bluuebunny/crossref_metadata_2025_split huggingface dataset:
Membrane Fluidity Is Related to the Extent of Glycation of Proteins, but not to Alterations in the Cholesterol to Phospholipid Molar Ratio in Isolated Platelet Membranes from Diabetic and Control Subjects
Authors: Winocour Peter D , Watala Cezary , Kinglough-Rathbone Raelene L 
Published: July 2018

SummaryPlatelets from diabetic subjects are hypersensitive to aggregating agents in vitro. Membrane fluidity modulates cell function and we previously reported reduced membrane fluidity associated with hypersensitivity to thrombin in intact platelets from diabetic subjects. Reduced membrane fluidity and hypersensitivity to agonists has also been reported in platelets from non-diabetic subjects whose platelets have an increased cholesterol/phos-pholipid molar ratio. Glycation of platelet membrane proteins is enhanced in diabetic subjects, and could contribute to the decreased membrane fluidity in these platelets. We examined the relation among fluidity, cholesterol/phospholipid molar ratio, and glycation of proteins in isolated platelet membranes from diabetic and control subjects. Seven poorly controlled diabetic subjects were compared with 7 age- and sex-matched control subjects. The mean steady-state fluorescence polarization value in 1,6-diphenyl-1,3,5-hexatriene-labeled isolated platelet membranes from diabetic subjects (0.184 ± 0.004) was significantly greater than from control subjects (0.171 ± 0.004, p <0.01); thus, fluidity in platelet membranes from diabetic subjects is decreased. Reduced fluidity in platelet membranes from diabetic subjects could not be attributed to changes in the cholesterol/phospholipid molar ratio. Total or very low density (VLDL), low density (LDL), or high density (HDL3) lipoprotein cholesterol concentration in plasma was not significantly different between groups, but the ratio of VLDL + LDL to HDL2 + HDL3 cholesterol was significantly greater in diabetic subjects (4.79 ± 0.73) than in control subjects (2.54 ± 0.30, p <0.02). Proteins were glycated significantly more extensively in platelet membranes from diabetic subjects (25.5 ± 0.9 nmol glucose/mg protein) than those from control subjects (21.0 ± 0.6 nmol glucose/mg protein, p <0.001). The extent of glycation of proteins in platelet membranes correlated significantly with the steady state fluorescence polarization values for platelet membranes from diabetic and control subjects (r = 0.715, p <0.01). More extensive glycation of proteins appears to be associated with reduced fluidity of platelet membranes from diabetic subjects, but not with increased cholesterol/phospholipid molar ratio.
Monoclonal Antibody-Based Enzyme-Linked Immunosorbent Assays (ELISA) for the Measurement of Vitamin K-Dependent Protein S: The Effect of Antibody Immunoreactivity on Plasma Protein S Antigen Determinations
Authors: D’Angelo Silvana Vigano , Tombesi Sandra , Marcovina Santica , Albertini Alberto , Valle Patrizia Della , D’Angelo Armando 
Published: July 2018

SummaryTwo monoclonal antibodies (Mabs) specifically directed to human protein S (PS) - named 5E9E9 and 3B10.25 - were produced and their properties compared to those of 2 previously characterized anti-PS-Mabs (HPS-2 and S10). 3B10.25, similar to S10, was directed to the calcium-free conformation of PS and had virtually identical affinity for free and C4b-binding protein (C4b-BP)-bound PS; 5E9E9 similar to HPS-2, had no calcium-dependency and was selectively directed to free PS. All Mabs were equally reactive to freshly purified and thrombin-cleaved PS. To evaluate the influence of C4b-BP bound PS on PS antigen determinations, ELISA systems employing the four Mabs individually as capture antibody (Ab) and peroxidase-conjugated polyclonal anti-PS IgG as detecting Ab were developed and compared to immunoelectrophoresis (EIA) and to an ELISA employing polyclonal anti-PS IgG as capture and detecting Ab, in the determination of PS in purified systems and in plasma. With all the ELISAs there was parallelism of dilution cuiyes obtained with normal plasma and purified PS; however, supplementation of plasma with purified C4b-BP resulted in loss of parallelism when employing the Mabs directed to free PS as capture Ab. Influence of high C4b-BP on PS antigen determinations was confirmed in a series of plasma samples from patients with C4b-BP levels ranging from 70% to over 200%. Compared to the values obtained with the S10- or 3B10.25 - based ELISAs - which were similar despite a 10-fold difference in sample dilution -plasma PS was underestimated by the ELISAs employing 5E9E9 or HPS-2 while it was overestimated by EIA. In addition, plasma PS and C4b-BP levels were significantly correlated only when it was measured by EIA or the ELISAs employing S10, 3B10.25 or polyclonal IgG. These results highlight the potential influence of high C4b-BP on plasma PS antigen determination. Accurate measurement of PS by ELISA requires selection of antibodies with identical affinity for all plasma PS forms.
Heparin Binding to Plasma Proteins, an Important Mechanism for Heparin Resistance
Authors: Young Edward , Prins Martin , Levine Mark N , Hirsh Jack 
Published: July 2018

