bluuebunny/crossref_metadata_2025 huggingface dataset:
The In Vitro Production of Thromboxane B2 by Platelets of Diabetic Patients Is Normal at Physiological Concentrations of lonized Calcium
Authors: Falcon Cristina R , Cattaneo Marco , Ghidoni Alberto , Mannucci Pier Mannuccio
Published: July 2018

Platelets of patients with diabetes and no evidence of macroangiopathy produce normal amounts of thromboxane (Tx) B2 in vivo, whereas they usually show increased production in vitro. Since in vitro studies have been usually performed in citrated PRP, we tested the hypothesis that the discrepancy between in vivo and in vitro studies is due to the low concentration of plasma ionized calcium ([Ca
2+
]o) that is present in citrated PRP. In fact, low [Ca
2+
]o artifactually potentiates the platelet TxB2 production in vitro. Forty patients with diabetes mellitus and 37 matched Controls were studied. Blood was anticoagulated with citrate, the thrombin inhibitor D-phenylalanyl-l-prolyl-l-chloromethylketone (PPACK) or both anticoagulants. Platelet aggregation, release of 14C-serotonin and TxB2 production were induced in platelet rieh plasma (PRP) by several agonists. The following results were obtained: i) Citrated PRP: Arachidonic acid induced aggregation (p <0.01) and TxB2 production (p <0.02) were significantly greater in patients than in Controls. No statistically significant differences were found with other agonists. ii) PPACK PRP: No statistically significant difference was found between diabetic platelets and Controls, iii) PPACK plus citrate PRP: The results were not different from those obtained with citrate alone. Therefore, our results show that diabetic platelets produce normal amounts of TxB2 in vitro when the [Ca2+]o is physiological.
Pharmacokinetics of Recombinant Factor VIIa in the Rat – A Comparison of Bio-, Immuno- and Isotope Assays
Authors: Thomsen Mads Krogsgaard , Diness Viggo , Nilsson Povl , Rasmussen Søren Nørgaard , Taylor Trevor , Hedner Ulla
Published: July 2018

Recombinant human factor VII a (rFVIIa) is an activated coagulation factor for intravenous use as a haemostatic agent in haemophiliacs who generate antibodies against factor VIII or IX. Plasma kinetic studies are important for the understanding of the action of rFVIIa which is exerted in the vascular compartment of the body, more specifically on the vessel walls at the site of injury. In the present study, rats were dosed 100 or 500 μg/kg 125I-rFVIIa i. V., without any side effects being observed, and the plasma profile of rFVIIa was studied by 3 different assays that were shown to correlate well at early times post-dose: trichloroacetic acid (TCA)-precipitable drug-related radioactivity, rFVIIa antigen determination by ELISA technique, and the assay of clot activity which is the only clinically applicable assay. The plasma concentration curve could be resolved into 1-3 exponentials, depending on the FVIIa detection principle that was employed. Initially, there was a short (ca. 10 min) phase of increasing concentrations before the attainment of C
max. This was followed by a plasma recovery (C
max × plasma volume/dose) in the vicinity of one half of the administered dose. The initial volume of distribution (V
1) corresponded to the vascular compartment whereas the volume of distribution at steady state (V
ss) was somewhat larger. Whole body clearance (CL-B) of rFVIIa was approx. 1 ml/min per kg, and mean residence time (MRT) and the half-life assumed to be associated with the loss of biological activity was approx. 1 h and 20-45 min, respectively. From these plasma data, rFVIIa appears to be a low clearance compound with limited tissue distribution and a short half-life. Tissue distribution studies showed that high 125I levels, assumed to be rFVIIa-related, included mineralised bone and well-perfused organs such as the liver which suggested that this organ was responsible for a major proportion of CL-B. Finally, mass balance studies showed that almost 90% of the administered radioactivity could be accounted for following an i. v. dose, predominantly as non drug-related radioactivity, even though a small amount of TCA-precipitable radioactivity was excreted via the biliary route. In conclusion, dose- or sex-dependent plasma kinetics and tissue distribution within a dose range of 100 to 500 μg/kg of rFVIIa was not observed. In the early and pharmacologically relevant phase after rFVIIa administration there appears to be good agreement between the various plasma assays employed in the study, indicating that the clot assay yields useful information in studies of rFVIIa plasma pharmacokinetics.
Heparin-Induced Thrombocytopenia (HIT): An Overview of 230 Patients Treated with Orgaran (Org 10172)
Authors: Magnani H N
Published: July 2018

Heparin-induced thrombocytopenia (HIT) with thrombosis occurs in about 1 in 2,000 heparin-treated patients. The arterial or venous thromboses may threaten life and limb hence alternative anticoagulation is needed. Some alternative treatments are possible i.e. LMWH, Ancrod, prostacyclin analogues, Dextran, aspirin and oral anticoagulants, but each has its drawbacks. This report reviews treatment of HIT patients with Orgaran (Org 10172), a low molecular weight heparinoid.

