bluuebunny/crossref_metadata_2025 huggingface dataset:
IN VITRO IMMUNE PARAMETERS OF MONOCLATEH, A, MONOCLONAL ANTIBODY PURIFIED HUMAN PLASMA FACTOR VIII:C THERAPEUTIC PREPARATION
Authors: Schreiber A B , Gillette R , Hrinda M E
Published: June 2019

MonoclateR is a highly purified human factor VIII:C preparation obtained by immunoaffinity chromatography of plasma cryoprecipitat followed by chromatography on aminohexyl sepharose. The resulting product is devoid of alloantigens, can be stabilized in the absence of extraneous human proteins and has a specific activity higher than 3000 U/mg.

To ascertain product integrity, we set out to compare MonoclateR preparations with either unfractionated human plasma, cryoprecipitateor commercially available lyophilized antihemophilia factor (AHF) preparations by Western blots. As probes, we utilized a series of monoclonal antibodies to defined factor VIII:C epitopes. The MonoclateR VIII:C chain composition appeared indistinguishable from that in the native state of in commercial, clinically useful preparations.

Patients with hemophilia are often afflicted with an impairment of cell-mediated immunity, when treated repeatedly with plasma cryoprecipitate preparations. Those preparations, containing at best 0.2% factor VIII:C in protein content, were found in our hands to dramatically inhibit both human mixed lymphocyte reactions and the phytohemagglutinin induced human lymphocyte mitogenesis. We obtained 50% inhibition at about 1 mg/ml protein concentration. These effects were not due to cytotoxicity nor related to procoagulant potency.

MonoclateR, in contrast, was totally devoid of immunosuppressive activity at all concentrations tested. This in vitro finding may be related to clinical observation of corrections in immune impairment in adult hemophiliacs treated with MonoclateR. With this immunological profile, MonoclateR represents in our opinion, a promising novel treatment modality for hemophilia A.
THE INFLUENCE OF L-LYSINE ON FACTOR VIII ELUTED FROM MATRIX BOUND DEXTRAN SULPHATE
Authors: Harrison P , Saundry R H , Savidge G F
Published: June 2019

Factor VIII/vWF displays high affinity for matrix bound sulphate groups. Linear salt and pH gradient elution from Dextran Sulphate Sepharose at 4°C resulted in complete separation of the complex from major protein contaminants with typical yields of 70-90% vWF:Ag and vWF (RCoF). Elution in physiological calcium concentrations gave VIII and VIII:Ag yields of 35% and 65% respectively. Addition of L-Lysine (1.0M) to all buffers inhibited VIII/vWF binding, although lysine gradients (0-1.0M) gave comparable binding and elution profiles as C-1.0M NaCl, but with impaired resolution between protein moieties. However, in the presence of L-Lysine, yields of VIII and VIII:Ag were significantly improved to 80% and 100% respectively when assay standardizations with appropriate lysine concentrations were performed. Furthermore, the binding of fibrinogen could be inhibited by 0.15M L-Lysine, 0.075M NaCl in the equilibrating buffer.

The presence of enzymic activity, as assessed by S-2222 and S-2238 in the eluting fractions could be abolished by the application of recryoprecipitated material pre-adsorbed with Al(OK)3 and celite. Incorporation of lysine to the buffers with the associated increase in yields further supports the potential viability of the separation principle for large scale purification of Factor VIII.
MONOCLONAL ANTIBODY BINDING TO FACTOR VIII:C
Authors: Berkner J A , Mitra G , Bloom J W
Published: June 2019

The interactions of monoclonal antibodies with highly purified Factor VIII:c have been studied utilizing the ELISA technique. ELISA plates were coated with Factor VIII:c, protein A purified monoclonal IgG was then added and bound antibody detected with peroxidase labeled antimouse IgG. A Scatchard-Sips plot approach to data analysis was used to calculate binding constants. The binding constants for four antibodies designated BD10, AD7, C7F7 and 39MH8 were as follows: BD10, KO = 7.1 x 108 M-1, n = 1.1 (moles antibody/moles ligand); AD7, KO = 3.1 x 108 M-1, n = 2.7; C7F7, KO = 3.6 x 1011M-1, n = 0.03; 39MH8, K = 6.0 x 1011 M-1, n = 0.03. The binding constants for C7F7 to the purified carboxy-terminal (residues 1649-2332) 80 kD functional region of the Factor VIII:c molecule were also determined: KO = 1.0 x 1011 M-1, n = 0.55. On the basis of these results the following conclusions can be drawn: 1) the antibodies can be divided into two groups: high affinity (suitable for use in immunopurification), C7F7 and 39MH8; low affinity: BD10 and AD7; 2) the antibodies in the low affinity group have valance values two orders of magnitude higher than the high affinity antibodies, C7F7 and 39MH8. The difference might be explained by the high affinity antibody epitopes on the immobilized Factor VIII:c being less exposed to the solution; 3) C7F7 binding to the 80 kD polypeptide, compared to the whole Factor VIII:c molecule, gave virtually identical Kc values, but dramatically different valance values. This suggests that the C7F7 epitope is more accessible on the 80 kD polypeptide.
USE OF ALBUMIN AND TWEEN AS STABILIZERS TO PREVENT ACTIVITY LOSS DURING CLOTTING ASSAYS OF COAGULATION FACTOR IX AND X CONCENTRATES
Authors: Miekka Shirley I
Published: June 2019

