bluuebunny/crossref_metadata_2025 huggingface dataset:
Subcutaneous Low-Molecular Weight Heparin or Oral Anticoagulants for the Prevention of Deep-Vein Thrombosis in Elective Hip and Knee Replacement?
Authors: Hamulyák Karly , Lensing Anthonie W A , van der Meer Jan , Smid Willem M , van Ooy André , Hoek Jaap A ,   for the Fraxiparine Oral Anticoagulant Study Group
Published: July 2018

Objective. To compare efficacy, safety, and feasibility of adjusted- dose oral anticoagulants (OAC) versus fixed-dose subcutaneous low molecular weight heparin (LMWH) for the prevention of deep venous thrombosis (DVT) in patients who have undergone elective hip or knee replacement.

Design. Multicentre, single blind randomised trial. OAC (acenocoumarol, target International Normalised Ratio, 2.0-3.0) and LMWH (nadroparine, 60 aXa IU/kg once daily) were started preoperatively and continued for 10 days. All outcome measures were adjudicated by an independent committee unaware of treatment allocation.

Subjects. 672 consecutive patients scheduled for elective hip or knee replacement surgery. All patients wore bilateral graduated compression stockings.

Main outcome measures. The endpoint for the assessment of efficacy was venography confirmed DVT or confirmed symptomatic pulmonary embolism. The endpoint for the assesment of safety was clinically important bleeding during study treatment or within 48 h of the end of treatment.

Results. Among the 517 patients with interpretable venograms, 391 had a hip replacement and 126 had a knee implant. DVT was demonstrated in 50 (20%) of 257 patients allocated to OAC and 43 (17%) of 260 patients allocated to nadroparine (p = 0.45), for an absolute difference in DVT incidence of 2.9% in favour of nadroparine (95% Cl, -3.7-9.5). Clinically important bleeding occurred in eight (2.3%) of the 342 oral anticoagulant treated patients and in five (1.5%) of the 330 nadroparine treated patients (p = 0.62), for an absolute difference in favour of nadroparine of 0.8% (95% Cl, -1.3-2.9).

Conclusion. Patients who undergo major orthopaedic operations have a high risk of venous thromboembolism. Once daily fixed-dose subcutaneous nadroparine is at least as efficacious and safe as daily adjusted OAC for prophylaxis against DVT after hip or knee implantation but is more simple to administer.
Multi-Center Calibration of the Third BCR Reference Material for Thromboplastin, Rabbit, Plain, Coded CRM 149S
Authors: van den Besselaar A M H P
Published: July 2018

In a collaborative study by eleven laboratories performed within the framework of the European Community Bureau of Reference (BCR), a third reference material for thromboplastin, rabbit, plain, has been calibrated against RBT/90, the current WHO international reference preparation for thromboplastin, rabbit, plain. This third reference material (coded CRM 149S) has a mean International Sensitivity Index (ISI)of 1.257 with a standard error of the mean of 0.013.

The previous BCR reference material for thromboplastin, rabbit, plain (coded CRM 149R) and the reference material for thromboplastin, bovine, combined (coded OBT/79) were also included in the trial for assessment of the long-term stability of the ISI values. The relationship between ISI values determined in the present study was nearly identical to that of the historical values. These results offer reassurance with regard to the long-term stability of these reference materials.
The Pharmacokinetics and Lipoprotein Fraction Distribution of Intramuscular vs. Oral Vitamin K1Supplementation in Women of Childbearing Age: Effects on Hemostasis
Authors: Hagstrom J Nathan , Bovill Edwin G , Soll Roger F , Davidson Kenneth W , Sadowski James A
Published: July 2018

Prenatal maternal vitamin K1 supplementation to improve the hemostatic status of the fetus may depend upon the route of administration and subsequent presentation at the placental barrier. We investigated intramuscular (IM) vs oral (PO) vitamin K1 supplementation in eight healthy, nonpregnant women of childbearing age. Pharmacokinetics were studied in each subject after a 5 mg IM dose and after a 5 mg oral dose of vitamin K1 approximately one month later. Plasma collected at the peak vitamin K level for each treatment was separated into very low density lipoproteins (VLDL), low density lipoprotein (LDL), high density lipoprotein (HDL) and lipoprotein-free fractions by density gradient ultracentrifugation. Vitamin K1 was measured in the plasma and lipoprotein fractions using HPLC. The concentration of vitamin K1, in plasma reached a peak 2 h after an IM dose and remained high throughout the 30 h course of the study. In contrast, the oral dose of vitamin K1 peaked at 4 h and rapidly decreased to near baseline by 18 to 30 h. The distribution of vitamin K1 in the lipid fractions was different for IM compared to PO. The percentage of vitamin K1 in the VLDL fraction at the peak for an oral dose was significantly higher than for an IM dose (80.8% ± 3.5 vs 10.8% ± 6.5, p <0.0001). After the oral absorption stage, the subjects took 5 mg of vitamin K1 orally, once a day, for 12 days. No significant differences were observed for the following coagulation proteins and hemostatic markers measured immediately before and after long-term oral vitamin K supplementation: factor II, factor VII, protein C, and thrombin-antithrombin III complex. In conclusion, physiological processing of supplemented vitamin K1 differs in the IM vs PO routes of administration and 12 days of oral vitamin K1does not alter the concentration of selected vitamin Independent coagulation proteins or thrombin-antithrombin complex generation
Cultured Human Mesangial Cells Produce both Type 1 and Type 2 Plasminogen Activator Inhibitors
Authors: Colucci Mario , Gesualdo Loreto , Montemurro Pasqualina , Cavallo Luca G , Conese Massimo , Mascolo Elisa , Ranieri Elena , Di Paolo Salvatore , Schena Francesco P , Semeraro Nicola
Published: July 2018

