bluuebunny/crossref_metadata_2025 huggingface dataset:
Influence of Six Mutations of the Protein C Gene on the Gla Domain Conformation and Calcium Affinity
Authors: Gaussem Pascale , Gandrille Sophie , Duchemin Jérôme , Emmerich Joseph , Alhenc-Gelas Martine , Aillaud Marie-Françoise , Aiach Martine
Published: July 2018

The protein C Gla domain was studied in six families presenting a type II hereditary deficiency characterized by low activity in a coagulation assay and normal activity in an amidolytic assay. Five of these mutations, previously described by our group, affected Arg-5, Arg-1, Arg 229 and Ser 252. We report here the first natural Glu 7 to Asp mutation in a sixth family.

We evaluated the binding of the mutated protein C to Hn, a monoclonal antibody (mAb) known to recognize the sequence Phe 4 to Arg 9 of the Gla domain; the presence of calcium ions suppresses the recognition of this epitope by Hn. Mutation of Arg 229 to Gin and Ser 252 to Asn did not modify the inhibition of protein C binding, whereas the Arg-1 to His mutation resulted in a loss of inhibition in the presence of CaCl2. This suggests that the protein C of this patient shows impaired carboxylation.

The protein C from patients bearing the mutations Arg-5 to Trp, Arg-1 to Cys and Glu 7 to Asp bound poorly to Hn mAb, even in the absence of calcium ions. The calcium affinity of the Gla domain was studied by pseudo-affinity chromatography, in which protein C was successively eluted from a Mono Q column by CaCl210 mM and NaCl 0.6 M. Protein C from the patient bearing the Arg-5 to Asp mutation had a normal elution profile, suggesting that a modification of the propeptide cleavage site impairs the conformation of the Gla domain but not carboxylation. Conversely, the patient bearing the mutation Arg-1 to Cys had an abnormal elution profile, showing that calcium binding affinity was impaired probably due to a defective carboxylation (the behaviour of the Glu 7 to Asp mutant lacking one Gla residue validated our experimental approach).

These results confirm that mutations in the serine protease domain do not modify the conformation of the N-terminal part of the Gla domain. Arg-1 to His, Arg-1 to Cys and Glu 7 to Asp mutations strongly modified affinity for calcium ions, whereas the Arg-5 to Trp mutation probably modifies the conformation of the Gla domain.
Kinetic Aspects of the Interaction of Blood Clotting Enzymes
Authors: Hemker H C , Kop J , Willems G M
Published: July 2018

It is shown that in a system where thrombin acts on fibrinogen, the clotting time can be used to assess coagulation velocity because clotting time and clotting velocity are inversely proportional.
The Effect of Prostacyclin (PGI2) on Platelet Behaviour. Thrombus Formation in Vivo and Bleeding Time
Authors: Ubatuba Fernando B , Moncada Salvador , Vane John R
Published: July 2018

Prostacyclin (PGI2) infused intravenously into anaesthetized rabbits inhibited electrically-induced thrombus formation in the carotid artery, increased bleeding time and inhibited ex vivo platelet aggregation induced by ADP or arachidonic acid. The increase in bleeding time and the inhibition of ex vivo platelet aggregation lasted for as long as the infusion of PGI2 was maintained but rapidly disappeared after infusion was stopped.

Prostacyclin is a more potent inhibitor of platelet function, in vivo than prostaglandin E1 (PGE1) or prostaglandin D2 (PGD2).

The effects of prostacyclin on all parameters studied except blood pressure were potentiated by the concomitant administration of theophylline, a phosphodiesterase inhibitor.
Mode of Action of Ticlopidine in Inhibition of Platelet Aggregation in the Rat
Authors: Ashida Shin-Ichiro , Abiko Yasushi
Published: July 2018

Ticlopidine, when orally administered to rats, resulted in activation of basal and prostaglandin E1 (PGE1)-stimulated adenylate cylase activity through increase in affinity of the cyclase in platelet membrane to PGE1, although it failed to affect adenosine- or sodium fluoride-stimulated activity of the enzyme. In washed platelets, Ticlopidine also activated basal and PGE1-stimulated activity of the cyclase and prevented reduction in the cyclase activity caused by low concentrations of PGE2. Furthermore, Ticlopidine inhibited malondialdehyde formation in platelets induced by thrombin but failed to inhibit that caused by exogenous arachidonic acid. Adenosine 3’,5’-cyclic monophosphate (c-AMP): phosphodiesterase activity of platelet lysate was not significantly affected by Ticlopidine treatment.

These findings indicate that Ticlopidine inhibits platelet aggregation and prostaglandin synthesis from endogenous substrate through activating basal and PGE1-stimulated activity of the cyclase, preventing PGE2-induced depression of the cyclase activity and thus increasing platelet c-AMP level.
Effects of Adenosine Diphosphate, Colchicine and Temperature on Size of Human Platelets
Authors: Laufer N , Grover N B , Ben-Sasson S , Freund H
Published: July 2018

An improved measuring system based on the Coulter principle and developed in our laboratory is used to size human blood platelets.

The mean volume of platelets in 24 healthy subjects is found to be 8.45 μm3 with a standard deviation of 1.07 μm3; the typical size-distribution curve is unimodal and asymmetrical, with a marked skew to the right.

The effects of different reagents on platelet size (shape factor × volume) are evaluated. Platelets increase in size by 23% following suspension in isotonic, phosphate-buffered saline and incubation with 10 μM adenosine diphosphate; no change is observed when the suspending medium is autologous plasma. Cooling the platelets to 0-4° C results in a size increase of 25%; rewarming to 37° C restores them to their initial size within 2 hr. A similar increase occurs when the platelets are incubated with 1-10 mM colchicine.