Because of its proven antithrombotic activity Orgaran was used to treat 230 HIT patients. One hundred and fifty, nine patients presented with at least one thrombotic problem, which in 88 was due to the heparin use. 92.8% of the patients were considered to have adequately responded to Orgaran during the treatment period. Fifty-nine deaths (25.7%) occurred of which 7 (3.0%) were attributed to Orgaran use. The remaining 52 deaths, 27 of which occurred after Orgaran treatment was successfully terminated, were due to the severe underlying disorders in these patients. These results and the lower cross-reactivity rate (≈10%) with the heparin-induced antibody compared with that of the LMWH (>90%) suggest that although problems remain, Orgaran can be a valuable alternative treatment for patients who suffer from HIT and who require anticoagulation.
Influence of Platelet Membrane Sialic Acid and Platelet-Associated IgG on Ageing and Sequestration of Blood Platelets in Baboons
Authors: Kotzé H F , Wyk V van , Badenhorst P N , Heyns A du P , Roodt J P , Lötter M G
Published: July 2018

Platelets were isolated from blood of baboons and treated with neuraminidase to remove platelet membrane sialic acid, a process which artificially ages the platelets. The platelets were then labelled with 111In and their mean life span, in vivo distribution and sites of Sequestration were measured. The effect of removal of sialic acid on the attachment of immunoglobulin to platelets were investigated and related to the Sequestration of the platelets by the spleen, liver, and bone marrow. Removal of sialic acid by neuraminidase did not affect the aggregation of platelets by agonists in vitro, nor their sites of Sequestration. The removal of 0.51 (median, range 0.01 to 2.10) nmol sialic acid/108 platelets shortened their life span by 75 h (median, range 0 to 132) h (n = 19, p <0.001), and there was an exponential correlation between the shortening of the mean platelet life span and the amount of sialic acid removed. The increase in platelet-associated IgG was 0.112 (median, range 0.007 to 0.309) fg/platelet (n = 25, p <0.001) after 0.79 (median, range 0.00 to 6.70) nmol sialic acid/108 platelets was removed (p <0.001). There was an exponential correlation between the shortening of mean platelet life span after the removal of sialic acid and the increase in platelet-associated IgG. The results suggest that platelet membrane sialic acid influences ageing of circulating platelets, and that the loss of sialic acid may have exposed a senescent cell antigen that binds IgG on the platelet membrane. The antibody-antigen complex may then provide a signal to the macrophages that the platelet is old, and can be phagocytosed and destroyed.
Functional Analysis of the Arg91GIn Substitution in the Factor VIII Binding Domain of von Willebrand Factor Demonstrates Variable Phenotypic Expression
Authors: Lavergne J M , Piao Y , Ribba A S , Girma J P , Siguret V , Piétu G , Boyer-Neumann C , Schandelong A , Bahnak B R , Meyer D
Published: July 2018

An Arg91Gln substitution in the mature von Willebrand factor (vWF) has been associated with defective binding of vWF to factor VIII (FVIII). We studied four families with members initially classified as having type I von Willebrand disease (vWD) who were either homozygous or heterozygous for the Arg91Gln change. The first family was the original case described by Nishino et al. (1) where three members were homozygous for the Gln91 allele. They had a low FVIII coagulant activity:vWF antigen (VIIIC:vWFAg) ratio, from 0.29 to 0.44, and the ability of their plasma vWF to bind FVIII was markedly decreased. All the heterozygous members had normal vWF and FVIII levels but the capacity of their plasma vWF to bind FVIII was reduced and intermediate between the homozygous members and normals. The affected individual from the second family was heterozygous for the Gln91 allele and demonstrated a VIIIC:vWFAg ratio of 0.98. The FVIII binding assay confirmed the heterozygous status indicating that the moderately low levels of vWF were due to reduced expression of both alleles. The propositus from the third family was also heterozygous and had below normal levels of vWF as well as a low VIIIC:vWFAg ratio of 0.34; however, FVIII binding to her plasma vWF was similar to that of the homozygous individuals suggesting that Gln91-vWF was the major circulating form. Her daughter who has type I vWD inherited the allele without the Gln91 mutation indicating that the expression of this allele was indeed impaired. The heterozygous patient in the fourth family had a vWF level of 24 U/dl but an VIIIC:vWFAg ratio greater than 2. Her plasma vWF showed normal FVIII binding implying that the Gln91 mutation was linked to a defect that repressed the expression of the allele. The Arg91Gln substitution appears to decrease but not eradicate FVIII binding suggesting a critical concentration of vWF be attained before the defect becomes phenotypically important.
Surgery and Environmental Influence as Risk-Factors for the Development of Deep Vein Thrombosis in Animal Experiments
Authors: Hoffmann R , Schimmer R C , Largiadèr F
Published: July 2018

The aim of this experimental study was to determine whether deep vein thrombosis (DVT) in animals occurs after osteosynthesis of the femur, and whether a postoperative drop in air pressure increases the frequence of DVT Thus, osteosynthesis of the femur was performed in 10 New Zealand rabbits. Postoperatively a drop in air pressure of 300 hPA was induced. After 40 h in the pressure cabin ascending phlebography was performed (Group A). In a control-group of 10 other rabbits surgery was performed without postoperative drop in air pressure (Group B) while in a second control only the drop in air pressure was induced without surgery (Group C).

The phlebographic studies showed DVT in 4 out of 10 rabbits in group A. In group B there was no demonstrable DVT while in group C one ease of DVT was seen. The difference between group A and group B and C is statistically significant. In conclusion, a rapid drop in air pressure seems to have significant impact on the pathogenesis of DVT in rabbits after osteosynthesis of the femur.
Biochemical and In Vivo Properties of High Purity Factor IX Concentrates
Authors: Berntorp Erik , Björkman Sven , Carlsson Maj , Lethagen Stefan , Nilsson Inga Marie
Published: July 2018

The purified factor IX concentrates Nanotiv (Kabi Pharmacia), Immunine (Immuno), Factor IX VHP (Bio-transfusion), Alphanine (Alpha) and Mononine (Armour) have been studied in vitro and compared with the prothrombin complex concentrates (PCCs) Preconativ (Kabi Pharmacia) and Prothromplex TIM4 (Immuno). The measured values for factor IX coagulant activity (IX: C) were in good agreement with the manufacturer’s label values. In contrast to the PCCs, most of the purified concentrates were virtually devoid of other vitamin K-dependent coagulation factors, the inhibitors protein C and S as well as fibrinogen, fibronectin and immunoglobulins. Indicators of thrombin generation, namely prothrombin fragments 1 and 2 (F 1 + 2) and thrombin-antithrombin complex (TAT), were present in varying amounts in all preparations. The specific activity in the purified concentrates exceeded that in the PCCs by a factor of 50-100. Some differences in purity were found between the purified concentrates.

