bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of Type 1 Iodothyronine Deiodinase by Bisphenol A
Authors: da Silva Maurício Martins , Gonçalves Carlos Frederico Lima , Miranda-Alves Leandro , Fortunato Rodrigo Soares , Carvalho Denise P. , Ferreira Andrea Claudia Freitas
Published: June 2019

Plastics are ubiquitously present in our daily life and some components of plastics are endocrine-disrupting chemicals, such as bisphenol A and phthalates. Herein, we aimed to evaluate the effect of plastic endocrine disruptors on type 1 and type 2 deiodinase activities, enzymes responsible for the conversion of the pro-hormone T4 into the biologically active thyroid hormone T3, both in vitro and in vivo. Initially, we incubated rat liver type 1 deiodinase and brown adipose tissue type 2 deiodinase samples with 0.5 mM of the plasticizers, and the deiodinase activity was measured. Among them, only BPA was capable to inhibit both type 1 and type 2 deiodinases. Then, adult male Wistar rats were treated orally with bisphenol A (40 mg/kg b.w.) for 15 days and hepatic type 1 deiodinase and brown adipose tissue type 2 deiodinase activities and serum thyroid hormone concentrations were measured. In vivo bisphenol A treatment significantly reduced hepatic type 1 deiodinase activity but did not affect brown adipose tissue type 2 deiodinase activity. Serum T4 levels were higher in bisphenol A group, while T3 remained unchanged. T3/T4 ratio was decreased in rats treated with bisphenol A, reinforcing the idea that peripheral metabolism of thyroid hormone was affected by bisphenol A exposure. Therefore, our results suggest that bisphenol A can affect the metabolism of thyroid hormone thus disrupting thyroid signaling.
A Photocatalyst-Free, SET-Mediated Photochemical Approach for the Synthesis of Dumbbell-Like Amine-Functionalized Bis-C60 Fullerene through C–C Bond Formation
Authors: Atar Amol Balu
Published: June 2019

A novel method for the synthesis of dumbbell-like amine-functionalized bis-C60 fullerene from simple bis-α-silyl tertiary benzyl amines and C60 fullerene is described. The photoreactions between bis-α-silyl tertiary benzyl amines and C60 furnished single-bonded bis-aminomethyl-1,2-dihyrofullerenes and double-bonded 1,2,5-trisubstituted bis-fulleropyrrolidines through 1,3-dipolar cycloaddition reactions of azomethine ylides under mild conditions.
SYNTHETIC PEPTIDE ANTIBODIES RECOGNIZE PLASMA AND RECOMBINANT FVIII
Authors: Pancham N , Dumas M , Brown J , Michaud T C , Knowles W J
Published: June 2019

Monoclonal antibodies were raised against synthetic peptides corresponding to the N-termini of the 90kd and 80kd subunits of human FVIII. Preliminary screening was performed directly against the peptides (linked to albumin) coated onto polystyrene wells. IgG was purified by Protein A-Sepharose and affinity purified using the immunogen peptides linked to Sepharose. Immunoreactivity with both plasma and recombinant FVIII was compared by Western blotting, two-site ELISA employing a neutralizing rabbit anti-FVIII IgG as capture antibody, and inhibition in a fluid phase FVIII activity assay. None of the antibodies neutralized clotting or amidolytic activities associated with either FVIII protein. All of the antibodies immunoblotted intacl or thrombin digested FVIII polypeptides according to their anticipated epitope recognition sites; this was useful in determining identity between the two types of FVIII protein. Furthermore, when either FVIII protein was bound to polyclonal IgG coated onto polystyrene microtiter wells, the 80kd antipeptide antibody was capable of detecting both antigens with equal affinity. These results, coupled with results using the 90k antipeptide suggest that epitope accessibility for these antipeptide antibodies is the same for plasma and recombinant FVIII under the conditions tested.
BIOCHEMICAL PROPERTIES OF COMMERCIAL, VIRUS-INACTIVATED FACTOR VIII CONCENTRATES
Authors: Berntorp E , Lethagen S , Nilsson I M
Published: June 2019

As all commercial factor VIII concentrates in current use have been subjected to some form of virus-inactivation, we wanted to compare their in vitro biochemical characteristics. Of the 10 concentrates studied, inactivation takes the form of dry heat treatment, varying from 60°C for 30 h to 72°C for 68 h, in six products (Octonativ, KabiVitrum; Hemofil T, Hyland; Factorate HP, Armour - heated two different ways; Monoclate, Armour; Nordiocto, Nordisk Gentofte). In the 4 remaining concentrates, inactivation is either by steam treatment (Kryobulin Tim 3, Immuno), heating at 60°C for 20 h as dry material slammed in heptane (Profilate, Alpha), wet-heating at 60°C for 10 h (Hemate, Behring), or by the New York Blood Center (solvent/detergent) method (OCTA-V. I., Octopharm). The variables studied were VIII:C by one-stage and chromogenic assay, and VI11 :Ag, vW:Ag, by electroimmunoassay, immunoradiometric assay (IRMA), crossed immunoelectrophoresis and SDS agarose gel electrophoresis followed by staining with radioactive antibody and fibrinogen. VIII :C activity values ranged from 20-53 IU/ml in all products but Monoclate (94 IU/ml). All products gave higher values of VIII:Ag than of VI11 :C, indicating partial inactivation of VIII :C during preparation; the ratios ranged from 1.2 to 1.3 for Kryobulin, Nordiocto, OCTA-V. I., and Hemofil T, and from 2 to 4 for the other products, being highest in Profilate and Monoclate. Specific activity was higher in Monoclate, Nordiocto, Hemate, OCTA-V. I. and Factorate HP (16.0, 7.4, 7.1, 4.8 and 3.3 IU/ml protein, respectively) than in the other products (1.3-2.3 IU/mg). All concentrates contained vW:Ag, and non-parallel dose-response curves indicated abnormality in the vWF molecule in all cases; and thus multimeric sizing failed to demonstrate the largest multimers of the vWF in any product. We conclude that although virus-inactivated F VIII concentrates are general ly comparable with regard to VIII :C content, they vary considerably in the degree of VIII :C inactivation during preparation, and in specific activity. No concentrate tested here contained native vWF. In vivo studies have shown VIII :C recovery and half-life to be comparable in heat-treated and non-heat-treated concentrates.
HEAT INACTIVATED HIGHLY PURIFIED FACTOR VIII CONCENTRATE IN THE TREATMENT OF HEMOPHILIACS
Authors: Koller U , Pabinger I , Lechner K , Knapp W
Published: June 2019

