Ratnoff O.P.
Published: May 2019

The mechanism of action of Hageman factor (HF, Factor Xll) in surface-mediated conversion of plasminogen to plasmin was investigated, assaying plasmin with H-D-valy 1-L-leucy1-L-1ysi ne p-nitroanalide·2HCL. Unexpectedly, amidolytic activity for this substrate evolved in the presence of kaolin in mixtures of purified preparations of plasminogen and HF, both devoid of detectable prekallikrein and high MW kininogen. The same result was obtained when plasminogen preparations were incubated in the absence of kaolin with HF-fragments prepared by digestion of HF with insoluble trypsin or with gel-filtered HF fragments (approximate MW 30,000). High MW kininogen did not enhance amidolysis in mixtures of plasminogen and HF with kaolin or HF fragments, but both amidolysis and fibrinolysis were enhanced by the fraction of normal plasma not absorbed at low ionic strength to QAE-Sephadex gels at ph 7-5-The analogous fraction of Fletcher trait (prekallikrein deficient) plasma, did not enhance amidolysis or fribrinolysi s. Kinetic study of the HF-kaolin-plasm inogen interaction indicated a direct enzymatic role for the HF moiety acting on plasminogen as a substrate.

These data suggest that, contrary to earlier observations, HF or HF-fragments may play a direct role in the activation of plasminogen.
An Aorta Intima Inhibitor of Platelet Aggregation
Authors: Heyns A.duP. , Badenhorst C.J. , Retief F.P.
Published: May 2019

The interaction of platelets and the vessel wall is of importance in the pathogenesis of intravascular thrombosis and atherosclerosis. We have previously described the presence of an inhibitor of platelet aggregation and a low Km ADPase in aorta intima extracts. The inhibitor was further characterized.

Human aorta intimas were homogenized, centrifuged and supernatants used. Platelet aggregation was measured in a aggregometer.ADPase activity was measured by incubating 14C-ADP with intima, separating metabolites by high voltage electrophoresis and quantitated.

Aggregation induced by ADP, collagen, Ristocetin and polylysine was inhibited by the intima extract. The inhibition was still evident in aspirin treated platelets. The inhibitor is present in the supernatant of 100 000g centrifuged extracts. This inhibitor appears not to be ADPase or the recently described prostacyclin (PGX) :- it is stable and not destroyed by incubation at 37°C, blocking of prostaglandin synthesis by pre-incubation of the intima with indomethacin has no effect on inhibitor activity.ADPase is inactivated by KCN whereas the inhibitor is not.

The inhibitor of platelet aggregation and to a lesser extent ADPase, in the intima may be important regulators of platelet-vessel wall interaction.
Characterization of the Fibrinolytic Components Specified by Vascular Endothelial Cells
Authors: Loskutoff D.J. , Edgington T.S.
Published: May 2019

Vascular endothelial cells derived from rabbit vena cava and maintained in continuous culture retained properties characteristic of intact endothelium and were employed to characterize its fibrinolytic components. Cells growing on 125I-fibrin-coated petri dishes synthesized and secreted both: a) a plasminogen activator (PA) and, b) an inhibitor of fibrinolysis. Cell fractionation studies revealed that all PA was associated with a membrane fraction, while the inhibitor appeared to exist free in the cytosol. The PA was active following SDS-polyacrylamide gel electrophoresis and migrated as a single molecular species with a mol. wt. ~50,000. Similar activity and mol. wt. profiles were obtained when intact tissues (superior vena cava, aorta and kidney) and urine were extracted and analyzed. The inhibitor was inactivated upon brief exposure to mildly acidic conditions and had a mol. wt. ~80,000. The cells respond to external stimuli (phorbol-myristate-ester) by increased production of both activator and inhibitor, suggesting that the synthesis of these molecules is coordinate regulated. The response is rapid with intracellular changes often apparent within 1-2 hours, and is initiated at the level of gene expression. Changes in extracellular activities appeared 6-8 hours later. The presence of both a PA molecule and a fibrinolytic inhibitor within the same cell, although restricted to different cellular compartments, introduces a previously unappreciated level of complexity to cellular fibrinolytic phenomena and raises difficulties for the interpretation of observations concerning the presence or absence of PA in tissues.
Plasma Membrane Vesicles from Mouse 15091A Tumor Cells: Agent of Platelet Aggregating Activity
Authors: Gasic G.J. , Catafalmo J.L. , Gasic G.P. , Shattil S.J. , Stewart G.J.
Published: May 2019

