bluuebunny/crossref_metadata_2025 huggingface dataset:
The Action of Immobilized Thrombin on Factor VIII, Fibrinogen and a Synthetic Tripeptide
Authors: Brown J E , Carton C L , Dietz A S , Baugh R F , Hougie C
Published: July 2018

Bovine thrombin was insolubilized by attachment to cyanogen bromide-activated Sepharose (Sepharose-thrombin) or to activated (Affi-Gel 10) agarose containing a 10 Å long arm (Affi-Gel-thrombin). Coupling in both instances approximated 7,000 units of thrombin per ml packed gel as determined by 125I-thrombin incorporation. The thrombin beads hydrolyzed the synthetic tripeptide Bz-Phe-Val-Arg-pNA (S-2160) at different rates, with the Sepharose-thrombin more active (220 esterase units per ml) than Affi-Gel thrombin (20.4 units per ml). The Km was significantly higher for the insolubilized thrombins (2×10-3M) than uncoupled thrombin (Km = 8×10-5M). The Sepharose- thrombin activated factor VIII significantly more rapidly than Affi-Gel-thrombin. Neither matrix-bound thrombin clotted a fibrinogen solution or liberated significant amounts of fibrinopeptides over 48 hr. This data indicates that a proteolysis of factor VIII, rather than a complex with thrombin, is the method of activation of factor VIII and that factor VIII is more accessible to the action of immobilized thrombin than is fibrinogen.
An International Collaborative Assay of Factor VIII Clotting Activity
Authors: Barrowcliffe T W , Kirkwood T B L
Published: July 2018

An International Collaborative Study was organised to replace the first International Standard for factor VIII. A freeze-dried concentrate, 73/552, and a freeze-dried plasma, 75/510, were assayed against the International Standard, and also compared to fresh normal plasma and local standards.

In assays of the concentrate 73/552 against the first I.S. the mean potency was 1.14 i.u./ ampoule and there was no significant difference between one-stage and two-stage methods. When assayed against average fresh normal plasma, the potency was 1.05 “normal plasma units” per ampoule. It was agreed by the participants that the potency of 73/552 be regarded as the mean of these two figures, i.e. 1.10 i. u./ampoule.

In assays of the freeze-dried plasma, 75/510, against the first I.S. the mean potency was 0. 68 i. u./ampoule, but the one-stage assays gave significantly higher potencies (mean 0.74 1. u./ampoule) than the two-stage assays (mean 0.59 i. u./ampoule). The same trend was also seen in the fresh normal plasmas, and in the local plasma standards. This finding has important implications for the standardisation of factor VIII.

Stability studies on the concentrate 73/552 gave a predicted loss of 0.02% per year at – 20° C. All participants agreed that the material was suitable to serve as an International Standard, and at the 26th meeting of the Expert Committee on Biological Standardisation of the World Health Organization, the material in ampoules coded 73/552 was established as the 2nd International Standard for factor VIII, with a potency of 1.10 i. u./ampoule.
Thromboplastin Calibration
Authors: Loeliger E A , Hoeff-van Halem R. van der , Halem-Visser L P van
Published: July 2018

Standardization of the thromboplastin most commonly used for anticoagulant control in The Netherlands has been achieved by the Dutch Reference Laboratory for Anticoagulant Control. The system established for control indicates the performance characteristics of each batch of thromboplastin in patients relative to a national calibration batch. Calibration constants were assessed according to an ICTH/ICSH proposal and with a reproducibility of approximately 2% (CV). With the use of a given calibration constant of the International Reference Preparation of Thromboplastin (1.0) and after assessment of the calibration constant of the National Reference Preparation in international terms (0.93), the accuracy of calibration with the various modifications of the calibration procedure is of the order of magnitude of 3% (CV). On the basis of the calibration protocol, physicians will be provided with correlation tables containing, for their convenience, prothrombin (thromboplastin) times in terms of the reference thromboplastin (uniform, i. e., batch-independent, prothrombin times), conventional prothrombin activity (percentages), and in due course with International Calibrated Ratios.* With this approach current practice in the prescription of oral anticoagulants will be maintained. Opportunity is given, however, to aim at internationally proposed therapeutically optimal target values.
Activation of Bovine Factor X in the Presence of Calcium, Magnesium, Barium or Manganese ion
Authors: Yue Robert H , Gertler Menard M
Published: July 2018

The binding of divalent metal ions to bovine factor X, factor Xa and the coagulant protein in Russell’s viper venom was studied by the technique of fluorescence quenching. Titration of factor X with Ca+2, Mg+2 or Ba+2 revealed that these metal ions can bind to factor X. A tightly binding site(s) was observed with Kd of 79 and 98 μM for Ca+2 and Mg+2 respectively. A loosely binding site(s) was evident with Kd of 0.55, 0.50 and 0.35 mM for Ca+2, Mg+2 and Ba+2 respectively. The quenching phenomenon was also observed when Mn+2 was used as titrant but factor X precipitated out when the concentration of Mn+2 was 10 mM. The binding of Ca+2, Mg+2, Ba+2 or Mn+2 to bovine factor Xa or to the purified coagulant fraction of Russell’s viper venom was very weak in each case.

In the absence of Ca+2, the coagulation fraction of Russell’s viper venom could not activate bovine factor X. Activation of factor X was achieved when Ca+2 was replaced by either Mg+2, Ba+2 or Mn+2. When the concentration of these ions were 5 mM, the efficiency of factor Xa generation was estimated to be: Ca+2> Mg+2> Ba+2> Mn+2. Higher concentration of Mg+2, Ba+2, or Mn+2 retarded the activation process. However, Ca+2, Mg+2, Ba+2 or Mn+2 has little or no influence on the esterase activity of factor Xa or purified Rusell’s viper venom.