SummaryHeparin dosage requirements vary widely among patients with venous thromboembolism. In this study, we measured the proportion of anticoagulantly-active heparin which was reversibly bound and neutralized by plasma proteins (defined as reversible heparin neutralization) in the pre-treatment plasma (in vitro) and in the 6 h post-treatment plasma (ex vivo) of patients with venous thromboembolism treated with a fixed dose of heparin. Reversible heparin neutralization was assessed by comparing the heparin levels measured as anti-factor Xa activity before and after the addition of low affinity heparin which is essentially devoid of antifactor Xa activity, in order to displace heparin bound to plasma proteins. The results indicate that reversible heparin neutralization due to binding to plasma proteins is a major determinant of the anticoagulant response to a fixed dose of standard heparin 6 h post-treatment and of the eventual heparin dose required to achieve a therapeutic anticoagulant effect on days 3-5 of heparin treatment.
The Role of Phospholipids and the Factor VII Gla-Domain in the Interaction of Factor VII with Tissue Factor
Authors: Wildgoose Peter , Jørgensen Tony , Komiyama Yutaka , Nakagaki Tomohiro , Pedersen Anders , Kisiel Walter 
Published: July 2018

SummaryWhether or not the factor VII Gla-domain is involved in the high-affinity interaction of factor VII and tissue factor via calcium-dependent interactions with surrounding phospholipids is unknown. To investigate this, we have purified the factor VII Gla-peptide (FVII-GP) from digested recombinant human factor VII a and assessed its effect on factor VII: tissue factor interactions. FVII-GP inhibited the activation of factor X by factor Vila in the presence of either soluble or cell surface tissue factor halfmaximally at 0.5 μM and 2.7 μM, respectively. However, FVII-GP failed to inhibit the specific binding of factor Vila to cell-surface tissue factor, and did not inhibit the ability of tissue factor to stimulate the amidolytic activity of factor Vila. Unrelipidated tissue factor apoprotein stimulated the amidolytic activity of factor Vila to the same extent as relipidated tissue factor apoprotein. These findings suggest that the factor VII Gla-domain does not directly interact with tissue factor, but rather is important for calcium binding and concomitant expression of other factor VII epitopes necessary for tissue factor recognition and binding. To test this hypothesis, we have prepared a monoclonal antibody against a putative factor VII epitope that participates in the interaction of factor VII with cell-surface tissue factor (peptide 195-206) and assessed its ability to bind to factor VII in the presence and absence of calcium. Binding of this monoclonal antibody (PW-4) to intact factor VII a was calcium-dependent and could be inhibited in a dose-dependent manner by peptide 195-206. The antibody reacted with Gla-domainless factor Vila, but only 37% as compared to intact factor Vila. In addition, PW4 as well as its Fab’ fragment, inhibited factor Vila binding to cell-surface tissue factor. These studies indicate that the factor VII Gla-domain does not provide structural elements that contribute to the formation of a stable factor VII/VII a-tissue factor binary complex. The factor VII Gla-domain appears to be necessary, however, in binding calcium ions and inducing a calcium-dependent conformational change in factor VII/VII a that expresses one or more neoepitopes that participates in the interaction of factor VII/VII a with the extracellular domain of tissue factor apoprotein.
Comparison of Recombinant Plasminogen Activator Inhibitor-1 and Epsilon Amino Caproic Acid in a Hemorrhagic Rabbit Model
Authors: Racanelli Adrienne L , Diemer Mark J , Dobies A Cathy , Dubin Joan R , Reilly Thomas M 
Published: July 2018