Assays for clotting activities of Vitamin K-dependent coagulation factors in Factor IX complex concentrates are IcnovTn to give variable results depending on the composition of the sample diluent. Higher potency values are obtained when deficient plasma is used for sample pre-dilution compared with dilution in buffer. This discrepancy is more pronounced in assays of higher purity Factor IX (FIX) or Factor X (FX) concentrates. We have found that addition of a mixture of bovine albumin (0.1% w/v) and Tween 20 (0.01% v/v) (BAT) to the dilution buffer can eliminate the discrepancy, giving clotting times and plot slopes equal to chose obtained upon dilution in deficient plasma. Less protection was obtained with either albumin or Tween added separately. Polyethylene glycol 8000 (0.1% w/v), commonly used to stabilize thrombin solutions, gave variable results. BAT had no effect on clotting times of whole plasma or of FIX or FX samples pre-diluted in deficient plasma. Neither deficient plasma nor BAT had any effect when added after sample dilutions were prepared: activity of a FIX concentrate was 137 U/ml when pre-diluted in Factor IX-deficient plasma and 1312 U/ml diluted in BAT, compared with 49 U/ml diluted in buffer alcne; addition of deficient plasma or BAT to the dilutions of sample in buffer gave activities of only 36 a).id 34 U/ml, respectively. Similar results were obtained with FX samples. Furthermore, when a solution of FX (pre-diluted to 1 U/ml in buffer without stabilizer) was merely transferred from one test tube to another without further dilution, clotting times increased progressively and activity decreased by 85% after 8 transfers. By contrast, an identical sample diluted to 1 U/ml with BAT remained essentially unchanged after 8 serial transfers. These results indicate that Vitamin K-dependent coagulation factors are very susceptible to surface adsorption or inactivation after dilution of concentrates, and that either BAT or deficient plasma will prevent this loss. The use of albumin and Tween as stabilizers provides a simpler, .less expensive alternative to prevent nonspecific surface adsorption and achieve more accurate measurement of clotting activities.
FACTOR IX CONCENTRATES ARE THROMBOGENIC AT HIGH DOSES IN DOGS
Authors: Ferguson J , Dawes J , Prowse C V , Foster P R , Feldman P A , Smith J K
Published: June 2019

A canine model has recently been established to assess the potential thrombogenicity of intravenously infused blood products. Elevated plasma levels of fibrinopeptide A (FpA) were identified as the most sensitive indicator of a thrombogenic response, and this was the only parameter to change significantly when issued batches of factor IX (II + X) concentrate were infused at a dose of 100 iu/kg. After infusion of 200 iu/kg, however, plasma FpA concentrations and FDP titres rose, the APTT was prolonged, and the platelet count and fibrinogen level fell. At this dose, therefore, FIX concentrates which were not identified by in vitro tests as potentially thrombogenic induced a response when infused into dogs.

A batch of FIX concentrate which failed the criteria for in vitro thrombogenicity and was therefore not issued for routine use was infused at 100 iu/kg. Plasma FpA levels rose as did the FDP titre, and fibrinogen concentrations fell, but the APTT was only slightly prolonged. The thrombogenic response to 200 iu/kg of issued FIX concentrate was at least as severe as that following infusion of 100 iu/kg of this rejected batch.

Thus, there is a threshold dose of FIX concentrate above which a severe thrombogenic response can ensue, and the current in vitro tests may not be a reliable indicator of potential thrombogenicity when FIX concentrates are infused at high doses. This should be taken into account when administering unusually high doses, particularly to patients who may have reduced levels of circulating protease inhibitors.
SITE SPECIFIC MUTAGENESIS WITHIN THE GLA-DOMAIN OF HUMAN FACTOR IX
Authors: Ashok Kumar A , Insley Margaret , Gambee Jay , Busby Sharon J , Berkner Kathleen L
Published: June 2019

Factor IX, a plasma protein, plays a critical role in blood coagulation. The biological activity of factor IX as well as several other plasma proteins depends on the presence of gamma-carboxy glutamic acid (Gla) residues in their amino terminal region. In vitro mutagenesis has been used to selectively replace Gla residues of factor IX with aspartic acid (Asp) residues in order to establish the contribution of individual as well as paired Gla residues to the normal functioning of the protein. These substitutions were made at positions 7, 15, 20 and 26 in human factor IX. In addition, residue number 18, a cysteine has been changed to serine in an attempt to disrupt the highly conserved disulfide bond in the gla-domain. The gla-domain mutants will be produced in mammalian cells and compared with native recombinant factor IX. A rapid immunoaffinity purification procedure, which has been used to obtain recombinant factor IX produced in the presence or absence of vitamin K, is being used to purify the mutants. Protein sequence analysis has been used to confirm complete processing and proper gamma-carboxylation of recombinant factor IX. The properties of these mutants as compared to human factor IX will be discussed.
IMPORTANCE OF LARGE VON WILLEBRAND FACTOR (vWF) MULTIMERS IN vWF INTERACTION WITH PLATELET GLYCOPROTEIN IIb/IIIa
Authors: Yamamoto M , Ando Y , Watanabe K , Iri H , Araki Y , Murata M , Murakami H , Satoh K , Ikeda Y
Published: June 2019

Recently it has been reported that, in addition to binding to glycoprotein (GP) lb, vWF also interacts with GPIIb/IIIa, although the physiological relevance of this interaction is not completeley clear. In this paper, we have investigated the role of different size of vWF multimers in vWF-mediated platelet aggregation. Different size of vWF multimers were purified from human plasma through Sephacryl S-1000 column according to the method of Fowler et al. Fractions were analysed by SDS-agarose gel electrophoresis by the method of Ruggeri et al. When each fraction was examined for ristocetin cofactor activity (RCo), only larger multimers exhibited significant RCo. The maximum extent of ristocetin-induced platelet agglutination by larger multimers (10 μg/ml) was 80%, while that of intermediate and lower multimers at the same concentration was 20% and 0%, respectively. Each fraction was then added to washed platelet suspensions in the presence of 10 μM ADP and 0.3 mM CaCl2. Only larger multimers induced platelet aggregation, while intermediate and lower multimers failed to induce platelet aggregation. The maximum extent of aggregation in the presence of larger multimers (10 μg/ml) was 70% of that in the presence of fibrinogen instead. Similar experiments were peformed using platelet-rich plasma from a patient with afibrinogenemia in stead of washed normal platelets. ADP caused significant aggregation only when purified vWF larger multimers or fibrinogen was added. This vWF-mediated aggregation was completely inhibited by monoclonal antibody to GPIIb/IIIa (1 μg/ml) and synthetic peptide, Arg-Gly-Asp-Ser, (1 mM).

