bluuebunny/crossref_metadata_2025 huggingface dataset:
CHANGES IN ANTI-CARD10LIPIN ANTIBODY TITER IN SERA OF I TP PATIENTS AFTER TREATMENT WITH INTRAVENOUS IMMUNOGLOBULIN
Authors: Cunningham-Rundles C , Bussel J , Lipscombs A
Published: August 2018

Antibodies to cardiolipin have previously been demonstrated in the sera of patients with idiopathic thrombocytopenia purpura (ITP). We questioned whether the levels of anti-cardiolipin in the sera of patients with ITP would be altered after treatment with intravenous immunoglobulin (IVGG). Using flexible polyvinyl chloride microtiter plates coated with bovine cardiolipin, we measured IgG and IgM anti-cardio1ipin levels by ELISA before and at multiple intervals during and after IVGG treatment for 17 patients who had ITP. We found that within 7-10 days of treatment, 16 of 17 patients had further increases of IgG anti-cardiolipin antibody. This is probably due to the infusion of IVGG concentrates, which we found contained substantial amounts of IgG anti-cardio1ipin. Increases in platelet counts had significant correlation with increased IgG anti-cardio1ipin (p>0.01), presumably also due to the infusion of IVGG. Surprisingly, 16 of 17 patients had a marked increase in serum levels of IgM anti-cardiolipin 7-10 days post IVGG treatment. Since IVGG does not contain IgM anti-cardiolipin and our assay is specific for this isotype, this antibody represents de novo synthesis. We previously reported and have also confirmed here, that 16 of these 17 patients had increased serum IgM levels after IVGG treatment. The increase in serum IgM anti-cardio1ipin antibody may explain part of the increased total serum IgM observed. IVGG has been believed to be potentially suppressive of various immunologic activities; our data shows that immunologic stimulation also occurs. The biologic effect of increased serum IgG and IgM anti-cardiol ipin after IVGG treatment in ITP, particularly since cardiolipin may be a constituent of platelet membranes, is still uncertain.
PROTHROMBIN TOKUSHIMA: A REPLACEMENT OF ARGININE-418 BY
TRYPTOPHAN THAT IMPAIRS THE FIBRINOGEN CLOTTING ACTIVITY OF DERIVED THROMBIN TOKUSHIMA
Authors: Miyata T , Morita T , Inomoto T , Kawauchi S , Shirakami A , Iwanaga S
Published: August 2018

Recently, we identified a Japanese family having an abnormal prothrombin, designated “prothrombin Tokushima”. The propositus of this family is a 10-year-old female suffering from bleeding tendency. She is a double heterozygote transmitted from both the maternal dysprothrombinemia and the paternal hypoprothrombinemia. The purified prothrombin Tokushima exhibited the following properties: (i) factor Xa-catalyzed proteolysis of the abnormal prothrombin in the presence of factor Va, phospholipids and calcium ions yielded a two-chain thrombin, fragment 1 and fragment 2, (ii) thrombin derived from the abnormal molecule exhibited 21.5 % clotting activity relative to normal thrombin and reduced platelet aggregation activity, (iii) the apparent Km of thrombin Tokushima towards Boc-Val-Pro-Arg-MCA was 8-fold larger than that of normal thrombin, and the kcat value was approximately one-half of that of the normal enzyme, and (iv) its active site was fully titrable using P-nitrophenyl-P’-guanidinobenzoate.

Subsequently, the structural studies on the abnormal thrombin were performed to identify the difference responsible for its reduced fibrinogen clotting activity upon conversion to thrombin. Amino acid sequence analysis of a peptide isolated from a lysyl endopeptidase digest of the abnormal thrombin indicated that Arg-418 (equivalent to Asn-101 in the chymotrypsin numbering system) had been replaced by Trp. This amino acid substitution can result from a single nucleotide change in the codon for Arg→4l8 (CGG→TGG). The Arg→Trp replacement found in the thrombin portion of prothrombin Tokushima appears to reduce its interaction with various substrates including fibrinogen and platelet receptors, and accounts for the recurrent bleeding episode observed in the propositus.
STIMULUS-DEPENDENT CHANGES IN THE SURFACE EXPRESSION OF GPIIb/IIIa AND FIBRINOGEN RECEPTORS. RELATIONSHIP TO PLATELET AGGREGATION
Authors: Niiya K , Hodson E , Bader R , Byers-Ward V , Plow E F , Ruggeri Z M
Published: August 2018

Platelet stimulation altered the binding of three monoclonal antibodies (monovalent Fab’ fragment) directed against the glycoprotein (GP)IIb/IIIa complex. We found that 47,600-60,300 molecules of antibody bound per platelet before stimulation, as compared to 89,200-146,500 molecules per platelet after thrombin stimulation. These changes were observed in parallel with a small but significant increase in the dissociation constant (Kd) of two antibodies. In contrast, no statistically significant changes were observed with ADP-stimulated platelets. The increased binding of LJ-CP3, but not of the other two antibodies, to activated platelets decreased by 3040% in the presence of EDTA at 22-25°C, suggesting the occurrence of divalent-cation mediated, activation-dependent changes in the corresponding GPIIb/IIIa epitope. Platelets stimulated by thrombin bound more fibrinogen than those stimulated by ADP, and significant differences in the extent but not in the affinity of fibrinogen binding were observed with different platelet agonists. When the pool of GPIIb/IIIa molecules exposed on the surface of unstimulated platelets was reacted with monoclonal antibody LJ-CP3 to block ADP-induced fibrinogen binding and platelet aggregation, thrombin stimulation still induced substantial binding and aggregation. This effect of thrombin required exposure of platelets to the active agonist and was not mediated by molecules released by thrombin into the medium. Therefore, platelets activated with “strong” agonists exhibit increased number of surface-oriented epitopes associated with GPIIb/IIIa. The GPIIb/IIIa molecules bearing these newly exposed epitopes are functional in that they bind fibrinogen and mediate platelet aggregation.
BIOSYNTHESIS AND ASSEMBLY OF PLATELET GPIIbIIIa
Authors: Duperray A , Troesch A , Berthier R , Chagnon E , Marguerie G
Published: August 2018

