bluuebunny/crossref_metadata_2025 huggingface dataset:
LOCALIZATION AND CHEMICAL SYNTHESIS OF A DIVALENT CATION REGULATED EPITOPE IN PLATELET MEMBRANE GPIIb
Authors: LOFTUS J C , Plow E F , Frelinger III A L , Smith M A , D’ouza S , Ginsberg M H
Published: August 2018

Platelet membrane glycoprotein (GP)IIb-IIIa is a component of a common adhesive protein receptor for fibrinogen, fibronectin, and von Willebrand factor. A monoclonal antibody, PMI-1, defines a divalent cation dependent regulation of the surface orientation of the heavy chain of GPIIb. Exposure of the PMI-1 epitope inversely correlates with the capacity of platelets to bind fibrinogen and aggregate. We have now localized and chemically synthesized this epitope. A 1.1 Kb cDNA clone which directs the synthesis of a fusion protein which bears the PMI-1 epitope was isolated from a lambda gt 11 expression library constructed from mRNA from the human erythroleukemia (HEL) cell line. The position of the N-terminal sequence of the light chain of GPIIb in the deduced amino acid sequence of the clone defined the orientation of the light and heavy chains of GPIIb. Analysis of the amino acid sequence corresponding to the heavy chain of GPIIb identified a single region with a high likelihood of containing a continuous epitope. A synthetic 17 residue peptide, corresponding to the predicted antigenic site, inhibited the binding of PMI-1 to platelets. Two uM peptide was required to inhibit binding 50% in the presence of 1 uM PMI-1, indicating an approximate dissociation constant of 1.5 uM for the peptide-antibody complex. This figure should be compared to a Kd of 0.95 uM (JCI 78:1103, 1986) for PMI-1 binding to GPIIb. A second peptide, corresponding to the region immediately adjacent to the predicted antigenic site, failed to inhibit PMI-1 binding. Neither peptide inhibited the binding of two other monoclonal anti GPIIb-IIIa’s to platelets. The peptides had similar effects on PMI-1 interaction with purified GPIIb-IIIa in detergent solution. These data localize the PMI-1 epitope to a 17 amino acid region located near the carboxyl terminal of the heavy chain of GPIIb. Thus, they chemically define a region of GPIIb whose surface expression reflects the competence of GPIIb-IIIa as a component of a platelet receptor for adhesive proteins.
MOLECULAR CLONING OF PLATELET GPIIb FROM HEL CELLS AND HUMAN MEGAKARYOCYTES
Authors: Uzan G , Lajmanovich A , Prandini M H , Frachet Ph , Duperray A , Marguerie G
Published: August 2018

Platelet GP IIb-IIIa is an heterodimer which functions as a receptor for fibrinogen, fibronectin and Von Willebrand factor and is implicated in platelet adhesive reactions. To study the structure function relationship of this glycoprotein, a recombinant DNA approach was initiated. cDNA expression libraries were constructed in » gtll vector, from erythro-leukemia cells (HEL) and megakaryocytes mRNA. The human megakaryocytes were isolated from patients with chronic myeloid leukemia. The HEL library was initially screened with polyclonal antibodies anti GPIIb IIIa. One clone, λIIbI, containing a 1.65 kbp insert reacted with a panel of different polyclonal antibodies anti GPIIb IIIa and a monoclonal antibody anti GPIIb. To further characterize this clone the synthesis of the fusion protein was induced by IPTG. The bacterial protein was then blotted onto nitro cellulose and incubated with antisera anti GPIIb-IIIa. Antibodies that specifically bound with the fusion protein were eluted and tested on platelet membrane extracts. The selected antibodies produced a positive signal at the GPIIb position similar to the signal produced by the monoclonal antibody anti GPIIb on the same membrane extract. Finally on western blotting, a protein of Mr= 170kD reacted with the monoclonal antibody anti GPIIb. λIIbI insert was used to screen the megakaryocyte library and 3 clones, λIIb2,λIIb3 and λIIb4 were isolated. The size of HEL cells and megakaryocytes GPIIb mRNA was estimated by northern blotting. Only one species of 3.9 kb was identified in both cells. The four different clones accounted for 50% of the coding sequence of this mRNA.Sequencing of these cDNAs indicated that the plasmatic domain of GPIIb contains a cystein rich region. The sequence of these clones will allow the study of the adhesines genetic diversity in different cellular systems.
AN IMMUNORADIOMETRIC ASSAY (IRMA) FOR HUMANTHROMBOMODULIN
Authors: Giddings J C
Published: August 2018

Thrombomodulin was separated from detergent-soluble fractions of human placenta using a combination of DEAE-Sepharose and thrombin-Sepharose chromatography. The final product demonstrated one major band (Mr approximately 100000) and three minor bands (Mr 40000 - 80000) on SDS-polyacrylamide gel electrophoresis. The purified protein markedly enhanced the rate of activation of human protein C by thrombin in the presence of calcium ions. Polyclonal antibodies to the isolated thrombomodulin were raised in rabbits and were shown to inhibit thrombin co-factor activity. Immunofluorescence of fibroblasts and of human umbilical vein endothelial cells in culture demonstrated that thrombomodulin antigen was specifically located in endothelial cell membranes. Affinity purified antibody was labelled with 125I and was used to establish an immunoradiometric assay (IRMA). The method was sensitive to approximately 10.0μg purified thrombomodulin per litre. The concentration of thrombomodulin in detergent-solubilised endothelial cells obtained at intervals during primary culture was proportional to the number of cells harvested for assay and appeared to reflect cell growth. Confluent cells from four experiments (approximately 2 × 106 cells per culture dish) contained on average 8.4ng thrombomodulin. Incubation of confluent endothelial cells with medium containing 1 unit per ml human α-thrombin for 10 mins at 37°C reduced the amount of cellular thrombomodulin detected in this assay by up to 30%. Assays of human plasma confirmed that low levels of thrombomodulin are present in normal circulating blood. A mean level of 160μg per litre was detected in 20 normal donors. The levels of thrombomodulin antigen in 10 normal serum were not significantly different from those in the corresponding normal plasma. Preliminary results illustrated that increased levels of thrombomodulin might be found in the plasma of some patients with a variety of clinical disorders. The data suggest that quantitative assays of thrombomodulin might provide a useful index of endothelial disturbances in vivo.
IMMUNOCHEMICAL ANALYSIS OF FACTOR VIII (FVIII) IN PLASMA AND HEAT-TREATED CONCENTRATES
Authors: Edwards J S , Kembal-Cook G , Barrowcliffe W T
Published: August 2018

