bluuebunny/crossref_metadata_2025 huggingface dataset:
CROSS-REACTIV7E Fl/III :C IN THE RABBIT: A POTENTIAL ANIMAL MODEL FOR FUIII:C STUDIES
Authors: Czerchaujski L , Hornsey V , Prowse C , Bessos H
Published: June 2019

A one step Enzyme-linked immunosorbent assay (ELISA) incorporating two anti-human FVIII :Ag monoclonal antibodies (MAbs) was used to determine FUIIIrAg in rabbits. Initial examinations showed the presence of cross-reactive FUIII:C in rabbit serum, plasma, and homogenates of normal rabbit liver, lung and spleen. Detailed investigation of normal rabbit plasma, and plasma depleted with Sephacryl S1000-anti FVIII :C MAbs and irrelevant MAbs (as control) using the ELISA, a chromogenic Fl/III:C activity assay, an immunoradiometric assay (IRMA) using human antibody, and fast protein liquid chromatography (FPLC) showed conclusively that the ELISA was specific for FVIII:C. The cross reactive FVIII:Ag was found to be most prominent in rabbit plasma, followed by serum, liver, lung and spleen. The ELISA should enable potential use of the rabbit as an animal model for FVIII:C studies, such as the enhancement of homologous FVIII:C with drugs or following tissue transplantation.
FACTOR VIII:C, 80 KD AND 90-210 KD POLYPEPTIDE QUANTITATION BY SLISA
Authors: Bloom J W , Berkner J  A , Mitra G
Published: June 2019

Purified Factor VIII:c is generally found proteolyzed into nultiple polypeptides. An 80 kD polypeptide appears to form a metal-linked complex with each of a series of 90-210 kD polypeptides and separation of these complexes results in loss af Factor VIII:c activity. To quantitate total Factor VIII:c content - active and inactive - three sandwich ELISA's were developed using an immunopurified polyclonal antibody as the capture antibody. This antibody was shown by a Western blot of Factor VIII:c to interact with all the 80-210 kD polypeptides. Utilizing biotinylated polyclonal antibody, an ELISA was developed that detected both the purified carboxy terminal (80 kD) and amino terminal (90-210 kD) polypeptides. Thrombin treated 80 kD polypeptide and Factor VIII:c were also detected by this ELISA. A second ELISA was developed with a biotinylated monoclonal, designated C8, that detected purified 90-210 kD polypeptides, but not the 80 kD polypeptide. A third ELISA was developed with a biotinylated monoclonal, designated C7F7, that detected only the purified 80 kD functional subunit of Factor VIII:c. Thrombin treated 80 kD polypeptide and Factor VIII:c were not detected by this ELISA. These three ELISA methods combined allow estimates of amounts of denatured or thrombin degraded Factor VIII:c to be determined, information not obtainable by the coagulation activity assays.
IN VIVO RECOVERY AND HALF-LIFE OF A STEAM-TREATED FACTOR IX (FIX) CONCENTRATE
Authors: Mörsdorf S , Seified E , Köhler M , Fasco F , Hellstern P , Pindur G
Published: June 2019

The introduction of heat treatment of FVIII or FIX concentrates has reduced the risk of infection, however, has raised the question of a reduced haemo-statical effect. Therefore, the in vivo recovery and half-life of a steam-treated FIX concentrate (S-TIM4, Immuno) were investigated in 10 haemophilia B patients from two haemophilia centers. Patients mean age was 33 y (range 17-51 y) and the mean body weight (BW> was 67 kg (range 44-81 kg). Basal FIX levels ranged from 0.007 to 0.03 (median 0,007) U/ml. The patients had not received FIX concentrate at least 7 d prior to the study. Patients 1-4 received 4 different lots, patients 5-10 received one single lot. Blood was drawn before and after 15, 30 min, 1h, 4 h, 8 h, 10 h, 12 h, 24 h and additionally 48 h in patients 1-4. FIX levels were measured using FIX deficient plasma from Immuno (patients 1-10) in center 1, additionally in patients 5-10 using FIX deficient plasma from MerzDade. In vivo recovery and half-life were calculated according to Allain (1980, 1984) and given in % and h, respectively. Results: The table shows the dose and the calculated in vivo recovery and half-life, according to the FIX measurements in center 1 (C1) or center 2 (C2).

Although the apparently longer half-life of patients 1-4 may in part be explained by the longer period of FIX measurements in center 1, the exclusive use of one single lot of FIX concentrate suggests an influence of the lot transfused in these patients. However, laboratory signs of DIC were not present.
ALLOANTIBODIES IN HEMOPHILIA B BINDING TO MULTIPLE FACTOR IX (IX) EPITOPES
Authors: Thompson A R
Published: June 2019

A high titer, boost responding inhibitor was present in a patient and his nephew, both with severe hemophilia B. On digests of their DNA, Southern blots hybridized to a cDNA were normal. They had no detectable IX antigen, including immuno-radiometric assays with a calcium-requiring polyclonal antibody fraction, in either serum or urine (less than 0.03 U/dl).

