bluuebunny/crossref_metadata_2025_split huggingface dataset:
Single-Chain and Two-Chain Tissue-Type Plasminogen Activator (t-PA) Bind Differently to Cultured Human Endothelial Cells
Authors: Aerts Rob J , Gillis Karin , Pannekoek Hans 
Published: June 2018

SummaryIt has recently been shown that the fibrinolytic components plasminogen and tissue-type plasminogen activator (t-PA) both bind to cultured human umbilical vein endothelial cells (HUVEC). After cleavage of t-PA by plasmin, “single-chain” t-PA (sct-PA) is converted into “two-chain” t-PA (tct-PA), which differs from the former in a number of respects. We compared binding of sct-PA and tct-PA to the surface of HUVEC. Removal of t-PA bound to HUVEC by a mild treatment with acid and a subsequent quantification of eluted t-PA both by activity- and immunoradiometric assays revealed that, at concentrations between 10 and 500 nM, HUVEC bind about 3-4 times more sct-PA than tct-PA. At these concentrations, both sct-PA and tct-PA remain active when bound to HUVEC. Mutual competition experiments showed that sct-PA and tct-PA can virtually fully inhibit binding of each other to HUVEC, but that an about twofold higher concentration of tct-PA is required to prevent halfmaximal binding of sct-PA than visa versa. These results demonstrate that sct-PA and tct-PA bind with different affinities to the same binding sites on HUVEC.
Monoclonal Antibodies Directed to the Calcium-Free Conformation of Human Protein S
Authors: Marcovina Santica , Coppola Raffaella , Valsecchi Carla , Zoppo Adele , Gelfi Cecilia , Mannucci Pier Mannuccio 
Published: June 2018

SummaryFour mouse hybridomas secreting monoclonal antibodies specific for human protein S (PS) have been generated. The antibodies, all of the IgG1 subclass, were designated S2, S3, S8, and S10. In a fluid phase radioimmunoassay, the binding of monoclonal antibodies to PS was about 30% greater in the presence of EDTA and totally inhibited in presence of Ca2+. Using the same technique, we performed displacement curves of 125I-labeled PS by purified PS, thrombin-cleaved PS, normal plasma, plasma from a patient on warfarin therapy, and plasma from a patient with no free PS and only PS bound to C4b-binding protein. The slopes of the curves show that the monoclonal antibodies reacted equally with all the tested forms of PS indicating that the antigenic site(s) to which the monoclonal antibodies are directed are present and exposed in free and bound PS, in thrombin-cleaved PS, and in the coumarin form of the protein. Each EDTA-dependent antibody, immobilized on Sepharose 4B-CNBr was used to purify PS from the barium citrate-absorbed, ammonium sulphate-soluble fraction of plasma. The fraction eluted from the immunoabsorbent with a buffer containing 4 mmol/1 CaCl2 and analysed by SDS-PAGE, contained two bands, one migrating with conventionally purified PS and the other with purified C4b-binding protein. Homogeneous PS was obtained by chromatography of the barium citrate absorbate on a DEAE-Sephadex column. The protein peak containing the bulk of PS was subsequently applied to the immunoadsorbent and eluted with 4 mmol/1 CaCl2. These studies demonstrate that EDTA-dependent monoclonal antibodies can simplify the isolation of PS from human plasma.
Von Willebrand Factor Multimer Patterns in Pregnancy-Induced Hypertension
Authors: Brenner B , Zwang E , Bronshtein M , Seligsohn U 
Published: June 2018

SummaryMicroangiopathy and disseminated platelet aggregation have been reported in thrombotic thrombocytopenic purpura (TIP) and pregnancy-induced hypertension (PIH). Since unusually large von Willebrand factor (vWF) multimers have been implicated in the evolvement of TTP, we analyzed factor VIII/vWF parameters in patients with PIH. Mean vWF: Ag level was significantly higher in 27 patients with PIH as compared to 20 matched healthy pregnant women (358 ± 160 u/dl vs. 274 ± 125 u/dl, p < 0.05). Moreover, plasma vWF: Ag levels and the ratio of vWF: Ag to factor VIII were found to be linearly correlated to the severity of PIH. In contrast, no significant differences in mean levels of factor VIII and ristocetin cofactor were observed between these groups. Crossed immunoelectrophoresis of vWF revealed a higher incidence of a pre-peak and an increased migration index in the PIH group as compared to the control group (60% vs. 44% and 1.27 ± 0.26 vs. 1.19 ± 0.18, p < 0.01 respectively). Analysis of plasma vWF multimer patterns by 1.4% agarose electrophoresis in 0.1% SDS revealed excessive amounts of large, medium and small size multimers in the PIH patients. Conceivably, the quantitative changes in vWF multimers reflect endothelial injury and may play a role in the microangiopathy observed in PIH.
Plasminogen Activator Inhibitor 1: Biosynthesis and mRNA Level Are Increased by Insulin in Cultured Human Hepatocytes
Authors: Kooistra T , Bosma P J , Töns H A M , van den Berg A P , Meyer P , Princen H M G 
Published: June 2018

