bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ex Vivo Inhibition of Platelet Aggregation in Patients with Severe Limb Arteriopathy Treated with BAY U3405, a Specific IX A2 Receptor Antagonist
Authors: Bellucci S , Kedra W , Groussin H , Jaillet N , Molho-Sabatier P , Andreassian B , Tobelem G 
Published: July 2018

SummaryA double-blind, placebo-controlled randomized study with BAY U3405, a specific thromboxane A2 (TX A2) receptor blocker, was performed in patients suffering from severe stade II limb arteriopathy. BAY U3405 or placebo was administered in 16 patients at 20 mg four times a day (from day 1 to day 3). Hemostatic studies were done before therapy, and on day 2 and day 3 under therapy. On day 3, BAY U3405 was shown to induce a highly statistically significant decrease of the velocity and the intensity of the aggregations mediated by arachidonic acid (56 ± 37% for the velocity, 58 ± 26% for the intensity) or by U46619 endoperoxide analogue (36 ± 35% for the velocity, 37 ± 27% for the intensity). Similar results were already observed on day 2. By contrast, such a decrease was not noticed with ADP mediated platelet aggregation. Furthermore, plasma levels of betathrombo-globulin and platelet factor 4 remained unchanged. Peripheral hemodynamic parameters were also studied. The peripheral blood flow was measured using a Doppler ultrasound; the pain free walking distance and the total walking ability distance were determined under standardized conditions on a treadmill. These last two parameters show a trend to improvement which nevertheless was not statistically significant. All together these results encourage further in vivo studies using BAY U3405 or related compounds on a long-term administration.
A P-Selectin/CD62P Monoclonal Antibody (LYP-20), in Tandem with Flow Cytometry, Detects in vivo Activated Circulating Rat Platelets in Severe Vascular Trauma
Authors: Chignier Elza , Parise Maud , McGregor Lilian , Delabre Caroline , Faucompret Sylvie , McGregor John 
Published: July 2018

SummaryP-selectin, also known as CD62P, GMP140 or PADGEM, is present in platelet a-granules and endothelial cell Weibel-Palade bodies and is very rapidly expressed on the surface of these cells on activation. In this study, an anti P-selectin monoclonal antibody (LYP20) was used, in tandem with flow cytometry, to identify activated platelets at the site of induced vascular trauma or in peripheral blood. Moreover, electron microscopy was performed to characterize sites of vascular trauma and quantify the number of adhering platelets. The same induced vascular trauma was observed to result into animals responding in 2 different ways (Group I, Group II) following the degree of platelet activation. Five rats, out of 14 with induced vascular trauma, had more than half of their circulating platelets expressing P-selectin when drawn at the site of the trauma (67.4% ± 3.44) or in peripheral blood (78.5% ± 2.5) (Group I). In the remaining 9 animals a much smaller proportion of circulating platelets expressed P-selectin when assayed from trauma sites (18% ± 3.34) or in peripheral blood (18.0% ± 4.30) (Group II). Enhanced P-selectin expression by circulating platelets in Group I, compared to Group II, appears to be linked to the degree of activated platelets adhering at sites of trauma (171 ± 15 × 103 platelets versus 48 ± 31 × 103 platelets per mm2). In the 5 control animals, that were not operated on, platelets expressing P-selectin when drawn at the site of a mock trauma (7.0% ± 1.84) or in the peripheral blood (11.2% ± 3.30) showed little activation. In addition, no platelet adhesion was seen on the vascular bed of these animals. Results from this study show that analysis of P-selectin (CD62P) expression, in circulating platelets, is a valuable and rapid marker of platelet activation following severe vascular trauma induced in rats. However, activated platelets were not detected to the same extent in the peripheral blood of all animals having undergone vascular trauma. It is conceivable that platelets, depending on the degree of activation, may be actively sequestered in organs and prevented from circulating. Alternatively, P-selectin may be rapidly endocytosed, or not expressed, by activated circulating platelets depending on the type of agonists implicated in vivo activation.
Association of a Variable Number of Tandem Repeats (VNTR) in Glycoprotein Ibα and HPA-2 Alloantigens
Authors: Simsek S , Bleeker P M M , van der Schoot C E , von dem Borne A E G Kr 
Published: July 2018