Using the method of Weinstein et al. (Proc.Natl.Acad.Sci. USA# 78, 5137-41, 1981) the FVIII polypeptide distribution in wet and dry heated concentrates, a monoclonal-purified concentrate and fresh plasma was examined. Samples were incubated for 2 hours at 37°C (in the presence of polyethyleneglycol 4000 to aid complex formation) with 125I-Fab' fragments prepared from a polyclonal human anti-FVIII :C antibody. The complexes were elec-trophoresed in a 3-9% polyacrylamide gradient gel, in the presence of SDS, under non-reducing conditions and visualised by autoradiography.

Fresh plasma slowed a range of peptide bands of apparent M.Wt. 80-280 kD, wicn a major band at 280 kD. FVIII concentrates showed a similar range of bands and, for one manufacturer's product (product E), an additional strong band of 40-50 kD. The proportion of total EVIII antigen in the 280 kD band was estimated by densitometry to be 20-40% in concentrates, can-pared with 65% in fresh plasma. Severe haemophilic plasma had no bands, confirming the specificity of the technique. FVIII antigen in 'wet' heated concentrates was shown to be more degraded (increase in low molecular weight forms) than in dry heated concentrates.

Fresh plasma incubated at 37°C for 24 hours shewed increased amounts of FVIII antigen in a low molecular weight form (90 kD).

Treatment of concentrates and plasma with thrombin resulted in a change of the peptide band pattern, which was dependent upon thrombin concentration and incubation time. Loss of the 280 kD band and intensification of a 90 kD band was observed, which correlated with an increase in FVIII :C by one-stage assay. Further proteolysis resulted in a band of inactive material of 40-50 kD, with identical mobility to the band seen in product E. FVIII :C activity in product E was higher by one-stage than by two-stage assay, and these results suggest more extensive thrombin degradation in this product.

The results show that the molecular form of FVIII in concentrates is dependent upon storage of plasma, the methodof concentrate preparation and the type of heat treatment.
BLOOD BANK PRODUCTION OF HIGH YIELD, HIGH PURITY, HEAT TREATED F VIII CONCENTRATE FROM HEPARINIZED BLOOD
Authors: Wallevik K , Inger-slev J , Stenbjerg Bernvil S
Published: August 2018

Applying trivial blood bank equipment we have developed a method to produce a heat-treated factor VIII concentrate from heparinized blood with an over-all yield between 40 and 50%, a specific activity around 2 i.u./mg and a low fibrinogen content (3-4 g/1). Electrophoretic multimer analysis of the von Wille-brand factor reveals a band pattern close to that of native plasma. The in vivo recovery is 99±25% and t/2 11.4±2.4 h (n=5). The method includes 3 precipitations in commercial "blood-bag" systems: (1) The classical cryoprecipitation, (2) Precipitation at 0°C of "cold insoluble globulins" including F VUI/vWf, (3) Precipitation of non F VIII/vWf related proteins at 10°C, pH 7.0 and specified ionic conditions.

The F VIII rich supernatant is stabilized by amino acids and citrate, freeze dried, and heated to 68°C for 24 h.

In our blood bank with ∼40,000 donations per year we have in a year produced ∼1,4 mill i.u. of the High Purity F VIII concentrate. This means that around 16,000 donations per year are collected in heparin, and that F VIII depleted plasma which we deliver for clinical use, either as liquid plasma or as a freeze-dridd product, contains solely heparin (∼4 i.u./ml) as anticoagulant.

The method is technically simple which together with the high yield and high purity of the F VIII concentrate makes the production economically profitable. The method is applicable in areas with limited technical resources.

We find it also important that F VIII concentrate, manufactured locally from healthy voluntary donors with personal attachment to their blood bank reveals the lowest risk for transmission of diseases to the patients.
CHARACTERIZATION OF SERUM PROSTACYCLIN BINDING DEFECTS IN THROMBOTIC THROMBOCYTOPENIC PURPURA (TTP)
Authors: Wu K , Manner C , Tsai A
Published: August 2018

To understand further the pathophysiologic significance of PGI2 binding defects in TTP, we measured serum PGI2 binding activity in 12 TTP patients and matched controls. Serum binding of PGI2 was measured by Sephadex G-25 gel filtration. The mean binding activity in 33 healthy subjects ages 20-40 years was 39.9 ± S.D 4.4%. The mean value of TTP (n=12) was significantly lower (26.2 ± 4.1, P <0.01). Serum from 5 severe ITP, 5 DIC and 5 thrombocythemia exhibited normal binding ctivity. To determine the binding kinetics we utilized 3H-iloprost in a gel filtration method described by Hirose and Kano (Biochim. Biophys. Acta. 751:376, 1971). To 50 mg of Sephadex G-50, 0.435 ml of 50mM Tris buffer (pH 7.4) was added. After swelling of the gel was completed 0.195 ml of the buffer solution containing serum and 3H-iloprost was added. The sample was mixed and the protein and ligand concentration was determined. The computer fitting of the binding isotherm according to the originally proposed equation yielded a binding curve consistent with a single class of binding sites. The Kd value of normal serum was 70 μM and the B 48 nmol/ml. Acute TTP serum exhibited a reduced bindingXaffinity (Kd 236 μM) and a slightly elevated capacity (Bmax 85 nmols/ml). The binding parameters improved following successful treatment but the Kd remained subnormal (120 μM). These data indicate that reduced PGI2 binding activity is due to lower affinity of the PGI2 binding proteins. The relationship between defective PGI2 binding activity and PGI2 production was then evaluated. Serial serum and 24 hour urine were collected. Urinary samples were extracted and their 6-Keto-PGF1α (6KP) and thromboxane TXB2 levels were measured by RIA. TTP patients in remission had normal levels of urinary 6KP and TXB2 while urinary 6KP and TXB2 were elevated in relapsing TTP. Defective binding was noted when relapse began to occur while elevated 6KP and TXB2 were noted 48 hrs later. Both 6KP and TXB2 were normalized when the disease was controlled. Our findings indicate that defective PGI2 binding plays an important role in causing excessive platelet activation and platelet-vessel wall interaction in TTP.
RHEOLOGY AND ISCHAEMIA
Authors: Dormandy J
Published: August 2018