It is proposed that these reagents, which are known to produce changes in the orientation of the marginal bundle of microtubules, cause platelets to undergo disc-sphere transformations.

Calculations are made which show that such transformations increase platelet size by 27% as measured electrically, and we conclude that the so-called volume changes reported in the literature reflect shape changes only and that no true volume increase actually takes place.
Measurement of the Heparin Neutralizing Capacity of Protamine
Authors: Graham David T , Pomeroy Alan R , Smythe David B
Published: July 2018

Several methods of estimating the heparin neutralizing capacity of protamine were investigated for their reliability and practicability. The results from two chemical methods were compared with those from two in vitro biological assays, one of which was the method of the British Pharmacopoeia (1973). An in vivo method using mice was used to assess the accuracy of the in vitro test methods. Three standard heparin preparations were tested against the W.H.O. 1st International Reference Preparation for Protamine. Two of the heparin preparations were of mucosal origin, one of which was the 3rd International Standard, and the third heparin preparation was of lung origin (the 2nd International Standard for Heparin).

The mean neutralization values (all methods) of heparin by protamine for a House Reference Preparation and the 3rd and 2nd International Standards for Heparin were 95.8, 109.8 and 89.9 units per mg of Protamine, respectively. All the methods read the House Reference Preparation to have a lower value than the 3rd International Standard, which had a higher value than the 2nd International Standard for Heparin. There was a constant relationship between the results of any one method and those of another. The chemical and in vitro biological methods gave results of comparable precision although the latter required a greater degree of technical skill and time to perform.
Factor XII-Dependent Fibrinolysis: A Double Function of Plasma Kallikrein and the Occurrence of a Previously Undescribed Factor XII- and Kallikrein-Dependent Plasminogen Proactivator
Authors: Kluft C , Trumpi-Kalshoven M M , Jie A F H , Veldhuyzen-Stolk E C
Published: July 2018

Fibrinolytic studies in euglobulin fractions of Fletcher trait plasma (deficient in prekallikrein) revealed reduced activities as compared to normal plasma. A quantitative assay for total plasminogen activator plus proactivator in plasma showed that the amount in Fletcher trait patients is about half of normal (normal = ± 100 blood activator units [BAU]/ ml). Plasma kallikrein partially purified in a high and low molecular weight form exerted plasminogen activator activity amounting to 10–15 BAU/ml plasma. So, the absence of kallikrein in the deficient plasma can not fully account for the reduction in activator activity. Additions of kallikrein preparations or normal plasma fractions resulted in additional activator activity in Fletcher trait plasma which was assessed at 30–40 BAU/ml. This activity was assumed to originate from a previously undescribed plasminogen proactivator whose activation is kallikrein- and factor XH-dependent.

Fractionation experiments demonstrated the presence of two major activities and a minor activity caused by kallikrein in normal plasma.

It is concluded that plasma kallikrein has two functions in the generation of factor XII- dependent fibrinolytic activity: one as a direct plasminogen activator and another as a factor in the activation of a major factor XH-dependent plasminogen proactivator.
Abnormalities of Platelet Adenine Nucleotides in Patients with Myeloproliferative Disorders
Authors: Nishimura Junji , Okamoto Seiki , Ibayashi Hiroshi
Published: July 2018

Platelet aggregation and adenine nucleotides in platelets have been studied in thirteen patients with myeloproliferative disorders. ADP induced aggregation was abnormal in two patients, but collagen induced aggregation was impaired in 11 patients. The concentrations of ATP and ADP in resting platelets in the patients with abnormal aggregation were significantly less than those in normal subjects. Marked reduction of the amounts of both nucleotides released into plasma was also observed after stimulation of collagen in these patients. Platelets in the patients with normal functions contained almost normal amounts of adenine nucleotides. We discussed the relationship between platelet dysfunction and adenine nucleotides in platelets of myeloproliferative disorders and concluded that platelet dysfunction was mainly attributable to reduction of releasable ADP.
Outcome of Disseminated Intravascular Coagulation in Relation to the Score when Treatment was Begun
Authors: Wada Hideo , Wakita Yoshihiro , Nakase Tutomu , Shimura Minori , Hiyoyama Katsuyo , Nagaya Shousaburou , Mori Yoshitaka , Shiku Hiroshi ,   the Mie DIC study group
Published: July 2018

We examined 395 patients with disseminated intravascular coagulation (DIC) divided into two groups: non-leukemic and leukemic. In 58% of the patients as a whole, treatment of DIC resulted in complete or partial remission, while exacerbation and death occurred in 31%. The efficacy of DIC treatment in the non-leukemic group was less than that in the leukemic group, indicating that the outcome of DIC depended, in part, on the underlying disease. We examined hemostatic indicators in relation to DIC score: prothrombin time (PT) ratio, FDP, platelet count, and fibrinogen levels were found to be important indicators for the diagnosis of DIC, but not for Pre-DIC. Plasma levels of fibrin-D-dimer, thrombin-antithrombin complex (TAT), and plasmin- plasmin inhibitor complex (PPIC) were significantly increased in pre-DIC. The efficacy of treatment in relation to the DIC score when the treatment was begun showed that greater efficacy was achieved in pre-DIC than in DIC patients. The outcome was poorer with increasing DIC score, suggesting that early diagnosis and early treatment are important. On examining the relationship between outcome and hemostatic indicators, we found that the PT ratio and the levels of antithrombin, plasminogen, PPIC, the PPIC/TAT ratio, and thrombomodulin were related to outcome, suggesting that very high consumption of blood coagulation factors, liver dysfunction, hypofibrinolysis, or organ failure caused a poor outcome. Although the outcome in DIC patients may not depend substantially on plasma levels of TAT and fibrin-D- dimer, we can use these indicators to treat DIC patients at an early stage.
The International and “NIH” Units for Thrombin - How Do They Compare?
Authors: Gaffney Patrick J , Edgell Tracey A
Published: July 2018

