bluuebunny/crossref_metadata_2025 huggingface dataset:
Factors Influencing the Deaggregation of Chymotrypsin-Treated Human Platelets Aggregated by Fibrinogen
Authors: Kinlough-Rathbone R L , Mustard J F , Packham M A , Harfenist E J
Published: July 2018

Washed human platelets aggregated by ADP can be deaggregated by EDTA or PGE1; provided the release reaction does not occur; when the release reaction occurs platelets deaggregate less readily. Platelets treated with chymotrypsin are aggregated by fibrinogen indicating that fibrinogen receptors may be permanently exposed by this treatment. Fibrinogen-induced aggregation of chymotrypsin-treated platelets provides another method of bringing platelets into close contact with each other. Although EDTA deaggregated chymotrypsin-treated platelets aggregated by fibrinogen in a medium containing a physiological concentration of Ca2+, EDTA did not deaggregate these platelets if they were in a medium without calcium in which the release reaction occurs. In this medium, when ASA was added to prevent the release reaction, EDTA caused deaggregation. More fibrinogen associated with platelets in the medium without calcium than in the calcium-containing medium. In both media, EDTA displaced approximately half of the fibrinogen indicating that deaggregation is not solely dependent on dissociation of fibrinogen from its receptors. Thus when platelets undergo the release reaction, a form of platelet-to-platelet adhesion occurs that is not disrupted by chelation of divalent cations and is therefore not likely to involve only fibrinogen or thrombospondin and fibrinogen since the association of fibrinogen with its receptor requires Ca2+ and the binding of thrombospondin to platelets that have undergone the release reaction is also dependent on Ca2+.
Influence of Plasma Fibronectin on the Response to Infusion of Thrombin and Adenosine Diphosphate
Authors: Kaplan J E , Snedeker P W , Baum S H , Moon D G , Minnear F L
Published: July 2018

An important physiological anti-thrombotic function has been suggested for plasma fibronectin and the reticuloendothelial system. The current study evaluated the effects of specific immunological fibronectin depletion and fibronectin supplementation upon resistance to infusion of thrombin and ADP in terms of mortality, mean arterial blood pressure and circulating levels of platelets, fibrinogen and fibrin degradation products. Mortality data indicated that rats with reduced fibronectin levels had reduced resistance to thrombin or adenosine diphosphate infusion. The increased lethality was characterized by reduced circulating levels of fibrinogen and platelets while fibrin degradation product levels rose. The infusion of ADP or thrombin in fibronectin supplemented rats failed to elicit higher mortality or reduce fibrinogen or platelet levels further. These data are consistent with the hypothesis that resistance to excessive coagulation and/or platelet aggregation is correlated to circulating plasma fibronectin levels. It is suggested that by direct effects and/ or acting as an opsonin for RES phagocytosis, plasma fibronectin acts as an important anti-thrombotic mechanism.
A Standardized Method for Measuring Anti-F VIII:C Inhibitors in Haemophilia A by Coagulation Inhibition in Agarose Gel
Authors: Jorquera J I , Carmona E , Aznar J A , Peiró A , Sánchez-Cuenca J M
Published: July 2018

Antibodies against factor-VIII coagulant activity can appear in haemophilic patients and, although infrequently, can affect individuals not suffering from Haemophilia A. The Oxford and Bethesda methods are presently the most commonly used techniques for measuring these antibodies. Both methods are time-consuming and not suitable for the screening of large risk groups.

An appropriate method for screening these coagulation inhibitors is that described by P. Bird in 1975. It is based on inhibition of the coagulation produced when plasma samples containing inhibitors diffuse in agarose gels mixed with normal platelet rich plasma (PRP). However, this technique is highly dependent on the variability derived from the use of PRP (amount of coagulant factor-VIII, number of platelets, etc.).

In an attempt to avoid these disadvantages, Bird’s method has been modified by using standardized commercial reagents (lyophilized plasma with 100% factor-VIII coagulant activity, purified fibrinogen, and platelet Factor 3) instead of PRP. The sensitivity reaches 0.8 Bethesda units and the correlation with the Bethesda method is r = 0.964, p <0.001. This newly developed method is as simple as Bird’s, and appears to be at least, as accurate and reproducible as the Bethesda method.
Fibrinogen Bergamo I (Aα16Arg → Cys): Susceptibility Towards Thrombin Following Aminoethylation, Methylation or Carboxamidomethylation of Cysteine Residues
Authors: Reber P , Furlan M , Beck E A , Finazzi G , Buelli M , Barbui T
Published: July 2018

An abnormal fibrinogen, denoted as “fibrinogen Bergamo I”, has been characterized. Its defect consists in an exchange of arginine by cysteine in position 16 of the Aα-chain, thus corresponding to that found in a number of other fibrinogen variants. The abnormal fibrinopeptide A cannot be split off by thrombin from intact fibrinogen Bergamo I. We describe three different chemical modifications of the cysteine Aαl6, i.e. aminoethylation, methylation and carboxamidomethylation, and their effects on the susceptibility of fibrinogen Bergamo I towards thrombin attack. S-aminoethylation of the Aαl6Cys renders the peptide bond Aαl6–17 cleavable by thrombin. Following methylation or carboxamidomethylation, the Aαl9-arginyl bond becomes accessible for thrombin. The chemically modified extended fibrinopeptide A can be readily separated from the normal fibrinopeptide A by HPLC. The latter two modifications are suitable alternative procedures for detecting the molecular defect Aαl6Arg → Cys of fibrinogen.
Antithrombin III Activity and Concentration in Diabetes Mellitus
Authors: Patrassi G M , Picchinenna R , Vettor R , Cappellato G , Coccarielli D , Girolami A
Published: July 2018