While the previous presentations have dealt with the experimental evidence linking flow patterns and shear stress to thrombosis and atherogenesis, this presentation will concentrate on the clinical evidence linking Theological abnormalities to macro and micro-circulatory ischaemia. Whole blood viscosity undoubtedly influences blood flow along larger vessels as suggested by Poiseuille. The two important determinants of whole blood viscosity are the red cell concentration and plasma fibrinogen.There is overwhelming epidemiological evidence that the red cell concentration is a primary risk factor for the development of essential hypertension, myocardial, cerebral and leg ischaemia. It is also a secondary risk factor in patients who already had a clinical episode of ischaemia in any of these territories. There is similar evidence in relation to a high plasma fibrinogen.Furthermore therapeutic haemodilution or defibrinogenation are increasingly used in the prevention and treatment of ischaemia.

Haemorheological factors in the microcirculation are probably equally important but more difficult to assess clinically. The concept of a 'vicious viscous spiral' in ischaemic tissue is gaining increasing support. Whatever the initial cause of the ischaemia, important secondary haemorheological changes occur which can perpetuate or aggravate the ischaemia. Most of the changes known to occur locally in ischaemic tissues such as hypoxia, acidosis, release of serotonin and platelet activation have been shown to impair the deformability of blood cells. This will be particularly important if the perfusion pressure is also decreased and may result in capillary plugging and uneven distribution of flow in the microcirculation. Abnormal blood cell filtrability, just as whole blood viscosity, has been shown to be associated with acute as well as chronic ischaemia in most territories. Furthermore there is a correlation between the magnitude of the haemorheological changes measured and the subsequent clinical course of the patient following an ischaemic injury.

The newest aspect of haemorheology to attract clinical attention is the role of the white cell in ischaemia. Epidemiological as well as recent experimental and clinical studies suggest that the Theologically activated white cell may be the most dangerous component of blood in terms of perpetuating and extending tissue ischaemia.

The assessment of the Theological properties of blood should form an integral part of studies looking at the causes and possible therapy of all forms of acute and chronic ischaemia.
CARRIER DETECTION IN JAPANESE FAMILIES WITH HAEMOPHILIA A USING FACTOR VIII GENE PROBE(F8A) AND ST 14-1 PROBE
Authors: Nishino M , Nishimura T , Naka H , Mikami S , Yoshioka A , Fukui H
Published: August 2018

Recently, the gene structure for human F.VIII protein was clarified, and F.VIII DNA probes have been used for carrier detection and prenatal diagnosis ofhaemophilia A. In order to make sure that the phenomena are universal, we have analysed the RFLPs of F.VIII gene in 16 Japanese families with haemophilia A, including a female haemophiliac case, using an intragenic F.VIII DNA probe(F8A) and an extragenic(linked) DNA probe(Stl4-1).

The probe F8A revealed two variant bands after digestion by Bel I. Of normal 60 X chromosomes (females) examined, about 85% bore the 879-bp fragment and 15%the 1165-bp fragment. Five of sixteen mothers of hemophiliacs, definite carriers, were found to be heterozygous for Bel I polymorphism. Since the relationship between Bel I alleles and hemophilia gene has been identified in the 5 families in which the mothers were heterozygous, we could diagnose the carrier status of two women whose brothers are hemophiliacs. Onthe other hand, we could identify that one "haemophilic woman" with less than 10% of F.VIII:C was a carrier status when we analysed the Bel I alleles in theother members of the family.

The probe DNA(ST 14-1) revealed seven variant bands ranging from 5.5 kb to 3.4 kb after digestion by Taq I. In 6 out of 16 families, the RFLPs of ST 14 locus were informative for carrier detection.

From these data, it was concluded that the Bel I polymorphism of F.VIII gene and the Taq I polymorphism of ST 14 locus were informative for carrier detection in 8 out of 16 families with haemophilia A
THE RELATIVE EFFICACY OF GENETIC ANALYSIS AND COAGULATION TESTING IN THE DIAGNOSIS OF CARRIERS OF HEMOPHILIA A
Authors: Lillicrap D , Giles A R , Holden J J A , White B N
Published: August 2018

This study has assessed the relative benefits of restriction fragment length polymorphism (RFLP) linkage and coagulation testing in the diagnosis of carriers of hemophilia A. 221 samples from 55 families have been studied for intragenic and flanking RFLPs. All samples were tested for the Factor VIII intragenic Bell RFLP and for the flanking marker St 14. 83% of obligate carrier females were heterozygous at oneor both of these two polymorphicsites. However, only38% of these women were heterozygous at the intragenic site and might safely be offered prenatal diagnosis using this marker for the hemophilia mutation. Carrier diagnosis was obtained in 52% of 81 potential carriers tested. Diagnosis wasbased on intragenic RFLP information in only 48% of these cases. Genetic diagnosis was possible in 27 atrisk women from families with no prior history of hemophilia. Four of these women were diagnosed as carriers on the basis of a gross Factor VIII gene deletion and the remaining 23 women were identified as non-carriers by the Bell (11) and Stl4 (12) RFLP data. 39 women remained undiagnosed after gene analysis studies. 23 of these women were female relatives of sporadic hemophiliacs and thus RFLP segregation analysis was inappropriate. A further 9 potential carriers were undiagnosed because of homozygosity in key individuals in their families. In 31 potential carriers we have quantitated Factor VIII:C (one stage assay) and vWf:Ag (Laurell and ELISA) and derived probabilities for carrier status. In 3 women there was conflicting genetic and coagulation data. Meanwhile, in 12 undiagnosed women from sporadic families, carrier diagnostic probabilities of > 0.9 were obtained. These studies indicate that optimal carrier detection for hemophilia A requires more intragenic and closely linked RFLPs and the continuance of coagulation testing to assist women from sporadic families.
DETECTION OF MUTATIONS IN HEMOPHILIA A
Authors: Higuchi M , Kochhan L , Schwaab R , Brackmann H H , Egli H , Olek K
Published: August 2018