The current International Standard (IS) for α-thrombin was established in 1991. It was related in unitage with the 1st IS for Thrombin established in 1975 and contains 100 IU of thrombin activity. The National Institute of Health (NIH) standard (Lot J) is in common use for the calibration of commercial thrombin reagents and this study reports on a comparison of the thrombin units defined by these 2 standards. This study has indicated that one “NIH” unit is equivalent to 1.1 to 1.3 IU of thrombin, depending on the influence of PEG in the assay. A unitage ratio figure of 1.15 was recommended following analysis of data obtained in the presence and absence of PEG in the comparative assays. This was confirmed by amidolytic assay data.
A Monoclonal Antibody against a peptide sequence of Fibrinogen Gamma Chain Acts as an Inhibitor of factor XIII-Mediated Crosslinking of Human Fibrin
Authors: Taubenfeld stephen M , Song Yeqing , Sheng DeQuan , Ball Evelyn L , Matsueda R Gary
Published: July 2018

Recurrent hemorrhage has been reported in humans as a result of acquired antibody inhibitors which interfere with the crosslinking of fibrin by factor XIII. One type of these inhibitors (Type III) prevents activated factor XIII from acting on fibrin. We have generated an anti- fibrin monoclonal antibody, called mAb 4A5, which binds to a peptide sequence at the carboxyl-terminus of human fibrinogen γ-chains. MAb 4A5 acts like a Type III inhibitor and prevents proper factor XHI-mediated crosslinking. Pre-incubation of fibrinogen or pooled human plasma with mAb 4A5, but not mAb D2 (specific for the carboxyl terminus of fibrin α-chains), resulted in clots which are soluble in either 5 M urea or 1% monochloroacetic acid. SDS-PAGE and immunoblotting analysis of these clots confirmed that mAb 4A5 inhibited γ-chain crosslinking in plasma clots and fibrin clots. Results from a factor XIII activity assay demonstrated that biotinylcadaverine cross-linking into fibrin by factor XIII could be inhibited by mAb 4A5 but not mAb D2, arguing that mAb 4A5 acted by binding the crosslinking site of factor XIII. Studies of the immunoreactivity of these mAbs with 12 different animal species showed that the γ-chain epitope recognized by mAb 4A5 was more conserved than the α-chain epitope recognized by mAb D2. The species fibrinogens, recognized by mAb 4A5 in binding assays, also showed impaired crosslinking when mAb 4A5 was present during the clotting reaction.
Human Anti-Streptokinase Antibodies Induce Platelet Aggregation in an Fc Receptor (CD32) Dependent Manner
Authors: Lebrazi Jamal , Helft Gérard , Abdelouahed Mustapha , Elalamy Ismaïl , Mirshahi Massoud , Samama Michel Meyer , Lecompte Thomas
Published: July 2018

Exposure to streptokinase (SK) elicits anti-SK antibodies (Abs), which inhibit fibrinolysis and induce platelet aggregation. The mechanism of the latter is not fully understood, although it seems to involve platelet binding by a plasminogen streptokinase and anti-SK ternary complex. Anti-SK Abs were purified by affinity chromatography from serum of patients having received SK for acute myocardial infarction (AMI), and were shown to be of the IgG type. Their effects were studied with (i) human platelets in citrated plasma in the presence of SK or acetylated plasminogen-SK activator complex (APSAC), and (ii) in washed platelets, resuspended in Tyrode buffer after lowering the ionic strength, in the presence of APSAC (which provides both SK and plasminogen). An antibody concentration-response curve was obtained, showing a plateau in the presence of 0.1 mg/ml IgG. By increasing the concentration of APSAC, we obtained a unimodal response curve, the optimal concentration of APSAC being 0.05 U/ml. Aggregation was suppressed by chelating calcium with EDTA, blocking fibrinogen binding by the synthetic peptide Arg-Gly-Asp-Ser (RGDS), and raising intraplatelet cAMP with Iloprost (a prostacyclin analogue). Aggregation required the interaction of the anti-SK Ab Fc domain with the platelet Fc-gamma receptor type II, also known as CD32, since: (i) it was blocked by the monoclonal antibody IV-3 directed against CD32, (ii) it did not occur with F(ab)’2 fragments, which block the response to the intact IgG. The clinical relevance of these platelet-activating anti-SK antibodies remains to be determined. Two factors might influence clinical outcome: (i) the amount and type of pre-existing anti-SK Abs; (ii) the known interindividual variability of the platelet response to binding and activation by IgG involving the CD32 molecule.
Comparison of Six Commercial Plasma References for Factor VIII, Factor IX and von Willebrand Factor
Authors: Kasper Carol K , Aronson David L , Davignon George , Foster Paul , Hillman-Wiseman Carol , Lusher Jeanne M , Manco-Johnson Marilyn , Meade James B , Montgomery Robert R , Rickies Frederick R , White Gilbert C
Published: July 2018

Six brands of normal reference plasma produced in the United States, with assigned assay values for factor VIII and IX and, in four instances, ristocetin cofactor and von Willebrand antigen, were assayed in nine coagulation laboratories in academic institutions in the same country. Differences in mean assays of reference plasmas, as a percent of labelled potency, were significant and were greater than differences among laboratories. Standard methods of assigning potency to commercial reference plasmas are recommended.
Influence of Various Heparin Preparations on Lipoproteins in Hemodialysis Patients: A Multicentre Study
Authors: Kronenberg Florian , König Paul , Neyer Ulrich , Auinger Martin , Pribasnig Andreas , Meisl Thomas , Pinter Georg , Dieplinger Hans
Published: July 2018

Recent studies have indicated controversial effects of low molecular weight heparin (LMWH) on lipid metabolism in patients on chronic hemodialysis as compared to unfractionated heparin (UFH).

