bluuebunny/crossref_metadata_2025_split huggingface dataset:
Recombinant Human Soluble Thrombomodulin Delivers Bounded Thrombin to Antithrombin III: Thrombomodulin Associates with Free Thrombin and Is Recycled to Activate Protein C
Authors: Aritomi Masaharu , Watanabe Naoko , Ohishi Rika , Gomi Komakazu , Kiyota Takao , Yamamoto Shuji , Ishida Torao , Maruyama Ikuro 
Published: July 2018

SummaryRecombinant human soluble thrombomodulin (rhs-TM), having no transmembrane domain or chondroitin sulfate, was expressed in Chinese hamster ovary cells. Interactions between rhs-TM, thrombin (Th), protein C (PC) and antithrombin III (ATIII) were studied. Equilibrium between rhs-TM and Th had no detectable time lag in clotting inhibition (K
d = 26 nM) or PC activation (K
d = 22 nM), while ATIII inhibited Th at a bimolecular rate constant = 5,200 M-1s-1 (K
d <0.2 nM). A mixture of ATIII, Th and rhs-TM showed that ATIII reacted with Th slower than rhs-TM, whose presence did not affect the reaction between ATIII and Th. In a mixture of rhs-TM, ATIII and PC, the repeated addition of Th caused the repeated activation of PC; which was consistent with the Simulation based on the assumption that rhs-TM is recycled as a Th cofactor. From these results, we concluded that upon inhibition of the rhs-TM-Th complex by ATIII, rhs-TM is released to recombine with free Th and begins to activate PC, while the Th-ATIII complex does not affect rhs-TM-Th equilibrium.
Evidence that the Protein C Activation Pathway Amplifies the lnhibition of Thrombin Generation by Recombinant Human Thrombomodulin in Plasma
Authors: ohishi Rika , watanabe Naoko , Aritomi Masaharu , Gomi Komakazu , Kiyota Takao , Yamamoto Shuji , lshida Torao , Maruyama Ikuro 
Published: July 2018

SummaryThrombomodulin (TM) is a cofactor for the thrombin-catalyzed activation of anticoagulant protein C. However, we have no evidence that thrombomodulin actually activates protein C during blood coagulation processing, nor do we know whether this activated protein C acts as an anticoagulant. We studied the inhibitory action of recombinant human soluble TM (rhs-TM) on thrombin generation in whole plasma. Human plasma was activated with small amounts of tissue factor using phospholipid vesicles in place of activated platelets. Thrombin generation was observed. The addition of only 2 nM of rhs-TM prevented rapid generation of thrombin and reduced the total amount of thrombin generated. In order to study the influence of the protein C activation pathway on this inhibitory action of rhs-TM, protein C-depleted plasma was used. rhs-TM had little inhibitory effect on protein C-depleted plasma. However, the addition of protein C caused a delay in thrombin generation and a reduction of the maximum thrombin concentration. We concluded that the anticoagulant activity of rhs-TM was amplified by the protein C activation pathway.
Evaluation of Factor Vlll Deficient Plasmas
Authors: Barrowcliffe T W , Tubbs Jill E , Wong Man Yu ,   On behalf of the Haemostasis and Thrombosis Task Force of the British Committee for Standards in Haematology
Published: July 2018

SummaryImmunodepleted plasmas from Organon Teknika, Dade, Stago, Diagen and the Scottish National Blood Transfusion Service (SNBTS), and haemophilic plasma from Immuno were compared by several laboratories with haemophilic plasma from local donors as substrates in one-stage factor VIII assays. Five clinical plasma samples and four concentrates were assayed against British Standard plasma and International Standard concentrate.Potencies of all plasma samples were not significantly different from those with local haemophilic plasma for Immuno, Organon Teknika, Stago and SNBTS substrates. Dade differed from haemophilic on one sample and Diagen on three. Buffer blank times and slopes of Standard lines were similar with all substrates. A positive drift between the beginning and end of the assay was found with the Immuno substrate and a negative drift with the Organon Teknika substrate.In the concentrate study results for all substrate plasmas were not significantly different from haemophilic on the intermediate purity and conventional high purity products. On the monoclonal and recombinant products, there was a tendency for the immunodepleted substrates to give lower potencies than the haemophilic, and significant differences were found with Dade and Stago on the monoclonal concentrate, and with Dade, Stago and Diagen on the recombinant concentrate.Overall, this study indicates that most commercially available substrate plasmas are suitable as replacements for locally collected haemophilic plasma in one-stage assays of clinical samples, and of intermediate purity and conventional high purity concentrates. For assays of very high purity concentrates (monoclonal and recombinant), haemophilic plasma is preferable as some immunodepleted plasmas give low results.
Cross-Linked αsγt-Chain Hybrids in Plasma Clots Studied by 1D- and 2D Electrophoresis and Western Blotting
Authors: Grøn B , Filion-Myklebust C , Bjørnsen S , Haidaris P , Brosstad F 
Published: July 2018