Hemophilia A (HA) is a x-linked bleeding disordercaused by lack or abnormality of factor VIII:C. Because of the heterogeneity of the clinical picture thedisease might result from many different molecular lesions.

We examined 202 patients of HA from 160 families by restriction analysis with Taq I, Msp I and EcoR I using three subcloned cDNA fragments of factor VIII:C.We detected 13 mutations within the factor VI11:C gene. All of these patients suffer from the severe form of HA.

Table I shows six deletions which we characterized. We also identified seven point mutations on four different exons using restriction enzyme Taq I. The results are shown in Table II.

That means in approximately 3.8% of the patients we found deletions and in 4.4% we found point mutations.

This confirms the results of Youssofian et al (1986):These authors consider the CpG-dimer as a mutation hotspot in the factor VI11:C gene.
THE ORIGIN OF THE MUTATION IN FAMILIES WITH AN ISOLATED CASE OF HAEMOPHILIA A
Authors: Bröcker-Vriends A H J T , Briët E , Dreesen J C F M , Bakker E , van de Kamp J J P , Pearson P L
Published: August 2018

Approximately one third of the patients with haemophilia appears to have no affected relatives. The proportion of cases due to a new mutational event as well as the gamete origin of the mutation has been much debated. The objective of this study was to define the origin of the mutation in families with an isolated case by DNA analysis. We investigated 22 families with an isolated case of haemophilia A. Intragenic (Bell, Xbal) and extragenic (BglII/DX13, Taql/Stl4) RFLPs were investigated for. If necessary, paternity was tested by DNA fingerprint patterns obtained with the 33.15 mini satellite probe.

In seven of the 22 families it could be demonstratedthat the abnormal X-chromosome of the haemophiliac was derived from the normal maternal grandfather. In six of these 7 families the mother of the patient had a high probability of carriershio on the basis of clotting factor VIII assays, so that, probably, the mutation had occurred in the paternal gamete. As a consequence, carriership could be excluded for aunts, nieces and more distant female relatives of the patient.

In three families the abnormal X-chromosome was derived from the maternal grandmother, while, sofar, in the remaining 12 families no conclusions as to the origin of the mutation could be drawn.

In contrast with earlier findings, these results illustrate that at least thirty percent of the isolatedcases of haemophilia A are due to a recent de novo mutation.
ABSENCE OF A BLEEDING TENDENCY IN SEVERE ACQUIRED DEFICIENCY OF PLASMA VON WILLEBRAND FACTOR (vWf)/F. VIII WITH NORMAL PLATELET vWf/F. VIII INDICES
Authors: Drouin J , Lillicrap D , Giles A R , Izaguirre C A , Windsor S , Hoogendorn H
Published: August 2018

A 67 year old male with IgA myeloma has been investigated for a severe deficiency of plasma vWf/F. VIII but normal platelet vWf/F. VIII. He has no personal history nor family history of bleeding problems. He was initially investigated for a prolonged APTT of 43 secs. (25-40) obtained in a preoperative clotting screen. During this investigation he was found to have IgA myeloma. In retrospect, an APTT prior to uneventful coronary artery bypass surgery two years previously had been prolonged. Routine investigation has shown that platelet count and bleeding time have been repeatedly normal. Plasma F. VIII:C is 0.08 u/ml., F. VIII:Cag 0.07 u/ml., vWf:Ag 0.05 u/ml. and ristocetin cofactor 0.05 u/ml. In contrast, platelet values for vWf:Ag of 53 units/109 platelets and F. VIII:Cag of 176 units/109 platelets are within the normal ranges for our laboratory. The platelet lysate vWf multimer pattern is also normal. Patient's plasma shows inhibitory activity against vWf:Ag but not against either F. VIII:C or ristocetin cofactor activity. When patient plasma is incubated for 60 mins at 37°C with vWf and analysed by crossed Immunoelectrophoresis (CIE) for vWf:Ag, a double arc precipitin line is observed with marked retardation of the first arc. A similar vWf:Ag CIE double precipitin arc is seen following the infusion of cryoprecipitate. T 1/2 for F. VIII:C and vWf:Ag are both reduced following the infusion of cryoprecipitate - F. VIII:C 2 hrs, vWf:Ag 3 trs. No secondary rise in F. VIIIrC is seen at 24 hrs. Despite severe deficiency of plasma vWf/F. VIII, this man does not have a clinical bleeding tendency. We postulate that his plasma vWf/F. VIII deficiency is the result of complexing of his IgA myeloma protein with vWf, resulting in premature clearance of the vWf/F. VIII complex. This case further emphasizes the role of platelet associated coagulation factors in maintaining normal haemostasis.
EXTRINSIC PATHWAY INHIBITOR (EPI) DURING ELECTUVE SUGERY : A COMPARISON WITH OTHER COAGULATION INHIBITORS
Authors: Sandset P M , Anderson T R , Ødegaard O R , Larsen M L
Published: August 2018

Hypercoagulation after surgical trauma is probably induced by tissue thromboplastin (TP) released into the circulation. EPI, in conjuction with activated factor x (FXa), is a potent inhibitor of the IP-factor VII complex. EPI levels (chromogenic substance assay) were compared to other inhibitors; antithrombin (AT), heparin cofactor II (HCII), andprotein C (PC) in patients undergoing cholecystectomy(n=4), hip prosthesis operation (n=5),and aortic grafts operationa(n=5) Mean AT and PC levels parallelled the decrease in albumin. HCII decreased more suggesting a real consumption ofsurgery. The changes in EPI levels depended on the type ofsurgery. In hip surgery, the decline in EI levels was marked and parallelled HCII. In constrast to the other inhibitors, EPI levels stayed low over the first week after surgery:

In cholecystectomy, changes were less marked and all inhibitors behaved similar. During aortic operations, EPI increased from mean 110% preoperatively to 252% peroperatively. It decreased to 86% the first day after operation. It then increased similar to the other inhibitors, but the level stayed higher than expected from the preoperative value. The patients received 3000 IU heparin peroperatively.