We reported that cells from most tumors display platelet aggregating activity (PAA) in heparinized plasma and that this activity contributes to metastasis.Recently, we demonstrated that PAA can be used as a marker of cell transformation in virally infected rat cells. The material responsible for PAA is shed into culture medium. Characterization revealed a material which is particulate and sedimentable at 50,000x g for 60 min.; it contains proteins and lipids with a free cholesterol to phospholipid ratio of 0.556.Delipida-tion as well as complete solubilization abolished PAA.SDS-ME PAGE, 7.5% slab gels, revealed 20 bands.

EM studies of 50,000x g pellets shed by 15091A cells indicated they contained numerous vesicles, some solid bodies, numerous free or vesicle associated small particles, and some amorphous material. Discontinuous sucrose density gradient centrifugation of the 50,000x g pellet yielded at the 1.07-1.17 g/cm3 interface a predominantly vesicular fraction which was the most active interfacial material. The vesicles, visible with phase contrast microscopy, resemble those produced by artificial plasma membrane vesiculation in various cell systems, including normal cells. Since PAA is only shown by transformed cells, vesicles from these must be different or much more numerous. Spontaneous vesiculation by tumor cells may be potentially important in understanding cell transformation and tumor metastases.
Tissue Thromboplastin in Leukocytes from Various Leukemias and its Localization by Immuno-Peroxidase Staining Method
Authors: Maekawa T. , Gonmori H. , Kobayashi N. , Ueno T. , Tanaka H.
Published: May 2019

Tissue thromboplastin(TTP) in the leukocytes from various types of leukemia was studied. Washed leukocyte suspensions were prepared by centrifugation and hypotonic lysis techniques from 11 acute myeloblastic leukemias(AML), 5 acute promyelocytic leukemias (APL), 6 chronic myelocytic leukemias and 20 healthy subjects. The TTP activity was estimated by Nemerson’s two stage method and effects on clotting times of various deficient plasmas were studied by Quick’s one stage method. The distribution of the activity in the homogenate of APL leukocytes was studied by differential centrifugation. Anti-placenta(P)TTP was produced and used for immunological studies and immunoperoxidase staining method(lPS). Homogenates of APL leukocytes showed significant TTP activity as much as 152 units per 10 million cells on the average, while homogenates of other blood cells, except a case of AML which had an activity of 3-8 units per 10 million cells, showed little activity. APL homogenates demonstrated striking shortening of the clotting times of Factor VIE and IX deficient plasmas but did not show this effect on Factor VII and X deficient plasmas. This activity sedimented mainly at TOO G in the centrifugation study and was neutralized time-dependently by anti-PTTP like brain(B) and PTTP. By Ouchterlony method, desoxycholate extract of APL leukocytes showed single precipitin line against anti-PTTP, which fused into the lines formed by purified B- and PTTP. APL leukocytes were deeply stained by IPS, while the other blood cells were not stained or, if stained, only faintly. In conclusion, APL leukocytes have strong TTP similar to B- and PTTP in antigenicity. The development of DIC may be predicted by these techniques, especially by IPS.
Vitamin K and Experimental Tumour Metastases
Authors: Hilqard P.
Published: May 2019

Evidence has accumulated that anticoagulants can reduce the incidence of metastases from malignant tumours. Coumarin derivatives have been found most effective in various experimental systems, decreased blood coagulability and direct effects on tumour cells have been considered as their mode of action.

In the studies described a syngeneic, spontaneously metastasising tumour (Lewis-lung carcinoma) was used in C57/BL mice. Long-term anticoagulation with phenprocoumon resulted in a significant reduction of pulmonary metastases. In contrast, controlled anticoagulation with heparin or ancrod was ineffective in the same tumour-host system. In short-term experiments, employing intravenously inoculated tumour cells, it could be demonstrated that the antimetastatic effect of the coumarin-derivative was independent of the effect on blood coagulation and of the drug itself. Under various experimental conditions diet induced vitamin K deficiency was equally effective in reducing metastases as well from primary tumours as from intravenously injected tumour cells.