Platelet GPIIbIIIa is a calcium-dependent heterodimer which is constituted of two proteins subunits GPIIb and GPIIIa. The GPIIb is itself made of two disulfide-linked subunits IIba and IIbβ. GPIlia is a single chain protein. GPIIbIIIa serves as a receptor for fibrinogen, fibronectin and von Willebrand factor and is implicated in platelet adhesive reactions. This protein is a member of an adhesion receptor protein family for which the name “cytoadhesins” has been proposed. As a preliminary step in the study of the genetic diversity of the members of this family, we have analysed the biosynthesis and assembly of GPIIbIIIa in human megakaryocytes and in a human megakaryocytic cell line : LAMA-84. Megakaryocytes were isolated from liquid cultures of cryopreserved blood cell concentrates from patients in the chronic phase of chronic myeloid leukemia. Using these cell preparations, we have shown that the a and 3 subunits of GPIIb derive from a common precursor, the pro-GPIIb, which associated in an early step with GP IIIa. In a second set of experiments, we have analysed the expression of the GPIIbIIIa complex in LAMA-84. Only a minority of the native cells were reacting with the anti GPIIIIIa antibodies as tested by immunofluorescent labeling. In contrast, the expression of GPIIbIIIa in these cells was amplified in the presence of the phorbol ester TPA. Metabolic labeling experiments indicated that a large quantity of pro-GPIIb was synthesized in the native cells, while very little of the mature forms of GPIIb and IIIa were detected. After TPA induction, the expression of GPIIIa was greatly enhanced with a simultaneous increase in mature GPIIb. These data indicate that a deficit in GP Ilia results in the biosynthesis of a non-associated pro-GPIIb which cannot be further processed, suggesting that the GPIIIa subunit is a regulatory component in the biosynthesis of the GPIIbIIIa complex.
PREPARATION OF ANTI-HUMAN THROMBOMODULIN ANTIBODIES AND THEIR APPLICATION FOR STUDY ON PLASMA THROMBOMODULIN
Authors: Kazama M , Ishii H , Tsubouchi J , Nakano M , Hiramoto N , Abe T , Majerus P W
Published: August 2018

Thrombomodulin is an endothelial cell membrane protein that is cofactor required for rapid activation of plasma protein C. Recently, thrombomodulin was found also in plasma in normal subject. However, the information of the soluble thrombomodulin in plasma is very poor. We now report the preparation of polyclonal and monoclonal anti-human thrombomodulin and their application for study on plasma thrombomodulin.

Thrombomodulin was extracted from human placenta by 0.5% of Triton X-100 and purified by DIP-thrombin column chromatography. Polyclonal anti-thrombomodulin antibody was obtained from rabbit serum. The three types of monoclonal anti-human thrombomodulin antibodies were obrained from hybrid cell of BALB/C spleen cell and SP2 mouse myeloma cell. The type 1 monoclonal antibody inhibited the thrombomodulin activity. The type 2 of the antibody did not inhibit the activity. The type 3 of antibody cross reacted with rabbit lung thrombomodulin. The IgGs of these antibodies were separated by protein A Sepharose. The plasma thrombomodulin was separated using polyclonal anti-thrombomodulin IgG Sepharose. The apparent molecular weight of plasma thrombomodulin was estimated by immunoblot analysis using monoclonal anti-thrombomodulin IgG. When run with mercaptoethanol, plasma thrombomodulin migrated mainly at Mr=85,000 against Mr=105,000 of tissue thrombomodulin. The plasma thrombomodulin had an apparent Km for 1 nM compared with 0.3nM for tissue thrombomodulin. The apparent Km for protein C was same between tissue and plasma thrombomodulin.
PLATELET FACTOR H REGULATES THE ACTIVITY OF THE ALTERNATIVE PATHWAY OF COMPLEMENT ON THE SURFACE OF NORMAL AND PAROXYSMAL NOCTURNAL HEMOGLOBINURIA PLATELETS
Authors: Devine D V , Rosse W F
Published: August 2018

Paroxysmal nocturnal hemoglobinuria (PNH)-is frequently complicated by thrombosis. It has been suggested that the abnormal interactions of PNH platelets with complement contribute to thrombosis. Using purified complement proteins, we have previously demonstrated that the platelets from some patients with PNH do not demonstrate elevated activity of C3bBb, the alternative pathway C3 amplification enzyme complex, even though they lack the C3bBb regulatory protein, decay accelerating factor (DAF). As measured by fluorescence flow cytometry, washed platelets from both normal donors and PNH patients released the fluid phase C3bBb regulatory protein, factor H, in response to the deposition of purified complement proteins. Platelet factor H was localized to the alpha granules by immunocytochemical techniques. A quantitative radioimmunoassay demonstrated that normal platelets released 54 ± 6 ng factor H/108 platelets in response to thrombin stimulation. PNH platelets contained less factor H (22 ±7 ng/108 platelets) than normal platelets. Thrombin stimulated platelets from patients with elevated C3bBb activity released less than half of the factor H measured in detergent extracts. However, thrombin stimulated platelets from PNH patients exhibiting normal C3bBb activity released nearly all their factor H. The release of factor H from normal platelets was blocked by treating the platelets with metabolic inhibitors. In the absence of factor H release, the activity of the C3bBb complex increased three-fold. In addition, the number of molecules of 1251-factor B bound per C3b increased from 0.40 to 0.92 when factor H release was blocked. The inhibition of DAF by anti-DAF had no effect on the activity of C3bBb if factor H could be released from the platelets. However, when factor H release was blocked by treatment with metabolic inhibitors, the inhibition of DAF by anti-DAF increased the activity of C3bBb by 40%. Therefore, in the absence of DAF, platelets can regulate complement activation by the alternative pathway via the release of platelet factor H. Since factor H is an alpha granule protein, platelet release in the presence of activated complement may contribute to the occurrence of thrombosis.
The Effect of Changes in the Leader Sequence of Human Protein C on Biosynthetic Processing and Gamma-Carboxylation
Authors: Foster D , Schach B , Rudisky M , Berkner K , Kumar A , Kumar A , Sprecher C , Hagen F , Davie E W
Published: August 2018