Plasmas from the patient and his nephew had 12 and 25 NIH U/ml inhibitor titers, respectively. They were fractionated over IX-agarose with calcium. Unlike fractionation of rabbit polyclonal antibodies, EDTA eluates did not bind IX. Purified patient inhibitors were eluted at low pH and contained detectable IgG1, IgG2 and IgG4 but not IgG3, IgA or IgM by radial immunodiffusion. On immunoradiometric assay, each patient's 125I-inhibitor bound to IX on the same solid phase inhibitor, indicating recognition of more than one epitope. Factor IX Ag was readily detected in these assays. The patients' inhibitors competed with binding of 125I-IX binding to each of 3 monoclonal antibodies. Each monoclonal antibody blocked 125I-IX binding to its own, insolubilized species. Similar results were obtained on an inhibitor plasma provided by H. Reisner (Chapel Hill, NC) which, unlike the present cases, contained a calcium-requiring antibody fraction (Briet E, et al Prog Clin Biol Res 150:123-139, 1984). Our patient's inhibitor was tested with two rabbit polyclonal fractions; the calcium-dependent fraction (epitopes in the light chain of IXa) and a specific heavy chain-binding fraction. The latter was from immunoaffinity purification of the non-calcium binding rabbit polyclonal fraction over an insolubilized synthetic peptide containing residues 256 through 269 of the IX sequence. Both fractions, in fluid phase, inhibited binding of 125I-IX to their own but not the other solid phase fraction. The patient's inhibitor did not block 125I-IX binding of the calcium-requiring fraction but did compete with the fraction prepared from the peptide column. These data suggest that the defect in our patient is a mutation between the Gla domain's 2nd or 3rd exons and the second growth factor-like region in the 5th exon.
THE CATALYTIC TRIAD RESIDUES (HIS221, ASP269, SER365) AND THE BINDING POCKET RESIDUE (ASP359) IN FACTOR IXBm ELSINORE (IXBmLE) ARE NOT ALTERED
Authors: Spitzer S G , Usharani P , Roser A D , Kasper C K , Bajaj S G
Published: June 2019

Previous studies suggested that the defect in IXBmLE (a nonfunctional variant of human IX) is either in the catalytic triad or at the site(s) of interaction with the macromolecular substrates (antithrombin III, factor VII or factor X). To distinguish between these possibilities, we isolated a complete IX cDNA clone from a human liver cDNA library. We also constructed a genomic library (in phage EMBL3) using DNA of the BmLE patient. The library was screened with normal IX cDNA and with synthetic oligonucleotides. The positive clones containing the exons coding for IX were plaque purified. Two clones which contained the coding sequence of the catalytic domain, i.e., His221 (exon VII), and Asp269, Asp359, and Ser365 (exon VIII) were selected for further studies. The phage containing exon VIII was first digested with Sail and EcoRI and a 2-Kb fragment, which hybridized with the segment of cDNA containing exon VIII, was gel purified. The 2-Kb fragment was further digested and the subfragments were cloned into M13; the length and direction of the fragments used in sequencing are shown below:

The phage containing exon VII was digested with PstI and SalI, and a 1-Kb fragment that hybridized with the 19-mer His221 probe was subcloned into M13 phage for sequencing. The sequence starting with residue Vall96 through residue Arg403 was found to be normal. Thus, none of the residues in the catalytic domain of IXBmLE are different from that of normal IX. These data provide strong indirect evidence that the noncatalytic aminoterminal portion of IX plays a significant role in the structural recognition of the macromolecular substrates. The sequence of this region of IXBmLE should provide information about the putative residue(s) essential for this recognition.
PRODUCTION OF ANTIBODIES TO HUMAN VON WILLEBRAND FACTOR IN LAYING HENS. ISOLATION OF IMMUNOGLOBULINS AND APPLICATIONS TO THE DETECTION OF MOLECULAR DEFECTS OF VON WILLEBRAND FACTOR
Authors: Toti F , Stierlé A , Wiesel M L , Schwartz A , Freyssinet J M , Cazenave J P
Published: June 2019

Von Willebrand disease (vWD) is an inherited disorder of primary hemostasis caused by deficiency or structural abnormalities of von Willebrand factor (vWF). VWF circulates in plasma and is also present in platelets. Plasma vWF, the carrier protein for factor VIII, is a large multimeric glycoprotein composed of identical subunits linked by disulfide bridges. Plasma and platelet vWF display distinct multimeric electrophoretic patterns. The different vWD subtypes can be classified either by the determination of vWFantigen (vWFíAg) and/or by multimer distribution. Antibodies to human vWF were raised in laying hens by intramuscular injections of purified human vWF. Immunoglobulins were isolated from egg yolks by selective polyethylene glycol and ammonium sulfate precipitations. These antibodies appeared to be monospecific, as they did not react with the plasma proteins of a patient with severe vWD. The pullets received weekly 50 μg vWF for 4 weeks and then had monthly injections. The antibodies occurred as early as the third injection, the yield being 300 to 500 mg of immunoglobulin per week (6-7 eggs). The titre could be constant over periods greater than 1 year. These immunoglobulins to vWF were tested in vWFíAg electroimmunoassays and for the multimer analysis of plasma and platelet vWF by electrophoresis and immunoblotting techniques. In no case could a difference be detected between assays performed with rabbit monospecific antiserum or with yolk immunoglobulins to human vWF. Ten to 12 multimers could be revealed for normal plasma vWF and up to 12 to 14 bands for normal platelet vWF (1.7% agarose). In the case of vWD, the electrophoresis patterns were identical with both antibodies. Thus, antibodies to vWF raised in laying hens are a suitable tool to detect and to characterize vWD. Although they do not interact with protein A, yolk antibodies are certainly advantageous to produce, as they do not contain IgM or IgA. Immunoglobulin fractions can contain up to 10 % of specific antibodies. Since they are available in larger quantities and are easy to isolate, larger homogeneous batches of antibodies can be obtained. This method may easily be applied to develop antibodies to a variety of antigens.
ABNORMAL vWF IN CONGENITAL CARDIAC DISEASE: EVIDENCE FOR FRAGMENTATION
Authors: Gill J C , Baganz S , Endres-Brooks J , Montgomery R R
Published: June 2019

We previously reported the presence of an acquired von Willebrand factor (vWF) abnormality characterized by absence of the highest molecular weight (raw) multimers in 12 children with non-cyanotic congenital cardiac lesions. In order to determine the prevalence and define the mechanism of the vWF defect, a prospective series of 17 children were studied at the time of cardiac catheterization. In addition to standard coagulation and vWF assays, vWF multimeric patterns were determined by SDS agarose electrophoresis using two agarose concentrations, low concentration (0.65%) and high concentration (3.0%). Nine of the 17 children had absence of high mw vWF multimers on 0.65% agarose gels as follows:

