bluuebunny/crossref_metadata_2025 huggingface dataset:
Genotype Assignment of Haemophilia A by Use of Intragenic and Extragenic Restriction Fragment Length Polymorphisms
Authors: Bröcker-Vriends A H J T , Briët E , Quadt R , Dreesen J C F M , Bakker E , Claassen-Tegelaar R , Kanhai H H H , van de Kamp J J P , Pearson P L
Published: July 2018

We performed DNA analysis in 20 families with haemophilia A in order to evaluate its usefulness for carrier detection and prenatal diagnosis.

The polymorphic Bell site within intron 18 of the factor VIII gene and the extragenic TaqI and Bglll polymorphic sites which are detected by the random DNA probes designated St 14 and DX13, respectively, were investigated for.

Two events of recombination were found between the St 14 and the haemophilia A locus in 51 informative meioses. In one of these recombinant meioses crossing over had also occurred between the DX13 and the haemophilia A locus. No further crossovers between the DX13 and the haemophilia A locus were found in 20 informative meioses.

Segregation analysis of the polymorphic markers and the deleterious mutation within the families allowed a diagnosis at the gene level for 52 out of 57 potential carriers. The new method considerably decreased the uncertainty about carriership for seventeen of the nineteen women with a probability of carriership between 5% and 95% based on pedigree analysis and factor VIII assays.

In seven cases chromosome and DNA analysis of a chorionic villus biopsy was carried out. Three of the fetuses were female, four were male. Three of the male fetuses had inherited the normal maternal X-chromosome and were, therefore, not affected. For another male fetus no diagnosis at the gene level was possible since the mother was homozygous for all the known restriction fragment length polymorphisms within or closely linked with the haemophilia A locus.
Molecular Basis and Clinical Aspects of Hereditary Megakaryocyte and Platelet Membrane Glycoprotein Disorders
Authors: Scharf R. E.
Published: July 2018

Specific membrane glycoproteins (GP) expressed by the megakaryocyte-platelet system, including GPIa-lla, GPIb-V-IX, GPIIb-llla, and GPIV are involved in mediat-ing platelet adhesion to the subendothelial matrix. Among these glycoproteins, GPIIb-llla plays a pivotal role since platelet aggregation is exclusively mediated by this receptor and its interaction with soluble macromolecular proteins. Inherited defects of the GPIIb-llla or GPIb-V-IX receptor complexes are associated with bleeding disorders, known as Glanzmann's thrombasthenia, Bernard-Soulier syndrome, or platelet-type von Willebrand's disease, respectively. Using immuno-chemical and molecular biology techniques, rapid advances in our understanding of the molecular genetic basis of these disorders have been made during the last few years. Moreover, analyses of patients with congenital platelet membrane glycoprotein abnormalities have provided valuable insights into molecular mechanisms that are required for structural and functional integrity, normal biosynthesis of the glycoprotein complexes and coordinated membrane expression of their constituents. The present article reviews the current state of knowledge of the major membrane glycoproteins in health and disease. The spectrum of clinical bleeding manifestations and established diagnostic criteria for each of these dis-orders are summarized. In particular, the variety of molecular defects that have been identified so far and their genetic basis will be discussed.
Hemodynamic and Platelet Response to the Bolus Intravenous Administration of Porcine Heparin
Authors: Bjoraker David G , Ketcham Thomas R
Published: July 2018

There is considerable evidence that under some conditions intravenous heparin infusion may cause or at least enhance platelet aggregation in vivo. Reports of heparin-induced vasodilatation and decreases in arterial blood pressure have not been accompanied by simultaneous observations of the platelet response. In this study both the hemodynamic and platelet response to the bolus administration of porcine intestinal mucosa sodium heparin were monitored in 24 cardiac and 12 vascular surgery patients. Mean arterial blood pressure decreased 7.1 ± 0.8 mmHg as a result of a 247 ± 34 dyne · sec/cm5 decrease in systemic vascular resistance. Platelet count, platelet volume distribution, and beta-thromboglobulin levels did not change with heparin infusion. These responses did not differ when comparing the 155 unit/kg group and the 400 unit/kg group or the 400 unit/ kg groups treated with different commercial preparations. The single patient who did have a decrease in platelet count and a severe rise in beta-thromboglobulin with heparin died intraoperat- ively of a massive myocardial infarction. Large increases in platelet factor 4 with heparin administration were not associated with platelet release but were dependent on whether or not the patient was treated with preoperative subcutaneous or intravenous heparin. There was no evidence that heparin-induced vasodilatation was mediated by platelet aggregation and release.
Intracoronary Platelet Release in Patients with and without Coronary Artery Disease
Authors: Verheugt F W , Serruys P W , Vliet H van , Spijkers A , Hugenholtz P G
Published: July 2018

Intracoronary platelet release of beta-thromboglobulin (BTG) was measured in 18 patients with a history of chest pain by analysis of aortic and coronary sinus blood samples in rest and during atrial pacing. Eleven proved to have coronary artery disease, while seven had normal coronary arteriograms and served as controls. In both groups no statistically significant transmyocardial gradient of platelet and plasma BTG content was observed under basal conditions, although the aortic plasma BTG levels in the patients (60.4 ± 4.1 ng/ml) were significantly (p <0.05) higher than in the controls (47.5 ± 2.6 ng/ml). However, also during atrial pacing no significant gradient could be demonstrated in the patients nor in the controls. It is concluded, that in stable coronary artery disease measurable intracoronary platelet release does not occur in rest nor atrial pacing and, therefore, does not seem to contribute to the development of exercise-induced myocardial ischaemia.
Heterogeneity of Sugar Composition of Factor VIII/ von Willebrand Factor in von Willebrand's Disease: Analysis by Crossed Affinoimmunoelectrophoresis Using Lectin (Ricinus communis Agglutinin-120)
Authors: Mikami S , Ueda M , Yasui M , Takahashi Y , Nishino M , Fukui H
Published: July 2018