Protein C is the precursor to a serine protease in plasma which contains gamma-carboxy glutamic acid and functions as a potent anticoagulant. Protein C shows considerable structural homology with the other vitamin K-dependent coagulation factors including prothrombin, factor VII, factor IX and factor X. Sequence analysis ofthe cDNAs for these proteins has revealedthe presence of a prepro leader sequence that contains a pre sequence or hydrophobic signal sequence and a propeptide containing a number of highly conserved amino acids. The pre region is removed from thegrowing polypeptide chain by signal peptidase, while the pro region is subsequently removed from the protein prior to secretion. Deletion mutants have been constructed in the propeptide portion of the cDNAfor human protein C in order to test the possibility that the propeptide portion of the 42 amino acid leader sequence serves as a molecular signal for gamma-carboxylation.Accordingly, these mutants containthe pre-peptide (hydrophobic leader) plusportions of the pro-peptide at the amino terminus of the light chain. These deletions include the removal of 4, 9, 12, 15, 16 or 17 amino acids from the carboxyl end of the leader sequence of 42 amino acids. The mutant proteins were expressed in carboxylation-competent mammalian cells and were then examined by Western blotting, barium citrate adsorption and precipitation, amino acid sequence analysis, and biological activity and compared with the native protein present in normal plasma. These studies have shown that deletions inthe pro-peptide region interfere with gamma-carboxylation and removal of the pro-peptide. Deletion of residues -1 through -12 had little effect on the carboxylationor secretion. Deletion of -1 through -17 completely abolished gamma-carboxylation, but had no measurable effect on secretion.Amino terminal sequence analysis of thelatter mutant showed that the light chainbegan with Thr-Pro-Ala-Pro... This corresponds to a sequence in the prepro leader starting at -24. This indicates that the signal peptidase cleavage site for human protein C is between residues -25 and -24 and removal of the pro-peptide had been blocked by the deletion.Furthermore, during biosynthesis and secretion, the amino-terminal region of the propeptide (residues from about -12 through -17) are important for carboxylation of protein C, while the carboxyl-terminal portion of the peptide (residues -1 through -4) are important for the removal of the proleader sequence by proteolytic processing.
PRODUCTION OF PLATELETS FOR TRANSFUSION - CURRENT METHODS AND PROBLEMS TO BE SOLVED
Authors: Elias M K , Smit Sibinga C Th
Published: August 2018

Initially, whole blood or platelet rich plasma were used as sources of platelets. Nowadays the methods of platelet concentrates (pc) production adopted in Blood Banks include the traditional method of platelet preparation by differential centrifugation of units of whole blood, besides the much more sophisticated technique of extracorporeal collection of pc with improved immunological compatibility.

Manually pc are produced by the platelet rich plasmamethod, the buffy coat method and multiple bag plateletapheresis. The machine collection of pc is done by plateletapheresis or platelet elutriation, with different degrees of automation.

The standard manual method remains quantitatively the most important source of platelets.However, there are major concerns:

-the need of multiple donors

-The high contamination with white cells, predominantly lymphocytes

-these pc are depleted from larger and more active platelets, as these are sedimented with the red cells

-increased risk of bacterial contamination. To solve these problems there are some potention solutions:

-use of single donor collectioon techniques

-depletion of leucocytes by:

a.elutriation of platelets from the buffy coat

b.filtration of random pc through cotton wool column

c. prostacyclin inhibition of platelet aggregation followed by cellulose acetate filtration

d.filtration on elutriated platelets through cotton wool

-use of a platelet synthetic medium void of glucose for resuspension and storage of pc to prevent lactate accumulation and pH fall

-use of closed sterile harness systems to collect platelets by surge plateletapheresis, which allows extended storage of leucocyte depleted pc.

Selection of the most appropriate platelet concentrate depends on the interrelationship of many factors:1) yield 2) function 3) viability after storage 4) afety 5) purity 6) potency 7) efficacy (recovery, survival and haemostatic capacity).
CARRIER DETECTION OF HEMOPHILIA A BY DNA ANALYSIS IN AFFECTED JAPANESE FAMILIES
Authors: Suzuki N , Iizuka A , Nagao T , Nakahori Y , Yamada M , Nakagome Y
Published: August 2018