We therefore conducted a cross-sectional multicentre study comparing 153 patients treated with LMWH and 153 patients with UFH, matched for sex, age and diabetes mellitus. Both groups have been treated with LMWH or UFH for six months or longer (14.9 vs.23.4 months). We observed no differences between the UFH and LMWH treatment groups for total cholesterol, LDL cholesterol, triglycerides, apoB, apoA-IV or Lp(a). The only significant differences were seen for HDL cholesterol and the corresponding apolipoprotein apoA-I, which were significantly higher in the UFH group (HDL cholesterol: 0.97 ± 0.35 mM/l vs. 0.87 ± 0.37 mM/l, p<0.05; apoA-I 1.23 ± 0.27 g/l vs. 1.15 ± 0.27 g/l, p <0.05).

We conclude that the results of studies investigating the influence of LMWH on lipid metabolism are as heterogeneous as the substances themselves. This challenges the beneficial influence supposedly had by LMWH preparations on lipid metabolism.
The Factor V Leiden Mutation which Predisposes to Thrombosis Is Not Common in Patients with Antiphospholipid Syndrome
Authors: Dizon-Townson Donna , Hutchison Christopher , Silver Robert , Branch D Ware , Ward Kenneth
Published: July 2018

Antiphospholipid syndrome is associated with venous, arterial, and placental thrombosis, possibly through autoantibody impairment of phospholipid-dependent protein C activation. Recently, a missense mutation in the factor V gene (1691 G → A) has been identified that results in an abnormal factor V product (1). This mutation, known as the Leiden mutation, causes an amino acid substitution of glutamine for arginine at position 506 in the factor V molecule and renders the protein resistant to proteolytic inactivation by activated protein C and thus predisposes to thrombosis (2, 3). We hypothesized that some individuals with antiphospholipid syndrome may also carry the Leiden mutation, and thus have a “second hit” predisposition to thrombosis. To test this hypothesis, allele-specific hybridization and allele-specific restriction analysis were used to test for the Leiden mutation in thirty women with the antiphospholipid syndrome, 10 of whom had a history of thrombosis. None of the women were heterozygous or homozygous for the factor V mutation. We conclude that the presence of the factor V Leiden mutation is not a prerequisite for the thrombotic events in patients with antiphospholipid syndrome, due to the occurrence of thrombosis seen in patients lacking the factor V mutation.
The Efficacy and Safety of Oral Anticoagulation in Patients with Cancer
Authors: Bona Robert D , Sivjee Khalil Y , Hickey Amy D , Wallace Donna M , Wajcs Selby B
Published: July 2018

Objectives: To compare the complication rate (bleeding and thrombosis) of oral anticoagulation in a cohort of patients with cancer to a cohort without cancer

Design: Prospective cohort study

Setting: Outpatient anticoagulation clinic in a community hospital

Patients: Consecutive patients enrolled in an anticoagulation clinic: 44 with cancer, 64 without cancer

Interventions: Patients received prophylactic doses of Warfarin (target INR 2–3 in the majority of instances) and complication rates were assessed

Measurements: Major bleeding (strictly defined), minor bleeding, recurrent thrombosis, proportion of time with therapeutic INR, frequency of clinic visits

Results: The rates of major bleeding, minor bleeding, and recurrent thrombosis were not statistically significantly different in the two groups of patients. Therapeutic INR’s were more difficult to sustain in the cancer patients as compared to the non-cancer patients (43.3% vs 56.9%, p <0.0001). There was a non significant trend towards more frequent monitoring for the cancer patients compared with the noncancer patients (4.6 vs 3.5 visits per treatment month, p = 0.14)

Conclusions: Oral anticoagulation is safe and effective in the patient with cancer. It is more difficult to sustain a therapeutic INR in the cancer patients and they may need more frequent monitoring to achieve a low complication rate
Inversion of Intron 22 in Isolated Cases of Severe Hemophilia A
Authors: Baiget Montserrat , Tizzano Eduardo F , Domènech Montserrat
Published: July 2018

Inversion involving intron 22 is the commonest type of mutation causing severe hemophilia A (HA). We investigated 15 families with isolated cases and 5 families with two or three brothers as the only affected members with hemophilia A, in order to determine the carrier status of the mothers, the origin of the mutation and the presence of germinal mosaicism. Our results show that all mothers tested were carriers of the inversion. In addition, three families whose unique hemophilic member was not available for analysis, were screened for the inversion. In one of these last families, the mother was diagnosed as a carrier and her sister and her niece as non-carriers. DNA haplotype analysis in 8 families with grandparents available for study demonstrated that the inversion originated almost exclusively in male germ cells. These findings have important relevance for genetic counselling in families with an isolated case or to exclude germinal mosaicism. Inversion analysis should constitute the first step in molecular diagnosis of severe hemophilia A.
Occlusive Thrombus Formation on Indwelling Catheters: In Vitro Investigation and Computational Analysis
Authors: Friedrich Peter , Reininger Armin J
Published: July 2018