Antithrombin III (AT III) levels have been reported to be low, normal, and high in diabetes mellitus. Furthermore, a dicrepancy between AT III activity and antigen concentration was reported. We have evaluated the behaviour of AT III activity and antigen level both in type 1 and type 2 diabetes, either in uncontrolled or in well controlled patients. AT III activity and antigen levels showed values similar to normal. No difference was seen between type 1 and type 2 diabetes. Similar results were observed in the group of well controlled diabetic patients. AT III activity and antigen did not correlate with blood glucose and glycosylated haemoglobin (HbA1). No difference was observed between AT III activity and antigen levels in any group. Therefore the hypercoagulable state found in diabetes mellitus does not depend on AT III modifications. A discrepancy between AT III activity and antigen was not confirmed. A dysantithrombinaemia, explained on the basis of an inactivation of protein glycosylation in diabetes mellitus has not been confirmed.
Inhibition of Platelet Aggregation by Human Placenta
Authors: Dembélé-Duchesne M J , Lahlou A Laghchim , Thaler-Dao H , de Paulet A Crastes
Published: July 2018

Human placental cytosol inhibits platelet aggregation induced by high doses of collagen. The aim of this study was to investigate whether this anti-aggregating activity was caused only by the presence of various activities already described in the placenta (an ADP-consuming enzyme, a fatty acid cyclooxygenase inhibitor, and a thromboxane synthetase inhibitor) or whether another factor was present.

Heating the cytosol at 50° C for 6 min destroyed the inhibitor of collagen-induced aggregation. ADPase and the AA pathway inhibitors were not modified by this treatment. We therefore show the presence of an additional anti-aggregating factor: it is destroyed by heating at 50° C.

We also tested for the presence of an inhibitor of AA release in the placental cytosol using three different methods (rabbit platelets in PRP, washed rabbit platelets, and NRK fibroblasts) but no inhibition could be evidenced.

We conclude that this new anti-aggregating factor, which is probably a protein, acts neither through AA release inhibition nor AA cascade inhibition.
Increased Euglobulin Fibrinolytic Potential in Women on Oral Contraceptives Low in Oestrogen – Levels of Extrinsic and Intrinsic Plasminogen Activators, Prekallikrein, Factor XII, and C1-Inactivator
Authors: Jespersen Jørgen , Kluft Cornelis
Published: July 2018

Components of the fibrinolytic system were studied in samples of plasma from 15 normal, young women and from 11 women taking oral contraceptives containing 30 μg ethinyl oestradiol and 150 μg levo-norgestrel. Fibrinolytic activity of euglobulins precipitated at pH 5.9 was higher than normal in the hormone group, with significant fluctuations related to the cycle. Normal women showed only minor fluctuations. The concentration of C1-inactivator was lower in euglobulins of the hormone group. However, the difference in fibrinolytic activity was retained, when C1-inactivator was inactivated with sodium flufenamate. Fluctuations of the extrinsic (tissue-type) plasminogen activator (t-PA) activity parallelled those of the euglobulin activity.

The intrinsic plasminogen activator activity (dextran sulphate precipitated euglobulin) was significantly increased in the hormone group and the cyclic pattern differed from that of the normal group. The increased activity was factor XII-dependent. Plasma prekallikrein did not differ. The factor XII level was increased in the hormone group but this could not explain the increased intrinsic fibrinolytic activity, suggesting an increase in the quantity of an additional factor XII-dependent proactivator.
Carrier Detection of Haemophilia B by Using an Intragenic Restriction-Fragment Length Polymorphism
Authors: Bröcker-Vriends A H. J. T , Briët E , Quadt R , Bertina R M , van der Linden I K , van de Kamp J J P , Pearson P L , Veltkampt J J
Published: July 2018

We analysed DNA from individuals of five families with haemophilia B, including nineteen potential carriers. A genespecific probe was used to reveal a TaqI restriction-fragment length polymorphism. Segregation analysis of the polymorphic marker and the deleterious mutation within families allowed diagnosis at the gene level for 16 out of the 19 potential carriers, two proving to be carriers and 14 non-carriers. The obvious advantage is that lyonisation, which is a limiting factor when gene product (clotting factor IX) measurements are used for carrier detection, does not interfere with this procedure and that the result is a definitive diagnosis instead of a risk estimate. The method also permits prenatal diagnosis on chorionic villi in the first trimester of pregnancy.

Restriction-fragment length analysis, based upon the probe and restriction enzyme used in this study, will be informative for approximately 45% of the individuals at risk of carrying or transmitting the haemophilia B mutation.
Enhancement of Factor VIII-von Willebrand Factor Ristocetin Cofactor Activity by Monoclonal Antibodies
Authors: Hornsey V , Micklem L R , McCann M C , James K , Dawes J , McClelland D B L , Prowse C V
Published: July 2018

Five monoclonal antibodies to human von Willebrand factor were selected for characterization from 18 produced in murine hybridomas. All showed a high and specific affinity for human von Willebrand factor (vWf) but exhibited little if any crossreaction with sera from other species. The antibodies defined four epitopes on vWf, none of which were involved in platelet binding. Binding of two distinct antibodies at one of these epitopes was associated with enhancement of the rate of vWf-dependent platelet agglutination in the presence of ristocetin. This effect was more noticeable when cryosupernatant plasma was used in place of normal plasma as the source of vWf, and was not explicable simply in terms of antibody-induced cross-linking of vWf.
Closure of a Recurrent Urethrovaginal Fistula in a Girl with Cloacal Anomaly Using Deflux Injection
Authors: Said Hanan , Syed Salahuddin , Zeinelabdeen Ali , Fayez Mohamed
Published: July 2018