SummaryClinical studies have shown that plasma insulin levels are closely related to plasma plasminogen activator inhibitor 1 (PAI-1) levels. To investigate a possible involvement of hepatocytes we have studied the effect of insulin on PAI-1 production by primary cultures of human hepatocytes. We have isolated human hepatocytes from seven left liver lobes. PAI-1 activity measured in 24 hours conditioned medium varied considerably between the various hepatocyte preparations (from 2.9 to 8.5 units per 5 cm2of cells) possibly as a result of interindividual variability in basal PAI-1 production by hepatocytes from different donors. In all cases, however, the relative extent, time profile and dose-dependency of the insulin-induced increase in PAI-1 synthesis were consistent. Up to about 7 nM, insulin dose-dependently increased both PAI-1 activity and PAI-1 antigen production. The increase in PAI-1 synthesis became measurable between 4 and 8 hours after addition of the hormone, and maximally reached twofold control values. The increase in PAI-1 synthesis could be fully explained by a concomitant increase in PAI-1 mRNA levels. The effect of insulin seems fairly specific for the synthesis of PAI-1: overall protein synthesis and mRNA levels of some control proteins (albumin and fibrinogen) did not markedly change after insulin addition. These results, obtained with primary cultures of human hepatocytes, are fully comparable with those obtained with the hepatocellular carcinoma cell line Hep G2. They strengthen the suggestion that the elevated level of PAI-1 in high insulin plasma might be the result of increased hepatic synthesis of PAI-1.
Poor Compliance Is a Major Factor in Unstable Outpatient Control of Anticoagulant Therapy
Authors: Kumar S , Haigh J R M , Rhodes L E , Peaker S , Davies J A , Roberts B E , Feely M P 
Published: June 2018

SummaryControl of oral anticoagulant therapy in outpatients is often unsatisfactory. The contribution of poor compliance with prescribed warfarin to unstable anticoagulant control was investigated prospectively using low-dose phenobarbitone as an indicator of compliance in 30 out-patients, 15 with stable and 15 with unstable control. Following entry to the study, there was no significant change in anticoagulation (p = 0.36) in the group with stable control. In the group who previously had unstable control, there was a significant change in INR (p = 0.0045) and anticoagulant control greatly improved. It appears that the considerable fluctuation in INR seen in many of the latter patients before the study was due to poor compliance and that entering them into the study modified their behaviour. Two patients in this group who continued to have unstable anticoagulant control were shown to be poorly compliant using the phenobarbitone indicator. The results suggest that, in outpatients, poor compliance is the major cause of unstable anticoagulation with warfarin.
Changes of Plasma Coagulation and Fibrinolysis in Response to Mental Stress
Authors: Jern Christina , Eriksson Elsa , Tengborn Lilian , Risberg Bo , Wadenvik Hans , Jern Sverker 
Published: June 2018

SummaryTo study the effects of standardized mental stress (arithmetic and the Stroop color word test) on plasma coagulation and fibrinolysis, blood samples were obtained before, during, and after 20 minutes of mental stress from 10 healthy, non-smoking young males aged 22 to 30 years. Reactions were compared with those observed during physical exercise and infusion of adrenaline. Both von Willebrand factor antigen and factor VIII coagulant activity increased significantly in response to mental stress (95 ± 28 vs 123 + 56%; p <0.05 and 125 ±54 vs 217 ± 170%; p <0.05, respectively). There was also a significant increase of factor VII coagulant activity (86 ± 31 vs 108 + 51%; p <0.05). Furthermore, mental stress caused an activation of the fibrinolytic system with an elevation of tissue plasminogen activator activity and tissue plasminogen activator antigen (0.80 ± 0.48 vs 1.23 ± 0.96 IU/ml; p = 0.076 and 4.38 ± 1.87 vs 5.78 ± 2.58 IU/ml; p <0.01). Fibrinogen concentration increased during stress (1.95 ± 0.29 vs 2.11 ± 0.27 g/1; p <0.05). Similar but more pronounced responses were observed during exercise and adrenaline infusion. Parallel to the increases in coagulation and fibrinolytic factors there were significant increases in heart rate, and systolic and diastolic blood pressure. It is concluded that mental stress has significant effects on plasma coagulation and fibrinolysis, and that it could thus affect important risk factors for cardiovascular disease.
Circadian Fluctuations of Plasminogen Activator Inhibitor and Tissue Plasminogen Activator Levels in Plasma of Patients with Unstable Coronary Artery Disease and Acute Myocardial Infarction
Authors: Huber Kurt , Rosc Danuta , Resch Irene , Schuster Ernst , Glogar Dietmar H , Kaindl F , Binder Bernd R 
Published: June 2018

