bluuebunny/crossref_metadata_2025 huggingface dataset:
Conformational Changes in Low Molecular Weight Kininogen Alters Its Ability to Bind to Endothelial Cells
Authors: Hasan Ahmed A K , Zhang Jie , Samuel Mathew , Schmaier Alvin H
Published: July 2018

The plasma kininogens, high (HK) and low (LK) molecular weight kininogens, are the parent proteins for bradykinin, a potent vasoactive peptide that locally influences vascular biology. Binding of both HK and LK to the endovascular wall contributes to bradykinin delivery. Recently, we found one preparation of LK (LKd) which had reduced inhibition of biotin-HK binding to endothelium. The functional defect in LKd was not merely due to bradykinin loss because two preparations of bradykinin-free LK blocked biotin-HK binding. However, using two different particular monoclonal antibodies to bradykinin, LKd, but no other preparation of LK, had its epitope to bradykinin exposed on nonreduced samples on immunoblot. These data suggested that LKd had an altered conformation which exposed the amino terminal arginine of bradykinin to antigenic detection. The altered conformation of LKd allowed it to be more susceptible to trypsin proteolysis. On circular dichroism, the percentage of α-helix was significantly increased, indicating an alteration in the protein. This alteration in LKd was not due to a loss of molecular mass of the protein. On laser desorption mass spectroscopy, the molecular mass of LKd was similar to the other preparations of LK. Investigations were performed to ascertain the mechanism by which LKd had altered ability to bind to cells. LKd was found to be proteolyzed by an unknown protease at the beginning of domain 2 between threonine119and alanine120. Reduction of functional LK with dithiothreitol to expose its bradykinin epitope did not produce the LKd defect. Proteolysis of functional LK with plasma kallikrein, elastase followed by plasma kallikrein, chymotrypsin, or bromelain also did not produce the defect seen in LKd. These combined data indicated that LK maintains a particular conformation that allows the protein to orient itself such that it can bind to endothelial cells. Proteolysis in the surface exposed region between domains 1 and 2 probably allows for the protein to unfold and contributes to its lost ability to bind to endothelial cells.
Orientation of the Carboxy-terminal Regions of Fibrin γ Chain Dimers Determined from the Crosslinked Products Formed in Mixtures of Fibrin, Fragment D, and Factor XIIIa
Authors: Siebenlist Kevin R , Meh David A , Wall Joseph S , Hainfeld James F , Mosesson Michael W
Published: July 2018

There are two schools of thought regarding the orientation of the intermolecular ∈-amino-(γ-glutamyl) lysine isopeptide bonds formed between γ chains in the D domains of assembled fibrin fibers. Some investigators believe that these bonds are oriented parallel to the direction of fiber growth (longitudinally) at the contacting ends of fibrin D domains (‘DD-long’), whereas others believe that these bonds are oriented across the two-stranded fibril, between D domains in opposing strands (‘DD-transverse’). To distinguish between these two possibilities, the structure of crosslinked products formed in mixtures of fibrin, plasmic fragment D, and factor XIIIa were analyzed, based upon this rationale: Complex formation between D fragments and a fibrin template depends upon the non-covalent ‘D:E’ interaction between each fibrin E domain and two D fragments (‘D:fibrin:D’). If carboxy-terminal γ chains in the D:fibrin:D complex become aligned in a DD-long configuration, only crosslinked fragment D dimers (‘D-D’) will result and the fibrin ‘template’ will not become crosslinked to the associated D fragments. If instead, γ chain crosslinks form transversely between the D fragments and fibrin, covalently linked D-fibrin complexes will result.

SDS-PAGE of factor XIIIa crosslinked mixtures of fibrin and fragment D demonstrated products of a size and subunit composition indicating D-fibrin and D-fibrin-D formation. Small amounts of D dimers were also formed at the same levels as were formed in mixtures of fragment D and factor XIIIa alone. Electron microscopic images of D-fibrin-D complexes prepared under physiological buffer conditions demonstrated that the D fragments were associated with the central E domain of the fibrin molecule, but that they could be dissociated from this non-covalent association in 2% acetic acid. These findings indicate that γ chain crosslinks occur transversely in D:fibrin:D complexes and permit the extrapolated conclusion that γ chain crosslinks are also positioned transversely in an assembled fibrin polymer.
Magnesium Modifies the Responses of Platelets to Inhibitory Agents which Act Via cAMP
Authors: Hardy E , Glenn J , Heptinstall S , Rubin P C , Horn E H
Published: July 2018

This study has investigated the interaction of raised extracellular magnesium and agents which act via cAMP with respect to inhibition of platelet aggregation and effects on platelet cAMP accumulation.

Iloprost (3 ng/ml) and PGD2 (0.2 μg/ml) each caused time dependent increases in platelet cAMP which were significantly greater in the presence of 3 mM added MgS04 (p <0.01). Addition of ADP (5 μM) resulted in a fall in cAMP which remained higher in the presence of MgS04 (p <0.01).

Forskolin (5 μg/ml) and DN9693 (100 μM) also caused increments in platelet cAMP but these responses were not influenced by added MgS04. Addition of ADP resulted in a further increase in cAMP which was augmented by MgS04 (p <0.03). This increase was abolished by adenosine deaminase (1.2 U/ml).