51 severe hemophiliacs who had previously been treated with not virus-inactivated intermediate purity factor VIII concentrates were divided into two groups according to their immunological status. Group A (n=23) consisted of patients with CD4/CD8 (helper/suppressor) T cell ratio of 1.0, group B (n=28) of . patients with a ratio of 1.0. In patients of group A treatment was switched in May 1983 to highly purified heat inactivated factor VIII concentrate (BEHRINGWERKE GmbH, Marburg) whereas patients of group B continued to receive intermediate purity factor VIII concentrate. In both groups laboratory tests (clinical investigation, routine liver function tests, differential blood count, lymphocyte subpopulations and quantitative immunoglobulin analysis) were performed in May 1983 and repeated 6, 12 and 18 months thereafter. In group A a significant reduction (p 0.005) of CD8 positive cells from 10587/μl (median) to 540/μl (18 months) was observed; no significant changes of CD8 positive cells occurred in group B. CD4/CD8 ratio rose from 0.58 to 0.86 in group A (p = 0.005) and remained unchanged in group B (1.38 versus 1.23). Serum IgG decreased in both groups but was more pronounced in group A. Thus treatment with heat inactivated highly purified factor VIII improved the immunological status of hemophiliacs with an inverse ratio. Retrospective analysis of antibodies to HIV, however, showed that most of the patients of group A were antibody positive (n=21), but the 2 negative patients remained negative. In group B of the 10 HIV negative patients one became positive, all others did not change. Whether this improvement of immunological laboratory findings is of clinical relevance, remains to be established, particularly with respect to the high incidence of antibody positive patients within group A.
FACTOR VIII MEDIATES BINDING OF FACTOR IX TO STIMULATED PLATELETS
Authors: Muntean W , Leschnik B
Published: June 2019

In previous work we have shown that factor VII! binds to phospholipids of the membrane of stimulated platelets and that von Willebrand factor is not required for binding of factor VIII to platelets. Since factor VIII is a cofactor in the activation of factor X by factor IX, we investigated whether factor VIII enhances binding of factor IX to the platelet surface.

Factor VIII and factor IX were purified by immunoadsorbent chromatography using specific rabbit antibodies. Washed human platelets (250/nl final concentration) stimulated by human thrombin and collagen were incubated with barbiturate buffer, or with purified factor IX (1 U/ml final concentration), or with factor IX (1 U/ml) in the presence of factor VIII (1 U/ml). Washed and stimulated platelets were also incubated with factor VIII and IX as above in the presence of different amounts of CaCl2. Platelets were then washed again and lysed by sonication. Factor VIII:Ag (immunoradiometric assay) and factor IX:Ag (ELISA) were measured in the platelet lysate prior to and after incubation of the lysate with phospholipase C.

Platelet bound IX:Ag was significantly higher after incubation of stimulated platelets with factor IX in the presence of factor VIII than after incubation of platelets with buffer or with factor IX alone. CaCl2 proved to be essential for binding of factor IX to platelets even in the presence of factor VIII, but CaCl2 was not required for binding of factor VIII to platelets. Measurable VIII :Ag and IX:Ag increased significantly after incubation of the platelet lysate with phospholipase C.

Our data suggest that factor VIII mediates binding of factor IX to phospholipids or receptors containing phospholipids on the membrane of stimulated platelets and thereby contributes to the assembly of the factor X activating complex on the platelet surface.
SEARCH FOR AND USE OF RESTRICTION FRAGMENT LENGTH POLYMORPHISMS (RFLPs) IN AND AROUND THE HUMAN FACTOR IX GENE
Authors: Liddell M B , Anson D S , Lillicrap D P , Peake I R
Published: June 2019

5 previously described RFLPs within the factor IX gene have been used for family studies (carrier detection) in 10 haemophilia B kindred. In all DNA from 91 individuals, including 25 obligate or possible carriers, was analysed by digestion with TaqI and XmnI and probing with the intragenomic probe VIII (all probes were provided by Professor G. G. Brownlee, Oxford). When noninformative, additional RFLPs (DdeI;probe XIII and MspI;probe II) were used. Of 12 possible carriers, 11 were diagnosed (6 as carriers, 5 normal). Of the confirmed carriers (6 diagnosed, 13 obligate) 15 were informative (heterozygous and phase known), and the overall incidence of heterozygosity was 72%. The recently reported BamHI RFLP was not found to be useful ( <1.0% frequency).