Cultured human mesangial cells (HMC) derived from normal kidneys have been shown to synthesize tissue-type plasminogen activator (t-PA) and excess amounts of PA inhibitor type 1 (PAI-1). Conflicting results have been obtained concerning the production of urokinase-type PA (u-PA) and efforts to show PA inhibitor 2 (PAI-2) met with failure. We evaluated the fibrinolytic profile of cultured HMC lines obtained from 12 patients with renal carcinoma and one cadaveric kidney donor. Subconfluent cells (third passage) were incubated overnight in serum-free medium. t-PA, u-PA, PAI-1 and PAI-2 antigens were assayed by ELISA methods and PA and PAI activities by amidolytic methods both in conditioned medium (CM) and cell extracts (CE). Besides PAI-1, PAI-2 antigen was detected in all but one HMC lines. At variance with the former, which was largely released in the culture medium, PAI-2 was mainly cell-associated. t-PA antigen was found in all but two cell lines while u-PA antigen was detected in relatively high concentrations in 8 cell lines. PA activity, identified as u-PA by functional and immunological criteria, was measured in CM of six of the eight u-PA producing cell lines, whereas PAI activity was undetectable or very low in CM of all cell lines, suggesting that PAI-1 was largely inactive. Functional assays of cell extracts demonstrated the presence of PA activity, again identified as u-PA, only in samples (five lines) containing u-PA antigen in excess over PAI-2. PAI activity was found instead in the extracts in which the inhibitor was higher than the activator (six lines) and was identified as PAI-2, as it inhibited u-PA but not single-chain t-PA and was neutralized by a polyclonal anti-PAI-2 antibody. The heterogeneous fibrinolytic pattern of HMC lines was confirmed by mRNA analysis of three representative lines. Results were similar when HMC lines at passage five were used, except that the u-PA content was significantly reduced both in CM and CE. These findings indicate that the fibrinolytic profile of cultured HMC is more complex than previously reported. The production of large amounts of PAI-2 may represent an additional control mechanism of proteinase activity.
Inhibitors of Plasminogen Activator in Neutrophils and Mononuclear Cells from Septic Patients
Authors: Haj Montaser A , Robbie Linda A , Adey Gillian D , Bennett Bruce
Published: July 2018

Leucocytes, both polymorphs and mononuclear cells, play a variety of roles in the evolution of human response to sepsis, both local and generalised. In this study, inhibitors of plasminogen activator were measured in leucocytes from normal and septic patients. Plasminogen activator inhibitor-1 (PAI-1) was identified in polymorphs from normal individuals and levels rose significantly in polymorphs from septic patients: neutrophils from normal subjects did not contain PAI-2 but this protein was detectable in significant quantities in polymorph preparations from septic patients. In contrast, mononuclear cells from normal and septic patients contained no detectable quantities of PAI-1. Significant amounts of PAI-2 were present in normal mononuclear cells, and the levels rose significantly in monocytes from septic patients. PAI-2 is thus here identified in human subjects, distinct from those with pregnancy or malignancy, as playing a role in a pathological process. The increased levels of both inhibitors produced by leucocytes may clearly contribute directly to the persistence of fibrin, a characteristic feature of the response to infection, local or general; they may thus participate in successful localisation of infections (abscess formation etc.) and in the evolution of the major systemic complications of disseminated sepsis characterised by microvascular occlusion by fibrin such as renal failure, shock lung or digital ischaemia.
A New Method for the Assay of Exposed Platelet Fibrinogen Receptor Using a Chemiluminescent Label
Authors: Katoh Makoto , Chishima Susumu , Kiuchi Nobukazu , Ikeo Tomihiro , Sasaki yasuhiko
Published: July 2018

Assay of the platelet fibrinogen-binding receptor glycoprotein (GP) IIb/IIIa is widely performed using 125I-labeled fibrinogen (125I-fibrinogen). We successfully devised a receptor binding assay system with high selectivity and sensitivity using a stable chemiluminescent acridinium derivative I-labeled fibrinogen (acridinium-fibrinogen).

Human fibrinogen in saline was labeled with equimolar acridinium dissolved in dimethylformamide, and allowed to react with gel-filtered human platelets in the presence of ADP. Acridinium-fibrinogen binding to GPIIb/IIIa was assayed by measuring chemiluminescence emitted on addition of 0.1 N NaOH containing 0.06% H202 in a luminometer. Non-specific binding was measured in the presence of 10 mM EDTA. Acridinium-fibrinogen binding to human platelets was rapid and reversible, specific and saturable, and dependent on ADP concentrations. Scatchard plot analysis revealed one class of binding sites with a Kd of 326 nM and Bmax of 7.8 pmol/108 platelets. These values were comparable to the data obtained by using 125I-fibrinogen. Unlabeled fibrinogen, RGDS, and HHLGGAKQAGDV (fibrinogen γ-chain 400-411) displaced acridinium-fibrinogen from its binding site with Ki values of 322 nM, 9.2 μM and 31.3μM, respectively. Thus, this binding assay system may be useful in measuring the binding between platelet GPIIb/IIIa and fibrinogen without using a radioisotop.
The Effect of Cigarette-Smoking on Cardiovascular Risk Factors: a Study of Monozygotic Twins Discordant for Smoking
Authors: Calori Giliola , D’Angelo Armando , Della Valle Patrizia , Ruotolo Giacomo , Ferini-Strambi Luigi , Glusti Cristina , Errera Antonino , Gallus Giuseppe
Published: July 2018