SummaryA rabbit ear model of blood loss was developed to compare the effects of an active form of recombinant plasminogen activator inhibitor-1 (rPAI-1) with epsilon amino caproic acid (EACA) in antagonizing tissue-type plasminogen activator (r-tPA)-induced blood loss. The antagonism of both rebleeding, which occurs as a result of hemostatic plug degradation, and r-tPA-induced hemorrhage, where rabbits lose approximately 30% of their blood volume, was studied. rPAI-1 (1 mg/kg i. v.) or EACA (70 mg/kg i. v.) antagonized the rebleeding induced by r-tPA (10 μg kg-1 min-1) to a similar extent. In the hemorrhagic studies, rPAI-1 effectively antagonized the r-tPA-induced hemorrhage with an ED50 of 3 mg/kg i. v., while the ED50 obtained for EACA was 230 mg/kg i. v. rPAI-1 may be of value in reversing r-tPA-induced blood loss during thrombolytic therapy or in clinical situations where excessive fibrinolysis contributes to bleeding.
Platelet Interaction with Collagen Fibrils in Flowing Blood
Authors: Baumgartner Hans R. , Tschopp Thomas B. , Weiss Harvey J. 
Published: July 2018

SummaryAnticoagulated whole blood from patients and control subjects was circulated through an annular perfusion chamber in which the fibrillar collagen of α chymotrypsin-digested subendothelium and intact subendothelium were exposed. The blood flow conditions corresponded to those in arteries (830 sec–1 wall shear rate). Platelet surface interaction was measured mor-phometrically.Decreased adhesion to fibrillar collagen associated with normal spreading and normal adhesion-induced formation of platelet thrombi was found with blood of patients with von Willebrand’s disease and the Bernard Soulier Syndrome, indicating a defect in the initial attachment reaction of platelets with collagen. Platelets of patients with thrombasthenia did normally adhere to the collagen fibrils and also lost their subcellular organelles during this reaction, but they totally failed to adhere to each other. In storage pool disease platelet thrombus formation was consistently inhibited whereas adhesion and spreading was inhibited in some patients and normal in others. In contrast adhesion was always normal after ingestion of aspirin which consistently caused a marked inhibition of platelet thrombi. These findings correspond – in essence – to those previously described on intact subendothelium. However, the observed defects are more pronounced on the fibrillar collagen than on intact subendothelium.
A Comparative Ex Vivo Study of Plasminogen Activators and Proteases for Fibrinolytic Activity and Side Effects in Rabbits
Authors: Janik B. A , Papaioannou S. E 
Published: July 2018

SummaryUrokinase, streptokinase, Brinase, trypsin, and SN 687, a bacterial exoprotease, have been evaluated in an ex vivo assay system. These enzymes were injected into rabbits and the fibrinolytic activity as well as other coagulation parameters were measured by in vitro techniques. Dose-response correlations have been made using the euglobulin lysis time as a measure of fibrinolytic activity and the 50% effective dose has been determined for each enzyme. Loading doses, equal to four times the 50% effective dose, were administered to monitor potential toxicity revealing that Brinase, trypsin, and SN 687 were very toxic at this concentration.Having established the 50% effective dose for each enzyme, further testing was conducted where relevant fibrinolytic and coagulation parameters were measured for up to two days following a 50% effective dose bolus injection of each enzyme. Our results have demonstrated that urokinase and streptokinase are plasminogen activators specifically activating the rabbit fibrinolytic system while Brinase, trypsin and SN 687 increase the general proteolytic activity in vivo.The advantages of this ex vivo assay system for evaluating relative fibrinolytic potencies and side effects for plasminogen activators and fibrinolytic proteases have been discussed.
In Vitro and in Vivo Measurement of Total Antiplasmin Activity
Authors: Matsuo Osamu , Mihara Hisashi 
Published: July 2018

SummaryTotal antiplasmin was measured in vitro and in vivo. In the former case, rabbit plasma was mixed with various concentrations of Urokinase (UK) and the least concentration for appearance of fibrinolytic activity was estimated. This concentration was multiplied by the plasma volume of the rabbit to give the in vitro total antiplasmin. The mean value for 14 rabbits was 4,068.6 units.In order to estimate the total antiplasmin in vivo, UK solution was infused into rabbits. The infusion speed was multiplied by the time of the first appearance of fibrinolytic activity to give the total antiplasmin, although when the infusion speed was low, fibrinolytic activity did not appear during infusion. The mean in vivo total antiplasmin calculated for 6 cases where the infusion speed was high and fibrinolytic activity was observed, was 28,699.8 units, i.e. about 7 (range, 3-11) times the in vitro value.
Comparative Studies of Coagulative and Fibrinolytic Faculties between the Japanese Monkey and the Human
Authors: Suzuki K , Egawa H , Hashimoto S 
Published: July 2018