Further RFLPs in and flanking the factor IX gene were sought by two procedures. Firstly cosmid pCHIXα, containing a 40kb insert including the 3' end of the factor IX gene and stretching some 35kb 3' to the gene was used as a large probe, with repetitive sequences being blocked by preannealing the probe with an excess of sonicated, denatured human DNA (Litt and White, PNAS 82, 6206). Results with 25 restriction enzymes (covering an estimated 1038 nucleotides) and DNA from 7 unrelated females were obtained, but only one low frequency PvuII RFLP (frequency about 1%) was identified. Similar experiments with further cosmid probes 3' to the gene are underway. The second technique was developed to analyse small DNA fragments (<1.0kb) generated by frequently cutting restriction enzymes. These fragments were separated on 3.5% polyacrylamide/0.5% agarose composite gels and then electroblotted onto hybond-N. Fragments of 150bp were readily visualised by this procedure. 3 frequently cutting enzymes have been used (Hinfl, Rsal and Mbol), and the blots probed with a factor IX c-DNA probe, or a unique sequence subclone of cosmid pCHIXα. To date no RFLPs have been identified. This search for further useful RFLP has illustrated the paucity of detectable sequence variation within this region of the X-chromosome.
EFFECT OF ELASTASE INDUCED CLEAVAGE OF VON WILLEBRAND FACTOR (vWf) ON ITS STRUCTURE AND FUNCTION
Authors: Howard M A , Coghlan M , Firkin B G
Published: June 2019

A proteolytic product of vWf termed fast migrating protein (vWf:FMP) has been identified using crossed immunoelectrophoresis (CIE) in normal serum and in the plasma from patients with disseminated intravascular coagulation (DIC). A fragment of vWf antigen (vWf:Ag) migrating to a similar position on CIE as vWf:FMP results from digestion of vWf:Ag with polymorphonuclear cells (PMNC). Since parallels exist between the conditions for generation of vWf:FMP by PMNC and elastase release by these cells the effect of purified elastase from porcine pancrease on vWf was investigated.

VWf was purified from plasma using polyethylene glycol, ammonium sulphate and zinc acetate precipitation, high speed centrifugation and elution from column of Sepharose 4B-CL. A fraction rich in vWf was radiolabelled with 125-Iodine to spike the purified preparation of vWf in order to increase the sensitivity of the protein detecting systems.

A mixture of radiolabelled and non-labelled purified vWf was incubated with elastase at concentrations ranging from 2.5 to 40 U/ml for periods of 0-48 hours. Modifications of the structure were assessed by SDS-agarose multimeric analysis, SDS-polyacrylamide electrophoresis and CIE. Alterations of function were quantitated by antigen levels, ristocetin (RCof) and botrocetin (BCoF) cofactor assays and a binding assay to fixed washed platelets in the presence of ristocetin or botrocetin.

These investigations show, 1. all but the highest molecular weight multimers of vWf are present when elastase has cleaved vWf such that no intact 240K subunit is present. 2. an intact 240K subunit is not essential for RCoF and BCoF activity or for ristocetin or botrocetin induced binding to platelets.
INCREASED ADHESIVE PROPERTIES OF THE CARBOHYDRATE-MODIFIED VON WILLEBRAND FACTOR (CHO-VWF): CRITICAL ROLE OF THE MULTIMERIC STRUCTURE AND CORRELATION WITH PLATELET AGGREGATION
Authors: Federici A B , De Romeuf C , De Groot P G , Mannucci P M , Samor B , Lombardi R , D'Alessio P , Mazurier C , Sixma J J
Published: June 2019

We have reexplored the role of the carbohydrate moiety (CHO) on the von Willebrand Factor (vWF) structure and function by critically evaluating its different purification steps and modifications in CHO content by specific enzymes. Structural and functional assays have been evaluated separately in each laboratory (Milano and Lille) and jointly in Utrecht during several organized experiments. Under our conditions, the CHOVWFs obtained were characterized by less than 5% of sialic acid "(Neu)asevWF" and about 45% of D-Galactose "(Neu-Gal)ase-vWF" remaining, by increased electrophoretic mobility without any significant losses of the high molecular weight multimers and by their capacity to induce spontaneous aggregation in normal platelet rich plasma (PRP). Platelet adhesion to these different CHO-vWFs was tested in the flat chamber devised by Sakariassen in the presence of different subendothelial matrices and data expressed as the percentage of the surface covered by platelets. The blood reconstituted with different plasma samples showed the following percentual values of surface coverage (mean ± SD):

- Normal plasma = 15 ± 3.8

- Severe vWd plasma = 4 ± 1.9

- SvWd pl + Native vWF = 14 ± 2.8

- SvWd pl + (Neu) ase-vWF = 23 ± 3.5

- SvWd pl + (Neu-Gal) ase-vWF = 19 ± 2.9

This significantly increased adhesion to the subendothelium of the CHO-vWFs corresponded to the spontaneous aggregation present in normal PRP but it disappeared when the multimeric structure was damaged by in vitro proteolysis and/or by storage conditions (changes in temperature and freezing and thawing). From these results we may conclude that removal of terminal sugars enhances not only platelet-vWF interactions, but also platelet adhesion to the subendothelium.
LOCALIZATION OF THE PLATELET-BINDING AND HEPARIN-BINDING DOMAINS OF BOVINE VON WILLEBRAND FACTOR
Authors: Kirby Edward P , Mascelli Mary Ann , Silverman Carol , Karl Daniel W
Published: June 2019

Bovine von Willebrand Factor (vWF) binds directly to human platelets and also to heparin-agarose. Cleavage of vWF with Protease I, a metalloenzyme isolated from the venom of the western diamondback rattlesnake, produces two major fragments with apparent Mr of 250 kD and 200 kD. The 200 kD fragment competes with native vWF for binding to the GPIb-associated vWF receptor on formalin-fixed human platelets and has weak platelet-agglutinating activity. It is composed of three polypeptide chains of apparent Mr of 97 kD, 61 kD, and 35 kD. Monoclonal antibodies #2 and H-9, which inhibit binding of vWF to a GPIb-associated receptor of platelets, recognize the 200 kD fragment.

Modification of vWF with ^5x-la.beled. Bolton-Hunter reagent (I*-BHR) causes inhibition of platelet-agglutinating activity at very low levels of incorporation. Modification of less than 2% of the amino groups in vWF causes 50% loss of platelet agglutinating activity and a decreased affinity of vWF for binding to platelets. Labeling with I*-BHR does not block binding to heparin-agarose, even when 5-10% of the amino groups are modified. Differential labeling at pH 7.0 and pH 8.5, followed by proteolytic fragmentation with Protease I, suggests that it is the modification of amino groups on the 200 kD fragment which is responsible fpr the decrease in platelet binding activity. Modification of the 97 kD peptide chain is best correlated with this loss of platelet binding activity.