SummaryFibrinogen and fibrin related chains in reduced human plasma as well as the bonds interlinking partially cross-linked fibrin from plasma clots have been studied by means of 1D- and 2D electrophoresis and Western blotting. Immunovisualization of reduced plasma or partially cross-linked fibrin with monoclonal antibodies specific for the α-chains or the γ-chains have shown that several bands represent material belonging to both chains. In order to decide whether these bands constitute αγ-chain hybrids or superimposed α- and γ-chain dimers, the cross-linked material was separated according to both isoelectric point (pI) and molecular weight (MW) using Pharmacia’s Multiphor II system. Western blotting of the second dimension gels revealed that partially cross-linked fibrin contains αsγt-chain hybrids and γ- polymers, in addition to the well-known γ-dimers and α-polymers. The main αsγt-chain hybrid has a pI between that of the α- and the γ-chains, a MW of about 200 kDa and contains Aα-chains with intact fibrinopeptide A (FPA). It was also observed that soluble fibrinogen/fibrin complexes as well as partially cross-linked fibrin contain degraded α-dimers with MWs close to the γ-dimers. These findings demonstrate that factor XIII-catalyzed cross-linking of fibrin is a more complex phenomenon than earlier recognized.
The Platelet Glycoprotein IIb/IIIa Complex Is lnvolved in the Adhesion of Activated Platelets to Leukocytes
Authors: Spangenberg Peter , Redlich Helge , Bergmann Iris , Lösche Wolfgang , Götzrath Matthias , Kehrel Beate 
Published: July 2018

SummaryThe adhesion of activated platelets to leukocytes (rosette formation) seems to be mediated by CD62 on platelets and its counterreceptor (CD 15 or a sialic acid-containing glycoprotein) on polymorphonuclear leukocytes (PMNL). However, neither treatment of platelets with an anti-CD62 antibody or fucoidan nor treatment of PMNL with anti-CD15 antibody or neuraminidase are able to inhibit completely the adhesion. Therefore, we have studied the platelet GPIIb/IIIa complex (CD41a) for its involvement in the adhesion of activated platelets to PMNL. The following evidences point to a participation of CD41a in the adhesion of activated platelets to leukocytes: a) inhibition of adhesion by monoclonal antibodies (mab) raised toward CD41a, b) inhibition of adhesion by peptides such as RGDS and echistatin, c) inhibition of adhesion by dissociation of the CD41a complex with EGTA, and d) inhibition of rosette formation using platelets from a thrombasthenic patient which have almost no CD41a in the surface membrane but a normal expression of CD62. It is likely that fibrinogen is involved in the adhesion of platelets to PMNL via CD41a, since fibrinogen increases the rosette formation of ADP-stimulated platelets. Furthermore, the incubation of unstimulated platelets with fibrinogen and an antibody raised against glycoprotein III a which stimulates fibrinogen binding to the platelet surface results in an enlarged rosette formation.
New Bleeding Time Devices with Retractable Blades Evaluated in Children, Healthy Volunteers and Patients with Prolonged Bleeding Time
Authors: Lethagen Stefan , Kling stefan 
Published: July 2018

SummaryThe Simplate II-R and the Simplate Paediatric, are two new Simplate bleeding time devices with retractable blades. In this study we compared the Simplate II-R with the Simplate II bleeding time and found a good correlation when performed by the same technician both in 30 healthy volunteers and in 7 patients with prolonged bleeding time. The same normal ranges may be applied for the Simplate II-R as for the Simplate II bleeding time. The Simplate II-R bleeding time was highly reproducible in healthy volunteers when performed by the same technician. We found no correlation between age and bleeding time, and no significant difference in bleeding time between males and females.We performed the Simplate Paediatric bleeding time test and calculated the normal ranges in 84 healthy children in 4 age groups: 3 days, 3 months, 1 year and 3–6 years of age. The Simplate Paediatric bleeding times were shorter in the children than the Simplate II-R bleeding times in the adults. We found no significant correlation between bleeding time and the inverse platelet count, and no significant difference in bleeding time between boys and girls. The retractable blades in the Simplate II-R and the Simplate Paediatric reduce the risk of accidental cuts. Both devices yield reproducible bleeding time measurements and can be used instead of older Simplate variants.
Comparison of the Non-Specific Binding of Unfractionated Heparin and Low Molecular Weight Heparin (Enoxaparin) to Plasma Proteins
Authors: Young Edward , Cosmi Benilde , Weitz Jeffrey , Hirsh Jack 
Published: July 2018