The nature of sugar chain of factor VTII/von Willebrand factor in plasma of normal subjects and patients with von Willebrand’s disease (vWd) was examined by crossed affinoimmunoelectrophoresis using anti-human factor VIII rabbit serum, with inserted Ricinus communis agglutinin-120 (RCA-120) agarose layer (RCA – CIE). Molecular weights of factor VlU-related antigen (VIIIR: Ag) were estimated by SDS polyacrylamide gel electrophoresis — crossed affinoimmunoelectrophoresis (SDS PAGE – RCA – CIE).

VIIIR :Ag, in normal plasma and in classical form of vWd, showed two precipitin peaks on RCA – CIE. The slower moving component of VIIIR :Ag with molecular weights over 3×106 daltons from normal subjects and patients with classical form of vWd showed a high affinity for RCA-120. The faster moving component of VIIIR: Ag below 3×106 daltons from the abovementioned subjects and patients with a variant form (Type IIA) showed a very weak affinity for RCA-120.

These results suggested that all of VIIIR: Ag in these variant cases may have a deficiency of galactose residues reactive with RCA, in addition to an incomplete polymerization of VIIIR: Ag, similar to that of the faster moving component of normal subjects.
Platelets, Coagulation and Fibrinolysis in Patients with Diabetic Retinopathy
Authors: Brooks A M V , Hussein S , Chesterman C N , Martin J F , Alford F P , Penington D G
Published: July 2018

Twenty control subjects, 12 insulin treated and 10 non-insulin treated diabetics were studied. All diabetics had retinopathy documented by fluorescein angiography and fluorophotometry. Factor VUIRiAg and plasma fibrinogen concentrations were elevated in both diabetic groups, but more so in the insulin treated patients. Within this latter group the plasma fibrinogen was also correlated with the degree of retinopathy.

Platelets separated on linear isosmolar Percoll gradients showed an increase in intraplatelet βTG content and concentration and a slight increase in volume of the lightest platelets in the insulin treated diabetics. Plasma platelet factor 4 and antithrombin III concentrations were normal and plasma βTG levels were elevated only in those patients with renal insufficiency. Platelet aggregometry was performed in 18 diabetic subjects and found to be normal.

It is concluded that abnormalities of coagulation and platelets in diabetes are determined by metabolic factors rather than the severity of microvascular disease per se.
Antithrombin III Infusion in Patients Undergoing Peritoneovenous Shunt Operation: Failure in the Prevention of Disseminated Intravascular Coagulation
Authors: Büller Harry Roger , ten Cate Jan W
Published: July 2018

Five patients with chronic liver disease and acquired antithrombin III (AT III) deficiency undergoing peritoneovenous (LeVeen) shunting for ascites, resistant to medical therapy, were studied prospectively for the development of disseminated intravascular coagulation (D.I.C.) after selective correction of the plasma AT III activity. This was accomplished by continuous infusion of purified human AT III concentrate beginning one day prior to surgery and continuing five to seven days post-operatively. This rigorous transfusion scheme of AT III concentrate could not prevent D.I.C. and bleeding.
Platelet Abnormalities in Muscular Dystrophy
Authors: Yarom R , Meyer S , More R , Liebergall M , Eldor A
Published: July 2018

Platelets which have complex membranes and calcium shifts similar to those in muscles were investigated in 14 patients with muscular dystrophy and 20 suitable controls. In 4 Duchenne and one limb-girdle dystrophy aggregations were done and found to be depressed with adrenaline and ADP. Electron microscopic and chemical examinations revealed an increased number of dense bodies, changed permeability and/or binding of cations and elevated intracellular calcium in all the 9 cases of Duchenne dystrophy while the 2 limb-girdle and 3 myotonic dystrophies varied.

A two phase polymer separation system applied to fixed platelets of all patients and controls showed no abnormality of surface negative charge.
Detection of Carriers in Glanzmann's Thrombasthenia
Authors: Zonneveld G T E , van Leeuwen E F , Sturk A , ten Cate J W
Published: July 2018

Quantitative glycoprotein (GP) analysis of whole platelets or platelet membranes was performed by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) and periodic acid Schiff staining in the families of two unrelated Glanzmann’s thrombasthenia (GT) patients. Each family consisted of two symptom free parents, a symptom free daughter and a GT daughter. All symptom free members had a normal bleeding time, clot retraction and platelet aggregation response to adenosine 5’-diphosphate (ADP), collagen and adrenalin. Platelet Zw* antigen was normally expressed in these subjects. GT patiens, classified as a type I and II subject, showed reduced amounts of GP lib and of GP nia. Analysis of isolated membranes in the non-reduced state, however, showed that the amount of GP Ilia was also reduced in three of the four parents, whereas one parent (of the GT type I patient) and the two unaffected daughters had normal amounts of GP Ilia. Quantitative SDS-PAGE may therefore provide a method for the detection of asymptomatic carriers in GT type I and II.
Comparative Studies on Thrombin Inhibitors in Experimental Microthrombosis
Authors: Markwardt F , Nowak G , Hoffmann J
Published: July 2018