In a girl born with cloaca, both hemivaginae and rectum were located above the bladder neck, and both ureters were connected to the hemivaginae. After diverting colostomy and cystovaginoscopy on the second day of life, the repair of cloaca was performed at 10 months of age by posterior sagittal anorecto vaginoplasty (PSARVP), including laparotomy and bilateral ureteric reimplantation. Eight months after the surgery, she developed a vesicovaginal fistula, which was repaired and closed by open surgery through the bladder. Three months after this procedure, a tiny urethrovaginal fistula was noticed, which was closed at the age of 2 years using hook diathermy to refresh the edges and was then closed by Deflux injection. The proper closure of the urethrovaginal fistula was confirmed by radiology and cystoscopy 3 months after the surgery. This report shows that injection of Deflux into a tiny urethrovaginal fistula following refreshing the edges may be a valid treatment option in selected cases.
A Kinetic Model Describing the Interaction of Bovine Prothrombin Fragment 1 with Calcium Ions
Authors: Kabis Charles W , Sarasua Martha M , Gottschalk Karen E , Bourne Carolyn D , Pedersen Lee G , Jackson Craig M , Hiskey Richard G , Koehler Karl A
Published: July 2018

A kinetic model is derived for the interaction of bovine prothrombin fragment 1 with calcium ions. The model requires binding of a minimum of two calcium ions for induction of the observed biphasic fluorescence decrease as a function of time. The model is shown to be consistent with experimental kinetic and equilibrium data by fitting theoretical curves for the biphasic fluorescence change to the data through exact solution of the nonlinear differential rate equations derived from the model. The rate constants for the binding of these two required calcium ions are calculated from the solutions as best fit parameters. The thermodynamic equilibrium constants, K1 and K2, for the binding of these two calcium ions are calculated from ratios of the forward and reverse rate constants as 0.6 × 104 and 5.4 × 104, respectively. Thus, the model correctly predicts positively cooperative calcium ion binding for at least the two calcium ions required to induce fluorescence quenching.
The Effect of Histamine (H2) Receptor Antagonists on the Fibrinolytic Activity In Vitro
Authors: Stadnicki Antoni , Kacperek Teresa
Published: July 2018

The effect of cimetidine and ranitidine on the fibrinolytic activity in vitro was tested. The inhibition rate of spontaneous lysis rose proportionally to the increasing drug doses. However, a similar effect was obtained with two other drugs of different chemical structures and pharmacological properties. Streptokinase activated fibrinolyse was not affected by either of the drugs at any concentration. Nor was clot formation affected in these experiment conditions.

The results of the tests in vitro indicate that inhibition of euglobulin fibrinolysis by H2-receptor antagonists is not specific for these drugs and not connected with their pharmacological properties. It is possible to assume that, during therapeutic use, cimetidine and ranitidine have no antifibrinolytic effect. Possible explanation and relevance of these observations are discussed.
Binding of Fibrinogen to Cultured Bovine Endothelial Cells
Authors: Delvos Ulrich , Preissner Klaus T , Müller-Berghaus Gert
Published: July 2018

The binding properties of purified bovine fibrinogen to bovine aortic endothelial cells have been examined in a tissue culture system. Endothelial cells bound 125I-fibrinogen in a calcium dependent fashion. Removal of calcium by EDTA instantaneously detached most of the cell-associated fibrinogen. Binding of fibrinogen to the endothelial cells was not saturable with time and dosage. Competition studies and displacement experiments did not indicate the involvement of a specific receptor site for the fibrinogen-endothelial cell-interaction. Bovine serum albumin provided in the physiological ratio of 20:1 to fibrinogen competed as effectively as unlabelled fibrinogen for 125I-fibrinogen binding to the endothelial cells. And furthermore, internalization of cell- associated tracer into the endothelial cells that could be demonstrated in the presence of serum free medium did not occur in the presence of albumin. These data suggest that fibrinogen binding to intact bovine endothelial cells is unspecific and presumably negligible in the presence of physiological albumin concentrations.
Phosphorus Labeling of Proteins and Phospholipids in Intact Platelets in Response to pH 5.3
Authors: Mürer Erik H , Daniel James L
Published: July 2018

Previous studies had shown that when gel-filtered or washed human platelets were incubated at pH 5.3, the cells secreted their granule-stored materials suggesting that low pH can act as a platelet activator. We determined here whether the effects of low pH on platelet protein phosphorylation and on platelet lipid metabolism were consistent with this view. When washed human platelets were incubated for 20 min at pH 5.3 and electrophoresed on SDS-PAGE, there was a great increase in 32P-label in the 20,000 and 47,000 dalton protein bands. There was also an increase in the labeling of phosphatidic acid and a small decrease in phosphatidyl inositol. When the platelets were returned to pH 7.6, the 32P labeling of the 20,000 and 47,000 dalton bands was greatly reduced, and that of phosphatidic acid reduced to the control value, while the labeling of phosphatidyl inositol was increased above control. Incubation at pH 5.3 for 60 min gave the same pattern, but return to pH 7.6 resulted in only partial reversal of labeling of the two protein bands and little decrease in the label associated with phosphatidic acid, but the radioactivity in phosphatidyl inositol was greatly increased. The changes in the 32P-labeling of phospholipids and proteins after incubation of platelets at pH 5.3 may reflect an increase in cytoplasmic Ca++ resulting from leakage of Ca++ from intracellular storage sites, a process which becomes irreversible after longer time exposure to the low pH. The activation of human platelets at pH 5.3 is a slow process which may not be directly comparable to the fast events in the normal stimulation-response coupling, but which is accompanied by changes common to platelet activation. Most interesting in this respect is that a return to physiological conditions will reverse these events, although prolonged exposure will make them irreversible. These studies may therefore present a way to study the boundary between reversible and irreversible processes in platelets.
Quantitative and Continuous Analysis of ATP Release from Blood Platelets with Firefly Luciferase Luminescence
Authors: Higashi Terumasa , Isomoto Akio , Tyuma Itiro , Kakishita Eizo , Uomoto Michiko , Nagai Kiyoyasu
Published: July 2018