The results suggest that complexation of divalent metal ion with factor X is prerequisite in the activation process. The binding of Mg+2, Ba+2 or Mn+2 to these loosely binding sites might have altered the geometrical configuration as well as the electrostatic environment on factor X significantly. Thus, it is more difficult to form the binary complex and a slower generation of factor Xa results. Therefore, divalent metal ion serves as a dual role in the activation of factor X to factor Xa depending upon the ionic concentration.
Comparative Plasma Heparin Levels after Subcutaneous Sodium and Calcium Heparin
Authors: Low Joyce , Biggs J C
Published: July 2018

Comparative plasma heparin levels were measured in normal subjects injected subcutaneously with 5,000 units of the sodium and calcium salts of heparin. Plasma heparin levels were measured up to 7 hr post-injection by an anti-factor Xa assay (Denson and Bonnar 1973). Preliminary studies indicated that heparin levels were reproducible in subjects who received two injections of the same heparin. Peak plasma concentrations (Cmax) and the time at which peak concentration was reached (Tmax) varied greatly from subject to subject. In one group of subjects (15) two commonly used heparins, a sodium heparin (Evans) and a calcium heparin (Choay) were compared. Peak heparin concentrations were not significantly different. However the Tmax for the sodium heparin (1.5 hr) was significantly earlier than the Tmax for the calcium heparin (3 hr) and this was not due to a difference in the volume of the two heparin injections. No significant difference could be detected in the plasma clearance rate and the molecular weight distribution of the two heparins.

In two other groups of subjects, sodium and calcium preparations from two manufacturers were compared. In general, the sodium salts gave rise to significantly higher plasma concentrations, which could be interpreted as a greater bioavailability of sodium salts. These results indicate that the salt of the heparin can influence the plasma concentration achieved after subcutaneous injection.
Heparin-Neutralizing Activity, Total Progressive Antithrombin and the Ethanol Gel Test in Acute Chest Pain
Authors: Raper C G L , Love H
Published: July 2018

92 patients admitted to a cardiac monitoring unit with chest pain were investigated. The mean heparin-neutralizing activity of the 34 patients with severe myocardial infarction was found to be significantly different from the mean of 70 normal controls. However, there was considerable overlap between individual observations and the normal range. Only 5 of the 34 patients with severe myocardial infarction had antithrombin levels outside the normal range. In patients with a severe myocardial infarct tested on the 2nd and 3rd day after infarction there was a significant increase in the number of positive ethanol gel tests compared with the other patients studied.
An Assay of the Antithrombotic Action of Warfarin: Its Correlation with the Inhibition of Stasis Thrombosis in Rabbits
Authors: Wessler Stanford , Gitel Sanford N , Bank Harry , Martinowitz Uri , Stephenson Ronald C
Published: July 2018

No assay of the antithrombotic action of warfarin has been available. Experiments were performed to determine whether Xa inhibitory activity - the reaction rate between activated factor X (Xa) and antithrombin III - could serve this function. 105 warfarin-treated patients demonstrated a significant 18% increase in Xa inhibitory activity compared to 51 controls, p <0.001, without any correlation between this activity and the prothrombin times in the treated patients. A similar increase in Xa inhibitory activity was obtained in rabbits treated with 2 mg of warfarin per day compared to control animals, p <0.001. Employing an assay which routinely produced venous thrombosis after clotting proteases were infused into warfarin-treated and control rabbits, three observations were made. 1. The extent of stasis thrombosis induced by injection of thrombin, Xa or activated factor IX, was significantly reduced in warfarin-treated rabbits compared to control animals, independent of alteration in the four established vitamin K-dependent zymogens. 2. In the rabbit, significant changes in prothrombin times and prothrombin and factor X activities preceded by 5 days both the increase in Xa inhibitory activity and the antithrombotic effect which became significant on the sixth day. 3. The correlation between Xa inhibitory activity of warfarin-treated rabbits and the extent of stasis thrombosis induced by Xa was significant, p<0.05. Xa inhibitory activity is one measure of the antithrombotic action of warfarin.
Clues for Understanding the Structure and Function of a Prototypic Human Integrin: The Platelet Glycoprotein IIb/IIIa Complex
Authors: Calvete Juan J
Published: July 2018

The glycoprotein (GP) IIb/IIIa, a Ca2+-dependent heterodimer, is the major integrin on the platelet plasma membrane. On resting platelets GPIIb/IIIa is maintained in an inactive conformation and serves as a low affinity adhesion receptor for surface-coated fibrinogen, whereas upon platelet activation signals within the cytoplasma alter the receptor function of GPIIb/IIIa (inside-out signalling), which undergoes a measurable conformational change within its exoplasmic domains, and becomes a competent receptor for soluble fibrinogen and some other RGD sequence-containing plasma adhesive proteins. Upon ligand binding, further structural alterations trigger the association of receptor-occupied GPIIb/IIIa complexes with themselves within the plane of the membrane. The simultaneous binding of dimeric fibrinogen molecules to GPIIb/IIIa clusters on adjacent platelets leads to platelet aggregation, which promotes attachment of fibrinogen-GPIIb/IIIa clusters to the cytoskeleton (outside-in signalling). This, in turn, provides the necessary physical link for clot retraction to occur, and generates a cascade of intracellular biochemical reactions which result in the formation of a multiprotein signalling complex at the cytoplasmic domains of GPIIb/IIIa. Glycoprotein IMIIa, also called αIIbβ3 in the integrin nomenclature, plays thus a primary role in both platelet adhesion and thrombus formation at the site of vascular injury. In addition, the human glycoprotein Ilb/IIIa complex is the most thoroughly studied integrin receptor, its molecular biology and major features of its primary structure having been elucidated mainly during the last six years. Furthermore, localization of functionally relevant monoclonal antibody epitopes, determination of the cross-linking sites of inhibitory peptide ligands, proteolytic dissection of the isolated integrin, and analysis of natural and artificial GPIIb/IIIa mutants have recently provided a wealth of information regarding structure-function relationships of human GPIIb/IIIa. The aim of this review is to summarize these many structural and functional data in the perspective of an emerging model. Although most of the interpretations based on structural elements of this initial biochemical model require independent confirmation, they may help us to understand the structure-function relationship of this major platelet receptor, and of other members of the integrin superfamily, as well as to perform further investigations in order to test current hypotheses.
Hypercoagulability and High Lipoprotein(a) Levels in Patients with Central Retinal Vein Occlusion
Authors: Bandello Francesco , Vigano’ D’Angelo Silvana , Parlavecchia Mariella , Tavola Alessandra , Della Valle Patrizia , Brancato Rosario , D’Angelo Armando
Published: July 2018