The influence of thrombin inhibitors, which differ both in their chemical structure and type of inhibition on thrombin-induced microthrombosis was studied in rat lungs. The antithrombotically effective doses and blood levels of the compounds correlated with their kinetic constants for inhibition of thrombin. In preventing microthrombosis, some of the synthetic inhibitors tested surpassed the effectiveness of heparin and approached that of the naturally occurring inhibitor hirudin.
Calibration of Reference Thromboplastins and Standardisation of the Prothrombin Time Ratio
Authors: Kirkwood T B L
Published: July 2018

Thromboplastins vary in their sensitivity to the haemostatic defect induced by oral anticoagulants. To provide a means of standardising prothrombin time tests, the World Health Organization adopted in 1977 a scheme for calibrating thromboplastins in terms of an International Reference Preparation. Unfortunately, the model on which this scheme was based does not always hold. A revised calibration model has therefore been developed and this has been tested in a recent collaborative study. The revised model, which retains fundamentally the same principle for standardising prothrombin time tests, has proved suitable for calibrating thromboplastins of different species and types and, moreover, has certain statistical advantages over its predecessor. In September 1982, the WHO Expert Committee on Biological Standardization adopted the revised model. This paper explains the nature and rationale of this change and considers its practical implications.
Comparison Between Subcutaneous and Intravenous DDAVP in Mild and Moderate Hemophilia A
Authors: De Sio L , Mariani G , Muzzucconi M G , Chistolini A , Tirindelli M C , Mandelli F
Published: July 2018

Sixteen patients with mild and moderate hemophilia were given Desmopressin (DDAVP) subcutaneously in the absence of any actual bleeding. The response to the drug – in terms of factor VIII coagulant activity rise – became apparent 15 min after the injection, reaching the maximal response after one hour (x̄ 3.2 times the baseline levels; SD 1.21). This response was not different from that elicited using the intravenous route in 18 hemophiliacs of comparable severity after the same time interval. No local or general side-effects were recorded after the subcutaneous administration of DDAVP. We therefore conclude that the subcutaneous route adds further evidence to the reliability of this alternative treatment in mild factor VIII deficiencies, thus making home treatment with this vasopressin analogue possible.
Studies of Alpha2-Adrenergic Receptors of Intact and Functional Washed Human Platelets by Binding of 3H-Dihydroergocryptine and 3H-Yohimbine-Correlation of 3H-Yohimbine Binding with the Potentiation by Adrenaline of ADP-Induced Aggregation
Authors: Lanza François , Cazenave Jean-Pierre
Published: July 2018

The binding of 3H-dihydroergocryptine and 3H-yohimbine to intact, discoid, functional, washed human platelets resuspended in Tyrode’s buffer containing Ca2+, Mg2+, human albumin and apyrase, was studied at 37° C. The binding of 3H-dihydroergocryptine was rapid, reversible and saturable (KD = 19.3 ± 4.2 nM, Bmax = 2590 ± 670 sites per platelet). The results were difficult to interpret because the bound ligand was not easily dissociated. In contrast, 3H-yohimbine bound in a rapid, reversible and saturable fashion to one class of sites (KD = 8.1 ± 1 nM, Bmax = 395 ± 35 sites/platelet) with the characteristics of alpha2-adrenergic receptors. Adrenaline alone did not aggregate intact platelets but potentiated ADP-induced aggregation. This effect of adrenaline was specifically inhibited by alpha2-antagonists such as yohimbine. The inhibition of 3H-yohimbine binding and the inhibition of the synergistic effect of adrenaline on ADP-induced aggregation by 16 different alpha- and beta-adrenergic compounds was significantly correlated (p <0.001). Thus, intact and functional washed human platelets can be used as a simple pharmacological model to screen alpha-adrenergic antagonists by measuring the inhibition of the potentiation of ADP-induced aggregation by adrenaline which is a direct reflection of the physiological effect of adrenaline on human platelet alpha2-adrenergic receptors. The inhibition constant derived from aggregation studies expresses the affinity of the ligand for its receptor as measured by more cumbersome binding studies with radioactive adrenergic antagonists such as 3H-yohimbine.
Effect of Ciprofibrate on Platelet Aggregation and Fibrinolysis in Patients with Hypercholesterolaemia
Authors: Simpson Iain A , Lorimer A Ross , Walker Isobel D , Davidson John F
Published: July 2018

The fibrinolytic and platelet effects of Ciprofibrate, a new lipid lowering drug, were studied in 7 patients with hypercholesterolaemia over a 12 week period. A significant reduction in plasma fibrinogen was noted and a lesser increase in fibrinolytic activity. However, no change in platelet aggregation was noted. As expected Ciprofibrate produced a significant hypolipidaemic effect in these patients.

