bluuebunny/crossref_metadata_2025 huggingface dataset:
FETAL PLATELET TRASFUSIONS IN THE MANAGEMENT OF ALLOIMMUNE THROMBOCYTOPENIA
Authors: Waters A H , Ireland R , Mibashan R S , Murphy M F , Millar D S , Chapman J F , Metcalfe P , de Vries L S , Rodeck C H , Nicolaides K H
Published: August 2018

Intracranial haemorrhage is the most serious complication of alloimmune neonatal thrombocytopenia (ANT). It has generally been assumed that this occurs during delivery, but evidence is accumulating that intracranial haemorrhage may have already occurred in utero. Management of the pregnancy at risk is therefore more exacting, and it has been suggested that intrauterine platelet transfusions may be of benefit (Daffos et al, Lancet, Li, 632. 1984). We have used this approach in two pregnancies in PlA1 negative mothers with PlA1 positive fetuses affected by ANT. Both were second pregnancies, the first in each case having produced a brain damaged infant due to CNS haemorrhage. First patient (CW): Ultrasound scans of the fetal head at 10,22,28 and 32 weeks were all normal. She was admitted at 35 weeks for fetal sampling and platelet transfusion. Ultrasonography showed dilated ventricles and a left anterior cerebral haematoma. The fetal platelet count was 12 × 109/1,rising after transfusion of PlA1negative platelets to 139 x 109/1. The baby was delivered by Caesarean section and the cord blood platelet count was 126 × 109/1.Subsequent clinical assessment by CT scanning and NMR indicated both recent (1-2 weeks) and older (>4weeks) cerebral haemorrhages (de Vries et al, in press). Second patient (CR): Platelet transfusions were started earlier in this pregnancy. At 26 weeks the fetal platelet count was 32 × 109/1, rising to 160 × 109/1 after platelet transfusion. This was repeated at 27 wk (25 to 280 × 109/1), 29 weeks (5 to 320 × 109/1) and regularly until birth. Before the third platelet transfusion, the mother received intravenous IgG 0.4 g/Kg/d for 5 days, which had no effect on the fetal platelet count. These cases illustrate the potential value of ultrasound-guided intravascular, umbilical cord transfusions of compatible platelets in raising the fetal platelet count in ANT, but emphasise the short duration of this effect (<1 week). As the procedure is so labour intensive, further studies are needed to identify the high risk pregnancies, to determine the optimal time for intervention and to assess the success of this approach.
INTERACTION OF POLYMORPHONUCLEAR LEUKOCYTES AND ENDOTHELIAL CELLS : FUNCTIONAL CONSEQUENCES
Authors: Williams T J , Rampart M , Nourshargh S , Hellewell P G , Brain S D , Jose P J
Published: August 2018

The mechanisms involved in the accumulation of polymorphonuclear leukocytes (PMNs) in an inflammatory reaction are complex. A key phase in this process is the attachment of the PMN to the microvascular (venular in most tissues) endothelial cell, initiated by the extravascular generation of a chemical mediator. Experiments in vitro suggest that mediators, such as C5a, may act in vivo by stimulating the increased expression of the CD18 complex on the surface of the PMN within the venule lumen (1), whereas IL-1 may act by causing the expression of an adhesive molecule on the endothelial cell (2). In vitro the former process is rapid whereas the latter is slow in onset. We have measured the local accumulation of intravenously-injected Ulln-PMNs in response to intradermally-injected mediators in the rabbit, in order to investigate possible mechanisms in vivo. PMN accumulation was found to be rapid in onset in response to C5a, the rate of accumulation falling progressively to low levels by 4 hours. In contrast PMN accumulation in response to IL-1 was slow in onset, reaching a peak rate at 3-4 hours. Intradermal injection of the vasodilator prostaglandins PGI2; PGE2 and the neuropeptides VIP and CGRP caused a marked potentiation of the rate of leukocyte accumulation. PMN accumulation induced by C5a was associated with increased microvascular permeability, as indicated by the leakage of intravenously-injected 125I-albumin with a time-course in parallel with the rate of PMN accumulation enhanced by intradermally-injected vasodilators. Depletion of circulating PMNs abolishes these responses to C5a (3). In contrast, leukocyte accumulation induced by IL-1 was associated with little plasma protein leakage, even in the presence of intradermal vasodilators. This observation indicates that PMN emigration itself does not lead to increased microvascular permeability. C5a, but not IL-1, may stimulate emigrating PMNs to secrete an endogenous factor that increases permeability by an action on endothelial cells (3). This factor does not appear to be the phospholipid PAF (4). In contrast to the enhancing effects of local PGI2, intravenously-infused PGI2 inhibited PMN accumulation induced by C5a and IL-1, and plasma protein leakage induced by C5a (5). This effect is probably mediated by elevation of cyclic AMP in intravascular PMNs. We have shown that C5a stimulation of PMNs in contact with endothelial cells in vitro induces endothelial cell PGI2 secretion (6). Thus, PGI2 may be part of a negative feedback system in vivo to control interactions between PMNs and endothelial cells.

These observations provide some clues to the intricacies of mechanisms of leukocyte accumulation in vivo.
POINT-MUTATION OF FACTOR VIII CODING SEQUENCES IN HAEMOPHILIA A
Authors: Matthews R J , Peake I R , Bloom A L
Published: August 2018

In order to study the molecular basis of haemophilia A, DNA from 26 haemophilia A patients (8 severe with inhibitors, 13 severe noninhibitors and 5 mild/moderate) was screened by the Southern blotting method with FVIII cDNA probe A (a i.7kb Kpnl cDNA fragment that spans exons 1 to 12) probe B (a 4.7kb EcoRI cDNA fragment that contains exons 14 to 25 and part of exon 26) probe C (a 1.8kb EcoRI cDNA fragment that contains the remainder of exon 26 and probe D (an Apal/EcoRI 783bp cDNA fragment that includes all of exons 22 to 25 and parts of exons 21 and 26). All cDNA probes were kindly provided by Genetics Institutes Inc.