In vivo, Nanotiv was compared with Preconativ and Immunine with Prothromplex TIM4 in crossover studies in patients with severe hemophilia B, and Mononine was tested in a single drug study. Most of the preparations yielded postinfusion increases in TAT, but not in FI + 2. Pharmacokinetic variables were analyzed with non-linear curve-fitting combined with model-independent methods. In retrospective comparisons, there were no apparent differences between Nanotiv, Preconativ and Mononine, whereas in vivo recovery seemed lower and the apparent clearance higher for Immunine and Prothromplex. Purified factor IX concentrates were successfully used as cover for surgery or in immune tolerance induction.
Multi-Center Study of Replacement of the International Reference Preparation for Thromboplastin, Rabbit, Plain
Authors: van den Besselaar A M. H. P
Published: July 2018

A multi-center study of the three W.H.O. international reference preparations (IRP) for thromboplastin has been performed. The main purpose of the study was the determination of the mean international sensitivity index (ISI) of a proposed replacement IRP for thromboplastin, rabbit, plain. Twenty laboratories in Europe, North America, and Australasia participated in the study. All clotting times were determined with the manual technique. The inter-laboratory variation of the ISI was considered as the relevant parameter for assessment of ISI precision. Low interlaboratory variation was observed in the calibration of rabbit thromboplastin against rabbit thromboplastin (2.9-3.0% coefficient of variation of calibration line slope). Relatively high interlaboratory variation was observed in the calibration of rabbit against human thromboplastin (3.7–7.2 CV). Intermediary interlaboratory variation was obtained in the comparison of rabbit or human plain thromboplastin with bovine combined thromboplastin (3.5-4.0% CV). These findings confirm the principle of like-to-like calibration in biological standardization.

The mean ISI values based on the IRP for rabbit thromboplastin (RBT/79) are in excellent agreement with those obtained with the IRP for bovine thromboplastin (OBT/79). In contrast, the mean ISI values determined with the IRP for human thromboplastin (BCT/253) were 6% lower than those with RBT/79. This bias may be caused by a difference in design of the two historical calibration studies. The mean ISI of the proposed replacement IRP for thromboplastin, rabbit, plain obtained by calibration against RBT/79 was 1.035 (standard error: 0.027). It is recommended to round the mean ISI off to 1.0.

Opportunity was also taken to compare a lyophilized normal pooled plasma with fresh individual normal plasmas. The mean PT-ratio of this lyophilized plasma obtained with RBT/79, and BCT/253 was 1.02, which was slightly but significantly higher than the mean PT-ratio obtained with OBT/79 (0.99).

INR equivalents were determined for a lyophilized pooled plasma derived from patients treated with oral anticoagulants. There were small but significant differences between the various thromboplastins, which may be caused by slight activation of the plasma or by the lyophilization process.
A Parent-Twin Study of Plasma Levels of Histidine-Rich Glycoprotein (HRG)
Authors: Boomsma D I , Hennis B C , van Wees A G M , Frants R R , Kluft C
Published: July 2018

Histidine-rich glycoprotein (HRG) is a non-enzymatic glycoprotein that acts as a modulator of several plasma proteins involved in coagulation and fibrinolysis. The contributions of genetic and environmental influences to inter-individual variation in plasma levels of HRG were studied in 160 Dutch families consisting of adolescent twin pairs and their parents. Results showed that 69% of the variance in plasma HRG concentrations could be accounted for by genetic factors. Heritability was the same in males and females and in parents and their offspring. There was no association between HRG levels of husband and wife and no evidence was found for the influence of shared family environment on the resemblance between relatives.
Inhibition of the Platelet Reaction by a High Molecular Weight Phosphoglycoprotein Isolated from Human Platelet Plasma Membranes
Authors: Apitz-Castro R , Fonseca G , Michelena V , Cruz M R
Published: July 2018

The effect of a phospho-glycoprotein (HMW-GP), obtained from human platelet plasma membranes, on the aggregation and secretion of human platelets was studied. Incubation of PRP with 4 to 16 μg/ml of HMW-GP results in inhibition of ADP-, Epinephrine-, Collagen-, and Thrombin-induced platelet aggregation. The effect is mainly reflected on the secondary wave of aggregation. The inhibitory effect is partially overcome by higher concentration of the inducers, however, even under these conditions, a clear tendency towards disaggregation is observed. 5HT release (Col-induced) is strongly decreased from 50% to 4.5. The inhibitory effect on Thrombin-induced aggregation is markedly dependent on external calcium, being maximal at 5 mM calcium. The HMW-GP does not bind ADP or Thrombin. Membrane conformation is markedly affected, as evidenced by the effect of HMW-GP on the iodination of surface polypeptides of intact platelets. It is suggested that interaction of HMW-GP with the platelet membrane blocks the signal(s) transmission that links stimulus to activation. The inhibition observed might just represent an experimental amplification of the endogenous modulatory function that has been proposed for this high molecular weight phosphoglycoprotein.
The Diagnosis of Mild Haemophilia by the Partial Thromboplastin Time Test. WFH/ICTH Study of the Manchester Method
Authors: O'Brien P F , North W R S , Ingram G I C
Published: July 2018

In two trials, various methods of performing the partial thromboplastin time test (PTT) were compared with the method described from Manchester by Poller and Thomson (1) and Poller et al. (2), for their ability to diagnose mild haemophilia. In the first trial, 64 laboratories in various countries compared their own methods and reagents with the Manchester method on three coded haemophilic plasmas distributed freeze-dried and on four local fresh normals collected in each laboratory. In the second trial, seven well-standardized commercial methods were tested in pairs against the Manchester method in seven experienced laboratories on 22 mild haemophilic samples and six normal samples each coded and distributed frozen. PTTs were related to factor VIII: C level.