A series of coagulation parameters and lipoprotein(a) (Lp(a)) were explored in plasma from 40 patients with central retinal vein occlusion (CRVO, non-ischemic type n = 12; ischemic type n = 28) free of local and systemic predisposing factors, 1 to 12 months after the acute event. Forty age- and sex-matched patients with cataract served as controls. Prothrombin fragment 1.2 (FI.2), D-dimer, FVII:C - but not FVII: Ag - were higher and fibrinogen was lower in CRVO patients than in controls. Patients with non-ischemic CRVO had higher FI .2 and FVII:C and lower heparin cofactor II than patients with ischemic CRVO. Lp(a) levels greater than 300 mg/1 were observed in 12 patients with CRVO and in 4 controls (30% vs 10%, p <0.025). Patients with high Lp(a) - consistently associated with the S2 phenotype - had higher FVII:C, FVII:C/Ag ratio, and fibrinogen than the remaining CRVO patients. Plasma FI.2 and D-dimer correlated fairly in controls (r = 0.41) and patients with normal Lp(a) levels (r = 0.55), but they did not in the group of patients with high Lp(a) (r = 0.19), where the latter parameter was negatively related to D-dimer (r = −0.55). There was no dependence of the abnormalities observed on the time elapsed from vein occlusion. The findings of activated FVII and high FI.2, D-dimer, and Lp(a) are not uncommon in patients with CRVO. Increased thrombin formation with fibrin deposition and impaired fibrinolysis may play a role in the pathophysiology of CRVO and require specific treatment
Comparative Thrombolytic Properties of Tissue-Type Plasminogen Activator and of a Plasminogen Activator Inhibitor-1 -Resistant Glycosylation Variant, in a Combined Arterial and Venous Thrombosis Model in the Dog
Authors: Collen Désiré , Stassen Jean-Marie , Yasuda Tsunehiro , Refino Canio , Paoni Nicholas , Keyt Bruce , Roskams Tania , Guerrero J Luis , Lijnen Henri R , Gold Herman K , Bennett William F
Published: July 2018

rt-PA-K, a variant of recombinant tissue-type plasminogen activator (rt-PA) with substitution of amino acids 296 to 299 with alanine (KHRR296-299AAAA) has increased fibrin-specificity and reduced sensitivity to plasminogen activator inhibitor-1; rt-PA-T, with threonine 103 replaced by asparagine has an additional glycosylation site and a reduced clearance; and rt-PA-N, with asparagine 117 mutagen-ized to glutamine lacks the high mannose carbohydrate side chain. We have investigated whether combination of these properties in a single molecule might yield an improved thrombolytic agent.

The thrombolytic potency and fibrin-specificity of the combination mutant rt-PA-TNK was compared with that of rt-PA in a combined venous whole blood clot model and platelet-rich arterial eversion graft thrombosis model in dogs given intravenous heparin and aspirin. Infusion of 0.125 to 1.0 mg/kg over 60 min in groups of 4 to 5 dogs produced dose-dependent fibrin-specific venous clot lysis. The thrombolytic potency (percent lysis per mg compound administered per kg body weight) of rt-PA-TNK was significantly higher than that of rt-PA as evidenced by a higher maximal rate of lysis of 480 ± 100% versus 140 ± 40% within the 2 h observation period per mg of compound administered per kg body weight (mean ± SEM, p = 0.004) and a significantly lower dose of 0.08 ± 0.01 versus 0.21 ± 0.04 mg/kg body weight at which the maximal rate of lysis was obtained (p = 0.004). This higher thrombolytic potency was the result of a significantly reduced clearance (240 ± 32 versus 540 ± 49 ml/min, p = 0.002) and a similar specific thrombolytic activity (percent lysis per |ig/ml plasma antigen level). Arterial reflow was obtained with 1 mg/kg rt-PA and with 0.5 mg/kg rt-PA-TNK, but with each agent recanalization was consistently associated with cyclic reocclusion and reflow. The frequency of arterial recanalization was somewhat higher with rt-PA-TNK (10/12) than with rt-PA (4/12) (p = 0.07) but the total patency times during a 2 h observation period were similar.
Transport Phenomena and Clot Dissolving Therapy: An Experimental Investigation of Diffusion-Controlled and Permeation-Enhanced Fibrinolysis
Authors: Wu Jung-He , Siddiqui Khalid , Diamond Scott L
Published: July 2018