SummaryThe human platelet alloantigen HPA-2 (Koa/Kob) system is involved in two clinical syndromes, neonatal alloimmune thrombocytopenia and platelet transfusion refractoriness. Wb have previously described that the human platelet alloantigens HPA-2a(Kob) and HPA-2b(Koa), are caused by a Thrl45Met amino acid polymorphism in the N-terminal globular domain of the human platelet glycoprotein (GP) Ibα. In the present study the question was addressed as to whether a genetic association exists between this Thrl45Met polymorphism and the recently described variable number of tandem repeat (VNTR) polymorphism in GP Ibα. Such an association has already been suggested by serological analysis (Ishida et al., 1991). This VNTR polymorphism results from a 13-amino-acid sequence repeat in the macroglycopeptide region of GP Ibα. Therefore, we developed a PCR method to analyze the VNTR region of 106 normal individuals who were also analyzed for the HPA-2 polymorphism. In this method genomic DNA derived from mononuclear cells was purified, the polymorphic region was amplified by PCR and was electrophoresed on agarose gels. Differences in the size of the PCR products made VNTR typing possible. Genotyping for the HPA-2 system was done by allele-specific restriction site analysis of PCR products with the restriction enzyme Sfa NI. The DNA derived from 12 HPA-2 (a-b+) subjects, contained only the B variant (with 3 repeats) of the VNTR polymorphism. The D variant (with 1 repeat) was only found in HPA-2a positive individuals. The C variant (with 2 repeats) was found to be strongly associated with HPA-2a. However, two members of a family with a HPA-2 (a+b+) genotype were found to be homozygous for the C variant of the VNTR polymorphism. This shows that the C variant can also be associated with HPA-2b. The A variant (with 4 repeats) was not encountered in the population studied. The strong association of HPA-2 and VNTR polymorphism, lying 761 bp apart on the GP Ibα gene, indicates linkage disequilibrium.
The Role of the Finger Domain of Tissue-type Plasminogen Activator (t-PA) and Plasminogen Activator Inhibitor 1 (PAI-1) in Initiation and Progression of Thrombolysis
Authors: Stringer Harold A R , van Swieten Peter , Horrevoets Anton J G , Smilde Annelies , Pannekoek Hans 
Published: July 2018

SummaryWe further investigated the role of the finger (F) and the kringle-2 (K2) domains of tissue-type plasminogen activator (t-PA) in fibrin-stimulated plasminogen activation. To that end, the action of purified (wt) t-PA or of variants lacking F (del.F) or K2 (del.K2) was assessed either in a static, human whole blood clot-lysis system or in whole blood thrombi generated in the “Chandler loop”. In both clot-lysis systems, significant differences were observed for the initiation of thrombolysis with equimolar concentrations of the t-PA variants. A relatively minor “lag phase” occurred in thrombolysis mediated by wt t-PA, whereas a 6.4-fold and 1.6-fold extension is found for del.F and del.K2, respectively. We observed identical lag-times, characteristic for each t-PA variant, in platelet-rich heads and in platelet-poor tails of thrombi. Since plasminogen activator inhibitor 1 (PAI-1) is preferentially retained in the platelet-rich heads, we conclude that the inhibitor does not interfere with the initial stage of thrombolysis but exerts its action in later stages, resulting in a reduction of the rate of clot lysis. A complementation clot-lysis assay was devised to study a potential interplay of del.F and del.K2. Accordingly, clot lysis was determined with combinations of del.F and del.K2 that were inversely varied in relation to equipotent dosage to distinguish between additive, antagonistic or synergistic effects of these variants. The isobole for combinations of del.F and del.K2 shows an independent, additive action of del.F and del.K2 in clot lysis. Under the conditions employed, namely a relatively high concentration of fibrin and Glu-plasminogen and a low concentration of t-PA variant, our data show: i) the crucial role of the F domain and the lack of effect of PAI-1 in initiation of thrombolysis, ii) the lack of importance of the fibrimbinding domains of t-PA and the regulatory role of PAI-1 in advanced stages of thrombolysis.
Neonatal Platelets Are Less Reactive than Adult Platelets to Physiological Agonists in Whole Blood
Authors: Rajasekhar Damodara , Kestin Anita S , Bednarek Francis J , Ellis Patricia A , Barnard Marc R , Michelson Alan D 
Published: July 2018