The present investigations indicate that vitamin K is involved at a cellular level during the early stage of metastasis formation. It is conceivable that vitamin K dependent γ-carboxyglutamic acid residues are located on the cell surface; their absence could substantially influence certain cell characteristics responsible for the lodgement of blood-borne tumour cells.
German-Austrian Multicenter two Years Prospective study on the Prevention of Secondary Myocardial Infarction by ASA in Comparison of Phenprocoumon and Placebo
Authors: Breddin K. , Überla K. , Walter E.
Published: May 2019

A multicenter clinical trial was conducted between Jan.1971 and March 1977 in seven hospitals in Germany and Austria on 945 patients who had survived a myocardial infarction for six weeks. The patients were randomly allocated within strata (hospitals, age, sex, secondary infarc-tion, decompensation, hyperlipemia) to ASA (1.5g/day) placebo or phenprocoumon (ph) treatment. The trial was double blind regarding placebo and ASA and open regarding ph. The data of patients dying within the two years’ observation period suffering secondary m.i. were reviewed by an expert panel of three cardiologists who did not participate in the study. There is no statistical relation between treatment and strata. 531 patients (56.1%) were followed throughout the study without complications, 12,2% had to be excluded for technical reasons, 10.5% did not continue the observation, 1.2% had intercurrent diseases. 6.3% dropped out because of side effects, 9.8% suffered a secondary infarction or died with sudden death, 3.9% died by other causes. 13 patients in the ASA group (317), 22 in the placebo (309) and 26 in the ph group (320) died after m.i. and by sudden death. 11 patients in the ASA group, 15 under placebo and 6 under ph survived a secondary m.i. 30 patients taking ASA dropped out of the study because of side effects, 17 in the placebo group and 13 in the ph group. The preliminary analysis of the data leads to the following conclusions: 1.under ph treatment more patients die than under ASA by m.i. plus sudden death. 2. Secondary m.i., death by m.i. and sudden death occured less frequent in the ASA group (24) compared with placebo (37) and ph (32) but side effects were more frequent in the ASA group.
Experimental Thrombocytopenia: Amelioration of Endothelial Pathology and Bleeding by Prednisone
Authors: Kitchens Craig S. , Weiss Leon
Published: May 2019

Experimental thrombocytopenia results in endothelial alterations associated with bleeding. In this study, we show that prednisone (Pred) may prevent and reverse these changes supporting inference that Pred decreases capillary fragility. Rabbits (3-4 kg) given busulfan developed a 98-99% reduction in platelet count and hemorrhaged profusely. Oral Pred (0.2mg/kg or 1.Omg/kg/day) reduced hemorrhaging despite persistent thrombocytopenia. Tongue biopsies on the third Pred day were examined by EM. Normal animals served as control, one normal group received 1.0mg/kg Pred daily, one thrombocytopenic group received no Pred, and one group received Pred while thrombocytopenia was induced.100 consecutive capillaries or venules of each preparation were examined for fenestrations, “thin spots”, (<800 Å thick), and mean thickness as determined by planimetry.

All preparations demonstrated normal endothelial junctions. We hypothesize that the bleeding of thrombocytopenia is caused by altered capillary and venule endothelium and that diminished bleeding observed with Pred administration is caused by alteration of these endothelial changes.
The Role of Factor IXa and Factor VIII in the Activation of Factor X.
Authors: Davie Earl W. , Vehar Gordon , Fujikawa Kazuo , Di Scipio Richard
Published: May 2019

Factor IXa and factor VIII participate in the middle phase of blood coagulation. These two proteins convert factor X to factor Xa in the presence of calcium ions and phospholipid. The coagulant activity of factor VIII is increased 50-100 fold by the addition of thrombin, and this activity is stabilized in the presence of CaCl2. The activated product (tentatively identified as activated factor VIII) was readily inhibited by diisopropyl phosphorofluoridate or antithrombin III, suggesting that it is a serine enzyme. The exact role of this enzyme in the conversion of factor X to factor Xa, however, is not known. When factor X (bovine or human) is converted to factor Xa, an activation peptide is cleaved from the amino-terminal end of the heavy chain. This gives rise to a new amino-terminal sequence of Ile-Val-Gly-Gly-in the heavy chain. No change occurs in the light chain during the activation reaction. These data indicate that the basic mechanism involved in the conversion of human and bovine factor X to factor Xa appears to be essentially identical and probably involves the formation of a charge relay system characteristic of the pancreatic serine proteases.