bluuebunny/crossref_metadata_2025_split huggingface dataset:
Quantification of Platelet-associated IgG for Differential Diagnosis of Patients with Thrombocytopenia
Authors: Schlenke P. , Hennig H. , Kirchner H. , Klüter H. , Hagenström H. 
Published: March 2018

SummaryImmune thrombocytopenia is due to platelet destruction by circulating glycoprotein-specific antibodies and is found in various disorders. Methods for the detection of platelet-associated IgG (PAIgG) are generally sensitive but unspecific, whereas glycoprotein-specific assays are highly specific but less sensitive. Usefully, a sensitive screening method for PAIgG detection would also provide information for differential diagnosis. We developed a quantitative direct Platelet Immunofluorescence Test (PIFT) by flow cytometry and studied 79 thrombocytopenic patients with immune thrombocytopenia and other disorders. The sensitivity of the assay was 94%, its specificity 66% for the detection of a clinically obvious immune thrombocytopenia. PAIgG levels of patients with immune thrombocytopenia differed significantly from those of other patients with low platelet counts (p <0.001). The quantitative PIFT proved to be a sensitive method for PAIgG detection and should therefore be used as a screening method. In addition, it could be helpful for differential diagnosis in marked thrombocytopenia where a MAIPA is not feasible.
Effect of Lipid-Lowering Treatment on Factor VII Profile in Hyperlipidemic Patients
Authors: Di Febbo Concetta , Amore Concetta , Di Castelnuovo Augusto , Baccante Giovanna , Donati Maria , Cuccurullo Franco , Iacoviello Licia , Porreca Ettore 
Published: March 2018

SummaryA link has been suggested between blood lipids and hemostatic activation. Factor VII (FVII) is a coagulation factor which plays a pivotal role in fibrin generation and thrombus formation. Clinical trials have demonstrated that inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase greatly reduce cardiovascular events in patients with and without coronary artery disease but few data, at this time, are available on the effects of lipid-lowering treatment on factor VII levels. We studied thirty-six IIA and IIB type hyperlipidemic patients who, after a preliminary period of lipid-lowering diet, added atorvastatin (20 mg/daily) or continued dietary treatment alone until they achieved LDL-C recommended levels (< 4 mmol/L). Four to six weeks of lipid lowering treatment with diet plus atorvastatin, produced a significant reduction in FVII coagulant activity (FVIIc) and antigen (FVIIAg). No significant changes were observed in activated FVII (FVIIa). The lipid-lowering treatment with diet alone induced an improved lipid pattern, but no significant changes in FVII profile. Our study suggests a significant effect of lipid-lowering treatment on FVII levels. A possibile nonlipid mechanism that modifies FVII pathway may be suggested.
Blocking the Initiation of Coagulation by RNA Aptamers to Factor VIIa
Authors: Lyerly H. , Lawson Jeffrey , Rusconi Christopher , Yeh Alice , Sullenger Bruce 
Published: March 2018

SummaryThe tissue factor/factor VIIa complex is thought to be the primary initiator of most physiologic blood coagulation events. Because of its proximal role in this process, we sought to generate new inhibitors of tissue factor/factor VIIa activity by targeting factor VIIa. We employed a combinatorial RNA library and in vitro selection methods to isolate a high affinity, nuclease-resistant RNA ligand that binds specifically to coagulation factor VII/VIIa. This RNA inhibits the tissue factordependent activation of factor X by factor VIIa. Kinetic analyses of the mechanism of action of this RNA suggest that it antagonizes factor VIIa activity by preventing formation of a functional factor VII/tissue factor complex. Furthermore, this RNA significantly prolongs the prothrombin time of human plasma in a dose dependent manner, and has an in vitro half-life of ∼15 h in human plasma. Thus, this RNA ligand represents a novel class of anticoagulant agents directed against factor VIIa.
Effect of Stabilizing versus Destabilizing Interactions on Plasminogen Activator Inhibitor-1
Authors: Vleugels Nele , Leys John , Knockaert Isabelle , Declerck Paul 
Published: March 2018