We have investigated the effects of diffusive and convective transport on fibrinolysis. Using a constant pressure drop (ΔP/L) from 0 to 3.7 mmHg/cm-clot to drive fluid permeation, various regimes of lytic agents were delivered into fine and coarse fibrin gels (3 mg/ml) and whole blood clots. Using plasmin (1 μM) delivered into pure fibrin or urokinase (1 μM) delivered into glu-plasminogen (2.2 μM)-laden fibrin, the velocity at which a lysis front moved across fibrin was greatly enhanced by increasing ΔP/L. Lysis of fine and coarse fibrin clots by 1 μM plasmin at ΔP/L of 3.67 and 1.835 mmHg/cm-clot, respectively, led to a 12-fold and 16-fold enhancement of the lysis front velocity compared to lysis without pressure-driven permeation. For uPA-me-diated lysis of coarse fibrin at ΔP/L = 3.67 mmHg/cm-clot, the velocity of the lysis front was 25-fold faster than the lysis front velocity measured in the absence of permeation. Similar permeation-enhanced phenomenon was seen for the lysis of whole blood clots. Without permeation, the placement of a lytic agent adjacent to a clot boundary led to a reaction front that moved at a velocity dependent on the concentration of plasmin or uPA used. Overall, these studies suggest that transport phenomena within the clot can play a major role in determining the time needed for reperfusion during fibrinolysis.
Tissue Extract and the Contact Reaction in Blood Coagulation
Authors: Biggs Rosemary , Nossel H. L
Published: July 2018

1. The addition of dilute brain extract to normal silicone collected plasma or to celite exhausted normal plasma produces an acceleration of thrombin formation similar to that of kaolin on normal plasma.

2. When dilute brain extract is added to PTA or Hageman deficient plasma samples, the concentration of brain required to simulate the kaolin activated normal contact mechanism is similar to that required for normal plasma. When very dilute brain extract is used the reaction of the Hageman deficient sample is defective.

3. When added to factor VIII or factor IX deficient plasma samples about ten times as much brain extract is required to produce the type of thrombin generation curve typically found on the addition of kaolin to silicone collected normal plasma.

4. It is suggested that in normal haemostasis the contact phase may be initiated, in part at least, by weak tissue extracts liberated at a site of injury.
The Effect of Dextran of Various Molecular Weight on the Coagulation in Dogs
Authors: Bergentz Sven Erik , Eiken Oddvar , Nilsson Inga Marie
Published: July 2018

1. Infusions of low molecular weight dextran (Mw = 42 000) to dogs in doses of 1—1.5 g per kg body weight did not produce any significant changes in the coagulation mechanism.

2. Infusions of high molecular weight dextran (Mw = 1 000 000) to dogs in doses of 1—1.5 g per kg body weight produced severe defects in the coagulation mechanism, namely prolongation of bleeding time and coagulation time, thrombocytopenia, pathological prothrombin consumption, decrease of fibrinogen, prothrombin and factor VII, factor V and AHG.

3. Heparin treatment of the dogs was found to prevent the decrease of fibrinogen, prothrombin and factor VII, and factor V otherwise occurring after injection of high molecular weight dextran. Thrombocytopenia was not prevented.

4. In in vitro experiments an interaction between fibrinogen and dextran of high and low molecular weight was found to take place in systems comprising pure fibrinogen. No such interaction occurred in the presence of plasma.

5. It is concluded that the coagulation defects induced by infusions of high molecular weight dextran are due to intravascular coagulation.
Zur Reaktionskinetik des Kalziums in der Blutgerinnung
Authors: Göttinger E
Published: July 2018

1. Die Abhängigkeit der Fibrinogenpolymerisation von der Kalziumionenkonzentration kann nur in Plasmen exakt untersucht werden, die durch Ionenaustausch entkalkt oder durch ÄDTA stabilisiert werden.

2. Die Fibrinbildung ist von der vorhandenen Kalziumionenkonzentration quantitativ abhängig. 1 Teil zugesetztes Kalzium entspricht bis zu 1500 Teilen gebildeten Fibrins.

3. Die Blutgase beeinflussen die Reaktion des Kalziums mit dem Gerinnungssystem zum Teil in typischer Weise.

4. Andere Erdalkaliionen beeinflussen die Kalziumwirkung in charakteristischer Weise.

5. Die zur Fibrinogenpolymerisation benötigten Mengen Kalzium und Thrombin liegen in der gleichen Größenordnung.
Nachweis eines Antiheparinfaktors in der gesunden und der arteriosklerotischen Gefäßwand
Authors: Hoffmeister Kl , Perlick E
Published: July 2018

1. In wäßrigen Extrakten aus menschlichen Aortenwänden konnte eine deutliche Antiheparinaktivität nachgewiesen werden. Diese war jedoch erst nach einer Inkubation der Extrakte mit Heparin bei 37° C über 15 Minuten voll wirksam und konnte durch Erhitzen zerstört werden. Sie war bei —20° C mehrere Wochen lang lagerungsstabil.