We have described effects of Ciprofibrate which may have potential benefits in this high risk patient group.
Reactivity of Small Molecular Forms of Human Factor VIII/von Willebrand Factor with Botrocetin and Anti-Factor VIII-Coated Latex Particles
Authors: Furlan M , Perret B A , Beck E A
Published: July 2018

Two recently developed tests for measurement of factor VIII/von Willebrand factor (FVIII/vWF), i.e. platetelet agglutination by botrocetin and a kinetic latex antigen assay, were compared with ristocetin cofactor and electroimmunoassay, in respect with FVIII/vWF size-distribution. FVIII/vWF was measured in six cases of atypical von Willebrand’s disease (type II), in gel-filtered fractions of normal cryoprecipitate and in the course of depolymerization of purified normal FVIII/vWF by disulfide reduction. Small molecular forms of FVIII/vWF from normal and variant type II plasma, and those derived by disulfide reduction of high-molecular weight FVIII/vWF, showed remarkably decreased reactivity in ristocetin-, botrocetin- and latex-assay. We conclude that for botrocetin-induced platelet agglutination, as well as for agglutination of antibody-coated latex particles, multiple interactions with repeating subunits of FVIII/vWF are required. As a practical consequence, the combined measurement of FVIII/vWF by the latex test and electroimmunoassay provides a simple tool for discriminating between the classical von Willebrand’s disease and its variants.
Purification and Characterization of Tissue-Type Plasminogen Activator in Maxillary Mucosa with Chronic Inflammation
Authors: Hamaguchi Yukiyoshi , Ohi Masuichi , Sakakura Yasuo , Miyoshi Yasuro
Published: July 2018

Tissue-type plasminogen activator (TPA) was purified from maxillary mucosa with chronic inflammation and compared with urokinase. Purification procedure consisted of the extraction from delipidated mucosa with 0.3M potassium acetate buffer (pH 4.2), 66% saturation of ammonium sulfate, zinc chelate-Sepharose, concanavalin A-Sepharose and Sephadex G-100 gel filtration chromatographies.

The molecular weight of the TPA was approximately 58,000 ± 3,000. Its activity was enhanced in the presence of fibrin and was quenched by placental urokinase inhibitor, but not quenched by anti-urokinase antibody. The TPA made no precipitin line against anti-urokinase antibody, while urokinase did.

All these findings indicate that the TPA in maxillary mucosa with chronic inflammation is immunologically dissimilar to urokinase and in its affinity for fibrin.
Questions and Answers on Prothrombin Time Standardisation in Oral Anticoagulant Control
Authors: Loeliger E A , Poller L , Samama M , Thomson J M , Van den Besselaar A M H P , Vermylen J , Verstraete M
Published: July 2018

One of the reasons why oral anticoagulants fell into disrepute is the absence of internationally accepted standardised procedures for controlling the level of anticoagulatiori. This deplorable situation resulted in over- and under-coagulation and uncertainty in the therapeutic range. International conformity can now be obtained by using an International Normalised Ratio (INR) which is derived from the individual result obtained in a given plasma sample and the International Sensitivity Index (ISI) of the tissue thromboplastin reagent used. Any thromboplastin reagent can be calibrated against an international primary or secondary W.H.O. reference preparation, so as to obtain its International Sensitivity Index. The new system of reporting the level of anticoagulation was designed and can only safely be applied in patients taking oral anticoagulants.
Further Studies on the Hypercoagulable State of Patients with Cushing’s Syndrome
Authors: Patrassi G M , Zanon R Dal Bo , Boscaro M , Martinelli S , Girolami A
Published: July 2018

Factors XII, XI, IX and VIII, plasminogen and alpha2-antiplasmin levels were found to be increased in a group of patients affected by Cushing’s syndrome. High activity of these coagulation factors could be due to their increased release and synthesis mediated by cortisol. A significant correlation between the main arterial pressure and either factor VIII antigen, ristocetin cofactor or factor XII activity was found. Moreover a similar correlation between factor XII activity and either factor VIII antigen or ristocetin cofactor was seen. In conclusion, the presence of a hypercoagulable state in Cushing’s syndrome seems confirmed. Synergic release of factor XII and factor VIII from endothelium could be due to high blood vessel tone secondary to hypercorticism. Finally, decreased fibrinolytic activity was suspected according to plasminogen and alpha2-antiplasmin increase.
Similar Mechanism of Various Lupus Anticoagulants
Authors: Exner Thomas
Published: July 2018

Potent lupus inhibitors from various patients were mixed with platelet free normal plasma and were compared in activated partial thromboplastin time (APTT), dilute prothrombin time (dil. PT), kaolin clotting time (KCT), contact product clotting time (CPCT), and Russell viper venom clotting time (RWCT) tests. In the last three tests platelets and platelet lipid substitutes were avoided to enhance the sensitivities of these tests for the lupus anticoagulant. Correlations between the KCT and the other tests were mostly good, indicating that different lupus inhibitors functioned by a similar mechanism. There was no significant trend between particular clinical symptoms and individual coagulation test combinations. The KCT was found to be the most sensitive test for the lupus inhibitor, followed by the CPCT, RWCT, dil. PT and APTT tests. Activated platelets tended to correct the APTT lupus inhibitor defect in all except the strongest inhibitor cases.
Reliability and Clinical Impact of the Normalization of the Prothrombin Times in Oral Anticoagulant Control
Authors: Loeliger E A , Besselaar A M H P van den , Lewis S M
Published: July 2018

In 1983, the World Health Organization (WHO) published recommendations for normalization, in oral anticoagulant control, of the prothrombin time (PT). The common denominator is the International Sensitivity Index (ISI) of a thromboplastin, obtained by means of thromboplastin calibration. The common scale is that of the International Normalized Ratio (INR). The INR is the PT ratio Patient PT/normal PT which would have been found normal PT with the WHO primary international reference preparation (IRP) 67/40. The reliability of the INR depends on the extent of calibration precision, patient-specific influences, as well as interlaboratory variation in the PT determination. Under well-controlled conditions the overall coefficient of variation (CV) of the INR is 11-13.5%, if thromboplastins of ISI ≃ 1 are used. For so-called low-sensitivity thromboplastins (ISI ≃ 2-2.5), the overall variation is larger due to a large between-laboratory variation of the measured PT-ratios

The user of thromboplastin will be provided with a chart or graph enabling him to convert the conventional terms used for expressing PTs into INRs. For quality assurance, and to prepare his own calibration chart if necessary, he should check normalization by means of control plasmas to which INRs have been assigned.