An analytical method was devised to determine the entire course of the adenosine-triphosphate (ATP) release from blood platelets based on a continuous measurement of firefly luciferase luminescence. The equation of the release reaction was derived after due consideration of substrate (ATP) consumption and product (oxyluciferin) inhibition on the luciferase reaction as follows: C, = vt × (Km/Vmax) × (I + It/Ki) + It where Ct is the total concentration of the released ATP at time t, vt is the velocity of the luciferase reaction at time t and is directly measured, It is the concentration of oxyluciferin at time t, and Vmax, Km and Ki are constants. C, of the gel-filtered platelet suspension (GFP) could be determined by substituting vt and It as functions of t. The effects of albumin and temperature on the reaction were also studied.
Effects of Oral Stanozolol Used in the Prevention of Postoperative Deep Vein Thrombosis on Fibrinolytic Activity
Authors: Sue-Ling H M , Davies J A , Prentice C R M , Verheijen J H , Kluft C
Published: July 2018

Plasminogen, fibrinogen, antithrombin III, euglobulin lysis time, tissue plasminogen activator (t-PA) and fast-acting t-PA inhibitor were measured in 21 patients receiving either stanozolol (10 mg orally given for 14 days preoperatively) or subcutaneous heparin, during a continuing comparative trial in the prevention of postoperative deep vein thrombosis.

Stanozolol treatment resulted in significant (p <0.01) increases between the 14th and 1st preoperative days in the plasma concentrations of plasminogen (3.4 to 4.9 Cu/ml) and antithrombin III (107% to 132%); t-PA levels did not increase significantly (6.0 to 16.0 mU/ml; p >0.1). There were significant (p <0.02) falls in fast-acting t-PA inhibitor (132% to 75%) and fibrinogen (2.4 to 1.8 g/1).

Surgery reversed the changes in fibrinolytic activity seen preoperatively in the stanozolol-treated patients, and similar changes were seen in the heparin-treated group. In this dosage, stanozolol does not appear to prevent the fibrinolytic shutdown which occurs after elective major surgery.
In Vitro Studies on the Fibrinolytic, Thrombolytic and Fibrinogenolytic Properties of a Tissue Plasminogen Activator from Guinea Pig Keratocytes
Authors: Electricwala A , Ling R J , Sutton P M , Griffiths B , Riley P A , Atkinson T
Published: July 2018

The fibrinolytic and thrombolytic properties of a tissue plasminogen activator (tPA) purified from the conditioned medium of an established guinea pig keratocyte (GPK) cell line were investigated in in vitro systems and compared with urokinase. Using the fibrin clot lysis assay, GPK activator appears to be similar to human melanoma tPA and not to human urokinase. GPK activator also caused negligible fibrinogen breakdown, when incubated with human plasma at 37° C over 23 hr. Urokinase on the other hand caused significant fibrinogenolysis, under similar conditions. Comparison of the lysis of plasma clots by GPK activator and human urokinase have shown that GPK activator was a much more effective fibrinolytic agent than urokinase, especially at lower concentrations (<50 IU/ml). Studies on the thrombolytic effect of GPK activator on the lysis of aged and cross-linked whole human blood clots and plasma clots hanging in artificially circulating human plasma suggest that GPK activator can lyse both these types of clots equally well. The lysis is dose dependent, attaining complete lysis within 3–6 hr with the concentration of GPK activator in the range of 1–5 μg/ml plasma. It is concluded that GPK activator has a higher fibrinolytic and thrombolytic activity and lower fibrinogenolytic activity than urokinase.
A Human Pharmacological Study Comparing Conventional Heparin and a Low Molecular Weight Heparin Fragment
Authors: Bratt G , Törnebohm E , Lockner D , Bergströ G
Published: July 2018

The pharmacokinetics of a heparin fragment of low molecular weight (LMWH) of 4000-5000 D and unfractioned standard heparin (UFH) have been studied after i. v. injections of different doses and infusions in 8 humans.

The heparin activity was significantly higher and the effect on APTT lower after LMWH fragment as compared to UFH in the same doses.

The half-life of heparin activity was about 1 hr for UFH and about 2 hr for LMWH. LMWH was found to be eliminated according to first order kinetics and there were no signs of dose dependency.
The Effect of Platelet Number and Haematocrit on Whole Blood Thromboxane Synthesis
Authors: Carter A J , Hanley S P
Published: July 2018

Whole blood, allowed to clot at 37° C in glass tubes, synthesized thromboxane A2 (TxA2) as determined by radioimmunoassay for thromboxane B2 (TxB2). The time course for TxB2 synthesis showed no further increase after 60 min and the concentration of TxB2 in serum obtained from 60 normal subjects positively correlated with the whole blood platelet count in EDTA anticoagulated blood from the same donor.