These experiments show that MgS04 can modify the cAMP responses produced by iloprost and PGD2 and by forskolin and DN9693 when ADP is present. It appears that as well as inhibiting, ADP can also stimulate cAMP production under certain experimental conditions. This appears to be due to breakdown of ADP to adenosine.
Minimum Fragments of the Heparin Molecule Able to Produce the Accumulation and Change of the Sulfation Pattern of an Antithrombotic Heparan Sulfate from Endothelial Cells
Authors: Pinhal Maria A S , Santos Isabel A N , Silva Irani F , Dietrich Carl P , Nader Helena B
Published: July 2018

Heparin and low molecular weight heparins stimulate two to three fold the accumulation of an antithrombotic heparan sulfate secreted by endothelial cells in culture. This led us to search for the minimum structural requirements of the heparin molecule able to elicit the enhancement of the heparan sulfate. Fragments were prepared from heparin by degradation with bacterial heparinase and heparitinases. A heparin pentasulfated tetrasaccharide was shown to be the minimum structural sequence able to enhance two to three fold the secretion of heparan sulfate by endothelial cells. The stimulation is specific for the endothelial cell, is concentration dependent and the effect is already noticed afterone hour of exposure of the cells to heparinand the tetrasaccharide. Degradation of the [35S]-heparan sulfate synthesized in the presence of heparin or the tetrasaccharide has shown a higher degree of sulfation of its iduronic acid residues.
Estimation of the Carbohydrate Moiety of von Willebrand Factor in the Plasma of Patients with Subtypes 2a and 2b of von Willebrand Disease
Authors: Romeuf Christophe de , Samor Bruno , Mazurier Claudine
Published: July 2018

Von Willebrand disease (vWD) results from quantitative (types 1 and 3) or qualitative (type 2) deficiency of von Willebrand factor (vWF). This glycoprotein present in plasma is involved in platelet adhesion at the site of vascular injury and serves as the carrier of antihaemophilic A factor (FVIII). Whereas recent studies have identified mutations in patients suffering from type 2 vWD, the integrity of the carbohydrate moiety of vWF in these patients is still matter of debate. In order to analyse in the plasma milieu the carbohydrate content of plasma vWF from various well-characterized type 2 vWD patients, we developed a colorimetric assay in microtiter plate based on the use of peroxidase- conjugated lectins specific for either α 2-6 sialic acid or β 1-4 galactose. Removal of sialic acid from purified plasma vWF induced significant changes in the reactivity of both lectins. The analysis of various normal plasmas showed no influence of the blood groups and allowed us to compare various vWD patients. The reactivity of lectins for plasma vWFs from two type 2A and six type 2B vWD patients harbouring different mutations was not statistically different from that of a pool of normal plasmas. We conclude that the α 2-6 sialic acid and β 1-4 galactose content of plasma vWF is not altered in these patients affected with types 2A and 2B vWD.
Localization of Topically Applied TFPI Binding Sites in an Intimectomized Microvessel
Authors: Ornberg Richard L , Deune Gene E , Ozbek Mustafa R , Wun Tze-Chien , Khouri Roger K
Published: July 2018

Tissue factor pathway inhibitor, TFPI, has been shown to be highly effective as a topically applied antithrombotic in an arterial model of vascular thrombosis. To elucidate the mechanism and site of TFPI action, recombinant TFPI was conjugated to 30 nm diameter gold particles and used to localize the sites of TFPI binding in a traumatized microvessel by transmission electron microscopy. The model, the central artery of the rabbit ear, was transected, denuded of endothelial lining (intimectomized), and re-anastomosed. Prior to the restoration of blood flow, TFPI-gold or unconjugated gold particles in solution were applied by irrigation to the intimectomized vessel lumen. After 10 min of blood flow, the artery was harvested for electron microscopy. TFPI-gold binding was localized to the fine strands of fibrin that lined the lumen of the intimectomized section of the artery. Little or no binding was found on platelets, exposed smooth muscle, cell membrane fragments, or uninjured vessel segments. The TFPI-gold binding could be competed with native TFPI. TFPI-gold was inhibitory, although less potent than native TFPI, in a prothrombin time assay. Unconjugated gold exhibited very little binding in the vascular model. Hence, the TFPI-gold conjugate behaved like native TFPI. Our observations have identified the fibrin complex as an in vivo binding site for TFPI and suggest that this is an in vivo site of action for TFPI as a topical antithrombotic agent.
The Effects of KBT-3022, a New Anti-platelet Agent, on Hemorheological Properties in Guinea Pigs
Authors: Yamamoto Noriko , Yokota Koichi , Yamashita Akira , Oda Minoru
Published: July 2018

Using guinea pigs, a study was conducted on the effects of KBT-3022, a new anti-platelet agent, on hemorheological properties in various tests including blood filterability, blood viscosity, shear stress-induced red blood cell (RBC) deformability and contents of ATP and 2,3-diphosphoglycerate (2,3-DPG). Oral administration of KBT-3022 at 1 and 10 mg/kg significantly increased blood filterability, and significantly reduced blood viscosity at 10 mg/kg without changing the hematocrit, plasma fibrinogen concentration or plasma viscosity. KBT-3022 (10 mg/kg, p.o.) improved RBC deformability in response to shear stress, which was evoked by passing the blood through a thin tube. This dose of KBT-3022 also increased the contents of ATP and 2,3-DPG in RBC. These findings indicate that KBT-3022 may reduce blood viscosity as a sequel to improvement of RBC deformability through direct action on RBC. The increase in the intracellular levels of ATP and 2,3-DPG was considered to be involved in this improvement of hemorheological properties. These hemorheological effects of KBT-3022 appear to be promising for the treatment of patients with ischemic vascular disease.
A Novel Mechanism for Exposure of Fibrinogen Binding Sites on GPIIb-IIIa by a Monoclonal Antibody
Authors: Hato Takaaki , Watanabe Akito , Nakatani Shingo , Minamoto Yoko , Fujita Shigeru
Published: July 2018