SummaryPlasminogen activator inhibitor-1 (PAI-1) is a unique member of the serpin family, as it spontaneously converts into a latent conformation. However, the exact mechanism of this conversion is not known. Previous studies reported that neutralizing monoclonal antibodies as well as reversal or removal of charges on the s3C-s4C turn results in a destabilization of PAI-1 leading to an accelerated conversion to its latent form.In this study the effect of the reversal or removal of charges in this “gate region” (R186E/R187E, H190E/K191E, H190L/K191L and R356E) on a stable PAI-1-variant (PAI-1-stab) was investigated. Whereas PAI-1-stab has a half-life of 150 ± 66 h, PAI-1-stab-R186ER187E, PAI-1-stab-H190E-K191E, PAI-1-stab-H190L-K191L and PAI-1-stab-R356E have a strongly decreased half-life (p< 0.005 versus PAI-1-stab) of 175 ± 48 min, 75 ± 34 min, 68 ± 38 min and 79 ± 16 min, respectively. Wild-type PAI-1 (wtPAI-1) had a half-life of 55 ± 19 min. These data indicate that the stabilization induced by the mutated residues in PAI-1-stab is counteracted by the additional mutations, resulting in half-lives similar to that of wtPAI-1, thereby suggesting that the stabilizing and destabilizing forces act mainly independently in these mutants. Extrapolation of these data to other (stable) serpins leads to the hypothesis that the s3C-s4C turn and the distal hinge region of the reactive site loop plays a role for the stability of serpins in general.
Identification and Biological Activity of the Active Metabolite of Clopidogrel
Authors: Savi P. , Pereillo J. M. , Uzabiaga M. F. , Combalbert J. , Picard C. , Maffrand J. P. , Pascal M. , Herbert J. M. 
Published: March 2018

SummaryLike ticlopidine, the ADP receptor antagonist clopidogrel is inactive in vitro and must be administered i.v. or orally to exhibit antiaggregatory and antithrombotic activities. We have previously shown that hepatic metabolism is necessary for activity. This study demonstrates that an active metabolite can be generated from human liver microsomes incubated with clopidogrel. Using several analytical methodologies (LC/MS, NMR, chiral supercritical fluid chromatography), we have identified its structure. In vitro, this highly unstable compound, different from that formed from ticlopidine, exhibited all the biological activities of clopidogrel observed ex vivo: Irreversible inhibition of the binding of 33P-2MeS-ADP to washed human platelets (IC50 = 0.53 µM), selective inhibition of ADP-induced platelet aggregation (IC50 = 1.8 µM) and ADP-induced adenylyl cyclase down-regulation. The irreversible modification of the ADP-receptor site which is responsible for the biological activity could be explained by the formation of a disulfide bridge between the reactive thiol group of the active metabolite and a cysteine residue of the platelet ADP receptor.
Abbreviations: ADP: adenosine 5’diphosphate; 2-MeS-ADP: 2-methylthioadenosine-5’-diphosphate; Bmax: maximum binding capacity; IC50: concentration which inhibits 50% of the activity; Kd: dissociation constant; LC/MS: Liquid chromatography coupled to mass spectrometry; NMR: Nuclear magnetic resonance
The Cleaved Peptide of PAR1 Results in a Redistribution of the Platelet Surface GPIb-IX-V Complex to the Surface-Connected Canalicular System
Authors: Furman Mark , Nurden Paquita , Berndt Michael , Nurden Alan , Benoit Stephen , Barnard Marc , Ofosu Frederick , Michelson Alan 
Published: March 2018