The association of cigarette smoking with the development of occlusive vascular disease is firmly established. Unfavourable changes in a series of variables held independent risk factors for the development of vascular lesions (HDL-cholesterol, haematocrit, white blood cell count, fibrinogen and plasminogen activator inhibitor-1 (PAI-1)) are thought to be directly influenced by cigarette smoking. However, the role played by the genotype in the effect of smoking on the above parameters has not been investigated. To control the genotype, we studied the relationship between cigarette smoking and a series of cardiovascular risk factors in 27 monozygotic twin pairs (7 male and 20 female pairs, mean age ± SD: 47.4 ± 12.9 yrs) with a life-long discordance for smoking. Smoking twins had a life-long dose of smoking (Brickman index) of 287.3 ± 241.5. Body mass index, blood pressure, haematocrit, haemoglobin and red blood cell counts, total cholesterol levels and the acute phase reactants α-acid glycoprotein and C-reactive protein were similar in smokers and non-smokers. Triglyceride was higher by 12.6% (9.5-35%, 95% confidence interval, p = 0.02) and HDL-cholesterol lower by 7.5% (0.2-15%, p = 0.04) in the smoking co-twins, who also had 8.4 % (-0.2-17%, p = 0.06) higher white blood cell counts and 4.1% (1.2-7%, p <0.01) larger mean platelet volume. There was no significant difference in clottable fibrinogen (by two methods) or in the activity of plasminogen activator inhibitor-1 between the two groups, nor was the within-pair difference in these parameters related to the smoking dose. Echo-doppler examination of the carotid arteries of 24 twin pairs showed mostly minor atherosclerotic lesions in 46% and 42% of the smoking and non-smoking co-twins. After adjustment for age, systolic blood pressure and platelet count and volume were the only variables significantly associated to the presence of vascular lesions. Cigarette smoking is associated with an atherogenic lipid profile and with changes in platelets and white cells potentially reflecting endothelial cell damage. When controlling the genotype, fibrinogen and PAI-1 activity levels did not seem directly influenced by cigarette smoking.
Detection of New Polymorphic Markers in the Factor V Gene: Association with Factor V Levels in Plasma
Authors: Lunghi B , Iacoviello L , Gemmati D , Dilasio M G , Castoldi E , Pinotti M , Castaman G , Redaelli R , Mariani G , Marchetti G , Bernardi F
Published: July 2018

Three novel polymorphisms were found in the repeated region of the large exon 13 of factor V gene, one giving rise to a codon dimorphism (Serl240) and two causing aminoacid substitutions (Hisl299Arg, Leul257Ile). An increasing frequency of the Argl299 (R2 allele) correlated with a decreasing mean plasma factor V activity in the groups of subjects under study, which included 26 unrelated subjects with partial factor V deficiency. Family studies supported the co-inheritance both of low factor V activity and of R2 allele. The reduction of factor V activity associated with the R2 allele was not clinically symptomatic even in the homozygous condition and was characterized by a parallel reduction of antigen in plasma, in which abnormal molecules were not detected. Data suggest that the R2 allele represents a marker in linkage with an unknown defect rather than a functional polymorphism.

These studies provide the first evidence of a genetic component in determining factor V levels in plasma and of a genetic linkage between the factor V gene and factor V deficiency. They also define specific haplotypes which are associated with factor V deficiency or with APC resistance (Arg506Gln) and are valuable fools for the study of factor V defects.
Substitution of Serine or Threonine at Position 473 of Tissue-type Plasminogen Activator Increases its Stability in Plasma
Authors: Alibhai Murtaza , Chow Alice M , Paoni Nicholas F
Published: July 2018

Inactivation by slow acting inhibitors in plasma is of little consequence for thrombolysis with wild type t-PA, since it is rapidly cleared from the blood stream and constantly replenished through infusion. However, it becomes increasingly important as the clearance rate of t-PA is reduced, through mutagenesis, to enable the molecule to be long acting and administered by a single bolus injection. The substitution of serine for alanine at position 473 substantially reduced the slow inactivation that occurs at pharmacological levels of t-PA in plasma. Approximately 70% of the activity of A473S remained after 4 h incubation in human plasma compared to approximately 25% for wild type t-PA. Wild type t-PA and A473S showed the same stability in alpha-2-antiplasmin depleted plasma, indicating that the resistance of A473S to inactivation is a result of reduced reactivity towards alpha-2-antiplasmin, the primary slow acting inhibitor of t-PA. The second order rate constant for the inactivation of A473S by purified alpha-2-antiplasmin was approximately 4 fold less than that of wild type t-PA, which is consistent with the results obtained in plasma. Substitution of threonine at position 473 also produced inhibitor resistance, but glycine did not. The substitution of charged or bulky residues at position 473 destroyed enzymatic activity. The mechanism of inhibitor resistance for A473S and A473T appears to be a reduced reactivity towards substrates with arginine at the PI position. The A473S mutation adds well to T103N, a mutation that causes an approximate 9 fold reduction in the clearance rate of t-PA. The double mutation variant, T103N, A473S had normal plasma clot lysis activity, and was stable in plasma over a 4 h incubation period at 37° C in vitro.
Fibrinolytic Response to lnterferon-a in Healthy Human Subjects
Authors: Corssmit E P M , Levi M , Hack C E , ten Cate J W , Sauerwein H P , Romijn J A
Published: July 2018

Interferons (IFNs) are used for a variety of disorders. It has been postulated that part of the effects of IFN may be mediated by IFN-induced modulation of endothelial cells. Since the principal activating and inhibiting factors of the fibrinolytic system are synthesized and stored in endothelial cells, we have studied the effects on fibrinolysis and coagulation of the administration of recombinant IFN-α (5 × 106U/m2) to healthy human subjects (n = 8) in a randomized controlled cross-over study. IFN-α significantly increased plasma levels of tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA). Simultaneously, plasma levels of the inhibitor of plasminogen activation, PAI-1, sharply increased. The net effect on plasma plasminogen activator activity (PA-activity) was a modest increase to 116% of baseline, however without a significant effect on plasmin generation, as reflected by plasma levels of plasmin-α2-antiplasmin complexes. IFN-α had no effect on the plasma levels of thrombin-antithrombin III (TAT) complexes.