Conformational changes in platelet membrane glycoprotein (GP) IIb-IIIa, whose nature is not defined, lead to exposure of fibrinogen binding sites. We have reported previously that F(ab’)2fragments of a monoclonal antibody, PMA4, directed against the GPIIb-IIIa complex- specific domain, induced binding of fibrinogen to platelets without causing intracellular activation, whereas Fab did not. In this study, we examined the mechanism responsible for the difference in the ability of PMA4 F(ab’)2and Fab to expose fibrinogen binding sites. PMA4 Fab had affinity for GPIIb-IIIa similar to that of PMA4 F(ab’)2. Addition of F(ab’)2goat anti-mouse Fab antibody to cross-link PMA4 Fab-bound GPIIb-IIIa molecules induced fibrinogen binding. There was a direct correlation between the number of molecules of PMA4 F(ab’)2and the amount of fibrinogen bound. PMA4 did not recognize ligand-induced binding sites (LIBS). These results suggest that the cross-linking of special sites on the GPIIb-IIIa complex-specific domain by bivalent antibody alters the conformation of GPIIb-IIIa to a state competent to bind soluble fibrinogen and that conformational changes in non-LIBS are involved in the mechanism for exposing fibrinogen binding sites on GPIIb-IIIa.
Identification of Novel Peptide Antagonists for von Willebrand Factor Binding to the Platelet Glycoprotein Ib Receptor from a Phage Epitope Library
Authors: Ricca George A , South Victoria , Searfoss George H , French Stephen , Cheadle Christopher , Murray Edward , Howk Richard , Jaye Michael
Published: July 2018

We have constructed a fusion phage epitope library in the filamentous bacteriophage fuse5. The library was made by inserting a degenerate oligonucleotide which encodes 15 variable amino acids into the NH2-terminal region of the phage gene III protein. This library, containing over 107different epitope bearing phage, has been used in an attempt to identify inhibitors of the von Willebrand factor (vWF)-platelet Glycoprotein lb interaction. The library was screened with a monoclonal antibody (RG46) that recognizes the GPIb binding domain of vWF (amino acids 445-733). A total of 30 clones falling into 8 classes have been identified that react with the RG46 antibody. Isolates from all 8 classes are positive by immunoblot analysis. The amino acid sequence of the gene III fusion protein from positive clones showed a strong homology to the known RG46 epitope. Peptides identified from the screen were synthesized and used to demonstrate that some of the synthetic peptides exhibited inhibitory activity towards ristocetin induced binding of vWF to the GPIb receptor. Thus, we have demonstrated that screening a fusion phage epitope library with a monoclonal antibody that inhibits vWF binding to the GPIb receptor can be a useful tool not only for mapping antibody recognizing determinants, but also can serve as a source for identifying novel peptides that are antagonists for vWF binding to the platelet GPIb receptor.
Definition of the Bleeding Tendency in Factor XI-Deficient Kindreds–A Clinical and Laboratory Study
Authors: Bolton-Maggs P H B , Patterson D A , Wensley R T , Tuddenham E G D
Published: July 2018

Individuals with severe factor XI deficiency are prone to excessive bleeding after injury or surgery, but the existence of a haemorrhagic tendency in partial factor XI deficiency is controversial. In this study, 172 members of 30 kindreds (20 non-Jewish) transmitting factor XI deficiency in North West England were interviewed and a bleeding history questionnaire completed. Blood was taken for coagulation assays. The questionnaires were categorised independently by two assessors to determine presence or absence of a bleeding tendency, in the absence of information about the factor XI level or family history. Analysis shows that 48% of heterozygotes have a bleeding tendency. Eighteen (60%) families came to attention because of bleeding problems in heterozygotes. Comparison of histories between partially deficient and non-deficient individuals demonstrated a higher incidence of menstrual problems, an increase in significant bruising, and an increased likelihood of excessive bleeding after tonsillectomy and dental extractions.

The incidence of von Willebrand’s disease was not increased, but individuals with heterozygous factor XI deficiency who were bleeders tended to have lower levels of factor VIIIc and von Willebrand factor, and were more commonly of blood group 0. These features may contribute to the bleeding tendency. There was no evidence of alteration in factor VII activity (as defined by the ratio of activity to antigen) between the bleeders and non-bleeders.

This is convincing evidence for abnormal bleeding in factor XI deficiency which is not confined to severely deficient patients.
Platelet Consumption in Thrombocythemia Complicated by Erythromelalgia: Reversal by Aspirin
Authors: Genderen Perry J J Van , Michiels Jan J , Strik Roel Van , Lindemans Jan , Van Vliet Huub H D M
Published: July 2018

The involvement of platelets in the pathogenesis of erythromelalgia, a frequent and characteristic microvascular thrombotic manifestation in patients with primary thrombocythemia and polycythemia rubra vera, was investigated by measuring the survival and turnover of 51Cr labeled autologous platelets in 10 patients with thrombocythemia complicated by erythromelalgia, in 10 asymptomatic thrombocythemia patients and in 6 subjects with reactive thrombocytosis. The mean platelet survival time of the erythromelalgia patients was 4.2 ± 0.2 days, which is significantly decreased as compared with asymptomatic thrombocythemia patients (6.6 ± 0.3 days, p <0.001) and patients with reactive thrombocytosis (8.0 ± 0.4 days, p <0.001). The mean platelet survival time of asymptomatic thrombocythemia patients was significantly decreased (p <0.01) as compared with reactive thrombocytosis patients. Treatment of erythromelalgia with aspirin increased the mean platelet survival time from 4.0 ± 0.3 days to 6.9 ± 0.4 days (p <0.001) and was associated with an elevation of the platelet count of 216 ± 30 × 109 platelets per liter (p <0.001). Coumadin failed to improve platelet survival or symptoms caused by erythromelalgia. The increased platelet consumption in erythromelalgia is attributed to the formation of platelet thrombi in the arterial microvasculature. This conclusion is supported by the ability of aspirin to interrupt platelet consumption and clinical features of erythromelalgia.
Complete Inhibition of Endotoxin-Induced Coagulation Activation in Chimpanzees with a Monoclonal Fab Fragment against Factor VII/VIIa
Authors: Biemond Bart J , Levi Marcel , ten Cate Hugo , Soule Howard R , Morris Lori D , Foster David L , Bogowitz Cheryl A , van der Poll Tam , Büller Harry R , ten cate Jan Wouter
Published: July 2018