SummaryThe only known function of the 41 amino acid cleaved peptide (TR1-41) of the seven transmembrane domain thrombin receptor (PAR1) is to activate platelets (as determined by aggregation, surface P-selectin, and fibrinogen binding to activated GPIIb-IIIa). We now demonstrate that TR1-41 results in a concentration-dependent decrease in the platelet surface expression of each component of the GPIb-IX-V complex, as determined by flow cytometry with a panel of monoclonal antibodies (including 6D1, directed against the von Willebrand factor binding site on GPIbα, and TM60, directed against the thrombin binding site on GPIbα). TR1-41 also decreased ristocetin-induced platelet agglutination. Immunoblotting after incubation of platelets with TR1-41 revealed neither a loss of platelet GPIb nor increase in supernatant GPIb fragments. As demonstrated by immunoelectron microscopy, TR1-41 resulted in a redistribution of GPIb, GPIX, and GPV from the platelet surface to the surface-connected canalicular system (SCCS). In summary, the cleaved peptide (TR1-41) of PAR1 results in a redistribution of the platelet surface GPIb-IX-V complex to the SCCS, thereby negatively regulating the GPIbα binding sites for von Willebrand factor and thrombin.
Titanium Is a Highly Thrombogenic Biomaterial: Possible Implications for Osteogenesis
Authors: Hong Jaan , Andersson Joakim , Nilsson Ekdahl Kristina , Elgue Graciela , Axén Niklas , Larsson Rolf , Nilsson Bo 
Published: March 2018

SummaryTitanium has superior osteointegrating properties compared to other biomaterials. The mechanism for this is unknown. During the initial phase of bone implantation the biomaterial comes into direct contact with whole blood. In this study we use a newly developed in vitro chamber model to compare different commonly used biomaterials in contact with whole blood. These materials were selected with respect to their different osteointegrating properties in order to correlate these properties with the response to whole blood. In the presence of 3 IU/ml of heparin only titanium induced macroscopic clotting. This was reflected by the generation of thrombin-antithrombin which was much increased in blood in contact with titanium compared with steel and PVC. The coagulation activation caused by titanium was triggered by the intrinsic pathway because the generation of FXIIa-AT/C1 esterase inhibitor paralleled that of thrombin-antithrombin, and both thrombinantithrombin complex and FXIIa-AT/C1 esterase inhibitor generation were abrogated by corn trypsin inhibitor, which is a specific inhibitor of FXIIa. The binding of platelets was increased on the titanium surface compared to the other biomaterial surfaces and the state of platelet activation was much more pronounced as reflected by the levels of β-thromboglobulin and PDGF. This study indicates that titanium is unsuitable as a biomaterial in devices which are in direct contact with blood for a prolonged period. Furthermore, PDGF and other α-granule proteins e.g. TGF-β, are known to be potent promotors of osteogenesis which suggests that the pronounced thrombogenic properties of titanium might contribute to the good osteointegrating properties.
Involvement of Lys 62[217] and Lys 63[218] of Human Anticoagulant Protein C in Heparin Stimulation of Inhibition by the Protein C Inhibitor
Authors: Shen Lei , Villoutreix Bruno , Dahlbäck Björn 
Published: March 2018

SummaryInhibition of activated protein C (APC) by protein C inhibitor (PCI) is stimulated by heparin, whereas inhibition by α1-antitrypsin (AAT) is heparin-independent. Three lysine residues located in a positively charged cluster in the serine protease domain of protein C (PC) were mutated to probe their involvement in the heparin stimulation of inhibition by PCI. These mutations were selected after analysis of the three-dimensional structure of APC and of molecular models for PCI and the APC-PCI complex. A double mutant, K62[217]N/K63[218]D, a single mutant, K86[241]S, and wild-type PC were expressed in embryonic human kidney 293 cells. Heparin stimulated the rate of inhibition of wt-APC by PCI approximately 400-fold, with second order rate constants (k

2

) in the absence and presence of heparin of 0.72 × 103 M–1s–1 and 2.87 × 105 M–1s–1, respectively. In contrast, heparin only yielded a 52-fold stimulation of the rate of inhibition of the double mutant APC by PCI as the rate constants in the absence and presence of heparin were k

