bluuebunny/crossref_metadata_2025 huggingface dataset:
Intervention durchführen und auswerten – Professionelles Reasoning in der Pädiatrie – Teil 2
Authors: Strebel Helen , Zillhardt Cornelie
Published: June 2019

In den letzten 20 Jahren entstand unter anderem das professionelle Reasoning, das den klinischen Entscheidungsprozess unterstützt. Helen Strebel und Cornelie Zillhardt durchlaufen Intervention und Outcome eines Fallbeispiels aus moderner Sicht und aus der Sicht von vor 20 Jahren. Heutzutage stehen zum Beispiel die Anliegen eines Kindes wie Rollerfahren im Zentrum der Therapie.
Steuerpflichtige Leistungen – Umsatzsteuer in der Ergotherapie
Authors: Päper Mirko
Published: June 2019

Heilbehandlungen sind in Deutschland grundsätzlich umsatzsteuerfrei – vorausgesetzt sie wurden vom Arzt oder einem Heilpraktiker verordnet. Anders sieht es mit Präventionsleistungen und Anschlussbehandlungen aus. Hier fallen Steuern an, die der Praxisinhaber auf seine Preise aufschlagen sollte. Steuerberater Mirko Päper erklärt, worauf Therapeuten beim Thema Umsatzsteuer achten müssen.
Erste Ergebnisse der transperinealen Ligatur des Fisteltrakts bei rektovaginalen Fisteln
Authors: Schwandner Oliver , Falch Claudius , Reisenauer Christl
Published: June 2019

Hintergrund Die chirurgische Therapie der rektovaginalen Fistel stellt trotz vielfältiger Operationsverfahren weiterhin eine Herausforderung dar. Es war Ziel dieser prospektiven Studie, die Ergebnisse einer innovativen Operationstechnik zu evaluieren.

Material und Methoden Es erfolgte eine transperineale Ligatur des Fisteltrakts ohne Fistulektomie oder Sphinkterplastik. Es wurden ausschließlich tiefe rektovaginale Fisteln für die Operationsmethode ausgewählt. Standardisierte Ein- und Ausschlusskriterien wurden definiert.

Ergebnisse Innerhalb eines 16-Monate-Zeitraums wurden an 2 Zentren 7 Patientinnen mit einer tiefen rektovaginalen Fistel über einen transperinealen Zugang operiert. Die Genese der rektovaginalen Fisteln war postpartal, operativ-iatrogen, bei Z. n. Bartholin-Abszess sowie bei Morbus Crohn. In allen Fällen konnte die Fistel identifiziert werden und zwischen einer Ligatur durchtrennt werden. Intra- und postoperative Komplikationen traten nicht auf. Nur eine Operation erfolgte unter Stomaschutz. Nach einem mittleren Follow-up von 9 Monaten kam es zu einer Heilung bei 4 von 7 Patientinnen (Erfolgsrate 57%).

Schlussfolgerung Die ersten Ergebnisse einer transperinealen Ligatur des Fisteltrakts bei der rektovaginalen Fistel scheinen vielversprechend. Um den objektiven Stellenwert zu beurteilen, müssen weitere Erfahrungen, Studien mit größerer Fallzahl und Langzeitdaten abgewartet werden.
RENAL FUNCTION AND MORPHOLOGY IN HEMOPHILIACS WITH PREVIOUS GROSS HEMATURIA
Authors: Tramonti G , Donadio C , Bartolai S , Rossi L , Panicucci F , Bianchi C
Published: June 2019

In hemophiliacs gross hematuria is often observed. The aim of this study is to evaluate renal functionand morphology in young hemophiliacs with at least one previous gross hematuria. Forty patients with hemophilia A or B were studied (age 8-49 yrs, mean 21.3). Neither renal nor urinary tract disease was referred. The following tests were performed: urinalysis, plasma creatinine and urea, glomerular filtration rate (GFR),effective renal plasma flow (ERPF),renal scans, isotopic renography and renal ecography. Urinalysis was normal in all but 6 pts. Plasma creatinine and urea were normal in all pts. GFR was reduced (< 90 ml/min) in 16 pts (mean 76.1±9.5),while it was normal (>90 ml/min) in the remaining 24 pts (mean 119.6±17.1). ERPF was < 350 ml/min in 11 pts (mean 290.3±51.7)and it was >350 ml/min in the remaining 24 pts (mean470.0±107.8). In 17 of these pts GFR has been measured a second time after 1-3 yrs. The second GFR decreased of more than 20% in 8 pts (mean decrease 35.3%)and increased of more than 20% in 3 pts (mean increase 52.9%). It was unchanged in the remaining 6 pts. In pts with lower GFR renal scans were abnormal in 5/12 cases, while in subjects with normal GFR renal scans resulted abnormal in only 4/22 cases. Isotopic renography demonstrated pathological findings in 5/9 pts with lower GFR,while in subjects with normal GFRthe abnormal renograms were 2/18. Renal ecography showed kidney abnormalities in 2/7 pts with low GFR while it was normal in 4/4 pts with normal GFR.

The above results indicate that a relevant number ofyoung hemophiliacs with previous gross hematuria have a clinically significant impairment of renal function, and some abnormalities of renal morphology.
TYPE III PRO-COLLAGEN PEPTIDE IN LIVER DISEASE IN HAEMOPHILIA
Authors: Evely R S , Preston F E , Triger D R , Hay C R M , Greves M C , Underwood J C E
Published: June 2019