Gram-negative sepsis is oftentimes complicated by activation of coagulation with disseminated intravascular coagulation and microthrombosis. This may contribute to the associated morbidity, multiple organ failure and death. Recent studies have established that the tissue factor-dependent pathway of blood coagulation has a significant participatory role in the initial endotoxin-induced activation of coagulation. Tissue factor (TF), expressed on the surface of activated monocytes and endothelial cells forms cell surface complexes with free circulating factors VII and VIIa. The latter complex proteolytically activates factors X and IX. Recent in vivo experiments have shown that a rapidly neutralizing TF monoclonal antibody prevents and arrests the endotoxin- induced activation of coagulation and similar studies have shown to reduce mortality in baboons. In this study we describe the preparation of a factor VII/VIIa neutralizing monoclonal Fab fragment and characterize its effect on in vivo activation of coagulation during experimental endotoxemia in chimpanzees.

Four chimpanzees received a bolus intravenous injection of 4 ng/kg endotoxin in combination with Fab fragments of a factor VII/VIIa neutralizing murine monoclonal antibody (12D10) at a dose of either 50 μg/kg (n = 2) or 100 μfig/kg (n = 2). Four control animals received a bolus injection of endotoxin alone. Administration of the 12D10 Fab fragments, immediately preceding the endotoxin bolus injection, effectively blocked the endotoxin-induced activation of coagulation. Plasma levels of products of in vivo activation, namely F1+2, TAT complexes and FpA remained at baseline values. The administration of 12D10 resulted in a rapid decline in factor VII/VIIa antigen levels which remained below 5 ng/ml for 180-240 min, followed by a rapid return to baseline levels. Endotoxin administration resulted in activation of the fibrinolytic system as reflected by a rapid increase in plasma plasmin- α2-antiplasmin complexes. Administration of 12D10 was without effect on the endotoxin-induced fibrinolytic activation.

In conclusion, this study confirmed the importance of the TF: VII complex in the initial, endotoxin-induced activation of coagulation which was completely blocked by neutralizing all free and tissue factor-complexed factor VII/VIIa by a specific monoclonal Fab fragment. Activation of fibrinolysis was not influenced. Therefore, neutralization of factor VII/VIIa might be a promising therapeutic option in preventing endotoxin-induced microthrombosis during Gram-negative sepsis.
Internalization and Degradation of Recombinant Human Coagulation Factor VIIa by the Human Hepatoma Cell Line HuH7
Authors: Chang Glenn T G , Kisiel Walter
Published: July 2018

Previous studies demonstrated that several normal and transformed cultured human cell lines specifically bind human coagulation factors VII and VIIa via tissue factor. In the present study, we show that 125I- radiolabeled recombinant human factor VIIa (125I-rFVIIa) binds to a human hepatoma cell line (HuH7). In the presence of rabbit polyclonal anti-human tissue factor apoprotein IgG, binding of 125I-rFVIIa to the HuH7 cells was decreased ∼60%, suggesting the presence of tissue factor-independent binding sites for 125I-rFVIIa on these cells. The binding isotherm of 125I-rFVIIa for the HuH7 cells in the presence of anti-tissue factor IgG exhibited a hyperbolic profile and was time-, temperature- and calcium-dependent. Furthermore, binding at 4° C was specific, dose-dependent and saturable. Scatchard analysis of the binding data demonstrated a single class of binding sites with a dissociation constant (Kd) of 3.2 nM and 27,000 binding sites per cell. At 4° C, 125I-rFVIIa bound to, and eluted from, the cell was indistinguishable from offered 125I-rFVIIa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography.

The molecular properties of the tissue factor-independent binding protein were studied by using the cleavable cross-linking agent 3, 3’- dithiobis(sulfosuccinimidylpropionate). A cross-linking product of 125I-rFVIIa and a cell surface protein with an apparent Mr ∼100,000 was observed. The cross-linking reaction was strongly inhibited by a 100-fold molar excess of unlabeled rFVIIa, but not by rabbit polyclonal anti-human tissue factor apoprotein IgG, indicating that cross-linking does not involve the extracellular domain of tissue factor.

After binding, internalization of 125I-rFVIIa by the HuH7 cells was observed at 37° C in the presence of rabbit polyclonal anti-human tissue factor apoprotein IgG. Internalization of 125I-rFVIIa proceeds at a rate of 0.4 fmol 125I-rFVIIa/min/106 cells, reaching a steady state after 2 h. Following binding and internalization, 125I-rFVIIa reappeared in the culture medium within 30 min, at approximately the same rate as internalized 125I-rFVIIa. This 125I-rFVIIa probably represents degradation of 125I-rFVIIa into small peptides, since it could not be precipitated by rabbit polyclonal affinity-purified anti-FVII IgG or by trichloroacetic acid. Chloroquine treatment of the HuH7 cells inhibited the degradation of 125I-rFVIIa, suggesting that degradation presumably occurs via a lysosomal-dependent pathway. These studies demonstrate that the liver may play an important role in the clearance mechanism(s) of FVIIa.
Antinociceptive Properties of Protein C in a Model of Inflammatory Hyperalgesia in Rats
Authors: Pichler L , Schramm W , Ulrich W , Varadi K , Schwarz H P
Published: July 2018