In the first trial, the better standardized methods gave more consistent results than others; but even so there were large differences between laboratories even on normal plasmas. The second trial established a reasonably linear relationship between log PTT and log VIII :C in the range 15-150 iu/dl; but different methods did not greatly vary in their ability to detect a mild haemophilic defect; this was confirmed by calculating the index Slope/(Residual S. D.) which was used to provide a rank order. Scatter diagrams suggested that a proportion of mild haemophilic samples would be likely to have PTTs within the normal range by any method. It is concluded that the Manchester method does not detect mild haemophilia with greater discrimination than the other methods studied; that each laboratory must establish its own normal range even if using a well-standardized method; and that in screening patients a PTT must be supplemented by a discriminating history.
Common Antigenic Sites in Human, Bovine and Porcine Fibrinogens
Authors: Cierniewski Czeslaw S
Published: July 2018

Specific antisera to the Aα, Bβ and γ chains of porcine fibrinogen were used to characterize an antigenic homology of human, bovine, and porcine fibrinogens. Antigenic determinants shared by these fibrinogens were mostly formed by the Aα chain. However, in the case of bovine and porcine fibrinogens they were also found in the Bβ and γ polypeptide chains. The results reported here show that the Aα chain determinants exposed on the intact fibrinogen molecule are conserved to a considerably larger extent than those of the Bβ and γ chains.
Dependence of Human Platelet Functional Responses on Divalent Cations: Aggregation and Secretion in Heparin- and Hirudin-Anticoagulated Platelet-Rich Plasma and the Effects of Chelating Agents
Authors: Lages Bruce , Weiss Harvey J
Published: July 2018

The dependence of ADP- and epinephrine-induced platelet aggregation and secretion on extracellular divalent cations was examined by quantitating these responses in citrate-, heparin-, and hirudin-anticoagulated platelet-rich plasma. ADP-induced 14C-5HT secretion in heparin-PRP and hirudin-PRP was generally decreased, relative to that in citrate-PRP, without corresponding reductions in aggregation, whereas in response to epinephrine, both aggregation and secretion were decreased in heparin-PRP, and abolished in hirudin-PRP. In heparin-PRP, but not in hirudin-PRP, the degree to which these responses were altered was highly variable among normal subjects, and was dependent on the anticoagulant concentration. Addition of citrate restored the extent of ADP-induced secretion and of epinephrine-induced aggregation and secretion in heparin-PRP to that observed in citrate-PRP, and increased the extent of ADP-induced secretion in hirudin-PRP. Addition of EDTA or EGTA, however, had no effect on ADP-induced secretion in heparin-PRP. These results suggest that ADP-induced aggregation and secretion, as well as responses to ADP vs. epinephrine, have different dependencies on extracellular or surface-bound divalent cations. The variable responses observed in heparin-PRP may reflect direct interactions of heparin with platelets, and this variability may account for the conflicting results of previous studies.
Genetic Basis and Carrier Detection of Hemophilia B of Chinese Origin
Authors: Lin Shu-Wha , Shen Ming-Ching
Published: July 2018

We have characterized the genetic defects of 17 hemophilia B patients of Chinese origin by means of the polymerase chain reaction (PCR) and direct sequencing. The single-strand conformation polymorphism (SSCP) was used as an initial screening method to analyze the entire coding region and the flanking introns of each individual’s factor IX gene. The abnormal exons were subsequently amplified and the nucleotide sequence determined. Of the 17 patients studied, 16 had single point mutations and one had a gross gene deletion of exons VII and VIII of factor IX. Among these 16 factor IX variants with point mutations 13 were missense and two were nonsense mutations. The remaining one had a nucleotide deleted, resulting in frame shifting at amino acid residue 97. A total of ten novel mutations, including the one with gross gene deletion, are reported in this study which have not been described previously. Five of the remaining seven variants were missense mutations with novel amino acids substituted for residues 127, 132, 180, 207, and 215, respectively. Mutations containing different amino acid residues at those positions have been reported. The last two are variants that have already been described to contain mutations at amino acid residues 333 and 365, respectively. To evaluate the efficiency of SSCP analysis in assessing the mutated exons and to further confirm our results we sequenced the entire exons of all 17 factor IX genes. The mutations detected by SSCP method were indeed the only mutation identified in each factor IX variant. The SSCP analysis and direct sequencing have also allowed us to circumvent the difficulties of carrier determination for Chinese by direct detection of the abnormal factor IX alleles inherited by the females.
Human Platelets Possess Tyrosylprotein Sulfotransferase (TPST) Activity
Authors: Sane David C , Baker Marshall S
Published: July 2018

