bluuebunny/crossref_metadata_2025 huggingface dataset:
Platelet and Fibrinogen Survival in Calves Implanted with Artificial Heart and Ventricular Assist Device -Correlation with Autopsy Findings
Authors: Al-Mondhiry Hamid , Pae Walter E , Miller Cynthia A , Pierce William S
Published: July 2018

The two main ingredients of blood clots formed on artificial surface are platelets and fibrinogen. In this study, we measured platelet and fibrinogen survival in calves implanted with total artificial heart (TAH) and left ventricular assist device (LVAD), and correlate these data with autopsy findings. Platelet survival with autologous 111In-labeled platelets was performed on nine calves implanted with TAH and five with LVAD. Fibrinogen survival with 131I-labeled homologous fibrinogen was performed on six calves with TAH and three with LVAD. Platelet survival was significantly shortened in both groups of animals: 5.89 ± 0.52 days, control 6.46 ± 0.31 days, p = 0.0013; fibrinogen survival was normal: 8.79 ± 1.20 days, control 8.64 ± 1.16 days. At autopsy two calves with TAH had multiorgan thromboembolism. Two other animals with TAH and four with LVAD had focal renal infarcts. Most animals had minor clot formation within the prosthetic device. Major septic complications occurred in four calves with TAH and one with LVAD. Continuous platelet activation by artificial surface probably explains the shortened platelet survival and thromboembolic complications.
Inhibition by Glaucocalyxin A of Aggregation of Rabbit Platelets Induced by ADP, Arachidonic Acid and Platelet-Activating Factor, and Inhibition of [3H]-PAF Binding
Authors: Bin Zhang , Kun Long
Published: July 2018

Glaucocalyxin A is a new diterpenoid isolated from the ethereal extract of the leaves of Rabdosia japonica (Burm f) Hara var glaucocalyx (Maxim) Hara (Labiatae) collected in the northeastern China. When it was incubated with washed rabbit platelets, glaucocalyxin A inhibited ADP- or arachidonic acid-induced platelet aggregation with IC50 values of 4.4 μmol/1, 14.1 μmol/1 respectively. Glaucocalyxin A also inhibited PAF-induced aggregation of rabbit platelets which were refractory to ADP and arachidonic acid with an IC50 value of 13.7 μmol/1. Analysis of [3H]-PAF binding showed that glaucocalyxin A prevented [3H]-PAF binding to intact washed rabbit platelets with an IC50 value of 8.16 μmol/1, which was consistent with its inhibition of PAF-induced platelet aggregation.
Human Vascular Endothelial Cells Catabolise Exogenous Glycosaminoglycans by a Novel Route
Authors: Dawes Joan , Pepper Duncan S
Published: July 2018

Heparin and other anticoagulant glycosaminoglycans were radiolabelled with 125I and their catabolism by human vascular endothelial cells in culture was studied. Heparin, heparan sulphate and pentosan polysulphate were associated with the cellular fraction and incorporated into the subendothelial matrix, but dermatan sulphate was not found in either fraction. High molecular weight, fully desulphated carbohydrate chains were major catabolic products of all those glycosaminoglycans which were taken up by the cells. Pentosan polysulphate was not degraded further, but the catabolism of heparan sulphate, and to a lesser extent that of heparin, also yielded small oligosaccharides. Thus the first step in catabolism of exogenous glycosaminoglycans by human vascular endothelial cells appears to be complete desul-phation, which destroys their biological activity, followed by depolymerisation of the carbohydrate chain. This alternative to the sequential action of lysosomal exoenzymes is dependent upon binding to the cell; thus dermatan sulphate, which is not associated with the cellular fraction, is not catabolised.
Autoantibodies to Thrombomodulin: Development of an Enzyme Immunoassay and a Survey of their Frequency in Patients with the Lupus Anticoagulant
Authors: Gibson John , Nelson Margaret , Brown Ross , Salem Hatem , Kronenberg Harry
Published: July 2018

In order to investigate the possibility that autoantibodies to thrombomodulin (TM) may exist in patients with the lupus anticoagulant (LA) and perhaps be implicated in the pathogenesis of recurrent thrombosis seen in such patients, we developed an enzyme-immunoassay to screen serum samples for anti-human TM activity. The major technical problem encountered in developing this assay was to reduce the non-specific binding of serum components from both the LA positive and the negative population. Considerable reduction of non-specific binding was achieved by use of a phosphate/citrate buffer at pH 8.0 and the use of an optimal sample dilution of 1/40. In addition, samples were always tested in parallel in blank wells and results are expressed as an OD ratio. Samples from 113 patients with the LA were assayed and compared to 78 patients referred for LA testing but found to be negative. The mean OD values for the LA positive patients (± SD) was 1.36 (0.44) with a range of 0.78-2.57. This was virtually identical to the values for the LA negative population (1.38 ± 0.40, range 0.76-2.77). The results of this study indicate that there is no evidence for the presence of a significant autoantibody activity to TM in patients with the LA when compared to LA negative patients. If such autoantibodies do exist their frequency must be quite low.
The Use of the D-Dimer Test in Combination with Non-Invasive Testing Versus Serial Non-Invasive Testing Alone for the Diagnosis of Deep-Vein Thrombosis
Authors: Heijboer Harriët , Ginsberg Jeffrey S , Büller Harry R , Lensing Anthonie W A , Colly Louisa P , ten Cate Jan Wouter
Published: July 2018

We studied the usefulness of the determination of plasma D-dimer levels (using an ELISA) in combination with non-invasive testing with impedance plethysmography (IPG) or real-time ultrasonography (US) for the diagnosis of deep-vein thrombosis (DVT), in outpatients with clinically suspected DVT This combined approach was compared to serial non-invasive testing alone in these patients. The sensitivity of a positive D-dimer test (>300 μg/1) for the presence of DVT was 100% (70/70 patients; 95% C. I.: 95-100%), whereas the specificity was 29% (69/ 239 patients; 95% C. I.: 23-34%). The proportion of patients in which a definitive decision about the presence or absence of DVT could be made on the day of referral, was calculated for both approaches. When applying the combined approach, in 42% of all referred patients the diagnosis of DVT could either be established or refuted on entry, as opposed to only 19% of patients using serial non-invasive testing alone.