To determine the hydrodynamic parameters involved in thrombus formation on indwelling catheters, fibrin coagulation was investigated in an in vitro model and compared with the prevailing flow field. Computational analysis was used to simulate clot growth and identify the variables which determine its localization. The fibrin clot grew from the distal end of the catheter along the interface between the bulk stream and the annular vortex located immediately downstream of the catheter tip, finally filling the entire vortex. The calculations of flow parameters for three different growth stages showed that regions of clot formation always correlated with regions exhibiting the longest residence time. Fibrin clotting could be simulated by a mathematical model using residence time as the only variable parameter. Our results demonstrate that the residence time is the single most important flow factor for fibrin coagulation and thus probably for thrombus formation. When the residence time equals the clotting time for the fluid, thrombus formation is initiated. Conversely, it ceases when the area of long fluid residence times is replaced by a fibrin clot. Hence the fibrin clot represents a hydrodynamic optimization of the previously disturbed flow.
The Role of Fibrinolysis in the Pathogenesis of the Haemorrhagic Syndrome Produced by Virulent Isolates of African Swine Fever Virus
Authors: Villeda C J , Gómez-Villamandos J C , Williams S M , Hervás J , Wilkinson P J , Viñuela E
Published: July 2018

The activity of several proteins involved in fibrinolysis and the morphological changes in the blood vessel walls of pigs infected with highly virulent (Malawi’83) and moderately virulent (Dominican Republic ‘78-DR’78) ASF virus isolates were determined. Pigs infected with the Malawi’83 virus developed an increased fibrinolytic activity due to high plasma levels of tissue-plasminogen activator (t-PA) of 71.3 ± 22.8 IU/ml (mean ± SD), which correlated well with an increased activation of interstitial capillary endothelial cells and high levels of 1150 ± 73.6 nM of fibrin monomer in the circulation. Animals infected with DR’78 virus, in contrast, showed an inhibition of fibrinolysis in the late stages of disease with almost a 5-fold increase of plasminogen activator inhibitor (PAI) activity of 196.0 AU/ml. These results suggest that activation of the fibrinolytic system in pigs infected with the Malawi’83 virus is probably due to increased formation and deposition of fibrin in the circulation, contributing to an increased bleeding tendency and higher mortality. On the contrary, animals infected with DR’78 virus developed an inhibition of fibrinolysis and thus a reduction in bleeding.
Effects of Two Different Doses of Hirudin on APTT, Determined with Eight Different Reagents
Authors: Monreal Manuel , Monreal Luis , de Gopegui Rafael Ruiz , Espada Yvonne , Angles Ana Maria , Monasterio Jasone
Published: July 2018

The APTT has been considered the most suitable candidate to monitor the anticoagulant activity of hirudin. However, its use is hampered by problems of standardization, which make the results heavily dependent on the responsiveness of the reagent used. Our aim was to investigate if this different responsiveness of different reagents when added in vitro is to be confirmed in an ex vivo study.

Two different doses of r-hirudin (CGP 39393), 0.3 mg/kg and 1 mg/kg, were administered subcutaneously to 20 New Zealand male rabbits, and the differences in prolongation of APTT 2 and 12 h later were compared, using 8 widely used commercial reagents. All groups exhibited a significant prolongation of APTT 2 h after sc administration of hirudin, both at low and high doses. But this prolongation persisted 12 h later only when the PTTa reagent (Boehringer Mannheim) was used. In general, hirudin prolonged the APTT most with the silica- based reagents.

In a further study, we compared the same APTT reagents in an in vitro study in which normal pooled plasma was mixed with increasing amount of hirudin. We failed to confirm a higher sensitivity for silica- containing reagents. Thus, we conclude that subcutaneous administration of hirudin prolongs the APTT most with the silica-based reagents, but this effect is exclusive for the ex vivo model.
The Nijmegen Modification of the Bethesda Assay for Factor VIII:C Inhibitors: Improved Specificity and Reliability
Authors: Verbruggen Bert , Novakova Irena , Wessels Hans , Boezeman Jan , van den Berg Marijke , Mauser-Bunschoten Eveline
Published: July 2018

Antibodies against factor VIII coagulant activity can appear in haemophiliacs who are treated with factor VIII preparations but also spontaneously in non-haemophiliacs. The Bethesda assay is the most commonly used method to detect these antibodies, but it lacks specificity especially in the lower range resulting in unreliable data.

Two modifications are proposed and tested to resolve the imperfections:

1. Buffering the normal plasma used in the assay- and control mixture with 0.1 M imidazole to pH 7.4.

Replacing the imidazole buffer in the control mixture by immunodepleted factor VIII deficient plasma.

These modifications allow better discrimination between positive and negative samples and improve reliability.
Limited Proteolysis of Human α-Thrombin by Urokinase Yields a Non-Clotting Enzyme
Authors: Bezeaud Annie , de Raucourt Emmanuelle , Miyata Toshiyuki , Bouton Marie-Christine , Angles-Cano Eduardo , Guillin Marie-Claude
Published: July 2018

Limited proteolysis of human α-thrombin by various proteases has been efficiently used to demonstrate the importance of two insertion loops located on the surface of this molecule. In the present study, we demonstrate that two-chain urokinase (tcu-PA) specifically cleaves the B chain of α-thrombin giving rise to a transient derivative, consisting of two non-covalently linked subunits. Although the thrombin derivative conserves its activity towards the synthetic substrate S-2238 (Km = 8.4 μM and kcat = 145 s-1 versus respectively 4.5 μM and 149 s-1 for α-thrombin), most of its coagulant activity is lost (140 NIH u/mg versus 3000 NIH u/mg) and its ability to activate platelets is considerably reduced (threshold for full platelet aggregation 2.5 nM versus 0.25 nM). The thrombin fragments were separated by HPLC and after reduction and S-carboxyamidemethylation were digested with a lysylendopeptidase; the resulting peptides were separated by HPLC and sequenced. One fragment corresponded to B chain fragment 1-73 and the second to B chain fragment 74-259 covalently linked to the A chain, indicating that tcu-PA cleaves selectively the peptide bond Arg 73-Asn 74 in the B chain. The proteolytic derivative obtained, designated βu-thrombin, is therefore identical to the transient proteolytic derivative, αt-thrombin, produced by trypsin. Prolonged incubation with tcu-PA resulted in further conversion in a derivative analogous to γt-thrombin.