Tyrosine sulfation is a widespread modification of secreted proteins including several coagulation proteins synthesized in the liver. Since factor V, a tyrosine-sulfated protein, is also synthesized in megakaryocytes, we determined whether platelets posses tyrosylprotein sulfotransferase (TPST) activity. Using the synthetic substrate EAY, substantial TPST activity (0.405 ± 0.049 pmol EAY-SO4 formed min−1 mg−1) was detected in platelet homogenates. This activity could not be accounted for by contaminating leukocytes, erythrocytes or plasma. The K
m of platelet TPST for EAY was 3.7 μM and V
max 0.09 pmol/min. For the cofactor 3' phosphoadenosine 5' phosphosulfate (PAPS) the K
m was 1.7 μM and V
max 0.11 pmol/min. The PAPS analogue 3', 5'-adenosine diphosphate inhibited platelet TPST with an IC50 of 15.4 μM. These findings suggest that tyrosine sulfation of factor V will occur in megakaryocytes. Platelets may be a useful source for further study of TPST.
Effect of the Infusion of OKY-046, a Thromboxane A2 Synthase Inhibitor, on Urinary Metabolites of Prostacyclin and Thromboxane A2 in Healthy Human Subjects
Authors: Morio H , Hirai A , Terano T , Tamura Y , Yoshida S
Published: July 2018

The influence of OKY-046, a selective thromboxane synthase inhibitor, on prostanoid formation in healthy human subjects was studied. Vehicle (5% glucose solution) or OKY-046 in 5% glucose solution at 15 μg kg−1 min−1 was intravenously administered to five male healthy volunteers for 6 h. Platelet aggregation and thromboxane B2 (TXB2) formation induced by collagen and arachidonic acid were suppressed by the infusion of OKY-046, while both were not affected by the infusion of vehicle. Urinary excretion of 11-dehydro-thromboxane B2, one of major urinary metabolites of thromboxane A2 (TXA2) was decreased by the infusion of OKY-046, while that of 2,3-dinor-6-keto-prostaglandin F1α, one of major urinary metabolites of prostacyclin (PGI2) was increased. The present study demonstrated that the infusion of OKY-046 improved the balance of TXA2/PGI2 into antithrombotic state in healthy subjects. It was also suggested that endogenously produced (probably platelet-derived) endoperoxides could be redirected into prostacyclin in vivo.
Trimucytin: A Collagen-Like Aggregating Inducer Isolated from Trimeresurus mucrosquamatus Snake Venom
Authors: Teng Che-Ming , Ko Feng-Nien , Tsai Inn-Ho , Hung Man-Ling , Huang Tur-Fu
Published: July 2018

Trimucytin is a potent platelet aggregation inducer isolated from Trimeresurus mucrosquamatus snake venom. Similar to collagen, trimucytin has a run of (Gly-Pro-X) repeats at the N-terminal amino acids sequence. It induced platelet aggregation, ATP release and thromboxane formation in rabbit platelets in a concentration-dependent manner. The aggregation was not due to released ADP since it was not suppressed by creatine phosphate/creatine phosphokinase. It was not either due to thromboxane A2 formation because indomethacin and BW755C did not have any effect on the aggregation even thromboxane B2 formation was completely abolished by indomethacin. Platelet-activating factor (PAF) was not involved in the aggregation since a PAF antagonist, kadsurenone, did not affect. However, RGD-containing peptide triflavin inhibited the aggregation, but not the release of ATP, of platelets induced by trimucytin. Indomethacin, mepacrine, prostaglandin E1 and tetracaine inhibited the thromboxane B2 formation of platelets caused by collagen and trimucytin. Forskolin and sodium nitroprusside inhibited both platelet aggregation and ATP release, but not the shape change induced by trimucytin. In quin-2 loaded platelets, the rise of intracellular calcium concentration caused by trimucytin was decreased by 12-O-tetradecanoyl phorbol-13 acetate, imipramine, TMB-8 and indomethacin. In the absence of extracellular calcium, both collagen and trimucytin caused no thromboxane B2 formation, but still induced ATP release which was completely blocked by R 59022. Inositol phosphate formation in platelets was markedly enhanced by trimucytin and collagen. MAB1988, an antibody against platelet membrane glycoprotein Ia, inhibited trimucytinand collagen-induced platelet aggregation and ATP release. However, trimucytin did not replace the binding of 125I-labeled MAB1988 to platelets. Platelets pre-exposed to trimucytin were resistant to the second challenge with trimucytin itself or collagen. It is concluded that trimucytin may activate collagen receptors on platelet membrane, and cause aggregation and release mainly through phospholipase C-phosphoinositide pathway.
Influence of Acute Myocardial Infarction and rt-PA Therapy on Circulating Fibrinogen
Authors: Seifried E , Oethinger M , Tanswell P , Nobel E Hoegee-de , Nieuwenhuizen W
Published: July 2018