Also, the costs per DVT diagnosed were calculated for the two diagnostic approaches. For the diagnosis of DVT the costs using serial IPG were comparable to the costs using the combination of IPG and the D-dimer test. The same conclusion holds for the comparison of serial US with the combination of US and D-dimer testing.

We conclude that for the diagnosis of DVT in symptomatic outpatients the combination of non-invasive testing with the D-dimer test might be preferred over serial non-invasive testing alone, although the safety of such an approach remains to be established in future management studies.
Venous Thrombosis during Pregnancy: Leg and Trimester of Presentation
Authors: Ginsberg J S , Brill-Edwards P , Burrows R F , Bona R , Prandoni P , Büller H R , Lensing A
Published: July 2018

In order to determine the relative frequencies of left and right leg venous thrombosis during pregnancy and the frequencies of venous thrombosis during the three trimesters, a cohort study of 60 consecutive patients with a first episode of venous thrombosis during pregnancy was performed. Fifty-eight women had isolated left leg thrombosis, two patients had bilateral venous thrombosis and no patient had isolated right leg venous thrombosis. Thirteen patients had venous thrombosis during the first trimester (21.7%), 28 during the second trimester (46.7%) and 19 during the third trimester (31.7%). These findings indicate that patients with symptoms in the right leg rarely have venous thrombosis. Because leg pain and swelling occur most frequently during the third trimester but venous thrombosis is relatively equally distributed during all three trimesters, patients presenting earlier during pregnancy are more likely to have venous thrombosis than patients presenting later during pregnancy.
In Vivo Significance of Kinetic Constants of Tight Binding Reversible Proteinase Inhibitors
Authors: Longstaff Colin
Published: July 2018

For a reaction between an enzyme and inhibitor, rate constants and equilibrium constants are both widely used as measures of inhibitor potency. Which one of these two parameters should be used depends on the mechanism of the reaction. Moreover, for biological studies it is not always the overall effectiveness of an inhibitor that is important, but also the rate at which it acts in conditions that may be encountered in vivo. In vitro investigations may be performed to determine the kinetic and thermodynamic constants in an enzyme inhibitor system and to investigate the influence of various other effector biomolecules. However, in translating these results into physiological conclusions, care must be taken that the in vitro experiments have been properly designed and data analysed correctly. This article is an attempt to indicate some of the problems that can be encountered in this process.
Effect of Leukocyte Proteinases on Tissue Factor Pathway Inhibitor
Authors: Petersen Lars C , Bjørn Søren E , Nordfang Ole
Published: July 2018

The effect of human neutrophil elastase and cathepsin G on recombinant tissue factor pathway inhibitor (TFPI) was investigated. A weak inhibition by TFPI of both elastase (Kx
= 0.4 μM) and cathepsin G (Kx
= 0.1 μM) was observed. Neutrophil elastase rapidly cleaved TFPI at the Thr87-Thr88 bond situated at the link between Kunitz domains I and II. Cleavage of TFPI by cathepsin G was also observed, but the reaction was much slower and resulted in a number of fragments. Proteolytic cleavage by both elastase and cathepsin G resulted in destruction of inhibitor function with respect to TFPI’s inhibition of factor Xa. Cleavage by neutrophil elastase was capable of restoring factor Xa amidoly-tic activity after its initial inhibition by TFPI. Inhibition of cathepsin G by TFPI was strongly augmented by stoichiometric amounts of factor Xa. However, the augmentation was temporary, presumably due to concomitant cleavage of TFPI by cathepsin G. These observations may have implications for the putative effect of neutrophil leukocyte stimulation on the regulation of the tissue factor-mediated coagulation pathway. Conversely, formation of a factor Xa/TFPI complex may reduce or modulate the proteolytic potential of stimulated leukocytes by temporary inhibition of cathepsin G.
Measurement of Markers of Activated Coagulation in Antithrombin III Deficient Subjects
Authors: Demers Christine , Ginsberg Jeffrey S , Henderson Penny , Ofosu Fred A , Weitz Jeffrey I , Blajchman Morris A
Published: July 2018

Functional antithrombin III levels were measured by factor Xa inhibition in 63 members of a large family with type 2 antithrombin III deficiency and individuals were classified as antithrombin III deficient or non-deficient according to the results. FI+2 and TAT complexes were measured using an ELISA and FPA levels were measured by radioimmunoassay.

Thirty subjects (48%) were classified as antithrombin III deficient and 33 (52%) as antithrombin III non-deficient. The mean level of FI+2 was significantly higher in the deficient adults (0.87 ± 0.26) compared to both the non-deficient adults (0.70 ± 0.21) (p = 0.03) and the deficient adults receiving warfarin (0.16 ± 0.01) (p <0.001). The differences in the mean values of TAT complexes and FPA between deficient and non-deficient individuals were not statistically significant.