In conclusion, hip operations produce a sustained drop in EPI activity.In aortic operations injection of heparin induced a dramatic, shortlived increase peroperatively followed by a restoration to high normal levels.
LCNG-TEFM SUBSTITUTION WITH Cl-INACTIVATOR IN PATIENTS WITH HEREDITARY AND ACQUIRED Cl-INH DEFICIENCY AND LIFE-THREATENING ANGIOEDEMA
Authors: Bork K , Witzke G
Published: August 2018

Patients with hereditary car acquired cl-inactivator (Cl-INH) deficiency suffer frcm repeated episodes of oedema of the skin and internal organs, which often constitute a threat to life (laryngeal, pulmonary and brain oedama). The treatment which is rapidly effective in the other, much more common, forms of angio-oedema (idiopathic, allergic or anaphylactoid, often associated with urticaria) has little or no effect in these cases. This applies particularly to treatment with antihistamin agents and corticosteroids. However, in patients with hereditary angiooedana (HAE) Danazol is effective for long-term prophylaxis and the substitution of Cl-INH for acute treatment. In isolated cases long-term prophylaxis, which is generally reliable, cannot be used because of intolerability reactions. Moreover, it is ineffective in the oedema of acquired Cl-INH deficiency.

As other forms of therapy did not achieve the desired response, long-term substitution with a concentrate of Cl-inactivator (Behringwerke AG, Marburg) was carried out in 2 patients (H.W., 47 yrs, male, hereditary angiooedana, 9 siblings died from angiooedana; W.K., 55 yrs, male, professional truipeter, suffering from angiooedana of unknown origin for 5 years, without other underlying disorders). The initial values were

Substitution was carried out according to the clinical symptoms. Patient H.W. required 500 U Cl-inactivator every 4th day and patient W.K. 1000-1500 U every 5th day until. During substitution therapy (which in pat. H.W. has so far been carried out for 8 months and in pat. W.K. for 6 months) there was a rise in Cl-INH and C4 with an almost total absence of clinical syirptans. No undesirable effects were observed.
KALLIKREIN-KININ SYSTEM IN PATIENTS WITH SHOCK. COMPARISON BETWEEN SEPTIC AND CARDIOGENIC SHOCK
Authors: Martínez-Brotóns F , Oncins J R , Mestres J , Amargós V , Reynaldo C
Published: August 2018

Alterations of the kallikrein-kinin system (KKS) consistent with activation and increased consumption have been reported in septic patients and it has been suggested that this activation could contribute to the development of septic shock. As similar alterations have been found in other critically ill patients, many of them prone to shock, we wonder if activation of the KKS could be a consequence rather than a cause of the hemodynamic instability. To answer this question we compared 12 patients with septic shock (8 fatal) with 10 cases of cardiogenic shock secondary to myocardial infarction (8 fatal) as a model of non septic shock. In adition 25 episodes of uncomplicated sepsis and 10 intra-intensive care unit controls were studied. A functional measure of factor XII, high molecular weight kininogen (HMWK) (coagulative methods), prekallikrein (PK), Cl-inhibitor (Cl-INH), α2-macroglobulin (α2-M)- Antithrombin III (AT-III) and kallikrein inhibitor activity (KIA) (chromogenic methods) was performed

RESULTS: Patients with septic shock, specially in fatal cases, showed a highly significant decrease in activities of factor XII (P<0.001), PK (P<0.0001), HMWK (P<0.005), α2-M (P<0.001), AT-III (P<0.0001) and KIA (P<0.005). Cl-INH activity was increased in uncomplicated sepsis (P<0.001) but came back to normal or was slightly decreased in septic shock. Components and inhibitors of the KKS were within normal limits in all patients with cardiogenic shock.

Our findings support the idea of a contribution of the KKS to the development of septic shock but this system neither seems to play a role in the pathogenesis of cardiogenic shock nor to be altered as a consequence of it.
MODERATE DECREASE OF FACTOR VII AND PROTEIN C WITHOUT OCCURRENCE OF HEPATIC VENO-OCCLUSIVE DISEASE AFTER ALLOGENEIC BONE MARROW TRANSPLANTATION IN 18 PATIENTS TREATED WITH LOW DOSE HEPARIN
Authors: CARON C , JOUET J P , HIMPENS J , HIVES P , GRUSON H , GOUDEMAND J
Published: August 2018

Decrease of factor VII (F VII) and protein C (PC) has been said to allow an early detection of hepatic veno-occlusive disease (VOD) (VILMER, Path. Biol. 1986, 34 : 79), that represents a serious complication of bone marrow transplantation (BMT). In this purpose, F VII (activity) and PC (antigen) have been measured in 18 patients (aged 9 to 45 yr- m : 26 yr) who underwent allogeneic bone marrow graft for chronic myelogenous leukemia (9 cases), acute lymphocytic leukemia (7 cases) acute myelogenous leukemia (2 cases). All patients received as preparation for BMT total body irradiation (mean dose = 10 Gy) along with cyclophosphamide (120 mg/Kg). All were given low dose heparin (100 UI/Kg/24 hr) from days -7 to +30. None of the patients developed VOD but graft-versus-host disease occurred in 13 out of them between days 18 and 52. Moreover, coagulation studies performed from days 1 to 28 detected a moderate decrease of F VII and PC (maximum on day 11). These parameters were normalized on day 28. The level of the other vitamin K-dependent factors was not significantly changed.