Heparin inhibits the agglutination of human platelets by bovine vWF. The 200 kD fragment of vWF binds both to platelets and to heparin-agarose. These observations suggest that the heparin-binding and platelet-binding domains of vWF, although distinct from one another, reside in the same region of the vWF molecule. The platelet-binding domain contains a small number of very reactive amino groups which are required for vWF binding to human platelets.

These studies were supported by a grant from the National Institutes of Health (#HL27993).
UNIQUE ACTIVATION BY SHEAR STRESS OF GLYCOPROTEIN IIb/IIIa + VON WILLEBRAND (VW) FACTOR CAUSES AGGREGATION: FILTER BLOCKAGE & THE LONG BLEEDING TIME IN VW DISEASE
Authors: O'Brien J R , Salmon G P
Published: June 2019

A filter made of fine glass fibres with tortuous capillarysized pores permitted a detailed study of the effect of varying the shearing forces by altering the pressure. Native, heparinized, citrated and EDTA blood and platelet-rich plasma were forced through the filter. Normal and VW blood were studied and the effects of antibodies to platelet glycoproteins (GPr) and of membrane-active drugs. Normally the filter blocked at 40mmHg but not at 5mmHg. At 40mmHg EM of the filter showed blockage by platelet aggregates. Initially the transit time through the filter was 8 millisecs. Platelet retention in the filter occurred in two phases: 0-3 secs required only high shear, VW factor (VWF) and GPr. IIb/IIIa. At 10-20 secs retention required high shear, VWF, GPr. IIb/IIIa and also divalent cations, and this phase only was inhibited by some membrane-active drugs. Thus VWF must act as the ligand in two different ways. It is known that ADP- or thrombin-induced aggregation requires GPr. IIb/IIIa and fibrinogen. Shear-induced blocking of the filter by blood with a normal concentration of fibrinogen requires GPr. IIb/IIIa and VWF. This indicates a unique method of exposing GPr. IIb/IIIa. The long bleeding time in VW disease highlights the absolute requirement for VWF and for the unique method of exposing GPr. IIb/IIIa induced by shear stress which evidently is required in normal capillary haemostasis.
VON WILLEBRAND FACTOR MULTIMERS IN CULTURED HUMAN ENDOTHELIAL CELLS: COMPARISON BETWEEN CELLULAR STORAGE POOL AND SUPERNATANT
Authors: Piovella F , Lombardi R , Vigotti M , Federici A B , Mannucci P M , Ascari E
Published: June 2019

The multimeric composition of von Willebrand factor (vWf) from cultured human endothelial cells (h.e.c.) has been compared with the multimeric composition of vWf from h.e.c. culture supernatant, human normal platelets and plasma. H.e.c. were derived from umbilical cord veins by collagenase digestion, seeded in culture flasks and grown to confluence in TC 199 culture medium, supplemented with 20% foetal calf serum (FCS). At confluency, cells were harvested by rubber policeman, resuspended in 500 |o.l HBSS with protease inhibitors and stored with culture media until assay. H.e.c. and platelet lysates were obtained by freezing-and-thawing (5 times) and by the addition of 1% Sodium Dodecyl Sulphate (SDS) before centrifugation at 10,000 x g for 20 min at 20°C. Plasma, culture supernatants and cell lysates supernatants were run under the same conditions in an SDS 1.4%, LGT-agarose electrophoresis system using a discontinous buffer. Multimeric patterns were shown by radiolabelled affinity purified rabbit anti-human polyclonal antibody. In all the experiments cultured h.e.c. supernatants exhibited all the set of multimers of vWf usually observed in normal plasma. When vWf from cultured h.e.c. extracts was analyzed, a set of multimers with higher molecular weight was shown, with a pattern very similar to platelet's. We conclude that vWf stored in h.e.c. compartments is characterized by higher molecular weight multimers than culture supernatants. This behaviour recalls the differences recorded between plasma and platelet vWf.
IDENTIFICATION AND FURTHER CHARACTERIZATION OF TYPE IIE VON WILLEBRAND DISEASE
Authors: Triplett D , Muagrave K , Daniels T , Bowie E J W
Published: June 2019

Type IIE von Willebrand disease has previously been identified in a single family with two affected members. We have identified a second family with five affected individuals. The pattern of inheritance is autosomal dominant and the clinical picture is characterized by mild mucocutaneous bleeding. Laboratory results for affected patients: VIII:C ranged from .62 u/ml to 1.0 u/ml, VWD antigen 2.0 u/ml to 4.75 u/ml, and ristocetin cofactor activity 0.04 u/ml to .32 u/ml. Bleeding times, PT and APTT results were within normal ranges.

Multimeric analysis of both citrated plasma and platelet lysates utilizing sodium dodecyl sulfate-polyacrylamide electrophoresis was characterized by a decrease of the largest oligomers and the individual multimers were abnormal with a single identifiable band. Plasma samples collected in the presence of a variety of inhibitors had no effect on the multi-meric pattern. Intravenous infusion of DDAVP (.3 ug/kg of body weight) resulted in a slight increase in the highest molecular weight oligomers but no change in the ristocetin cofactor activity or individual multimeric patterns.
HYPOTHYROIDISM AND ACQUIRED VON WILLEBRAND'S DISEASE
Authors: Preston F E , Greaves M , Sampson B , Kernoff P B A , Savidge G , Bax N
Published: June 2019

A diagnosis of type IA von Willebrand's disease was made in three patients presenting with a mild bleeding tendency. Previously unrecognised hypothyroidism was also confirmed in two patients. In the third, hypothyroidism was diagnosed four years after initial presentation. In all three patients, thyroxine therapy was associated with correction of the haemostatic defect and resolution of the bleeding tendency.