Patients with chronic renal failure produced less serum TxB2 than age- and sex-matched controls; they also had lower haematocrits. After re-calculating TxB2 production as a function of platelet count and haematocrit all but one of the patients fell in the range of values obtained for controls. These results suggest that chronic renal failure may not be associated with a cyclooxygenase defect and that clotted whole blood TxB2 production should be expressed as a function of platelet count and haematocrit.
Urokinase Quality: Analysis of Different Preparations
Authors: Rogatti W , Yokoigawa K , Kobayashi T
Published: July 2018

Five commercially available German Urokinase (UK) preparations were examined for purity, fibrinolytic activity, and molecular weight composition. With the exception of one preparation (B) all samples showed comparable fibrinolytic activity (91-107% of the activity declared). Preparations A, D, and E contained almost exclusively high-molecular-weight UK (HMW-UK), i.e., 82-86%. However, preparation C (from kidney tissue cultures) contained 95% of low-molecular-weight-UK (LMW-UK), and sample B consisted of nearly equal amounts of both HMW-UK and LMW-UK (47:53). Purity criteria (coagulative and lysozyme activity, endotoxin content, protease activity, HBS antigen) were fulfilled by preparation E, and, with certain restrictions, also by preparations A and C.
Defibrination During Warfarin Therapy in a Man with Protein C Deficiency
Authors: Francis Robert B , McGehee William G
Published: July 2018

A 57 year old man presented with apparently spontaneous lower extremity deep vein thrombosis and pulmonary embolism. He was treated in conventional fashion with intravenous heparin and oral warfarin. After 4 daily doses of warfarin the prothrombin and proconvertin (P+P) time was within therapeutic range, and heparin was stopped. Over the next six hours complete defibrination occurred, associated with severe bleeding complications. Functional protein C measured after normalization of routine coagulation tests averaged 40% of normal, and was only 3.5% of normal immediately prior to the episode of defibrination. We conclude that the very low functional protein C levels seen immediately prior to defibrination were caused by a combination of pre-existent protein C deficiency and warfarin therapy, and directly predisposed to defibrination once heparin was stopped, despite “therapeutic” warfarin anticoagulation. Exacerbation of intravascular coagulation should be considered a potential prothrombotic effect of warfarin therapy in protein C deficient individuals.
Hereditary Protein S Deficiency and Venous Thrombo-Embolism
Authors: Broekmans A W , Bertina R M , Reinalda-Poot J , Engesser L , Muller H P , Leeuw J A , Michiels J J , Brommer E J P , Briët E
Published: July 2018

Protein S, a vitamin K-dependent coagulation factor, is involved in the regulation of the anticoagulant activity of activated protein C.

Using an immunoradiometric assay for total protein S in plasma we identified 14 patients (7 male and 7 female) in three unrelated Dutch families as fulfilling the criteria for an isolated protein S deficiency. In 9 patients who were not receiving oral anticoagulant treatment the mean total protein S antigen concentration was 0.50 ± 0.08 U/ml (± S.D.) and the calculated free protein S concentration was 0.15 ± 0.01 U/ml (± S.D.). In the five patients who were on oral anticoagulant treatment the mean total protein S antigen was 0.23 ± 0.05 U/ml (± S.D.).

Seven of the 14 patients had a history of venous thromboembolism occurring at a mean age of 25 years and often without an apparent cause. Protein S deficiency is inherited as an autosomal dominant trait.
Heparin Assays and Bleeding Complications in Treatment of Deep Venous Thrombosis with Particular Reference to Retroperitoneal Bleeding
Authors: Holm H Asbjørn , Abildgaard Ulrich , Kalvenes Sigmund
Published: July 2018

Bleeding complications occurred in 30 (11%) out of 280 patients who received continuous heparin infusion for deep venous thrombosis (DVT). 22 (8%) had minor while 8 patients (3%) had major bleeding complications (1 intrathoracic [fatal], 2 gastrointestinal and 5 retroperitoneal). Heparin activity, in daily drawn blood samples, was determined by four assays (chromogenic substrate [CS] assay, activated partial thromboplastin time [APTT], thrombin time with citrated plasma [CiTT] and thrombin time with recalcified plasma [CaTT]). The differences in median heparin activity between patients with minor bleeding and patients with no bleeding did not reach significance for any of the tests. In patients with major bleeding, the differences were significant with the CS (p = .011) and the CaTT (p = .030) assays. Patients with retroperitoneal bleeding had significantly increased median activity judged by all four assays: CS (p = .002), CaTT (p = .003), APTT (p = .010), CiTT (p = .029). The difference was most pronounced after four days of heparin treatment, but there was a considerable overlap with patients without bleeding.
Endothelial Cells Produce a Lipoxygenase Derived Chemo-Repellent which Influences Platelet/Endothelial Cell Interactions – Effect of Aspirin and Salicylate
Authors: Buchanan M R , Butt R W , Magas Z , Ryn J Van , Hirsh J , Nazir D J
Published: July 2018

We performed experiments to determine whether endothelial cells synthesize phospholipid metabolites via the lipoxygenase pathway and whether these metabolites influence platelet/vessel wall interactions. Monolayers of cultured human endothelial cells were incubated with 14C-arachidonic acid and their cyclo-oxygenase and lipoxygenase metabolites were extracted and identified by radioimmunoassay, thin layer chromatography and high performance liquid chromatography. We found that in addition to the membrane-associated production of PGI2, endothelial cells synthesized a cytosol-associated metabolite, LOX, which was presumably derived through the lipoxygenase pathway. Inhibition of LOX was associated with an increase in PGI2 production and inhibition of PGI2 with an increase in LOX production. Under either condition, platelet adhesion to cultured endothelial cells was significantly decreased. In contrast, when both PGI2 and LOX production were inhibited, platelet adhesion to endothelial cells was enhanced. Furthermore, when LOX was bound to a thrombogenic surface, platelet adhesion was significantly decreased whereas when arachidonic acid or 12-HETE was bound to the surface, platelet adhesion was increased. We conclude that endothelial cells produce not only a cyclo-oxygenase metabolite, but also a lipoxygenase metabolite, both of which influence platelet/endothelial cell interactions.
Bioimmunoassay (BIA) of Tissue Plasminogen Activator (t-PA) and Its Specific Inhibitor (t-PA/INH)
Authors: Mahmoud M , Gaffney P J
Published: July 2018