These results show that the interactions of α-thrombin and u-PA are not limited to the inactivation of scu-PA by α-thrombin previously reported by other groups, but that reciprocally tcu-PA, the fully active form of scu-PA, may induce the loss of α-thrombin clotting and platelet stimulating activities. Furthermore, these data underscore the functional importance of the integrity of the peptide bond Arg 73-Asn 74 of the B chain on the recognition of macromolecular substrates by α-thrombin.
Protein Tyrosine Kinase Activation Is Required for LPS and PMA Induction of Tissue Factor mRNA in Human Blood Monocytes
Authors: Ternisien C , Ollivier V , Khechai F , Ramani M , Hakim J , de Prost D
Published: July 2018

Tissue factor (TF) is a transmembrane glycoprotein which assembles with factor VIIa on cell surfaces to form a proteolytically active cofactor-enzyme complex; the TF/VIIa complex initiates the coagulation protease cascade. In response to bacterial lipopolysaccharide (LPS) and phorbol-12 myristate 13-acetate (PMA), monocytes synthesize and express TF on their surface. However, the mechanisms by which LPS and PMA activate TF synthesis by human blood monocytes are not fully understood. As it has been established that LPS and PMA activate protein tyrosine kinase (PTK) in monocytes, we studied the role of PTK in LPS and PMA induction of TF by human blood monocytes. Both LPS-and PMA-induced TF activity was inhibited in a concentration-dependent manner by the protein tyrosine kinase-specific inhibitors herbimycin A and genistein. TF antigen determination confirmed that LPS- and PMA-induced cell surface TF protein levels decreased in parallel to TF functional activity under herbimycin A and genistein treatment. Northern blot analysis of total RNA from LPS- and PMA-stimulated monocytes showed a concentration-dependent decrease in TF mRNA levels in response to herbimycin A and genistein. The rate of decay of LPS-induced TF mRNA, evaluated after the arrest of transcription by actinomycin D was not affected by genistein and herbimycin A, suggesting that the inhibitory effects occur at least partly at the transcriptional level. We conclude that LPS- and PMA-induced TF production by human monocytes is dependent on tyrosine kinase activation.
Thrombotic Status Analyser
Authors: Gorog Diana A , Kovacs Iren B
Published: July 2018

A physiologically relevant global in vitro test is described which allows the overall assessment of both thrombotic and thrombolytic activities of blood. In principle, native blood is drawn in pulses through a capillary tube where haemodynamic forces induce a platelet reaction culminating in vessel occlusion. Dislodgement/disintegration of the stabilised thrombus under pressure is a reflection of thrombolysis. Evidence is presented for the platelet-rich nature of the occlusion and that disruption of the thrombus and re-established patency is the result of thrombolysis, that is fibrinolysis with significant contribution from platelets.

This test sensitively detects hypercoagulability (stasis); platelet hyperreactivity (coronary artery disease); anti-platelet effect (aspirin, prostacyclin) and the thrombolytic effect of, thrombin generation by, and resistance to streptokinase. Therefore, this overall assessment of thrombotic status could be of great diagnostic and therapeutic benefit in clinical practice.
A Structural/Functional Domain on Human CD36 Is Involved in the Binding of Anti-Nakα Antibodies
Authors: Daviet Laurent , Morel-Kopp Marie-Christine , Kaplan Cécile , McGregor John L
Published: July 2018

The human CD36 antigen is an integral membrane glycoprotein expressed by platelets, monocytes, endothelial cells and various tumor cell lines. CD36 acts as a receptor for thrombospondin, collagen, Plasmodium falciparum-infected erythrocytes and oxidized low- density lipoprotein. Individuals possessing the Naka-negative phenotype do not express CD36 and risk developing anti-CD36 isoantibodies upon blood transfusion or during pregnancy. In the present study, we have examined the interaction of an anti-Naka serum with recombinantly expressed CD36. Results obtained show that five functional CD36 monoclonal antibodies (OKM5, FA6-152, L103, ESIV-C7 and 10/5) prevent the binding of the anti-Naka serum whereas a single monoclonal antibody (13/10) has no effect. Consistent with this result, an epitope map of CD36 generated using cross-blocking experiments, indicates that the inhibitory monoclonal antibodies recognize closely- related epitopes whereas 13/10 reacts with a distinct CD36 determinant. Furthermore, we have demonstrated, in a recent study, that OKM5, FA6-152, L103 and 10/5 bind to the same CD36 domain defined by amino acids 155 to 183. Taken together, our results indicate that the 155-183 sequence is important for the binding of the anti-Naka serum to CD36 and may represent a surface-exposed, immunogenic and presumably functional region on human CD36.
Differences in the Interactions of Lupus Anticoagulant IgG with Human Prothrombin and Bovine Prothrombin
Authors: Mohan Rao L Vijaya , Hoang An D , Rapaport Samuel I
Published: July 2018