Our results indicate that larger multimers of vWF play major roles in vWF interaction with GPIIb/IIIa.
STUDY OF IIA VON WILLEBRAND'S DISEASE (VWD) VARIANTS TO DETERMINE DEGREE AND TYPES OF HETEROGENEITY
Authors: Enayat S M , Hill F G H , Sultan Y , Williams C W
Published: June 2019

Thirty four IIA vWD patients (16 from kindred I, 2 from kindred II and 17 unrelated patients) from 19 families were studied to compare multimer patterns using discontinuous SDS gel electrophoresis on a variety of agarose gels. Platelet multi-mers and effect of EDTA on plasma multimers were also studied in some.

The large kindred and 9 other patients showed identical multimer and triplet abnormalities. The 11 other patients showed different multimer patterns either by having intermediate multimers or different triplet patterns. The second kindred had a similar triplet abnormality to kindred I but had intermediate multimers. Two other patients showed similar patterns except on 2% agarose gels when differences in the lowest multimer was seen. Of the 3 patients of YS, one showed the common IIA pattern but also had intermediate multimers, another had an unusually faint upper triplet band, while the third in addition to a faint upper triplet band with ESVWF 2+ had no identification of minor or major bands with ESVWF 10+ Another patient lacked high and some intermediate multimers but had a normal triplet pattern. The pattern we have seen in Kernoff's patient (1) still appears unique. In kindred II abnormal triplets persisted and high multimers appeared in EDTA plasma. In kindred I (and similar patients) intermediate multimers and a change in triplet pattern was observed in EDTA while lysed platelets showed an abnormal pattern different to the plasma one.

This emphasizes the heterogeneity of IIA vWD and the need to consider multimer deletion, triplet pattern, platelet multimers, effect of EDTA in trying to subclassify in order to study structure function relationships of vWF.

1. Kernoff PBA, Gruson R, Rizza CR. (1974) A variant of factor VIII related antigen. Br. J. Haematol. 26: 435.

+ESVWF 2 and 10 are monoclonal antibodies to vWF epitopes.
BLEEDING TIME IN TREATED PATIENTS WITH SEVERE VON WILLEBRAND DISEASE IS NOT CORRECTED ONLY BY GIVING NORMAL MULTIMERIC PLASMA VON WILLEBRAND FACTOR
Authors: Mannucci P M , Moia M , Altieri D , Monteagudo J , Castillo R
Published: June 2019

Even though it is generally held that cryoprecipitate (cryo) and fraction I-0 correct the prolonged bleeding time (BT) in patients with von Willebrand disease (VWD), perusal of reported data indicates that the correction is usually short lasting and often partial. We decided to do a controlled study of the relationship between the multimeric structure of von Willebrand factor (VWF) in 5 patients with severe VWD after infusion of three plasma concentrates : "wet" cryo, lyophilized (lyo) cryo, and fraction 1-0 given in random order. The dosage of concentrates was tailored to achieve post infusion levels of RiCof above the lower normal limit (50 U/dL) for at least 3 hours. The post-infusion BT values are shown in the table.

These findings indicate that the attainment of a normal BT is the exception rather than the rule after infusion of three plasma fractions used for treatment of severe VWD. In all the concentrates the proportions of large VWF multimers, calculated by scanning the electrophoretic gels, were the same as in normal standard plasmas. An intact multimeric structure was recovered in post-infusion plasma of patients treated with wet cryo, whereas there was post infusion loss of large multimers after lyo and fraction I-O. In conclusion, an intact multimeric structure in post infusion plasmas is necessary but not sufficient to sustain a normal BT in VWD patients.
HETEROGENOUS RESPONSES TO CRYOPRECIPITATE AND DDAVP IN TYPE IIA VON WILLEBRAND'S DISEASE (VWD)
Authors: Chediak J , Maxey B , Eldridge J , Telfer M C
Published: June 2019

Von Willebrand's disease is an autosomal dominant disorder characterized by excessive mucocutaneous bleeding, prolonged bleeding time (BT), and reduce amounts of ristocetin cofactor activity (RiCof). The Von Willebrand factor antigen (VWF:Ag) shows either reduced amounts or no multimers (Types I and III), or a selective reduction of high molecular weight multimers (HMW) (Type IIA and IIB). Variable responses to DDAVP have been reported in IIA VWD suggesting that IIA patients (pts) are a heterogenous group. Some IIA pts may show RiCcf activity after DDAVP infusion even though no HMW multimers are found.

Von Willebrand factor antigen, RiCof and BT were analyzed in five pts (2 females and 3 males) known to have Type IIA VWD. Baseline values shov/ed marked reductions of RiCof (less than 13% of normal), BT greater than 15 minutes, faster immunoelectrophoresis of VWF:Ag and absent HMW multimers. Immunoelectrophoresis of VWF:Ag by the Laurell technique gave variable amounts ranging from 10 to 125% or normal. Three adult pts received DDAVP (Stimate) or cryoprecipitate (cryo) and the responses on abnormal parameters were assessed up to 48 hours. In two pts the BT corrected with cryo, whereas in the third patient the correction was minimal. The three pts showed a normal decay of both VWF:Ag or RiCof after cryo. However, after DDAVP the decay of VWF:Ag and RiCof was similar to that after cryo in one patient and more rapid in two patients.