Of 5 children with absence of high mw vWF multimers on 0.65% agarose gels, 4 had 3% agarose gel patterns similar to that seen in patients with Type IIA von Willebrand disease (vWd). Two of 3 studied had the presence of vWF:frag on crossed immunoelectrophoresis. The patterns were not different when samples were drawn into a "cocktail" of proteolytic inhibitors. There was no correlation of abnormal vWF multimers with elevated B thromboglobulin, low dose ristocetin induced platelet aggregation or abnormal platelet vWF subunits. Thus, abnormal vWF multimers are commonly associated with non-cyanotic congenital cardiac lesions, particularly VSD. The lack of abnormality in platelet vWF multimers, absent low dose ristocetin aggregation, and presence of vWF:frag suggests that these alterations may be secondary to fragmentation or are of endothelial cell origin.
INVESTIGATIONS ON MULTIMERIC STRUCTURE OF PLATELET VON WILLE-BRAND FACTOR IN PATIENTS WITH HEREDITARY DISORDERS OF PLATELET FUNCTION
Authors: Vigh Zs , Scharrer I
Published: June 2019

Von Willebrand factor (vWF), a multimeric glycoprotein, plays an essential and multifunctional role in the hemostatic process. It is well known that platelet glycoproteins IB, IIB and IIIA contain receptors for vWF. Von Willebrand factor was also found in alpha granules of platelets. Therefore we investigated the multimeric structure of platelet vWF in 12 patients with different inherited disorders of platelet function. The patients had the following diagnosis: Hermansky Pudlak syndrome, Thrombasthenia and up to new undefined hereditary disorders of platelet function. The method is based upon:

1) washing of platelets 2) release of platelet vWF 3) separation of vWF multimers by SDS-agarose electrophoresis 4) subsequent blotting of vWF mul timers onto nitrocellulose 5) staining by peroxidase conjugated antibodies.

The investigations were repeated 3 times and compared to those of normal platelets. In 2 patients with Hermansky-Pudlak syndrane no multimeric structure could be detected in platelets whereas the multimeric pattern of plasma of these patients was normal. Also in one patient with the tentative diagnosis: thrombasthenia we couldn't find any multimeric structure in platelets compared to the normal multimeric composition of plasma. In 2 patients with giant platelets the multimeric distribution was normal. In the remaining 6 patients we observed multimeric structure which was different from that seen in vWd variants and in healthy volunteers. In 1 patient we found normal multimeric pattern in plasma and platelets.

Based on our findings it can be assumed that the analysis of multimeric structure of platelet vWF can be helpful for the diagnostic approach and for the insight in pathogenesis of inherited disorders of platelet function.
A NEW PLATELET DISFUNCTION CHARACTERIZED BY SPONTANEOUS PLATELET AGGREGATION AND ENHANCED von WILLEBRAND FACT0R-PLATELET INTERACTION
Authors: Casonato A , De Marco L , Del Ben M G , Mazzucato M , Fabris F , Ruggeri Z M , Girolami A
Published: June 2019

The proposita is a 48 years old woman who presents mild bleeding tendency and a bleeding time of 7 min. (n. 6 min ± 1.4). Her platelet count is within normal limits and has normal plasma Factor VIII coagulant activity, Ristocetin cofactor activity, von Willebrand Factor (vWF) antigen and vWF multimeric pattern. Spontaneous platelet aggregation (SPA) is observed when citrated PRP is stirred in an aggregometer cuvette. SPA is completely abolished by an anti glycoprotein IIb/IIIa monoclonal antibody and slightly affected by the addition of an anti GPIb monoclonal. The proposita shows an increased ristocetin-induced platelet aggregation although to a lesser extent than that observed in type I IB von Willebrand disease. The distinct feature of this patient, which is also present in two family members studied, is the strong platelet aggregation observed upon the addition of purified vWF at concentrations ranging from 60 to 120 μg/ml f.c. In several normal individuals the addition of vWF up to 300 μg/ml always failed to induce platelet aggregation. The vWF induced platelet aggregation is abolished by anti GPIb and anti GPIIb/IIIa monoclonal antibodies and by EDTA (5 mM). Apyrase (5 ATPase U/ml) inhibits the second wave of aggregation. Patient′s platelets in PRP are 6-6 times more reactive to asialo vWF (AS vWF) induced platelet aggregation than normal platelets. We were not able to demostrate any increased binding of vWF to the patient′s platelets in the presence or in the absence of ristocetin, while we have demonstrated enhanced binding of As vWF, compared to normal, to platelets in PRP. Patient′s platelets do not show an abnormality of GPIb as detected by SDS page and immunoblot analysis using antibody LJIb 10 which reacts with denaturated and reduced GPIb alfa chain. The number of GPIb molecules detected by monoclonal antibody is 17,870 (n. 19,796 ± 3,791). Unlike the previous reported patients with pseudo or platelet type von Willebrand disease, our patient has normal vWF parameters.
FURTHER STUDIES ON TYPE IID VON WILLEBRAND'S DISEASE (vWD) VARIANTS
Authors: Hill F G H , Enayat M S , Williams C W , Short P E
Published: June 2019

Five individuals from a single kindred and 3 other unrelated patients with type IID vWD have been studied. Multimeric analysis patterns obtained using discontinuous gel electrophoresis were identical both in major and minor band positions and distribution. In 2 where lysed platelets were studied, platelet and plasma multimeric analysis patterns were identical. EDTA plasma when compared with citrate plasma had more intense bands but there was no increase in intermediate or high molecular weight multimers. When fresh, frozen and thawed plasma or plasma left at 4°C for 24 hours were studied using multimeric analysis with autoradiography using 2 monoclonals (ESVWF 2 and 10) against vWF epitopes, significantly more intense autoradiographs were obtained with non-fresh rather than fresh plasma. These results suggest that freezing and thawing or storing plasma initiate a conformational change with the exposure of more epitope sites in these variants.