Lupus anticoagulant (LA) IgGs have been reported to inhibit more effectively and consistently the Xa/Va/phospholipid complex-catalyzed activation of human prothrombin than the Xa/Va/phospholipid complex-catalyzed activation of bovine prothrombin. This led us to carry out studies to determine whether the ability to inhibit the activation of prothrombin of LA IgGs, separated from the plasma of 15 patients by protein A affinity chromatography, could be related to the ability of the LA IgGs to bind to prothrombin under various experimental conditions. Of 14 LA IgG preparations tested all prolonged to a variable but substantial extent the dilute Russell’s viper venom time (dRVVT) of human plasma but only minimally prolonged the dRVVT of bovine plasma. In a purified prothrombin activation system with a rate limiting concentration of phospholipid, all 15 LA IgG preparations inhibited the activation of human prothrombin with the majority showing >50% of inhibition. In contrast, only one LA IgG markedly inhibited (>50%) the activation of bovine prothrombin and five others moderately inhibited (25-40%) the activation of bovine prothrombin. Nevertheless, the majority of LA IgG preparations bound to immobilized bovine prothrombin on a Western blot and also to immobilized bovine prothrombin on a microtiter well. In an ELISA in which phosphatidylserine (PS) was immobilized on microtiter wells, bovine prothrombin supported the binding of 10 of 15 LA IgG preparations to PS. However, the extent of binding was lower than that observed with human prothrombin. In experiments with 125I-human prothrombin or 125I-bovine prothrombin in a solution containing Ca2+, the addition of PS/PC vesicles enhanced the binding of both human and bovine prothrombin to some LA IgG preparations. The enhanced binding was particularly evident for bovine prothrombin. Although seemingly related for some preparations, the ability of a LA IgG to bind to bovine prothrombin, either in the presence or absence of PS, and the ability of that LA IgG to inhibit the activation of bovine prothrombin was not consistently related for all preparations.
The Thrombin Activation Pathway Modulates the Assembly, Structure and Lysis of Human Plasma Clots In Vitro
Authors: Torbet Jim
Published: July 2018

Thrombin activation of the soluble plasma protein fibrinogen is vital for successful haemostasis. Thrombin is generated from prothrombin by the prothrombinase complex which also includes factor Xa, factor Va, Ca2+ and a procoagulant membrane surface. Factor X activation is catalysed in a complex including either factor Vila and tissue factor, or factor IXa and factor Villa. Factor IXa can be generated either by the factor Vlla/tissue factor complex or by factor XIa which is in turn produced by the contact phase reactions in vitro. Once activated, fibrinogen develops into the fibrin polymeric matrix at the site of injury. It is not known to what extent the properties of this haemostatic plug are sensitive to the pathway leading up to thrombin generation. Here static human plasma is studied in vitro using magnetically induced birefringence. It is shown that the contact phase/factor XIa pathway gives rise to linear fibrin assembly progress curves whereas the factor Vlla/tissue factor activation of factor X provokes largely sigmoid assembly. The latter pathway also causes the formation of significantly thicker fibres even although assembly is more rapid. This result is the inverse of that anticipated from the study of simple model systems. Whilst the streptokinase activated lysis of both types of clot exhibits similar biphasic kinetics, an exponential main phase followed by a sigmoidal tailing off, the data suggest that clots produced by the contact phase/factor XIa pathway are more recalcitrant to lysis. These results demonstrate that the profile of thrombin generation not only determines the kinetics of assembly but also influences the rate of lysis and structure of the haemostatic plug.
Soluble Thrombomodulin Purified from Human Urine Exhibits a Potent Anticoagulant Effect In Vitro and In Vivo
Authors: Takahashi Yasuo , Hosaka Yoshitaka , Niina Hiromi , Nagasawa Katsuaki , Naotsuka Masaaki , Sakai Kiyoshi , Uemura Akio
Published: July 2018

We examined the anticoagulant activity of two major molecules of soluble thrombomodulin purified from human urine. The apparent molecular weights of these urinary thrombomodulins (UTMs) were 72,000 and 79,000, respectively. Both UTMs showed more potent cofactor activity for protein C activation [specific activity >5,000 thrombomodulin units (TMU)/mg] than human placental thrombomodulin (2,180 TMU/mg) and rabbit lung thrombomodulin (1,980 TMU/mg). The UTMs prolonged thrombin-induced fibrinogen clotting time (>1 TMU/ml), APTT (>5 TMU/ml), TT (>5 TMU/ml) and PT (>40 TMU/ml) in a dose-dependent fashion. These effects appeared in the concentration range of soluble thrombomodulins present in human plasma and urine. In the rat DIC model induced by thromboplastin, administration of UTMs by infusion (300-3,000 TMU/kg) restored the hematological abnormalities derived from DIC in a dose-dependent fashion. These results demonstrate that UTMs exhibit potent anticoagulant and antithrombotic activities, and could play a physiologically important role in microcirculation.
Regulation of Platelet Aggregation by Post-fibrinogen Binding Events
Authors: Peerschke Ellinor I
Published: July 2018

A variety of data suggest that fibrinogen binding is necessary but not sufficient for platelet aggregation: post fibrinogen binding events may play an important role. The present study compared fibrinogen binding and platelet aggregation in response to dithiothreitol (DTT) and ADP. DTT induced saturable and specific fibrinogen binding (Kd 0.07 + 0.02 μM, Bmax 15,000 + 3000 molecules/platelet) which supported complete platelet aggregation as determined by single platelet counting. The aggregates were small, however, and more readily dissociated by EDTA than their ADP-treated counterparts, despite quantitatively similar fibrinogen binding. Unlike fibrinogen bound to ADP-stimulated platelets, fibrinogen bound to DTT-treated platelets remained sensitive to dissociation by EDTA over a 3 h time course, retained its ability to support aggregation, even when aggregation was induced 60 min after the initial platelet exposure to fibrinogen, and remained accessible to polyclonal antibodies and plasmin. Confocal scanning laser microscopy showed only surface clustering of fibrinogen bound to DTT-treated platelets over the 3 h time course compared to rapid fibrinogen clearing from the surface of ADP-stimulated platelets. These data suggest that post fibrinogen binding events involved in the stabilization of fibrinogen binding and/or the redistribution of bound fibrinogen may play important roles in regulating platelet aggregation.
Vessel Wall Injury and Arterial Thrombosis Induced by a Photochemical Reaction
Authors: Saniabadi Abby R , Umemura Kazuo , Matsumoto Nobuteru , Sakuma Sadayuki , Nakashima Mitsuyoshi
Published: July 2018