In 12 patients treated with 100 mg rt-PA/3 h for acute myocardial infarction (AMI), serial fibrinogen levels were measured with the Clauss clotting rate assay (“functional fibrinogen”) and with a new enzyme immunoassay for immunologically intact fibrinogen (“intact fibrinogen”). Levels of functional and “intact fibrinogen” were strikingly different: functional levels were higher at baseline; showed a more pronounced breakdown during rt-PA therapy; and a rebound phenomenon which was not seen for “intact fibrinogen”. The ratio of functional to “intact fibrinogen” was calculated for each individual patient and each time point. The mean ratio (n = 12) was 1.6 at baseline, 1.0 at 90 min, and increased markedly between 8 and 24 h to a maximum of 2.1 (p <0.01), indicating that functionality of circulating fibrinogen changes during AMI and subsequent thrombolytic therapy. The increased ratio of functional to “intact fibrinogen” seems to reflect a more functional fibrinogen at baseline and following rt-PA infusion. This is in keeping with data that the relative amount of fast clotting “intact HMW fibrinogen” of total fibrinogen is increased in initial phase of AMI. The data suggest that about 20% of HMW fibrinogen are converted to partly degraded fibrinogen during rt-PA infusion. The rebound phenomenon exhibited by functional fibrinogen may result from newly synthesized fibrinogen with a high proportion of HMW fibrinogen with its known higher degree of phosphorylation. Fibrinogen- and fibrin degradation products were within normal range at baseline. Upon infusion of the thrombolytic agent, maximum median levels of 5.88 μg/ml and 5.28 μg/ml, respectively, were measured at 90 min. Maximum plasma fibrinogen degradation products represented only 4% of lost “intact fibrinogen”, but they correlatedstrongly and linearly with the extent of “intact fibrinogen” degradation (r = 0.82, p <0.01). In contrast, no correlation was seen between breakdown of “intact fibrinogen” and corresponding levels of fibrin degradation products. We conclude from our data that the ratio of functional to immunologically “intact fibrinogen” may serve as an important index for functionality of fibrinogen and select patients at high risk for early reocclusion. Only a small proportion of degraded functional and “intact fibrinogen”, respectively, is recovered as fibrinogen degradation products. There seems to be a strong correlation between the degree of elevation of fibrinogen degradation products and the intensity of the systemic lytic state, i.e. fibrinogen degradation.
Prothrombin Synthesis in Rat Hepatoma H4IIEC3 Cells: Response to the Proposed “Coagulopoietin Factor”
Authors: Shah D V , Engelke J A , Suttie J W
Published: July 2018

Based on studies in intact animals, the presence of humoral factors, “coagulopoietins”, which regulate the synthesis of vitamin K-dependent plasma proteins has been proposed. These proposed factors are produced in response to vitamin K deficiency, coumarin treatment, or specific antibody depletion of vitamin K-dependent clotting factors. The production of pro-thrombin by rat hepatoma H4IIEC3 cells has now been shown to be dependent on the source of bovine serum in the media. Cells grown in serum from cows treated with dicoumarol produce about 20% more prothrombin in 24 h than those cells grown in control serum. The humoral factor causing this response is present early in the course of dicoumarol treatment, and the increase in prothrombin production is dependent on the amount of serum from a dicoumarol-treated cow in the media. Based on membrane filtration studies, the factor appears to be associated with the protein fraction of serum.
A Sensitive Peroxidase Staining Immunoblotting Method for Measuring Total Protein S in Human Plasma
Authors: Li Xiangan , Hatanaka Kaoru , Guo Ling , Tsushima Motoo , Kitamura Yukihiko , Yamamoto Akira
Published: July 2018

In plasma, protein S is found in its free form and as a complex with C4b-binding protein. After 125I-protein S was added tonormal human plasma and applied to SDS-8% polyacrylamide gel electrophoresis, the autoradiogram of the gel showed only one single band at free protein S position. Applying this evidence, we have developed a peroxidase staining Western Blotting method to quantitate total protein S in human plasma which consists of sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by blotting to nitrocellulose membrane and a sensitive avidin-biotinylated peroxidase staining method (ABC technique). The measurement of protein S by the immunoblotting was reproducible and the coefficient of variation was 7%. As little as 1 ng of protein S could be detected. C4b-binding protein did not affect the measurement of protein S. Compared to other immunoassays, this peroxidase staining immunoblotting method has the advantage of directly estimating the apparent molecular weight of protein of interest, eliminating nonspecific stain and having high sensitivity without using radioisotope.
Binding of Anisoylated Lys-Plasminogen Streptokinase Activator Complex to Cells in Culture
Authors: Vasudevan Jayanand , Humphries John E , Gonias Steven L
Published: July 2018

Anisoylated Lys-plasminogen streptokinase activator complex (APSAC) was purified from Eminase® by chromatography on Superose-12. Purified APSAC did not significantly deacylate within 4 h at 4° C in solution as determined by hydrolysis of D-Val-L-leu-L-lys-p-nitroanilide HCl (S-2251). At 37° C, maximum amidase activity developed in 120 min; ε-amino-n-caproic acid (EACA) did not affect the apparent rate of APSAC deacylation but stabilized the streptokinase-plasmin(ogen) complex (SkPl) which formed. APSAC bound to C6 glioma cells and human umbilical vein endothelial cells (HUVECs) in culture. Binding was completely inhibited by EACA suggesting an essential role for the plasminogen kringle domains. Cell-associated APSAC deacylated to form active SkPl which hydrolyzed S-2251 and D-Val-Leu-Lys-7-amino-4-methyl coumarin. The rate of APSAC deacylation was increased when the APSAC was cell-associated. APSAC that was initially bound to C6 cells or HUVECs also activated 125I-plasminogen. This activity may have reflected cell-associated APSAC or APSAC that dissociated into solution. Plasmin was recovered bound to cells and in solution. These studies demonstrate that APSAC associates with cell-surfaces and retains activity. In the circulation, cell-surfaces may provide a significant pharmacologic compartment for intravenously administered APSAC.
Antithrombin III, the Major Modulator of Intravascular Coagulation, Is Synthesized by Human Endothelial Cells
Authors: Chan T K , Chan Vivian
Published: July 2018