No large structural alterations of the FVIII gene were detected in any of the patients. However altered TaqI restriction sites within the coding regions of 3 patients were observed. DNA from patient 1 with severe haemophilia (VIIIAg < 0.1 u/dl, inhibitor negative) when probed with probe C showed a substitution of the normal 2.6kb TaqI and 4.5kb EcoRI fragments with novel 12kb TaqI and 11.5kb EcoRI fragments respectively. In addition he showed the normal 4kb Bglll fragment with probe C. A point mutation or small deletion (50bp is suspected to be present within exon 26.

Patient 2 had severe haemophilia and a FVIII inhibitor of 12 units (Bethesda). DNA from patient 2 when probed with probe B revealed a novel 5.0kb TaqI fragment instead of the normal 2.2kb and 2.8kb fragments.The location of the altered Taq I restriction site within the coding region of exon 18 was confirmed with intragenomic probe pi 14.12 that includes exons 17and 18 (kindly provided by Genentech Inc.) A family study with this mutation specific fragment showed the patients sister and mother to be carriers.

DNA from Patient 3 (severe haemophilia, factor VIII inhibitor 33 units) when probed independently with probes B and D revealed the absence of the normal 2.4kb and 1.4kb TaqI fragments and the generation of a novel 3.8kb TaqI fragment suggesting an alteration of the TaqI site within the coding region of exon 23.

The detection of altered TaqI restriction sites in 3of our patients is further evidence that 'CG' dinucleotide sequences might be relative hot-spots for mutation when occurring within coding sequences of genes.
HEMOPHILIA B IN CANINES IS DUE TO A POST-TRANSCRIPTIONAL DEFECT
Authors: High K A , Evans J P , Ware J L , Stafford D W , Roberts H R
Published: August 2018

Factor IX is a vitamin K dependent plasma proteinsynthesized in the liver; a deficiency of Factor IX results in hemophilia B. An animal model for hemophilia B exists in dogs; affected animals have severe disease, with activity levels of less than 1%. The purpose of the current study is to determine the molecular basis of canine hemophilia. In previous work, we had shown that the Factor IX gene in hemophilic dogs appeared to be at least partly intact; thus, genomic DNA from normal, carrier and hemophilic dogs, when probed with sequences from the 4th, 7th, and 8th exons of the human gene, gave identical patterns on Southern blot. We have now completed the mapping of the hemophilic gene, using probes from the first, second, third and sixth exons, and have shown it to beentirely intact, that is, free of any large deletions or rearrangements, as determined by Southern blotting. In addition, using the guanidinium thiocyanate technique, we h^ve prepared total RNA from normal and -hemophilic dog livers add analyzed these samples by Northern blotting. The results show that the hemophilic dog synthesizes a Factor IX transcript of approximately 3 kilobases, that is, of the same size asthe normal dog. In addition, baaed on signal intensity, the transcript appears to be produced in roughlyequivalent amounts in the normal and hemophilic dogs.We conclude that the defect responsible for canine hemophilia B interferes with the production of the normal Factor IX protein at a post-transcriptional level. Moreover, since the hemophilic dogs produce Factor IX mRNA it should be possible to elucidate the gene defect in the hemophilic animals by preparing normal and hemophilic canine liver cDNA libraries and isolating and characterizing the respective Factor IX cDNAs.
STRUCTURAL AND FUNCTIONAL PROPERTIES OF A PROTHROMBIN ACTIVATOR FROM THE VENOM OF BOTHROPS NEUWIDI
Authors: Govers-Riemslag J W P , Knapen M H J , Tans G , Zwaal R F A , Rosing J
Published: August 2018

The prothrombin activator from the venom of Bothrops neuwidi has been purified to homogeniety by gelfiltration on Sephadex G-100, ion-exchange chromatography on DEAE-Sephacel and metal-chelate affinity chromatography on an Epoxy-activated Sepharose 6B column loaded with ZnCl . The overall purification was about 200-fold, which indicates that the crude venom contains about 0.5 weight % of the prothrombin activator. The venom activator is a single chain protein with an apparent molecular weight of 60,000 dalton. It readily activated bovine prothrombin with a Km of 37.7 uM and a Vmax of 120 umoles prothrombin activated per min/mg of purified venom activator. Venom-catalyzed prothrombin activation was not accelerated by the accessory components of the prothrombinase complex i.e. phospholipids plus calcium-ions and Factor Va. The venom activator does not require added calcium-ions for the expression of its prothrombin-converting activity. Calcium ions do, however, affect the catalytic activity of the venom activator. At 2 mM CaCl there is a 2-fold increase of the rate of venom-catalyzed prothrombin activation. However, at higher CaCl concentrations there is a gradual decrease of the activity of the venom activator. Gelelectro-phoretic analysis of prothrombin activation indicated that the venom activator only cleaved the Arg 323-Ile 324 bond of bovine prothrombin since meizothrombin was the only product of prothrombin activation. The activator did not hydrolyze the chromogenic substrates S2222, S2337, S2238, S2366, S2302 or chromozym TH and its prothrombin converting activity was not inhibited by benza-midine, phenylmethylsulfonylfluoride, dansyl-glu-gly-arg-chloro-methylketone and soybean trypsin inhibitor. However, chelating agents such as EDTA, EGTA and o-phenanthroline strongly inhibited the enzymatic activity of the venom activator. The activity of chelator-treated venom activator could, however, be restored by the addition of an excess CaCl . These results indicate that the enzyme from Bothrops neuwidi does not belong to the serine proteases but has the properties of a metal proteinase. Thus, the activator differs remarkably from Factor Xa, but strongly resembles the prothrombin activator from the venom of Echis carinatus, both structurally and functionally.
PURIFICATION AND CHARACTERIZATION OF THE PROCOAGULANT OF THE VENOM OF TROPIDECHIS CARINATUS
Authors: Masci P P , Whitaker A N , Morrison J J , Bennett E A
Published: August 2018