The association of von Willebrand's disease and hypothyrodism prompted us to examine the relationship between thyrotrophin (TSH), T3, T4 and components of the factor VIII complex in 12 patients with clinical and biochemical hypothyroidism. Factor IX was also studied. Mean VIII:C (measured by 2 stage assay) was 0.90 u/ul (range 0.55 - 1.14); mean vWF:Ag 0-83 u/ul (range 0.44 - 1.64); mean VIII:Rcof 0.75 (range 0.45 - 1.55); mean factor IX 0.72 (range 0.39 - 1.19). Multimeric analysis of vWF:Ag performed in samples from 8 patients was normal. VIII:Rcof levels were significantly lower than those of normal controls. A significant inverse correlation was obtained between TSH and factor IX and T4 and vWF:Ag. Although there is a definite inverse relationship between TSH and factor IX, this is not evident with respect to factor VIII and a different mechanism is probably responsible for the modest reduction of vWF:Ag and the occurrence of clinically-evident von Willebrand's disease which we have demonstrated in a small proportion of hypothyroid patients.
EFFECTS OF VASOPRESSIN AND ADRENALINE ON FIBRINOLYSIS: SYNERGISTIC OR ADDITIVE?
Authors: Grant P J , Wiles P G , Boothby M , Davies J A , Prentice C R M
Published: June 2019

Vasopressin (aVP) andadrenalineboth enhance plasminogenactivator activity when infused at low doses in man and probably act asphysiological regulators of fibrinolysis under certain conditions. Therelative contributions of these hormones to changes in fibrinolysis are unknown. This study was carried out to investigate whether aVP and adrenaline act synergistically on plasminogen activator in man. Four normal volunteers were infused with (1) aVP (1. OU/h), (2) adrenaline (420 μg/h), (3) aVP and adrenaline and(4) 0.9% saline for 1h. Saline (0.9%) wasinfused for 30 min before and afterinfusion. There was a minimum of 2 weeks between infusions andthe subjects were not aware of the contentsof the infusate. Samples were takenafter 30 min saline infusion and every 30 min for 1 1/2h foreuglobulinclot lysis time (ECLT), factor VIII:C, aVP and adrenaline. During (1) and (3) plasma aVP rose from (median)0.9 pg/ml to 13.2 pg/ml after 1h. During (2) and (4) plasma aVP remained constant at 0.9 pg/ml. Plasma adrenaline rose from 0.21nmol/1 to 0.62 nmol/1 after 1h during (2) and (3) and remained unchanged at 0.20 nmol/1 during(1) and (4). Plasminogen activator activity (106 /ECLT2 ) rose (%) from 100 to 150% at 1h during adrenalineinfusion,(p < 0.05), 100 to 148% during aVP (p < 0.05), 100 to 252% (p < 0.05) when both wereinfused and from 100 to 123% (N. S) during saline infusion. Factor VIII remained unchanged. The results indicate that at low physiological concentrations, aVP and adrenaline have additive effects on fibrinolysis but are probably not synergistic. This is consistent with the view that aVP and adrenaline mediate this response by different receptors.
INACTIVATION OF HEPATITIS VIRUSES AND HIV IN PLASMA AND PLASMA DERIVATIVES BY COMBINED TREATMENTWITH β-PR0PI0LACT0NE/UV-IRRADIATION
Authors: Stephan W , Dichtelmüller H , Prince A M , Gürtler L , Deinhardt F
Published: June 2019

A combined treatment of plasma andplasma derivatives by β-Propiolactone (β-PL) /UV-irradiation is inuse at Biotest for the preparation ofthe virus safe serum preserve Bisekd® and coagulation factor concentrates.

The efficacy of this sterilization procedure has been demonstrated for HAV(> 8.2 log10), HBV ( 7.0 log10) and HNANB (> 4.5 log10). As HIV has become a major problem tne inactivation of HIV by β-PL/UV in human plasma was tested. Pooled human plasma was spiked with 104.2 infectious units per ml of the Gallo strain of HIV/ HTLV-III and sterilized with 0.25 % β-PL, 60 min at pH 7.2 and subsequently UV-irradiated (4 × 20 W). After treatment with β-PL alone orβ-PL/UV no infectious HIV was detectable by reverse transcriptase assay in inoculated H-9 cultures after 14 days of cultivation (> 4.2 log10 inactivation). When the virucidal efficacy of ft-PL and UV was tested separately, β-PL inactivated > 3.5 log10, UV-irradiation another 2.5 log10 of HIV, as demonstrated by immunofluorescence tests in H-9 cultures 27 daysafter inoculation.

When cryoprecipitate/F VUI-concentrate was sterilized by ft-PL and UV, > 4.5 log10 of HIV were inactivated by UV and > 3.5 log10 by α-PL. The results indicate, that the combined treatment by β-PL/UV inactivates all potentialtiters of HIV, which can be expected inscreened and pooled human plasma orcryoprecipitate, used for the preparation of virus safe plasma derivatives.
VIRAL SAFETY OF SOLVENT/DETERGENT-TREATED AHF IN PATIENTS WITH HEMOPHILIA
Authors: Horowtz M S , Hilgartner M W , Lipton R A , Rooks C , Horowitz B
Published: June 2019

The safety of an Antihemophilic Factor concentrate (Factor VIII-SD) treated with the organic solvent tri(n-butyl) phosphate (TNBP) and sodium cholate is being assessed with respect to transmission of non-A, non-B hepatitis virus (NANBHV) and human immunodeficiency virus (HIV). TNBP/cholate treatment has been previously shown to inactivate at least 10,000 infectious doses each of hepatitis B virus (HBV) and NANBHV using a chimpanzee model, and 30,000 tissue culture infectious doses of HIV.