Several DNA probes have been isolated to detect Factor VIII gene and a DNA segment which locates veryclose to the gene.
They have been successfully used to detect carriers and patients of hemophilia A.We analyzed DNA samples of Japanese population to see whether these probesare also useful for carrier detection of hemophilia A in affected Japanese families, since the size and frequency of allelic fragments detected by a DNA probe are sometimes different in various ethnic groups.A probe of St14 (DXS52) is thought to be one of the best probes for such analysis in Caucasian population because it detects very polymorphic DNA fragments containing a minisatellite. When Taq I digests of Japanese DNA samples were hybridized with Stl4, several DNA fragments with a range from 1.7 kb to 5-5 kb were detected, where .at least 6 fragments were polymorphic. A notable difference between Japanese and Caucasian was that a band of 5-5 kb was variable in Japanese while it was constant in Caucasian. We have so far detected 10 alleles, and about 60% of Japanese women were heterozygous. Using these informationsabout Japanese population, we could detect carriers in several families. Other RFLPs data are necessary to increase information content. Similar studies arein progress using different probes i.e. an extragenic probe ; DX13/Bgl II, and two intragenic probes ; exon 14-26/Bcl I and exon 26/Bgl I. We thank Mandel J.L., Strasbourg, Davies K., Oxford and Genetics Institute, Cambridge for probes.
THE INFLUENCE OF N-LINKED GLYCOSYLATION AND BINDING PROTEIN (BiP) ASSOCIATION IN THE SECRETION EFFICIENCY OF COMPLEX GLYCOPROTEINS
Authors: Kaufman Randal J , Bole David G , Dorner Andrew J
Published: August 2018

We have studied the role of Binding Protein (BiP) or glucose regulated protein, GRP 78) in the processing and secretion of factor VIII (fVIII), von Willebrand factor (vWF), and tissue plasminogen activator(tPA) expressed in Chinese hamster ovary cell lines.fVIII is a 300 kDa protein which has a heavily glycosylated internal domain containing 20 clustered potential N-linked glycosylation sites.A significant proportion of the expressed fVIII is bound to BiP in the endoplasmic reticulum (ER) in a stable complex andnever secreted. Deletion of the heavily glycosylatedregion results in a lesser degree of association with BiP and increased secretion. Tunicamycin treatmentof cells producing the deleted form of fVIII resultsin stable association of the unglycosylated fVIII with BiP and inhibition of efficient secretion. vWF contains 17 potential N-linked glycosylation sites which are scattered throughout the molecule. vWF is transiently associated with BiP in the ER, demonstrating that CHO cells are competent to saecrete a complex glycoprotein. tPA, which contains 3 utilized N-linked glycosylation sites, exhibits low level association with BiP and is efficiently secreted. Disruptionof normal N-linked glycosylation of tPA, by site directed mutagenesis of the 3 Asn residues to Gin residues or by tunicamycin treatment of the tPA expressing CHO cells, results in reduced levels of secretion and increased association with BiP. This effect is enhanced by high levels of expression. The findings suggest that occupancy of glycosylation sites may effect protein folding and alter secretion efficiency by influencing the extent and stability of association with BiP.
HUMAN HEPATOCYTES CONTAIN HIGH MOLECULAR WEIGHT POLYPEPTIDES OF FACTOR VIII
Authors: Ingerslev J , Sloth Christiansen B , Bukh A , Stenbjerg S , Munck Jørgensen T , Munck Petersen C
Published: August 2018

Human hepatocytes were isolated by the two-step collagenase technique applied on distal left liver lobe. Homogenous and large cells were isolated revealing hepatocyte characteristics by light-microscopy. Hepatocytes were washed repeatedly in albumine buffer (5%), resuspended in the same buffer and sonicated using a cell density of 0.75 × 106 cells/ml. In some cases cells were separated from non-viable cells by flotation on a linear Percoll gradient. Supernate material after sonication was subjected to ELISA for VIII:Ag using human antibodies and vWf:Ag by polyclonal antibodies. Freshly isolated cells contained at least 0.25 IU/ 0.75 × 106 hepatocytes, whereas the vWf:Ag was below 0.01 IU/ 0.75 × 106 cells. The material obtained from sonication was further studied using fast protein liquid chromatography by Mono-Q HR 5/5 revealing a single peak of VIII: Ag eluting in the same position as the high molecular weight polypeptides of VIII :Ag of high purity FVIII derived from the plasma source. Isolated hepatocytes also were cultivated at 37°C in medium RPMI 1640 supplemented with Ultroser G (4%), glutamine and antibiotics. Cells secreted increasing quantities of albumin, fitrinogpn and protease-inhibitors. The supernatants also contained VIII: Ag in quantities ranging from 0.04 - 0.17 IU/ml after 24 hours, but no further secretion was observed. No vWf: Ag could be detected. Cells harvested and sonicated after 30 hours of culture only contained 0.04 IU/ 0.75 × 106 cells. Our results shows, that VIII :Ag is present in freshly isolated human hepatocytes and that only traces of vWf:Ag is found. A hepatocyte site of production of VIII is speculated. These very preliminary findings do not permit conclusions concerning active synthesis of VIII in hepatocytes. Further studies are underway.
RELATIONS BETWEEN APROTININ CONCENTRATIONS AND HEMODYNAMICS IN EXPERIMENTAL ACUTE PANCREATITIS
Authors: Ruud T E , Müller-Esterl W , Fritz H , Stadaas J O , Aasen A O
Published: August 2018

Acute pancreatitis (AP) was induced in juvenil pigs by injection of Na-taurocholate into the pancreatic duct. Eight animals remained untreated (group A), while 7 pigs (group B) received 60000 KIU/kg aprotinin (Tras-ylol, Bayer AG Leverkusen, West Germany) intravenously during 30 min before the induction of AP, thereafter 10000 KIU/kg/h during a 6 h observation period. Seven pigs (group C) received 60000 KIU/kg aprotinin during 30 min starting 3 h after the induction of AP, thereafter 10000 KIU/kg/h. The total infusion volume was the same in all 3 groups (5 ml/kg/h). Using an enzym-linked immunosorbent assay for aprotinin, maximum plasma concentration of aprotinin in group B was found 30 min after start of the infusion (2.8(1.9-5.4)umol/l). The aprotinin concentration thereafter remained elevated for the rest of the observation period (1.4(0.4-1.9) umol/1 after 6 h). The aprotinin concentration gradually increased in the peritoneal exudate during ongoing aprotinin infusion. After 1 h the aprotinin conc. in plasma and the exudate were within the same range. Similar results were observed in group C experiments, where the aprotinin conc. in plasma and peritoneal exudate both were approximately 2 umol/1 1 h after start of the infusion. Assayed by the chromogenic peptide substrate S-2302 (Kabi Vitrum AB, Stockholm, Sweden), markedly elevated values of plasma kallikrein inhibition were detected both in plasma and peritoneal exudate after start of aprotinin infusion in group B and C.