Tropidechis carinatus is a venomous elapid snake distributed throughout Eastern Queensland. It has been considered as a tropical relative of Notechis scutatus and, similarly, the crude venom contains an indirect prothrombin activator, which will clot plasma provided that Factor V is present. Myotoxins and neurotoxins are also present. Envenomated patients regularly develop disseminated intravascular coagulation. The crude whole venom of T.carinatus was shown to have five major components by gel filtration, SDS PAGE and HPLC, and even more components by isoelectric focusing. The procoagulant eluted with a molecular weight of 55,000, being found in peak II on gel filtration on Sephadex-G150. The procoagulant was purified using a combination of Sephadex-G150 chromatography and ion-exchange on DEAE Sephadex-A50 and shown to migrate as a single band of molecular weight 55.000 by SDS PAGE. On reduction by β -mercaptoethanol this component was resolvec into u heavy chain of molecular weight 30.000 and a light chain of 25,000. The procoagulant was shown to bind to con A-Sepharose 4B and Blue Sepharose 4B. Coagulation studies using this purified procoagulant confirmed a factor Xa-like activity activating prothrombin in the presence of factor V. The purified fraction is unstable in buffer solutions at 4°C, probably because of trypsin - like autodigestion. Ouchterlony studies of the procoagulants of T.carinatus and N.scutatus show both lines of homogeneity and spurring, indicating similarities but also significant differences between the two proteins. The purified procoagulant was lethal to adult rats, an IV injection of 10 μg killing in 1 - 2 minutes. Death was prevented by prior heparinization, suggesting that the procoagulant is toxic in the absence of neurotoxin and other components.
COMPOSITION AND ANTICOAGULANT PROPERTIES OF GLYCOSAMI-NOGLYCANS EXTRACTED FROM NORMAL AND VARICOSE VEINS AND FROM AORTIC WALLS
Authors: Capel P , Vichhi M , Janssens A , Mandelbaum I , Barroy J P , Fondu P
Published: August 2018

We studied the concentrations of total and individual gluco-saminoglycans (GAG) extracted from different vessel walls, and their anticoagulant effects on APTT and thrombin time (TT of normal plasma. The vessels considered were varicose veins, veins presumably normal (autologus saphene crosses, various veins taken during autopsy), and aortas (autopsy).

The results (mean, sd) are presented in the Table.

No important differences were observed between normal veins from different localization, even in veins presenting higher risk of thrombosis like the veins of the lower limits.

It is concluded that there is a marked difference in the composition and anticoagulant properties of aortic GAG as compared with venous GAG. Varicose veins showed higher GAG content and stronger anticoagulant properties as compared with normal veins. This latter observation may be a reflect of defense mechanism against thrombotic stress of the higher pressure present in these abnormal veins.
THE EFECT OF CMTI-I INHIBITOR ON HUMAN BLOOD CLOTTING SYSTEM
Authors: Furmaniak-Kazmierczak E , Jagielski J , Wilusz T
Published: August 2018

Polipeptyde inhibitors for different serine proteases have been isolated from a variety of plants. Among them there are the inhibitors from squash seeds of molecular weight about 3 300 /1/. The experiments were carried out to determine the effect of one of the squash inhibitors /CMTI-I/ on human blood clotting system. The 0.1 ml of inhibitor /O,8-100 ug/ was added to 0,1 ml of normal intact plasma and incubated 0,5, 15, 30 and 60 minutes at 37°C. It was found that partial tromboplastin time /PTT/ and activated partial thromboplastin time /APTT/ were prolonged. CMTI-I did not show a progressive mode of action upon prolonged time of incubation. There was no effect of CMTI-I on prothrombin time /PT/, thrombin time /TT/ and Stypven-cephalin time /ScT/. The influence of CMTI-I on APTT of factor-XII and factor-XI deficient plasmas as well as on a plasma without factor-XII and factor-XI /exhausted plasma/ was studied. The APTTs of the factor-XII and factor-XI deficient plasmas were prolonged while the APTT of the "exhausted plasma" was unchanged. The performed experiments shown that CMTI-I inhibitor blocks the clot-promoting activity of contact activated plasma. This inhibitory action is stronger in the case of factor XI than of factor XII.

1. Wieczorek M., et al., 1985, Biochem. Biophys. Res. Commun. 126:646-652.
A NEW PEPTIDE THROMBIN INHIBITOR FROM STREPTOMYCES GRISEUS
Authors: Liu Hua-zhen , Chen Wei , Liu Qi-ying , Zhang Xia , Wang Li-xiu , Chi Cheng-wu
Published: August 2018

A new peptide thrombin inhibitor was found in the Streptomyces griseus strain 254 isolated from a soil sample from Tongan, Fujian province, China, the inhibitor being a secondary metabolic product. The production of the inhibitor reached a maximum after 3 days culture of bacteria at 28°C in a rotary shaker. The inhibitor excreted in the culture filtrate was purified by absorption on macroporous resin, followed by ion exchange chromatography on DEAE-52, CM-32 cellulose, affinity chromatography on the immobilized thrombin and high performance liquid chromatography. The amino acid composition of the inhibitor was determined to be Val(2), Met(l), Ile(l), Leu(2) and Arg (1), similar to that of the amino acid residues around the reactive site of human antithrombin III, the critical plasma inhibitor of thrombin. The NH2-terminal residue of the inhibitor seems to be blocked by the alkyl group due to the negative reaction to ninhydrin, whereas the COO-terminal residue is most likely to be arginal because of that Arg was not found in the amino acid analysis, unless the peptide was oxidized by performic acid before acid hydrolysis. The chromogen substrates Bz-Phe-Val-Arg-PNA and Bz-Gly-Pro-Lys-PNA were used to determine the thrombin and plasmin activities, respectively. Besides thrombin, the purified inhibitor also exhibits a weak inhibitory activities on trypsin and much weak on plasmin, but not on chymotrypsin and other protein-ases.
EFFECT OF A FIBRIN-DERIVED PEPTIDE ON PULMONARY ANGIOTENSIN CONVERTING ENZYME (ACE) ACTIVITY AND ON PRESSURE RESPONSES TO BRADYKININ (BK)
Authors: Saldeen K , Moalli R , Saldeen T
Published: August 2018