During the past 10 years we have carried out liver biopsies on haemophiliacs with biochemical evidence of chronic liver disease (CLD). To date 44 biopsies have been obtained from 35 patients. Histological diagnoses are Chronic Persistent Hepatitis (CPH) 24, Chronic Aggressive Hepatitis (CAH) 11 and Cirrhosis 9. Serial biopsies indicate that progressive liver disease is now a serious problem in haemophilia. Liver biopsy is not without risk and therefore it is important to identify factors which may be of value in predicting the nature of the liver disease or its progression. Since intra-hepatic fibrosis is a feature of CLD we measured Type III amino terminal propeptide of pro-collagen (PC III) by radio-immunoassay on samples taken within a mean of 4.8 months of the liver biopsy. A normal range was established as 4.3 - 15.7ng/ml on healthy subjects (median 7.0). Median values and ranges for patients with CPH (N=13), CAH (N=5) and cirrhosis (N=5) were 8 (5.4 - 23.4), 14.2 (7.2 - 19.8) and 14.2 (11.2 - 23.0)ng/ml respectively. Although pro-collagen III values tended to be higher in progressive liver disease (CAH and cirrhosis) this did not reach statistical significance. It would, therefore, appear that unlike serum IgG, pro-collagen III will not be a valuable predictor of progressive liver disease in haemophilia. A larger study is necessary to clarify this.
THE BENEFITS OF MODERN SUBSTITUTION THERAPY IN HEMOPHILIA
Authors: Smit C , Varekamp I , Rosendaal F , Bröcker-Vriends A , Suurmeijer T , Briët E
Published: June 2019

Coagulation factor preparations became available in the treatment of hemophilia about twenty years ago, followed by the introduction of prophylactic therapy and home-treatment. The purpose of our longitudinal study was to quantify the impact of these treatment modalities on the medical and social situation of hemophiliacs.

We carried out three mail surveys (1972, 1978 and 1985) among Dutch hemophiliacs. In 1985 we sent questionnaires to 1162 of the estimated total of 1300 patients with a response of 81%. Eighty-six percent of the respondents had hemophilia A, 14% hemophilia B; 41% had severe hemophilia (<1%) 19% moderately-severe (1-5%) and 40% mild hemophilia (>5%). Growth of prophylactic therapy and home-treatment for severe hemophilia was The mean age increased from 21 yrs in 1972 (n=435) to 29 yrs in 1985 (n=935)(general male population: 34 yrs). The number of manifest bleedings decreased from 25 in 1972 to 15 in 1985, with a corresponding decrease in the number of transfusions for acute bleedings. Hospitalization per 100 patients with severe hemophilia decreased from more than 2100 days in 1972 to 440 days in 1985. Non-attendance at school caused by hemophilia dropped from 6 to 2 weeks per year, and sickleave from work from 35 to 15 days per year, so that it now equals sickleave among the general male population. Unemployment figures for hemophiliacs were similar to those for the general population, but disability figures are still higher. Our study shows in a quantitative way that the benefits of modern hemophilia treatment are impressive and that its costs are more than justified.
ANALYSIS OF HUMAN FACTOR VIII INHIBITOR EPITOPES TO FACTOR VIII POLYPEPTIDES BY IMMUNOBLOTTING
Authors: Yoshioka A , Shima M , Tanaka I , Fujiwara T , Nakai H , Fukui H
Published: June 2019

In order to clarify human factor VIII inhibitor epitopes to factor VIII (F. VIII), we analyzed the inhibitor IgG developed in the patients with hemophilia A and autoimmune disease using immunoblotting of purified F. VIII and thrombin-degraded F. VIII. IgG fractions were obtained from 6 cases of severe hemophilia A and one autoimmune disease. The titer of the inhibitor plasma ranged from 50 to 3,000 Bethesda units/ml. Purification of F. VIII from commercial F. VIII concentrate was performed by immunoadsorbent column chromatography using anti-von Willebrand factor monoclonal antibody and anti-fibrinogen, anti-fibronectin and anti-IgM goat antibodies and subsequently by Aminohexyl Sepharose column chromatography essentially according to the method of Fulcher et al.. The specific activity of the purified F. VIII was 2,700 units/mg. On sodium-dodecylsulfate (SDS) 5 to 10% gradient polyacrylamide gel electrophoresis (PAGE), 80 kDa of a main fragment and a series of 90 to 210 kDa fragments were visualyzed by Coomassie blue staining. Immunoblotting of 5-10 ug of each of unreduced purified F. VIII and thrombin-degraded F. VIII. followed by reaction with inhibitor IgG samples, monoclonal antihuman IgG-3 and IgG-4 antibodies and radiolabeled rabbit antimouse IgG.

All inhibitor IgG samples reacted with both purified F. VIII and thrombin-degraded F. VIII. The pattern of reactivity of inhibitor antibodies was divided into three groups; 1) inhibitor IgG which reacted with 80 and 70 kDa derived from carboxy-terminus of the F. VIII molecule, 2) inhibitor IgG which reacted with 90 to 210 kDa and 54 and/or 44 kDa derived from amino-terminus of the F. VIII, and 3) inhibitor IgG which reacted with both of N- and C-terminus of the F. VIII. One inhibitor IgG belonged to group 3 strongly reacted with a series of higher molecular polypeptides of F. VIII ranged from 180 .to 210 kDa. One autoantibody IgG from the patient with autoimmune disease was also belonged to group 3. There was no relationship between the titer and the pattern of reactivity of each inhibitor plasma. In summary, there is a heterogeneity of F. VIII inhibitor epitopes to F. VIII molecules.
A MURINE MODEL FOR FACTOR VIII ANTIBODY ANTI-IDIOTYPE REAGENTS
Authors: Moffat E H , Furlong R H , Bloom A L , Giddings J C
Published: June 2019

The regulation of factor VIII antibody (FVIIIAb) production in haemophilic and non-haemophilic patients may be effected by anti-idiotype (Aid) antibodies which specifically react with FVIIIAb. Aid antibodies (reagents) were prepared from rabbits immunised with murine monoclonal FVIIIAb. Immuno fluorescent microscopy and cell culture studies were performed using murine hybridoma cells which secreted the FVIIIAbs.

Immuno fluorescence studies examined the ability of the Aid reagents to bind to acetone fixed FVIIIAb secreting hybridoma cells. Positive surface membrane and intra-cytoplasmic staining patterns were seen with the Aid reagent when incubated with the corresponding murine hybridoma cell line. This reaction was blocked subsequent to the addition of the corresponding monoclonal FVIIIAb but was preserved when unrelated monoclonal FVIIIAb was incubated with the hybridoma cells. No fluorescence was observed when Aid reagent was incubated with unrelated FVIIIAb secreting hybridoma cultures.