In group A, 5 out of 8 animals died in a circulatory collapse, while the animals in group B and C remained hemodynamic stable and all survived the observation.

Aprotinin concentrations of 1-3 umol/1 in plasma and peritoneal exudate improves the outcome during experimental acute pancreatitis in pigs.
THE EFFECT OF THE VENOM OF THE ORIENTAL HORNET ON COAGULATION FACTORS
Authors: Kornberg A , Kaufman S , Silber L , Ishay J
Published: August 2018

The extract from the venom sac of Vespa orientalis (VSE) inactivates exogenous and endogenous thromboplastin (Joshua and Ishay, Toxicon, 13:11-20,1975). The prolongation of both prothrombin time (PT) and recalcification time suggests inactivation of other factors. The aim of the present study is to investigate the effect of VSE on clotting factors. A lyophilized VSE with protein concentration of 5 mg/ml was used. Studies were performed in vitro with human plasma and in vivo in cats. Routine methods were employed for the assay of PT, activated tissue thromboplastin (APTT), thrombin time (TT), fibrinogen degradation products (FDP), fibrinogen and factors V,VII,VIII,IX,X. Human plasma was incubated with various concentrations of VSE (0,1,5,10,50,100 μg/ml) for 60 min and for various incubation times (0,5,15,30,+ 60,90,120 min) with 50 μg/ml VSE (n=8). 1 μg/ml VSE prolonged PT from 13.5 to 16 sec (p<0.05) and APTT from 62 to 180 sec. PT was maximal (17.7 sec) with 10 μg/ml and APTT (442 sec) with 50 μg/ml VSE. Factors V,VII,X decreased gradually from 94-105% to 11%,11% and 29% with 100 μg/ml VSE and VIII and IX to 1% even with 1 μg/ml VSE. After 5 min with constant concentration of VSE (50 μg/ml) PT was 14.9 sec (normal 13 sec) and APTT 165 sec (normal 54 sec). Both were maximal (17.5 and 298 sec) after 60 min. Factors VII and X decreased to 13% and 32% and VIII and IX to >1% after 60 min of incubation. Injection of 5 mg/kg VSE to cats (n=6-8) resulted in prolongation of PT from 9.4 to 11.2 sec and of APTT from 19.5 to 63 sec after 5 min. Both were maximal after 90 min (12.3 and 127 sec). Factors V,VII and X decreased from 100% to 7.6%, 13% and 37% and VIII and IX to 1% after 10 min. In all experiments TT and plasma fibrinogen were not affected and FDP were normal. Heating of VSE for 5 min at 80°C abolished completely the anticoagulant activity but dialysis for 24 hr at 4°C had no effect on it. The activity was eluted on Sephadex-25 both in void and post void volumes. The results show that VSE has a potent anticoagulant activity against various factors. Factors VIII and IX are markedly decreased. The effect on V, VII and X is moderate. Plasma fibrinogen is not affected. The nature and clinical significance of the anticoagulant activity merit further investigation.
ENHANCED GLOMERULAR PROCOAGULANT ACTIVITY AND FIBRIN DEPOSITION IN RATS WITH MERCURIC CHLORIDE-INDUCED AUTOIMMUNE NEPHRITIS
Authors: de Prost D , Kanfer A , Guettier C , Nochy D , Hinglais N , Druet P
Published: August 2018

The mechanism involved in glomerular fibrin deposition was investigated during HgCl2-induced autoimmune glomerulonephritis in the Brown Norway rat. To ascertain whether the local hemostatic system was activated secondarily to the immunological conflict, the ability of glomerular lysates to induce coagulation in vitro was measured in treated and control rats. On day 12 (latent phase of the disease), 20 (acme) and days 32 and 42 (recovery phase) after the first mercury injection proteinuria, glomerular procoagulant activity (PCA) and the incidence of glomerular fibrin deposits were assessed, the latter with a fluoresceinated anti-rat fibrinogen serum. All three parameters peaked on day 20. Glomerular PCA was characterized as thromboplastin. Results are as follows :

The number of la positive cells detected in glomeruli from HgCl2-injected rats by monoclonal 0x6 antibody was not different from the control number at any phase of the disease ; the number of macrophages per glomerular section detected by electron microscopy at day 20 in HgCl2-injected rats was 1.8±0.60 vs 0.3±0.11. No correlation was found between glomerular PCA and either the number of macrophages or of la positive cells present in the glomeruli.

Since glomerular PCA was maximal at the onset of fibrin formation in the glomeruli and then decreased toward its basal level, and since the fibrin disappeared, it is concluded that increased production of thromboplastin by glomeruli, with activation of the extrinsic coagulation pathway, is a factor which may contribute to intraglomerular fibrin deposition in HgCl2-induced glomerulonephritis.
HEPARIN COFACTOR II: A SIMPLE ASSAY METHOD
Authors: Vinazzer H , Pangraz U
Published: August 2018