We investigated the role of human protein C in an animal model of inflammatory hyperalgesia. Pain was induced by intraplantar injection of carrageenan (3 mg) into the hind paw of rats. The pain threshold was measured by exerting increasing amounts of pressure (in mmHg) on the paw until a struggle reaction was observed. Protein C (8-800 IU/kg) was administered intravenously immediately after carrageenan. Controls received either intraplantar injections of saline (100 µ1) instead of carrageenan or carrageenan alone. Effects on pain threshold were expressed in percent of the pretreatment value. Carrageenan alone lowered the mean pain threshold after 3 h to 33.2 ± 2.2% of the pretreatment level. Addition of protein C resulted in a dose-dependent rise in pain threshold towards the level observed in control animals treated with saline instead of carrageenan (pain threshold after 800 IU/kg protein C = 62.9 ± 2.3% of pretreatment level), demonstrating an antinociceptive effect. Protein C had no effect in animals not preconditioned with intraplantar carrageenan. Thus protein C clearly antagonized the inflammatory pain induced by carrageenan. The antinociceptive action of protein C was antagonized by injection of a monoclonal antibody against protein C, providing additional evidence that the effect was protein C-mediated.
ARIC Hemostasis Study-IV. Intraindividual Variability and Reliability of Hemostatic Factors
Authors: Nguyen Nghia D , Ghaddar Habib , Stinson Valarie , Chambless Lloyd E , Wu Kenneth K ,   for the Atherosclerosis Risk in Communities (ARIC) Study Investigators
Published: July 2018

We have recently reported the short-term intraindividual variability of several coagulation factors and inhibitors included in the ARIC study (Chambless et al. Ann Epidemiol 1992; 2:723). In this paper, we reported the intraindividual variability results of additional hemostatic factors. Blood samples were collected for hemostatic assays three times at 1-2-week intervals from 39 subjects recruited from 4 ARIC field centers. The contributions of within-person, processing and assay (designated “method”) and between-person variances to the total variance were estimated and from them the reliability coefficient, R, was computed as the proportion of total variance in the between-person component. The R value was high for (β-thromboglobulin and tissue- plasminogen activator: 0.83 and 0.81, respectively; and intermediate for D-dimer and plasminogen activator inhibitor-1: 0.73 and 0.72, respectively. Protein S (total and free) and platelet factor 4 had low repeatability (R<0.50) derived mostly from “method” variability while low R value (0.03) for fibrinopeptide A was attributed to high “method” and “within-person” variability. Gender, age and the level of hemostatic factors did not influence the intraindividual variability.
Up-Regulated Expression of Plasminogen Activator Inhibitor-1 in Hep G2 Cells: Interrelationship between Insulin and Insulin-Like Growth Factor 1
Authors: Anfosso F , Alessi M C , Nalbone G , Chomiki N , Henry M , Juhan-Vague I
Published: July 2018

Insulin resistance represents a situation with a high risk of athero-thrombosis and is accompanied by increased plasma plasminogen activator inhibitor-1 (PAI-1) levels. Fasting insulin level is highly correlated with PAI-1 levels in plasma. It has been shown that insulin increases PAI-1 synthesis by the human hepatoma cell line Hep G2. Moreover when Hep G2 cells expressing a down-regulation of insulin receptors by incubation with 10-7 M insulin, were stimulated by 10-9 M insulin, an overexpression of PAI-1 synthesis was observed despite a reduced number of insulin receptors. Insulin-like growth factor 1 (IGF-1) shares many properties with insulin. The aim of the present study was to evaluate the effect of IGF-1 on PAI-1 synthesis by Hep G2 cells down-regulated either by insulin or IGF-1.

Incubation of Hep G2 cells with increasing doses from 10-9 to 10-7 M IGF-1 induced a dose-dependent stimulation of PAI-1 synthesis up to 4.5-fold the control level. When cells were first pre-incubated with 10-7M IGF-1 for 18 h, acid washed, and then stimulated with 10-9 M IGF-1, the expression of IGF-1 receptors was greatly reduced (up to 70%). In contrast PAI-1 secretion was increased 3.4-fold the level of control cells and by 1.9-fold the level of cells first stimulated with 10-9M IGF-1. Both transcripts of PAI-1 mRNA were also increased. The overexpression of PAI-1 synthesis was observed irrespective of the hormone used in the down-regulation step (i.e. 10-9 M insulin or IGF-1) or in the stimulation step (i. e. 10-9 M insulin or IGF-1). The results showed an interrelationship between insulin and IGF-1 on PAI-1 synthesis in down-regulated Hep G2 cells. They also suggest that in the insulin resistant state, IGF-1 would be able to participate in the increase in PAI-1 plasma levels by stimulating down-regulated insulin target cells.
Acute Dynamic Exercise Increases Fibrinolytic Activity
Authors: Rankinen Tuomo , Väisänen Sari , Penttilä llkka , Rauramaa Rainer
Published: July 2018