2
= 2.44 × 103 M–1s–1 and k

2
= 1.26 × 105 M–1s–1, respectively. The double mutant K62N/K63D eluted at approximately 10% lower NaCl concentration from a heparin Sepharose column than the K86S mutant or wt-APC. These data suggest K62 and K63 in APC to be part of a heparin binding site which is important for heparin-mediated stimulation of inhibition of APC by PCI.
Abbreviations: APC, activated protein C; PC, protein C; PCI, protein C inhibitor, AAT, α1-antitrypsin also called α1-proteinase inhibitor, AT, antithrombin; TM, thrombomodulin. The chymotrypsinogen nomenclature for APC (1) is used in the text while the PC numbering is indicated between brackets whenever appropriate. P1, P2... and P1’, P2’.. designate inhibitor residues amino- and carboxy-terminal to the scissile peptide bond, respectively, and S1, S2.. and S1’, S2’.. the corresponding subsites of the protease (2). The antitrypsin numbering (3) for PCI is used along this article while the PCI numbering is mentioned between brackets whenever appropriate.
Evidence that Modifications of Lp(a) In Vivo Inhibit Plasmin Formation on Fibrin
Authors: Soulat Thierry , Loyau Stéphane , Baudouin Veronique , Durlach Vincent , Gillery Philippe , Garnotel Roselyne , Loirat Chantal , Anglés-Cano Eduardo 
Published: March 2018

SummaryIn the present study we have investigated the effect of individual variations in the concentration of Lp(a) on plasmin formation at the surface of fibrin. The plasma Lp(a) concentrations from 20 nephrotic children were high at flare-up of the disease (0.43 ± 0.45 g/l) and decreased progressively with remission at both 6 weeks (0.28 ± 0.24 g/l) and 6 months (0.24 ± 0.288 g/l). In contrast, the concentration of plasminogen showed an inverse variation, with low values at flareup (1.27 ± 0.34 μM) and normal values at remission (1.66 ± 0.17 μM at 6 weeks and 1.99 ± 0.21 μM at 6 months). An increase in plasmin formation (from 0.62 ± 0.49 to 0.73 ± 0.61, and 0.84 ± 0.75 pmol/well) and a decrease in apo(a) binding (from 5.45 ± 2.42 to 4.54 ± 2.12, and 3.93 ± 1.51 fmol/well) on the surface of fibrin, were concomitantly observed from flare-up to remission at 6 weeks and at 6 months, respectively. Values for plasmin formation parallel the amount of plasminogen bound. The low concentration of plasminogen found at flare-up may also have contributed to the increased binding of Lp(a) as indicated by a decrease in the maximal amount of Lp(a) bound (Bmax) to fibrin as a function of plasma plasminogen concentrations. Bmax was 1.51 fmol in the absence of plasminogen and decreased to 1.1 fmol and 0.93 fmol at respectively 1 and 2 μM of plasminogen. Altogether, these data constitute the first quantitative evidence indicating that plas-min formed at the surface of fibrin may vary with modifications of the concentration of Lp(a) in vivo.
A Soluble Fibrin Standard: Comparable Dose-response with Immunologic and Functional Assays
Authors: Marder Victor , Kanouse Joel , Francis Charles , McCarron Bonnie 
Published: March 2018

SummaryA soluble fibrin (SF) preparation has been developed as a potential standard by the Scientific and Standardization Committee for use in assays evaluating in vitro preparations and patient plasma samples. The SF standard was prepared by reaction of factor XIII-free fibrinogen with thrombin, followed by neutralization with hirudin and solubilization of the fibrin in acetic acid. As characterized by SDS-PAGE, the polypeptide chain structure shows the anticipated loss of fibrinopep-tides and lack of sγ or α chain crosslinking. The standard was added to pooled normal plasma at concentrations from 12.5 μg/ml to 340 μg/ml and tested with four commercially available assays based on immunologic reactions using ELISA or latex agglutination or on t-PA cofactor activity for plasminogen to plasmin conversion. Absolute “soluble fibrin” concentrations were calculated using the manufacturers’ calibrators and showed distinct dose-response relationships for each assay. Expression of the results following log-transformation produced a series of parallel lines, indicating that this SF preparation can serve as a standard, effectively normalizing the disparate proprietary internal calibrators currently used for each assay.
Coexistence of Factor V Leiden and Factor II A20210 Mutations and Recurrent Venous Thromboembolism
Authors: D’Andrea Giovanna , Colaizzo Donatella , Cappucci Giuseppe , del Popolo Annamaria , Brancaccio Vincenzo , Ciampa Antonio , Grandone Elvira , Di Minno Giovanni , Margaglione Maurizio 
Published: March 2018