SummaryIn the present study, the coagulative and the fibrinolytic faculties of the Japanese monkey (Macaca fuscata) were investigated and compared with those of the human.Regarding the coagulative faculty, the plasmic fibrinogen level, prothrombin times, and partial thromboplastin times of the Japanese monkey were similar to those of the human, but the antithrombin level in the monkey was higher than that in the human.Regarding the fibrinolytic faculty, the simian plasminogen level was significantly higher than the human, but the simian, plasma clot and the euglobulin clot lysis times were extremely prolonged, which means that the Japanese monkey has a great fibrinolytic potential, but that it is difficult to activate.In addition, the human and simian reactivities of plasminogen to streptokinase were also investigated and compared.
Assay of the Esterase Activity of Thrombin, Plasmin and Trypsin with a Chromogenic Substrate p-Nitrobenzyl p-Toluenesulfonyl-L-Arginine
Authors: Aogaichi Tadashi , Plaut Gerhard W. E 
Published: July 2018

Summaryp-Nitrobenzyl p-toluenesulfonyl-L-arginine has been synthesized. A number of trypsin-like enzymes can catalyze the hydrolysis of this ester leading to formation of p-nitrobenzyl alcohol. After separation from the ester and p-toluenesulfonylarginine by extraction into chloroform, the p-nitrobenzyl alcohol liberated can be measured spectrophotometrically at 271 nm. Under the conditions of the assay, the hydrolysis of 1 μmol/ml of the ester is equivalent to an absorbance change of 4.45 cm–1 at 271 nm. With 0.10 mM p-nitrobenzyl p-toluenesulfonyl-L-arginine in 0.1 M Tris-HCl at pH 8.4 and 30°, the enzymatic hydrolysis is linearly proportional to time up to consumption of 60% of the ester. Product formation is proportional to enzyme concentration with 0.05 to 0.2 NIH clotting units/ml for bovine or human thrombin, 0.005 to 0.02 CTA units/ml for human plasmin, and 0.01 to 0.04 μg/ml protein for bovine pancreatic trypsin. In 0.1 M Tris-HCl at pH 8.4 and 30°, Kmis 14 μM and Vmax is 0.037 μmol/min/NIH unit/ml for bovine thrombin, Km is 78 μM and Vmax is 0.31 μmol/min/CTA unit/ml for human plasmin, and Km is 12 μM and Vmax is 138 μmol/min/mg protein/ml for bovine trypsin. With bovine thrombin, activities at pH 7.3 and at pH 9.2 were 30% lower and 40-50% higher than the rate at pH 8.4. Samples of bovine and human thrombin ranging in specific clotting activity from 59 to 2133 NIH units/mg protein showed esterase activities varying from 0.15 to 0.4 μmol p-nitrobenzyl alcohol formed/10 min/NIH unit.
Cigarette-Smoking Effect on Platelet Function
Authors: Grignani G , Gamba G , Ascari E 
Published: July 2018

SummaryThe immediate effect of cigarette-smoking on ADP-induced platelet aggregation and on platelet adhesiveness was investigated in 12 normal subjects aged 20 to 40, in 10 normal subjects aged 43 to 72 and in 10 patients with cerebrovascular disease aged 45 to 75.All the subjects were heavy smokers (more than 20 cigarettes a day).After smoking 2 cigarettes a significant increase in ADP aggregation and platelet adhesiveness was found in the group of young heavy smokers, while in the old subjects with or without cerebrovascular disease the increase in platelet activity was never significant.These data were discussed and some hypotheses for this higher reactivity of platelets from young people were suggested.
Characterisation of Serum Fibrinogen Degradation Products Using Gel Chromatography and Radioimmunoassay
Authors: Ratky S. M , Sykes A , Cooke E. D , Gordon Y. B 
Published: July 2018

SummaryFibrin(ogen) degradation products (FDP) are detected by radioimmunoassay in the serum of all subjects. Some of these FDPs have considerable biological activity in vivo, and hence may play a significant part in the process of coagulation. Chromatography of serum samples from patients including those with pulmonary emboli and deep venous thrombosis indicates that the majority of non-clottable fibrin(ogen)-related antigen present is of high molecular weight- greater than that of fibrinogen – and that in pathological situations there is a quantitative increase not a qualitative alteration in the molecular species present.
Early Venous Thrombosis
Authors: Day T. K , Cowper S. V , Kakkar V. V , Clark K. G. A 
Published: July 2018

SummaryThe passage of a minimal electric charge was used to initiate thrombosis in rabbit femoral veins, and the events occurring during formation of the thrombus were observed using scanning electron microscopy. Thrombosis began to occur within ten minutes after passage of the charge, upon an apparently unaltered endothelium. The first event was the laying down of a fibrin meshwork and this was shortly followed by the appearance of regularly arranged platelet clumps.
Inherited Bleeding Syndromes in Iraq
Authors: Al-Mondhiry Hamid A. B 
Published: July 2018