In these two patients, the data would be compatible with the rapid proteolysis of endogenoue VWF released by the patient's endothelial cells, whereas the exogenous VWF given as cryo showed normal survival in the patient's blood.
CYCLOSPORIN A (CyA) INDUCED ENDOTHELIAL CELL INJURY
Authors: Zoja C , Furci L , Ghilardi F , Zilio P , Benigni A , Remuzzi G
Published: June 2019

The chronic administration of CyA to animals and humans to prevent graft rejection may induce renal arteriolar damage resembling hemolytic uremic syndrome (HUS). This is a syndrome of vascular damage with thrombotic occlusions of the microcirculation. Endothelial damage is considered the first event in the pathogenetic cascade leading to HUS. We have used bovine aortic endothelial cells in culture to address the issue of CyA-induced arteriolar damage. CyA-induced a time (1-24 hours) and dose (1-50 μM) dependent cell damage. CyA-induced injury was characterized by an early cell detachment followed by lysis as documented by the increase in LDH and Cr release. 1 μM CyA did not induce cell detachment and lysis was evident only after prolonged incubations. 10 and 50 μM CyA both induced marked cell detachment and lysis: lysis started 3 hours after incubation of endothelial cells with CyA and was maximal at the end of 24 hour incubation (LDH release, percent specific increase over control values: 10 μM CyA, 47%; 50 μM CyA, 70%; 51 Cr release, percent specific increase over control values: 10 μM CyA, 28%; 50 μM CyA, 34%). CyA-induced injury was associated with dose- and time-depedent increase in prostacyclin (PGI2) and thromboxane A2 (TxA2) release by endothelial cells exposed to 10 and 50 μM CyA. CyA-induced generation of PGI2 and TxA2 was inhibited when the incubations were carried-on in the presence of acetyl salicilic acid (500 μM). These studies indicate that CyA exerts a direct toxic effect on endothelial cells and might help to understand the pathogenesis of CyA-induced vascular damage.
HAEMOSTATIC CHANGES IN CIRRHOSIS AFTER REPEATED DOSES OF DESAMINO D-ARGININE VASOPRESSIN (DDAVP)
Authors: Burroughs A , Taylor L , Sprengers D , Hatton R A , McIntyre N , Kernoff P BA
Published: June 2019

Eight patients with stable cirrhosis received DDAVP (0.3ug/kg I. V. of Desmopressin acetate, Ferring Pharmaceuticals) and this dose was repeated after 4h and 24h. Blood for haemostatic studies was collected immediately before and lh after each dose. Results, shown below as medians (expressed in u/dl), with ranges in parentheses, were analysed using the Wilcoxon signed rank test for paired data. One hour after the first dose, von Willebrand factor antigen (VW:ag) and Ristocetin co-factor activity (Ricof) rose from 380 (182-1060) to 502 (230-1000), p<0.01 and from 400 (154-1200) to 494 (180-1600), p<0.01 respectively. This rise was sustained at 4h but increased further lh after the second dose: VW:ag to 540 (305-1000) and Ricof to 570 (420-1150), p<0.01. Levels were significantly above baseline (p<0.01) at 24h but rose again after the third dose: VW:ag to 540 (300-1180), p=<0.01 and Ricof to 479 (277-625). A similar but less marked response was seen in factor VIII:C from 220 (112-300) to 300 (128-360) p= 0.01 at lh, which was maintained up to 4h but not 24h. The rise after the second and third dose was not significant. Multimeric analysis of VWF showed an increase in the very high MW oligomers after DDAVP. Irrespective of the basal value, the euglobulin lysis time shortened consistently after DDAVP and platelet aggregation showed an enhancement with ristocetin, a decrease with adrenaline and no change with adenosine diphosphate and collagen. The bleeding time shortened in half the patients but did not correlate with other changes. We conclude that rises in VW:ag and Ricof are sustained with repeated doses of DDAVP despite the high initial levels in cirrhotics, whereas rises in VIII:C and fibrinolytic activity are transient.
DIABETICS DEMONSTRATE A BLUNTED VON WILLEBRAND FACTOR RESPONSE TO DESMOPRESSIN INFUSION
Authors: Grant M B , Daniels T , Claire D , Lottenberg R
Published: June 2019

The increase in von Willebrand factor (vWF) following desmopressin (DDAVP) (1-desamino-8-D-arginine vasopressin) infusion was markedly blunted in severe hemophiliacs who had high vWF levels after treatment with vWF rich plasma concentrates. Diabetics with microangiopathy appear to have disease-induced elevation of vWF. In the current study, the vWF response to DDAVP infusion was measured in 30 diabetics (12 type I, 18 type II) and 16 controls, matched for age, sex and weight. Extent of nephropathy, macroangiopathy, and neuropathy was evaluated. Diabetic retinopathy was assessed by indirect ophthalmoscopy and fluorescein angiography (n=8 proliferative retinopathy, n=6 background retinopathy, n=16 no retinopathy). Plasma samples were collected in the supine, overnight-fasted state. DDAVP (0.3 μg/kg) was infused over 30 min and samples obtained at 0-60 min. vWF antigen was assayed by Laurell rocket electrophoresis. Tissue plasminogen activator (t-PA) activity was measured by a coupled chromogenic substrate assay. Results; Basal vWF levels for type I diabetics (124±16%, MeantSEM) and type II (178±14%) were increased as compared to controls (94±6%), p<.05 and p<.005, respectively. vWF levels for diabetics with proliferative retinopathy (194±29%) were significantly higher than for diabetics with background retinopathy (106±13%) p<.01. Diabetics with elevated basal levels of vWF (>150%) showed less of an increase in vWF following DDAVP infusion than diabetics with normal basal levels (p<.01). The percent increase in vWF following DDAVP administration inversely correlated with basal vWF levels (type I, r=.51; p<.01 and type II, r=.46; p<.01). The basal vWF level was the significant determinant of DDAVP response, not the presence or absence of diabetic complications. Peak t-PA levels showed no difference in controls or diabetics. In contrast to the vWF response, a normal t-PA response to DDAVP infusion was observed in diabetics. Conclusion: Diabetics with microvascular complications and high circulating levels of vWF demonstrate a blunted vWF response to DDAVP. This supports the existence of a negative feedback mechanism as previously reported for the transfused hemophiliacs.
THE EFFECT OF EARLY BLOOD TRANSFUSION ON THE OUTCOME OF GASTROINTESTINAL HAEMORRHAGE
Authors: Blair S D , Javanvrin S B , McCollum C N , Greenhalgh R M
Published: June 2019

It has been suggested that mortality due to upper gastrointestinal haemorrhage may be reduced by restricting blood transfusion [1], We have assessed whether this is due to an anticoagulant effect in a prospective randomised trial.