Following the addition of Aid reagent to FVIIIAb secreting hybridoma cultures and incubation for 19 hours, the resultant hybridoma supernatants were examined for FVIIIAb content using an immunoradiometric technique. The Aid reagent failed to inhibit FVIIIAb secretion by hybridoma cells. Thus, although Aid reagents were capable of binding to fixed FVIIIAb secreting cells, they failed to inhibit FVIIIAb secretion from hybridoma cultures. The conjugation of Aid reagent with immunotoxin may however have cytotoxic potential. The murine model provides a method for the study of Aid regulation of FVIIIAb production in haemophilia.
INFLUENCE OF HEPARIN ON FACTOR VIII (FVIII) ASSAY
Authors: Tran T H , Zuhlke U , Hauert J , Duckert F , Marbet G A , Wagenwoord R
Published: June 2019

Thrombin-activated FVIII accelerates the conversion of factor X to activated factor X (FXa) by activated factor IX, phospholipids and calcium ions. In plasma, FVIII is activated by initial traces of thrombin, which, in the presence of heparin, is rapidly inhibited by binding to anti thrombin III and heparin cofactor II. To avoid the effect, we have experienced with increasing amounts of exogenous thrombin. We were able to match the heparin cofactors concentration in diluted plasma with thrombin, so that the presence of heparin did not affect the formation of FXa, whose activity was assessed with a chromogenic substrate. Indeed, addition of heparin at any concentration to citrated plasma showed no significant deviation from the FVIII control value. Levels of FVIII in heparinized plasmas similar to those in citrated plasmas further confirmed the finding. Patients plasmas showed comparable FVIII levels before and after heparin infusion, though plasma PTT was clearly prolonged after in vivo heparinization. FVIII chromogenic assay was correlated with the one-stage clotting assay by measuring FVIII levels in 60 hemophiliacs A and carriers, in patients with von Willebrand disease (27) and other congenital deficiencies (4), high risk of thrombosis (15), bleeding tendency (20), disseminated intravascular coagulation (4) and circulating anticoagulants (2), and commercial concentrates. There was a highly significant correlation between both techniques (N=127, r=0.97, Y=0.91X + 4, range 1-380%). Three severe hemophiliacs with were detected with both methods. Data obtained from both techniques were also in good agreement in the range of 1-20% FVIII.

Thrombin was added both to activate instantaneously FVIII and to neutralize heparin cofactors in samples. It thus abolishes the incubation time needed to generate in situ traces of thrombin and the influence of heparin on our FVIII assay. An eventual fibrin formation does not affect the FXa formation and the reading. The technique is also suited for automation.
EFFECT OF COLD ACTIVATION, AL(OH)3 ADSORBTIGN, TISSUE FACTOR AND PLATELET ON ONE/TWO STAGE CEROMOGENIC ASSAYS OF F. VIII
Authors: Seghatchian M J , Dembinski M J
Published: June 2019

Two stage Coatest assay of F. VIII is reportedly insensitive to pretreatment of F. VIII with thrombin. Since thrombin is formed rapidly, during the incubation step, a one stage method was tried by incorporating S2222, containing thrombin inhibitor (1-2581) in the incubation system, thus making the assay highly specific to F. Xa-induced activation of F. VIII and allowing to monitor directly the formation of paranitroaniline in microtray plate at 2-5 min. intervals. Initial comparative analyses performed on cold activated and/or adsorbed/non-adsorbed samples (FFP, cryoprecipitate and hypo- or hypercoagulable state) revealed that in all cases the lag phase was prolonged (2-3 fold) in the one stage method. Cold activation had little effect on the lag phase/reaction rate, whereas AL(0E)3 decreased up to 50% F. VIII like activity, prolonged the lag phase and dose-response curves become non-parallel. Substituting phospholipid (pL) by tissue factor (TF) or addition of diluted TF (1/500) to reaction mixture increased synergistically the rate of F. Xa generation in both adsorbed and non-adsorbed system. In contrast washed platelets (PLT), up to 3000 x 109/1, were less effective on both TF or PL-induced F. Xa generation. The presence of 1-2581 in this system prolonged the lag phase to Ih. Substitution of the conventional O. D. reading by the time required to achieve a fixed absorbancy (O. D. =0.5) make the one stage coatest F. VIII equivalent to the APPT-type assay. Based on these results it is concluded that thrombin is involved in increasing F. VIII catalytic activity. TF and F. VII contribute to the shortening of the lag phase and increased F.a generation. The Kinetic property of cell surface-bound F. VIII is not the same in the presence or absence of thrombin. The reported insensitivity coatest F. VIII to thrombin is probably due to the fact that thrombin activated F. VIII is a good substrate for F. Xa and is cleaved by F. Xa which is produced in abundance in the two-stage chromogenic assays. A new procedure for monitoring various pathways of F. Xa generation, based on the coatest reagent is provided, which is particularly suitable for large scale screening of blood donors and blood products.
THE EFFECT OF CALCIUM ON THE STABILITY OF PURIFIED FACTOR VIII
Authors: Qanz P R , Tackaberry E S , Palmer D S , Malchy B , Rock G
Published: June 2019