Decreased ACE activity is a common finding in patients with adult respiratory distress syndrome (ARDS) and in animal models of lung injury. The nature of this effect is unknown. Fibrin, also a common finding in lung injury, is degraded to small peptides by proteolytic enzymes. Peptide 6A, corresponding to amino acid residues 43-47 of the BB-chain of fibrin (cgen), is produced by plasmin degradation of fibrin and has been shown to inhibit ACE in vitro. The purpose of the present investigation was to study whether peptide 6A inhibits pulmonary ACE in vivo and, if so, determine its effect of hemodynamic changes induced by bradykinin (BK) in the rabbit. We investigated the effect of peptide 6A an the hydrolysis of a synthetic ACE substrate, benzoyl-phe-ala-pro (BPAP) in anesthetized rabbits and in isolated lungs. Peptide 6A caused a reversible, dose-dependent inhibition of BPAP hydrolysis. The ID 50 for peptide 6A inhibition of ACE hydrolysis of BPAP was approximately 1 micrcmole.

In both isolated rabbit lupgp and in the intact animal, BK injection elicited a dose dependent increase in pulmonary arterial pressure. In intact animals, this was accompanied by a dose dependent decrease in systemic arterial pressure.

In both preparations, responses to BK were potentiated by addition of 1 micromole of peptide 6A. In isolated lungs, co-injection of peptide 6A significantly increased the pulmonary artery pressure response to every dose of BK except the highest (1 mg). In the anesthetized rabbit, 1 micromole of peptide 6A significantly (p<0.05) increased the pulmonary hypertensive response to 100 and 200 nanograms of BK and significantly (p<0.05) decreased the systemic hypotensive response following 100 , 200 , 300 and 400 nanograms of BK. The amount of BK needed to increase pulmonary arterial pressure was 1000-fold greater in the isolated lungs than in the intact animals. Peptides of this type might contribute to decreased ACE activity in patients with ARDS and may potentiate BK induced pulmonary hypertension and systemic hypotension in these patients.
ANTI-THROMBINneo IgG IN AN ASYMPTOMATIC PATIENT WITH A BENIGN MONOCLONAL GAMMOPATHY
Authors: Coller B S , Jesty J
Published: August 2018

A 68 year old male hospitalized for cardiac disease was found to have an elevated prothrombin time (18.5/12.4 s) and aPTT (59.6/25.9s). He had no history of excessive bleeding or bruising. Subsequent evaluation revealed: thrombin time >500/34.1 s; fibrinogen 260 mg/dl functional and 522 mg/dl immunologic; reptilase 25.6/18.1 s; thrombin-induced platelet release of ATP (patient=0 and control=14.6 nmoles/109 platelets at 0.5 U/ml); AT-III 89% functional and 36.5 mg/dl immunologic; and prothrombin 167%. Mixing experiments showed the presence of an inhibitor of the thrombin time, and purification of IgG by protein A affinity chromatography showed the inhibitor of fibrin formation to reside in the IgG fraction. When coupled to Affi-gel 10, patient IgG (but not control IgG) removed purified thrombin from solution; the same gel did not remove prothrombin. The patient's IgG did not inhibit thrombin’s cleavage of a chromogenic substrate (Chromozym TH). Studies on the patient's serum revealed: IgG 2,360 mg/dl, IgA 371 mg/dl, and IgM 107 mg/dl. Serum protein electrophoresis and immunoelectrophoresis showed a monoclonal IgG lambda protein with probably normal amounts of normal IgG. Other parameters (hematocrit, albumin, calcium, bone marrow histology, bone X-rays) indicated that the patient has a benign monoclonal gammopathy, not multiple myeloma. We conclude that our patient is producing an IgG inhibitor that reacts with a neo-antigen produced by the cleavage of prothrombin to thrombin; the IgG can prevent the interaction of thrombin with fibrinogen and the thrombin receptor on platelets, but not small synthetic substrates. We suspect that his monoclonal IgG is the inhibitor and find it remarkable that he has no increase in bleeding.
INDUCTION OF ANTITHROMBIN III (AT III) ANTIBODIES BY IMMUNIZATION WITH SYNTHETIC PEPTIDES
Authors: Stüber W , Pelzer H , Heimburger N
Published: August 2018

The primary structure of AT III was examined in respect of potential antigenic sites. The topics were the determination of the hydrophilicity, hydropathy, acrophilicity and the propensities for alpha-helices, B-turns and 13-sheets. The peptides AT III 21-34, 21-42, 129 - 140, 226 - 240 and 343 -363 were synthesized using the solid phase peptide synthesis methode. The subsequent purification of the crude peptides was achieved by h.p.I.e. or by ion exchange chromatography. The peptides were coupled to keyhole limpet hemocyanine (KLH) via thioether bonds. Antisera against KLH-peptides were raised in rabbits (n = 25) and tested with AT III-coated polystyrene tubes; bound antibodies were detected with anti-rabbit-IgG-peroxidase. Obtained antisera were further purified by immuno-adsorption using immobilized AT III. Polystyrene tubes were coated with purified peptide antibodies and binding of AT III was studied with enzyme immunoassay technique (EIA) using anti-AT III-peroxidase.