These findings suggest that untreated antithrombin III deficient subjects generate more thrombin than their non-deficient family members and that warfarin inhibits this thrombin formation. In this cross-sectional study, it is not possible to correlate the levels of the surrogate makers with future clinical outcome.
Tumor Necrosis Factor Induces von Willebrand Factor Release in Healthy Humans
Authors: van der Poll Tom , van Deventer Sander J H , Pasterkamp Gerard , van Mourik Jan A , Büller Harry R , ten Cate Jan W
Published: July 2018

Tumor necrosis factor (TNF) is an important mediator of the host responses to systemic infection. To investigate the effect of TNF on the release of von Willebrand factor (vWF) into the circulation, we performed a controlled study in six healthy men, sequentially measuring the plasma concentrations of vWF antigen after a bolus intravenous injection of recombinant human TNF (50 μg/m2). TNF induced a marked increase in vWF antigen plasma levels, becoming significant after 45 min and peaking after 4 h (percentage increase from base line: 351 ±46; p <0.0001, TNF versus saline). The multimeric organization of circulating vWF was not affected by TNF. We conclude that TNF may be an early mediator of increased vWF secretion in systemic infection.
A Novel Sensitive Assay for Functional Factor XII Based on the Generation of Kallikrein-C1-lnhibitor Complexes in Factor Xll-Deficient Plasma by Glass-Bound Factor XII
Authors: Dors D M , Nuijens J H , Huijbregts C C M , Hack C E
Published: July 2018

We developed a very sensitive assay for functional factor XII. This assay is based on the property of glass-bound factor XII to activate prekallikrein (PK) into kallikrein in factor XH-deficient plasma, which is assessed by measuring the formation of kallikrein- C1 -inhibitor complexes in this plasma by radioimmunoassay.

Incubation of varying amounts of factor XII in glass tubes led to a dose-dependent increase in kallikrein-C1-inhibitor complexes in factor XH-deficient plasma with a lower limit of detection of ± 20 pg of factor XII. The specificity of the assay for factor XII was demonstrated by experiments with plasmas deficient for factor XII, PK or high molecular weight kininogen (HK) and by incubation of factor XII with Polybrene or with a monoclonal antibody that inhibits the amidolytic and procoagulant activity of factor XII. The high sensitivity of the assay appeared to be due to the inability of Cl-inhibitor to inhibit factor XII bound to glass, which resulted in a molar ratio of generated kallikrein to glass-bound factor XII of at least 100:1.

This assay for factor XII may be a feasible tool in studies on structural and functional aspects of (recombinant) factor XII species synthesized by cultured cells.
Comparison Between a One-Point Dilute Phospholipid APTT and the Dilute Russell Viper Venom Time for Verification of Lupus Anticoagulants
Authors: Alving Barbara M , Barr Charles F , Johansen Lawrence E , Tang Douglas B
Published: July 2018

In the present study, the dilute Russell viper venom time (RVVT) and the dilute phospholipid activated partial thromboplastin time (PL-APTT), which are two assays used for the verification of lupus anticoagulants (LA), were modified to increase standardization. The modified assays were then compared with respect to sensitivity and specificity in detecting LA in plasmas from 72 patients with a prolonged APTT. Modifications included utilizing a single dilution of phospholipid that was either bovine brain thromboplastin (Thrombofax) or liposomes comprised of phosphatidylcholine/phosphatidylserine, and expressing the results as a ratio of the clotting times of the mixture of patient and normal plasma/clotting time of normal plasma. In the RWT, the correlation coefficient between assay results for liposomes and Thrombofax was 0.88 and in the PL-APTT, the correlation was 0.68. A positive test for LA was defined as a ratio of ≥1.3 for the PL-APTT with liposomes and ≥1.2 for the PL-APTT with Thrombofax and the RWT with Thrombofax or liposomes. Regardless of the phospholipid source in the test system, the PL-APTT demonstrated higher sensitivity and the RWT showed greater specificity in detecting patient plasmas that contained antiphospholipid antibodies.
G4120, an Arg-Gly-Asp Containing Pentapeptide, Enhances Arterial Eversion Graft Recanalization with Recombinant Tissue-Type Plasminogen Activator in Dogs
Authors: Lu Hua Rong , Gold Herman K , Wu Zaomin , Yasuda Tsunehiro , Pauwels Patrick , Rapold Hansjörg J , Napier Mary , Bunting Stuart , Collen Désiré
Published: July 2018

The effects of G4120, a cyclic Arg-Gly-Asp (RGD) containing peptide which inhibits fibrinogen binding to the platelet receptor GPIIb/IIIa, on thrombolysis with recombinant tissue-type plasminogen activator (rt-PA) were investigated in a combined arterial and venous thrombosis model in heparinized dogs. The arterial thrombus model consisted of a 3 cm everted (inside-out) carotid arterial segment inserted into a transsected femoral artery which occludes within 30 min with platelet-rich material and which is resistant to recanalization with 0.5 mg/kg rt-PA. The venous thrombus was a 125I-fibrin labeled whole blood clot produced in the contralateral femoral vein.