So the moderate decrease of F VII and PC found in the post-graft period was not associated with hepatic VOD. However, as none of the patients developed this complication, these results do not exclude that a major decrease of these parameters could serve as an early diagnosis of VOD. On the other hand, a prophylactic effect of low dose heparin cannot be ruled out.
A STUDY OF THE EARLY HAEMOSTATIC CHANGES FOLLOWING RUSSELL'S VIPER BITE IN HUMANS
Authors: Than Than , Ei Han Khin , RA Hutton , Lwin Mvint , Nu Swe Tin , DA Warrell
Published: August 2018

Amongst its many actions, Russell's viper (RV) venom activates factors X and V and enhances fibrinolysis, leading to defibrination which contributes to the clinical sequelae of RV bite. Early administration of antivenom may be life-saving, but not all of those bitten become sufficiently envenomed to require treatment. In an attempt to predict at an early stage those subjects who will progress to defibrination, we have serially monitored the haemostatic changes in 20 bite victims using the PT, APTT, thrombin time, platelet count, assays for factors X and V and fibrinogen and fibrin(ogen) degradation products (FDP).

In five patients, no evidence of defibrination was seen at any time and none of these developed obvious clinical symptoms. In a further six subjects, slight prolongation of the PT (16-21/14s), APTT (39-51/38s) and thrombin time (16-25/14s) occurred concomitantly with a moderate fall in factor X (20-80%), factor V (30-66%) and fibrinogen (0.6-2.Og/1), but FDP never exceeded 40ug/ml. In the remaining nine subjects who all eventually defibrinated completely, moderate coagulation factor deficiency and thrombocytopenia developed as early as 1-2h after bite. The most pronounced and consistent changes were a rise in FDP to above 80ug/ml (80-640ug/ml) and a fall in factor V (2-50%), these results being obtained on admission, 1-12h after bite. We conclude that an FDP level of 80ug/ml or more is highly suggestive of impending defibrination and could be regarded as a criterion for commencing antivenom therapy.
A HEP ARINE-LIKE ANTICOAGULANT IN A PATIENT WITH WEGENER’S GRANULOMATOSIS
Authors: Chrobák L , Rozsíval V , Herout V
Published: August 2018

In a 23-year-old man with Wegener’s granulomatosis and mild bleeding coagulation studies revealed a significant prolongation of the coagulation time (CT) prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time (TT), failure of TT and aPTT to correct in a 1:1 mixture with pooled normal plasma (PNP), correction of the prolonged TT with toluidine blue and correction of TT and aPTT both in vitro and in vivo^protamine sulphate (P.S.).

All other coagulation tests, i.e.,bleeding time, platelet count? fibrinogen level, euglobulme lysis time were within normal limits. The patient did not receive heparine. TT after administration of 5ml of protamine sulphate i.v. to the patient became normal - 18,2 s (19,7 s).

In summary a patient with Wegener s granulomatosis associated with an endogenous heparine-like anticoagulant is reported. The anticoagulant could be corrected both in vitro and in vivo by protamine sulphate.
ALTERED COAGULATION IN CEREBRAL ISCHEMIA PATIENTS
Authors: Fisher M , Francis R
Published: August 2018

We investigated coagulation changes in a group of patients with cerebral ischemia, ranging from transient ischemic attacks to cerebral infarction. Patients were studied acutely (within 72 hours of onset of ischemia) and again approximately 2 months following the initial examination. We evaluated platelet activation, fibrin generation, and fibrinolysis by measuring plasma beta-thromboglobulin (BTG), fibrinopeptide A (FPA), and fibrinopeptide B-beta 1-42 (FPB), respectively. We compared measurements in cerebral ischemia patients with a group of age -and sex-matched neurological inpatients without vascular disease ("patient controls") and a similarly matched group of normal volunteers ("normal controls"). BTG levels for 90 patients studied acutely were not significantly different compared to 58 of the same patients studied 2 months later, 16.4 ± 11.3 ng/ml (mean ± SD) versus 17.5 ± 10.2 ng/ml. Both values were significantly increased (p< .05) compared to normal controls (12.2 ± 6.5 ng/ml, n = 44); patient controls (13.2 ± 7.6 ng/ml, n = 18) were not significantly different from normals. In contrast, FPA measurements were significantly increased in acute patients compared to normals (3.3 ± 5.8 versus 1.0 ± 1.7 ng/ml, p< .05) while FPA levels 2 months post-ischemia (0.6 ± 0.9 ng/ml) were no different than normals. FPB measurements were not significantly different among either acute patients (6.5 ± 2.4 pmol/ml) or patients 2 months post-ischemia (4.8 ± 1.5 pmol/ml) compared to normals (6.5 ± 1.8 pmol/ml).

In summary, we have found, among cerebral ischemia patients, sustained increases in BTG, acute increases in FPA, and normal FPB. These findings are compatible with a model of cerebral ischemia consisting of acutely increased fibrin generation without concomitant increased fibrinolytic activity, superimposed on a background of increased platelet activation.
STRUCTURE ACTIVITY RELATIONSHIPS OF DERMATAN SULFATE : EFFECT OF MOLECULAR SIZE AND CARBOXYL GROUP ESTERIFICATION ON HEPARIN C0FACT0R-II ACTIVATION
Authors: Mardiguian J , Corgier M , Jouany M
Published: August 2018