Despite bleeding times in excess of 20 minutes ristocetin and botrocetin induced platelet aggregation and ristocetin and botrocetin cofactor assays were normal on fresh plasma. Both cofactor activities fell by 25-30% when fresh plasma was frozen and thawed once, whereas vWF:Ag fell by 20%.

These results suggest that in IID vWD vWF:Ag is unstable and easily undergoes conformational change with loss of ristocetin and botrocetin cofactor activities and exposure of increased numbers of some epitopes.
NEW VARIANT FORMS OF von WILLEBRAND'S DISEASE
Authors: Matthews K B , Kernoff P A , Tuddenham E G D , Goodall A H
Published: June 2019

Two unusual variants of von Willebrand's Disease (vWD) have been observed; one resembling Type Ic vWD and one an unclassified variant form of the disease. Both are associated with a history of mild bleeding and with a prolonged bleeding time.

Two related patients (father and daughter) presented with reduced vWF activity (RiCoF) and vWF:Ag. Multimer analysis showed the presence of all molecular weight multimers but with a lack of triplet structure. This was confirmed in 3% agarose gels and resembled the pattern of multimers previously described as Type Ic vWD. Both patients responded well to DDAVP.

In the second case the patient had a lowered vWF activity (RiCoF) but normal vWF:Ag, analogous to a Type Ila vWD pattern. Mutimer analysis however demonstrated the presence of all MW multimers with a normal triplet structure. This defect was not detected with two monoclonal antibodies that recognise the GPIb binding site on vWF and is thought to represent a minor abnormality in the vWF molecule in this patient.
THE EFFECTS OF CYCLOSPORIN A (CyA) ON PLATELET FUNCTION IN HUMAN RENAL ALLOGRAFT RECIPIENTS AND NZW RABBITS
Authors: Cohen H , Mackie I J , Patel R , Neild G H , Machin S J
Published: June 2019

CyA therapy is associated with microvascular thrombotic complications both in human allograft recipients and experimental animals. We have assessed evidence for increased platelet reactivity, i.e. presence of spontaneous aggregation, responses in platelet rich plasma (PRP) to ADP, total platelet nucleotide content and release to 20μg/ml collagen, and sensitivity to ZK 36,374, a prostacyclin analogue, serially in 21 human renal allograft recipients for 1 year post-transplantation and in 15 O NZW rabbits which received CyA 25mg/kg (n=5), or 'placebo' (the carrier for CyA) (n=5), or N saline (n=5) i.v. for 10 consecutive days. In the humans, spontaneous aggregation was observed on 10 occasions in 5 patients and responses to low doses of ADP, 0.5 and 1.0 μM, were significantly increased compared to normal controls up to 1 year (p<0.002) post transplantation. Total platelet nucleotide (ATP + ADP) content was significantly decreased (p<0.002) up to 3 months post-transplantation, indicative of in vivo activation, as was nucleotide release. Platelet sensitivity to ZK 36,374 decreased after 2 months (p<0.01) and this persisted at 1 year (p<0.02) compared to sensitivity at 1 week post transplantation (paired t tests). In the animal model, CyA or 'placebo' administration were unassociated with spontaneous aggregation, responses to ADP 0.5-5.0 μM and collagen 2.0-4.0 μg/ml remained unchanged as did sensitivity to ZK 36,374. In conclusion, CyA-treated renal allograft recipients exhibit spontaneous platelet aggregation, hyperaggregability to low doses of ADP, in vivo activation and decreased sensitivity to ZK 36,374, with abnormalities persisting up to one year posttransplantation. These abnormalities are not reproducible in an animal model.
EFFECT OF DDAVP ON PRIMARY HEMOSTASIS WITH CONGENITAL AFIBRINOGENEMIA
Authors: Taki M , Inagaki M , Miura T , Saito N , Meguro T , Yamada K
Published: June 2019

It has been reported recently that DDAVP might be an useful tool in the therapy and prevention of bleeding in patients with congenital afibrinogenemia (CA).

To study the mechanism of its efficacy, changes in the platelet functions of a patient with CA were examined prior to, and one hour after, the infusion of DDAVP (0.4 μg/Kg). A patient with Glanzmann's thrombasthenia (GT) was also examined, to allow a study of the role of platelet membrane glycoprotein IIb/IIIa (GP IIb/IIIa), a deficient platelet in GT, in the resulting effects of the drug. When both patients were infused with DDAVP, the level of plasma von Willebrand factor (vWF) increased two- to fourfold, accompanied by an enhancement of ristocetin-induced platelet agglutination. The level of plasma fibrinogen was never changed.

The prolonged bleeding time observed was markedly improved only in the CA patient, remaining unchanged in the GT patient, after the infusion of DDAVP. This indicates that DDAVP is effective in diminishing the bleeding tendency in CA, but not in GT. Among the platelet functions tested, only the platelet retention rate on glass beads, ADP-induced platelet aggregation and collagen-induced platelet aggregation improved in CA, each remaining unchanged in GT. In particular, collagen-induced platelet aggregation was markedly improved in the CA patient. However, the platelet adhesion to collagen (50 μg/ml)-Sepharose remained normal, both before and after the infusion of DDAVP in CA.