SummaryThis paper presents data on the occurrence and pattern of inherited bleeding syndromes (IBS) in Iraq, a hitherto unexplored problem. During the first fourteen months of a prospective on-going study at a major university center, 116 patients from 62 families were diagnosed as having IBS. All patients were referred because of moderate to severe bleeding diatheses. They included 62 haemophiliacs, 32 patients with von Willebrand’s disease (VWD), 9 with Christmas disease (CD), 6 with afibrinogenemia, 1 with prothrombin deficiency, and 6 were thought to have platelet dysfunction. 32 other bleeders (16 hemophiliacs, 14 VWD, and 2 CD) were also recognized among the pedigrees studied but were not available for full investigations. The clinical and laboratory features of the patients observed in Iraq do not seem to be significantly different from those of patients in Western Europe or North America. Although the absolute incidence and relative distribution of these disorders in the entire population cannot yet be determined, the rate of occurrence per segment population is likely to be high, most likely due to the high rate of consanguinity and large number of births per family, phenomena still prevalent in this country.
Purification and Properties of a Neutral Protease from Soil Bacterium WM 122
Authors: Papaioannou S. E , Marsheck W. J 
Published: July 2018

SummaryAn extracellular protease SN 687, secreted by the soil bacterium isolate WM 122, has been purified by means of gel filtration, ammonium sulfate precipitation, DEAE-Sephadex and hydroxylapatite chromatography. Apparent homogeneity was ascertained by Polyacrylamide gel electrophoresis. The protease was inactivated by ethylenediamine tetracetic acid (EDTA) but not by diisopropylfluorophosphate (DFP), and it was partially inhibited by serum inhibitors. SN 687 was shown to be of high specific activity against casein and fibrin, but it did not hydrolyze L- lysine -methyl ester dihydrochloride (LME), p-tosyl-L-arginine-methyl ester hydrochloride (TAME) and N-benzoyl-L-tyrosine-ethyl ester hydrochloride (BTEE) synthetic substrates. The optimum pH for hydrolysis of casein was 7.5 and the molecular weight, as determined by gel filtration, was 31,000.
Protease Nexin 1 Is Expressed in the Human Placenta
Authors: White Elizabeth A , Baker Joffre B , McGrogan Michael , Kitos Paul A 
Published: July 2018

SummaryProtease nexin 1 (PN1), a serine protease inhibitor that inactivates thrombin, urokinase, and plasmin, is produced abundantly in cultures of human fibroblasts and rat and human glioma cells. The major sites of PN1 synthesis in vivo and the specific physiological function(s) of this serpin are unknown. Using Northern blot analysis and a full-length PN1 cDNA probe we demonstrated the presence of PN1 mRNA in human term placentas. In situ hybridization of placental tissue with a PN1 riboprobe showed that PN1 mRNA is present throughout the placenta and is also abundant in the placental membranes. Immunohistochemical analysis with an anti-PN1 antibody showed co-localization of PN1 and its mRNA within the placenta.
Plasma Factor V Activation Is Prevented by Activated Protein C in the Presence of Phospholipid Vesicles, Not Platelets
Authors: Solymoss Susan , Nguyen Kim Thi Phu 
Published: July 2018

SummaryActivated protein C (APC) is a vitamin K dependent anticoagulant which catalyzes the inactivation of factor Va and VIIIa, in a reaction modulated by phospholipid membrane surface, or blood platelets. APC prevents thrombin generation at a much lower concentration when added to recalcified plasma and phospholipid vesicles, than recalcified plasma and platelets. This observation was attributed to a platelet associated APC inhibitor. We have performed serial thrombin, factor V one stage and two stage assays and Western blotting of dilute recalcified plasma containing either phospholipid vesicles or platelets and APC. More thrombin was formed at a given APC concentration with platelets than phospholipid. One stage factor V values increased to higher levels with platelets and APC than phospholipid and APC. Two stage factor V values decreased substantially with platelets and 5 nM APC but remained unchanged with phospholipid and 5 nM APC. Western blotting of plasma factor V confirmed factor V activation in the presence of platelets and APC, but lack of factor V activation with phospholipid and APC. Inclusion of platelets or platelet membrane with phospholipid enhanced rather than inhibited APC catalyzed plasma factor V inactivation. Platelet activation further enhanced factor V activation and inactivation at any given APC concentration.Plasma thrombin generation in the presence of platelets and APC is related to ongoing factor V activation. No inhibition of APC inactivation of FVa occurs in the presence of platelets.