Dermatan is a high molecular weight glycosaminoglycan which has been shown to enhance the inhibition of thrombin by heparin-cofactor II. The aim of this study was to establish the influence of the molecular size and the role of the carboxyl group on the in vitro activity of Dermatan Sulfate. Pig skin Dermatan Sulfate was fractionated according to molecular size by gel-chromatography on Ultrogel Ac 44. Each fraction was characterized by its sulfur content and by its mean molecular weight measured on a TSK - 4000 column in reference to standard heparin fractions. Methyl esters of the unfractionated Dermatan Sulfate with varying degree of esterification, where prepared via activation of the carboxyl groups with a carbodiimide and reaction with methanol. The results of this study show that the heparin - cofactor II mediated anti-thrombin activity of Dermatan Sulfate is increasing with the molecular weight and is abolished by esterification of the carboxyl groups. Moreover, it can be speculated that each fraction contains the same amount of high affinity fraction and that, like heparin, the potency of the high affinity component is increasing with the molecular weight.
ANTITHROMBIN III GLASGOW: A VARIANT WITH INCREASED HEPARIN AFFINITY AND REDUCED ABILITY TO INACTIVATE THROMBIN, ASSOCIATED WITH FAMILIAL THROMBOSIS
Authors: Lane D A , Lowe G DO , Flynn A , Ireland H , Thompson E , Erdjument H
Published: August 2018

A functional anti thrombin III CAT III) deficiency has been identified in three generations of a family with a high incidence of deep venous thrombosis. The deficiency presented as ~50% reduction in its heparin cofactor activity compared to its antigen concentration. No abnormality was detected by crossed immunoelectrophoresis of plasma in the presence or absence of heparin. Plasma from the propositus was precipitated with dextran sulphate, applied to heparin-Sepharose and the AT III stepwise eluted with NaCl . The AT III had a reduced ability to inactivate thrombin, when this was monitored by substrate hydrolysis or by SDS PA gel e1ectrophoresis. Its mobility was normal when examined by the latter technique on 10-20X gels under reducing and non-reducing conditions. Patient AT III was reapplied to heparin-Sepharose and eluted with a NaCl gradient. A minor active pool eluted in the same concentration range, 0.5-1.2M, used to purify normal AT III, while predominantly inactive AT III eluted at higher NaCl concentrations, 1.2-2.0M. Further purification of variant AT III was achieved by passage of heparin-Sepharose eluates through a thrombin-Sepharose column and structural studies are being undertaken on this material. It is concluded that AT III Glasgow has increased affinity for heparin but reduced ability to inactivate thrombin. It can be differentiated from two other AT III variants that bind heparin with high affinity, Milano and Northwick Park, but has similar features to those of AT III Chicago.
A NEW ASYMPTOMATIC TYPE III VARIANT OF ANTITHROMBIN III WITH DECREASE HEPARIN COFACTOR ACTIVITY : AT III AMIENS
Authors: ROUSSEL B , DIEVAL J , GROSS S , CLAISSE J F , DELOBEL J
Published: August 2018

A qualitative abnormality of AT III suggested by the discrepancy between a normal level of AT III antigen (0,33 g/1) and a decreased heparin cofactor activity (60 % of normal) was discovered in a 37 years old woman during a routine laboratory examination for oral contraceptive. The propositus was asymptomatic as she did not developpe any thrombo-embolic disease during three previous pregnancies. There was no familial history of thrombo-embolism. The AT III level measured by radial immuno-diffusion was within the normal range. The progressive anti factor lia and anti factor Xa activities (chromogenic substrates CBS 3 447 and CBS 3 139) were normal (92 % and 100 %). Plasma and serum crossed immunoelectrophoresis (CIE) showed a normal pattern. In the presence of heparin, anti factor Xa and anti factor Xa activities were decreased (60 % and 45 %); Plasma and serum crossed immunoelectrophoresis showed an abnormal slow moving peak exhibiting the inhability of the molecule to bind completely to heparin. CIE with various other glycosaminoglycans are on experiments.

Familial study revealed that the daughter of the propositus was carrying the same molecular abnormality.

We conclude that AT III Amiens is an hereditary type III variant.
STUDIES ON THE DIRECT PROTEOLYTIC ACTION OF HUMAN TISSUE PLASMINOGEN ACTIVATOR ON HUMAN FIBRONECTIN AND VITRONECTIN
Authors: Wasi S , Juodvalkis S , Alles P , Aubin J E
Published: August 2018

The ability of cells to make or break specific attachments to extracellular matrix (ECM) and other cells is important in cell migration, proliferation and wound repair. Specific attachment proteins believed to be involved in mediating these interactions comprise functional domains joined by protease sensitive segments. Proteases can conceivably modulate cellular interactions by releasing functional domains from parent molecules. Tissue plasminogen activator (t-pA) is known to participate in various pathophysiological processes. That t-pA may also act directly on structural proteins has not been investigated. We have studied the direct proteolytic action of melanoma t-pA on fibronectin (FN), vitronectin (VN) and laminin (LN). These were incubated with t-pA for 0 to 48 h in 50 mM Tris HCi, pH 7.4. The cleavage products were separated on polyacrylamide slab gels and blotted onto nitrocellulose strips. FN and VN fragments with cell attachment properties were identified by incubating the strips with human gingiva fibroblasts and staining with Amido black. Monoclonal antibodies to FN were used to identify heparin, cell and gelatin binding fragments. VN was converted to a major 55 Kd product as a function of time. Lower molecular weight species migrating at 45 Kd, 30 Kd and 15 Kd positions were also identified. Most of these fragments possessed cell attachment properties. LN became susceptible to t-pA digestion after dénaturation with H2O2. The catalytic activity of t-pA could be inhibited in the presence of nitrophenyl-p-guinidino benzoate (a synthetic inhibitor of plasminogen activator), whereas O-phenanthroline (a metalloprotease inhibitor), α 2-antiplasmin and trasylol had no effect. A monoclonal IgG preparation (HI 72 A1, kindly provided by Dr. David J. Loskutoff) that specifically inhibits t-pA also inhibited the protelyotic action of t-pA on FN. These data suggest that direct proteolytic action of t-pA on adhesive proteins may modulate cellular behaviour in various normal and pathological conditions which involve dynamic interactions between cells and ECM and where plasminogen activator levels are elevated either transiently or permanently, for example during tissue remodelling, wound-related repair and thrombolytic therapy.
PRIMARY STRUCTURE OF TETRANECTIN. SEQUENCE HOMOLOGY WITH ASIALOGLYCOPROTEIN RECEPTORS AND WITH PROTEOGLYCAN CORE PROTEIN FROM CARTILAGE
Authors: Fuhlendorff J , Clemmensen I , Magnusson S
Published: August 2018