The involvement of calcium and phospholipid in the activation of Factor X to Xa by Factor IXa and Factor VIII has been well documented. Although we and others have shown that maintenance of physiological concentrations of calcium has a positive effect on the stability of Factor VIII in plasma, calcium’s role in the structure and function of Factor VIII remains to be fully elucidated. To this end, we examined the effect of calcium on the stability of highly purified Factor VIII. Homogeneous Factor VIII (specific activity approximately 5,200 U/mg) was prepared from heparinized blood using a six-step purification procedure including cryoprecipitation, polyethylene glycol precipitation, Affi-Gel Blue, Aminohexyl, polyelectrolyte E5 and immunoaffinity chromatography. This yielded a single chain high molecular weight species of approximately 260,000. The protein was tested for stability using the one stage assay over 6h of incubation at 4°C in buffers containing 0 mM, 5 mM, and 10 mM CaCl2. Addition of 5 mM and 10 mM CaCl2 to desalted, purified Factor VIII resulted in an immediate 12% (for 5 mM CaCl2) and 23% (for 10 mM CaCl2), enhancement of procoagulant activity compared to samples containing no added calcium. The calculated half-life (T1/2) of activity of Factor VIII in buffers containing no added calcium was 3.8h, whereas the Tl/2 for preparations incubated in the presence of 5 mM and 10 mM CaCl2 were increased to 5h and 5.5h respectively. Although the addition of calcium improved the recovery of activity over the first 0.5h of incubation, at later times the rate of decay in the calcium containing preparations was similar to Factor VIII preparations without added calcium. Our results suggest that removal of calcium from the microenvironment of purified Factor VIII by desalting, results in an immediate loss of procoagulant activity, which can be partially restored within the first 0.5h following readdition of calcium. The decay in Factor VIII activity observed at later times in the 0 mM, 5 mM and 10 mM CaCl2 containing buffers likely reflects calcium-independent denaturation of the protein.
THE CHANGES OF FACTOR VIII ANTIGEN DURING THE COAGULATION PROCESS
Authors: Tanaka I , Yoshioka A , Fujiwara T , Nakai H , Fukui H
Published: June 2019

The changes of factor VIII (F. VIII) during blood coagulation process is still controversial. We analyzed the F. VIII antigen (F. VIII:Ag) at various intervals of in vitro blood clotting by immunoassays using polyclonal and different kinds of monoclonal antibodies to F. VIII.

We used two immunoassays, an immunoradiometric assay (IRMA) and an enzyme-linked immunosorbent assay (ELISA). The IRMA was performed by the method of Peake et al. using high-titer allo-antibodies to F. VIII. The ELISA was performed by two-site solid phase system consisting of alloantibodies as the first and one of three kinds of monoclonal antibodies (NMC-VIII/1, -VIII/2 or C5*) as the second antibody. Using the immunoblotting technique, it had been shown that NMC-VIII/1 recognized the 80/79 kDa derived from C-terminus and both NMC-VIII/2 and C5 recognized 54 kDa derived from N-terminus.

The mean levels of F. VIII:Ag in 20 normal plasmas and sera were 0.97±0.23 U/ml and 0.68±0.21 U/ml, respectively using the polyclonal IRMA. The mean levels of F. VIII:Ag in normal sera were 0.14±0.05 U/ml (NMC-VIII/1), 0.71±0.21 U/ml (NMC-VIII/2), and 0.012±0.02 U/ml (C5) using the monoclonal ELISAs. In the initial phase of whole blood coagulation in vitro, the increase of F. VIII: Ag was observed by the polyclonal IRMA as F. VIII:C assayed by a one-stage clotting method increased. On the other hand, the F. VIII:Ag assayed by NMC-VIII/1 or C5 monoclonal ELISA progressively decreased to the serum level within 30 min. The F. VIII:Ag by NMC-VIII/2 declined to the serum level at a slow rate. In order to study the influence of thrombin on F. VIIIrAg during blood clotting, a synthesized selective thrombin inhibitor (MD-805, Mitsubishi Chemical Ind.) was previously added to the whole blood tested. The changes of F. VIII:Ag with MD-805 by the monoclonal ELISAs were almost the same as those without MD-805.

It is suggested that in the whole blood coagulation process the antigenicity of F. VIII molecule changes in the initial phase (within 30 min.), but that thrombin does not play the main role of the phenomenon in physiological concentration.

*C5 was kindly supplied from Dr. C. Fulcher (Scripps Clinic and Research Foundation, USA)
DIFFERENTIAL RESPONSES OF DIFFERENT FACTOR VIII MOLECULAR FORMS TO THROMBIN AND EDTA
Authors: Poon M-C , Chow K-S , Low S , Sinclair G D
Published: June 2019

Prevailing data suggest that activation of factor VIII (VIII) results in the enzymatic cleavage of the molecule into two chains bound by Ca++. When Ca++ becomes critically involved is not clear. We have investigated this process by studying the relationship of thrombin activation of VIII and the EDTA enhancement of VIII antigen (VIII:Ag). VIII:Ag was assayed by a specific and sensitive (<0.7 mU/ml) ELISA using microtiter plates coated with human anti-VIII IgG and probed with a mouse monoclonal antibody to VIII:Ag. Unlike VIII activities, plasma VIII:Ag levels detected by this ELISA were found to be relatively constant during in vitro thrombin activation. However, in the presence of EDTA (at pH 7.0), the apparent VIII:Ag values were enhanced by 1.6-1.8 fold. Sera devoid of VIII activity, on the contrary, already had VIII:Ag values about 1.6-fold higher than the corresponding plasma values, and the raised serum VIII:Ag values could not be further enhanced in the presence of EDTA. Fractionation of VIII from cryoprecipitates by aminohexyl-agarose chromatography in buffers containing Ca++ and protease inhibitors revealed the heterogeneous nature of VIII molecules. The different VIII fractions showed differential responses to activation by thrombin, and VIII:Ag enhancement by EDTA. Thrombin activation studies showed that the earlier fractions were most activatable, with the thrombin activatability falling progressively with successive fractions. When EDTA enhancement of VIII:Ag was studied, the reverse was observed: the enhancement increased progressively with successive fractions. Thus, aminohexyl-agarose chromatography resulted in fractionation of VIII into a continuous spectrum of molecular forms. We postulate that the earlier eluting fractions contained mainly "native" molecules which responded maximally to thrombin and minimally to EDTA. Subsequent fractions contained mainly the "active" form of VIII which responded minimally to thrombin but maximally to EDTA. The enhancement of VIII:Ag by EDTA is likely due to the removal of Ca++ with exposure of additional antigenic determinants. Our data suggest that Ca-^ participates mainly in the conformation of "active" VIII molecules. In serum, the molecule had been degraded with maximum exposure of the antigenic determinants, so that EDTA had no further enhancement effect.
THROMBIN-ACTIVATED FACTOR VIIIa IS COMPOSED OF A NONCOVALENT 73/51 kD DIMER
Authors: Fay P J
Published: June 2019