As a result, immunoreactivity of rabbit antisera against synthetic peptides of AT III could be achieved. The obtained antibodies against the individual synthetic peptides as well as their mixtures exhibited specific binding to AT III when tested with EIA.
PRODUCTION AND CHARACTERISATION OF A MONOCLONAL ANTIBODY AGAINST HUMAN ANTI-THROMBIN III
Authors: Rathjen Deborah A , Geczy Carolyn L
Published: August 2018

To study the role of anticoagulants, particularly antithrombin III (AT III) and heparin, on the activation of coagulation by monocytes/macrophages which have been stimulated with a soluble lymphocyte activation product, macrophage procoagulant inducing factor, we have prepared monoclonal antibodies (MAbs) to human AT III.

In fusion experiments, in contrast to wells containing peritoneal feeder cells, positive hybrids were only found in wells containing medium conditioned by the macrophage cell line J774 (Rathjen and Geczy, 1986). Of 5 hybrids which initially produced antibody, only one hybrid, showed stable Ab production. The MAb, designated 22/23, was not cross-reactive with either 1 antitrypsin or ovalbumin and did not inhibit the biological activity of AT III in chromogenic assays which measured inhibition of thrombin and Factor Xa by AT III. An immunoadsorbent prepared using MAb 22/23 depleted AT III activity from a purified AT III preparation. Reduction and alkylat ion of the disulphide bonds of the protein portion of AT III completely abbrogated MAb binding indicating that the native configuration of AT III was important. Isoelectric focussing of AT III, followed by transfer of the focussed protein to nitrocellulose by diffusion and probing with MAb 22/23, revealed at least 8 bands in the region of pH 5.2 to 5.85. Coomassie blue staining of a gel run in parallel showed 9 bands in this region. The MAb provides a useful tool for the detection of AT III on both cultured cells (bovine aortic endothelial cells are positive by immunofluorescence) and tissue sections.

Rathjen, D.A. and Geczy, C.L. Hybridomo. 5s 255-261 (1986)
COMPARATIVE STUDIES ON ANTITHROMBIN III AFFINITY OF LOW MOLECULAR WEIGHT HEPARIN AND UNFRACTIONATED HEPARIN
Authors: Sakuragawa N , Shimotori T , Takahashi K
Published: August 2018

Purpose: Low molecular weight(LMW)heparin shows stronger antifactor Xa(F-Xa) and weaker anti-thrombin(TH) activities compared with unfractionated(UF) heparin, and shows less bleeding tendency in the cases of clinical use. These characteristics were surmised to be depend on antithrombin III(AT-III) affinity of the heparin. Materials and methods: LMW heparin(Kabi and Pharmuka), UF heparin (Novo) and heparin cofactor II(HC-II) purified by our method were used. AT-III affinity column chromatography with 0.1 M Tris-buffer (pH 7.4)-NaCl 0.02 to 2.5 M linear gradient was performed. From the point of AT-III affinity strength, non-affinity(Na), low affinity (La) and high affinity(Ha) were separated, and aPTT, anti-F-Xa and anti-TH activities were assayed on each fractions. HC-II was assayed by biological activity.

Results: (1) Kabi-LMW heparin; Na 34.5%, La 39.3%, Ha 26.2%, Pharmuka-LMW heparin; Na 58.0%, La 24.1%, Ha 17.3%. Novo; Na 0%, La 50%, Ha 50%. (2) APTT; Na showed no effect, but Ha showed the strongest prolonging effects on aPTTs even having less amount of uronic acid, and more prominent effects were observed in UF(Novo)-heparin than LMW heparins. (3) La showed higher activity of anti-F-Xa and anti-TH activities than Ha. (4) Anti-TH activity of AT-III was observed in both fractions of La and Ha, but that of HC-II was observed in each fractions including Na.

Conclusion: It was surmised that the differences of the characteristics between LMW heparin and UF heparin were depend on to the strength of AT-III. The different characteristics of HC-II from AT-III to anti-TH were observed and surmised to be depend on the binding ability to the fractions.
INTERACTION BETWEEN CULTURED ENDOTHELIAL CELLS AND ABNORMAL ANTITHROMBIN III "TOYAMA"
Authors: Sakuragawa N , Saitoh S , Takahashi K
Published: August 2018

Purpose: Abnormal antithrombin III(AT-III)Toyama showed non-affinity to heparin and heparinoid to show loss of immediate antithrombin activity. On the endothelial cells, there are heparinoids including heparan sulfate. We investigated on the interaction between cultured endothelial cells and abnormal AT-III"Toyama" from the viewpoint of antithrombin activity.

Materials and methods: (1) Endothelial cell culture:^125I-labelled normal and abnormal AT-III were placed on the washed endothelial cultured cells in 0.2 ml of RPMI-1640 medium for 15 min at 37°C. The medium was suctioned off and the cell layer was washed with Hank's balanced salt solution. The cells were incubated with 1 ml of heparin(3 ug/ml) for 15 min at 4°C. The radioactivity in the supernatant was counted, and represented AT-III which bound to the cells surface. (2) Antithrombin activity: 0.23 ml of thrombin solution^ U/ml) and 0.03 ml of normal or abnormal AT-III plasma were mixed, and incubated on the cultured cell surface for 5 min at room temperature. The residual thrombin activity was assayed by 0.3 ml of the substrate (S-2238) solution(0.8mM)for 5 min. After these procedures,2 ml of 2% citric acid solution was added to stop the reaction, and 0D(405 nm) was recorded.

Results: Abnormal AT-III showed reduced binding-activity to cultured cells to one fifth compared with normal AT-III, and the residual thrombin activity in the abnormal was higher compared with that in normal plasma.