A photometric assay method for heparin cofactor II (HC II) is described. In a first step antithrombin III (AT III) in plasma is blocked by an anti human AT III immunoglobuline from goats. After dilution of this plasma with Tris buffer pH 8.4 containing 3 IU/ml heparin and addition of thrombin the remaining thrombin activity is measured by use of the chromogenic substrate S-2238 Kabi. The following preliminary experiments were carried out: Variation of the amount of anti-AT III added to plasma resulted in complete inactivation of 1.25 units AT III by 1.0 ml of the inhibitor. Incubation of 1 ml anti AT III with 1 ml purified AT III ( 1 U/ml} or with 1 ml normal plasma completely abolished AT III activity within 60 sec. Incubation of the reaction mixture with thrombin resulted in maximum inactivation after 180 sec. This is in contrast to AT III activated by heparin which immediately inactivates thrombin. Anti-Xa activity after depletion of AT III was assayed in a similar way by addition of factor Xa to the reaction mixture and measuring the remaining Xa activity by the substrate S-2222. In these tests no anti Xa-activity was found after AT III depletion. From these experiments there was assumed that the anti thrombin activity measured under the following conditions was due to the action of HC II:

Plasma ( 50 μl) was mixed with anti AT III (50 μl) and was incubated for 60 sec. Tris buffer with heparin pH 8.4 (900 μl) was added. From this mixture 200 μl was pipetted into a cuvette at 37°C followed by 200 μl thrombin ( 2 IU/ml). After an incubation time of 180 sec 200 μl S-2238 ( 2 mmol/1) was added and the difference in OD/min was determined at 405 nm. A calibration curve was made by series of dilutions of normal AT III depleted plasma from 20 healthy individuals. The following preliminary results ofrHC II activity as a percentage were obtained:
STRUCTURE OF HUMAN α2-PLASMIN INHIBITOR DEDUCED FROM THAT OF cDNA
Authors: Sumi Y , Nakamura Y , Sakai M , Muramatsu M , Aoki N
Published: August 2018

The complete amino acid sequence of α2-plasmin inhibitor (α2PI) was determined by cDNA cloning. A Agt 10 cDNA library was prepared from poly(A)+mRNA isolated from cultured human liver cells. The labeled oligonucleotides, corresponding to the reported partial amino acid sequences of α2PI, were used as probes to screen the library. One of the positive clones was subcloned into plasmid pUC8. A 2.2 kilobase cDNA clone thus isolated contains a region coding for a portion of a leader sequence, the mature protein, a stop codon (TGA), a 3' noncoding region (733 nucleotides), and a poly(A)tail (37 nucleotides). The amino acid sequence deduced from the cDNA is composed of 452 amino acids starting with an amino-terminal sequence of Asn-Gln-Glu-Gln and ending with a carboxyl-terminal sequence of Gly-Ser-Pro-Lys. The sequence shows approximately 30% homology with those of other plasma serine protease inhibitors. However, α2PI extends 50-52 amino acids beyond the carboxyl-terminal ends of the other inhibitors. This 50-52 carboxyl-terminal amino acid sequence is therefore specific to α2PI, and contains the sequence that is exactly the same as that of the peptide containing the plasminogen binding site. There are three lysine residues possibly involved in the binding to plasminogen in this region. From the homology with the other inhibitors, the inhibitor's reactive-site peptide bond was suggested to be Met-Ser and the same as that of ai-antitrypsin. The Met residue is located at the 362 position from the amino-terminal end.
EFFECT OF THE LIPOPROTEIN FRACTION FROM PLASMA-EUGLOBULIN ON THE PLASMINOGEN ACTIVATION
Authors: Deguchi K , Suzuki H , Wada H , Shirakawa S , Iga Y , Kameyama S
Published: August 2018

The effect of the lipoprotein fraction on fibrinolysis was assessed in an in vitro system containing plasminogen and tissue plasminogen activator(t-PA). The fraction were separated from plasma-euglobulin by the sucrose density gradient ultracentrifugation at 90,000 g, 5°C for 48 hours. The fraction corresponding to high density lipoprotein(HDL), especially its subtraction HDL~ (1.125 d 1.121), did not promote any amidolytic activity on S-2251.However, it induced amidolytic activity in the presence of t-PA and even stronger activity in the presence of plasminogen and t-PA. the potentiating effect of this subtraction on the plasminogen activation by t-PA or urokinase was also evidenced on the plasminogen free fibrin plate. The HDL, fraction may contain plasminogen and a potentiating substance involved in the conversion of plasminogen to plasmin by t-PA or urokinase.

The maximum level of plasminogen in the HDL3 fraction was 30 % in both its activity evaluated by chromogenic assay and its antigen evaluated by the immunodiffusion method. And the pattern of the plasminogen in this subfraction on the crossed immunoele-ctrophresis agaist anti-human plasminogen rabbit serum was similar to that in normal plasma. Furthermore, the latex aggregation test revealed the presence of fibrinogen and/or its degradation products in this subtraction, their levels has ho effect on plasminogen activation by t-PA. The HDL~ fraction treated with heat at 56°C for 15 min. retained the activity.

We conclude from the above findings that the fraction corresponding to HDL,especially HDL3,from plasma-euglobulin contains a substance that potentiates tne plasminogen activation by t-PA or urokinase, in addition to plasminogen.
LEUKOTRIENES INDUCE THE ACUTE RELEASE OF TISSUE-TYPE PLASMINOGEN ACTIVATOR FROM PERFUSED RAT BLOOD VESSEL WALLS
Authors: Tranquille N , Emeis J J
Published: August 2018

In a previous publication (Blood 66, 86, 1985) we suggested, on the basis of inhibitor experiments, that lipoxygenase metabolites might be involved in the release of tissue-type plasminogen activator (t-PA) from vessel walls. To test this suggestion, isolated rat hindlegs were freed of blood with Tyrode-BSA solution, and subsequently perfused with Tyrode-BSA containing various lipoxygenase metabolites. The perfusate was collected at timed intervals and assayed for t-PA activity by a spectrophoto-metric procedure. Of the compounds tested (see Table) 5-HETE did not induce PA release. However, leukotriene (LT) C4 and LTD4 dose-dependently (10-200 nM) induced the release of t-PA, which plateaued at 160 and 200 nM, respectively. Peak levels of t-PA activity were found at one minute, although the amount of t-PA released was less than that induced by PAF-acether. The PA activity released proved to be t-PA by functional and immunological criteria. Release of t-PA induced by LTC4 and LTD 4 was inhibited by the LT receptor antagonist FPL-55712 (10 μM).