In 5 dogs given an intravenous bolus of 0.05 mg/kg G4120 followed by a continuous infusion of 0.05 mg/kg per hour for 3 h (group I), arterial occlusion persisted throughout a 4 h observation period and was still present at 24 h in all dogs; the extent of venous clot lysis after 120 min was 27 ± 7%. In 5 dogs given the same infusion of G4120 in combination with 0.5 mg/kg rt-PA over 60 min, recanalization of the arterial graft occurred in all dogs, within 13 ± 2 min and persisted throughout the observation period of 4 h (p = 0.01 versus G4120 or rt-PA alone); at 24 h, however, all grafts were occluded. Venous clot lysis in this group was 75 ± 8% (p = 0.002 versus G4120 alone andp NS versus rt-PA alone). Pathologic analysis revealed platelet-rich or mixed thrombus with platelet-rich and erythrocyte-rich zones. The last 6 dogs were given a reduced dose of G4120 consisting either of a 0.05 mg/kg bolus followed by an infusion of 0.05 mg/kg over 1 h in 3 dogs (group III) or of a single 0.05 mg/kg bolus in 3 dogs (group IV), both given in combination with 0.5 mg/kg rt-PA infused over 60 min. These protocols produced recanalization within 15 ± 2 and 34 ± 8 min, respectively, which was maintained throughout the 4 h observation period. Venous lysis in these groups was 63 ± 4 and 97 ± 1% respectively. Bleeding times prolonged from 1 to 2 min to >30 min with G4120, but returned towards baseline within 2 h after the end of the infusion. Platelet aggregation with ADP was completely inhibited with G4120 but partially recovered within 1 h after the end of the infusion. No fibrinogen breakdown was observed in association with the rt-PA infusion.

Thus, G4120, a synthetic GPIIb/IIIa receptor antagonist, enhances and accelerates lysis of platelet-rich arterial thrombosis with rt-PA and prevents reocclusion during and within 3 h after the infusion. It may be useful for the conjunctive use with thrombolytic agents in patients with arterial thromboembolic disease.
The Effect of Intravenous Adenosine Diphosphate on the Number of Circulating Platelets in Experimental Animals: Inhibition by Prostaglandin E1, Dipyridamole, SH-869 and VK-774
Authors: Holmes Ian B. , Smith Gordon M. , Freuler Franz
Published: July 2018

The number of circulating platelets was monitored in anaesthetized animals by a continuous flow technique, using a Technicon Autocounter®. Intravenous infusions of adenosine diphosphate (ADP) produced transient, dose-dependent falls in circulating platelet numbers in rabbits, dogs, rats, pigs and squirrel monkeys. The rat was the most sensitive of the species investigated.

In the rabbit, the effect of a submaximal dose of ADP was inhibited in a dose-dependent manner by intravenous infusions of prostaglandin E1 (PGE1), dipyridamole, and two derivatives of dipyridamole (SH-869 and VK-774). The dose-response curves for PGEl, SH-869 and VK-774 were approximately parallel, whereas that for dipyridamole was considerably less steep. PGE1 was the most potent inhibitor, but the duration of action was very short. Dipyridamole and SH-869 produced inhibition of long duration. The duration of action of VK-774 was intermediate.

All inhibitors produced marked and often long-lasting hypotension. The fact that no inhibition of ADP effects could be demonstrated with dibenzyline and hexamethonium, which also produced marked hypotension of long duration, indicated that inhibition of the ADP effect by the four antagonists studied was not due to changes in blood pressure.
In Vivo Labeling of Platelets with 75Se – Selenomethionine in Patients with Hepatic Cirrhosis and Thrombocytopenia.
Authors: Mayer Manfred , Herrmann Ilse , Kempgens Uwe , Queisser Wolfgang
Published: July 2018

Labeling of platelets in vivo by 75Se – Selenomethionine (75Se-M) was performed in nine cases of hepatic cirrhosis and thrombocytopenia for evaluation of the kinetics of platelet maturation. Folic acid and vitamin B12 deficiency was excluded by pretreatment of the patients with these agents. The platelet maturation time – time between the injection of the isotope and maximum radioactivity of separated blood platelets – was shortened to 7.7±1.1 days (mean±SD) compared to the normal of 9.1±1.4 days. For explanation a disturbance of megakaryocyte maturation and/or platelet release from the bone marrow is suggested.
Soluble Fibrin Complexes: Separation as a Function of pH and Characterization
Authors: Benabid Y , Concord E , Suscillon M
Published: July 2018

1. The influence of the pH on the separation of high molecular weight derivatives obtained by a limited action of thrombin on fibrinogen was studied by agarose gel chromatography. When the pH of the elution buffer was 8.5, non crosslinked associations were easily separated in two peaks eluted prior to the fibrinogen peak: one contained a dimer, the other several high polymers. At pH 6.5, only the fibrinogen peak appeared: the fibrinogen molecule proteolysed by thrombin formed no stable associations at this pH and was eluted with the intact fibrinogen molecule. In the presence of factor XIII and Ca++, numerous associations were obtained which are independant of the pH.

2. The polypeptide chain composition of the different species separated at pH 8.5 was studied by SDS-polyacrylamide gel electrophoresis. This technic showed Aα, Bβ and γ chains in the fibrinogen peak, whereas in the chromatographic fractions containing the dimer four bands corresponding to Aα, α, Bβ and γ chains were found. In the peak containing the high polymers, only the presence of α, Bβ and γ chains was demonstrated.