These results suggest that an increase in the plasma vWF level and the existence of platelet membrane GPIIb/IIIa may be necessary for the improvement of primary hemostasis, after the infusion of DDAVP. The vWF-mediated platelet aggregation by collagen or ADP may produce this effect in the CA patient.
SUCCESSFUL SURGICAL MANAGEMENT OF A PATIENT WITH COMBINED FACTOR V AND VIII DEFICIENCY WITH DDAVP + FFP
Authors: McKenna R , Cole E R , DOOLAS A
Published: June 2019

A 59 year-old white male with a life long history of severe bleeding following trauma or surgical procedures was documented to have a combined factor V and factor Vlll-C deficiency. His baseline factor V ranged between 16% - 30% and factor VIII-C was between 20% - 30%. His APTT ranged between 50 - 56 seconds (21-31 N) with a prothrombin time activity between 33% - 40% of normal ( ≥ 70% N). Factor II, Vll/X, X, IX, XI, XII, template bleeding time and platelet function studies were normal. There was no severe factor XIII deficiency.

Since the response of these patients to DDAVP is unknown and the patient was admitted with a large hematoma in the subolecranon bursa, DDAVP was infused in a dose of 0.5 μ g/kg body weight over 15 minutes. The baseline factor VIIl-C of 20% rose to 55% at 5 minutes after termination of DDAVP and to 62% and 66% at 3 and 6 hours respectively. The factor VIII-C level dropped to 45% and 36% at 12 and 24 hours respectively after a single dose of DDAVP. As measured by a sensitive 125I-fibrin assay, this dose of DDAVP caused a net rise in plasminogen activator activity of 2.43 CTA U/ml.

The patient had worn an inguinal truss for approximately 20 years for two large oblique inguinal hernias, one of which entended to the level of the mid-thigh. Pre and peri-operative management of the right inguinal herniorrhaphy consisted of DDAVP in a dose of 0.5 μg/kg Q 12 hourly for two doses, FFP at 9 ml/kg Q 12 hourly for three doses, and Amicar for 48 hours starting post-operatively. This regimen maintained the factor VIIl-C at ∼ 50% with factor V between 44% - 50% for a period of three days. On the 4th postoperative day a left inguinal herniorrhaphy was accomplished with DDAVP and FFP (dosage similar to previous) administered Q 12 hourly for three doses, then once in the next 24 hours, and Amicar for three days. A 4 cm wound hematoma noted on the first day of the second surgical procedure was evacuated, and was due to the presence of a bleeder since Vlll-C and V levels were higher than the values indicated on the first procedure. No red cell transfusions were given; fluids were restricted to 600 ml per day for 24 hours after the last dose of DDAVP. Successful bilateral inguinal herniorrhaphies without significant hemorrhages was achieved with exposure to a minimal volume of blood products.
HEMOPHILIAC MENINGITIS WITH PARTICULAR MANIFESTATION AND HIV ANTIBODIY IN SPINAL FLUID
Authors: Abe T , Terao T , Nakayama Y , Kato Y , Kazama M , Takahashi I
Published: June 2019

A 25-year-old patient with hemophilia A, who had had thrombocytopenia since 1985, was admitted to a hospital in August 1986 because of headache and slight disturbance of consciousness. Diagnosis of cerebral bleeding was made based on brain CT scan. Lymphadanopathy was noted then, and after relief of the symptoms S. aureus sepsis developed. Examination of serum for HIV antibody performed at this time gave a positive result. He was transferred to our hospital for evaluation of AIDS-relatedimmunologic disorder. The ratio of T4/T8 was 0.16, but no definitive opportunistic infections were found. Soon later, he developed headache and fever. There were no other neurological signs. Spinal fluid examination revealedlymphocytic pleocytosis without bacilli, fungi or antibodiesto viruses commonly causing encephalomeningitis. However, antibodyto HIV was detected in the spinalfluid. By a supportive care with useof antibacterial agents thesymptoms were alleviated with diminishing of the pleocytosis in spinal fluid. Lentinan, an antitumor polysaccharide, was administered with some effect on his immunologic functions. Three months later, meningitis recurred which wasalsorelieved by a supportive care.

HIV is known to cause meningeal and CNS diseases. In this case the presence of HIV antibody in the spinal fluid was interpreted to be due to HIV invasion of nervous system. Furthermore, this casehas some particular feature in consideration of the common pictureof neurological involvements of AIDS, indicating that HIV induced a variety of meningeal and CNS manifestations.
HTLV III/LAV ANTIBODY STATUS, AND IMMUNOLOGICAL ABNORMALITIES IN A HEMOPHILIC POPULATION FROM PORTUGAL Campos
Authors: Campos M , Santos L , Lobato J , Melo C , Justica B
Published: June 2019

An immunological study in a population of 46 hemophiliacs (A and B)under replacement therapy mainly with cryo, plasma and eventually withccmercial concentrates was carried out to detect changes related to HTLV III infection and those due totreatment. We followed the patients during 85-87 and we evaluate the abnormalities over a period of 2 years . Methods. Ig's (nephelometry): total lymphocytes, T3, T4, T8 subsets and B cells (IF microcopy):delayed hypersensivity skin tests (DHST) ,7 antigens: serology for HTLV III (ELISA and WB) (β 2 microglobulin (ELISA) Results. In 85,10 patients had anti HTLV III(28%) and all these received concentrates.9 out 46 had never been treated with concentrates and all were negative for HTLV III . Most of the HTLV III +ve patients showed lymphopenia, low T4 and total B cells, anergy or depressed DHST,increase Ig levels, mainly IgG and IgM. Among the HILV -ve patients there were no significant diferences between tose treated withconcentrates and most showed only an increased Ig's levels (IgG and IgM During 85 and 86 all the patients were treated with plasma, cryo and heat-treated concentrates. In the end of 86 none of the seronegative showed seroconversion. Among the 10 seropositives in 85,1 died (AIDS) ,1 is in ARC and 8 are ssymptomatic . All theseroposi tive patients shows high levels of β2 microglobulin.