SummaryThe non-specific binding of anticoagulantly-active heparin to plasma proteins may influence its anticoagulant effect. We used low affinity heparin (LAH) essentially devoid of anti-factor Xa activity to investigate the extent and possible mechanism of this non-specific binding. The addition of excess LAH to platelet-poor plasma containing a fixed amount of unfractionated heparin doubled the anti-factor Xa activity presumably because it displaces anticoagulantly-active heparin from plasma proteins. Although dextran sulfates of varying molecular weights also increased the anti-factor Xa activity, less sulfated heparin-like polysaccharides had no effect. These findings suggest that the ability to displace active heparin from plasma protein binding sites is related to charge and may be independent of molecular size. In contrast to its effect in plasma containing unfractionated heparin, there was little augmentation in anti-factor Xa activity when LAH was added to plasma containing low molecular weight heparin (LMWH), indicating that LMWH binds less to plasma proteins than unfractionated heparin. This concept is supported by studies comparing the anticoagulant activity of unfractionated heparin and LMWH in plasma with that in buffer containing antithrombin III. The anti-factor Xa activity of unfractionated heparin was 2-fold less in plasma than in the purified system. In contrast, LMWH had identical anti-factor Xa activity in both plasma and buffer, respectively. These findings may be clinically relevant because the recovered anti-factor Xa activity of unfractionated heparin was 33% lower in plasma from patients with suspected venous thrombosis than in plasma from healthy volunteers. The reduced heparin recovery in patient plasma reflects increased heparin binding to plasma proteins because the addition of LAH augmented the anti-factor Xa activity. In contrast to unfractionated heparin, there was complete recovery of LMWH added to patient plasma and little increase of anti-factor Xa activity after the addition of LAH. These findings may explain why LMWH gives a more predictable dose response than unfractionated heparin.
Evaluation of Whole Blood Flow Cytometric Detection of Platelet Bound Fibrinogen on Normal Subjects and Patients with Activated Platelets
Authors: Janes Sarah L , Wilson Darren J , Chronos Nicolas , Goodall Alison H 
Published: July 2018

SummaryActivated platelets can be detected by measuring platelet-bound fibrinogen in a whole blood, flow cytometric assay, using a fluorescently-conjugated polyclonal antibody.Fibrinogen binding to unstimulated platelets from normal subjects was low in this assay, as was expression of the CD63 antigen. Single cell counting of samples prepared for flow cytometric analysis showed platelet aggregates do not form during the assay procedure. Immune complexes were not seen, and fibrinogen binding to the platelets was unaffected by the CD32 MAb, IV.3. Artefactual activation of the unfixed samples could be minimised by control of phlebotomy, time and temperature of incubation. Variations in platelet count in the range 140–430 × 109 1-1 and in plasma fibrinogen in the range 2–6 g 1-1 did not affect the assay results.Comparison of fibrinogen binding with expression of CD63 antigen on normal platelets, stimulated with agonists in vitro, demonstrated that fibrinogen binding detects an earlier stage of platelet activation.Platelet bound fibrinogen was shown to be sensitive in detecting small numbers of activated platelets in clinical samples in twelve patients on intensive care, four undergoing haemofiltration. The patients had a significantly higher median percentage of circulating platelets with bound fibrinogen (p <0.005), but fibrinogen binding was significantly lower (p <0.02) in response to 10-5 M ADP, compared to twelve age-matched normal Controls.
Heparin Cofactor II Deficiency in Patients Infected with the Human Immunodeficiency Virus
Authors: Toulon P , Lamine M , Ledjev I , Guez T , Holleman M E , Sereni D , Sicard D 
Published: July 2018