Conclusion: Abnormal AT-III showed less binding activity to the cultured endothelial cells, and less thrombin neutralizing activity to show thrombogenic tendency.
ANTITHROMBIN III GENEVA : AN HEREDITARY ABNORMAL ANTITHROMBIN III (AT III) WITH DEFECTIVE HEPARIN COFACTOR ACTIVITY
Authors: de Moerloose Ph , Reber G , Minazio Ph , Bouvier C A
Published: August 2018

A 43-year old man presented a pulmonary embolism. Despite a negative family history for thromboembolic disorders, the unusual circumstances of apparition and the relatively young age of the patient prompted us to study carefully the coagulation parameters. Routine coagulation tests, as well as plasminogen, alpha-2-anti-plasmin, protein C and protein S were all within normal range. Biological and immunological assays of AT III were performed on 12 members of the family and showed a low AT III activity in the propositus and other members of this family (mean 50%), but normal immunologic levels. Crossed immunoelectrophoresis in absence of heparin showed a normal pattern, but in presence of heparin showed an abnormal peak as compared with controls. Kinetics experiments showed a normal inhibition of Xa and 11a in absence of heparin, but abnormal in presence of heparin. An affinity chromatography on heparin Sepharose revealed two populations of AT III, one of which was devoid of heparin cofactor activity.

The toponym AT III Geneva is proposed for this new familial abnormal AT III with defective heparin cofactor activity. This family confirms the low incidence of thromboembolic events reported in this type of AT III variant.
FIBRINOLYTIC ACTIVITY (FA) OF NORMAL HUMAN PERIPHERAL BLOOD MONOCYTES (MC)
Authors: Grau E , Moroz L A
Published: August 2018

FA of blood encompasses a large cellular phase in addition to a fluid (plasma) phase. Polymorphonuclear neutrophils (PMN) have been implicated in this cellular activity, and MC have demonstrated fibrinolytic potential. Using a solid phase radiofibrin assay, we have examined FA of normal blood and plasma, and of purified PMN and MC alone, and with purified plasminogen (PLG), mini-plasminogen (mPLG) produced by gMN elastase digestion, or autologous plasma. PMN alone (0.5 x 106/mL) had striking activity (292 ± 25 SEM ng fibrin lysed/h), (n=10 normal subjects) while MC alone (0.5 x 106/ml) had mean FA of 32 ± 4 ng/h, all of which could be accounted for by contaminating PMN in the MC preparations (36 ± 8 ng/h). In comparison, mean whole blood FA wgs 72 ± 4 ng/h, and plasma FA was 22 ± 4 ng/h. When MC (0.5 x 106/ml) were assayed with PLG (2-40 yg/ml) or autologous plasma for 1 h, no significant FA was generated, indicating that neither intrinsic nor PLG-dependent (plasminogen activator, PA) activity of MC contribute significantly to the FA of whole blood as measured by routine 1 h assay, where 70% of measured FA involves the cellular phase. However, with longer assay times (2-6 h), there was time-dependent appearance of FA when MC were mixed with PLG or with autologous plasma. This FA was dependent upon interaction between MC and PLG, since no FA was generated by supernatants of MC preincubated alone, while FA was readily detected in the medium when MC and PLG were mixed. Comparing effects of PLG and mPLG, FA of MC (0.5 x 106/ml) with PLG (40 Ug/ml) was 447 ± 9 ng/3 h, while FA with mPLG (40 pg/ml, an approximate 3-fold molar excess) was 156 ± 5 ng/3 h, indicating a possible role for the N-terminal portion of the PLG molecule (containing kringle domains 1-4 absent in mPLG) in interaction ofg MC and PLG, or of MC-derived PA and PLG. FA of MC (0.5 x 106/ml) in autologous plasma (83±3 ng/3 h) was markedly reduced after lysine-Sepharose PLG depletion (14 ± 1 ng/3 h), and by addition of tranexamic acid (10 mmol/L) (5±1 ng/3 h). Thus, normal peripheral blood monocytes, like PMN, may contribute, albeit in a minor manner, to normal blood fibrinolytic activity, via PLG activation rather than direct proteolysis, and constitute an additional mechanism for interaction between cellular and fluid (plasma) phases in blood fibrinolytic activity.
HORMONE REGULATION OF THE FIBRINOLYTIC SYSTEM IN THE OVARY
Authors: Ohlsson M , Hsueh A J W , Ny T
Published: August 2018

In the ovary, the release of oocytes from graafian follicles during hormone-induced ovulation has been found to be associated with substantial increases in follicular plasminogen activator (PA) activity. Most of the PA activity comes from the granulosa cells that have been shown to produce tPA, uPA as well as the type-1 PA-inhibitor,(PAI-1).

We have studied the molecular mechanism of follicle stimulating hormone (FSH) and gonadotropin releasing hormone (GnRH) on the synthesis of tPA in primary cultures of rat granulosa cells. FSH and GnRH were both found to induce tPA in granulosa cells in a time and dose dependent manner. The effect of FSH and GnRH on the levels of tPA mRNA was also studied by northern and slot blot hybridizations. FSH and GnRH were both found to increase the level of tPA mRNA. The stimulation was up to 18 -fold compared to untreated cells.

The induction of tPA mRNA by FSH and GnRH was additive and the time courses of the stimulation by the hormones differed, suggesting that different cellular mechanisms are involved. Consistent with the ability of FSH to activate the cAMP dependent protein kinase A pathway, the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine further enhanced the FSH induction of tPA mRNA.

GnRH is known to activate the phospholipid-dependent protein kinase C pathway. Likewise the effect of GnRH can be mimicked by the kinase C activator, phorbol myristate acetate.