There is sufficient clinical evidence to express optimal therapeutic ranges in terms of INR. Manufacturers should revise and adapt their inserts where necessary in order to conform to these requirements.
Dynamics of Platelet Tubulin in Response to Changes in Free Sulfhydryl Groups
Authors: Steiner M
Published: July 2018

Pools of polymerized and total tubulin were measured in human platelets as a function of free sulfhydryl groups both in acid-soluble and acid-precipitable cell fractions. Changes in free thiols were produced either by storage of platelets at room temperature or by addition of the potent oxidizing agent diazene dicarboxylic acid (diamide) and were correlated with shifts in the dynamic equilibrium between assembled and disassembled microtubules and platelet aggregation. Diamide at concentrations of 0.5 to 5 mM depleted acid soluble SH groups and reduced protein thiols while causing a progressive decrease in polymerized tubulin. Similar changes, although not as severe, were initiated by storage of platelets at room temperature. Platelet aggregation especially that induced by collagen showed a positive correlation with the pool of polymerized tubulin. Our results indicate that the state of oxidation of sulfhydryl groups especially in the acid- precipitable fraction plays an important role in determining the position of equilibrium between polymerized and depolymerized tubulin.
A Role for Pericellular Proteoglycan in the Binding of Thrombin or Antithrombin III by the Blood Vessel Endothelium? – The Effects of Proteoglycan-Degrading Enzymes and Glycosaminoglycan-Binding Proteins on 125I-Thrombin Binding by the Rabbit Thoracic Aorta In Vitro
Authors: Hatton Mark W C , Moar Sue L
Published: July 2018

Rabbit thoracic aorta segments were treated with either proteoglycan-degrading enzymes or with glycosaminoglycan- binding proteins to examine the nature of the endothelial and subendothelial binding sites of 125I-thrombin. Treatment (5-30 min) with enzymes (heparitinase, chondroitinases AC or ABC) caused a decrease in 125I-thrombin binding by the endothelium (30-70%) and by the subendothelial (intima-media) layer (20-50%); a low- specificity protease destroyed endothelial binding almost entirely and reduced binding to the subendothelium by approximately 60% over a similar period. Of the glycosaminoglycan-binding proteins, pretreatment of the aorta wall with protamine caused a 30% decrease in thrombin binding to the endothelium whereas lipoprotein lipase (present during 125I-thrombin uptake) decreased binding by up to 40%. Pretreatment with antithrombin III did not significantly affect binding of either 125I-thrombin or 125I-FPR-inactivated thrombin. In contrast to thrombin, 125I- antithrombin III was not readily uptaken by the aorta segments. These observations indicate that, whereas the minimal binding by 125I-antithrombin III probably does not involve endothelial proteoglycan, a strong case can be made for endothelial and subendothelial proteoglycan binding sites for thrombin..
Prevention of Postoperative Thromboembolism in Sweden – The Development of Practice During Five Years
Authors: Bergqvist David
Published: July 2018

All climes in Sweden (223) dealing with general, urologie, orthopaedic, and gynaecologic surgery were sent a questionnaire concerning their policy about prophylaxis against postoperative thromboembolism in 1982. The praxis was also compared with that obtained in a similar survey in 1977. 96% replied. 91% used some sort of specific prophylaxis which is significantly more than in 1977. Also a significantly larger number of patients were operated on with prophylaxis than in 1977 (25 vs 19%). The dominating methods were dextran 70 and low-dose heparin. Compared with 1977 peroral anticoagulation, low-dose heparin and mechanical methods had decreased significantly, low-dose heparin in combination with dihydroergotamine increased significantly and dextran showed an unchanged use.
A Functional Plasminogen Assay Utilizing the Potentiating Effect of Fibrinogen to Correct for the Overestimation of Plasminogen in Pathological Plasma Samples
Authors: Gram Jørgen , Jespersen Jørgen
Published: July 2018

An overestimation of the plasminogen concentration occurs in patients with elevated levels of fibrin degradation products (fdp) and/or fibrinogen, when using assays based on the activation of plasminogen by streptokinase (Sk) followed by the hydrolysis of a synthetic chromogenic substrate. This source of error could be overcome by addition of fibrinogen in excess to the plasminogen assay thereby obtaining maximum stimulation of the Sk-plasminogen complex. The late degradation products of fibrinogen and fibrin, Dcate and Ddimer, respectively, had no potentiating effect on the Sk-plasminogen complex. The modified assay was designed as a manual method or for a centrifugal analyzer. The excellent reproducibility is illustrated by the coefficient of variation (CV) within series being 1.3% (n = 24) and from day to day being 1.9% (n = 20).
Platelet Regeneration Time After Aspirin Ingestion and Platelet Survival Time After Labelling with 51Chromium or 111Indium Oxine – A Comparative Study
Authors: Boneu A , Sié P , Bugat R , Caranobe C , Eber M , Cazenave J P , Boneu B
Published: July 2018