2. In makroskopisch normalen Aortenwänden war die Antiheparinaktivität am größten, sie ging mit zunehmendem Arteriosklerosegrad zurück. Die Untersuchungen weisen auf eine Abnahme der Gerinnungsaktivität der Gefäßwand bei zunehmender Arteriosklerose hin.
Zur Frage der Beeinflussung der Permeabilitätsverhältnisse im Bereich des Zentralnervensystems durch gerinnungshemmende und fibrinolytisch wirkende Stoffe
Authors: Liebaldt G , Matis P , Nagel W
Published: July 2018

Zusammenfassende Besprechung

Die therapeutisch üblichen Dosen der untersuchten Stoffe wurden unter Wahl eines feinstdispersen, gut schrankengängigen Farbstoffs bewußt überschritten. Die beobachteten Veränderungen dürfen deshalb nicht überbewertet werden. Sie zeigen aber, daß gerinnungshemmende und fibrinolytisch wirksame Körper im Zentralnervensystem die Permeabilitätsverhältnisse quantitativ, qualitativ und topisch unterscheidbar beeinflussen können. Die makroskopisch eindrucksvollen Unterschiede hinsichtlich der Färbung des Tubergebietes und der Umgebung (Liquemin streng lokalisiert; Marcumar, vor allem aber Actase und Streptokinase mehr diffus) deuten auf bestimmte Qualitätsunterschiede. Auffällig ist die besondere dyshorische Auswirkung des Heparins im Bereich des Ammonshorns gegenüber einer diesbezüglichen Bevorzugung der Tuberregion durch die anderen Präparate. Zwischen dieser organ-(region-)spezifischen Beeinflussung des Permeabilitätsverhaltens einerseits und einer organspezifischen Blutungsneigung bzw. einem organspezifischen Beitrag zur Blutstillung andererseits [s. hierzu (2)] dürften prinzipielle Beziehungen bestehen.

Die Dissoziation von ausgeprägter Gerinnungshemmung und (hier subpleuraler) Blutung ist besonders bei dem dicumarolanalog wirkenden Marcumar in Bestätigung früherer Befunde (4, 6, 8, 10) deutlich, desgleichen für Liquemin (1, 7, 9), aber nur angedeutet nachweisbar.

Aus unseren Untersuchungen ergibt sich 1. die Tatsache einer lokalen Spezifität der Schädigungsmöglichkeit innerhalb des ZNS, 2. die Notwendigkeit, diese Faktoren als Meßwerte zu berücksichtigen, und 3., daß gewisse Reaktionen von Versuchstieren, die zunächst kein befriedigendes pathologisch-anatomiches Äquivalent aufweisen (z. B. 3.), u. U. durch zentralnervöse Beeinflussung zu deuten sind.
Antiplasmin Deficiency in Polycythaemia: a Form of Thrombopathy
Authors: de Vries S. I , Braat-van Straaten M. A. J , Müller E , Wettermark M
Published: July 2018

A marked fibrinolytic activity could be demonstrated in the blood of 10 patients with polycythaemia. In these cases fibrinolysis was considered the result of a deficiency an antiplasmin. It could be demonstrated that this naturally occurring inhibitor of fibrinolysis is present in normal platelets but is lacking in the patient’s own thrombocytes. This factor is probably attached to the surface of the thrombocytes. It could be removed by repeatedly washing. Lack of antiplasmin is a dysfunction of platelets and a form of thrombopathy. It does not respond on treatment with ACA or prednisone. In primary polycythaemia busulfan (Myleran) was considered to be the treatment of choice, whereas in 1 case of secundary erythrocytosis (Fallot’s tetralogy) the reaction on unroasted peanuts was unmistakable. In 4 more patients an alcoholic extract of peanuts was given, and promising results were obtained in 3 of them. The importance of this “thrombopathic type’ of fibrinolysis has been pointed out especially in view of surgical interventions.
Studies on the Coumarin Anticoagulant Drugs : The Assay of Warfarin and its Biologic Application
Authors: O’Reilly Robert A. , Aggeler Paul M. , Hoag M Silvija , Leong Lois
Published: July 2018

A spectrophotometric method is described for the determination of warfarin in biologic fluids. The warfarin is extracted from the specimen with acidified ethylene dichloride, washed with mild alkali and recovered from the organic phase with strong alkali. The extinction is then measured spectrophotometri-cally at the ultraviolet absorption maximum for warfarin. Results of UV absorption spectrophotometry and countercurrent distribution analysis showed that the assay is specific for unchanged warfarin in plasma; in addition, it measures a probable metabolite of warfarin in urine. Further studies indicated that the metabolite may be hydroxy-warfarin.

The method was applied in determining the absorption and elimination of warfarin after oral and intravenous administration. Gastrointestinal absorption of the drug was rapid and complete. With both routes the plasma concentrations and prothrombinopenic response were similar, and the rate of disappearance from the plasma followed an exponential decay curve. A correlation was found between rate of elimination and biologic response — the slower the rate of elimination, the more pronounced the depression of prothrombin complex activity. The application of this dual approach, the simultaneous study of the metabolic fate of the drug and the prothrombinopenic response, should provide a more rational basis for the therapeutic use of anticoagulant drugs.
Effect of Lecithin on the Fibrinolytic System in Guinea-Pig Serum
Authors: Olesen E. S
Published: July 2018

Lecithin preparations from egg yolk or from soya beans added to guinea-pig serum which is subjected to isoelectric precipitation produce fibrinolytic activity in the precipitates in a manner similar to that previously reported for anionic polyelectrolytes. The possible physiological significance of this observation is discussed.
The Ultrastructure of Rabbit Blood Platelet Aggregates
Authors: Hovig Torstein
Published: July 2018

Different types of rabbit blood platelet aggregates formed in vitro were studied by means of electron microscopy.

1. Aggregates were produced by addition of small amounts of thrombin to citrate-PRP. The platelets, rich in pseudopods, were lying close together with no detectable fibrin between them. Fibrin was observed only in the periphery of the aggregate. Varying structural changes, with loss of granules and mitochondria, occured in the platelets. The appearance of these aggregates was similar to that of the haemostatic platelet plug formed in vivo.

In clots of PRP induced by greater concentrations of thrombin, fibrin was readily detectable as bundles of fibres. Platelets with complete loss of internal structure were scattered in the fibrin framework.