It is concluded that FSH and GnRH regulates tPA production by differnt molecular mechanisms, and that the increase in tPA activity is mediated via an increase in the levels tPA mRNA. Since both gonadotropins and GnRH cause ovulation in hyposectomized animals, similar stimulatory actions of these hormones on the tPA activity suggest a correlative relationship between this enzyme and the ovulatory process.
SINGLE-CHAIN UROKINASE TYPE PLASMINOGEN ACTIVATOR (SCU-PA) FROM HT-1080 HUMAN FIBROSARCOMA CELLS IS A GENUINE PROENZYME
Authors: Skriver L , Petersen L C , Lund L R , Nielsen L S , Danø K
Published: August 2018

U-PA is released from many cells as a single polypeptide chain (scu-PA) that is converted into its active two-chain form (tcu-PA) by limited proteolysis with plasmin. There is general agreement that scu-PA has an extremely low amidolytic activity, but different oppinions exist, as to whether scu-PA itself can activate plasminogen. We have reinvestigated the plasminogen activating activity of our scu-PA preparations by means of a direct [125]I-plasminogen conversion assay and two amidolytic assays for plasmin and u-PA activity. In the [125]I-plasminogen conversion assay in the presence of bovine pancreatic trypsin inhibitor (BPTI) the subsequent plasmin catalyzed conversion of scu-PA is blocked while the plasminogen activation is unaffected. In this assay with 3oo nM Glu-plasminogen and 15 pM BPTI, 4o nM scu-PA caused a low but significant plasminogen conversion, which could be fully inhibited by pretreatment of scu-PA with diisopropylfluorophos-phate (DFP). DFP-treated scu-PA was convertible to fully active tcu-PA. Rates of plasminogen activation in this type of assay for scu-PA activity was at least 4oo fold slower than that measured for tcu-PA. A coupled amidolytic assay with Lys-plasminogen, scuPA or tcu-PA, BPTI, and the high affinity plasmin substrate H-D-Val-Phe-LyspNA (S2390) was performed under conditions that ensures a low steady state concentration of free plasmin. In this assay the initial rate of Lys-plasminogen activation by DFP-treated scu-PA was at least 25o fold slower than that measured for tcu-PA. Finally, u-PA activity was measured in an assay with the chromogenic substrate <Glu-Gly-ArgpNA (S2444) (o.8mM) in the presence of highly purified Glu-plasminogen (3oonM) and DFP-treated scu-PA (2nM) in the absence of BPTI. Within the initial 15 min of incubation no detectable hydrolysis of S2444 occurred. Addition of tcu-PA (2pM) or plasmin (o.lnM) to the scu-PA/Glu-plasminogen mixture caused a significant reduction of the lag period before onset of the cascade reaction leading to scu-PA conversion and subsequent hydrolysis of S2444. We conclude that the low rates of plasminogen activation measured in these assays by scu-PA might be accounted for by the presence of trace amounts of tcu-PA in the scu-PA preparations, and that scu-PA meets the requirements for a genuine proenzyme
EXTRAHEPATIC METABOLISM OF RECOMBINANT TISSUE-TYPE PLASMINOGEN ACTIVATOR (tPA) IN DOGS
Authors: Fong K L L , Boyle K E , Crysler C S , Landi M S , Griffin H E , Lynn R K
Published: August 2018

Hepatic uptake has been proposed as the major mechanism of tPA clearance from systemic circulation. However, our recent studies demonstrated that tPA was rapidly Inactivated through complexation with protease Inhibitors in dog plasma In vitro, and that tPA-inhibitor complexes were present in plasma of dogs receiving tPA. Therefore, the present work was undertaken to differentiate hepatic from extrahepatlc clearance of tPA. Pharmacokinetics of tPA were determined in anesthetized beagle dogs with either Intact hepatic circulation or with Interrupted hepatic blood flow achieved by hepatic artery ligation and portal caval shunt.

Recombinant two-chain tPA was administered as an Intravenous bolus dose (80 μg/kg) and plasma active tPA concentrations were measured using a modified and validated S-2251 chromogenlc assay. Following tPA administration to Intact dogs, plasma active tPA concentration declined blexponentlally with time with 84% of the active tPA eliminated during the α-phase. The t1/2's of the a and β-phase were 1.76 ± 0.74 and 6.23 ± 1.56 min, respectively. The systemic clearance was 25.98 ± 1.13 ml/min/kg and the volume of distribution at steady state (VDss) was 73.9 ± 15.1 ml/kg. Upon the elimination of hepatic blood flow, the systemic clearance was reduced by 54% while VDss was unaffected. The contribution of plasma Inactivation of tPA to the systemic clearance was estimated from in vitro Inactivation studies In 37°C plasma. Based on the pseudo first order Inactivation rate constants of 0.184 min-1 and 0.095 min-1 at Initial tPA concentrations of 25 and 250 IU/ml respectively, clearance rates from 5.02 to 9.2 ml/min/kg were calculated. These data suggest that (1) in Intact dogs, 46% of the tPA clearance occurs extrahepatlcally and (2) Inactivation of tPA in plasma accounts for a major portion of the extrahepatlc clearance.
THE INTERACTION OF ONE AND TWO CHAIN TISSUE-TYPE PLASMINOGEN ACTIVATOR WITH INTACT AND DEGRADED FIBRINOGEN
Authors: Higgins Deborah L
Published: August 2018

Tissue-type plasminogen activator (t-PA) plays a central role in fibrinolysis in vivo. One-chain recombinant t-PA was isolated using lysine-Sepharose chromatography followed by chromatography on monoclonal antibody column which is specific for one chain t-PA. Two chain t-PA was produced by incubation of one chain t-PA with plasmin-Sepharose. Both forms of t-PA were incubated with plasminogen-free fibrinogen as well as fibrinogen that had been degraded with plasmin until it was -50% clottable. Thrombin was added to catalyze fibrin formation and the amount of free t-PA in the clot supernatants was measured by ELISA. A quantitative analysis to determine the dissociation constant (Kd) and moles of t-PA bound per mole of fibrin monomer (n) was performed using a computer fit of a nonlinear regression analysis. The data obtained gave the following results:

Plasmin degradation of the fibrinogen caused the formation of a new set(s) of sites with a significantly lower Kd(s), which caused an increase in binding to degraded fibrin when compared to intact fibrin. ε-amino caprioc acid (ε-ACA) (0.1M) was found to compete for t-PA binding to the loose sites in both intact and degraded fibrin, but not the tight sites formed by degradation. Significantly (>20%) more one chain t-PA than two chain t-PA bound to both intact or degraded fibrin. Differential binding of one and two chain t-PA as well as tighter binding to degraded fibrin may play a significant role in the regulation of fibrinolysis.
BINDING OF TISSUE-TYPE PLASMINOGEN ACTIVATOR AND PLASMINOGEN TO FIBRINOGEN AND ITS DEGRADATION PRODUCTS
Authors: Bosma P J , Rijken D C , Nieuwenhuizen W
Published: August 2018