A simultaneous investigation of platelet regeneration time (PRT) based upon malondialdehyde (MDA) recovery after a single oral intake of 500 mg of aspirin and of platelet survival time (PST) after labelling with 51chromium or 111Inindium oxine was performed in 25 cancerous patients. A pilot study conducted with 9 healthy volunteers demonstrated that the MDA assay was highly reproducible and specific for the platelet cycloxygenase activity. The pattern of MDA recovery after aspirin ingestion was linear in the healthy volunteers and in the patients presenting both a normal and an accelerated platelet turnover. PST were calculated using the four mathematical models recommended by the International Committee for Standardization in Hematology; the best fit was given by the multiple hit model in 22 cases and by the linear regression model in 3 cases. The mean results obtained in the patients investigated with the 51chromium were consistently shorter than those obtained in the patients investigated with the mindium oxine while the mean PRT were almost identical in the two groups. An excellent correlation between PRT and PST was observed after 111Indium oxine labelling and using the weighted mean method for PST determination. These results suggest that the 111Indium oxine technique is a better method for platelet labelling and that the results provided by the weighted mean method reflect more closely the in vivo platelet turnover than those provided by the multiple hit model.
Incorporation of Some Eicosaenoic Acids into Endothelial Cells – Effect on Platelet Inhibitory Activity and Prostacyclin Production
Authors: Sicard Béatrice , Lagarde Michel
Published: July 2018

Primary cultures of endothelial cells from human umbilical veins were grown until confluency. Then, dihomogammalinolenic acid (DHLA or 20:3n-6) and eicosapentaenoic acid (EPA or 20:5n-3), precursors of monoenoic and trienoic prostanoids, respectively, as well as 5,8,11-eicosatrienoic acid (20:3n-9), and isomer of DHLA, were incorporated into endothelial lipids. DHLA-rich endothelial cells had a decreased capacity of prostacyclin production. By contrast EPA- or 20:3n-9-rich endothelial cells were comparable to controls in this respect. DHLA and EPA were efficiently acylated into cell phospholipids and triglycerides at the opposite of 20:3n-9. It is suggested that both DHLA and EPA could alter the liberation of endogenous arachidonic acid for prostacyclin synthesis but this might be counterbalanced in EPA-rich endothelial cells by PGI3 production. We conclude that DHLA enrichment of endothelial cell lipids may impair the possible beneficial effect of the acid upon platelet functions whereas that of EPA would not be modified.
Isolation and Partial Characterization of Adenylate Cyclase-Enriched Membranes from Human Platelets
Authors: Shimada N , Tsubokura M , Kimura N
Published: July 2018

Isolation of adenylate cyclase-enriched membranes from human platelets was attempted using glycerol lysis technique followed by ultracentrifugation on discontinuous sucrose gradients composed of 24, 30, 34, 37, and 41% (w/w). Adenylate cyclase activity was enriched 4-fold in sample/24% sucrose interface, 7-fold in 24%/30% sucrose interface, and 4-fold in 30%/ 34% sucrose interface fractions with the recovery of 15-20% of the total activity. The enrichment and subcellular distribution of adenylate cyclase resembled in general those of phosphodiesterase and acid phosphatase with slight differences in each other. Protein profiles from SDS-polyacrylamide gel electrophoresis showed that the heavy chain of myosin (Mr = 200,000) was enriched in sample/24% sucrose interface and lower molecular weight proteins in 34%/37% sucrose interface and pellet. The interface fractions between 24 and 34% sucrose were, therefore, collected as adenylate cyclase-enriched membranes.

Adenylate cyclase associated with the membranes displayed high specific activity (0.1 and 1-2 nmol/min/mg protein in the absence and presence of stimulants, respectively), and possessed sensitivities to prostaglandins (E1, I2, and D2) as well as cholera toxin. Activation of adenylate cyclase by these compounds required added GTP, indicating that the contamination of the membrane preparations with GTP-like substance (s) was minimal, if at all present.
Determination of Soluble Fibrin in Plasma by a Rapid and Quantitative Spectrophotometric Assay
Authors: Wiman B , Rånby M
Published: July 2018

A rapid, sensitive and quantitative spectrophotometric assay of soluble fibrin in plasma samples has been developed. The method is based on the principle that fibrin stimulates the activation of plasminogen by tissue plasminogen activator (t-PA). A sample containing fibrin is incubated with t-PA, plasminogen and a plasmin-sensitive chromogenic substrate. An increase in absorbance which is dependent on the fibrin concentration in the sample is obtained. In plasma samples an initial lag-phase, due to the presence of alpha2-antiplasmin is observed. However, the change in A405 per square minute during the later part of the reaction was found to be proportional to the fibrin content and to generate linear standard curve intercepts at the origin. The detection limit of bathroxobin-digested fibrinogen added to plasma was about 5 nmol/L. Recovery experiments at 20 and 75 nmol/L levels in plasma samples from healthy individuals were excellent. The “within-run” variation (CV) was determined as 3.7%. The formation of fibrin after addition of minute amounts of thrombin to plasma could be monitored with the method. Plasma from 8 healthy individuals was found to contain about 9.2 ± 1.9 nmol/L fibrin. Plasma samples from 46 patients with a suspected haemostatic disturbance had higher levels of soluble fibrin (53 ± 62 nmol/L). Seven of the 46 samples had concentrations above 150 nmol/L.
The Reliability of Activated Partial Thromboplastin Time Methods and the Relationship to Lipid Composition and Ultrastructure
Authors: Stevenson K J , Easton Ann C , Curry A , Thomson Jean M , Poller L
Published: July 2018

Wide variations in procoagulant properties, lipid composition and ultrastructure of five commonly used APTT methods have been demonstrated. Performance of the methods with a range of coagulation abnormalities has been ranked. Most of the reagents obtained a high score with one or more defects, but a low score with others. A consistent good ranking throughout was only observed with one reagent.