2. The platelets were usually loosely packed in thrombin-induced aggregates of washed platelets, with no visible fibrin between them. Considerable structural changes of the platelets were observed. Rods or filaments of electron-dense substance were observed inside the platelets.

3. The platelets were lying close together in ADP-induced aggregates, but several free spaces could be observed between them. Small and inconsistent structural changes of the platelets appeared.

4. Aggregates induced by saline extract of tendons demonstrated marked structural changes of the platelets, with loss of granules and mitochondria. Platelet membranes were mostly preserved. Several free spaces were observed between the platelets. No electron-dense substance indicative of fibrin was detected.

The appearance of the different types of aggregates is compared and discussed in relation to the ultrastructure of the haemostatic platelet plug.
The Separation of Thromboplastin Formed from Pig’s Plasma in the Thromboplastin Generation Test
Authors: Cross M. J
Published: July 2018

1. Plasma thromboplastin has been formed from a mixture of pigs’ plasma, serum and platelets using a modification of the thromboplastin generation system of Biggs and Douglas (1953). The thromboplastic activity in the modified system was more stable than in the original system.

2. A sediment with considerable thromboplastic activity has been obtained by centrifugation. This sediment was free of platelets and contained very little thrombin.

3. The sediment when resuspended in buffer was fully active only in the presence of calcium and between pH 6.6 and 7.0. The activity slowly decreased at 0—4° C and rapidly at 65° C.

4. The sediment rapidly converted prothrombin to thrombin in the absence of factor V.

5. The activity of the sediment was unaffected when it was incubated with thrombin.
Factors which Influence the Bleeding Time
Authors: den Ottolander G. J. H , Bleijenberg A
Published: July 2018

A prolonged bleeding time can be due to a vascular, plasmatic, or thrombocytic cause. On the basis of investigations in 8 patients with a prolonged bleeding time, these different forms are discussed.

There appeared to be at least two plasmatic factors one of which is the bleeding factor of Nilsson. The demonstration of a thrombocytic cause is sometimes only possible with transfusions of normal platelet suspensions. A patient is described with a prolonged bleeding time due to an acquired thrombo-pathia, resulting from excessive haemorrhages and exchange transfusions.
Leucocyte Rheological Properties Are Altered in Patients with Diffuse Atherosclerosis
Authors: Galante Alberto , Pietroiusti Antonio , Magrini Andrea , Carta Sandro , Franceschelli Laura , Piccolo Paola , Pistolese Giuseppe Raimondo , Martelli Eugenio , Mastino Antonio , Matteucci Claudia , Grelli Sandro , Favalli Cartesio
Published: July 2018

To evaluate whether atherosclerosis may be associated with altered leucocyte rheology, we assessed leucocyte count (by Coulter counter), aggregation (by means of the leukergy test) and expression of adhesion molecules integrin LFA-1 and CD 44 (by means of immunofluorescence staining and flow cytometry) in 9 patients with carotid plus lower limb artery atherosclerosis (group A), 14 patients with carotid atherosclerosis only (group B) and 23 controls without atherosclerosis (group C). The level of LFA-1 (calculated as mean fluorescence channels - MFCs) on neutrophils, lymphocytes and monocytes was significantly higher (p <0.05) in group A and B patients than in controls (group A - mean ± SE : 383.77 ± 9.42 vs 295.45 ± 5.76; 474.22 ± 8.86 vs ± 7.84; 457.66 ± 12.03 vs 396.25 ± 4.37. Group B: 322.42 ± 6.36 vs 295.45 ± 5.76; 421.42 ± 7.21 vs 388.35 ± 7.84; 415.71 ± 7.73 vs 396.25 ± 4.37, respectively); furthermore, the MFC of LFA-1 on neutrophils was significantly different (p <0.05) between group A and B patients. The percentage of aggregated leucocytes was significantly higher (p <0.05) in group A patients (4.46 ± 1.07) than those in groups B (1.75 ± 0.38) and C (1.43 ± 0.25), whereas no significant difference was detected between groups B and C. Leucocyte number and expression of CD44 were not significantly different among the 3 groups. In conclusion, changes in leucocyte rheology are present in patients with atherosclerosis and may contribute to chronic ischaemia.
A Hemi-nested, Allele Specific, Whole Blood PCR Assay for the Detection of the Factor V Leiden Mutation
Authors: Sinclair Gary D , Low Sandra , Poon Man-Chiu
Published: July 2018

We describe a novel hemi-nested, allele specific whole blood PCR assay for detection of the factor V Leiden mutation associated with the plasma defect, activated protein C resistance. This assay utilizes 5 μl of whole blood without prior DNA extraction. The hemi-nested design, employing an outer primer pair in combination with nested, allele specific primers obviates the need for restriction enzyme digestion. PCR reactions are analysed directly on agarose or polyacrylamide minigels. The assay confirmed the genotypes of 50 individuals previously categorized by PCR and Mnll digestion, and has been subsequently utilized in the genotyping of 445 individuals referred for thrombosis studies.
The Role of a Decision Rule in Symptomatic Pulmonary Embolism Patients with a Non-high Probability Ventilation-perfusion Scan
Authors: Michel Bowine C , Kuijer Philomeen M M , McDonnell Joseph , Beek Edwin J R van , Rutten Frans F H , Büller Harry R
Published: July 2018