Patients enrolled in the study have had no previous exposure to blood products made from plasma pools, although some have received small quantities of single-donor products. They have normal alanine amino transferase (ALT) levels and no markers of prior HIV infection and have all been vaccinated against HBV. Each has been treated, as required, with an individual lot of Factor VIII-SD prepared at the New York Blood Center by an FDA-licensed procedure. ALT levels and HIV antibody have been monitored bi-weekly for two months and monthly until the end of six months. Seven patients who have received 475 -20,000 units of AHF(total units 38,255, median dose ∽3400 units) have been followed for at least three months. There has been no indication in these patients ofinfection with either NANBHV or HIV. An eighth patient who had an elevatedALT level prior to enrollment was followed for HIV antibody only. He remains HIV antibody negative through fivemonths of follow-up. Six additional patients have entered the study, but have not yet required treatment or have been followed for only a short time. These results suggest that the risk of virus infection associated with the use of AHF concentrates is significantly diminished by solvent/detergenttreatment.
COMPARATIVE INVESTIGATION OF CLOTTING AND CHROMOGENIC ASSAYS FOR HEPARIN AND LOW MOLECULAR WEIGHT HEPARIN-(OID) IN PLASMA OF VOLUNTEERS AND PATIENTS
Authors: ten Cate H , HENNY Ch P , Sturk A , Prins A , ten Cate J W
Published: June 2019

Two recently developed and simplified anti-Xa methods, a chromogenic assay and a clot-based assay (Heptest) were investigated on their accuracy and ex-vivo characteristics. The coefficients of variation (c.v.) for heparin, a heparin fragment (K 2165) and a LMW hepa-rinoid (Org 10172) were respectively: Heptest, within assay c.v.: 5.4; 4.5; 5.0 % between-assay c.v.: 7.8; 5.8; 9.5 %. Chromogenic Anti-Xa assay, within assay c.v.: 3.7; 5.6; 4.6 %, between assay c.v.: 6.6; 8.2; 12.1 %. In plasma samples obtained from volunteers and patients who participated in clinical studies using heparin, K 2165 and Org 10172, the ex-vivo correlation between both assays were determined. Generally, heparin and K 2165 induced anti-Xa activities correlated very well (0.83 < r< 0.99) and the slopes of the regression lines approached the value of 1. K 2165 in haemodial-ysis patients produced much higher Heptest values compared to the chromogenic anti-Xa assay, for which no explanation is provided. For Org 10172 the anti-Xa assay was more sensitive than the Heptest, although both tests detected anti-Xa activities at very low levels (± 0.02 U/ml). After s.c. injection of Org 10172 a poor correlation was found (r=0.49), which may be due to inter-individual differences in bio-availability. In conclusion, the chromogenic anti-Xa assay and the Heptest are accurate and sensitive assays. However, the tests give substantial differences in results depending upon the heparin preparation and patient category treated.
COMPARISON OF VARIOUS AMI DOLYTIC HEPARIN ASSAYS: REPORT OF A COLLABORATIVE STUDY
Authors: Sternberger A , Haas S , Breddin K , Blümel G
Published: June 2019

The authors performed a controlled study to compare commercially available amidolytic heparin assays based on anti-Xa or anti-I la systems. All participants (see below) were provided with the same batch of all heparin preparations and reagents including homogenous plasma preparation. In addition plasma was spiked with the various unfractionated (n=2) as well as lmwh- (n=5) and heparinoid preparation (n=l). The four university laboratories analyzed all heparin preparations that are registered or used in clinical trials in FRG (n=8). The two testkit producers analyzed registered heparin preparations and the heparin producer their brand only. The study was recently completed and the data analysed and collected. The first evaluation revealed that the amidolytic anti-I la assay is insensitive for lmwh and heparinoids. Determination of unfractionated heparins is not accurate below 0.5 units of the corresponding standard. The Xa-methods are more sensitive for lmwh and heparinoids, however these methods also reveal a high standard deviation. Estimation of concentrations below 0.5 units of all tested heparin preparations is questionable.

The authors are indented for active cooperation to Prof. Dr. K. Andrassy (Med. Univ. Heidelberg), Dr. M. Biegholdt (MIDY LABAZ), Dr. G. Dertinger (Sandoz AG), Dr. VJ. Feller (Braun Melsungen AG), Dr. P. Friberger (Kabi Vitrum Stockholm), Dr. P. Hell stern (Univ. Klinik Homburg), Dr. D. G. Meulemann (Organon Scientific Development Group), Dr. X. Muller (Nordmarkwerke), Dr. Zimmer (Boehringer Mannheim GmbH).
CIRCADIAN CHANGES IN THE ANTICOAGULANT EFFECT OF HEPARIN. PHARMACODYNAMIC AND PHARMACOKINETIC EFFECT
Authors: Decousus H A , Scully M F , Reynaud J , Arnaud-Crozat E , Boissier C , Barral R , Queneau P , Reynaud J , Parker C , Kakkar v v
Published: June 2019

Six patients, with thromboembolic arterial disease, were prospectively studied to assess the influence of the time of injection of a constant dose of calcium heparin (Choay ), given subcutaneously, on the level of heparin measured by APTT and anti-Xa assay. For each patient, the initial dose of heparin was adjusted by APTT 6h after a morning injection to 1.5 and 2.5 times control. Dose was then kept constant. Four randomized times of injection were tested (8am, 4pm, 8pm and 12pm), in each patient acting as his own control. Blood was sampled via a cannula, at Oh, 2h, 3h,4h,5h,6h, 8h, lOh and 12h after injection. The mean APTT and anti-Xa values for the evening injections (8pm and 12pm) were higher than for the morning injection (8am), at 2h until lOh after injection. These differences were significant (analysis of variance:p<0.001) and reached almost 30 seconds for mean APTT values measured 4h, 5h and 6h after injection. For the afternoon injection (4 pm) the mean APTT and anti-Xa values were intermediate but significantly different from all the other times of injection (analysis of variance: p<0.01).