One hundred patients with severe, acute gastrointestinal haemorrhage were randomised to receive either at least 2 units of blood during the first 24 hours of admission, or no blood unless their haemaglobin was lessthan 8g/dl or they were shocked. Minor bleeds and varices were excluded As hypercoagulation cannot be measured using conventional coagulation tests, fresh whole blood coagulation was measured by the Biobridge Impedance Clotting Time (ICT). Coagulation was assessed at 24 hour intervals and compared to age matched controls with the results expressed as mean ± sem.

The ICT on admission for the transfusion group (n=50) was 3.2±0.2 mins compared to 10±0.2 mins in controls. This hyper-coagulable state was partially reversed to 6.4±0.3 mins at 24 hours (p<0.001). The 50 allocated to receive no blood had a similar ICT on admission of 4.4±0.4 mins but the hypercoagulable state was maintained with ICT at 24 hours of 4.320.4 mins. Only 2 patients not transfused rebled compared to 15 in the early transfusion group (p<0.001). Five patients died, and they were all in the early transfusion group.

These findings show there is a hypercoagulable response to haemorrhage which is partially reversed by blood transfusion leading to rebleeding
ANTITHROMBOTIC ACTIVITY OF A SYNTHETIC HEPARIN PENTASACCHARIDE IN A RABBIT STASIS THROMBOSIS MODEL USING DIFFERENT THROMBOGENIC CHALLENGES
Authors: Walenga J M , Fareed J , Petitou M , Lormeau J C , Samama M , Choay J
Published: June 2019

The synthetic pentasccharide, representing the critical sequence required in heparin for binding to antithrombin III, provides a unique tool to study the question of whether an agent solely capable of inhibiting factor Xa but devoid of anti-factor Ila activity in vitro, has the capacity to produce an antithrombotic effect in vivo. We have previously demonstrated in a rabbit stasis thrombosis model using a human serum challenge, a significant antithrombotic effect of the pentasaccharide (Walenga et al., Thromb Res 43:243, 1986). To extend and confirm these studies, four modifications of the stasis thrombosis model were developed using more specified induction sites of thrombosis. The following thrombogenic challenges were selected: monkey brain thromboplastin, an activated prothrombin complex concentrate, a non-activated prothrombin complex concentrate administered simultaneously with Russell's viper venom, and factor Xa. Dose-dependent antithrombotic responses were obtained in all four systems with ED50 values between 25-43 ug/kg for pentasaccharide as compared to 16-47 ug/kg for heparin. Complete inhibition of induced thrombosis was obtained in all four systems for pentasaccharide. Ex vivo analysis revealed expected anti-factor Xa levels but no anti-factor IIa activity. It was also shown that pentasaccharide in the rabbit was capable of inhibiting the generation of thrombin without directly inhibiting formed thrombin. It is concluded that an oligosaccharide with high anti-factor Xa activity, devoid of anti-factor Ila activity, is capable of inhibiting thrombosis induced in rabbit stasis models, but that higher dosages than heparin are required for this effect-in terms of anti-factor Xa activity.
KINETICS OF LOW MOLECULAR WEIGHT HEPARIN IN MAN DETERMINED BY PROTAMINE CHLORIDE
Authors: Bode V , Franz R , Welzel D , Wolf H , Harenberg C
Published: June 2019

Low molecular weight (LMW) heparin is characterized by a higher affinity to antithrombin HI, less inhibition of thrombin and increased inhibition of factor Xa. The half life of the antifactor Xa activity of LMW heparin is doubled compared to normal heparin. However, these parameters reflect the pharmacodynamics rather than the kinetic of the compound. We, therefore, analyzed the kinetics of LMW heparin after i.v. injection in man using protamine chloride for gravimetric evaluation of LMW heparin in the plasma samples.

Six healthy adults received 100 units per kg body weight normal heparin or 100 anti Xa units per kg LMW heparin (Sandoz AG, Niimberg, FRG). To serial samples of venous blood protamine chloride was added in serial dilutions until the thrombin inhibition was antagonized. Since factor Xa inhibition of LMW heparin cannot be abolished completely by protamine chloride, two amounts of protamine chloride were added to the plasma samples ex vivo, until factor Xa was inhibited up to 0,2 and 0,04 units/ml. The following maximal plasma concentrations (C max) and half lives (T/2) were calculated (average values):

The pharmacokinetics of normal heparin show no differences on thrombin and factor Xa interaction. LMW heparin, however, interacts to 30 % with thrombin and to 100 % with factor Xa; the half life on factor Xa is twice as long as on thrombin; releases endogenous compounds with antifactor Xa activity, which are neutralized only hardly by protamine chloride;and these endogenous compounds mediate in part the longer half life.
A NEW MUCOPOLYSACCHARIDE FROM STICHPUS JAPONICUS(SEA CUCUMBER) AND ITS ANTICOAGULANT PRORETIES
Authors: Takamatsu J , Saito H , Kamiya T , Muranaka Y , Minami Y F , Fan H T
Published: June 2019

A new mucopolysaccharide(FGAG) has been isolated from the cell wall of Stichopus japonicua. The molecular weight of FGAG is about 50,000. The component sugar of the FGAG are identified as galactosamine, glucuronic acid, fucose and sulfate with the molar ratioof 1:0.94:0.84:3.60,respectively. The anticoagulant effects of FGAG were studied. At low concentration(lμg/ml), FGAG completely inhibited the rabbit plateletaggregation induced by thrombin and prolonged thrombin time of not only human plasma but purified fibrinogen solution to a similar extent, suggesting that action of FGAG is not depend on plasma component(antithrombin III and/or Heparin c.ofactor II).