3. These experimental results concerning the effect of pH on the formation of soluble complexes showed that the presence of fibrin monomers in fibrinogen solution was not sufficient to promote any associations. The formation of such derivatives is strongly dependent on the pH of the solution. This obviously can be explained by an influence of the pH either on the ionization of polymerisation sites and the intermolecular bonds between the complex units or on the unmasking of the polymerisation sites by a hypothetical pH induced conformational change of the fibrinogen molecule.
Soluble Fibrin Complexes in Early Fibrin Digests*
Authors: Nanninga Luddo B.
Published: July 2018

Early fibrin digests were analyzed in the ultracentrifuge and were found to contain compounds with molecular weights up to 3 x 106; these compounds are considered to be soluble fibrin complexes. Their similarity with intermediate polymers in inhibited clotting systems has been stressed. Their viscosity at low shear rates is extremely high; therefore, a rapid method to establish their presence in plasma would be the measurement of plasma and serum viscosity at low shear rates. Moreover, they shorten the clotting time (thrombin time) of fibrinogen considerably.
Controlled Trial of the Sequential Use of Streptokinase and Ancrod in the Treatment of Deep Vein Thrombosis of Lower Limb
Authors: Tibbutt D. A , Chesterman C. N , Williams E. W , Faulkner T , Sharp A. A
Published: July 2018

Treatment with streptokinase (‘Kabikinase’) was given to 26 patients with venographically confirmed deep vein thrombosis extending into the popliteal vein or above. Treatment was continued for 4 days and the patients were allocated randomly to oral anticoagulant therapy or a course of treatment with ancrod (‘Arvin’) for 6 days followed by oral anticoagulant therapy. The degree of thrombolysis as judged by further venographic examination at 10 days was not significantly different between the 2 groups. The majority of patients showed clinical improvement but there was no appreciable difference between the groups at 3 and 6 months. Haemorrhagic complications were a more serious problem during the period of treatment with ancrod than during the equivalent period in the control group.
Some Factors Influencing Metabolism and Distribution of Fibrinogen in Man and Rabbits
Authors: Chen Yi-Hsiang , Reeve E. B
Published: July 2018

To shed some light on the homeostatic regulation of plasma fibrinogen, metabolic studies were made in healthy females, and in normal, thyroidectomized, and thyroxine-treated rabbits. In females, compared with normal males, plasma fibrinogen concentration, plasma and interstitial fibrinogen decreased consequent to an increased fractional catabolic rate and a normal fibrinogen synthesis rate. The interstitial/plasma fibrinogen ratio remained unchanged. In normal rabbits, with increasing body weight fractional catabolic rate and catabolic rate decreased, while fibrinogen concentration and plasma fibrinogen remained constant owing to a simultaneous decrease in fibrinogen synthesis. In addition, fractional transcapillary transfer rate and transcapillary flux also decreased resulting in a shrinkage of interstitial fibrinogen. Thyroidectomy and thyroxine-injection markedly altered fibrinogen metabolism: thyroid hormone accelerated fibrinogen catabolism but also stimulated synthesis. The net result was an increase in plasma fibrinogen and fibrinogen concentration. The interstitial/plasma fibrinogen ratio decreased in thyroxine-treated, and increased in thyroidectomized animals. This study defines the variations of the fibrinogen system parameters in these physiologic and pathologic conditions, and illustrates some patterns of alterations in fibrinogen metabolism.
Properties of Washed Human Platelets
Authors: Kinlough-Rathbone Raelene L , Fraser Mustard J , Packham Marian A , Perry Dennis W , Reimers Hans-Joachim , Cazenave Jean-Pierre
Published: July 2018

We have shown previously that washed human platelets resuspended in Tyrode solution containing albumin and apyrase maintain their disc shape and their ability to aggregate upon the addition of low concentrations of ADP, providing fibrinogen is added to the suspending medium. We have now examined their responses to other aggregating and release-inducing agents. Collagen, arachidonate, thrombin, immune serum globulin, the ionophore A 23, 187 and phytohaemagglutinin from Phaseolus vulgaris caused aggregation and release of granule contents. The response to adrenaline was variable. Serotonin caused the platelets to change shape but no aggregation or release occurred. Addition of a small amount of plasma was necessary for ristocetin-induced aggregation. Polylysine caused immediate platelet-to-platelet adherence with little or no release of granule contents. Responses to collagen or thrombin were greater in a modified medium containing magnesium but no calcium; in this medium, aggregation caused by ADP or polylysine was followed by the release of granule contents whereas these agents caused aggregation without release in a medium with both calcium and magnesium. When protein was omitted from the suspending medium, platelet aggregation in response to ADP was variable. In this medium, collagen and thrombin caused more extensive release than in the albumin-containing medium. Aggregation by polylysine was accompanied by release and extensive lysis in the protein-free medium. Thus, the composition of the final resuspending medium has a major effect on the responses of washed human platelets to aggregating agents.
Enzymatic Properties of 5′-AMP Deaminase in Platelet Lysates
Authors: Holmsen H , Østvold A.-C , Pimentel M. A
Published: July 2018

Metabolic ATP is converted to hypoxanthine during platelet secretion, metabolic shock and slowly in the resting state. This conversion involves deamination of 5′-AMP to 5′-IMP which has been studied by incubating 5′-AMP (3–40 μM) with human platelet lysates and quantifying the metabolites formed. Deamination occurred only when EDTA was present or endogenous ATP absent, showing that 5’-AMP deaminase (EC 3.5.4.6) was the only enzyme attacking AMP. EDTA stimulated AMP deamination, probably by removal of endogenous heavy metals which were powerful inhibitors of deamination. The experiments were therefore performed with EDTA. 5′-AMP deaminase was soluble, and had optimal activity at pH 6.5; however, the rate of AMP deamination was highly dependent on the type of buffer used. Km was 0.92 × 10–3M and Vmax was 0.26 μmoles/min x mg protein. The deamination required presence of monovalent cation with Li+ = Na+ >K+ >NH4
+, a sequence distinctly different from that seen with erythrocyte and muscle deaminase. 50 mM Na+ gave maximal activity with [math]. Tris+ and K+ were competitive inhibitors with respect to Na+, a feature not reported for the enzyme from other tissues. Above 60 mM K+ there was no effect by Na+ (0–100 mM). PPi, GTP, ITP, CTP and UTP gave greater inhibition than Pi and phospho-esters. Unlike AMP deaminase from other tissues, the platelet enzyme was insensitive to fluoride. ATP counteracted Pi and GTP inhibition and activated with or without Na+; ATP-and Na+-activation were additive. The activity increased as the adenylate energy charge was lowered, but was linearly related to the AMP concentration, thus in sharp contrast to deaminase from the liver. It is suggested that in the intact platelet AMP deamination is regulated chiefly through variations in the AMP level and not likely through variations in the ATP/Pi ratio.
A Simple and Practical Method for Isolation of an Early Plasmin Degradation Product of Human Fibrinogen
Authors: Takagi Kimiteru , Kawai Tadashi
Published: July 2018

One of the earliest plasmin degradation products of human fibrinogen, so-called fragment A, was isolated by a simple method.