We conclude that despite considerable effects on endothelial cells, IFN-α does not significantly alter the coagulant-fibrinolytic balance, although the occurrence of such changes under pathological circumstances is not excluded.
Anti-thrombotic Effect of a PAI-1 Inhibitor in Rats Given Endotoxin
Authors: Abrahamsson T , Nerme V , Strömqvist M , Åkerblom B , Legnehed A , Pettersson K , Westin Eriksson A
Published: July 2018

The aim of this study was to investigate the anti-thrombotic effects of an inhibitor of the plasminogen activator inhibitor-1 (PAI-1) in rats given endotoxin. In studies in vitro, PRAP-1, a Fab-fragment of a polyclonal antibody against human PAI-1, was shown to inhibit PAI-1 activity in rat plasma as well as to stimulate clot-lysis of the euglobulin fraction derived from rat plasma. Endotoxin administered to anaesthetised rats produced a marked increase in plasma PAI-1 activity. To study fibrin formation and lysis in vivo after intravenous (i. v.) injection of the coagulant enzyme batroxobin, 125I-fibrinogen was administered to the animals. The thrombi formed by batroxobin were rapidly lysed in control animals, while the rate of lysis was markedly attenuated in rats given endotoxin. PRAP-1 was administered i.v. (bolus + infusion) to rats given endotoxin and batroxobin and the PAI-1 inhibitor caused a dose-dependent decrease in the 125I-fibrin deposition in the lungs. An immunohistochemical technique was used to confirm this decrease in density of fibrin clots in the tissue. Furthermore, PRAP-1 decreased plasma PAI-1 activity in the rats and this reduction was correlated to the decrease in lung 125I-fibrin deposition at the corresponding time point. It is concluded that in this experimental model the PAI-1 antibody PRAP-1 may indeed inhibit thrombosis in animals exposed to endotoxin.
Proteins of the Fibrinolytic System in Human Thrombi
Authors: Robbie Linda A , Bennett Bruce , Croll Alison M , Brown Paul A J , Booth Nuala A
Published: July 2018

The proteins of fibrinolysis have been quantified in human thrombi, to assess the balance between plasminogen activators and their major inhibitor PAI-1. The relative roles of PAI-1 and α2-AP were also examined since we have previously shown that both platelet PAI-1 and plasma α2-AP are important determinants of clot lysis in vitro. Extracts and sections were prepared from human thrombi for quantitative immunoassay and immunohistochemical staining respectively. PAI-1 and α2-AP were present at high concentrations. Levels of t-PA and t-PA-PAI-1 complex were relatively low. Staining confirmed the presence of abundant PAI-1, associated primarily with platelet material within the thrombus and also with fibrin. Staining for α2-AP was also intense and demonstrated strong association with fibrin. The α2-AP concentration was similar to its high plasma concentration, whereas PAI-1 levels were up to 30 times greater than that in circulating blood, suggesting that active recruitment of platelets contributes to the high PAI-1 concentration in thrombi.
Fibrin Binding of Plasminogen Coated Liposomes In Vitro
Authors: Heeremans J L M , Los P , Prevost R , Crommelin D J A , Kluft C
Published: July 2018

In this study, the fibrin binding properties of liposomes containing a number of plasminogen (Pig) molecules on the outside were compared to those of free (non-liposomal) Pig in an in vitro model system. Fibrin monolayer coated 96-wells plates were used, containing fibrin monomer at a density of around 3.4 to 3.9 × 10-4 nmol/cm2. These densities are similar to liposomal Plg-densities, thus allowing multivalent interactions to occur.

In the panel of experimental conditions that was chosen, binding of free Pig and liposomes with Pig showed three main differences in characteristics. Firstly, in the fibrin binding of Plg-liposomes not all Pig may be involved, but on the average 40% of the total amount of liposomal Pig. This was shown by lysing the liposomes after binding to the fibrin and estimation of truly bound Pig. With Plg-densities on the liposomes below the fibrin binding sites density, the maximal number of bound Pig molecules remains below the amount of available fibrin binding sites. Secondly, a higher binding rate by at least one order of magnitude was observed for liposomes with Pig compared to free Pig. Thirdly, liposomes with Pig exhibit a fibrin binding affinity which increases with Plg-density, because of the multivalent character of interaction. Liposomal Pig can successfully compete for fibrin binding sites with a 100 fold higher concentration of free Pig.

These in vitro findings indicate that in view of avid and rapid fibrin binding, liposomes with attached plasminogen may be suitable for in vivo targeting to fibrin based thrombi.
Heparin Reverses the Procoagulant Properties of Stimulated Endothelial Cells
Authors: Cadroy Y , Gaspin D , Dupouy D , Lormeau J C , Boneu B , Sié P
Published: July 2018

We examined the ability of unfractionated heparin to modulate the procoagulant activities of stimulated endothelial cells (EC). Confluent human venous umbilical EC were incubated with heparin before or after stimulation, then rinsed extensively to eliminate any heparin in the solution. EC, stimulated for 4 h with endotoxin and interleukin 1β, expressed tissue factor and prothrombinase activities. When EC were treated with heparin (6 and 60 μg/ml) during the last 10 min of the stimulation period, EC-related procoagulant activities were inhibited in a dose-dependent manner (80-90% inhibition at 60 μg/ml). The inhibition was antithrombin-dependent and it disappeared after heparin removal in less than 15 min at 37° C but persisted at 4° C.

