bluuebunny/crossref_metadata_2025_split huggingface dataset:
INACTIVATION OF FACTOR Va BY PIASMIN
Authors: Omar M N , Lee C D , Mann K G 
Published: August 2018

The inactivation of Factor Va by plasmin was studied in the presence and absence of phospholipid vesicles and calcium ions. The action of plasmin resulted in a rapid loss of the ability of Factor Va to serve as a cofactor to Factor Xa , as judged by clotting assays and . direct assays of prothrombin activation using the fluorophore, dansylarginine N-(3-ethyl-1,5-pentanediyl) amide (DAPA) . The rate of Factor Va inactivation catalyzed by plasmin was markedly enhanced by the addition of phospholipid vesicles (PCPS), suggesting that the action of plasmin on Factor Va may be a membrane bound phenomena. Both Factor Xa and prothrombin were capable of protecting Factor Va from inactivation by plasmin. SDS-PAGE was utilized to correlate plasmin catalyzed proteolysis of Factor Va with the concomitant loss of activity. Data obtained with Factor Va and the isolated chains of the cofactor indicated that the light chain (E) was cleaved by plasmin to yield products similar to those obtained with Factor Xa and Activated Protein C (APC). The heavy chain (D) was found to be degraded by plasmin to produce proteolytic fragments distinct from those produced by Factor Xa and APC. The action of plasmin on single chain Factor V was notable for an initial, transient increase in total Factor V activity, followed by subsequent loss of activity, indicating the transient formation of active intermediates. SDS-PAGE analysis revealed the degradation of Factor V by plasmin to final, inactive products via several transient, higher molecular weight intermediates. These findings may be of some significance in pathophysiologic states in which systemic fibrinolysis may occur, possibly contributing to the depletion of clotting factors. The identification of such ongoing processes may eventually be facilitated by the observation that the degradation of Factor Va by plasmin leads to end products which may be unique to this interaction.(Supported by NIH Grants HL-35058 and HL 34575)
INTRA-ARTERIAL THROMBOLYTIC THERAPY IN ACUTE MIDDLE CEREBRAL ARTERY STROKE
Authors: Del Zoppo G J , Otis S M , Zyroff J , Hacke W , Zeumer H , Harker L A 
Published: August 2018

18 patients presenting with acute carotid territory stroke, secondary to angiographically demonstrated occlusion of the middle cerebral artery (MCA), have been treated within 8 hours of the onset of acute symptoms by local intra-arterial infusion of urokinase or streptokinase. All patients were screened by baseline CT cerebral scan to exclude intracerebral hemorrhage as a cause of the acute stroke. 14 patients demonstrated complete, 2 partial, and 2 no recanalization (reopening) of the previously occluded artery following a 1 to 2 hour infusion of the fibrinolytic agent.10 of the 14 patients displaying complete recanalization had complete neurological recovery or improvement with residual neurological deficits, while the 2 patients who did not display recanalization did not improve clinically. No clinical improvement was observed in the absence of recanalization.Hemorrhagic transformation of cerebral ischemic areas may be classified as hemorrhagic infarction (minimal hemorrhage, no clinical deterioraton) and parenchymatous hemorrhage (mass effect, clinical deterioration). Minor infarction-related hemorrhages without detectable neurological sequelae (hemorrhagic infarctions) were found by CT scan in 4 patients; all displayed complete recanalization; and all hemorrhagic infarctions resolved.This uncontrolled prospective clinical experience suggests that early local infusion of thrombolytic agents in selected patients may be efficacious and safe.
VINCULIN ISOFORMS IN HUMAN BLOOD PLATELETS
Authors: Asijee G M , Bruin T , Sturk A , ten Cate J E , Muszbek L 
Published: August 2018

In several cells vinculin has been implicated in the interaction between the cytoskeleton and the outer ceil membrane. We have recently demonstrated that vinculin becomes associated with the Triton X-100 insoluble cytoskeleton of blood platelets upon thrombin-induced activation (Asijee et al, Exp Cell Res, 1987, in press). In the present study we demonstrate by SDS-PAGE and immunoblotting that vinculin is also present in the membrane skeleton of both non-activated and activated human blood platelets. The membrane skeleton was prepared by the method of Fox (J Clin Invest, 1985: 76, 1673), and platelets were stimulated 5 min at 37 °c with 0.1 U/ml thrombin. The association was specifically inhibited by DNase I-induced depolymerization of the actin filaments.Protein analysis by the O’Farrell technique (first dimension IEF, second dimension SDS-PAGE) and subsequent immunoblotting demonstrated purified vinculin to consist of 4 major isoforms (pI 6.8 - 7.2). These isoforms differed in subcellular distribution. Upon thrombin-induced platelet activation, cytoskeletal vinculin consisted of the 2 most acidic isoforms, and cytoplasmic vinculin of 2 more basic isoforms. The membrane skeleton-associated vinculin contained all 4 isoforms.We conclude that: 1. vinculin is a component of the membrane skeleton of both non-activated and activated human blood platelets, 2. similar to chicken gizzard smooth muscle cells, human blood platelet vinculin consists of several isoforms with differing subcellular distribution.
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.
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.
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 coatb.filtration of random pc through cotton wool columnc. prostacyclin inhibition of platelet aggregation followed by cellulose acetate filtrationd.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).
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.
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.
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:
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.