Blood was sampled also on two consecutive days at 12am and 12pm from eight patients receiving heparin subcutaneously for treatment of DVT (administered at 6am and 6pm respectively). Heparin levels by APTT, TT and three antifactor Xa methods (Heptest, Hepaclot, Chromogenic) were significantly higher at night than morning (analysis of variance p<0.005). Cosinor analysis confirmed these results are consistent with circadian variation as we have previously reported after continuous infusion of UF heparin (Br. Med. J., 1985, 290, 341-344). The observed variation was found to be a resultant of a pharmacodynamic effect (circadian variation in assay response to heparin) and pharmacokinetic effect (circadian variation in 99m Tc-heparin clearance). Such circadian variation should be taken into account when deciding heparin dosage.
THE EFFECT OF SHEAR ON OLIGOMER FORMATION; EFFECTIVE REMOVAL OF MONOMERS
Authors: Berk H R
Published: June 2019

Fibrin polymerization has been found to be influenced by shear flow conditions (Puryear,1980). In order to determine which mechanisms are responsible for theeffect reported it is necessary to look at the various stages in fibrin polymerization. It is known that the enzymatic attack of thrombin on fibrinogen is not influenced by shear (Sellers,1981). The next step, which is investigated in this study, is the oligomer-early protofibril stage.

Fibrinogen (human, Kabi) is reacted with thrombin (human, Sigma) under Couette flow conditions (volume-averaged shear 0-250sec-l) in a pH 6.8, 4mM CaC12, HEPES buffered solution (I. S.-.15). The reaction time is chosen so that 6% of fibrinopeptide A (FPA) is released. The reaction is stopped by a 1,6 hexandiol-hirudin solution. The effect of shear on oligomer population is measured using large angle 1ightscattering techniques.

In order to predict theoretical shear effects on oligomer formation, it is important to be able to predict the population size. This is done using Jamney’s (1983) predictions, for early reaction time, assuming q=16. Given a size distribution it is possible to apply low Reynold’s number hydrodynamic and Smoluchowski1 s( coagulation theories to predict possible shear affects.

Hydrodynamic theory predicts no effect of shear on oligomer formation; Peclet numbers are too small. Smoluchowski coagulation theory, on the other hand, predicts that for oligomer sized particles in the shear range studied, orthokinetic (shear induced) coagulation becomes more important than peri kinetic (Brornian) coagulation.

Results obtained from Zimm analysis show a dramatic increase in molecular weight, compared to the stagnant case, in the shear region corresponding to where orthokinetic coagulation dominates. The higher the thrombin concentration, the more extreme and earlier (i.e. lower shear) these effects are felt. After a peak is reached in molecular weight there is a sudden drop. This is caused by monomer exhaustion which shifts the population to a more homogeneous type. The concept of orthokinetic coagulation is important physiologically since it is advantageous to incorporate monomers onto fibers as quickly as possible.
COMPARISION OF THE DETECTION OF LUPUS ANTICOAGULANTS USING THREE DIFFERENT METHODS AND THE PRESENCE OF ANTI-CARDIOLIPIN ANTIBODIES
Authors: Criel A , Gilbert B , Van Hoof A , Hidajat M , Louwagie A
Published: June 2019

Lupus anticoagulant (LAC) is an antibody directed against phospholipids which prolongs in vitro clotting assays. Several detection methods have been described; however all give some different results. Recently ELISA and RIA assays have been developed which detect IgG and IgM anti-cardiolipin antibodies. The aim of our study was to compare three different LAC tests with an ELISA anti-cardiolipin test. The tests used were : kaolin clotting time (KCT or Exnertest), tissue thromboplastin inhibition test (TTI or Schleider test), activated partial thromboplastin time using a 50, 100, 200 fold dilution of the phospholipid preparation (APTT dilution test), and an IgG and IgM anti-cardiolipin ELISA test. 114 samples of patients suffering from diseases known to be accompanied with LAC antibodies (auto-immune diseases, recurrent abortion, thromboembolism, etc.) were studied. Positivity with one of the tests was found in 45 patients (39%). Patients with the diagnosis of SLE or otherimmune diseases showed the highest positivity (56%) whereas those with thromboembolism, recurrent abortion etc. were only positive in 27%.

Among these 45 positive patients the TTI was positive in 41 cases (91 %);however in 10 cases (24 %) this was the only positivity found. The KCT test and the APTT dilution test were both positive in 18 cases (40 %). Anti-cardiolipin antibodies were found in 21 patients (47 %): IgG only in 12 (27 %), IgM only in 5 (11 %), both IgG and IgM in 2 (4 %); in 19 of these 21 patientsthe TTI was also positive.

In our study the TTI test seems to be the most sensitive test but possibly also the test with the highest aspecific positivities. IgG and IgM anti-cardiolipin antibodies were less frequently found than expected.
FIBRINOLYTIC ACTIVITY AND OTHER COAGULATION PROTEINS IN PATIENTS WITH LUPUS ANTICOAGULANT
Authors: Borrell M , Fontcuberta J , Muñiz E , Grau E , Oliver A
Published: June 2019

Patients with lupus anticoagulant (LA) is a group with recognised risk of thrombosis. In order to study the possible causes of this tendency, we have studied the fibrinolytic activity and some other coagulation parameters considered as thrombotic markers in a group of 12 patients with LA. Half of them had presented thrombotic disease and/or recurrent abortions. 3 patients presented systemic lupus erythematosus and in the other 9 we couldn't detect any connective tissue disorder. The following tests were performed: tissue plasminogen activator (t-PA) activity, t-PA antigen, t-PA inhibitor, fibrin plates with and without kaolin, -antiplasmin, plasminogen, prekallikrein, antithrombin III, protein C, protein S. The results were analysed comparing the values obtained in LA group with respect to normal values, and among the LA group between patients who had suffered from thrombotic disease and those who did not.

The results obtained did not show significant differences between control group and LA patients except in t-PA activity which was found decreased in LA group: 1.86±0.89 versus 3.0±0.94 in control group (meantstandard deviation). Among this group there were no differences between patients who had suffered from thrombotic disease and those who didn’t.

