bluuebunny/crossref_metadata_2025_split huggingface dataset:
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.
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)
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 studiedThe 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
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.
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.
ABNORMAL STRUCTURE OF VON WILLEBRAND FACTOR IN MYELOPROLIFERATIVE SYNDROME IS ASSOCIATED WITH EITHER THROMBOTIC OR BLEEDING DIATHESIS
Authors: López-Fernández M F , López-Berges C , Martín R , Pardo A , Romos F J , Batlle J 
Published: June 2019

The multimeric and subunit patterns of plasma von Willebrand factor (vWF) were analyzed in eight patients with myeloproliferative syndrome (MS) in order to investigate the possible existence of heterogeneity in the "in vivo" proteolytic cleavage of the protein, previously observed in this entity. Six patients lacked large vWF multimers, five of them having normal bleeding times (BT) and clinically documented episodes of thrombotic origin, whereas one patient had long BT and bleeding symptoms. Seven patients showed an increase 176 kDa subunit fragment while the 189 kDa polypeptide was increased in only one. In addition, another patient (and prior to any therapy) showed the presence of a new fragment of aproximately 95 kDa which disappeared after Busulfan therapy. The collection of blood from these patients with proteinase inhibitors did not correct the abnormalities.The infusion of DDAVP to two patients with abnormal vWF was accompanied by: the appearence of larger vWF multimers which disappeared rapidly from plasma; an increase in the relative proportion of the satellite bands of each multimer and a further increase in the 176 kDa fragment. These data show some heterogeneity in the vWF abnormality present in MS which may be related in part to a variable degree of proteolysis of vWF ocurring "in vivo" rather than "in vitro", and which may be associated to either a bleeding or (even) a thrombotic diathesis. They also suggest that despite the presence of abnormal, already proteolyzed vWF, DDAVP-enhanced proteolysis occurs in MS to a similar extent as described in normal individuals.
LARGE SCALE PREPARATION OF VON WILLEBRAND FACTOR BY AFFINITY CHROMATOGRAPHY
Authors: Newman J , Farb D 
Published: June 2019

Treatment of bleeding in von Willebrand's disease usually consists of the infusion of cryoprecipitate or plasma. DDAVP is effective in some patients. Commercial concentrates for the treatment of Hemophilia A are ineffective as a source of von Willebrand Factor (vWF) replacement in von Willibrand's disease presumedly because of the absence of higher molecular weight forms of vWF protein. A vWF concentrate obtained during the course of preparation of an affinity purified Factor VIII may provide an alternative therapeutic agent without impacting the available Factor VIII supplies. The process used for the preparation of a highly purified Factor VIII concentrate (MonoclateTM, Armour Pharmaceutical Co.) from cryoprecipitate includes an affinity chromatography step which separates vWF/Factor VIII complex from other proteins in cryoprecipitate using an anti-vWF monoclonal antibody (C. A. Fulcher & T. S. Zimmerman, 1982). Factor VIII is then dissociated from the vWF remaining on the column and is eluted immediately by 3M sodium thiocyanante (NaSCN) as a step in the regeneration of the column. Unless the NaSCN is rapidly removed from the elutriant, the vWF activity as measured by platelet agglutination is destroyed. We have taken the NaSCN eluate and processed it immediately over an in-line G-10 or G-25 Sephadex column which removes the NaSCN while the vWF is eluted with a buffered isotonic solution. Alternatively, the vWF has been precipitated from the NaSCN by ammonium sulfate or polyethylene glycol. VWF prepared by any of these methods retains platelet agglutinating activity and has a distribution of vWF multimers similar to those of vWF in normal plasma.The potency of vWF prepared from cryoprecipitate by this process is 20 units/ml and the specific activity is 20 units/mg. In several fractionations of kilogram amounts of cryoprecipitate, vWF was isolated and subjected to lyophilization and heating at 68° for 30 hours without loss of bioactivity. Drug development in progress suggest that the combined effect of affinity chromatography and exposure to NaSCN may negate the need for a heating procedure to reduce the risk of viral transmittance.
ASYMPTOMATIC vWD VARIANT WITH ABSENT RISTOCETIN ACTIVITY BUT PRESERVED BOTROCETIN ACTIVITY AND A DAUGHTER WITH TYPE III (HOMOZYGOUS) vWD
Authors: Hill F G H , Williams C W , Enayat S M , Darbyshire P J 
Published: June 2019