The influence of acute bouts of dynamic exercise on plasma tissue plasminogen activator (tPA), plasminogen activator inhibitor (PAI) and fibrinogen was investigated on nine healthy non-smoking men aged 23 to 37 years. Subjects performed maximal and two submaximal (duration 30 min, intensity 50% [aerobic threshold] and 78% [anaerobic threshold] V02max) bicycle ergospirometry tests separated by seven days. The order of the submaximal tests was randomized. Blood samples were drawn before, immediately after and 24 h after each test. Plasma tPA and PAI activities were measured amidolytically, and fibrinogen concentration by thrombin method. All postexercise values were corrected for the change in plasma volume. The pre-exercise tPA and PAI activities and fibrinogen concentration were similar in each tests. tPA activity increased during each test (from 2.0 [SEM 0.24] IU/ml to 20.3 [3.14] during maximal, 2.0 [0.22] to 19.0 [2.59] during anaerobic threshold test, 1.8 [0.22] to 5.5 [0.82] during aerobic threshold; p <0.001 during all tests, p = 0.01 aerobic threshold vs maximal and anaerobic threshold). PAI activity decreased during maximal (from 6.6 [2.51] AU/ml to 2.0 [2.00], p <0.05) and anaerobic threshold (5.0 [2.07] to 0.2 [0.22], p <0.01) tests but not during aerobic threshold test (7.0 [3.69] to 4.5 [2.93], p = 0.123). One subject had clearly higher pre-exercise PAI activity and smaller tPA response to exercise as compared to other subjects. All 24 h post-exercise activities were similar to pre-exercise values. Plasma fibrinogen concentration did not change during any tests. Single acute bouts of dynamic exercise transiently increase tPA and decrease PAI activity without affecting plasma fibrinogen concentration in healthy young men.
Association of Variables of Coagulation, Fibrinolysis and Acute-phase with Atherosclerosis in Coronary and Peripheral Arteries and those Arteries Supplying the Brain
Authors: Heinrich Jürgen , Schulte Helmut , Schönfeld Rainer , Köhler Ekkehart , Assmann Gerd
Published: July 2018

We investigated the vessel status of coronary and peripheral arteries and those arteries supplying the brain in 929 consecutive male patients admitted to a coronary rehabilitation unit. The severity of coronary atherosclerosis was scored using coronary angiography. Changes in extracranial brain vessels and manifest cerebrovascular disease (CVD) were determined by B-mode ultrasound and Doppler examination. Peripheral arterial disease (PAD) was diagnosed using base-line and stress oscillography. We assessed variables of coagulation, fibrinolysis, and the acute phase response.

There was a significant increase in plasma fibrinogen, plasminogen, d-dimer and C-reactive protein (CRP) with increasing severity of coronary heart disease. Compared to men with unaffected arteries, men with 3 diseased coronary arteries had 58% greater d-dimer concentrations. Patients with CVD and PAD, respectively, also had significantly higher fibrinogen, d-dimer and CRP concentrations. We did not find an association between plasminogen activator inhibitor activity and the severity of coronary atherosclerosis.

In conclusion, plasma fibrinogen, d-dimer and CRP concentrations were significantly related to atherosclerosis in the coronary, peripheral and extracranial brain arteries.
Inhibition of Thrombin by Antithrombin III and Heparin Cofactor II In Vivo
Authors: Liu Longbin , Dewar Lori , Song Yingqi , Kulczycky Myron , Morris Blajchman A , Fenton John W , Andrew Maureen , Delorme M , Ginsberg Jeffrey , Preissner Klaus T , Ofosu Frederick A
Published: July 2018

The critical role of thrombin in the pathogenesis of venous and arterial thrombosis, and the effectiveness of glycosaminoglycans as antithrombotic drugs are well known. Antithrombin III is a major inhibitor of thrombin and augmentation of its inhibitory actions by heparin is the basis for the clinical uses of heparin. Recent clinical and experimental studies have demonstrated that another glycosaminoglycan, dermatan sulfate, is an effective antithrombotic drug. Dermatan sulfate catalyses the inhibition of thrombin by heparin cofactor II. The concentrations of heparin cofactor II are higher in the plasmas of individuals with congenital antithrombin III deficiency and pregnant women than controls. The role of heparin cofactor II as a physiologic thrombin inhibitor is unknown. Enzyme-linked immunosorbent assays were used to quantify thrombin-heparin cofactor II and thrombin-antithrombin III endogenous to the plasmas of adult antithrombin III-Hamilton deficient subjects, their siblings with normal antithrombin III levels, pregnant women at term and 3 to 5 days after delivery. Both thrombin-antithrombin III and thrombin-heparin cofactor II complexed with vitronectin were detected in all the plasmas. Significantly, the concentrations of thrombin-heparin cofactor II-vitronectin were higher in the plasmas of congenital antithrombin III deficient subjects and in pre- and post-delivery plasmas than those of normal subjects. In addition, the concentrations of thrombin-heparin cofactor II decreased 3 to 5 days after delivery, reflecting the disappearance of the catalytically active dermatan sulfate elaborated by the placenta. Thus, heparin cofactor II normally inactivates thrombin in vivo, with its role increasing in conditions associated with high levels of heparin cofactor II and/or dermatan sulfate.
Reproducibility of Fibrinolytic Response to Venous Occlusion in Healthy Subjects
Authors: Stegnar Mojca , Mavri Alenka
Published: July 2018