SummaryIn human plasma, heparin cofactor II (HCII) is a thrombin inhibitor, whose deficiency has been reported to be associated with recurrent thrombosis. The finding of two cases of low plasma HCII activity in two patients infected with the human immunodeficiency virus (HIV) led us to investigate this coagulation inhibitor in the plasma of a larger population of HIV-infected patients. The mean plasma HCII activity was significantly lower in 96 HIV-infected patients than in 96 age- and sex-matched healthy individuals (0.75 ± 0.24 vs 0.99 ± 0.17 U/ml, p <0.0001). HCII antigen concentration was decreased to the same extent as the activity. The proportion of subjects with HCII deficiency was significantly higher in the HIV-infected group than in healthy individuals (38.5% vs 2.1%). In addition, HCII was significantly lower in AIDS patients than in other HIV-infected patients, classified according to the Centers for Disease Control (CDC) on the basis of an absolute number of circulating CD4+ lymphocytes below 200 x 106/1. The link between HCII and immunodeficiency is further suggested by significant correlations between HCII activity and both the absolute number of CD4+ lymphocytes and the CD4+ to CD8+ lymphocyte ratio. Nevertheless, the mean HCII level was not different in the various groups of patients classified according to clinical criteria, except in CDC IVD patients in whom HCII levels were significantly lower. In addition, no correlation could be demonstrated between HCII and protein S activities, another coagulation inhibitor whose plasma level was also found to be decreased in HIV-infected patients. A similar prevalence of HCII deficiency was also found in a small series of 7 HIV-infected patients who developed thrombotic episodes, an unusual complication of the infection. This suggests that, in HIV-infected patients, HCII deficiency is not in itself the causative factor for the development of thrombosis.
Platelet Hyperactivation in Patients with Essential Thrombocythemia Is not Associated with Vascular Endothelial Cell Damage as Judged by the Level of Plasma Thrombomodulin, Protein S, PAI-1, t-PA and vWF
Authors: Bellucci S , Ignatova E , Jaillet N , Boffa M C 
Published: July 2018

SummaryThe occurrence of thrombotic events remains an important clinical problem in Essential Thrombocythemias (ET). Thus, hemostatic, fibrinolytic and vascular status was investigated in 16 patients (5 males and 11 females) with ET. Among them five presented thromboses in their past history. Platelet hyperactivation, as evidenced by a mean three-fold increase in plasma betathromboglobulin (βTG), was observed in 13 among 16 patients; surprisingly this activation was present even when the platelet count was normal (in two patients) or subnormal, below 600 x 109/1 (in 11 patients). The mean value was 104 ± 57 IU/ml significantly different from that of normal controls (35 ± 16.5 IU/ ml) (p <0.001). An artefactual in vitro platelet activation was ruled out by the concomitant measurement of platelet factor, 4 (PF4). D-dimers fibrin degradation products (D-Di FDP) were normal in all patients. Vascular endothelial cell function parameters were not markedly modified. The mean value of plasma thrombomodulin ™ was found slightly but not significantly increased (60.1 ±4.9 ng/ml versus 49.1 ± 10.0 ng/ml in controls). The values of plasma TM correlated neither with that of the platelet count nor with that of plasma βTG or plasma PF4. The mean values of plasma protein S, von Willebrand factor (vWF), plasminogen activator inhibitor type 1 (PAI-1), tissue plasminogen activator (tPA) were normal and were not correlated neither with that of plasma TM nor with that of plasma βTG. In spite of a significant increase in vWF in two patients who presented peripheral thromboses, the markers of vascular endothelial cell function were not significantly different in patients having presented or not thromboses. Thus, in ET, these data showed the presence of platelet hyperactivation, even in patients in remission; it was not associated with vascular endothelial cell damage which might account for a greater risk of thrombosis.
Comparison of a Standardized Procedure with Current Laboratory Practices for the Detection of Lupus Anticoagulant in France
Authors:   Working Group on Hemostasis of the Société Française de Biologie Clinique
Published: July 2018