SummaryPrevious studies have reported that the platelets of healthy term neonates have either diminished or normal reactivity compared to the platelets of adults. To circumvent the methodologic problems of previous studies, we used a whole blood flow cytometric method to study neonatal platelet reactivity to thrombin, a combination of ADP and epinephrine, and U46619 (a stable thromboxane A2 analogue). Inclusion in the assay of the peptide GPRP (an inhibitor of fibrin polymerization) enabled us to study platelet reactivity to human α-thrombin in whole blood. Umbilical cord blood and day 1 peripheral blood were collected from 30 healthy term neonates and compared to peripheral blood from 20 normal adults. In whole blood samples without added agonist, there were no significant differences between neonates and adults in the platelet binding of monoclonal antibodies 6D1 (GPIb-specific) or 7E3 (GPIIb-IIIa complex-specific). As determined by S12 (a P-selectin-specific monoclonal antibody), neither neonates nor adults had circulating degranulated platelets. However, in both cord and peripheral whole blood samples, neonatal platelets were significantly less reactive than adult platelets to thrombin, ADP/epinephrine, and U46619, as determined by the extent of increase in the platelet surface expression of P-selectin and the GPIIb-IIIa complex, and the extent of decrease in the platelet surface expression of the GPIb-IX complex. For example, as compared to maximal platelet surface P-selectin in adults (with thrombin 10 U/ml), thrombin 1 U/ml resulted in platelet surface P-selectin of 95 ± 2% (mean ± S.E.M.) in adult peripheral blood, but only 70 ± 4% in cord blood and 70 ± 3% in neonatal peripheral blood (p <0.0001). Thrombin 0.1 U/ml resulted in platelet surface P-selectin of 49 ± 4% in adult peripheral blood, but only 10 ± 2% in cord blood and 17 ± 2% in neonatal peripheral blood (p α0.0001). Similar results were obtained in a washed platelet system. In summary: 1) Compared to adult controls, neonatal platelets are hyporeactive to thrombin, a combination of ADP and epinephrine, and a thromboxane A2 analogue in the physiologic milieu of whole blood. 2) The hyporeactivity of neonatal platelets compared to adult platelets is the result of a defect intrinsic to neonatal platelets. 3) Whole blood flow cytometry is particularly advantageous for neonatal studies because only 5 μl of blood per assay is required.
Increased Levels of Soluble Adhesion Molecules in Type 2 (Non-Insulin Dependent) Diabetes mellitus Are Independent of Glycaemic Control
Authors: Steiner M , Reinhardt K M , Krammer B , Ernst B , Blann A D 
Published: July 2018

SummaryPatients with Type 2 (non-insulin dependent) diabetes mellitus are at increased risk of thrombosis and the premature development of atherosclerosis. This may be related to damage to the endothelium (which may be the primary target tissue for the disease process) resulting from a loss of normal glycaemic metabolic control. Thus changes in endothelial cell function, such as modified release of soluble leukocyte and platelet adhesion molecules, may be important. Accordingly, E-selec-tin, von Willebrand factor (vWf), vascular cell adhesion molecule (VCAM) and intercellular adhesion molecule (ICAM) were measured in serum from 60 patients and 76 controls. Raised levels of vWf (p = 0.0002), E-selectin (p <0.0001) and VC AM (p = 0.003) in patient’s samples failed to correlate with glycaemic control as assessed by levels of fructosamine and glycated haemoglobin, or with 24 h urine albumin. Levels of ICAM were not increased in our patients. Levels of the two endothelial cell products, vWf and E-selectin, failed to correlate although E-selectin correlated with low density lipoprotein cholesterol (p = 0.016). vWf correlated with VCAM (p <0.001) and hypertension (p = 0.032). We conclude that levels of soluble adhesion molecules vWf, E-selectin and VCAM are raised in Type 2 diabetes mellitus. The mechanisms for these changes appear to be independent of glycaemic control but may relate to concurrent hypertension and/or hypercholes-terolaemia.
The Effects of Insulin-Like Growth Factor-1 on Plasminogen Activator Inhibitor-1 Synthesis and Secretion: Results from In Vitro and In Vivo Studies
Authors: Padayatty S J , Orme S , Zenobi P D , Stickland M H , Belchetz P E , Grant P J 
Published: July 2018