Prostaglandin E1 and E2, prostacyclin and ZK 36374 did not induce acute t-PA release at 0.1-2.8 μM concentrations in our model. LTC4 and LTD4 also induced an acute increase of t-PA activity in vivo in rats at a dosage of 2 μg/kg i.v.

The data show that LTC4 and LTD4 can directly induce the acute release of t-PA, possibly by interacting with an endothelial LT receptor.
CULTURED HUMAN OMENTAL TISSUE MICROVASCULAR ENDOTHELIAL CELLS LYSE FIBRIN CLOTS IN CONTRAST TO UMBILICAL VEIN ENDOTHELIAL CELLS
Authors: Speiser W , Anders E , Müller-Berghaus G
Published: August 2018

Previously, we have shown that cultured human omental tissue microvascular endothelial cells (HOTMEC) produce about 100-fold more tPA antigen than cultured human umbilical vein endothelial cells (HUVEC). Free plasminogen activator inhibitor (PAI) activity, but not free tissue plasminogen activator (tPA) activity was detected in the conditioned media of both cell types. In the present study, the clot lysis activities of HOTMEC and HUVEC were compared with each other. For this purpose, 2 mg/ml purified human fibrinogen and 0.2 μM purified human plasminogen were added to serum free medium (SFM) on top of a confluent cell layer. After the addition of α -thrombin (1 NIH U/ml; final concentration), a clot generated within 2 min. The clots formed on the surface of HOTMEC were lysed within 6 h, whereas clots on the surface of HUVEC were not lysed within 72 h. To find out, whether tPA is involved in the activation of the fibrinolytic system, studies were performed in the presence of antibodies against tPA or urokinase (uPA). The fibrinolytic capacity of HOTMEC could be blocked by incorporation of anti-tPA IgG into the clot. Clot lysis by HOTMEC was neither affected by the incorporation of anti-uPA IgG nor of exogenous PAI derived from conditioned medium of HUVEC. Incubation of HUVEC with 1000 μl SFM containing 1 NIH U/ml α -thrombin for 1 h and consecutive cultivation in 5% fetal calf serum for 48 h resulted in a 2.5-fold increase in tPA production. The elevated tPA synthesis was accompanied by an equal increase in PAI production so that free PAI activity, measured in conditioned media, did not change after incubation with α -thrombin. In contrast to experiments with HUVEC, tPA and PAI production of HOTMEC were not influenced by α-thrombin. From these studies it is concluded that 1. the fibrinolytic capacity of HOTMEC is stronger than that of HUVEC, 2. tPA released from endothelial cells is protected from inactivation by PAI when fibrin is present, and 3. HOTMEC do not release higher concentrations of tPA and PAI after contact with (X.-thrombin as it is known for HUVEC.
INDUCTION OF PLASMINOGEN ACTIVATOR AND PLASMINOGEN ACTIVATOR INHIBITOR ACTIVITY DURING DIFFERENTIATION OF HL-60 CELLS
Authors: Krishnamurti C , Alving B M , Wright D G
Published: August 2018

While increased production of plasminogen activator(PA) has been associated with malignant growth and differentiation, the effect of cell maturation on plasminogen activator inhibitor (PAI) production has not yet been defined. We therefore determined the effect of phorbol myristate acetate(PMA), an agent that induces monocytoid cellular maturation, on the secretion of both PA and PAI in HL-60 cells, a promyelocytic leukemia cell line. Cells were seeded at 5x105/ml and grown in a 5% CO2 incubator at 37°C in a serum-free medium in the absence or presence of 1.6 nM PMA. Experiments were performed either in polypropylene tubes to which cells did not adhere, or in petri dishes that provided surfaces for cellular adherence. Supernatants were assayed for PA activity with a functional chromogenic assay using plasminogen and S-2251 with urokinase (UK) as a standard. PAI was measured by determining the inhibition of exogenous UK (0.05 U/ml final) after incubation with the supernatants at 22°C for 30 min. In the absence of PMA, supernatants of control cells grown in tubes or petri dishes had a PA activity of 2 mU/106 cells at 72 hr. After exposure to PMA for 72 hr, PA activity in supernatants of non-adherent cells grown in tubes increased 50-fold over control, while only an 8-fold increase was measured in supernatants of cells that had been incubated in petri dishes and allowed to undergo adherence. The PA had a Mr of 56 kD as determined by fibrin zymography, and functional activity was completely inhibited by antibodies to UK. While there was no inhibition of exogenous UK in supernatants of control cells grown in tubes or petri dishes, there was a 50% inhibition of UK added to the supernatants of the PMA-treated cells grown under non-adherent conditions in tubes. Inhibition of exogenous UK was 94% by supernatants from PMA-treated cells that had undergone adherence in petri dishes. The inhibitory activity was completely abolished by incubation of the supernatants with IgG purified from antiserum to PAI-2 (courtesy Dr. Kruithof). These data indicate that PMA induces the production of UK-like PA and also an inhibitor similar to PAI-2, which has been found in monocytes. Furthermore, the regulation of both PA and PAI production appears to depend on conditions that affect cellular adherence.
PATHOGENIC BACTERIA HAVE HIGH AFFINITY RECEPTORS SPECIFIC FOR PLASMIN
Authors: Broeseker T A , Boyle M D P , Lottenberg R
Published: August 2018