After intravenous injection into rabbits(lmg/kg),significant prolongation of activated partial thromboplastin time(APTT) was observed. Although the in vivo antithrombin activity of the FGAG was weaker than that of heparin.it lasted longer than that of heparin and was not inhibited by platelet factor 4.

These results suggested that FGAG is a new and unique mucopolysaccharide with antithrombin activity and may come into use for the treatment ofDiseminated Intravascular Coagulation.
THE ROLE OF Tc-99m RED BLOOD CELL (RBC) VENOGRAPHY IN PATIENTS WITH CLINICALLY SUSPECTED DEEP VEIN THROMBOSIS
Authors: Leclerc J R , Welfson T , Rush C , Lepanto L , Arzoumanian A , Raymond F , Rosenthall L
Published: June 2019

Although it has been available for a number of years, Tc-99m RBC venography has never been evaluated in an epidemiological study. We have compared Tc-99m RBC venography to impedance plethysmography (IPG) in 113 consecutive eligible patients with clinically suspected first episode of deep vein thrombosis (DVT). IPG was performed at initial presentation (day 0) and RBC venography within the next 72 hours.

RBC venography was performed by labelling in vitro 5ml of patients' red blood cell with 20 millicuries of Tc-99m. Patients with an initially abnormal IPG underwent contrast venography to rule out a falsely positive test result. Patients with an initially normal IPG had the test repeated at day 1, 3, 5 to 7 and 10 to 14. Anticoagulant treatment was witheld in all patients who remained normal by serial IPG testing. All patients underwent a 3 month follow-up period. RBC venography was considered abnormal if there was a 50% or greater decrease in isotope concentration in a deep vein compared to the same vein in the other leg. The sensitivity of RBC venography was 79% with 95% confidence limits (C. L.) from 59% to 92%. Specificity was 61% (95% C. L. from 51% to 71%). Positive and negative predictive values were 37% (95% C. L. from 25% to 51%) and 91% (95% C. L. from 81% to 96%) respectively. None of the patients who remained normal by serial IPG testing died from or had objectively documented venous thromboembolism during the follow-up period. We conclude that : 1)

RBC venography is non-specific and, 2) a decision to treat should not be made on the basis of the results of this test alone in view of its low positive predictive value.
THROMBOSIS PROPHYLAXIS IN TOTAL HIP ARTHROPLASTY PATIENTS USING A COMBINATION OF PHYSICAL METHODS
Authors: Caprini J A , Kudrna J C , Mitchell A S
Published: June 2019

Lowering the incidence of thromboembolic events in patients undergoing total hip arthroplasty remains a high priority and various drug and physical method protocols have been devised to achieve this goal. We report here our experience using full-length antiembolism stockings (TED), full-length sequential compression devices (SCD) and the continuous passive motion device (CPM) to prevent thrombosis in patients undergoing total hip procedures. 106 consecutive patients were enrolled in this protocol, including 80 primary and 26 revision hip arthroplasties. Cement was used in 40 primary and 12 revision procedures. Our population was composed of 46 males and 60 females ranging in age from 30 to 92 years. The prophylactic protocol included TED hose preoperatively and during surgery, with application of SCD and CPM in the Recovery Room. These patients had immediate leg elevation and were ambulated within 24-48 hours postoperatively. SCD/CPM devices but not the TED hose were removed for ambulation and discontinued when the patients were fully ambulatory. All patients had preoperative coagulation studies and were monitored postoperatively using doppler ultrasound, strain gauge plethysmography and daily radioiodinated fibrinogen scanning. Contrast venography was done if any clinical signs or symptoms appeared and to confirm or rule out thrombi in all those with equivocal or abnormal flow studies or scans. Patients receiving antiplatelet drugs and anticoagulants were excluded from the study. Deep vein thrombosis developed in 4 of 106 patients (3*8%) postoperatively, including 3/4 above-knee clots. 2/4 also had minor pulmonary emboli. All were successfully treated with anticoagulants and no fatalities occurred. Patient acceptance of these devices was very high and no one refused to use either the SCD or CPM appliances. Minor skin irritation occurred in about 3% and was the only side effect. Our results indicate that this combination of physical methods is highly effective in lowering the incidence of thromboembolism following total hip replacement without any serious side effects.
PRESENCE OF THE LUPUS ANTICOAGULANT IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS DOES NOT CAUSE INHIBITION OF PROSTACYCLIN PRODUCTION
Authors: Rustin M H A , Bull H A , Dowd P M , Isenberg D A , Snaith M L , Machin S J
Published: June 2019

The cause of the thrombotic tendency in patients having the lupus anticoagulant (LA) is unknown. Since inhibition of prostacyclin production by endothelial cells (EC) may be a pathogenetic factor, the effect of sera from normal subjects (NS, n=9), SLE (systemic lupus erythematosus) + LA (n=9) and SLE-LA (n=13) on the production of PGI2 by cultured human EC was studied.

Confluent 1° cultures of human umbilical vein EC were incubated with 1, 5, 10 and 20% sera from the above for 24 hours. After stimulation with thecalcium ionophore A23187, 6-keto-PGF1α(the stable metabolite of PGI2) in the supernatant was measured by radioimmunoassay.

A dose dependent inhibition of 6-keto-PGF1α was observed with all the sera but only the 10 and 20% sera from patients with SLE-LA produced a significantly greater inhibition than control sera. The mean production of 6-keto-PGFia (ng/104 cells) was 2.278 (NS), 2.6594 (SLE-LA) and 2.1418 (SLE + LA)after incubation with 1% sera for 24 hours. This decreased to 1.3647, 0.6517 and 0.942 respectively following incubation with 20% sera. This represented a 44% (NS), 71% (SLE-LA) and 62% (SLE + LA) inhibition of 6-keto-PGF1α production compared to serum free media.