Conclusions . The low incidence of seropositivity for HTLV III,is probably due to the scarce use of comercial concentrates before 85. The absence of seroconversion despite the use of heat-treated concentrates is probably due to the safe of the product s. We must emphazise the absence of clinical symptoms in 8 seropositive patientsduring two years. There is a positive correlation between the level of (β2 microgldbulin and the seropositivity. Theimmunological profile observed is related more to the parenteraladministration of blood products thanto the specific composition and/or method of preparation of the therapeutic products. The serological profile from the HTLV III +ve patients isprobably a result of the administration in the past of comercial concentrates virus infected.
HEMOSTATIC PARAMETERS DURING PREPARATION OF LEUKOCYTE-POOR BLOOD
Authors: Losito R , Sternbach M , Masson A , Paguin M , Boyer A , Trepanier A
Published: June 2019

Non-hemolytic febrile transfusion reactions are believed to be due to sensitization to granulocytes. Leukocyte removal can diminish or prevent the reaction. Use of the Pall filter (Pall Bio Medical Prod. Corp.) has resulted in some patient reactions. The purpose was to re-investigate this filter in vitro and compare it to the Imugard (Terumo) and the Erypur (Organon Teknika) filters and study their influence on hemostatic activation pathways. Method: RBC cones., aged 5 or 10 days, were centrifuged (5,200 R. P. M., 4°C, 15 m.). Cells were leftundisturbed for 6-24 hours before filtration. Hematological parameters analysed included: leukocyte, red cell and platelet counts, differential and scanning electron microscopy. Activation studies related to clotting, fibrinolysis, kinin and complement pathways including both biological and immuno-assays; they consisted of the recal time, PTT, pro time, Factors I, VIII, XII, HMW kininogen, kallikrein, F. D. P., AT-III, alpha 2-macroglobulin, alpha 1-antitrypsin, plasminogen, Cl inactivator, C3, C4 and C5. Over 75 filtrations have been performed. Results of cell recovery are illustrated in table below. Changes in tests related to activation were minimal with the Pall filter while a wide spectrum of derangements were common with the other twofilters.

Conclusion: it appears the Erypur filter gives optimal results in the removal of leukocytes and platelets, whereas there is good recoveryof red cells by the Pall and Imugard filters. Secondly, the Pall filter (nylon) does not appear to be involved with the contact pathways whereas Erypur (cellulose acetate) and Imugard (cotton) have an influenceon these pathways.
ANTITHROMBOTIC ACTIONS OF A SULFOMUCOPOLYSACCHARIDE MIXTURE (ATERIOD) IN ANIMAL MODELS
Authors: Cornelli U , Welena J M , Fareed J , Huan X , Hoppensteadt D
Published: June 2019

Ateriod obtained from beef mucosal lining is a sulfomuco-polysaccharide mixture of various glycosaminoglycans which contains derma tans, heparatans and traces of heparin. It has been used in the treatment ofatherosclerosis and related vaso-oclusive disorders. Ateriod is standardized in terms of its lipoprotein lipase activation actions. Ateriod contains signfi-cant in vitro anticoagulant and antiprotease (anti-factor Xa and anti-factor Ila) activities as measured by clot-based and chromr ogenic substrate methods. However, this in vitro activity is 7-10 times lesser than heparin. In order to study the antithrombotic actions of this agent in subcutaneous, intravenous and oral routes, we utilized a rabbit stasis thrombosis model with a prothrombin complex concentrate/Russell's viper venom thrombogenic challenge and prolonged stasis. The apparent ED50 for the antithrombotic action were found to be: IV (75-100 ug/ kg), SC (0.8-1.3 mg/kg) and oral (20-30 mg/kg). In both the IV- and SC studies, sustained anticoagulant and antiprotease actions were evident. The observed antithrombotic actions did not relate to the anti-factor IIa or anti-factor Xa actions. Pretreatment of Ateriod with equigravimetric amounts of protamine and platelet factor 4 did not neutralize the antithrombotic actions of this agent in the rabbit model. In a primate (Macaca mulatta) model of pharmacokinetics, ex vivo analysis following subcutaneously administered Ateriod showed sustained anticoagulant and antiprotease effects. The time course of the subcutaneously administered Ateriod was markedly different than heparin and a low molecular weight heparin. Treated animals were shown to resist induced hypercoagulability following injection of homologous serum as measured by FPA generation for extended periods. These studies suggest that Ateriod produces a strong antithrombotic action and that it has highly sustained pharmacokinetics. The antithrombotic activity appears to be primarily mediated via non-antithrombin - HI dependent events which may be related to heparin cofactor II and vascular/ cellular modifications.
ACTIVATED PARTIAL THROMBOPLASTIN TIME MONITORING OF HEPARIN THERAPY: COMPARISON OF INSTRUMENTS AND REAGENTS
Authors: Seveso M P , Macagni A , Viganò D’Angelo S , Dettori A G , Bonini P A , D’Angelo A
Published: June 2019

It is current clinical practice to monitor heparin therapy by maintainig the activated partial thromboplastin time (APTT) at 1.5-2.5 × control normal pool. There is however controversy regarding the choice of reagents with respect to their heparin sensitivity. Choice of instrumentation is also known to affect the results. We have compared two automatic coagulometers, the ACL (Instrumentation Laboratory), a laser-nephelometric centrifugal analyzer and the KOAGULAB 40A (Ortho Diagnostics), an optical automatic coagulometer, with the tilt tube technique for the performance of APTT. Seven commercial reagents have been used for APTT replicate determinations in 30 normals, 30 liver disease patients and 30 patients on heparin therapy (20-40,000 IU daily). The overall observed imprecision (C. V.)was 1.8% for ACL, 3.0% for KOAGULAB 40A and 2.3% for tilt tube technique. The F test for the two-way interaction of ratios was statistically significant (p<0.001) for the large majority of reagent/ technique combinations in normals, liver disease and heparin treated patients. The percentage of patients adequately, inadequately and excessively heparinazed obtained with the two automatic instruments and with the tilt tube technique were not sig nificantly different when using the same reagent. However, major differences were observed when comparing the different reagents. For instance, inadequately heparinized patients were 90% according to one reagent (Cephotest, Nyegaard) as opposed to 17% according to another reagent (APTT, Instrumentation Laboratory). These results stress the need for a standardized APTT reagent to provide effective laboratory monitoring of heparin treatment.
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FIBRINOLYTIC POTENCY OF NON ANTICOAGULANT, OXI-EEDUCED SLOW AND FAST MOVING HEPARINS
Authors: Porta R , Pescador R , Niada R , Mantovani M , Prino G
Published: June 2019