The number of significant correlations between the reagents’ procoagulant activities and lipid content confirms the view that the performance of an APTT method is largely dependent upon its lipid composition. Marked differences in concentration and distribution of phospholipids, fatty acids and neutral lipids were evident. The importance of the concentration of phosphatidyl serine in regulating the procoagulant activity of an APTT method has been demonstrated. Electron microscopy provides evidence of the contrasting composition of the reagents from the more discrete uniform liposomes present in the more reliable reagents, to more ill-defined components present in those reagents which performed less well. The study highlights the need for standardisation of the APTT.
An IgM Lupus Anticoagulant that Neutralizes the Enhancing Effect of Phospholipid on Purified Endothelial Thrombomodulin Activity-A Mechanism for Thrombosis
Authors: Freyssinet J-M , Wiesel Marie-Louise , Gauchy Josiane , Boneu B , Cazenave J-P
Published: July 2018

An anticoagulant activity was isolated from the plasma of a patient with a strong lupus-like anticoagulant using gel filtration by high performance liquid chromatography. IgM were detected in this anticoagulant fraction which exhibited specificity towards 50% phosphatidylcholine - 50% phosphatidylserine vesicles and cardiolipin. These phospholipids were able to produce an apparent 3-fold enhancement of purified human protein C activation by human a-thrombin in the presence of purified human placenta thrombomodulin. In the absence of phospholipid, the anticoagulant fraction had no effect on thrombomodulin activity. The anticoagulant fraction could neutralize the enhancement of thrombomodulin activity by phospholipid in a dose-dependent manner. This study suggests that the neutralization of phospholipid might result in a reduced activation of protein C which could be responsible for the occurrence of thrombotic complications in a proportion of patients with lupus anticoagulants.
Behaviour of the Contact Phase of Blood Coagulation in the Adult Respiratory Distress Syndrome (ARDS)
Authors: Velasco F , Torres A , Guerrero A , Andres P , Guerrero R , Aljama P , Alvarez F
Published: July 2018

In order to assess the role of the kallikrein-kinin (K-K) system in the pathogenesis of the adult respiratory distress syndrome (ARDS) we have prospectively determined coagulation contact phase, blood gas and hemodynamic parameters in patients with ARDS at 0, 36 and 72 h from diagnosis.

Compared to normal values, significantly lower mean levels of factor XII (71.4 ± 9.8%, p <0.0005), prekallikrein (PPK) (52 ± 5.7%, p <0.0005), high molecular weight kininogen (HMWK) (73 ± 2%, p <0.0005) and α2-macroglobulin (α2-M) (51 ± 7.1%, p <0.0005) were found in ARDS patients.

The functional kallikrein inhibitory activity (KKI) and Cr esterase inhibitor antigenic (CIINH) were significantly higher in these patients (113.2 ± 5, p <0.005 and 124.7 ± 7.6, p <0.0005 respectively) compared with normal values during the entire study period.

The KKI/CIINH ratio decreased significantly in our ARDS patients at 0, 36 and 72 h (p <0.025; p <0.05 and p <0.005 respectively).

We found a significant correlation between PPK levels and oxigenation index (r = 0.69, p <0.001), PPK and the static thoracic compliance values (r = 0.64, p <0.001). There was also a significant correlation between PPK levels and Qs/Qt (r = -0.89, p <0.001). ARDS patients that survived presented a stability in the PPK values in successive tests. Nevertheless non-survivors showed a progressive decrease in PPK levels during the follow-up period.

Our results suggest that the plasma kallikrein system becomes activated during ARDS and that this activation might increase the lung vessels’ permeability. In addition, PPK levels are in our opinion a useful prognostic parameter in predicting the outcome of ARDS patients.
Modulation of Arachidonic Acid Metabolism in Human Endothelial Cells by Glucocorticoids
Authors: De Caterina Raffaele , Weksler Babette B
Published: July 2018

To learn whether glucocorticoids inhibit prostaglandin (PG) production in vascular endothelial cells, we investigated the effects of glucocorticoids on PG synthesis by cultured human umbilical vein endothelial cells (EC). Pretreatment of EC with dexamethasone (DX, 10-9 to 5 x 10-5 M) caused a dose-dependent inhibition of PGI2 production when PG synthesis from endogenous arachidonate was stimulated by human thrombin (0.25-2 U/ml) or ionophore A 23187 (1-5 μM). The inhibition was detectable at 10-7 M DX and maximal at 10-5 M (4.0 ± 0.7 vs. control: 7.7 ± 1.9 ng/ml, mean ± S.D., P <0.01). The production of PGE2 and the release of radiolabelled arachidonate (AA) from prelabelled cells were similarly inhibited. Prolonged incubation of EC with glucocorticoids was required to inhibit PG production or arachidonate release: ranging from 8% inhibition at 5 h to 44% at 38 h. In contrast, prostaglandin formation from exogenous AA was not altered by DX treatment. When thrombin or ionophore-stimulated EC were restimulated with exogenous AA (25 μM), DX-treated cells released more PGI2 than control cells (5.7 ± 0.5 vs. 4.1 ± 0.6 ng/ml, P <0.01). Both the decrease in PGI2 production after thrombin/ionophore and the increase after re-stimulation with AA were blunted in the presence of the protein synthesis inhibitor cycloheximide (0.1-0.2 μg/ml). Thus, incubation of EC with glucocorticoids inhibits PG production at the step of phospholipase activation. The time requirement for these steroid effects and their blunting by cycloheximide are consistent with the induction of regulatory proteins, possibly lipocortins, in endothelial cells.
A Comparison of the Antithrombotic and Haemorrhagic Effects of a Low Molecular Weight Heparin (LHN-1) and Conventional Heparin
Authors: Diness V , Nielsen J I , Pedersen P C , Wolffbrandt K H , Østergaard P B
Published: July 2018