Background: In order to improve the use of information contained in the medical history and physical examination in patients with suspected pulmonary embolism and a non-high probability ventilation-perfusion scan, we assessed whether a simple, quantitative decision rule could be derived for the diagnosis or exclusion of pulmonary embolism. Methods: In 140 consecutive symptomatic patients with a non- high probability ventilation-perfusion scan and an interpretable pulmonary angiogram, various clinical and lung scan items were collected prospectively and analyzed by multivariate stepwise logistic regression analysis to identify the most informative combination of items. Results: The prevalence of proven pulmonary embolism in the patient population was 27.1%. A decision rule containing the presence of wheezing, previous deep venous thrombosis, recently developed or worsened cough, body temperature above 37° C and multiple defects on the perfusion scan was constructed. For the rule the area under the Receiver Operating Characteristic curve was larger than that of the prior probability of pulmonary embolism as assessed by the physician at presentation (0.76 versus 0.59; p = 0.0097). At the cut-off point with the maximal positive predictive value 2% of the patients scored positive, at the cut-off point with the maximal negative predictive value pulmonary embolism could be excluded in 16% of the patients. Conclusions: We derived a simple decision rule containing 5 easily interpretable variables for the patient population specified. The optimal use of the rule appears to be in the exclusion of pulmonary embolism. Prospective validation of this rule is indicated to confirm its clinical utility.
Physical Activity, Fibrinogen Plasma Level and Gene Polymorphisms in Postmenopausal Women
Authors: Rauramaa Rainer , Väisänen Sari , Nissinen Aulikki , Rankinen Tuomo , Penttilä IIkka , Saarikoski Seppo , Tuomilehto Jaakko , Gagnon Jacques , Pérusse Louis , Bouchard Claude
Published: July 2018

The relations between habitual physical activity (PA), fibrinogen gene polymorphisms and plasma fibrinogen were investigated in 191 postmenopausal women. Subjects who reported PA at least 4 times/ week had lower fibrinogen level (3.19 g/1; 95% Cl 3.10; 3.27) than women reporting PA 2-3 times/week (3.43 g/1; 3.29; 3.58) or sedentary subjects (3.64 g/1; 3.33; 3.94). There were no differences in plasma fibrinogen across the α-fibrinogen (Rsal, TaqI) or (β-fibrinogen (Mnll, Bell, Hindlll) genotypes, the frequencies of which were in a Hardy- Weinberg equilibrium. An interaction between Rsal, which was in complete linkage disequilibrium with TaqI, and PA on plasma fibrinogen was observed, even after adjustments for BMI, smoking and medication (p = 0.024). Among women homozygous for the common Rsal allele, the physically most active had lower fibrinogen level (p <0.001) compared to the physically less active subjects. These results suggest that, in postmenopausal women, the relation between physical activity and plasma fibrinogen is modulated by genetic variation in the α-fibrinogen gene.
Heat Stability of Two Candidate International Reference Preparations for Recombinant Human Tissue Factor
Authors: Besselaar A M H P van den , Rijn C J J van , Taberner D A , Goodman L J , Poller L , Clarke K
Published: July 2018

Adequate heat stability of international reference preparations (IRP) for thromboplastin (tissue factor) is an essential requirement. Accelerated degradation testing was performed by three laboratories on two candidate IRP for recombinant human tissue factor.

Heat treatment of these candidates resulted in slight shortening of the PT, contrasting with heat-induced prolongation of the PT observed with a conventional human brain derived IRP.

Heat stability of these candidates was improved when compared with the stability of previous recombinant human tissue factor preparations.

The PT-ratio did not change significantly when the candidates were stored for 28 days at 44° C. It’ can therefore be concluded that both candidates are acceptable with regard to stability.
The Migration of Human Smooth Muscle Cells In Vitro Is Mediated by Plasminogen Activation and Can Be Inhibited by α2-Macroglobulin Receptor Associated Protein
Authors: Wijnberg Monique J , Quax Paul H A , Nieuwenbroek Nancy M E , Verheijen Jan H
Published: July 2018

The plasminogen activation system is thought to be important in cell migration processes. A role for this system during smooth muscle cell migration after vascular injury has been suggested from several animal studies. However, not much is known about its involvement in human vascular remodelling. We studied the involvement of the plasminogen activation system in human smooth muscle cell migration in more detail using an in vitro wound assay and a matrix invasion assay. Inhibition of plasmin activity or inhibition of urokinase-type plasminogen activator (u-PA) activity resulted in approximately 40% reduction of migration after 24 h in the wound assay and an even stronger reduction (70-80%) in the matrix invasion assay. Migration of smooth muscle cells in the presence of inhibitory antibodies against tissue-type plasminogen activator (t-PA) was not significantly reduced after 24 h, but after 48 h a 30% reduction of migration was observed, whereas in the matrix invasion assay a 50% reduction in invasion was observed already after 24 h. Prevention of the interaction of u-PA with cell surface receptors by addition of soluble u-PA receptor or α2-macroglobulin receptor associated protein (RAP) to the culture medium, resulted in a similar inhibition of migration and invasion. From these results it can be concluded that both u-PA and t-PA mediated plasminogen activation can contribute to in vitro human smooth muscle cell migration and invasion. Furthermore, the interaction between u-PA and its cell surface receptor appears also to be involved in this migration and invasion process. The inhibitory effects on migration and invasion by the addition of RAP suggests an involvement of a RAP sensitive receptor of the LDL receptor family, possibly the LDL-receptor related protein (LRP) and/or the VLDL receptor.
Activation of Human Platelets Causes Post-translational Modifications to Cytoplasmic Dynein
Authors: Rothwell Stephen W , Calvert Valerie S
Published: July 2018