When EC were treated with heparin (60 μg/ml) for 24 h then extensively washed before stimulation, the anticoagulant effect was more modest (50% inhibition). The effect was antithrombin-dependent. Inhibition was maximum after 18-24 h of pretreatment of EC with heparin and was stable for at least 7 h. The cell surface displayed a “heparin-like” activity: treatment by heparin doubled the rate of thrombin-antithrombin complex formation and this effect was heparinase sensitive and chondroitinase ABC insensitive.

Thus, heparin modulates the procoagulant properties of stimulated EC according to two distinct mechanisms. The first one is rapid and transient, probably related to the presence of heparin molecules bound at the membrane surface. The second is delayed and persistent, and our results suggest that it is mediated by an increase in the membrane heparan sulfate molecules.
Platelet Density and Size in Inflammatory Bowel Disease
Authors: Järemo P , Sandberg-Gertzen H
Published: July 2018

Objective: The present study investigates platelet density and mean platelet volume (MPV) in active inflammatory bowel disease (IBD).

Experimental protocol: 27 patients with IBD (18 ulcerative colitis and 9 Crohn’s disease) were compared to 12 healthy volunteers. 18 subjects had active disease. Platelet counts and mean platelet volume (MPV) were determined. Thereafter, platelets were separated according to density on a linear preformed Percoll gradient. A computerized device was employed for scanning transmission variations and thus the platelet distribution in the gradient and the platelet peak were identified.
Monoclonal Antibodies to Human Fibrin: Interaction with other Animal Fibrins
Authors: Edgell Tracey , McEvoy F , Webbon P , Gaffney P J
Published: July 2018

Four monoclonal antibodies have previously been raised in our laboratory for possible use in thrombus imaging and the targeting of thrombolytic agents. These antibodies were raised to various human fibrin-related immunogens and each antibody had been selected for its specificity towards fibrin and not fibrinogen. To study further these antibodies in animal circulation models both in vivo and in vitro, their selectivity towards human fibrin as opposed to other animal fibrins was examined. In this study dissociation constants for each antibody with each of six species fibrins (human, baboon, pig, dog, sheep, and rabbit) were estimated using both fibrin clots and monolayers. Some limited data were also obtained with Sepharose-fibrin. Of the antibodies two (denoted 12B3B10 and 12B3A11) are seen to bind almost exclusively to human fibrin with dissociation constants of about 8 × 10−10 M using fibrin clots and monolayers. These same two mabs bound to baboon fibrin with a dissociation constant of 2 × 10−9 M, while neither displayed significant levels of binding to the fibrins from dog, pig, sheep and rabbit. The other two antibodies investigated (1H10 and 5F3) were found to bind well to fibrins of human, baboon, pig and dog, with dissociation constants in the range of 1.4-4.2 × 10−9 M. However neither 1H10 nor 5F3 displayed significant recognition of sheep and rabbit fibrins. Both 1H10 and 5F3 were also found by means of competitive ELISA’s to retain their selectivity to baboon, dog and pig fibrins in the presence of their respective fibrinogens.
The Lysosomal Granule Membrane Protein, Lamp-2, Is also Present in Platelet Dense Granule Membranes
Authors: Israels S J , McMillan E M , Robertson C , Singhroy S , McNicol A
Published: July 2018

Lysosomal Associated Membrane Protein-2 (LAMP-2) is an inherent component of lysosomal granule membranes in diverse cell types, including platelets. We examined platelets for evidence of LAMP-2 in dense granule membranes as CD63 has previously been shown to be present in both lysosomal and dense granule membranes. Immunological techniques were used to examine the localization of LAMP-2 in control platelets and those from an individual with Hermansky-Pudlak syndrome (HPS), a condition characterised by platelet dense granule deficiency. Immunoblotting studies demonstrated that LAMP-2 was enriched in a dense granule preparation. Flow cytometry of thrombin-stimulated control platelets was consistent with biphasic surface expression of LAMP-2. The early expression was accompanied by dense granule, but minimal lysosomal granule, release. The late expression was accompanied by additional lysosomal granule release only. Thrombin stimulation of HPS platelets showed only late, lysosome-associated LAMP-2 expression. Immunoelectron microscopy indicated the presence of LAMP-2 in the membranes of serotonin-containing granules as identified by an anti-serotonin polyclonal antibody. These data indicate that LAMP-2 is present in the membranes of platelet dense granules in addition to lysosomal granules, and has a similar distribution to CD63.
Platelet Aggregation and Adhesion during Dietary Copper Deficiency in Rats
Authors: Lominadze David G , Saari Jack T , Miller Frederick N , Catalfamo James L , Justus David E , Schuschke Dale A
Published: July 2018