Human factor VIII purified from plasma concentrates consists of a series of heterodimers composed of a light chain of 83 kD noncovalently bound to a heavy chain which varies in size from 93 to 170 kD. Previously, we showed that each of the purified heterodimers wasactivated by thrombin to a similar extent. Activation to factor VIIIa was correlated with proteolysis of the light chain generating a73 kD polypeptide and cleavage of the heavy chain(s) generating polypeptides of 51 and 43 kD, whereas subsequent inactivation of factor VIIIa occurred in the absence of further proteolysis (Biochim Biophys Acta 871:268-278, 1986). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of reduced or nonreduced samples showed similar polypeptide patterns indicating that there were no covalent linkages between the 51 and 43 kD chains. However, prior data does not distinguish between a factor VIIIa complex of the 73, 51 and 43 kD polypeptides and a subset of these chains. To identify factor VIIIa, thrombin- treated factor VIII at peak activity was subjected to rapid gel filtration on Superose 12. Factor VIII activity eluted as a single peak representing about 30% of the applied activity after correction for spontaneous inactivation. SDS-PAGE followedby silver staining showed that activity was correlated to fractionscontaining the 73 and 51 kD polypeptides, which co-eluted and which were separated from both the 43 kD fragment and thrombin. Densitometric scans of the stained gel indicated the stoichiometry of the 73:51 kD polypeptides in eachactive fraction to be 1:1. Addition of EDTA(50 mM) to a similar thrombin-factor VIII mixture resulted in rapid inactivation of factor VIIIa. Gel filtration followed by SDS-PAGE analysis of this sample showed that the 73 and 51 kD polypeptides eluted separately and were more included, while the elution position of the 43 kD polypeptide was unchanged. These results suggest that factor VIIIa is represented by a noncovalent dimer consisting of a 73 kD polypeptide derivedfrom the light chain plus a 51 kD polypeptide derived from the heavy chain.
ANALYSIS OF STRUCTURAL REQUIREMENTS FOR FACTOR VIII FUNCTION USING SITE-DIRECTED MUTAGENESIS
Authors: Pittman Debra D , Wasley Louise C , Murray Beth L , Wang Jack H , Kaufman Randal J
Published: June 2019

Factor VIII (fVIII) functions in the intrinsic pathway of coagulation as the cofactor for Factor IXa proteolytic activation of Factor X. fVIII contains multiple sites which are susceptible to cleavage by thrombin, Factor Xa, and activate) protein C. Proteolytic cleavage is required for cofactor activity and may be responsible for inactivation of cofactor activity. In order to identify the role ofthe individual cleavages of fVIII in its activation and inactivation, site-directed DNA mediated mutagenesis of fVIII was performed and the altered forms of fVIII produced and characterized. Conversionof Arg residues to lie residues at amino acid positions 740, 1648, and 1721 resulted in resistance to thrombin cleavage at those siteswith no alteration of in vitro procoagulant activity. Modification of the thrombin cleavage sites at either positions 372 or 1689 resulted in loss of cofactor activity suggesting that these sites are important for activation. Modification of the postulated activated protein C cleavage site at position 336 resulted in fVIII with a higher specific activity than wild type, possibly due to resistance toproteolytic inactivation.

DNA mediated mutagenesis was also used to study the role of post-translational biosynthetic modifications of fVIII. Structural characterization of recombinant fVIII suggested the presence of sulfated tyrosine residues within two acidic regions located between amino acid residues 336-372 and 1648-1689. Individual modification of theseTyr residues to Phe had negligible effect on synthesis and in vitrocofactor activity. The effect of combinations of these mutations onsecretion, cofactor activity, and vWF interaction will be presented.
A POINT MUTATION AND A GENE DELETION OF FVIII GENE IN SEVERE HAEMOPHILIA
Authors: Bernaedi F , Bertagnolo V , Bartolai S , Rossi L , Panicucci F , Conconi F
Published: June 2019

The presence of Factor VIII (FVIII) gene lesions has been investigated in 100 haemophilia A patients using cDNA probes for the 3'part of FVIII gene (exons 14-26 ).

In two related severe patients without inhibitor a deletion removesthe exon 26; the gene lesion has been confirmed with several restriction enzymes and has been shown by densitometry of the autoradiographic pattern in a woman of the same family. The complete deletionof the exon 26 has been described by Gitschier et al. in a patient with inhibitor. Thus the comparison of the end points of the two deletions could help to define the mechanism originating these gene lesions and the relation between gene lesions and the presence of antibody.

In a patient with severe Haemophilia and without inhibitor a mutation removing the TaqI site in the exon 24 and originating an abnormal band of 4.2 Kb has been found. A C→T transition in this TaqI site, originating a nonsense codon and a new Hindlll site, has been reported by Gitschier et al in a patient presenting inhibitor. The DNA from our patient tested with Hindlll shows a normal pattern thus indicating a C→T transition in the antisense strand. This mutation should causean aminoacid change (CGA→CAA, Arg→Gln) possiblyresponsible for the FVIII inactivation but that does not remove theantigenic determinants present in the COOH terminal part of FVIII.

In addition the same mutation has been observed in an unrelated (asdemonstrated by RFLPs analysis) Italian haemophilic patient confirming the observation of Youssoufian et al that TaqI sites are mutational hot spots in FVIII gene.
EFFECT OF DIFFERENT FVIII/vWF-CONTAINING THERAPEUTIC PRODUCTS ON PLATELET ADHESION TO COLLAGEN
Authors: De Romeuf C , Mazurier C , Goudemand M
Published: June 2019

In an attempt to predict by in vitro study the possible efficiency of various therapeutic products rich in FVIII/vWF in the treatment of patients with von Willebrand's disease, we have compared their ability to support platelet adhesion to human non fibrillar collagen type I, III.