The non-significant reduction in prostacyclin production by sera from patients with SLE and the lupus anticoagulant suggests that other factors are responsible for the thrombotic tendency in these patients.
PROTEINASE-INHIBITOR COMPLEXES (PIC) IN SEPTIC AND NON-SEPTIC SHOCK. COAGULATION; LEUKOCYTE AND BACTERIAL PROTEASE INHIBITION BY MEANS OF PLASMA-INHIBITOR REPLACEMENT
Authors: Egbring R , Seitz R , Wolf M , Lerch L , Menges T
Published: June 2019

In septic or cardiac shock antithrombin III-thrombin (AT III-Thr) and a1antitrypsin-elastase(a1AT-ELP) as well as a2antiplas-min-plasmin (a2AP-Pl) are found to be elevated to different extents. In cardiac shock AT III-Thr is predominantly increased, while in septic disorders a2AT-ELP as indicator of leukocyte stimulation is additionally found to be elevated. Stimuli for leukocyte activation are bacterial endotoxins, immune complexes, factor Xlla and others. The possible action of bacterial proteases during septic infections is only known in animal models. To stop hemorrhagic complications in disseminated intravascular coagulation (DIC) following septic (n=24) or non-septic (n=15) shock, we treated the patients with AT III concentrate and FFP in relatively high amounts containing a2macroglobulin (a2M), a1antitrypsin (a1AT) and others which are not available as concentrates. Subsequent to the procedure PIC's decreased, coagulation factors and inhibitors as well as thrombocyte counts increased. In in vitro models bacterial proteases have been shown to destroy a1AT, activate prothrombin and others. Only a2M may inhibit proteolytic activity of Staph aureus, N. meningitidis, P. aeroginosa and K1. pneumoniae and E. coli as our in vitro studies, using fibrin plates containing a2M, demonstrated. Not only bleeding or microthrombotic complications might be influenced by plasma derivative substitution, but also proteases released from bacteria
AN EXPERIMENTAL MODEL TO STUDY INTERRELATIONSHIP BETWEEN HYPER-COAGULABLE STATE AND ORGAN FAILURE IN SEPSIS
Authors: Tanaka T , Itoh T , Tsujinaka T , Sakon M , Kambayashi J , Mori T
Published: June 2019

Disseminated intravascular coagulation (DIC) and multiple organ failure (MOF) are critical consequences of septic patients but the pathogenesis has not been fully elucidated yet, because of the complexities of clinical cases. Thereby, attempts were made to establish animal model with DIC & MOF without injecting endotoxin, bacteria or procoagulants into circulation, which may result in misleading conclusions. Twenty four male Japan albino rabbits were divided into the following 4 groups; [A]-ligation of bile duct and instillation of fecal suspension ( appr. 2 X 107 gram negative rods)into the proximal bile duct. [B]-ligation of bile duct and instillation of sterile saline. [C]-laparotomy alone. [D]-[A] receiving intravenous heparin (50u/kg/h). Citrated blood from femoral vein was collected before and 3,6,9 hours after the surgical procedure. After the last blood collectioh, animals were sacrificed and lungs, liver and kidneys were harvested for histological evaluation. During 9h observation period,.the rabbits were kept anesthetized with pentobarbiturate and were hydrated with 4ml/kg/h saline. All animals survived during the period. The following findings were obtained in [A]. Platelet count was gradually decreased (p<0.05) and significant leukopenia was noted at 9h (p<0.01). APTT and PT were significantly prolonged after 3h (p㌼0.01) after 6h. Plasma creatinine and bilirubin were significantly increased than those of [B] (p<0.01). The amount of endotoxin in circulating blood, determined by colorimetric assay, was significantly elevated up to 200pg/ml (p<0.01) . Histological examination revealed glomerular thrombi and focal necrosis of the liver. In [B], bilirubin was slightly elevated and PT was prolonged but much abnormal values were noted in [A]. Endotoxemia was not detected at all in [B]. In [D], the value of PT and fibrinogen was comparable to that of [B] and also the value of bilirubin and creatinine was close to that of [B]. Thus, we established sepsis model with reproducible hypercoagulable state and organ failure. The prevention of organ failure by heparin administration indicates that hypercoagulable state is common etiologic factor in occurrence of DIC and MOF in sepsis
IN VIVO AND IN VITRO NEUTRALIZATION OF BACTERIAL TOXINES BY IGM ENRICHED AND CONVENTIONAL I. V. IMMUNOGLOBULINS
Authors: Dichtelmuller H , Stephan W
Published: June 2019

Severe septic phenomena are caused bybacterial toxins. We therefore investigated the neutralization of toxins derived from Staphylococcus aureus and Pseudcmonas aeruginosa by different i.v. irtmunoglobulin preparations using hemolysis inhibition tests and mouse protection tests. The efficacy of conventional i.v. immunoglobulin containing preparations were compared with an IgM enriched i.v. immunoglobulin (Pentaglobin).

For hemolysis inhibition tests sterile filtered supernatant of Staphylococcusaureus was prepared and given to human erythrocytes. When IgM enriched immunoglobulin was added, toxin depended hemolysis was inhibited. By addition of three different i.v. immunoglobulin preparationsno inhibition of hemolysis was observed. In order to confirm these results in vivo, mice were exposed to the toxic supernatant of Staphylococcus aureus intraperitoneally and treated with i.v. immunoglobulins (3.1 mg/animal) 30 min after toxin exposure. Significant protection of toxin exposed animals was achieved by IgM enriched i.v. immunoglobulin (92 % protection) but not by conventional i.v. immunoglobulin (17 % protection). Similar results were obtained when mice were exposed to toxic supernatant of Pseudcmonas aeruginosa instead of Staphylococcus aureus. We therefore conclude, that IgM is essential for neutralization of bacterial toxins and IgM enriched i.v. immunoglobulins are more effective in therapy of severe septic phenomena, compared to conventional i.v. immunoglobulins.
PLATELET FUNCTION AND KALLIKREIN SYSTEM IN PATIENTS WITH ESSENTIAL HYPERTENSION
Authors: Bodzenta-Lukaszvk A , Krupiński K , Bielawiec M
Published: June 2019