The role of dietary copper deficiency in platelet-to-endothelial cell adhesion and in platelet-to-platelet aggregation was studied in vitro. Platelets were obtained from male, weanling Sprague-Dawley rats fed purified diets which were either copper-adequate (CuA, 6.3 μg copper/g of diet) or copper-deficient (CuD, 0.3 μg copper/g of diet) for 4 weeks. The platelet adhesion study was performed by adding CuA or CuD platelets either suspended in homologous plasma or in Tyrode buffer salt solution (TBSS) to cultured rat endothelial cells. After a one hour incubation at 37° C non-adhered platelets were removed and counted in a microcytometer. Platelet aggregation in platelet rich plasma (PRP) samples was induced by adding ADP (2 × 10−4 M) and measured in a turbidometric platelet aggregometer. The content of von Willebrand factor (vWF) in platelets and in plasma and the content of fibrinogen in platelets was determined. Platelet adhesion to rat endothelial cells was significantly lower for platelets from CuD rats than for platelets from CuA rats. ADP induced platelet aggregation from CuD rats was significantly higher than platelet aggregation from CuA rats. The content of vWF in platelets and in plasma from CuD rats was significantly lower than in platelets and plasma from CuA rats. However, the amount of fibrinogen in platelets from CuD rats was about 4-fold higher than that in platelets from CuA rats while the plasma fibrinogen was lower in CuD rats than in CuA rats. These studies illustrate that copper deficiency diminishes platelet adhesion to endothelial cells but increases platelet aggregability. The results suggest that these physiological alterations may be the result of decreased platelet vWF and increased platelet fibrinogen during dietary copper deficiency.
Heparin Ablates Force Development during Platelet Mediated Clot Retraction
Authors: Carr Marcus E , Carr Sheryl L , Greilich Philip E
Published: July 2018

Although snake venom enzymes such as reptilase do not cause viscous metamorphosis, platelet secretion or clot retraction; when batroxobin and calcium are added to citrated blood significant platelet force development occurs. When this batroxobin-calcium system was applied to the study of platelet function during cardiopulmonary bypass (CPB), force development was found to be completely inhibited. After heparin reversal by protamine sulfate, significant recovery of force occurred. The present investigation was performed to evaluate the role of heparin in reducing force development during CPB. At concentrations above 0.10 U/ml, heparin totally suppressed force development in normal plasma. Addition of protamine sulfate to heparinized plasma caused complete recovery of force development. These concentrations of heparin had little effect on platelet aggregation by ADP or collagen. Possible direct effects of heparin on fibrin assembly and structure were studied by adding varying amounts of heparin to plasma and then inducing clot formation with batroxobin. At 1 U/ml, heparin reduced the size of fibrin fibers by 33%. Higher heparin concentrations had no additional effect. These results indicate that heparin may be responsible for a significant component of the decreased platelet force noted during cardiopulmonary bypass. To test whether heparin’s effect could be due to suppression of thrombin activity, the effects of the antithrombin hirudin on force development were measured. Hirudin also inhibited force development in a concentration dependent manner. Thus, heparin’s reduction of platelet force development may be due, at least in part, to suppression of thrombin activity.
Hypercoagulable State in Patients with Takayasu’s Arteritis
Authors: Akazawa Hiroshi , Ikeda Uichi , Yamamoto Keiji , Kuroda Toshio , Shimada Kazuyuki
Published: July 2018

Thrombus formation is recognized pathologically in the affected arteries and is supposed to play a major role in the pathogenesis of Takayasu’s arteritis; however, hemostatic conditions in this disorder have not been elucidated fully. We determined plasma levels of molecular markers for platelet activity (platelet factor 4; PF4, β-throm-boglobulin; βTG), thrombotic status (thrombin-antithrombin III complex; TAT, fibrinopeptide A; FPA), fibrinolytic status (plasmin-β2-plasmin inhibitor complex; PIC, D-dimer), and endothelial injury (von Willebrand factor antigen; vWF:Ag, thrombomodulin; TM) in 30 patients with Takayasu’s arteritis and in 20 age-matched control subjects. Plasma levels of PF4, βTG, TAT, FPA and D-dimer, but not PIC, in patients with Takayasu’s arteritis were substantially higher than those in normal control subjects. The levels of these markers were not different between the active and inactive stages of the disease. Plasma levels of vWF:Ag in patients with Takayasu’s arteritis did not differ significantly from those in normal subjects, and plasma levels of TM were significantly lower than those in normal subjects. In patients with Takayasu’s arteritis, platelet and coagulation activities are significantly increased, leading to hypercoagulable state and thrombus formation, although there is little, if any, endothelial damage.
Predicting Daily Maintenance Dose of Fluindione, an Oral Anticoagulant Drug
Authors: Cazaux V , Gauthier B , Elias A , Lefebvre D , Tredez J , Nguyen F , Cambus J P , Boneu B , Boccalon H
Published: July 2018

Due to large inter-individual variations, the dose of vitamin K antagonist required to target the desired hypocoagulability is hardly predictible for a given patient, and the time needed to reach therapeutic equilibrium may be excessively long. This work reports on a simple method for predicting the daily maintenance dose of fluindione after the third intake. In a first step, 37 patients were delivered 20 mg of fluindione once a day, at 6 p.m. for 3 consecutive days. On the morning of the 4th day an INR was performed. During the following days the dose was adjusted to target an INR between 2 and 3. There was a good correlation (r = 0.83, p<0.001) between the INR performed on the morning of day 4 and the daily maintenance dose determined later by successive approximations. This allowed us to write a decisional algorithm to predict the effective maintenance dose of fluindione from the INR performed on day 4. The usefulness and the safety of this approach was tested in a second prospective study on 46 patients receiving fluindione according to the same initial scheme. The predicted dose was compared to the effective dose soon after having reached the equilibrium, then 30 and 90 days after. To within 5 mg (one quarter of a tablet), the predicted dose was the effective dose in 98%, 86% and 81% of the patients at the 3 times respectively. The mean time needed to reach the therapeutic equilibrium was reduced from 13 days in the first study to 6 days in the second study. No hemorrhagic complication occurred. Thus the strategy formerly developed to predict the daily maintenance dose of warfarin from the prothrombin time ratio or the thrombotest performed 3 days after starting the treatment may also be applied to fluindione and the INR measurement.
Instrument Effect on the Activated Protein C Resistance Plasma Assay Performed by a Commercial Kit
Authors: De Stefano V , Paciaroni K , Mastrangelo S , Rutella S , Bizzi B , Leone G
Published: July 2018