Platelet adhesion was studied in steady flow conditions at the wallshear rate of 1200 sec−1 , using a flat perfusion chamber containingcollagen-coated coverslips.*

Perfusates were samples of reconstituted blood made up from washed red cells to an hematocrit of 40 %, washed aspirin-treated and 111In-oxine labelled platelets to 2.1011/1 and a vWF-deficient plasma containing various amounts (0.25 to 2 U vWF:Ag/ml of plasma) of therapeutic product. The extent of platelet adhesion was evaluated by radioactivity counting and expressed in percentage (100 % representing the value obtained with normal plasma, 0 % being the value obtained with vWF-deficient plasma).

Cryoprecipitate enabled us to obtain 50 % platelet adhesionat a concentration lower than 0.5 U vWF:Ag/ml. Two virus-inactivatedproducts derived from this cryoprecipitate were compared : Theheated FVIII/vWF concentrate supported 50% platelet adhesion at a concentration higher than 1.5 U vWF:Ag/ml whereas solvent-detergent treated concentrate failed to achieve 50 %adhesion at a 2 U vWF:Ag/ml. These results which may vary from one batch to another are correlated on the whole with the vWF multimeric composition.

In conclusion, provided that sufficient doses of vWF:Ag are infused, some virus-inactivated FVIII concentrates containing high molecular weight forms of vWF should be able to correct the bleeding time of patients with von Willebrand's disease
RECOVERY AND SURVIVAL STUDIES OF A NEW FACTOR VIII PRODUCT
Authors: Addiego J E , Bailey P , Bradley M , Courter S , Lee M
Published: June 2019

Lyophilized protein concentrates are the international treatment of choice to manage bleeding in hemophiliacs. These pooled plasma products, however, expose the recipient of treatment to an increased risk of viral infections. While current manufacturing techniques of these products appear to be effective in eradicating the human immunodeficiency virus (HIV), transmission of other viruses, especially non-A/non-B (NANB) hepatitis, is still a majoor complication of concentrate therapy. Hyland Therapeutics Division Travenol Laboratories, Inc., has developed a new process using the techniques of immunoaffinity chromatography and organic solvent/detergent treatment to prepare a high specific activity product; Antihemophilic Factor (Human), Method M (AHF-M); that may render it free of pathogenic viruses. To determine the recovery and half-life of factor VIII in this product five severe hemophiliacs in a nonbleeding state were given! 50 U/kg of M-AHF and 50 U/kg of a currently licensed factor concentrate (HEMOFILe CT) in a crossover blinded study with a seven day interval between the respective infusions. The table below shows the recovery and half-life results for the five patients studied

The factor VIII recoveries and half-lifes were similar for both products. No significant adverse clinical reactions were detected in any patient either during or after their infusions. This product appears to produce adequate circulating levels of factor VIII. It also appears to be safe for administration in humans. Further studies are on-going to test the overall efficacy of this product and to confirm that the manufacturing process is effective in eliminating pathogenic viruses
NO ANTI-HIV SEROCONVERSION AFTER REPLACEMENT THERAPY WITH STEAM-TREATED FACTOR VIII CONCENTRATE. A STUDY OF 60 PATIENTS WITH HEMOPHILIA A AND VON WILLEBRAND'S DISEASE
Authors: Schimpf Kl , Brackmann H H , Bock D , Landbeck G , Lechler E , Vinazzer H
Published: June 2019

Transmission of hepatitis viruses and HIV has proven to be a risk of replacement therapy. Since Dec. 1983 factor VIII concentrates in which viruses are inactivated by steam-treatment (Factor VIII TIM 3 or S-TIM 3) are available for therapy. As they are manufactured by 80% from US plasma it was necessary to prove that they do not transmit HIV. For ethical reasons it is not possible to treat control groups of patients with non-virus- inactivated concentrate. Non-transmission of HIV can, therefore, only be proven if anti-HIV seroconversion does not occur in larger groups of patients treated with this type of product. We collected data from 60 patients, who were “virgin” (24) or, if pre-treated, anti-HIV seronegative. Therapy with Factor VIII TIM 3 or S-TIM 3 was started between Sept. 1984 and April 1986. The median length of observation till the last anti-HIV testing was 12 (6 - 24) months. The median total dosage of Factor VIII was 56,500 (500 - 427,500) IU, the median patient age was 20 (1 - 61) years. In none of the patients anti-HIV seroconversion (ELISA test) was observed. According to the rule ofthree the upper 95% confidence limit for random sample of 60 cases with zero events would be 3/60 or 5%.
HUMAN AND PORCINE FVIII INHIBITOR RESPONSE FOLLOWING INFUSION OF HUMAN FVIII CONCENTRATE - MEASUREMENT USING AN AGAROSE GEL SYSTEM
Authors: Perry D J , Hill F G H
Published: June 2019

Conventional methods for determining inhibitor levels in haemophiliacs are time consuming and labour intensive. The agarose gel technique of Jorquera et al. (1) has been modified and standardized to measure human and porcine inhibitors to VIII:C.

26 samples from 12 haemophiliacs with inhibitors were analysed and in all cases antibody to human and porcine VIII:C was detected.

Six haemophiliac patients with 'high responder-type' inhibitors were studied using stored plasma and the rise in antibody titres to both human and porcine VIII:C was determined sequentially during treatment with human FVIII concentrate.

One patient (J. C.) had received a single treatment with porcine concentrate (Hyate:C) with no rise in porcine VIII:C.

In 5 of 6 patients both porcine and human inhibitor titres rose but the porcine levels were less than the human. However, in the remaining high responder patient (I. I.) with previously mild haemophilia and no exposure to porcine FVIII, the rise in porcine antibody titre greatly exceeded that of human. This patient has continued to be treated with human FVIII concentrate during which time the human inhibitor titres have fallen more than the porcine.