It is well known that heparin is able to induce an increase of fibrinolytic activity when i.v. administered in man and in several animal species. Nevertheless, its anticoagulant properties can cause serious problems of bleeding and this restricts the therapeutic use of heparin. An oxi-reductive process applied to heparin leads to a conpound with reduced anticoagulant activity. Moreover, heparin can be separated into slow moving (SM) and fast moving (FM) components on the basis of electrcphoretic properties. The SM and FM components display quantitatively different biological activities. The aim of this paper was to verify if the oxi-reduction could affect or not the fibrinolytic activities of SM and FM. SM and FM, prepared by ethanolic precipitation from parent heparin, were oxidized by periodate and stabilized by reduction (RO-SM and RO-FM). The USP, APTT and anti Xa titres were determined in vitro using sheep plasma and kits from Boehrirger Biochemia (APTT) and Sigpia (Anti Xa). Fibrinolytic activities were assessed ex vivo after i.v. injection into rabbits at different doses to find out the effective dose hundred (ED100). The euglobulin fraction obtained from plasma was applied on human fibrin plates and the lysis areas were measured. The Table gives the results

The oxi-reduction decreased dramatically the anticoagulant activities of SM and FM heparins while their fibrinolytic activities were practically unaffected. Tne oxi-reduced heparins could be helpful therapeutic agents in pathological conditions characterized by a diminished fibrinolytic activity. They could represent an effective alternative to heparin; the very lew anticoagulant activities reduce the risk of bleeding, specially in the high risk patients, while the good fibrinolytic activity, comparable to heparin, could allow the dissolution of fibrin clots.
HIGH CONCENTRATIONS OF HEPARIN ARE MORE INHIBITORY TO PLATE- AGGREGATION IN-VITRO THAN ARE LOW MOLECULAR WEIGHT HEPARINS AND HEPARINOIDS AT THE SAME CONCENTRATION
Authors: Messmore H , Griffin B , Seghatchian J , Coyne E
Published: June 2019

Other investigators have shown that heparin in the usual therapeutic range (0.1-0.5 units/ml) has an enhancing effect on ADP aggregation and an inhibitory effect on collagen and thrombin induced aggregation. The effects of low molecular weight heparin (LMWH)and heparinoids (dermatan sulfate, heparan sulfate) on platelet aggregation have not been as extensivelystudied.
We have utilized citrated platelet rich plasma (3.2%citrate-whole blood 1:9) drawn in plastic and adjusted to a final platelet count of 250,000/ul. A Bio-Data 4 channgl aggregometer was utilized with constantstirring at 37 C. The reaction was allowed to run for 20 minutes.
Platelet rich plasma was supplemented 1:9 with saline or heparin and various agonists were then added ifno aggregation occurred. ADP, collagen, thrombin, ristocetin and serum from patients with heparin inudced thrombocytopenia (HIT) were utilized as agonists. Heparin was substituted at concentrations of 0.1 to 500 units per ml and various LMWH and heparinoids were substituted in equivalent anti-Xa or gravimetric concentrations.
At low concentrations no inhibitory effect on any ofthe agonists was observed with any of the heparins or heparinoids.
At concentrations of heparin of 100 u/ml or greater, all agonists were inhibited. At equivalent concentrations of five different LMWH (Cy 216, Cy 222, Pk 10169, Kabi 2165 and pentasaccharide) inhibition did notoccur at all or at very high concentions only. Dermatan sulfate and heparan sulfate inhibited only at high concentrations. HIT serum could not aggregate platelets with dermatan sulfate or pentasaccharide atany concentrations, but it was a good agonist with the other heparins and heparinoids.
PRODUCTION AND CHARACTERISATION OF MONOCLONAL ANTIBODIES AGAINST HEPARIN Deborah
Authors: Rathjen A , Geczy Carolyn L
Published: June 2019

To complement the studies using MAbs to AT III and because of the reported ability of heparin to modulate several aspects of the cell-mediated immune response, we have prepared two mouse monoclonal antibodies (MAbs) to porcine mucosal heparin.

MAb 25/15 is an IgGl and MAb 26/7 is an IgM. Both MAbs have iso-electric points between pH5.85 and 6.55. The MAbs recognise porcine and bovine mucosal heparin and rat mast cell heparin. Heparins with both high and low affinitiesfor antithrombin III (ATIII) bound both MAbs but neither MAb altered the binding of heparin to AT III. These antibodies did not recognise other proteoglycans (chondroitin sulphate types A, B and C, keratan sulphate and hyaluronic acid) with the exception of heparan sulphate, (the cellular equivalent of heparin) and Arte- paron (Luitpold-Werk, Munchen; a synthetically poly- sulphated chondriotin sulphate), in competition and solid-phasebinding assays. Dextransulphate(Pharmacia) was also recognised by these MAbs. Cross-reactivity with Arteparon and dextran sulphate indicate that charged sulphate goups on the mucopolysaccharides may be importantBfor MAb binding. The Mabs described may beuseful probes for endogenous heparin at the cellular and tissue level and may allow further investigation of the many biological activitiesof heparin
PROPHYLAXIS AGAINST POSTOPERATIVE DEEP VEIN THROMBOSIS (DVT)- A DOUBLE-BLIND MULTICENTER TRIAL COMPARING A HEPARIN FRAGMENT GIVEN ON THE EVENING BEFORE SURGERY WITH CONVENTIONAL LOW DOSE HEPARIN
Authors: Bergqvist D , Frisel J , Hallböök T , Horn A , Lindhagen A , Ljungndér H , Ljungström K G , Mätzsch T , Onarheim H , Risberg B , Törngren S , Örtenwall P , Burmark U S
Published: June 2019