Human endothelial cells in culture is shown to synthesize antithrombin III (At-III). The endothelial cell At-III(EC-At-III) consists of a small fraction similar to plasma At-III and a larger fraction with decreased heparin-binding as tested by crossed immunoelectrophoresis. However, both the anti-Xa and thrombin-neutralizing activities of the EC-At-III were rapid and active even in the absence of added heparin. It is concluded that the major portion was probably bound to endogenous heparin-like substance, thus accounting for its decreased exogenous heparin binding. The presence of At-III and other antithrombotic factors in the vascular endothelium offer protection against thrombosis and possibly atherosclerosis.
The Evaluation of Plasmin and Streptokinase Activator Complexes in a New Rabbit Model of Venous Thrombosis
Authors: Dupe R J , English P D , Smith R A G , Green J
Published: July 2018

A new quantitative model for the examination of potential thrombolytic agents is described. A thrombus was formed in the inferior vena cava of the rabbit, using 125I-labelled fibrinogen mixed with a standard amount of thromboplastin. The thrombus was anchored securely by means of a woollen thread inserted longitudinally into the lumen of the vein. Thrombotic extension of the radioactive clot and rethrombosis during the experimental period (5 h) were prevented by the administration of intravenous heparin. In control animals, the thrombus remained stable and there was no rise in the radioactivity of the circulating blood during 5 h. In animals given thrombolytic agents, lysis could be followed continuously by measuring the rise in blood radioactivity. Total lysis was determined by counting the radio-label in the residual clot after treatment.

The model has been used to study the thrombolytic activity of streptokinase-human plasminogen complexes, activator-free plasmins of human and porcine origin, and human plasmin produced by streptokinase (SK) activation of human plasminogen. The role of activator complexes in the thrombolytic activity of SK-activated plasmin is discussed.
The Thrombolytic Activity of Streptokinase in the Rabbit
Authors: English P D , Smith R A G , Dupe R J , Green J , Hibbs M J
Published: July 2018

The activity of streptokinase (SK) has been examinEd in a rabbit model of venous thrombosis. Significant thrombolysis was observed at low doses of SK without evidence of systemic activation of the fibrinolytic system. The origidn of the specificity of SK-induced fibrinolysis in the rabbit was explored, using a chromogenic substrate coupled assay to measure the enhancement of plasmin generation in the presence of rabbit fibrin. The results obtained suggest that rabbit fibrin influences the fibrinolytic system at three points, viz: the assembly of an activator complex from rabbit plasminogen and SK; the activity of that activator complex towards further rabbit plasminogen; and the activity of the resulting rabbit plasmin in the presence of plasma inhibitors.
Effects of Suloctidil on Platelet Survival Time Following Cardiac Valve Replacement
Authors: Col-de Beys Chr , Ferrant A , Moriau M
Published: July 2018

Platelet functions and blood clotting parameters were examined in 23 patients with a shortened platelet survival time after cardiac valve replacement.

Treatment was given at random: for a first group antivitamins K with suloctidil and for the second group antivitamin K alone.

Six weeks of treatment with antivitamin K alone did not induce any significant change in the platelet survival time, platelet retention or platelet factor 4 in plasma.

In contrast, the shortened platelet survival time increased significantly after 6 weeks of treatment with suloctidil. Moreover the platelet retention and the amount of platelet factor 4 in plasma were significantly reduced, indicating that normalization of platelet survival is the result of decreased destruction, presumably at the level of the prosthetic surface. The clinical benefits expected from these results remain to be validated by a longer follow up period.
Severe Thrombocytopenia Delays but Does Not Prevent the Occlusion of an Arterial Prosthesis in Rats
Authors: Reyers I , Mussoni L , Donati M B , Gaetano G de
Published: July 2018

This study shows that experimentally-induced immune thrombocytopenia significantly delayed occlusion of an arterial prosthesis inserted in rat abdominal aorta. Thrombocytopenia was effective when induced several hours or shortly, or even several hours after the insertion of the prosthesis. Maintenance of severe thrombocytopenia by daily administrations of antiplatelet antiserum appeared to further delay thrombotic occlusion.

However, though delayed, occlusion eventually occurred in all rats, even in those with very low platelet count. This would imply that any attempt to prevent arterial prosthesis thrombosis solely by interfering with platelets is ultimately bound to fail.
Coagulation and Fibrinolysis Study in Systemic Lupus erythematosus: Haematological, Urinary and Tissue Parameters
Authors: Stratta P , Canavese C , Valmaggia P , Rotunno M , Levi E , Bulla A , Vercellone A
Published: July 2018

Haematochemical, urinary and tissue parameters were examined in the elaboration of the coagulation and fibrinolysis profile in 33 cases of systemic lupus erythematosus in different stages of the disease. Coagulation abnormalities varied from hypo- to hyper-coagulabitity, these being often associated in the same patient, either simultaneously or at different stages of the disease. Activation of coagulation, closely related to the immunological activity of the disease, was present in 80% cases in the acute stage, and 36% of those in the remission stage. The lupus-like anticoagulant was not much involved, and platelets were the prime figures in the haemostatic abnormalities of lupus, those being the preferred target of direct antibody activities, or possibly of immune complexes as well. Activation of the coagulatory cascade is not uncommonly accompanied by a thrombophilic tendency coupled with signs of consumption, this being the expression of a continuously stimulated haemostatic balance.
Radiolabeling of Fibrinogen Using the Iodogen Technique
Authors: Knight Linda C , Budzynski Andrei Z , Olexa Stephanie A
Published: July 2018