This method is simple to perform and has the advantage that both human and porcine inhibitor titres can be easily and quickly assessed so that the most appropriate therapy can be given.

1. Jorquera JI, Carmona E, Aznar JA, Peiro A and Sanchez-Cuenca JM. (1985) A Standardized Method for Measuring Anti-FVIII:C Inhibitors in Haemophilia A by Coagulation Inhibition in Agarose Gel. Thromb. Haemostas. 54(2): 377-380.
A MONOCLONAL ANTIBODY WHOSE BINDING IS INHIBITED BY DIVALENT CATIONS
Authors: de Serres M , Reisner H M , Monroe D , Roberts H
Published: June 2019

Factor IX (FIX), a vitamin K dependent coagulation protein, is functionally deficient or absent in patients with hemophilia B. Binding of Ca++ by the gammacarboxyglutamic acid residues (Gla) of FIX is necessary for coagulant activity. Antibodies havebeendes-cribed which selectively bind to FIX in the presence of Ca++ and appear to interact with Ca-H- stabilized epitopes of FIX. One IgM, Kappa murine monoclonal antibody (Mab), 129-1, has been found to react preferentially with FIX in the absence of Ca-H- or with FIX having a reduction in gammacarboxylation. 129-1 was characterized by direct binding ELISA assays, the standard assay buffer (.02M Tris - .15M NaCL pH 7.5) was supplemented with Ca-H- or ED TA to final concentrations to 5mM and lOmM respectively. In the presence of Ca-H-, titers ranged from 10 to 100 as compared to 100 K to 500 K in the presence of EDTA. Control Mab's, FIX-30 and 2D521 showed no such effect. Maximum inhibition occurred ≥2.5 mM Ca-H- concentration. Mg++, Sr-H- and Mn-H- were tested with similar results. Chemically degammacarboxylated FIXa was compared to FIX and FIXa controls in the presence of Ca-H- or EDTA, to determine the importance of Gla residues. In the presence of EDTA, Mab 129-1 had essentially equal reactivity with all these proteins (titers 100 K). In the presence of Ca-H-, binding to FIX and FIXa was inhibited (500 and 1 K respectively). However, the binding to Gla modified FIXa in the presence of Ca-H- was only slightly reduced (10 K), suggesting the importance of Gla residues in the Ca-H- mediated inhibition. FIX was purified from concentrate, coumadinized plasma and culture supernatant (produced in the absence of vitamin K) from the cell line PMN45 using HPLC affinity chromatography with Mab 2D521. Mab 129-1 binding to FIX immunopurified from concentrate and a control biochemically purified FIX protein preparation was significantly higher in the presence of EDTA (titers 500 and 5 K) than in Ca-H- (titers 10 and 10). FIX purified from coumadinized plasma or PMN45 cells showed equal low reactivity with 129-1 in the presence or absence of Ca-H- (titer 10 in all cases). FIX-30 and 2D521 controls showed no such reduction in binding. Therefore, Gla residues may play a role in the binding of 129-1 to FIX.
ANALYSIS OF FACTOR IX GENE IN NORMAL SUBJECTS AND HEMOPHILIA B PATIENTS IN JAPAN
Authors: Kojima T , Tanimoto M , Kamiya T , Obata Y , Kurachi K , Saito H
Published: June 2019

We have examined DNA samples from 25 hemophilia B patients (21 B- patients, 2 BR patients and 2 B+ patients) and 51 normal subjects with molecular probes (pHFIX and 2 genomic fragments). By structural gene analysis, 4 out of 7 patients who developed anti-factor IX antibodies were detected to have gross factor IX gene deletion. Although these four patients showed normal pattern of HPRT gene detected by pCDHPRT, the gene deletions were found to expand more than 34kb including with entire factor IX exons. Quantitative Southern blot analysis of factor IX gene of the patient's family members indicated that the gene deletion was inherited in one family, establishing the carrier status of 2 aunts, 2 cousins and one sister. The 'de novo' mutation of factor IX gene was also established in 2 families. Three patients with anti-factor IX antibodies and 17 patients without antibody to factor IX had normal pattern of factor IX gene by several restriction enzyme digestions. Analysis of factor IX gene of three patients with anti-factor IX antibodies and two B+ patients are now underway to detect the unique gene defects which may be responsible for the disease Phenotypes. Common RFLPs in factor IX gene were studied in normal Japanese subjects. More than 80 X chromosomes were analysed with BamHI, Ddel, MspI, TaqI or XmnI digestion, followed by hybridization with pHFIX. RFLPs produced by these enzymes were found to be uncommon or possibly absent in normal Japanese subjects. These results imply that racial differences in the frequency of gene polymorphisms should be seriously considered before initiating the gene counseling by the genetic probes.
PROPERTIES OF MONOCLONAL ANTIBODIES TO HUMAN FACTOR IX-PHAGE CAP-SID FUSION PROTEIN
Authors: Reisner H M , de Serres M , Lin S-W , Stafford D W
Published: June 2019