In order to localize the binding site(s) for tissue-type plasminogen activator (t-PA) in the fibrin(ogen) molecule, two-chain t-PA was immobilized onto microtitration plates and incubated with fibrinogen and various fibrinogen fragments. The extent of binding was quantified with an enzyme immunoassay. Hardly any binding to t-PA was observed with fibrinogen, fragments X, Y and E. A moderate binding was observed with fragments D(cate) and D(EGTA) and a strong binding with the cyanogen bromide fragment FCB-2 (Kd apparent = 65 nM). Results of control experiments, in which the binding of fibrinogen and its fragments to immobilized Lys-plasminogen was measured, using the same assay, were in line with literature data: hardly any binding was found with fibrinogen, fragments X and Y. A moderate binding was observed with fragments D and E and a strong binding with FCB-2 (Kd apparent = 100 nM). The stimulatory capacity of the various fragments on the Lys-plasminogen activation by t-PA, as studied in a spectrophotometric assay, was found to be absent for fragment E, low for fibrinogen, fragments X, Y and D, and high for FCB-2. It is concluded that the t-PA binding site in the fibrin (ogen) molecule resides in the distal domains from which fragments D and FCB-2 are derived. The site is apparently hidden in fibrinogen and early fibrinogen degradation products. Binding of both plasminogen and t-PA is required for stimulation of the plasminogen activation, as illustrated by fragment E which binds plasminogen and no t-PA, and has no stimulatory capacity.
ENHANCEMENT OF PLASMINOGEN ACTIVATION BY TISSUE-TYPE PLASMINOGEN ACTIVATOR IN THE PRESENCE OF ANCROD AND FIBRINOGEN
Authors: Reddy K N N , Cercek B , Ganz W
Published: August 2018

Ancrod, a thrombin-like enzyme from Malayan pit viper venom, when infused into experimental animals or man converts fibrinogen into fibrin micro-clots. In a recent study we have found that in dogs pretreatment with ancrod markedly enhanced thrombolysis by recombinant tissue-type plasminogen activator (rt-PA), presumably by depleting fibrinogen and preventing new fibrin uptake by the thrombus during lysis. The rapid clearance of large amounts of fibrinogen from the circulation and the appearance of fibrin degradation products during ancrod treatment is indicative of high fibrinolytic activity. In this study we found that ancrod enhances activation of plasminogen by rt-PA indirectly via plasmin mediated digestion of fibrin. To a reaction mixture containing lys-plasminogen, human fibrinogen and val-leu-lys-pNA (S-2251), ancrod was added followed by rt-PA. At time intervals plasminogen activation was followed by measuring amidolytic activity on S-2251 at 405 nm. Ancrod or fibrinogen alone had no significant effect on the activation of plasminogen by rt-PA. However, the amidolytic activity increased with time in reaction mixture containing ancrod and fibrinogen. When a small amount of alpha-2-antiplasmin was added to the reaction mixture prior to the addition of rt-PA (the inhibitor level was sufficient to inhibit only a fraction of plasmin generated during the assay period) the activation rate was very much reduced. Thus, a small amount of plasmin initiates digestion of fibrin which may result in the exposure of new sites on fibrin that enhance the rate of activation of plasminogen by rt-PA. The rapid clearance of fibrinogen during ancrod infusion may not be due to increased susceptibility of micro-clots to lysis but to increased rate of plasmin formation. These results further confirm the observations of other investigators about the role of plasmin in the activation of plasminogen by tissue plasminogen activator.
t-PA PROTEIN DETERMINATION: IMPROVED ACCURACY THROUGH COMPOSITION CONSIOUS AMINO ACID ANALYSIS (CCAAA)
Authors: Brändström A , Rånby M
Published: August 2018

Protein determination by composition consious amino acid analysis (CCAAA) is generally applicable to polypeptides with known amino acid composition.

Traditionally, in protein determination by AAA the mass of protein substance in a preparation is quantitated by simple summation of the mass of all amino acid residues found, although some amino acids e.g. Trp, Ser, Thr, Cys are unsatisfactorily determined. In this study we have explored the, possibility of improving protein determination by AAA in cases where the amino acid composition of the preparation is precisely known from sequence data. In CCAAA protein determination the molar amounts of amino acids that are known to be quantitatively recovered are divided by the number of residues known to be present in the polypeptide. The mean of these values is identified as the molar quantity of polypeptide in the sample. In addition, the standard deviation of the mean serves as identification and homogeneity control. In this study the accuracy of the method was checked gravimetrically.

CCAAA was applied on t-PA prepared from cultured Bowes melanoma cells by immunopurification on PAMl-Sepharose 6BFF and subsequent gelfiltration in 1 mol/L NH4HCO3 on Sephacryl SA-200. About 2 pg of lyophilized protein was hydrolysed in 6 mol/L HC1 under vacuum for 24 hours at 110°C and amino acid analysed. Amino acids selected for calculation of the molar quantity were (number of residues within parentheses) Asp+Asn(50), Glu+Gln(52), Pro(29), Ala(32.5), Val(25), Leu(39), His(16), Lys(21) and Arg(35.5). To obtain the gravimetric quantity the molar quantity was multiplied with the theoretical molecular weight as calculated from the sequence and with the carbohydrate content taken into account.

For t-PA the standard deviation of the mean molar quantity typically was less than 5%. The result from CCAAA was about 10% higher than that obtained from conventional AAA. The result from CCAAA was within 10% of that found gravimetrically. The absorbtivity of t-PA was estimated to 1.75 (g/L)-1cm-1.

In summary, composition consious amino acid analysis (CCAAA) is recommended as reference method for protein determination of well defined protein preparations where the amino acid composition is known.