In order to evaluate the influence of the coagulation instrument on the activated protein C (APC)-resistance plasma assay performed by a commercial kit, we tested 70 plasma samples on 4 different instruments during a simultaneous session run using a same lot of Coatest APC-resistance (Chromogenix). The results were analyzed employing three different modes of expression (aPTT prolongation in the presence of APC, APC-sensitivity ratio, normalized APC-sensitivity ratio) and three different diagnostic threshold values (below the control mean −2 standard deviations or the lowest control value or the 5th percentile of the control values). The inter-instruments variability in the mean values of the control individuals can be limited expressing the results as nor-malized-APC-sensitivity ratio (range 0.99-1.05). The overall diagnostic yield in thrombotic patients and their relatives depended mainly on the instrument employed and only in some cases on the mode of expression of the results and on the diagnostic threshold value. The sensitivity of the commercial assay on heterozygotes for factor V Leiden diagnosed by gene analysis was overall satisfactory (75-100%) but in some cases a lower diagnostic yield was noticed, depending on the type of instrument employed and/or the type of expression of the results and/or the diagnostic threshold values. Thus the instrument system adopted should be carefully considered in the interpretation of the results using the commercial kit.
Fibrinogen and Factor Vllag in Healthy Adolescents: The Floren-teen (Florence Teenager) Study
Authors: Prisco Domenico , Fedi Sandra , Brunelli Tamara , Cellai Anna Paola , Hagi Mohamed Isse , Gianni Roberto , Santoro Emilio , Cappelletti Carlo , Pepe Guglielmina , Gensini Gian Franco , Abbate Rosanna
Published: July 2018

At least five studies based on more than twenty thousand healthy subjects indicated that fibrinogen is an independent risk factor for cardiovascular events; less clear-cut is the relation between factor VII and risk for arterial thrombotic disorders, which was demonstrated in two of the three studies investigating this association. However, no study has investigated the behaviour of fibrinogen and factor VII in an adolescent population. In a study of Preventive Medicine and Education Program, fibrinogen (clotting method) and factor Vllag (ELISA), in addition to other metabolic parameters, life-style and familial history, were investigated in 451 students (313 females and 138 males, age 15-17 years) from two high schools of Florence. Fibrinogen levels were significantly higher in women than in men, whereas factor Vllag levels did not significantly differ. Both fibrinogen and factor Vllag significantly correlated with total cholesterol (p <0.05) while only fibrinogen correlated with body mass index (p <0.01). Factor Vllag was significantly correlated with systolic blood pressure (p <0.001). This study provides information on coagulation risk factors in a population of adolescents which may be of importance in planning coronary heart disease prevention programs.
Macrophages and Neutrophils Infiltrating into the Liver Are Responsible for Tissue Factor Expression in a Rabbit Model of Acute Obstructive Cholangitis
Authors: Higure Aiichiro , Okamoto Kohji , Hirata Keiji , Todoroki Hidekazu , Nagafuchi Yukihisa , Takeda Shigeaki , Katoh Hidenori , Itoh Hideaki , Ohsato Keiichi , Nakamura Shin
Published: July 2018

Acute obstructive cholangitis (AOC) is one of the most fatal outcomes in sepsis, and frequently complicates disseminated intravascular coagulation (DIC). Recently we found that the plasma tissue factor (TF) level increased and changed in parallel with plasma markers of DIC in patients with AOC. To elucidate the role of TF in the pathogenesis of coagulopathy in AOC, we investigated the plasma levels of TF and its localization by immunohistochemical staining in rabbit models of AOC. Plasma TF activity significantly increased 3 h after the insult (0.63 ± 0.19 U/ml; p <0.01) compared with that beforehand (0.05 ± 0.02 U/ml), then reached a maximum level at 6 h (0.94 ±0.16 U/ml). The fluctuations in plasma TF activity correlated with those of the coagulation parameters including platelet count, fibrinogen, prothrombin time, and antithrombin III activity. Immunohistochemically, enhanced expression of TF was mainly detected in macrophages and neutrophils that had infiltrated into the liver sinusoids and around the bile duct, but not in the sinusoidal endothelial cells. A double immunofluorescence study revealed the concomitant presence of TF and fibrin at sites where macrophages and neutrophils had conglomerated. However, we could not detect an apparent change in TF expression in the lung or kidney. These data suggest that macrophages and neutrophils infiltrating into the liver sinusoids and around the bile duct play a pivotal role in TF expression, leading to coagulopathy in the acute phase of obstructive cholangitis in rabbits.
Factor Xa Enhances the Binding of Tissue Factor Pathway Inhibitor to Acidic Phospholipids
Authors: Valentin Sanne , Schousboe Inger
Published: July 2018

In the present study, the interaction between tissue factor pathway inhibitor (TFPI) and phospholipids has been characterized using a microtitre plate assay. TFPI was shown to bind calcium-independently to an acidic phospholipid surface composed of phosphatidylserine, but not a surface composed of the neutral phosphatidylcholine. The interaction was demonstrated to be dependent on the presence of the TFPI C-terminus. The presence of heparin (1 U/ml, unfractionated) was able to significantly reduce the binding of TFPI to phospholipid. The interaction of TFPI with phosphatidylserine was significantly decreased in the presence of calcium, but this was counteracted, and even enhanced, following complex formation of TFPI with factor Xa prior to incubation with the phospholipid surface. Moreover, a TFPI variant, not containing the third Kunitz domain and the C-terminus, was unable to bind to phospholipid. However, following the formation of a TFPI/factor Xa-complex this TFPI variant was capable of interacting with the phospholipid surface. This indicates that the role of factor Xa as a TFPI cofactor, at least in part, is to mediate the binding of TFPI to the phospholipid surface.
An Experimental Model of Intracranial Hemorrhage during Thrombolytic Therapy with t-PA
Authors: Paoni Nicholas F , Steinmetz Hope G , Gillett Nancy , Refino Canio J , Badillo Julie M , Bunting Stuart
Published: July 2018