Tetranectin (Mr = 68,000) is a tetrameric blood plasma protein, which binds to plasminogen and also to the lysine-binding site of the isolated kringle 4 from plasminogen. Its four polypeptide chains, which are non-covalently bound, each consists of 181 amino acid residues. We have determined the complete amino acid sequence and the disulfide bonds. Each position corresponds to a single amino acid residue except 34 which contains Ala and Ser and 37 which contains Val and Met in equimolar amounts. The three disulfide bonds connect Cys-50 to Cys-60, Cys-77 to Cys-176 and Cys-152 to Cys-168. The sequence of tetranectin was found to be homologous, to an extent indicating common ancestry, with the extracellular part of the asialoglyco-protein receptors and with the C-terminal globular domain of the cartilage proteoglycan core protein. Conserved residues include the six half-cystines of tetranectin. Therefore, we can now propose disulfide bond patterns for the proteins homologous with tetranectin. Supported by NIH-grant HL-16238 (S.M.), the Danish Science and Medical Research Councils, the Danish Cancer Society and NOVO Foundation.
DIURNAL VARIATION OF COMPONENTS OF THE FIBRINOLYTIC SYSTEM
Authors: Grimaudo V , Kruithof E K O , Hauert J , Bachmann F
Published: August 2018

To elucidate the causes of the well known diurnal variations of the fibrinolytic activity we have studied severed parameters of the fibrinolytic system in 8 healthy male volunteers during a period of 24 h. Blood sanples were taken at 8, 10 and 12 a.m., 4 and 8 p.m. and at 8 a.m. next morning. Hie following parameters were analyzed: euglobulin clot lysis time (ECLT), fibrinolytic activity of euglobulins on fibrin plates (FPA), antigen concentrations of tissue-type plasminogen activator (t-PA) by ELISA and of urokinase (u-PA) and plasminogen activator inhibitor-1 (PAI-1) by RIA, the overall PA-inhibitor activity by the indirect chranogenic substrate assay (Verheijen et al; 1984) and the electrophoretic-zymogra-phic analysis of the euglobulins fraction.

Global fibrinolytic activity increased from 1.0 ±0.3 U/ml at 8 a.m. to 2.5 ±1.1 U/ml at 8 p.m. (p <0.002) as measured by ECLT (U=300/lysis time in minutes) and from 2.3 ±0.4 IU/ml to 4.7 ±1.1 IU/ml (p <0.001) as determined by FPA. In contrast, t-PA antigen decreased from 5.1 ±0.8 ng/ml at 8 a.m. to 2.9 ±1.5 ng/ml at 8 p.m. (p<0.01) and u-PA antigen from 8.1 ±1.2 ng/ml to 7.1 ±0.7 ng/ml (p<0.05). Most pronounced was the decrease of PAI-1 antigen from 23.5 ± 7.6 ng/ml in the morning to 8.8 ±2.6 ng/ml at 8 p.m. (p <0.001), while PAI activity decreased from 8.8 ±3.6 U/ml to 5.5 ±1.8 U/ml at 4 p.m. (p <0.002). During the entire study period the electro-phoretic-zymographic analysis did not reveal any free t-PA; the latter was present throughout in the form of the 110 kD t-PA/PAI-1 ccnplex. During the day the intensity of the 110 kD band decreased progressively.

These findings confirm the physiological diurnal fluctuation of the fibrinolytic activity. All components of the fibrinolytic system are involved in this fluctuation and participate in the modulation of fibrinolytic activity.

Our study demonstrates that the diurnal increase of fibrinolytic activity is due principally to a marked decline of the PAI-1 concentration during the day and not to an increase of PA antigen levels.
PURIFICATION AND CHARACTERIZATION OF TISSUE PLASMINOGEN ACTIVATOR PRODUCED BY IMR-90 CELLS
Authors: Morinaga T , Itagaki Y , Suzuki A , Yasuda H , Higashio K
Published: August 2018

Tissue plasminogen activator ( t-PA ) produced by IMR-90 ( human embryonic fibroblast ) cells cultured in the serum-free medium ( DMEM ) containing 1% proteos^e peptone and 1.6 - 3.6mM CaCl2 was purified by the procedure consisted of ultrafiltration, immunoadsorpt ion chromatography, HPLC and lysine-Sepharose chromatography. The yield of t-PA from the culture broth was approximately 47%. The purified t-PA migrated as a single band on SDS-polyacrylamide gels. The molecular weight of the t-PA was estimated to be 66,000 by SDS-polyacrylamide gel electrophoresis and 69,000 by gel filtration method. Purified t-PA had a specific activity of 36 × 104 IU/mg protein by fiblin plate method or 54 - 56 X 104 IU/mg protein by clot lysis method using t-PA obtained from WHO as a standard. The amino acid composition of fibroblast t-PA was very similar to those of melanoma t-PA and uterine t-PA. Isoelectric point of fibroblast t-PA ranged from 5-7 to 8.2. The t-PA had twice as much affinity for fiblin as did high molecular weight urokinase ( UK ). Both t-PA and UK had optimum temperature at 41°C and optimum pH between 8.0 - 9.0. The polyclonal and monoclonal antibodies raised against t-PA quenched t-PA activity but had no effect on UK activity. The inhibitors of serine proteases, difluorophos-phate and gabexate mesilate, strongly inhibited the activities of fibroblast t-PA and UK. The nucleotide sequence analysis of the t-PA cDNA isolated from the cDNA library prepared from IMR-90 mRNA revealed the nucleotide changes at two positions in the coding region as compared to that of melanoma t-PA cDNA. Neither of the changes replaced the coded amino acid. The N-terminal amino acid of fibroblast t-PA was determined to be valine, indicatig the structural similarity of fibroblast t-PA to uterine t-PA.