A bioimmunoassay (BIA) for tissue plasminogen activator (t-PA) is described which depends on the binding of t-PA and its inhibitor complexes to an immobilised IgG to t-PA and the subsequent assessment of the bound t-PA using glutamic acid- plasminogen (glu-plgn) and the chromogenic substrate, S-2251. This assay indicated that neither normal plasma nor its euglobulin precipitate contain any measurable free and functionally active t-PA. The BIA was also used to measure the level of the fast acting specific t-PA inhibitor (t-PA/INH) in plasma by assessing the residual t-PA activity in the plasma euglobulin fraction (pH 5.9), following the addition of a known amount of t-PA to the plasma.
Further Studies on the Mechanism of Action of Human Plasma in Stimulating Prostacyclin Production by Rat Smooth Muscle Cells
Authors: Vergara-Dauden Margarita , Balconi Giovanna , Breviario Ferruccio , Chiabrando Chiara , de Gaetano Giovanni , Dejana Elisabetta
Published: July 2018

Human normal plasma stimulates prostacyclin (PGI2) production by vascular cells. This plasma activity may be greatly modified in different pathological conditions. The purpose of this study was to characterize some aspects of the mechanism of action of plasma in modulating PGI2 release. Cultured rat aortic smooth muscle cells were used. Citrated plasma from healthy donors stimulated PGI2 production in a concentration-dependent way. Plasma-derived serum containing increasing concentrations of platelets had the same PGI2 stimulating activity as citrated plasma. Plasma stimulation of PGI2 production was accompanied by release of endogenously incorporated arachidonic acid (AA) from the cell membrane. Similarly to AA, plasma induced PGI2 synthesis only once, a second or third challenge producing a reduced response from the cells. Cells stimulated twice with plasma responded to AA like unstimulated cells while cells stimulated twice with AA were poorly responsive to subsequent stimulation with plasma. When the cells were repeatedly stimulated with AA in the presence of plasma no refractoriness was apparent.

This study suggests that plasma increases PGI2 synthesis by the release of endogenous substrate from the cell membrane and by protecting the cells from self-inactivation during AA conversion to prostaglandins.
A Modification of the Wu and Hoak Method for the Determination of Platelet Aggregates and Platelet Adhesion
Authors: Bowry Sudhir K , Prentice Colin R M , Courtney J M
Published: July 2018

The Wu and Hoak method for determining circulating platelet aggregates has poor reproducibility; problems have been reported with the composition of the buffer systems, haemolysis, the effects of blood collection technique and a divergence of the platelet aggregate ratio in blood for healthy donors from the theoretical value of 1. Our investigations suggest that the original technique is highly operator-dependent, especially the collection of blood and the method of counting platelets after centrifugation. We describe an improved modification of the Wu and Hoak technique; a new buffer system has been developed and the proportion of blood in the buffered EDTA and buffered EDTA- formalin solutions has been altered to obtain platelet rich plasma. The platelet aggregate ratio (PAR) by this modified method for healthy donors in two different studies was 0.97 ± 0.02 and 0.98 ± 0.01 respectively. Finally, the principle of Wu and Hoak was used to measure accurately platelet adhesion, without the role of platelet-platelet interactions (aggregation). Platelet adhesion and aggregation were then used to evaluate the thrombogenicity of various artificial surfaces, including silicone rubber and polytetra- fluoroethylene (PTFE) vascular grafts.
A Multicenter Study on Amidolytic Factor X Evaluation in Oral Anticoagulant Therapy
Authors: Berthier A M , Pommereuil M , Scarabin P Y , Conard J
Published: July 2018

For laboratory control of oral anticoagulation, amidolytic factor X (F X) determination may offer an alternative to standardization difficulties of prothrombin time (PT). In order to validate this amidolytic assay on a large scale, a multicenter study was undertaken in 6 French laboratories using the same chromogenic substrate (Stachrom X Stago) and different automated instruments. Intra and between laboratory reproducibility of factor X was estimated on fresh and lyophilized patients plasmas and was found to be highly satisfactory. Standardization of the method did not seem to depend on the chromogenic substrate used, as investigated in two different centers. Results of PT and factor X were compared in over 500 patients on a longterm stabilized oral anticoagulant treatment: there was a strong positive correlation between the 2 tests in each center. The therapeutic range for factor X was evaluated from therapeutic PT values reported by Duckert and Marbet for the different thromboplastin reagents: the estimated mean range was 21 to 32%.

Pooling the results of the six different centers a concordant information for prothrombin time and factor X amidolytic assay was found in 76% of patients and a fully discordant response was present in 0.6%. The results suggest that amidolytic factor X may be suitable for monitoring long-term anticoagulation. However, prospective trials are needed to evaluate its usefulness as compared to conventional methods.
Investigation of Alternative Mechanisms of Collagen - Induced Platelet Activation by Using Monoclonal Antibodies to Glycoprotein IIb-IIIa and Fibrinogen
Authors: Connellan J M , Thurlow P J , Barlow Bernadette , Lowe Melinda , McKenzie I F C
Published: July 2018

Anti-HuPl-ml (reactive with IIIa) and anti-C2G7 (reactive with the carboxyl terminal of the a chain of fibrinogen) were used to investigate platelet aggregation, fibrinogen binding and thromboxane synthesis induced by low dose collagen (LDC) or high dose collagen (HDC) in normal or aspirin treated platelets.