Multiple clinical trials have proven that thrombolytic therapy is an effective treatment for acute myocardial infarction. Spontaneous intracranial hemorrhage (ICH) occurs in a small percentage of patients as a result of the treatment. The etiology of the ICH is unknown and there is currently no established experimental model for this side effect. A model of ICH during thrombolytic therapy has been developed using spontaneously hypertensive rats (SHR). The SHR were made susceptible to ICH during thrombolytic therapy by bilateral ligation of the external jugular veins. This procedure produced asymptomatic hemorrhagic lesions in the brains of the animals in the hours preceding the administration of t-PA/heparin. The incidence of ICH following the administration of test substances was assessed by histological examination and by measuring the red blood cell count in a sample of cerebrospinal fluid taken from the atlanto-occipital space. t-PA administration produced a low frequency of ICH in this model. The incidence and severity of ICH were dramatically increased, and significant mortality at 24 h was observed, by combining heparin with the t-PA. No fewer hemorrhages were observed if the t-PA and heparin were administered sequentially rather than simultaneously. Furthermore, ICHs were observed whether the t-PA dose was administered over 4 h, 1 h, or as a double bolus 30 min apart. The potentiation of ICH by heparin was dose dependent and proportional to the prolongation of the aPTT. Although the precise mechanism of ICH during thrombolytic therapy is unknown, many similarities exist between the observations made in this model and in the human clinical experience.
Venous Thrombosis and the “Wessler Test”
Authors: Thomas Duncan P
Published: July 2018

The Wessler test in animals has been used extensively for over 40 years as a laboratory measure of in vivo hypercoagulability. The test, in which induced hypercoagulability is combined with local venous stasis, has contributed significantly to our understanding of the pathogenesis of venous thrombosis and pulmonary embolism. In addition, the Wessler test has proved invaluable for assessing the thrombogenicity of various blood products, as well as for assaying the effectiveness of heparin and heparin fractions.
Evaluation of the Fibrin Binding Profile of two Anti-fibrin Monoclonal Antibodies
Authors: Raut S , Gaffney P J
Published: July 2018

Two anti-fibrin monoclonal antibodies, MAbs 1H10 and 5F3, raised to human freeze-fractured fibrin and thrombin-treated N-terminal disulphide knot (T-NDSK), respectively, were compared for epitope binding to various domains of the fibrinogen/fibrin moiety. Using plasmin-mediated fibrinogen digests, immunoblots showed that both MAbs crossreacted strongly with fragments X and Y, weakly with fragment-E and not at all with fragment D. Purified fragments D and E used in an ELISA confirmed that MAbs 1H10 and 5F3 cross-reacted in a dose-response fashion with the isolated fragment-E, while there was no reaction with fragment-D. The two MAbs were similarly shown to react with fibrin-derived fragment-E. Surface Plasmon Resonance (SPR) technology, employed to further evaluate the epitopes in fibrin, showed that MAb 1 HI0 had a higher affinity for fragment-E (KD = 8.04 × 10-9 M) than MAb 5F3 (KD = 1.13 × 10-8 M). Individual association and dissociation rate constants of 7.97 × 105 M-1S-1 and 3.97 × 10-3S-1, respectively, for MAb 1H10, and 5.16 × 105 M-1s-1 and 3.62 × 10-3s-1, respectively, for MAb 5F3 were also obtained. A SPR inhibition assay confirmed that MAb 1H10 had a greater affinity for fragment-E than MAb 5F3. However individual isolated polypeptide chains of fibrinogen fragment E (E-Aα, E-Bβ, E-γ) showed no reaction with the two antibodies in ELISA, immunoblot or SPR analysis procedures. Furthermore, SPR pair-wise epitope mapping analysis revealed that MAbs 1H10 and 5F3 have in fact distinct epitopes on fragment-E. These distinct epitopes appeared to be a conformational amalgam of linear sequences in two or three of the polypeptide chains of fragment-E, or distinct conformational epitopes on one of the three subunit chains alone.
Liver Steatosis and Its Relation to Plasma Haemostatic Factors in Apparently Healthy Men - Role of the Metabolic Syndrome
Authors: Cigolini M , Targher G , Agostino G , Tonoli M , Muggeo M , De Sandre G
Published: July 2018

The relationship between liver steatosis, evaluated by ultrasonography, and various plasma haemostatic factors was examined in 64 apparently healthy males, aged 38 years. Plasma levels of factor VII clotting activity (F-VIIc), plasminogen activator inhibitor-1 (PAI-1) activity and antigen, tissue-type plasminogen activator (t-PA) activity significantly differed in men with liver steatosis (n = 31) as compared with those without steatosis (n = 33). No significant differences were found in t-PA antigen and F-VII antigen. The men with liver steatosis also had significantly higher body mass index (BMI), plasma triglyceride and 2 h post-load insulin concentrations. While the differences in plasma haemostatic factors were substantially unchanged after adjustment for BMI, they totally disappeared when further allowance was made for plasma triglyceride and 2 h insulin concentrations. In conclusion, these results indicate that liver steatosis correlates specifically with increased PAI-1, F-VIIc and decreased t-PA levels, and suggest that such a relation is largely mediated by concomitant alterations in plasma triglyceride and insulin concentrations.