SummaryA decrease in the fibrinolytic potential, mainly due to an elevation of plasminogen activator inhibitor (PAI), has been described in patients with stable coronary artery disease and a previous myocardial infarction. We investigated plasma levels of PAI and tissue plasminogen activator (t-PA) and their possible circadian variations in patients with unstable coronary artery disease (CAD). Sixty-three patients were studied for at least 2 consecutive days during their stay at the coronary care unit (CCU). Diurnal plasma fluctuations in PAI and t-PA and onset of further myocardial ischemic episodes were monitored. As controls we used 22 age-matched patients submitted to the clinic because of non cardiac chest pain or valvular disease who revealed no evidence of CAD. PAI levels were significantly elevated in patients with unstable CAD (p < 0.0001) but were not influenced by the extent of underlying CAD, history of previous myocardial infarction, known risk factors for CAD, or by extent of myocardial damage. The circadian variation of PAI levels with peak values between midnight and 6 A. M. found in controls was still present in patients but at a higher level. Preservation of circadian pattern in PAI plasma levels despite myocardial ischemic attacks indicates that elevation of PAI is rather not caused by a reactive phenomenon. On the other hand, elevated PAI levels and episodes of severe myocardial ischemia exhibiting a median time of onset at 10 A. M. seem to be closely related.
Synthesis of Factor VIII in Human Hepatocytes in Culture
Authors: Ingerslev J , Christiansen B Sloth , Heickendorff L , Petersen C Munck 
Published: June 2018

SummaryAlthough several investigators have attempted to identify the site of synthesis of factor VIII (FVIII), the cellular species responsible for maintenance of plasma FVIII has not been clearly defined. Indications point at hepatocytes and certain endothelial cells. The present study investigated the FVIII coagulant antigen (VIII : Ag) of hepatocytes obtained by two-step collagenase digests of human liver pieces. Following Percoll gradient centrifugation, less than 1% of cells harvested were non-parenchymal. Lysates of freshly isolated and purified hepatocytes contained 165–250 mU of VIII: Ag/106 cells as defined by a two-site ELISA employing a haemophilic antibody against human FVIII. This material contained a single peak of VIII: Ag polypeptides as jugded from the VIII: Ag ELISA profile of Mono-Q fast protein liquid chromatography fractions. A haemophilic antibody specific for epitopes of the light chain of FVIII, employed in immunoisolation of VIII : Ag in lysate of human hepatocytes, extracted a polypeptide pattern that was studied in a reduced SDS-PAGE electrophoresis gel and compared to that of immunoisolate from normal plasma. After electroblotting onto nitrocellulose and reaction with a monoclonal antibody towards the light chain of FVIII, the appearance of a doublet at 78–79 kDa in both these materials indicated the presence of the light chain of FVIII in human hepatocyte lysate. During culture, human hepatocytes secreted 20–80 mU of VIII: Ag per 1 × 106 cells per 24 hours. Further, a significant secretion of VIII: Ag was found in media of cultured human hepatoma cells, Hep-G2, whereas human blood monocytes and human fibroblasts did not secrete detectable VIII: Ag. In all of these cell cultures, vWf : Ag was indetectable or present as trace. Our results suggest that the human hepatocyte is a production site of FVIII.
Insulin Stimulates the Synthesis of Plasminogen Activator Inhibitor 1 by the Human Hepatocellular Cell Line Hep G2
Authors: Alessi M C , Juhan-Vague I , Kooistra T , Declerck P J , Collen D 
Published: June 2018