This peptide has a molecular weight of approximately 22,500, migrating electrophoretically at beta-area, and its amino acid composition shows a very high content of glycine, serine, threonine and proline, and a markedly low content of hydrophobic amino acids. This fragment does not react against anti-fibrinogen; however, the anti-serum of this fragment reacts strongly with fibrinogen.
Use of 125I-Fibrinogen Kinetic Data to Detect Disseminated Intravascular Coagulation and Deposition of Fibrin in Patients with Metastatic Cancer
Authors: Robson Elean B , Murawski Gail F , Bettigole Richard E
Published: July 2018

A technique using computerized data handling for following the fate of 125I-fibrinogen through various physiological compartments is presented. Its use in detecting fibrin build up in patients with metastatic tumors and cancer-caused DIC is explained. An increase in j3u (fractional catabolic rate as seen in the urine data) throughout the course of a study was found to be an important indicator of extravascular fibrin build up.
Interlaboratory Oral Anticoagulant Quality Assessment by the Netherlands Federation of Thrombosis Services
Authors: van Dijk-Wierda C. A , Hermans J , Loeliger E. A , Roos J
Published: July 2018

The 50 laboratories of the Netherlands Federation of Thrombosis Services, covering a population of 9 million and responsible for the laboratory control of approximately 150,000 patients under oral anticoagulation, have participated since 1974 in a voluntary external and internal quality control program. The external program comprises a monthly distribution to the member laboratories of a series of artificially prepared control blood samples, two of which are identical. The overall variation of the coagulation times found were 10% (CV) in 1974 and 8% (CV) in 1975. Performance improved rather abruptly at the beginning of 1975, after the application of a tight methodological standardization and improvement by the manufacturer of the thromboplastin preparation (Thrombotest) used by the great majority of the laboratories involved. The main source of variation was found to be random error in the Thrombotest determination, approximating 6%. Interbatch variation of Thrombotest and inter-aliquot variation of control blood samples both do amount to approximately 3% (CV). In terms of rabbit tissue thromboplastins, which have a lower sensitivity than Thrombotest (i. e., a flatter slope of the correlation between the PT and the anticoagulant effect), the total variation in the performance of the Dutch laboratories is 2.2-5.6% (CV), which is unusually low. The main reason for this is the fact that the laboratories can rely not only on the services of the manufacturer but also on a central information office and a reference laboratory responsible for the preparation of the control blood as well as the standardization (calibration) of thromboplastin.
Studies on the Tissue Thromboplastin During the Coagulation-Fibrinolytic Process – Ultrastructural Changes
Authors: Hasegawa Hiroshi , Nagata Hiroshi , Murao Makoto
Published: July 2018

An electron-microscopical study was made on morphological changes in which T-Tbp would undergo during clotting and fibrinolytic process. Morphological appearance of concentrically arranged membrane structure in T-Tbp remained nearly intact during blood coagulation process. T-Tbp, which existed in the sediments following dissolution of fibrin clot by the application of UK, showed an appearance of fine granules adhering to the surface of aggregates of particles through SEM. Through TEM, T-Tbp in the sediments was found to have retained its concentrically arranged membrane structures in most places, while, in some other places the appearance of fused membranes, smaller single vesicles and long sheets of membranes, and the formation of “blebs” etc. were observed. Various morphological changes caused by fibrinolytic substances accompanied the loss in coagulation activities.

Our results showed that coagulation activities of T-Tbp must be completely dependent upon the presence of the membrane structures.
Cytokine Regulation of the Synthesis of Plasminogen Activator Inhibitor-2 by Human Vascular Endothelial Cells
Authors: Zoellner Hans , Wojta Johann , Gallicchio Marisa , McGrath Katherine , Hamilton John A
Published: July 2018

Plasminogen activators are inhibited by plasminogen activator inhibitors-1 (PAI-1) and -2 (PAI-2). We describe the synthesis of PAI-2 by human vascular endothelial cells (EC) cultured from umbilical vein, saphenous vein and foreskin microvasculature in response to interleukin-1α (IL־1α) and tumour necrosis factor α (TNFα) and compare it with that of PAI-1. Both PAI-2 and PAI-1 were quantitated by ELIS As. PAI-2 was cell-associated while PAI-1 was secreted by EC. IL-lα and TNFα increased the synthesis of PAI-2 and PAI-1 by EC in a dose-dependent manner. IL-1α was a stronger stimulus for PAI-2 synthesis than TNFα, while both cytokines were equally effective for PAI-1. Northern blot analysis revealed similar changes in mRNA levels to those in antigen levels. PAI-2 synthesis by cytokine-stimulated EC may be important in thrombus formation and inflammation.
Anticoagulant Activity of Hirulog™, a Direct Thrombin Inhibitor, in Humans
Authors: Fox Irving , Dawson Adrian , Loynds Peter , Eisner Jane , Findlen Kathleen , Levin Elizabeth , Hanson Don , Mant Tim , Wagner John , Maraganore John
Published: July 2018

Hirulog™ (BG8967) is a direct thrombin inhibitor built by rational design using the protein hirudin as a model (Maraganore et al. [1990]; Biochemistry 29: 7095–101). In order to evaluate the therapeutic potential for hirulog in the management of thrombotic disease, the tolerability and anticoagulant activity of the agent were examined in a study of human volunteers.