Since the pathogenesis of hypertension is still discussed the aim of this study was to investigate behaviour of platelets and kallikrein system in patients suffering from this disease. In 30 patients with essential hypertension, aged 23-31 years and 20 normotensive healthy subjects, aged 21-35 years the following parameters of platelet function were studied: platelet aggregation induced with ADP,platelet activating factor (PAF) and arachidonic acid (AA) according Born's method, plasma beta-thromboglobulin (Beta-TG) and platelet factor 4 (PF4) , plasma thromboxane B2 (TXB2) and cyclic AMP using radioimmunoassay kits. The activity of kallikrein and factor XII was also determined using Chromo-zym PK (Boehringer Mannheim GmbH). The hypertensive patients demonstrated a greater platelet aggregability by ADP and PAF, elevated concentrations of Beta-TG and TXB2 as well as decreased level of cyclic AMP in comparison to normotensive subjects. No significantly changes in platelet aggregability by AA and activity of PF4 were found in the group of hypertensive patients. There were also significantly decreased plasma concentrations of kallikrein and factor XII in these patients. Obtained results have shown hyperfunction of blood platelets and depletion of kallikrein system in the patients with essential hypertension. These results suggest that among different pathogenic factors function of blood platelets and kallikrein system should be taken into consideration.
THE VITAMIN K DEFICIENCY IN INFANCY IN JAPAN -- THE SECOND NATIONWIDE SURVEY
Authors: Nagao T , Hanawa Y , Sawada K , Tsukimoto I , Ikeda I , Komazawa M , Shiraki K , Shirahata S , Tsuji Y , Terao T , Matsuyama E , Maki M , Mikami S , Murata B , Motohara K , Yamada K , Yamamoto Y
Published: June 2019

Questionnaires were sent to 1,218 hospitals with more than 200 beds, in order to know the incidence of hemorrhagic disease due to vitamin K deficiency in infancy beyond 2 weeks after birth, during 4 and a half years, i.e. from January 1981 to June 1985. Out of the 534 cases reported, 407 had no obvious reasons for vitamin K deficiency: "idiopathic vitamin K deficiency in infancy". Other 68 cases had bleedingepisodes due to vitamin K deficiency associated with hepatobiliary lesions (e.g.congenital bile duct atresia), chronic diarrhea, long term antibiotic therapy and so on: "secondary vitamin K deficiencyin infancy". The third group consisting of 59 cases was so called "near miss" type, in which hemorrhagic tendency was discovered at the time of mass screening tests for vitamin K deficiency or by chance withoutany clinical hemorrhage. In the idiopatic group, 345 cases (84.8%) developed their bleeding episodes between 21 and 59 days of age, and 368 cases (90.4%) were wholly breast-fed. Intracranial hemorrhage was seen in 338 cases (83.0%) of this group. In most cases of this series (97.3%),no vitamin K was supplemented after birth. Administration of vitamin K is an urgent routine procedure during the first one or two months of life for all newbornbabies, although the incidence of the idiopathic vitamin K deficiency in infancyhas not decreased significantly comparedto the results of the first nation-wide survey (Jan. 1978 - Dec. 1980). This study was sponsored by the Ministry of Health and Welfare of Japan.
PERNATAL MANAGEMENT OF FETAL THROMBOCYTOPENIA
Authors: Daffos F , Forestier F , Kaplan C , Muller J Y
Published: June 2019

Fetal thrombocytopenia resulting from alloimmunisation (NAIT] or from autoimmune pathology (ITP) may contribute to morbidity from hemorrhage particularly when bleeding occurs into the central nervous system.

Utilizing a safe procedure for in utero blood samplings i.e. directpuncture under ultrasound guidance, we are able to propose a prenatal management. Considering NAIT we haveuntil now treated 6 patients. We propose a screening protocol for highrisk group based on maternal antecedents and immunological grounds. Fetal blood sampling is performed at 20th week of gestation allowing platelet count and typing. If there isincompatibility between the fetus and his mother two ways can be consFdered : absence or presence of thrombocytopenia. If the platelet countis normal, nothing is done until 37th week of gestation. In the other case, frequent ultrasound examinations are done. At the 37e week, a fetal blood sampling is performed andin utero maternal platelet transfusion is done in the case of thrombocytopenia, before the delivery. It is possible with this prenatal treatment to have vaginal delivery. Considering ITP. when the maternal status permit it. fetal blood samplingslet us to know exactly the fetal platelet count. By this way. the indication of delivery can be documented.

This procedure offers a new possibility of easily taking iterative samples. until the end of pregnancyand represents a particular interest in the prenatal treatment of suchhemorrhagic disorders.
LEVELS OF TOTAL AND FREE PROTEIN S DURING NORMAL AND PATHOLOGICAL PREGNANCY AND IN POST-PARTUM
Authors: GRUEL Y , MOALIC P , DUROUCHET E , GUEROIS C , DELAHOUSSE B , LEROY J
Published: June 2019

Levels of total and free Protein S (PS) were measured on plasmas from 28 women during normal (n = 15, mean age = 25) and pathological pregnancy (Hypertension or preeclampsia, n = 13, mean age = 27). Prepartum (PreP) samples were obtained in the third trimester between the 30th and the 40th weeks of gestation, and postpartum (PostP) blood collected in the 5 days after delivery. Total PS level was determined using Laurel 1 rocket immunoelectrophoresis (Diagnostica Stago, Asnifcres-France). Free PS was measured using the same method after precipitation of C4b-BP-Bound-PS by polyethylene glycol. C4b-Binding Protein (C4b-BP) determinations were conducted by Laurel 1 method as well. Results were expressed as a percentage (Mean ± SD) of a normal adult pool (n = 15).

A significant decrease of free PS level was observed both in normal or pathological pregnancy and in postpartum. These data might be explained, by the increase of C4b-BP plasmatic level.

*= p<0.05 (as compared to normal controls), **= p<0.01, ***= p<0.001 (Student´s t test).