In our studies of human platelets we have detected the presence of the molecular motors kinesin and dynein. Dynein is present at a concentration (0.8 μg/g tissue) that is approximately 1/3 the concentration reported for neuronal tissue. Immunofluorescence microscopy of resting platelets shows that, while platelet microtubules are arranged in coiled hoops forming the marginal band in the cortical region of the platelet, dynein is distributed in a pattern of punctate staining throughout the cytoplasm of the platelets. Fractionation of unactivated platelets shows that dynein partitions to the soluble fraction. Stimulation of platelets with thrombin, ADP or epinephrine causes a partial translocation of dynein from the soluble fraction to the particulate fraction with thrombin being the most efficient agent at promoting this shift. Dynein intermediate chain recovered in the soluble fraction of disrupted platelets following activation displays a transient, time-dependent phosphorylation. In contrast, dynein intermediate chain recovered in the particulate fraction shows decreased phosphorylation. These results indicate that human platelets contain a complex microtubule-based system of motor proteins that is an integral part of the physiological changes occurring during platelet activation.
Plasma Derived von Willebrand Factor Preparations: Collagen Binding and Ristocetin Cofactor Activities
Authors: Chang Ping , Aronson D L
Published: July 2018

Five plasma preparations (11 lots) used in the treatment of von Willebrand’s disease (vWD) were evaluated. The collagen binding function of von Willebrand factor (vWF) containing preparations was compared with the ristocetin cofactor activity and the vWF antigen. Some preparations have higher ratio of functional activity (ristocetin cofactor and collagen binding) relative to the antigen than is found in normal plasma. The ristocetin cofactor activity and the collagen binding activity are tightly correlated (r = .95). Ultracentrifugal (UCF) analysis was used to compare the size distribution of vWf antigen, ristocetin cofactor and collagen binding activity. The sedimentation of all of the vWF parameters in the plasma products was slower than in plasma. In plasma products the ristocetin cofactor activity sediments the most rapidly, the collagen binding activity is slower and the antigen the slowest. The collagen/antigen ratio decreases with decreasing vWF size. Assignment of potency to vWF containing preparations utilizing the collagen binding activity may be more precise and as accurate as with the traditional ristocetin cofactor assay.
Endothelium-derived Nitric Oxide Decreases Polymorphonuclear Leukocyte Interaction with the Deeply Injured Arterial Wall under Intermediate and High Shear Conditions
Authors: Provost Patrick , Merhi Yahye
Published: July 2018

Previous studies have shown that nitric oxide (NO) inhibits specific agonist-induced polymorphonuclear leukocyte (neutrophil) and platelet aggregation in vitro. However, the inhibitory effects of NO on neutrophil interaction with the deeply injured arterial wall under conditions of flow is unknown. Therefore, we investigated the influence of NO derived from the endothelium on neutrophil and platelet interactions with the downstream arterial media under controlled flow conditions. Porcine aortic media, simulating deep arterial wall injury, was exposed to flowing porcine non-anticoagulated arterial blood for 5 min at intermediate (1006 s1) and high (3397 s1) shear conditions, and deposition of radiolabeled neutrophils and platelets was quantified. Neutrophil deposition on the exposed arterial media was reduced, by more than 30%, by pretreatment of the endothelium with the physiological precursor of NO, L-arginine, from 84.1 ± 13.7 to 57.4 ± 7.2 X 103/cm2 (p < 0.05) at 1006 s*1, and from 99.3 ± 9.8 to 65.5 ± 8.7 X KP/cm2 (p < 0.05) at 3397 s1 of shear rate, relative to control. Pretreatment of the endothelium with the inactive stereoisomer D-arginine had no effect on neutrophil deposition. These specific inhibitory effects of L-arginine were completely abolished by the inhibitor of NO synthesis, N^-nitro- L-arginine methyl ester (l-NAME) at both shear rates. Endothelial pretreatment with D-arginine, or with L-arginine, in the absence or presence of l-NAME, did not significantly influence platelet interaction with the thrombogenic arterial media at intermediate and high shear rates. These results indicate that NO derived from the endothelium can modulate and has a greater influence on neutrophil, than on platelet, interaction with the injured arterial wall exposing the media under conditions of flow typical to moderately and severely stenosed arteries.
Coagulation Factor VII, Serum-Triglycerides and the R/Q353 Polymorphism: Differences between Older Men and Women
Authors: Mennen Louise I , de Maat Moniek P M , Schouten Evert G , Kluft Cornelis , de Jong Paulus T V. M , Hofman Albert , Grobbee Diederick E
Published: July 2018

Coagulation factor VII activity (FVIIC) is a risk indicator for cardiovascular disease. It is related to serum-triglycerides and the R/Q353 polymorphism (alleles R and Q) in the gene coding for factor VII is strongly associated with factor VII. The association of serum- triglycerides with factor VII may differ between the genotypes, but the results of earlier studies were inconsistent and did not include older people. We studied FVII, triglycerides and the R/Q353 polymorphism in the Rotterdam Study.

In 1158 older subjects (489 men and 669 women) FVIIC, factor VII: Chr, serum-triglycerides and the R/Q353-genotype were determined.

In women triglycerides were positively associated with FVIIChr and FVIIC (FVIIChr: β = 12.4 % PP/mmol/L, CI: 10.3-14.5; FVII:C: β = 13.1% PP/mmol/L, CI: 10.4-15.8). These associations varied by genotype (FVII:Chr: RR: β = 11.7, CI: 9.6-13.8, RQ/QQ: β = 7.9, CI: 4.6-11.2; FVII:C: RR: β = 12.5, CI: 9.5-15.5, RQ/QQ: β = 6.4, CI: 1.4-11.4).

In men, the associations of FVII:Chr and FVII:C with triglycerides were weaker (FVII:Chr: β = 5.9, CI: 4.1-7.7; FVII:C: β = 8.7, CI: 6.2-11.2). There was no difference between the genotype groups.

These results suggest that only in older women the strength of the association of factor VII with serum-triglycerides varies according to genotype of the R/Q353 polymorphism.