Anti-HuPl-ml and anti-C2G7 inhibited LDC induced platelet aggregation almost completely whilst abolishing fibrinogen binding; thromboxane production although reduced, still occurred. Thus LDC activation was dependent on fibrinogen binding to Gp IIIa. Aggregation of platelets induced by HDC was inhibited with anti-C2G7 or anti-HuPl-ml by 15-35% in association with a modest reduction in thromboxane (TxB2) production (with anti-HuPl-ml) and total inhibition of fibrinogen binding. When anti-HuPl-ml was added to aspirin treated platelets, aggregation with HDC although substantially reduced was not totally abolished.

Collagen appears to activate platelets in a dose related manner in which there are at least three possible mechanisms via: (i) GpIIb-IIIa and associated fibrinogen binding; (ii) prostaglandin pathway; (iii) an alternate pathway responsible for ∼20%-30% of platelet aggregation.
Experimental Studies on the Antithrombotic Action of a Highly Effective Synthetic Thrombin Inhibitor
Authors: Kaiser B , Markwardt F
Published: July 2018

The antithrombotic action of the highly effective synthetic thrombin inhibitor Nα-(2-naphthylsulfonylglycyl)-4-amidino-phenylalanine piperidide was studied in various models of experimental thrombosis in rats. Intravenous infusion of the thrombin inhibitor caused a dose-dependent inhibition or prevention of stasis-induced venous thrombosis, of arterial thrombosis after electrically-induced damage of the vessel wall and of thrombotic occlusion of an extracorporeal arterio-venous shunt.
Monoclonal Antibodies to Human 54,000 Molecular Weight Plasminogen Activator Inhibitor from Fibrosarcoma Cells - Inhibitor Neutralization and One-Step Affinity Purification
Authors: Nielsen L S , Andreasen P A , Grøndahi-Hansen J , Huang J Y , Kristensen P , Danø K
Published: July 2018

Mouse monoclonal antibodies were derived against a plasminogen activator inhibitor with a mol.wt. of ∼54,000 (54 K) from the human fibrosarcoma cell line HT-1080. Screening for hybrids producing antibodies directed against the inhibitor was performed with enzyme-linked immunosorbent assay and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting. Four clones of hybridomas producing IgG1 antibodies were further characterized. The inhibitor was purified ∼50-fold to homogeneity from conditioned cell culture fluid with a yield of ∼85% by a one-step procedure using Sepharose-conjugated monoclonal antibody. In the 125I-fibrin plate assay one of the antibodies neutralized the effect of the inhibitor on urokinase-type plasminogen activator. Two of the antibodies bound complexes between urokinase-type plasminogen activator and inhibitor while the remaining two antibodies did not. The antibodies could be used for immunocytochemical localization of the inhibitor in HT-1080 cells. All four antibodies cross-reacted with a plasminogen activator inhibitor derived from cultured human umbilical cord endothelial cells.
The Systemic Fibrinolytic Effect of BRL 26921 During the Treatment of Acute Peripheral Arterial Occlusions
Authors: Earnshaw J J , Westby J C , Makin G S , Hopkinson B R
Published: July 2018

BRL 26921 is a new acylated streptokinase-plasminogen complex which may have a more specific local thrombolytic effect than streptokinase or urokinase. 34 patients with acute peripheral arterial occlusions were given eight hourly bolus injections of 5 mg BRL 26921 for up to 72 h. Systemic fibrinolysis was observed in all patients yet in only 24% was the occluding thrombus lysed. 44% of the patients had haemorrhagic complications and 24% suffered further thrombotic events during or soon after treatment. There was no correlation between the degree of systemic fibrinolysis produced and dissolution of the thrombi. The degree of systemic fibrinolysis did not affect the complication rate. There is no evidence from this study that BRL 26921 has a specific local thrombolytic effect.
Further Studies on Aggregation of Platelet-Type von Willebrand’s Disease Platelets by Human von Willebrand Factor
Authors: Takahashi H , Tatewaki W , Hanano M , Nagayama R , Shibata A
Published: July 2018

Platelet-type von Willebrand’s disease (vWD) is a bleeding disorder characterized by a heightened interaction between platelets and von Willebrand factor (vWF) as the result of an intrinsic platelet abnormality (probably in GPIb). Platelet aggregability was nearly normal in response to thrombin, wheat germ agglutinin and Ricinus communis agglutinin in this disorder. Unmodified platelets showed no aggregation upon the addition of peanut agglutinin. Partially purified human vWF induced little aggregation of washed patient platelets, but the aggregation was greatly enhanced in the presence of plasma devoid of vWF. Monoclonal antibodies directed against GPIb and GPIIb/IIIa as well as EDTA completely inhibited vWF-induced aggregation. These results indicate that human vWF induces aggregation of platelet-type vWD platelets in the presence of divalent cations and some plasma cofactor(s), and that both GPIb and GPIIb/IIIa are involved in this aggregation.
Monoclonal Antibodies Against Human High Molecular Weight Urinary Urokinase: Application for Affinity Purification of Urinary Prourokinase
Authors: Wojta J , Kirchheimer J C , Turcu Liselotte , Christ G , Binder B R
Published: July 2018