From these results we suggest that unpaired t-PA activity may contribute but not be the only responsible factor for the dvelopment of thrombosis in these patients.
IMMUNOHISTOCHEMICAL STUDIES ON THE LOCALIZATION OF ENDOTOXIN (LPS) IN VIVO BY USING HORSESHOE CRAB FACTOR C
Authors: Uragoh K , Sueishi K , Nakamura T , Iwanaga S , Tanaka K
Published: June 2019

The localization of LPS in vivo was studied with immunohistochemical method using Ig G against Factor C, which was extracted from hemocyte lysate of horseshoe crab and had the specific affinity to LPS. Organs of rats and guinea pigs were light microscopically investigated at different times after intravenous injection of LPS (E.coli; 0111:B4,026:B6 and salmonella typhosa). Tissues were fixed with buffered formalin and then embedded in paraffin. Deparaffinized sections were incubated with Factor C (lpg/ml) for 1 hr, and then with anti-Factor C Ig G for 1 hr, followed by immunoperoxidase method. The immunohistochemical specificity was examined by absorption of Factor C with LPS, binding competition between Factor C and anti-LPS factor which was extracted from hemocyte lysate of horseshoe crab as well as Factor C or using normal animal tissues and normal Ig G. The immunohistochemical specificity was revealed by these examinations. Immunohistochemically, LPS located
predominantly in liver and lung, especially in Kupffer cells and infiltrating monocytes and neutrophils, and aggregated platelets since 5 minutes after intravenous injection of LPS. On the endothelial surface of hepatic sinusoids, glomeruli and pulmonary vessels, LPS was also detected in early period. In addition, LPS was also shown within adrenocortical parenchymal cells, particularly of fascicular zone, later. LPS was not detected 3 days after injection of LPS in liver and lung, but remained during 3 days of observation in adrenocortical parenchymal cells. The present studies revealed that Factor C could be available for immunohistochemical demonstration of LPS in vivo, and reticuloendothelial system, macrophages/monocytes and neutrophils were important as the scavenger cells of LPS and might play a significant role on the development of multiorgan failure in endotoxemia.
THE ABSORPTION, EXCRETION AND TRANSPLACENTAL TRANSPORT OF VITAMIN K IN THE PERINATAL PERIOD
Authors: Suzuki S
Published: June 2019

It is a well-known fact that hemorrhages are observed in wholly breastfed infants beyond the neonatal period. In order to clarify vitamin K (VK)-deficiency, it is necessary to follow-up the absorption and excretion of VK2.1. To 128 cases of newborns. i) The activity of VK-dependent factors(II, VII, X) were determined by Hepaplastin test(HPT), ii) Using Latex-test, PIVKA-II was tested. We found values of HPT (Y) and PIVKA-II (X) to be inversely proportional in the relation. Y=6l.9 - 6.7 X (r= -0.3). 2. These 15 cases of hypoprothrombinemia, VK2 6mg, VK2 2mgwere given, and plasma VK2-concentration was measured by gas chromatography. After 3 hours VK2 6mgconcentration was 1030ng/ml; VK2 2mg, was 224ng/ml. This clearly shows a dose-response relation. 3. VK2 transplacental transport was also proved by using umbilical venous blood after Cesarean section. (Before Cesarean section, VK2 60mgwas given. ) In umbilical venous blood, relatively high doses of VK2 (50 - 120ng/ml) were demonstrated. Additionally, the y-carboxylglutamic acid-concentration in the urine of newborn, who received VK2-syrup was higherthan those who did not receive it.
AMNIOTIC FLUID FROM TERM GESTATION ENHANCES PLATELET PRODUCTION OF VASOCONSTRICTOR THROMBOXANE: POTENTIAL ROLE IN PERSISTENT PULMONARY HYPERTENSION IN THE NEONATE
Authors: Stuart M J , Wu J , Ganley C , Sunderji S
Published: June 2019

One of the causes of persistent pulmonary hypertension (PPH) in the neonate may involve the interaction of amniotic fluid (AF) and pits, with the release of thromboxane A2 (TXA2) causing an increase in pulmonary vascular tone. To prove or disprove this hypothesis, AF from term (n=6) and from 15 to 17 weeks gestation (n=6) were preincubated for 15 minutes with platelet rich plasma followed by the addition of thrombin or arachidonic acid, and plt. TXB2assessed by radioimmunoassay. AF from early gestation had no significant effect on the production of the potent pulmonary vasoconstrictor metabolite thromboxane. Mean plateletTXB2 formation in the presence of thrombin (0.2 unit per ml) was 0.5±0.26 (1SD) pmol per 106 pits, in control vs 0.65±0.4 pmol for paired prts preincubated with early gestation AF. However, plts.that had been preincubated with term AF prior to thrombin stimulation produced significantly greater amounts of TXB2 (0.79±0.4 pmol) compared to paired control aliquots (0.45±0.27; P<0.05). Similar results were observed using the pit. agonist arachidonic acid. The thromboxane enhancing capacity of term AF appeared to reside in its lipid fraction. When compared to a baseline production of 1.4±0.45 pmol TXB2 per 106 pits, in response to thrombin (1 unit per ml), unfractionated AF or its lipid fraction enhanced TXB2 production to 2.87±0.53 and 2.81±0.62 pmol respectively (P<0.01). Values for the aqueous extract however were no different from buffer control values (1.45±0.5). Following perinatal aspiration, and the absorption of AF into the pulmonary capillaries, in situ contact between AF and blood in the pulmonary vasculature could cause thrombin generation and the production of proaggregatory thromboxane. Recruitment of more pits and the production of the plt-fibrin thrombi that have been documented in the pulmonary microcirculation of neonates with perinatal aspiration and PPH could then occur. The use of thromboxane synthetase inhibitors in an experimental model of perinatal aspiration should be evaluated.