In a randomized, placebo-controlled study (n = 54), the intravenous infusion of hirulog over 15 min showed a rapid, dose-dependent prolongation of activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT). There was a corresponding dose-dependent increase in plasma hirulog levels. The peptide was rapidly cleared with a half-life of 36 min and a total body clearance rate for the peptide of 0.43 1 kg−1 h−1. Similar activity was observed following subcutaneous injection but with sustained pharmacodynamic and pharmacokinetic behavior. There was a significant correlation between pharmacokinetic and pharmacodynamic variables for both intravenous (r = 0.8, p <0.001) and subcutaneous administration (r = 0.7, p = 0.002).

To evaluate the possible interactions of aspirin on the tolerability and anticoagulant activity of intravenous hirulog, a cross-over design was employed in eight subjects. Aspirin administration did not modify the peptide’s activity. At the administered dose of 0.6 mg kg−1 h−1 for 2 h, hirulog infusion prolonged APTT from 230 to 260% baseline. The infusion of hirulog in subjects who had received aspirin was not associated with any significant changes in the template bleeding time.

The final phase of the study examined the activity and tolerability of hirulog in ten subjects during prolonged intravenous infusions for up to 24 h. The peptide (0.3 mg kg−1 h−1) exhibited sustained anticoagulant activity with no evidence for a cumulative effect. During hirulog infusion, APTT was prolonged from 210 to 250% baseline.

In all phases of the study, hirulog administration was generally well-tolerated.

Our observations show that hirulog is an active antithrombin agent with excellent tolerability in humans. As a direct thrombin inhibitor, hirulog provides a novel approach for the management of thrombotic disease.
Inflammatory and Procoagulant Mediator Interactions in an Experimental Baboon Model of Venous Thrombosis
Authors: Wakefield Thomas W , Greenfield Lazar J , Rolfe Mark W , DeLucia Alphonse , Strieter Robert M , Abrams Gerald D , Kunkel Steven L , Esmon Charles T , Wrobleski Shirley K , Kadell Amy M , Burdick Marie D , Taylor Fletcher B
Published: July 2018

Theoretic and in vitro evidence suggests that thrombosis and inflammation are interrelated. The purpose of the present study was to define the relationship between inflammation and deep venous thrombosis (DVT) in an in vivo model. Initiation of DVT was accomplished by administration of antibody to protein C (HPC4, 2 mg/kg) and tumor necrosis factor (TNF, 150 μg/kg); stasis; and subtle venous catheter injury. Thrombosis was assessed by thrombin-antithrombin assay (TAT), 125I-fibrinogen scanning (scan) over both the proximal and distal iliac veins, and ascending venography. Cytokines TNF, interleukin-6 (IL-6), monocyte chemotactic protein-1 (MCP-1), and interleukin-8 (IL-8) were measured along with differential white blood cell counts, platelet counts, fibrinogen (FIB), and erythrocyte sedimentation rates (ESR). Baboon pairs were sacrificed on day 3 (T + 3d), T + 6d, and T + 9d and veins removed. All animals developed inferior vena cava and left iliofemoral DVT by venography; no right DVT was found. TAT was elevated by T + 1hr and peaked at T + 3hrs. Left iliofemoral DVT was found at T + 1hr by scan and reached a 20% uptake difference between the affected left and nonaffected right side at T + 3hrs. TNF peaked at T + 1hr; MCP-1 peaked at T + 6hrs; IL-8 and IL-6 peaked on T + 2d; all cytokines declined to baseline. TNF and TAT elevations were found to correlate with all cytokines; elevations in IL-8 were correlated with elevations in MCP-1 and IL-6 (p <0.05). Correlation between cytokines and scan revealed a significant (p <0.05) relationship only between elevations in IL-6 and distal iliac fibrin accumulation; no significant correlation was found between IL-8 and MCP-1 and scan. Increased mature polymorphonuclear leukocytes were found by T + 2d; immature forms were prominent at T + 3hrs, T + 6hrs, and T + 2d. Increased monocytes were noted by T + 4d; increased lymphocytes and platelets by T + 8d. ESR and FIB were elevated by T + 3d. Histopathologic study revealed venous inflammation at T + 3d, with beginning thrombus organization by T + 6d. MCP-1 localized to areas of thrombus and phlebitis. The development of DVT in this model involves inflammatory as well as coagulant activity. We conclude that this model allows studies on the role of inflammatory mediators in the development and natural history of DVT.
A Database of Polymorphisms in the von Willebrand Factor Gene and Pseudogene
Authors: Sadler J Evan , Ginsburg David
Published: July 2018

Nucleotide sequence polymorphisms in the von Willebrand factor (vWF) gene are useful for genetic studies in von Willebrand disease (vWD). This database describes 33 known vWF polymorphisms distributed throughout the vWF gene. DNA sequence information is available for 21 of these sites. The most informative system is a tetranucleotide repeat polymorphism in vWF intron 40. Sixteen of these polymorphisms are within vWF exons, and approximately half of them also alter the encoded amino acid sequence. Many occur close to mutations that cause vWD. The high prevalence of vWF polymorphisms must be considered in the analysis of candidate vWD mutations. In addition to the vWF gene on chromosome 12, there is a partial unprocessed vWF pseudogene on chromosome 22 that corresponds to vWF exons 23 to 34. Three polymorphisms have been assigned to the vWF pseudogene. Because the vWF gene and pseudogene have diverged only ∼3.1% in DNA sequence, correct assignment of polymorphisms to either locus can be difficult in the region of homology. This problem has been solved in some cases by comparison of the published sequences and predicted restriction maps for the gene and pseudogene.
Ultraschalluntersuchungen der hirnversorgenden Arterien Bedeutung für Diagnostik und Therapie
Authors: Widder B.
Published: July 2018