Functional domains of the human factor IX (FIX) molecule may be expressed in E. coli as fusion proteins (FPs) with phage T7 capsid protein 10 (CP 10). Such proteins are synthesized in large amounts and, because of their insolubility can be easily purified Polyclonal antibodies raised to FIX FPs cross react with the native molecule, but the utility of FPs in the production of monoclonal probes of native FIX is unclear. Hence, we have characterized the specificity of murine hybridomas resulting from the immunization with a FP containing amino acids -3 to 49 of human FIX (FP4D) . FP4D proved to be a potent immunogen in mice. Using solid phase ELISA assays, 46% of 2,200 cultures screened reacted with the immunizing antigen. Most of the positive cultures reacted only with FP4D and/or CP 10 and were discarded. Fifty cultures reacted with native FIX and were further studied. None of the FIX reactive cultures inhibited FIX:C activity in clotting assays. Binding of more than half of the cultures to FIX was inhibited by the presence of 5mM Ca++ suggesting a similarity between FP4D and the non Ca++ bound steric state of FIX. Four cultures were subcloned 2X to insure monoclonality. These antibodies were further evaluated for binding to FIX, FP4D, and CP 10 in the presence or absence of Ca++. Three of the four Mabs reacted with FP4D and FIX and showed varying degrees of inhibition by Ca++ only with FIX. One antibody reacted equally with all three proteins in the presence or absence of Ca++. Two of the Mabs were detectable in a solid phase RIA and showed increased radioligand binding in the absence of Ca++. Most Mabs against FP4D appear to detect epitopes which are influenced by the presence of Ca++. Since the FIX amino acid sequence contained in FP4D is gammacarboxylated and capable of binding Ca++ in the native molecule, the epitopes detected by these Mabs may be masked or altered by Ca++ binding. These Mabs may prove to be useful probes in the study of Ca++ binding in the native FIX molecule.
MONOCLONAL ANTIBODY WITH PREFERENTIAL AFFINITY FOR LARGE MULTIMERS OF VON WILLEBRAND FACTOR
Authors: Ryu Tomiko , Nakayama Akiko , Oguchi Atsushi , Kinoshita Tadatoshi , Kazama Mutsuyoshi , Abe Takeshi
Published: June 2019

von Willebrand factor (vWF) consists of a series of multimers of 270,000 mol. wt. subunits. Ristocetin cofactor activity (vWF:RCo) and capacity of binding to platelets of vWF are associated with large multimers, and Type IIA von Willebrand's disease (vWD) is characterized by lack of the large multimers. The significance of multimeric structure in relation to vWF function remains unclear. We obtained a monoclonal antibody (MAb) to human vWF which inhibited ristocetin-induced platelet aggregation. This antibody proved to bind preferentially to larger multimers by the finding that the MAb-conjugated Sepharose adsorbed large multimers of vWF from cryoprecipitates, leaving small multimers unadsorbed. vWF:Ag levels of noraml subjects determined by ELISA using the MAb correlated well with those by ELISA using polyclonal antibody (PAb) to vWF. When the plasma from patients with Type IIA vWD and platelet type vWD were examined, the values obtained by the MAb ELISA had a good correlation with vWF:RCo, but were lower than the values obtained by the PAb ELISA. In gel filtration of factor VIII concentrate, vWF:Ag detected by the MAb and vWF:RCo were present in the void volume and large size fractions, whereas vWF:Ag detectable with PAb appeared broadly from the void volume to smaller size fractions. The MAb inhibited ristocetin-dependent binding of vWF to platelets, but did not affect ADP-induced binding of vWF to platelets. These findings suggest that the large multimers have a function-associated specific structure which is absent in the small multimers, and the MAb will be useful for the investigation of multimer-function relationship of vWF.
RAPID, SENSITIVE N0N-RADI0ACTIVE QUANTIFICATION AND ANALYSIS OF PLASMA VON WILLEBRAND FACTOR (vWF) MULTIMERS
Authors: Moake J L , Harris M A , Whitley C E , Alfrey C P
Published: June 2019

Assessment of plasma vWF abnormalities by clinical coagulation laboratories is difficult because the available test systems for vWF antigen quantification and multimer analysis are expensive, laborious, and require days, radioactive anti-vWF antibodies and autoradiographic methods. We have devised simple, rapid, sensitive alternative techniques for vWF quantification and multimer analysis that can be readily installed in clinical laboratories. Plasma vWF antigen quantification is by a 2 hour enzyme immunoassay that accurately detects levels as low as 0.23% of normal. Plasma vWF to be quantified is bound to polyclonal monospecific antihuman vWF attached to small glass beads, and anti-human vWF conjugated with alkaline phosphatase is added to make an insoluble "sandwich." A substrate solution consisting of phenylphosphate and 4-amino-antipurine is added, followed by potassium ferricyanide. Optical density (at 490-510 nm) of the red color that develops is directly proportional to the plasma concentration of vWF antigen. Plasma vWF multimeric analysis is by a one-day electrophoretic immunobiot procedure. Plasma vWF multimer forms are solubilized in SDS-urea-Tris-EDTA, separated by horizontal 1% agarose gel electrophoresis, and transferred to a cationic membrane. Other protein binding sites on the membrane are blocked with milk proteins, and the membrane is overlaid with anti-vWF IgG linked to alkaline phosphatase. vWF multimers are then displayed as blue bands by soaking the membrane in an alkaline solution of the histochemical stain, fast blue RR (commonly used for leukocyte alkaline phosphatase scoring) dissolved in naphtol AS-MX phosphate. These simple, non-radioactive procedures performed together permit the rapid distinction of classical (Type I) von Willebrand's disease (vWD), characterized by low vWF antigen and normal multimers, from the Type II vWD syndromes, characterized by a relative deficiency of the largest plasma vWF forms. Unusually large vWF multimers, present in remission plasma of patients with chronic relapsing thrombotic thrombocytopenic purpura (TTP), are also easily detected using this rapid system of multimer analysis.
VASCULAR DAMAGE AND FACTOR VIII RELATED ANTIGEN IN THE RHEUMATIC DISEASES
Authors: Belch J J F , Zoma A , Richards I M , McLaughlin K , Sturrock R D , Forbes C D
Published: June 2019

F. VIII is produced by the blood vessel wall. Noxious stimuli increase endothelial release of F. VIII Related Antigen (VIII R:Ag). It might be expected that the development of vasculitis would be associated with increased levels of VIII R:Ag. Eight patient groups have been studied; 25 patients with systemic sclerosis, 19 with systemic lupus erythematosus, 15 with rheumatoid arthritis (RA) plus vasculitis, 19 with systemic vasculitis and 14 with atherosclerosis. These were compared to 29 patients with primary Raynaud's Disease, 15 with RA without vasculitis and 50 controls. Results show that where there was evidence of vascular disease, VIII R:Ag was elevated.

VIII R:Ag appeared a more specific marker for vascular damage than ESR or CRP. Further longitudinal studies in 11 patients showed good correlation between progression of vascular disease and VIII R:Ag.