Binding of the key fibrinolytic enzyme, plasmin, to certain pathogenic group A streptococci was studied. In these experiments the ability of a group A streptococcal strain, 64/14, to bind either 125I-human plasminogen or the same label following activation with urokinase was measured. It was found that this strain bound <10% of the labeled plasminogen but >70% of labeled plasmin. This property distinguishes the plasmin receptor from streptokinase. These bacteria did not express a common serine protease receptor/inhibitor since they failed to bind labeled trypsin or urokinase. Maximal binding of plasmin occurred between pH 6.0 and 8.0 and in the ionic strength range of 50-200 mM salt. The Kd of plasmin binding to bacteria was approximately 10-10 M at pH 7.4 in 150 mM salt. This was determined by a non-linear least squares analysis of equilibrium binding data. Binding was reversibly inhibited by either epsilon aminocaproic acid (I50 of 0.2 mM) or lysine (I50 of 3.0 mM) suggesting the involvement of the high affinity lysine binding site of plasmin in its binding to bacteria. Bacterial bound plasmin retains its enzymatic activity, being capable of cleaving chromogenic substrates and solubilizing a fibrin- clot. The bacterial bound enzyme activity was inhibited by the low molecular weight inhibitors aprotinin and phe-pro-arg chloromethyl ketone but not by alpha-2 plasmin inhibitor. The ability of bacteria to acquire membrane associated proteolytic activity which cannot be physiologically inhibited may contribute to their tissue invasive properties.
REVERSIBLE BINDING OF THE TISSUE PLASMINOGEN ACTIVATOR WITH FIBRONECTIN
Authors: Xiong Yi-min , Wang Zhen-yi , Xu Ye-lu , Chi Chenq-wu
Published: August 2018

Fresh pig heart tissues were homogenized and deli-pidized with cold acetone. The dry acetone powder was extracted with 0.45 M potassium acetate pH 4.5, the extract was fractionated with ammonium sulfate to 60% saturation, successively followed by four chromatography steps: chromatography on CM-Sepharose CL-6B; gel filtration on Sephadex G-100; Fibrin-Sepharose affinity chromatography and chromatography on DEAE-Sepharose CL-6B. The fibrin plate method was used for the tissue plasminogen activator (t-PA) activity determination. The porcine t-PA purified was proved to be homogeneous either by SDS gel electrophoresis or by high performance liguid chromatography with a molecular weight of 67,000. Using the human melanoma t-PA antigen and its rabbit antiserum, the double immunodiffusion and immu-noelectrophoretic tests demonstrated that the porcine t-PA possessed a cross-reaction with the human t-PA. During the purification procedure, it was found that the porcine t-PA was contaminated and reversibly bound with the cell fibronectin. The binding ability depends on salt concentration. They could be separated from each other on Sepharose G-100 gel filtration at high salt concentration, this reversible binding was further confirmed by mixing these two purified components monitored on high performance liquid chromatography. The fibronectin-bound t-PA retained its ability to activate plasminogen, indicating that these two proteins may exist in a form of complex in the in vivo tissue. Some monoclonal antibodies against the porcine t-PA were obtained, being used to screen what domain of the enzyme is responsible for binding the cell fibronectin.
MUTANTS OF TPA AND THEIR CATALYTIC PROPERTIES
Authors: Chaudhuri B , Heim J , Meyhack B , van Oostrum J
Published: August 2018

Mutants of tPA were obtained by in vitro mutagenesis to study the effects of N-linked glycosylation, and to gain insight into the properties of pure single-chain tPA. The glycosylation mutants were obtained by modification of the last residue of the sequence Asn.X.Ser/Thr, . known to be specific for N-linked glycosylation, whereas the plasmin sensitive sequence Arg275 Ile276 was modified to Asp 275 Ile276 to obtain a plasmin insensitive tPA. Mutant proteins were expressed in yeast under the control of the repressible acid-phosphatase promoter, and in CHO-cells. The novel proteins were isolated mainly by affinity chromatography using the selective protease inhibitor DE-3. A tPA mutant containing a modification in the sequence Asnl84 Glyl85 Ser 186 showed similar results as compared to recombinant yeast tPA in the clot lysis test, a direct fluoresence assay (FU test), and the indirect double rate assay according to Verheyen (Verheyen test). A mutant with modifications in the sequences Asnll7 Ser 118 Serll9, Asnl84 Glyl85 Serl86, and Asn448 Arg449 Thr450, showed an increased clot lysis activity with no increase in activity in the FU test and Verheyen test. However, the stimulation by fibrinogen fragments in the Verheyen assay was found to be doubled as compared to yeast tPA. An even further increase in stimulation by fibrinogen fragments, upto 500% over yeast tPA, was observed with the plasmin insensitive tPA, although this mutant only showed 20% of the yeast tPA activity in the FU and Verheyen tests
CONSTRUCTION AND EXPRESSION OF HYBRID PLASMINOGEN ACTIVATOR GENES
Authors: Rajput B , Alaimo D , Asselbergs A M , Reich E
Published: August 2018

Hybrid plasminogen activator (PA) genes containing fragments of cDNA encoding the non-catalytic part of tissue-PA and the .catalytic domain of urokinase and vice versa were constructed and expressed in Chinese Hamster ovary (CHO) cells. The hybrid nature of the products in stably transformed cells was analyzed at the level of DNA and RNA using probes derived from different regions of the urokinase and tissue-PA cDNAs and at the protein level by means of polyclonal antibodies raised against tissue-PA and urokinase. The hybrid genes made hybrid RNAs and proteins of the expected sizes. The proteins were enzymatically active as determined by zymography and chromogenic enzyme assays and this activity was blocked by the appropriate antibodies. The effect on hybrid PAs of cyanogen bromide cleaved fibrinogen fragments, poly-D-lysine and heparin which are known to affect the activity of tissue-PA and urokinase differently was also studied