SummarySecretion of plasminogen activator inhibitor 1 (PAI-1) by cultures of human umbilical vein endothelial cells and human hepatocellular cell line Hep G2 was evaluated after insulin stimulation. The secretion of PAI-1 antigen and activity was measured in the conditioned medium and the cellular extracts after incubation of confluent cultures with 1% serum medium for 24 hours.Insulin induced a dose dependent increase of the PAI-1 secretion by Hep G2 cell line. At 10-8 M a two fold increase of PAI-1 antigen and activity were observed whereas a2 antiplasmin and fibrinogen were not significantly modified. No effect of insulin was observed on PAI-1 antigen and PAI activity production by human endothelial cells whereas endotoxin resulted in a two fold increase in PAI-1 secretion. In recent clinical studies we have demonstrated that the level of plasma insulin correlated with that of PAI-1. Thus we hypothesize that hepatocytes represent a physiological source of plasma PAI-1 which is modulated by plasma insulin level.
Platelet Agglutinating Protein p37 Causes Platelet Agglutination through its Binding to Membrane Glycoprotein IV
Authors: Lian Eric C-Y , Siddiqui Farooq A , Jamieson G A , Tandon Narendra N 
Published: July 2018

SummaryA 37 kDa platelet agglutinating protein (PAP p37) has previously been shown to be present in a subset of patients with thrombotic thrombocytopenic purpura and has been purified from their plasma. Using solubilized platelet membrane proteins from normal donors, it was shown by Western blotting that r2sI-p37 bound to a membrane protein of 97 kDa (red/unred). Furthernore, the same protein was identified by reverse immunoblotting in which purified p37 was electrophoresed, transferred to the nitrocellulose sheet and incubated with solubilized normal platelet membrane proteins. The complex formed between p37 and the membrane protein was identified by autoradiography using polyclonal and monoclonal (OKM5) anti- GPIV antibodies, but was not detected by polyclonal antibody to GPIIIa. Similar studies with purified platelet GPIV under both reducing and non-reducing conditions demonstrated the binding of 125I-p37. Polyclonal and monoclonal antibodies to GPIV completely inhibited the platelet agglutination induced by TTP plasma containing p37, however, normal rabbit IgG, rabbit anti- GPIIIa IgG, and murine monoclonal anti-GPIIb/IIIa (10E5) antibodies had no effect. These data indicate that platelet GPIV is the receptor site for PAP p37.
A Monoclonal Antibody to the Human Platelet Glycoprotein II b/III a Complex Induces Platelet Activation
Authors: Modderman Piet W , Huisman Han G , Mourik Jan A van , Borne Albert E G Kr von dem 
Published: June 2018

SummaryThe platelet glycoprotein (GP) IIb/IIIa complex functions as the receptor for fibrinogen on activated platelets. The effects of two anti-GPIIb/IIIa monoclonal antibodies on platelet function were studied. These antibodies, 6C9 and C17, recognized different epitopes, which were exclusively present on the undissociated GPIIb/IIIa complex. Whereas C17 inhibited the binding of fibrinogen to platelets and platelet aggregation induced by adenosine diphosphate (ADP) or collagen, 6C9 caused irreversible aggregation of platelets, both in the presence and absence of extracellular fibrinogen. When incubated with unstirred (nonaggregating) platelets, 6C9 induced release of alpha and dense granule-constituents as well as binding of 125I-fibrinogen to platelets. The latter was evidently mediated in part by platelet-derived ADP, since it was inhibited to a large extent by apyrase, the ADP-hydrolyzing enzyme. F(ab’)2 fragments of 6C9 did not induce platelet-release reactions but caused (slow) aggregation of platelets in the presence of extracellular fibrinogen.These results indicate that binding of an antibody to a specific site on the platelet GPIIb/IIIa complex may cause fibrinogen-mediated aggregation. The Fc part of the platelet-bound antibody appears to be involved in the induction of platelet release.
Platelet Function and the Bleeding Time in Progressive Renal Failure
Authors: Gordge M P , Faint R W , Rylance P B , Neild G H 
Published: June 2018