Fibrinolytic response to venous occlusion is used to assess the efficiency of the fibrinolytic system. Reproducibility of fibrinolytic variables after 20 min upper arm venous occlusion was investigated in 40 apparently healthy subjects tested twice in a period of 11-23 (mean 15) days. Resting and post-venous occlusion euglobulin clot lysis time, tissue-type plasminogen activator (t-PA) activity, t-PA antigen, plasminogen activator inhibitor (PAI) activity and plasminogen activator inhibitor type 1 (PAI-1) antigen determined on the two occasions did not differ significantly. Positive correlation coefficients for variables before (r=0.43-0.74, all p<0.01) and after venous occlusion (r=0.07-0.66) indicated low to moderate associations between repeated measurements. Differences between repeated measurements relative to the initial measurement were greater after venous occlusion than before venous occlusion and were for euglobulin activity 41 (0-826)%, t-PA activity 27 (2-398)%, and for t-PA antigen 27 (0-179)% (medians and ranges). Poor responses were defined by the lowest (euglobulin activity, t-PA activity and t-PA antigen) or the highest (PAI activity) fifth percentile of the distribution. Almost no agreement between poor responses was observed: poor responses at the first occasion were not determined in the same subjects when re-examined after two weeks. It was concluded that due to relatively low reproducibility of the variables measured after venous occlusion the test needs improvement in order to be potentially clinically useful.
Interaction of Lp(a) with Plasminogen Binding Sites on Cells
Authors: Miles Lindsey A , Fless Gunther M , Scanu Angelo M , Baynham Patricia , Sebald Matthew T , Skocir Pamela , Curtiss Linda K , Levin Eugene G , Hoover-Plow Jane L , Plow Edward F
Published: July 2018

Lp(a) competes with plasminogen for binding to cells but it is not known whether this competition is due to the ability of Lp(a) to interact directly with plasminogen receptors. In the present study, we demonstrate that Lp(a) can interact directly with plasminogen binding sites on monocytoid U937 cells and endothelial cells. The interaction of Lp(a) with these sites was time dependent, specific, saturable, divalent ion independent and temperature sensitive, characteristics of plasminogen binding to these sites. The affinity of plasminogen and Lp(a) for these sites also was similar (Kd = 1-3 μM), but Lp(a) bound to fewer sites (̴10-fold less). Both gangliosides and cell surface proteins with car- boxy-terminal lysyl residues, including enolase, a candidate plasminogen receptor, inhibited Lp(a) binding to U937 cells. Additionally, Lp(a) interacted with low affinity lipoprotein binding sites on these cells which also recognized LDL and HDL. The ability of Lp(a) to interact with sites on cells that recognize plasminogen may contribute to the pathogenetic consequences of high levels of circulating Lp(a).
Interactions of Staphylokinase with Human Platelets
Authors: Lijnen H R , Van Hoef B , Collen D
Published: July 2018

The interactions of recombinant staphylokinase (SakSTAR) with human platelets were investigated in a buffer milieu, in a human plasma milieu in vitro, and in plasma from patients with acute myocardial infarction (AMI) treated with SakSTAR.

In a buffer milieu, the activation rate of plasminogen by SakSTAR or streptokinase (SK) was not significantly altered by addition of platelets. Specific binding of SakSTAR or SK to either resting or thrombin- activated platelets was very low. ADP-induced or collagen-induced platelet aggregation in platelet-rich plasma (PRP) was 94 ± 2.7% or 101 ± 1.7% of control in the presence of 0.1 to 20 μM SakSTAR, with corresponding values of 95 ± 2.8% or 90 ± 4.6% of control in the presence of 0.1 to 4 μM SK. No effects were observed on platelet disaggregation. ATP secretion following collagen-induced platelet aggregation was 4.3 ± 0.26 μM for SakSTAR (at concentrations of 0.1 to 20 μM) and 4.4 ± 0.35 μM for SK (at concentrations of 0.1 to 4 μM), as compared to 3.4 ± 0.70 μM in the absence of plasminogen activator.

Fifty % lysis in 2 h (C50) of 60 μl 125I-fibrin labeled platelet-poor plasma (PPP) clots prepared from normal plasma or from plasma of patients with Glanzmann thrombasthenia and immersed in 0.5 ml normal plasma, was obtained with 12 or 16 nM SakSTAR and with 49 or 40 nM SK, respectively. C50 values for lysis of 60 μl PRP clots prepared from normal or patient plasma were also comparable for SakSTAR (19 or 21 nM), whereas SK was 2-fold more potent toward PRP clots prepared from Glanzmann plasma as compared to normal plasma (C50 of 130 versus 270 nM).

No significant effect of SakSTAR on platelet function was observed in plasma from patients with AMI treated with SakSTAR, as revealed by unaltered platelet count, platelet aggregation and ATP secretion.

Thus, no effects of high SakSTAR concentrations were observed on human platelets in vitro, nor of therapeutic SakSTAR concentrations on platelet function in plasma.
Vampire Bat Salivary Plasminogen Activator Evokes Minimal Bleeding Relative to Tissue-Type Plasminogen Activator as Assessed by a Rabbit Cuticle Bleeding Time Model
Authors: Mellott Michael J , Ramjit Denise R , Stabilito Inez I , Hare Timothy R , Senderak Edith T , Lynch Joseph J , Gardell Stephen J
Published: July 2018

Cuticle bleeding time (CBT) measurements in anesthetized rabbits were performed to assess the potential bleeding risks which may accompany the administration of tissue-type plasminogen activator (tPA) or vampire bat salivary plasminogen activator (BatPA). The dose of BatPA or tPA used in this study, 42 nmol/kg, was previously shown to be efficacious using a rabbit femoral artery thrombosis model (Gardell et al, Circulation 84:244, 1991). CBT was determined by severing the apex of the nail cuticle and monitoring the time to cessation of blood flow. CBT was minimally elevated (1.6-fold, p<NS) following bolus intravenous administration of BatPA; in contrast, bolus intravenous administration of tPA dramatically elevated CBT (6.2-fold, p<0.05). Rabbits treated with tPA, but not BatPA, displayed profound activation of systemic plasminogen and consequent degradation of Factor VIII and fibrinogen. Elevations in CBT after the administration of tPA were reversed by the replenishment of plasma Factor VIII activity to 40% of control, but were unaffected by complete replenishment of plasma fibrinogen. The results of this study suggest that the administration of BatPA, at a dose that promotes thrombolysis, may evoke a minimal bleeding risk, relative to an equi-efficacious dose of tPA. In addition, the tPA-provoked proteolytic consumption of Factor VIII may be a key contributor to the heightened bleeding risk.
Antithrombotic Activity in vivo of SDZ 217-766, a Low-molecular Weight Thrombin Inhibitor in Comparison to Heparin
Authors: Tapparelli Carlo , Metternich Rainer , Gfeller Patrick , Gafner Bernhard , Powling Michael
Published: July 2018