Bergen, Halmstad, Goteborg and KabiVitrum AB Stockholm, Sweden and Norway. At the Xth Int. Congress on Thrombosis and Haemostasis in San Diego results from a multicenter trial on 432 patients were presented, comparing a low molecular weight heparin (LMWH) fragment (Fragmin, Kabi) once daily with low dose heparin twice daily. Prophylaxis started 2 hours preoperatively. The frequency of postoperative DVT did notdiffer (6.4 % v. 4.3 %) but the onset of thrombosis was delayed in the LMWH group. Haemorrhagic complications occurred significantly more often in the LMWH group (11.6 % v. 4.6 ?o). The results were similar independent whether the analysis was made according to the intention to treat principle or based on patients with correct prophylaxis. On the basis of these data and newer knowledge regarding the pharmacokinetics of LMWH a second prospective randomized double-blind multicenter trial on patients.older than 40 years undergoing elective abdominal surgery was started. The only difference was that the first dose of 5000 ariti-factor Xa units of LMWH was given on the evening before surgery. Only conventional low dose heparin was given 2 h before surgery. The study is designed to include 1000patients, a number which will be obtained during the spring 1987. At the time of abstract deadline 799 patients have been included. The over-all frequency of DVT (fibrinogen uptake test) is 6.8 %, the frequency of haemorrhagic complications 3.6 % and mortality 1.8 % (one fatal pulmonary embolism). These frequencies remained unaltered after inclusion of 336, 576, 655 and 799 patients. Although the code has not been broken yet, it can be concluded that the new regimen with start of prophylaxis the evening before surgery has not altered the frequency of DVT or mortality whereas the frequency of haemorrhagic side effects has decreased considerably.
PROSPECTIVE STUDY ON DOSE SCHEDULE OF HEPARIN THERAPY FOR DIC COMPLICATION IN LEUKEMIA PATIENTS
Authors: Sadakata H , Iri H , Uchiyama T , Andoh K , Tanaka H , Kobayashi N , Maekawa T
Published: June 2019

From the retrospective analysis of correlation between the activity of tissue factor (TFA) of leukemia cells (LC) and DIC complication in patients with acute leukemia, we have already reported an adequate dose schedule of heparin treatment for DIC can be calculated in accordance with the TFA of LC To evaluate this dose schedule, the prospective analysis was designed. Prior to the remission induction chemotherapy, TFA of LC obtained from 67 patients with leukemia (ANLL: M1;5, M2;22, M;34, M4;8, M5;4, M6;3, CML-BC: 11) was measured by Nemerson's two-stage method reported previously. Regardless of DIC complication, continuous heaprin therapy with 0, 15,000 or 9,700X + 9,000 units/day (X: logarithm value of TFA) was started with chemotherapy in patinet with 0, 0.8−4.1 U or >4.1 U of TFA, respectively. The complete remission and significant decrease of LC were achieved in 16 patients with ANLL and 5 patients with CML-BC, respectively. In 20 patients whose LC had 0.8 U of TFA or more (group A), 15 and 1 patients were complicated by DIC before and after start of the chmotherapy, respectively. DIC was improved in all of these patients. Other 4 patients were not complicated by DIC. There was no major bleeding due to heparin administration. In 47 patients whose LC had less than 0.8 U of TFA (group B), 40 patients were not complicated by DIC throughout the observation period. Remaining 4 and 3 patients were complicated by DIC before and after start of the chemotherapy, respectively. Among these patients, only one, whose DIC was due primarily to endotoxinemia, failed in control of the DIC. Consequently, in 67 patients subjected to this study, only one patient died of complicated DIC, although 17 patients (group A:4, group B:13) died of various causes other than DIC during the observation periods. These results suggest that our dose schedule of heparin is appropriate for both prevention and treatment of DIC complication in leukemia patients.
SIDE EFFECTS OF ASCENDING VENOGRAPHY
Authors: Vigo M , Lensing A W A , Corbetti F , Biondetti P R , Tropeano P , Prandoni P
Published: June 2019

Two hundred and sixtyeight (268) consecutive out-patients with clinical features compatible with deep venous thrombosis (DVT) were referred to our Department for contrast venography, which was carried out according to standard methods, employing 120-160 ml of a non-ionic contrast medium (Iohexol). All side effects probably related to venography were recorded during the test, immediately after its execution, at one day and during long-term follow-up (1 week, 1 month and 6 months), including impedance plethysmography (IPG) evaluation in patients with normal venograms. Eighteen patients (7%) did not undergo venography because of severe edema of the dorsum of the foot (3), impossibility to find a vein (7), patient refusal (1), known hypersensibility to radiopaque dye (4) and allergic reactions after injection of contrast medium (3). Our analysis therefore included 250 patients. Hypersensitivity reaction to the contrast medium following the venography were encountered in 3 patients (1%) of whom two had severe reactions. Pain and tenderness of the foot and calf after the test was observed in 15 patients (6%). No clinical signs and symptoms of pulmonary embolism were observed during and after the procedure and all serum creatine levels, assessed before venography, at day 1 and day 7, remained unchanged. There were 7 instances of contrast extravasation (3%) which did not result in local skin or tissue damage. In none of the patients was there any evidence to suggest the presence of post-venographic phlebitis and no patients with negative venograms developed a positive IPG during the period of follow-up.