SummaryBleeding time and platelet function tests were performed on 31 patients with progressive chronic renal failure (CRF) due to non-immunological (urological) causes, and compared with 22 healthy controls. Patients were classified as mild (plasma creatinine <300 μmol/l), moderate (300-600 μmol/l) or severe renal failure (>600 μmol/l). Bleeding time was rarely prolonged in mild and moderate CRF and mean bleeding time significantly elevated only in severe CRF (p <0.005). Haematocrit was the only index which correlated with bleeding time (r = -0.40). Platelet counts, collagen stimulated thromboxane generation, and platelet aggregation responses to ADP, collagen and ristocetin were all either normal or increased in all three CRF groups, but thromboxane production in clotting blood was reduced. Plasma fibrinogen, C reactive protein and von Willebrand factor (vWF) were elevated in proportion to CRF. We found no evidence that defects in platelet aggregation or platelet interaction with vWF prolong the bleeding time in patients with progressive CRF.
Haemophilia A in a Female: Study of a Family Using Intragenic and Extragenic Restriction Site Polymorphisms
Authors: Piétu G , Thomas-Maison N , Sié P , Larrieu M J , Meyer D 
Published: June 2018

SummaryRestriction fragment length polymorphisms(RFLPs) were studied in a large Algerian family which includes 6 haemophiliacs and a previously described case of female haemophilia A. The female propositus is 66 years old with a normal karyotype. Her parents are first cousins. Her 3 sons are haemophiliacs and her 3 daughters with affected children are obligate carriers. The proband has an excessive bleeding tendency and markedly reduced levels of F. VIII (VIII C 0.03 U/ml, VIII Ag 0.01 U/ml) with elevated vWF Ag (2.30 U/ml), similar to the levels observed in affected males from the family. Four RFLPs can be identified by Southern blotting after digesting genomic DNA with the restriction enzymes Bcl I, Bgl I, Kpn I/Xba I and Taq I and hybridization with a 647 bp Stu I/Sca I F. VIII genomic probe, a 1.8 Kb EcoRI F. VIII cDNA probe, a 1.0 Kb EcoRI/Sst I fragment of intron 22 and the extragenic probe ST 14, respectively. With these four RFLPS, the propositus was found to be homozygous for the alleles segregating in this family with the abnormal X-chromosome. The carrier status was proven in a granddaughter and excluded in another. In conclusion, this RFLP linkage analysis is another argument to suggest that the propositus, a rare case of female haemophilia, is homozygous for the abnormal gene.
Investigation by HPLC of the Catabolism of Recombinant Tissue Plasminogen Activator in the Rat
Authors: Harris Roy , Frade Louis Garcia , Creighton Lesley J , Gascoine Paul S , Alexandroni Maher M , Poole Stephen , Gaffney Patrick J 
Published: June 2018

SummaryThe catabolism of recombinant tissue plasminogen activator (rt-PA) was investigated after injection of radiolabelled material into rats. Both Iodogen and Chloramine T iodination procedures yielded similar biological activity loss in the resultant labelled rt-PA and had half lives in the rat circulation of 1 and 3 min respectively. Complex formation of rt-PA was investigated by HPLC gel exclusion (TSK G3000 SW) fractionation of rat plasma samples taken 1-2 min after 125I-rt-PA injection. A series of radiolabelled complexes of varying molecular weights were found. However, 60% of the counts were associated with a single large molecular weight complex (350–500 kDa) which was undetectable by immunologically based assays (ELISA and BIA) and showed only low activity with a functional promoter-type t-PA assay. Two major activity peaks in the HPLC fractions were associated with Tree t-PA and a complex having a molecular weight of ̴ 180 kDa. HPLC fractionation to produce these three peaks at various timed intervals after injection of 125I-rt-PA showed each to have a similar initial rate half life in the rat circulation of 4-5 min. The function of these complexes as yet is unclear but since a high proportion of rt-PA is associated with a high molecular weight complex with a short half life in the rat, we suggest that the formation of this complex may be a mechanism by which t-PA activity is initially regulated and finally cleared from the rat circulation.
Anti-thrombin III and Heparin Clotting Times in Thrombosis and Atherosclerosis
Authors: O’Brien J. R 
Published: June 2018

SummaryA survey of the literature shows that when whole blood clots thrombin is formed and there is a decrease in anti-thrombin III (Anti-Th. Ill) and an increase in heparin neutralizing activity (HNA) which is probably identical to platelet factor 4 derived from platelets. Many studies of atherosclerosis and of arterial and venous thrombosis using various tests thought to measure Anti-Th. Ill and HNA report a decrease in Anti-Th. Ill or an increase in HNA or both. We have measured both in patients with atherosclerosis and survivors of myocardial infarction. The HNA was increased and the serum anti-thrombic activity was decreased relative to controls and there was an inverse correlation between the two measurements. All this evidence suggests that some kind of mild chronic intravascular coagulation may occur in atherosclerosis. These changes could be related to the cause or the result of atherosclerosis.