Arterial thrombosis may be initiated in an experimental animal by a photochemical reaction between transmural green light and i.v. administered Rose Bengal, a photosensitizer dye. In this study, scanning electron microscopy has been used to reveal the nature of vessel injury and the cellular composition of the photochemically induced thrombus. A 5 mm segment of the guinea pig femoral artery was occluded by a thrombus about 10 min after irradiation with green light in the presence of systemically administered Rose Bengal. Electron microscopy revealed that following photochemical reaction, endothelial cells first contract and, with further irradiation, become detached from the vessel wall, with their cell membrane being destroyed at the irradiated site where an occlusive platelet-rich thrombus was formed. Endothelial cell injury and vessel occlusion could be completely inhibited by the aminothiol, DL-cysteine administered i.v. 1 min after Rose Bengal. The mechanism of endothelial injury in this model appears to be by singlet molecular oxygen, 1O2 formed by energy transfer from the photo-excited dye to O2.
Surgery of a Posterior Communicating Artery Aneurysm with Fetal-Type Circulation
Authors: González Nicolás , Morales Manuel , Ravera Franco , Ruiz-aburto Arturo , Vásquez Juan , Muller José , Mosquera Jhon , Zapata Rodrigo
Published: July 2018

Inadvertent occlusion of a fetal-type posterior communicating artery in aneurysm surgery could result in posterior circulation infarction and neurological morbidity. The case of a patient with an unruptured posterior communicating artery aneurysm with lateral projection and a fetal-type posterior communicating artery is presented. The utility of the carotid-oculomotor window as a surgical corridor to safely find the fetal-type posterior communicating artery is discussed.
Zum Umgang mit kritischen Ereignissen in der Balintgruppe – ein Werkstattbericht
Authors: Langewitz Wolf , Amann Michael , Emrich Manfred , Guillaumier Colette , Leggemann Hanne , Winkelmann Daniel
Published: July 2018

Es wird über die Erfahrungen einer Leitergruppe berichtet, die an der letzten Balintwoche in Sils (www.sils-balintwoche.ch) mithilfe von Critical Incident Protokollen (CIP) über konkrete Erfahrungen mit Leiterinterventionen diskutiert hat. Dabei ging es um Momente, in denen Interventionen der Leitung als kritisch erlebt oder vermisst wurden. In den CIP wurden Situationen beschrieben, die den Teilnehmenden in lebhafter Erinnerung geblieben waren, entweder als Leiter einer Gruppe oder als ‚Geleiteter‘. In einem ersten Analyseschritt liessen sich die wesentlichen Probleme in der Leitung der Anfangsphase einer Gruppenarbeit (Fallvorstellung und erste Reaktionen der Teilnehmer) und dem Mittelteil (Arbeiten mit den Assoziationen und Kommentaren der Gruppenmitglieder) zuordnen. Im nächsten Schritt wurden mögliche Leiterinterventionen im Rollenspiel erprobt. Als wesentliches Charakteristikum der Balintarbeit wurde der Umgang mit nicht deterministischem Material, das sich nicht ohne Weiteres kategorial einordnen lässt, herausgearbeitet. Der Leiter hat daher die Aufgabe, vorzeitigen Festlegungen und Interpretationen entgegenzuwirken. Dazu gehört, zunächst und sorgfältig Wahrnehmungen am eigenen Leib entgegenzunehmen (z. B. Kältegefühl, Schwere, Wunsch, tief Luft zu holen), bevor diese in eigentliche Gefühle (z. B. Angst, Trauer) ‚übersetzt‘ und diese dann einzelnen Personen zugeschrieben werden. Hilfreiche Leiterinterventionen stammen aus dem Repertoire einer Gesprächsführung, die den diskursiven Raum offenhält (z. B. Spiegeln, Reflecting Back). Der richtige Zeitpunkt für Leiterinterventionen wurde als ein Moment der Überraschung identifiziert, wenn ein Votum etwas völlig Neues anspricht oder wenn ein widersprüchliches Votum im Gruppenprozess untergeht. Theoretisch einordnen liess sich die Auseinandersetzung mit dem Vagen und Unbestimmten und dennoch unendlich Reichen am ehesten mit dem Begriff der Situation aus der Neuen Phänomenologie, die vom Philosophen Hermann Schmitz entwickelt wurde
Extending the dynamic range of microchannel plate detectors using charge-integration-based counting
Authors: Gershman Daniel J. , Avanov Levon A. , Chornay Dennis J. , Rager Amy C. , Pollock Craig J. , Grubbs Guy , Mackler David A. , Tucker Corey J. , Paschalidis Nikolaos P.
Published: July 2018

Microchannel plate (MCP) detectors provide a mechanism to produce a measureable current pulse (∼0.1 mA over several nanoseconds) when stimulated by a single incident particle or photon. Reductions of the device’s amplification factor (i.e., gain) due to high incident particle flux can lead to significant degradation of detection system performance. Here we develop a parameterized model for the variation of MCP gain with incident flux. This model provides a framework with which to quantify the limits of high-flux MCP operation. We then compare the predictions of this model to laboratory measurements of an MCP’s response to a pulsed charged particle beam. Finally, we demonstrate that through integration of the MCP output current in pulsed operation, effective count rates up to ∼1 GHz can be achieved, more than an order of magnitude increase over conventional counting techniques used for spaceflight applications.