The antithrombotic effects after intravenous administration of a low molecular weight heparin (LHN-1) and conventional heparin were compared in a rabbit model of experimental thrombosis, where thrombus formation was induced by a combination of endothelial damage and stasis. Both compounds were able to prevent thrombosis completely. However, LHN-1 was significantly less potent than conventional heparin, the ratio between doses with the same antithrombotic effect being 2.4:1 on a weight basis. Bleeding times after administration of LHN-1 and conventional heparin were determined by tail transsection in anaesthetized rats and by template bleeding in the ear of conscious pigs. Given intravenously at a dose ratio of 2.4:1 (w/w), LHN-1 affected APTT less than conventional heparin, whereas the effects on haemostasis were not significantly different. In conclusion, it was found that after intravenous administration LHN-1 prevented experimental thrombosis as effectively as conventional heparin. However, the correlation between antithrombotic and haemorrhagic effects of LHN-1 was the same as that of conventional heparin. The corresponding relation in man remains to be determined.
The Effects of Modulation of Prostanoid Metabolism on the Thoracic Platelet Accumulation Induced by Intravenous Administration of Collagen in the Guinea-Pig
Authors: Butler K D , Shand R A , Wallis R B
Published: July 2018

The effects of intravenously administered collagen on the circulatory platelet count, TxB2, 6-keto PGF1α and 51Cr-labelled platelet accumulation in the thorax have been evaluated in the guinea-pig. Administration of collagen induced a dose-related peripheral thrombocytopenia and a concomitant increase in 51Cr-labelled platelets in the thorax. There was also a transient dose-related increase in plasma TxB2 but no change in plasma 6-keto PGF1α levels.

The thromboxane synthetase inhibitors tested, reduced the platelet accumulation, but only CGS 13080 significantly inhibited TxB2 production. In contrast all the cyclooxygenase inhibitors tested impaired the elevation of plasma TxB2 after collagen, but only diclofenac inhibited the 51Cr-labelled platelet accumulation.

The greater effect of thromboxane synthetase inhibitors compared to cyclooxygenase inhibitors on platelet accumulation in this system cannot be completely explained by the changes measured in the circulating prostanoids.
Heparin-Induced Osteoporosis in Rats
Authors: Matzsch Thomas , Bergqvist David , Hedner Ulla , Nilsson Bo , Østergaard Per
Published: July 2018

In order to study the effect of heparin in inducing osteoporosis, 30 female rats were divided in two groups and treated with daily injections of 2 IU heparin/g body weight for 33 and 65 days and compared with the same number of rats acting as controls. The mineral bone mass in the femora of the animals was measured quantitatively. A significant (p <0.001) reduction in bone mineral mass was found in the heparin-treated animals. This effect was present to the same degree after 33 days as after 65 days of treatment. It is concluded that heparin in this dose causes osteoporosis in rats after 33 days and that the described method can be used as an experimental model for further studies on this topic.
Comparative Turn-Over of Heparin Cofactor II and Antithrombin III in Baboons Influence of Heparin and Pentosan Polysulfate Administration
Authors: Sié P , Lansen J , Lacheretz F , Verschuere B , Boneu B
Published: July 2018

Heparin and pentosan polysulfate (PPS) interact in plasma with antithrombin III (AT III) and Heparin cofactor II (HC II) respectively. To assess the influence of heparin or PPS treatment on the metabolism of their respective cofactors, we performed a double tracer study in baboons receiving heparin or PPS. Purified AT III and HC II from human plasma were labelled with 131I and 125I respectively by the lactoperoxidase-glucose oxidase technique. The tracers had unchanged biological activities, were homogenous in SDS-PAGE, migrated as native proteins by crossed immunoelectrophoresis in the presence of heparin or PPS and virtually coeluted with endogenous baboon proteins from heparin-agarose. Nine animals were randomly allocated to receive, during the metabolic study, heparin (500 IU/kg/d, n = 3), PPS (5 mg/kg,d, n = 3) or a placebo (n = 3) given in 2 daily subcutaneous injections. Heparin levels and anticoagulant effects were similar in extent and duration to those usually achieved in man. The plasma concentrations of AT III and HC II did not vary under treatment. The half-life of the elimination phase in the placebo group ranged from 1.95 to 2.33 d for AT III and from 1.96 to 2.21 d for HC II. There was no significant difference in the half-lives of the 2 inhibitors between the placebo group and the animals receiving heparin or PPS. This suggests that clinical conditions associated with heparin treatment may be important for the effect of heparin on AT III metabolism previously reported in patients. When the data of the nine animals were pooled, a strong positive correlation (p <0.001) between the halflives of AT III and HC II was found, indicating similarities in the catabolic pathways of these two parent molecules.