The properties of human fibrinogen labeled with 125-Iodine using Iodogen (1, 3, 4, 6-tetrachloro-3α, 6α-diphenylglycoluril) as an oxidizing agent were compared with those of an iodine monochloride labeled counterpart. It was found that thrombin clottability, binding to staphylococci, the relative specific radioactivity of the Aα, Bβ, and γ chains and in vivo clearance from plasma in rabbits were the same in these two labeled fibrinogen preparations. Labeling efficiency was higher when iodogen was used. It is concluded that human fibrinogen labeled with radioiodine using the Iodogen technique is suitable for studies in vitro and in vivo.
Comparison of Progressive Antithrombin Activity and the Concentration of Three Thrombin Inhibitors in Nephrotic Syndrome
Authors: Boneu B , Bouissou F , Abbal M , Sie P , Caranobe C , Barthe P
Published: July 2018

In order to compare the plasmatic progressive antithrombin activity to the concentration of three thrombin inhibitors, antithrombin III (AT III), α2 macroglobulin (α2, M), α1 anti-trypsin (α1 AT) in nephrotic syndrome, a prospective study was carried out on a group of 28 children affected with the disease. A dramatic reduction of the level of AT III and of α1 AT, two inhibitors of molecular weight close to that of albumin, was observed. The decreased level of AT III was counterbalanced by an increase in α2 M. This phenomenon accounts for the increased progressive antithrombin activity observed in all the affected children. It is suggested that the above compensatory mechanism explains the absence of thrombotic accidents in this series and that the benefit of heparin therapy is doubtful in these conditions.
Studies of Phospholipid Reagents Used in Coagulation I: Some General Properties and Their Sensitivity to Factor VIII
Authors: Barrowcliffe T W , Gray Elaine
Published: July 2018

Some general properties of phospholipid reagents have been studied, particularly in relation to their use in detection and measurement of Factor VIII deficiency. There was wide variation among reagents in their total phospholipid concentration, and in the degree of contamination with tissue factor and oxidation products. Although clotting times of normal and haemophilic plasma were affected by change of phospholipid concentration, presence of oxidation products, and order of addition of reagents,
these factors did not appear to influence the efficiency of detection of the Factor VIII defect. The method of activation had a definite influence on sensitivity to Factor VIII deficiency, both the type of activator and the time of activation being important.

In Factor VIII one-stage assays, use of different phospholipids with the same substrate plasma gave significant differences in precision, but the estimates of potency of a Factor VIII concentrate against a plasma standard were not significantly different.
Enhanced Antithrombotic Mechanisms in Patients with Maturity-Onset Diabetes mellitus without Thromboembolic Complications
Authors: Grignani G , Gamba G , Geroldi D , Pacchiarini L , Solerte B , Ferrari E , Ascario E
Published: July 2018

Some haemostatic parameters have been evaluated in a group of rigorously selected patients with maturity-onset diabetes mellitus without thromboembolic complications and in apparently normal subjects of the same age before and after the venous occlusion test (VOT).

In basal conditions diabetics had higher levels of AT III as biological activity and higher fibrinolytic and antifibrinolytic activities than controls

After VOT, F VIII R:Ag increased significantly in both groups, more markedly in controls than in diabetics, while F VIII: C showed no modification.

Also AT III R:Ag increased after the test, but such variation was significant only in diabetics; on the contrary, the biological activity of AT III was always significantly decreased after the test.

After VOT there were also in both groups highly significant increases in the fibrinolytic and antifibrinolytic activities.

Finally, HbA1c levels directly correlated with AT III as biological activity before VOT, but with no other parameter either before or after the test.

These data suggest the existence in patients with diabetes mellitus without thromboembolic complications of an activated protective mechanism against intravascular clotting.
Enhancement of Thrombin-Induced Degradation of Phosphatidylcholine in Reserpinized Rabbit Platelets
Authors: Watanabe Yasuko , Soda Masako , Kobayashi Bonro
Published: July 2018

When 1-14C-arachidonic acid-labeled, washed platelets from the reserpinized rabbits were exposed to thrombin, the decrease in radioactivity of phospholipids was significantly stimulated as compared with the control platelets. The increase in radioactivity of fatty acids and their oxygenated metabolites was also stimulated. TLC analysis revealed that the stimulation of the decrease in radioactivity of phospholipids was almost exclusively attributed to that of phosphatidylcholine (PC), and that thrombin induced a slight but significant increase in radioactivity of phosphatidyl ethanolamine (PE) in the reserpinized platelets. The results suggest that thrombin-induced degradation of PC is enhanced in the reserpinized platelets, and the overproduced fatty acids would be metabolized to the larger amount of oxygenated products which result in the activation of the platelets. Thrombin-induced mobilization of PC to PE in the reserpinized platelet phospholipids was also suggested.
In Vitro Effect of Ethanol on ADP and Collagen-Induced Platelet Aggregation
Authors: Bengmark Stig , Elmér Olle , Göransson Göran , Zoucas Evita
Published: July 2018

Platelet aggregation was measured by Born’s method. Plasma from Sprague-Dawley rats and inbred albino rabbits was used. 20% ethanol in 0.9%(w/v) NaCl was added to plasma at final concentrations of 56.5 mM, 85.9 mM 171.5 mM and 343.1 mM or to whole blood at final concentrations of 8.7 mM, 17.4 mM, 52.5 mM, 171.5 mM and 343.1 mM. Experiments using 0.9% NaCl volumes equivalent to the added ethanol volume were also conducted. Dose-response aggregation tests showed that a decrease in ADP-and collagen-induced aggregation existed when ethanol was mixed with rat or rabbit blood prior to the preparation of platelet-rich plasma. There was no effect on ADP-induced platelet aggregation when ethanol was mixed with rat or rabbit plasma. Collagen-induced aggregation was impaired only in rat plasma when ethanol concentration reached 343.1 mM. These results suggested that ethanol modified platelet function possibly via red cells.