Monoclonal antibodies against urinary urokinase were obtained by immunizing mice with purified human high molecular weight urokinase. Five antibodies were selected and denominated MPW1UK, MPW2UK, MPW3UK, MPW4UK, and MPW5UK, respectively. All selected antibodies reacted with high and low molecular weight urokinase. Cleavage of the low molecular weight paranitroanilide substrate pyro-Glu-Gly-Arg-pNA by urokinase was not inhibited by the antibodies and only one antibody (MPW5UK) inhibited plasminogen activation by urokinase. The ability of MPW5UK to bind to coated urokinase was 100-fold higher than that of the other antibodies. MPW5UK was used to prepare an immunosorbent for the purification of urokinase antigen from freshly voided crude urine. One-chain prourokinase was separated from two-chain urokinase by chromatography of the urokinase antigen containing mixture on agmatine Sepharose. As judged by SDS gel electrophoresis one-chain prourokinase as well as two-chain urokinase were purified to apparent homogeneity by this two-step procedure; the yields were 18% and 47% for single-chain prourokinase and two-chain urokinase, respectively, as calculated from total urokinase antigen contained in the starting material.
Inhibition of Tissue-Type Plasminogen Activator in Plasma of Women Using Oral Contraceptives and in Normal Women During a Menstrual Cycle
Authors: Jespersen J , Kluft C
Published: July 2018

Tissue-type plasminogen activator (t-PA) inhibition in plasma was assessed in 15 normal women and in 10 women using oral contraceptives (OC) containing 30 μg ethinyl oestradiol and 150 μg levo-norgestrel. The levels of t-PA inhibition were significantly lower in the OC group with marked fluctuations related to the hormone cycle. Normal women had only minor fluctuations.
A Pasteurised Concentrate of Human Plasma Factor XIII for Therapeutic Use
Authors: Winkelman L , Sims G E , Haddon M E , Evans D R , Smith J K
Published: July 2018

A therapeutic concentrate of factor XIII containing both A and B sub-units has been prepared from 300 kg pools of human plasma. The process starts from a cold-ethanol fraction from cryoprecipitate supernatant and therefore does not interfere with the recovery of other clinically valuable plasma proteins. Factor XIII is purified approximately 600-fold from plasma by precipitation with sodium citrate and by the removal of fibrinogen by brief heating. The product has been pasteurised in sorbitol solution to inactivate blood-borne viruses, ultrafiltered to remove sorbitol, adsorbed with bentonite and freeze-dried in a formulation meeting requirements for intravenous injection.
Interaction of Vasopressin with Human Blood Platelets: Dependency on Mg2+
Authors: Roos Isabella , Ferracin Fabrizia , Pletscher Alfred
Published: July 2018

Arginine-vasopressin (AVP) in the presence of Mg2+ but not in the absence of bivalent cations led to accumulation of [32P]-phosphatidic acid ([32P]-PA) in human blood platelets. Mg2+ also enhanced the specific binding of [3H]-AVP to intact platelets. The concentrations of the cation which enabled AVP to cause half maximal rise of [32P]-PA and those inducing half maximal [3H]-AVP-binding were of the same order. It is concluded that the stimulation of phosphatidyl inositide breakdown by AVP in presence of Mg2+ is at least partially due to a Mg2+-induced enhancement of specific AVP-binding to the platelet membranes.
Characterization of Four Monoclonal Antibodies to Factor VIII Coagulant Protein and Their Use in Immunopurification of Factor VIII
Authors: Croissant Marie-Pascale , vande Pol Hendrik , Lee Helen H , Allain Jean-Pierre
Published: July 2018

Four monoclonal anti-VIII: C antibodies were obtained from the fusion of the splenocytes of one B alb/C mouse with a specific activity ranging from 2.3 to 45,000 U/mg when purified from ascitic fluid. Only one antibody was able to inhibit completely Factor VIII: C in normal plasma. The four antibodies could bind Factor VIII: CAg in plasma and commercial concentrate both in liquid and solid phase, and were suitable for immunopurification of Factor VIII :C.

Three antibodies competed with polyclonal anti-VIII: CAg Fab′ in a liquid phase IRMA, and all of them were able to displace their own binding to Factor VIII: CAg. Competition studies between monoclonal antibodies for the binding to Factor VIII: CAg were performed and showed the recognition of different epitopes and various functional impact. These studies indicate that at least one antibody, with the lowest anti-VIII:C titer clearly recognizes a different epitope of VIII: C than those recognized by the others. Affinity constants ranged from 109 to 1010 l/mole.
Prolonged Bleeding Time, Reduced Platelet Aggregation, Altered PAF-Acether Sensitivity and Increased Platelet Mass Are a Trait of Asthma and Hay Fever
Authors: Szczeklik A , Milner P C , Birch J , Watkins J , Martin J F
Published: July 2018

In a case control study 31 asymptomatic patients with asthma and/or hay fever and 31 normal controls had their bleeding time measured using the Simplate II device and venostasis. Mean bleeding time in the atopic group (527 s) was significantly prolonged compared to the controls (393 s) (p <0.0005). Platelet aggregation to collagen and ADP (but not to PAF-acether) was significantly depressed in the atopies. Mean circulating platelet mass was significantly greater in atopies than in the controls (p = 0.006). Stepwise multiple regression analysis showed that within the control group bleeding time was best predicted by platelet mass (p = 0.007). No such relationship was found in the atopies. However stepwise multiple regression analysis showed that bleeding time in the atopies (but not in the controls) was best predicted by PAF-acether induced platelet aggregability (p <0.05). In neither group was bleeding time related to collagen induced platelet thromboxane B2 production.

It is hypothesised that in respiratory atopy the depressed aggregatory function of platelets is not compensated for sufficiently by an increase in platelet mass, leading to prolongation of the bleeding time. This haemostatic imbalance, whose cause remains to be established, appears to constitute a trait of atopy.