SummaryIn vitro studies have shown that insulin and IGF-1 releases the fibrinolytic inhibitor plasminogen activator inhibitor-1 (PAI-1) from cells of hepatic origin. To investigate the effects of IGF-1 on fibrinolysis: 1) cultured hepatoma cells were grown in the presence of IGF-1 and media collected for secreted PAI-1 and cells probed for PAI-1 mRNA, 2) 8 hypopituitary patients were treated with recombinant human growth hormone (rhGH) and 3) 5 type 2 diabetic patients were treated with recombinant human IGF-1 (rhIGF-1). Treatment of Hep G2 cells with IGF-1 (1000 ng/ml) increased secretion of PAI-1 from a median value of 80 ng/106 cells (range 21-91) to 144 ng/106 cells (range 128-169) after 24 h (p <0.01). Synthesis of PAI-1 mRNA increased in a similar fashion. Treatment of hypopituitary patients with rhGH led to an increase in circulating IGF-1 from a mean value of 166 (range 41-324) ng/ml at baseline to 322 (77-575) ng/ml at 4 weeks and 259 (104-533) ng/ml after 8 weeks (p <0.02). Despite this, no changes in circulating PAI-1 or fibrinolysis occurred. Type II diabetic patients treated with rhIGF-1 showed an increase in circulating IGF-1 from a mean value of 120 ng/ml (range 109-196), at baseline to 823 ng/ml (585-894) after 5 days. This also was not associated with changes in circulating PAI-1 or in fibrinolysis. The results confirm that IGF-1 induces the synthesis of PAI-1 in Hep G2 cells. However, marked increases in IGF-1 had no effect on circulating PAI-1 or fibrinolysis.
Fluorogenic Fibrinogen and Fibrin Facilitate Macromolecular Assembly and Dynamic Assay of Picomolar Levels of Plasminogen Activators under Well Mixed Conditions
Authors: Wu Jung-He , Diamond Scott L 
Published: July 2018

SummaryFibrinogen labeled with fluorescein isothiocyanate (FITC) was tested for its ability to serve as a template for macromolecular assembly as well as to provide a fluorogenic signal to allow continuous monitoring of plasminogen activation and fibrinolysis. As dilute solutions of FITC-fibrinogen or FITC-fibrin fiber suspension were degraded during lysis, release of fluorescent fragments abolished proximity- based quenching and resulted in a 2.0- or 3.6-fold increase in fluorescence intensity, respectively. Addition of plasmin at a final concentration of 10 pM to FITC-fibrinogen (10 nM) produced a detectable level of fluorescence dequenching. The assay had sufficient sensitivity to detect plasmin activity in the presence of excess antiplasmin activity, indicating the dissociation of a reversible antiplasmin-plasmin complex. The detection limit of the reaction assay was 20 pM and 200 pM of recombinant tPA and urokinase, using 10 nM FITC-fibrin and 10 nM and 5 nM plasminogen, respectively. The 10-fold greater sensitivity of the assay for tPA was likely due to the molecular assembly of tPA and plasminogen on the FITC-fibrin. Addition of thrombin (1 U/ml) and plasmin (0.1 nM) to 10 nM FITC-fibrinogen produced fluorescence quenching at first due to fibrinogen polymerization followed by dequenching due to fibrinolysis. Addition of 10 mM ∈-aminocaproic acid to mixtures of thrombin and plasmin allowed the quenching assay of thrombin activity in the presence of active plasmin. FITC-fibrinogen could be copolymerized with recalcified platelet poor plasma (isolated from citrated whole blood) to yield fibrin that was fluorogenic. Dequenching was observed when plasmin was used to degrade the fibrin formed from the platelet poor plasma. Given the large signal generated upon degradation of the fluorogenic fibrin(ogen), at least 105 determinations can be run from 100 mg of FITC-labeled fibrinogen using a standard fluorimeter and 0.1 to 3.0 ml reaction volumes. The versatility of the fluorogenic fibrinogen substrate allowed the configuration of assays to detect and measure the activity of thrombin, plasmin, tPA, uPA, and α2-antiplasmin. The ability to assemble blood proteins on a fluorogenic fibrinogen or fibrin template provides unique opportunities for the dynamic study of binding and enzymatic events on the fibrin surface under well mixed conditions.
Von Willebrand Activity of Low Molecular Weight Human Factor VIII Increases by Binding to Gold Granules
Authors: Furlan Miha , Perret Beat A , Beck Eugene A 
Published: July 2018

SummaryHuman factor VIII/von Willebrand protein is a population of multimers which vary in size but contain apparently identical subunits. Large-molecular-weight forms possess higher ristocetin cofactor/von Willebrand activity than the native smaller oligomers. Disulfide reduction of large factor VIII multimers results in progressively decreasing molecular size and a loss of ristocetin cofactor activity. Small molecular forms of factor VIII were adsorbed onto gold granules (average diameter 20-30 nm) and thereby increased their ristocetin cofactor activity. The amount of adsorbed material and the extent of activation were dependent on the pH of the colloid suspension. The maximum recovery of von Willebrand activity was observed at pH 4.75. Aggregation of fixed human platelets by factor VIII-coated gold particles was dependent on ristocetin concentration and was not competitively inhibited by unbound low-molecular-weight factor VIII. These results suggest that the subunits of the native small factor VIII species possess potential binding affinity for platelet receptors, which is manifested following formation of large factor VIII polymers. We conclude that an optimal size of remarkably high molecular weight is required for efficient aggregation of platelets by factor VIII as occurs during the primary phase of hemostasis.
Blood Fibrinolytic System in Rana tigrina
Authors: Srivastava V M , Dube B , Dube R K , Agarwal G P , Ahmad N 
Published: July 2018