The propositus, female aged 1 year, was investigated because of severe bruising. Bleeding time (BT) was in excess of 20 mins, VIIIC <0.01 u/ml and vWF Ag and ristocetin cofactor absent. Family studies showed:-Multimeric analysis was not possible on the propositus, but was normal in the plasma of all other family members. The father of the propositus, however, had an abnormal multimer pattern on lysed platelets, in that the faint low molecular weight doublet is absent and a dense band in the position of the lower band of the doublet with anodal and cathodal trailing.The maternal grandmother and mother appear to have asynpto-matic type I vWD and the father possibly has asymptomatic type I together with an alteration in the biologically vWF site with loss of the ristocetin site. This abnormality is not seen in his parents and his daughter possibly has type III vWD. The propositus' father although having some similarities to the patient of Howard et al., is different in other aspects.1. Howard MA, Salem HH. et al. (1982) Variant von Willebrand's disease Type B - Revisited. Blood, 60: 1420-1428.
SEVERE VON WILLEBRAND'S DISEASE WITH ABNORMAL PLATELET AGGREGATION
Authors: Ihara A , Kobayashi Y , Aramitsu Y , Hara Y , Fujimura K , Kuramoto A 
Published: June 2019

A 17-year-old boy with a life long history of easy bruising, epistaxis, subcutaneous hematoma and prolonged bleeding time from minor injuries, was initially diagnosed as having von Willebrand's disese, when he presented epistaxis at 2 years of age. Since his initial diagnosis, he has been treated with cryoprecipitate on many occasions to correct his bleeding tendency. The laboratory findings in the patient and his family are summarized in the table. The patient's mother, father and one brother have not complained any bleeding tendency.Multimeric analysis of vWF using SDS agarose gels showed absence of large multimer in the patient's plasma, decreased large multimer in mother's and brother's plasma. When the patient's PRP was tested for aggregation and release of ATP by ADP, epinephrine and collagen, using lumi aggregometer, platelet aggregation was abnormal as shown by disaggregation and almost no ATP activity was detectable in the supernatant. ATP contents of patient's and father's platelet was about 50% of normal platelets. Washed platelets of the patient in normal plasma did not aggregate normally, but washed normal platelets in the patient's plasma aggregate normally. No inhibitor of vWF could be demonstrated in the patient's plasma. Clot retraction was normal. These findings suggest that our patient has inherited vWD from materal side and the platelet aggregation defect, probably a kind of storage pool disease, from the paternal side of the family.
EFFECTIVENESS OF DDAVP IN PATIENTS WITH VON WILLEBRAND'S DISEASE WITH SEVERELY REDUCED BASAL LEVEL OF F VIII/VWF AND NORMAL PLATELET CONTENT(TYPE I, PLATELET NORMAL) UNDERGOING TOOTH EXTRACTION
Authors: Rodeghiero F , Castman G , Di Bona E 
Published: June 2019

DDAVP has been used for hemorrhage prevention during dental extraction in patient (pts.) with von Willebrand's disease (vWd), usually of type I. However, only about 20 cases have been reported. Moreover, themajority of pts. had basal level ofVIII:C and vWf equal to or higher than 20% so that dental procedures could probably have been performed safely also without any specific treatment.We report the experience with DDAVP for hemorrhage prevention during dental extraction in 14 pts. withtype I vWd (confirmed byplasma multimeric analysis, Prof. Mannucci, Milan) and severely reduced basal level of VIII:C (mean 12.9 ± 3 UI%; range 8-18 UI%) and vWf (< 3 UI%), measured as ristocetin cofactor. VWf:Ag and vWf platelet content (UI%/109 platelets; platelets separated by Ficoll-Hypaque and lysed with TritonX) was in normal range (17-35 UI%and 22-68% in pts.; 17-37 UI% and 24-62 UI% in 18 controls). After DDAVPinfusion (0.4 μg/ml), VIII:Cand vWf activities increasedrespectively 8.5 folds (range 6.4-11.8) and 28.2 folds (range 16-40). Return to basal levels occured with an half-life of60 -80 minutes for all the activities. Bleeding time, prolongedin 8 pts., was ormalizedin 7 cases and shortened in 1. All the pts.referred of previous hemorrhage after tooth extractionperformedin the past. A total of 50 extractions were performed (3.67 teeth/pts). Mild bleeding occured in only 2pts.3-5 days after extraction and waspromptly stopped by repeating DDAVPinfusion.In conclusion, DDAVP has proved effective for dental extraction in type I vWd pts. with severely reduced VIII:C and vWf activities in plasma but normal platelet content.