Antithrombotic potency of SDZ 217-766, a potent inhibitor of thrombin and other trypsin-like serine proteases, was studied in comparison with heparin in rat models of thrombin induced lung platelet accumulation, of thrombosis in arterio-venous shunt, and of venous thrombosis induced by tissue factor. Thrombin-induced platelet accumulation in the lung was inhibited dose-dependently by SDZ 217-766 following intravenous (i.v.) administration of 0.03 mg/kg to 0.3 mg/kg as well as by intraduodenal (i.d.) administration of 3 mg/kg and 10 mg/kg. Comparable inhibitory effects were observed with heparin at 30 IU/kg and 100 IU/kg. In the rat arterio-venous shunt, following i.v. administration of SDZ 217-766, thrombus formation was inhibited by 40% at 0.1 mg/kg, by 60% at 0.3 mg/kg and was abolished at 1.0 mg/kg whilst APTT was prolonged 1.1 fold over the control value at 0.1 mg/kg and 2.7 fold at 1.0 mg/kg. Similar inhibitory effects were observed following i.d. administration of 10 and 30 mg/kg with only marginal (1.2 to 1.8 fold) APTT elevation. In the same model, heparin administered either i.v., 30-300 IU/kg, or subcutaneously, 100 and 300 IU/kg, inhibited thrombus formation dose dependently but in contrast to SDZ 217-766, the inhibitory effect was paralleled by 5-to >10 fold APTT elevation over baseline. In the venous thrombosis model, SDZ 217-766 infused at 10 μg/kg/min and 20 pg/kg/min, reduced thrombus formation by 35% and 70%, respectively. In comparison, thrombus formation was decreased by 22% when heparin was infused at 1 IU/kg/min, and abolished at 3 IU/kg/min. At the doses tested, APTT was prolonged much less with SDZ 217-766 (1.6 to 2.7 fold over baseline) than with heparin (2.8 to >10 fold over baseline).

The ability of SDZ 217-766, compared with heparin, to prevent thrombus formation with a minimum degree of anticoagulation together with its potential oral bioavailability make SDZ 217-766 an interesting candidate for the use in thrombosis prevention. For the use in thrombosis treatment, however, the restricted selectivity profile of SDZ 217-766 for thrombin over other serine proteases may limit its therapeutic potential.
Inhibition of Prothrombinase by Antithrombin-Heparin at a Macroscopic Surface
Authors: Speijer Han , Billy Didier , Willems George , Hemker H Coenraad , Lindhout Theo
Published: July 2018

The antithrombin-dependent inhibition of prothrombinase, assembled at a macroscopic surface, was studied under flow conditions utilizing a tubular flow reactor that consists of a phospholipid-coated glass capillary. Prothrombinase activity was determined from steady-state rates of thrombin production upon perfusion with prothrombin and from factor Va-associated factor Xa activity present in the flow reactor. The prothrombinase density was maintained at a low level (0.03 fmol/cm2) to assure that the rate of thrombin production reflected the amount of prothrombinase present in the capillary. Perfusion of the flow reactor with antithrombin resulted in an exponential decrease of prothrombinase activity in time. The second order rate constant (8.5 × 104 M-1min-1) is comparable with the rate of inactivation of free factor Xa. Inhibition was much faster when antithrombin was complexed with heparin. The second order rate constants of inhibition decreased with decreasing heparin chain length: 9.6 × 107, 4.5 × 107 and 0.39 × 107 M-1min-1 for unffactionated heparin, low molecular weight heparin and synthetic pentasaccharide heparin, respectively. In the presence of prothrombin (0.2 μM), however, the heparin-dependent rate of inhibition of prothrombinase was about 50-fold lower. The heparin-independent inhibition of prothrombinase by antithrombin (4 μM) in the presence of prothrombin (0.2 μM) was virtually negligible. At a 70-fold higher surface density of prothrombinase (2 fmol/cm2) prothrombinase activity was much faster inactivated. The rate of thrombin production, however, was not affected. In conclusion, at low prothrombinase densities, prothrombin efficiently protects prothrombinase from inhibition. At high densities, prothrombinase is much less protected but the higher rate of prothrombinase inactivation has no consequences for the thrombin production because of the transport-limited regime.
Cloning of an Avian Antithrombin: Developmental and Hormonal Regulation of Expression
Authors: Tejada Max L , Deeley Roger G
Published: July 2018

We present the complete amino acid sequence of a non-mammalian antithrombin together with a structural comparison to its mammalian homologs. In the majority of cases, but not all, the analyses indicate high degrees of primary structural identity within regions of predicted or demonstrated functional importance. We have also taken advantage of the avian model to define the ontogeny of antithrombin mRNA expression. Northern analyses indicate chicken antithrombin mRNA levels to be detectable in the liver early during development and increase to reach adult levels just prior to hatching. Although no sex-dependent differences in antithrombin mRNA levels were detected, the administration of estrogen to male roosters was observed to cause a 3-fold decrease in antithrombin mRNA levels, suggesting that the hormone may alter circulating antithrombin levels via a pre-translational mechanism.