SummaryFibrinolytic activity is well demonstrable in the blood of Rana tigrina. There occurs prompt lysis of diluted plasma; and the plasma euglobulin fraction shows lysis on both unheated and heated fibrin (human or bovine) plates, implying the presence of plasmin-like enzyme in this fraction. The fibrinolytic activity is remarkably inhibited by the erythrocyte-lysate and is moderately enhanced by leucocytes-thrombocytes. EACA suppresses the lysis of dilute cell-free plasma clots at concentration of l0-4M or more, possibly indicating the presence of plasminogen activator in the plasma. Activation of fibrinolysis by human urokinase and not by streptokinase, shows the probable presence of plasminogen and absence of proactivator.
Heterogeneity of Molecular Size of Factor Vlll/von Willebrand Factor in von Willebrand's Disease
Authors: Mikami S , Takahashi Y , Nishino M , Okubo Y , Fukui H 
Published: July 2018

SummaryPatterns of VIIIR:AG in the plasma and its fractions, cryoprecipitate and cryosupernatant, from various types of von Willebrand’s disease (vWd) were observed by SDS 1.5% polyacrylamide gel electrophoresis - crossed immunoelectrophoresis (SDS PAGE - CIE).
VIIIR:AG in normal cryoprecipitate showed several precipitin peaks which correspond to molecular weights ranging from 8 X 105 to 1 X 10
7 daltons and are similar to those in normal plasma. Normal cryosupernatant VIIIR: AG gave smaller molecular weights from 8 X 10
5 to 2 X 10
6 daltons.VIIIR:AG in the plasma and cryoprecipitate from 2 patients with classical vWd gave low precipitin peaks with molecular weights in normal range. VIIIR:AG from 2 patients with subgroup A variant which showed fast anodal migration on the conventional CIE, presented 3 peaks with molecular weights of 8 X 105 to 3 X 106 which are similar to those in normal cryosupernatant. VIIIR: AG from 2 patients with subgroup B variant which showed normal migration on the CIE, gave normal patterns through all fractions.
Substrate Recognition by Vitamin K-Dependent Carboxylase
Authors: Ulrich Magda M W , Soute Berry A M , van Haarlem L Johan M , Vermeer Cees 
Published: July 2018

SummaryDecarboxylated osteocalcins were prepared and purified from bovine, chicken, human and monkey bones and assayed for their ability to serve as a substrate for vitamin K-dependent carboxylase from bovine liver. Substantial differences were observed, especially between bovine and monkey d-osteocalcin. Since these substrates differ only in their amino acid residues 3 and 4, it seems that these residues play a role in the recognition of a substrate by hepatic carboxylase.
A Quantitative Enzyme Immunoassay for Primary Fibrinogenolysis Products in Plasma
Authors: Koppert P W , Kuipers W , Nobel B Hoegee-de , Brommer E J P , Koopman J , Nieuwenhuizen W 
Published: July 2018

SummaryWe have developed a two-step enzyme immunoassay (EIA) that allows the quantitation of degradation products derived from fibrinogen (FbgDP) and that does not detect degradation products derived from cross-finked (XDP) or noncrosslinked fibrin (fdp).The EIA is based on two monoclonal antibodies (FDP-14 and Y-18), developed in our institute. FDP-14 is used as catching antibody. It complexes exclusively with degradation products, irrespective whether these are derived from fibrinogen or from fibrin. It does not complex with intact fibrinogen or fibrin. Y-18 is reactive with fibrinogen and fibrinopeptide A-comprising fibrinogen fragments. It is used, conjugated with horse-radish peroxy-dase, as tagging antibody.The FbgDP-EIA is highly specific, accurate and sensitive. The coefficient of variation is between 3 and 8%; the lower detection limit is less than 0.025 μg/ml.The assay has been applied to plasma from patients with suspected disseminated intravascular coagulation (DIC), to plasma from patients undergoing streptokinase (SK) therapy for acute myocardial infarction and to plasma from newborn babies.DIC patients had no or very low levels of FbgDP, but high levels of other degradation products, SK-treated patients showed high levels of degradation products two hours after termination of the SK infusion. A considerable fraction of these degradation products was shown to be FbgDP. Plasma from newborn babies contained elevated levels of FbgDP associated with prolonged prothrombin times.