Die Dopplersonographie der extrakraniellen hirnversorgenden Arterien hat sich in den vergangenen Jahren als zuverlässige Methode zur Erkennung höhergradiger Stenosen und Verschlüsse bewährt. In Kombination mit der Schnittbild-Untersuchung (Duplex-Scan) können auch geringgradige Stenosen erfaßt werden, und die transkranielle Dopplersonographie erweitert das Untersuchungsspektrum auf die großen Hirnbasisarterien. In jüngster Zeit kommt den Ultraschallmethoden zunehmende Bedeutung bei der Therapieplanung zu: Die Bestimmung der Autoregulationsreserve mit dem Doppler-C02-Test eignet sich zur Erfassung hämodynamisch kritischer Verschlußprozesse der A. carotis, und die Einführung echomorphologischer Parameter bei der DuplexUntersuchung ermöglicht Aussagen über subintimale Blutungen bei Karotisstenosen mit dadurch erhöhtem embolischen Risiko. Weiterhin gibt die transkranielle Dopplersonographie Auskunft über die Funktionsfähigkeit intrakranieller Kollateralen, gibt Hinweise auf das Clamping-Risiko in der Karotischirurgie, und trägt bei Subarachnoidalblutungen durch Verlaufskontrollen des Vasospasmus zu einer individuell angepaßten operativen und konservativen Therapie bei.
Wirkung von Knoblauch (Allium sativum L.), insbesondere rheologische und hämostaseologische Effekte
Authors: Jung F. , Mrowietz C. , Wenzel E. , Kiesewetter H.
Published: July 2018

Es werden die Wirkungen von Knoblauch auf Theologische und hämostaseologische Variablen, Lipide und den Blutdruck sowohl aus eigenen Untersuchungen als auch aus der Literatur zusammengestellt. Erste deutlich dosisabhängige Effekte werden nach Einmaldosen von 300 mg bzw. Tagesdosen von 600 mg gefunden. Der maximale Wirkzeitpunkt liegt zwischen 4 und 6 Stunden nach der Einnahme. Knoblauch reduziert die spontane und die induzierte Thrombozytenaggregation, die Plasmaviskosität, den Hämatokritwert und läßt die fibrinolytische Aktivität, den Durchmesser präkapillärer Arteriolen und die Mikrozirkulation ansteigen. Eine signifikante lipidsenkende Wirkung ist bislang in kontrollierten klinischen Studien mit 423 Patienten belegt. Milde Blutdrucksenkungen sind in mehreren Untersuchungen beobachtet worden. Bei einer täglichen Dosis von 900 mg treten bei bis zu 25% der Patienten knoblauchbedingt Geruchsbelästigungen auf. Darüber hinaus werden von 5% der Patienten gastrointestinale Beschwerden, bei starker Überdosierung Blutdruckabfälle (in seltenen Fällen bis zum Kollaps) angegeben. Ein Wirksamkeitsnachweis im Sinne einer Antiatherosklerosewirkung liegt bisher nicht vor, eine Gehstrekkensteigerung konnte für die periphere arterielle Verschlußkrankheit in einer Pilotuntersuchung gezeigt werden.
Extra-Cranial Bleeding and Other Symptoms due to Low Dose Aspirin and Low Intensity Oral Anticoagulation
Authors: Meade T W , Roderick P J , Brennan P J , Wilkes H C , Kelleher C C
Published: July 2018

Data from the early stages of the thrombosis prevention trial (TPT) have been used to establish and quantify the risk of extracranial bleeding due to low dose aspirin (75 mg) and low intensity oral anticoagulation with warfarin (international normalised ratio, INR, 1.5) singly or in combination, in men aged between 45 and 69 who are at high risk of ischaemic heart disease (IHD). The design of the trial is factorial, the four treatments being combined low dose aspirin and low intensity anticoagulation (WA), low intensity anticoagulation alone (W), low dose aspirin alone (A) and double placebo treatment (P). The trial is being carried out through the Medical Research Council’s General Practice Research Framework, with participating practices throughout the United Kingdom. Results are based on the first 3,667 men entered. The risk of major gastrointestinal bleeding due to active treatment is probably about 1 in 500 man-years of treatment, there currently being no difference between the three active regimes (WA, W, A). Intermediate and minor bleeding episodes occur more frequently with WA than with W or A on their own, the excess being mainly due to minor nose bleeds and bruises. In turn, both W and A on their own cause more such minor episodes than placebo treatment, P. There is no evidence that any of the three active regimes increases the risk of peptic ulceration, nor do they increase reports of indigestion. Aspirin increases reports of constipation and reduces reports of blurred vision. Minor bleeding occurs less frequently in smokers than in non-smokers but is not influenced by age. The antithrombotic regimes used are feasible and acceptable. So far, combined treatment and treatment with warfarin alone are not associated with more frequent serious hazards than low dose aspirin on its own, a regime that is increasingly used in clinical practice. The results provide a measure of reassurance for further trials of low intensity oral anticoagulation with warfarin alone or in combination with low dose aspirin.