SummaryA multicenter study involving 13 laboratories was designed to compare a common procedure for screening lupus anticoagulants (LA) to the different practices currently in use in these laboratories. The common procedure combined 3 phospholipid- dependent assays, including mixing studies and a phospholipid neutralizing test. Due to the heterogeneity of LA expression, an abnormal result in at least one of the tests was sufficient to classify a sample as positive for LA. Consecutive samples referred for LA diagnosis were evaluated in parallel by each participant and the data found using the common procedure were analyzed independently according to mutually agreed cut-offs and criteria for sample classification. Within a period of 3 months, 535 samples were included, of which 147 were judged LA positive, 29 undetermined and 359 negative by the respective laboratories using their current practice. When using the common procedure, 149 plasmas were said to be positive, 38 undetermined and 348 negative. Absolute concordance occurred for 81% of the specimen population and absolute discordance (positive versus negative) for 7%. The level of agreement between the common procedure and the current practices, assessed by kappa indexes, indicated noticeable variations in the rates of detection from laboratory to laboratory. Among the different tests used in the common procedure, regular APTT was the least sensitive (about 50% detection) but none of the other tests alone recognized more than 73% of specimens from the LA positive population. This yield increased to about 90% with any combination of 2 sensitive tests. The common procedure should not be considered as a methodological gold standard but as an acceptable basis for the collaborative works required for the improvement and standardization of laboratory practices for screening LA.
Plasminogen-Activator in Human Early Milk: Its Partial Purification and Characterization
Authors: Okamoto Utako , Horie Noboru , Nagamatsu Yoko , Yamamoto Jun-Ichiro 
Published: July 2018

SummaryMilk plasminogen-activator was partially purified from human transitional milk collected at about 10 days after delivery, by a five-step procedure involving chloroform treatment, ammonium sulfate precipitation, and column chromatography on Sephadex G-150, CM Sephadex C-50 and DEAE Sephadex A-50. This gave milk-activator with a maximum purification factor of about 2,400-fold with respect to the skimmed milk. The CM Sephadex-step preparation showed, on polyacrylamide gel electrophoresis, a single plasminogen-activator activity band located between the bands of albumin and prealbumin of human serum. This preparation exhibited no kinin forming activity. The activator hydrolyzed acetyl-glycyl-L-lysine methyl ester with similar order kinetic constants to urokinase, and was inhibited strongly by diisopropyl-fluorophosphate. The molecular weight of the activator as estimated by gel filtration was approximately 86,000, the isoelectric points as estimated by gel isoelectric focusing were pH 7.2, 6.9 and 6.6, and the activator activity was not quenched by antiurokinase globulin, indicating that the milk-activator is a different entity from urokinase.
The Clinical Usefulness of the Platelet Aggregation Test for the Diagnosis of Heparin-Induced Thrombocytopenia
Authors: Chong B H , Burgess J , Ismail F 
Published: July 2018

SummaryThe platelet aggregation test is widely used for the diagnosis of heparin-induced thrombocytopenia (HIT), a potentially serious complication of heparin therapy. We have evaluated its sensitivity and specificity in comparison with those of the 14C-serotonin release test. The sensitivity of the platelet aggregation test was found to vary with the heparin concentration and the donor of the platelets used in the test. The optimal heparin concentrations were between 0.1 and 1.0 U/ml. Using these heparin concentrations, the mean sensitivity varied from 39% (with the least reactive platelets) to 81% (with the most reactive platelets). In comparison, the sensitivity of the release test ranged from 65% to 94%. The specificities of the platelet aggregation test were 82%, 90% and 100% for the following control groups: (1) non-thrombocytopenic patients given heparin, (2) patients with thrombocytopenia due to other causes, and (3) normal controls not given heparin, respectively. The corresponding specificities for the release test was 94%, 90% and 100%. The specificities can be further increased to 100% for all controls with the adoption of a two-point system which defines a positive result as one in which platelet aggregation occurs with a low heparin concentration (0.5 U/ml) but not with 100 U heparin/ml. For optimal results, a two-point platelet aggregation test should be performed with heparin concentrations of 0.5 and 100 U/ml and using platelets of more reactive donors.
Kinetic Analysis of Bovine Factor VIII in the Hemophilic Dog
Authors: Gentry Patricia A , Kirby Edward P , Gentry Rodney D 
Published: July 2018

SummaryBovine factor VIII, which did not contain platelet aggregating factor activity, was infused into hemophilic dogs. Factor VIII procoagulant (VIII: C) levels in the dogs increased dramatically, then decreased in a biphasic manner. The half-life of the longest component was 3-7 hrs. The infusions were hemostatically effective and also caused a prolonged shortening of the activated partial thromboplastin time. These studies demonstrate that the platelet aggregating factor activity of bovine factor VIII is not essential for its maintenance in the circulation and that preparations lacking this activity may be clinically useful. When concentrates of partially purified factor VIII: C (essentially free of both platelet aggregating factor and factor VIII-related antigen) were infused, marked increases in VIII: C levels were also observed, but the half-life was significantly shorter (T1/2 of approximately 1 hr).