SummaryPatients carrying the FV Leiden or the FII A20210 mutation have a high risk of venous thromboembolism. Among 542 patients with a documented diagnosis of deep venous thrombosis in one leg consecutively referred for a thrombophilic work-up, we have retrospectively assessed the rate of objectively documented previous recurrence in carriers of both FV Leiden and FII A20210 mutations. Eighty-two patients had experienced 115 episodes of recurrent venous thromboembolism. The rate of recurrent venous thromboembolism was 29.2% among subjects with and 14.5% in those without deficiencies of natural anticoagulant proteins (p = 0.055), and 24.6% among patients with and 14.0% in those without antiphospholipid antibodies (p = 0.036). The frequency of having a recurrent thromboembolism was 16.2%, 20.0%, and 36.4% among carriers of FV Leiden, FII A20210 mutation, or both gene defects, respectively, and 12.8% in subjects carrying neither mutation (p for trend = 0.004). When adjusted for age, sex, and thrombophilic risk factors, the rate was higher among patients with than in those without deficiencies of natural anticoagulant proteins (OR: 3.0; 95% CI: 1.2-7.5), aPL 2.5 (95% CI: 1.3-4.9), or both FV Leiden and FII A20210 gene mutations (OR 4.8; 95% CI: 1.9-12.2).The rate of previous recurrent venous thromboembolism was significantly higher in subjects carrying both FV Leiden and FII 20210 mutations and was comparable to that observed in subjects with deficiencies of natural anticoagulant proteins or antiphospholipid antibodies.
Fibrinogen Bastia (γ 318 Asp → Tyr) a Novel Abnormal Fibrinogen Characterized by Defective Fibrin Polymerization
Authors: Lounes Karim Chabane , Soria Claudine , Valognes Antoine , Turchini Marie France , Koopman Jaap , Soria Jeannette 
Published: March 2018

SummaryA new congenital dysfibrinogen, Fibrinogen Bastia, was discovered in a 20-year-old woman with no clinical symptoms. The plasma thrombin-clotting time was severely prolonged. The functional plasma fibrinogen concentration was low (0.2 mg/ml), whereas the immunological concentration was normal (2.9 mg/ml). Purified fibrinogen Bastia displayed a markedly prolonged thrombin-clotting time related to a delayed thrombin-induced fibrin polymerization. Both the thrombin-clotting time and the fibrin polymerization were partially corrected by the addition of calcium ions. The anomaly of fibrinogen Bastia was found to be located in the γ-chain since by SDS-PAGE performed according to the method of Laemmli two γ-chains were detected, one normal and one with an apparently lower molecular weight. Furthermore, analysis of plasmin degradation products demonstrated that calcium ions only partially protect fibrinogen Bastia γ-chain against plasmin digestion, suggesting that the anomaly is located in the C-terminal part of the γ-chain. Sequence analysis of PCR-amplified genomic DNA fragments of the propositus demonstrated a single base substitution (G → T) in the exon VIII of the γ chain gene, resulting in the amino acid substitution 318 Asp (GAC) → Tyr (TAC). The PCR clones were recloned and 50% of them contained the mutation, indicating that the patient was heterozygous. These data indicate that residue Asp 318 is important for normal fibrin polymerization and the protective effect of calcium ions against plasmin degradation of the C-terminal part of the γ-chain.
Synthesis and Expression of Tissue Factor Pathway Inhibitor by Serum-stimulated Fibroblasts, Vascular Smooth Muscle Cells and Cardiac Myocytes
Authors: Steer S. , Kuppuswamy M. N. , Kisiel W. , Bajaj S. P. , Bajaj M. S. 
Published: March 2018

SummaryTissue factor pathway inhibitor (TFPI) plays an important role in regulating tissue factor (TF)-initiated blood coagulation. Since serum stimulation of fibroblasts, vascular smooth muscle cells and cardiac myocytes in culture increases the expression of TF mRNA and antigen (Ag) in these cells, we hypothesized that serum may also induce increased synthesis of TFPI in these cells to regulate the TF-induced extravascular clotting at an injury site. To test this concept, we used primary isolates of the following human cell types – fetal and adult lung fibroblasts, pulmonary and aortic smooth muscle cells, and cardiac myocytes. Serum-stimulation of these cells resulted in an increased expression of TF mRNA and Ag (8 to10-fold). Upon serum stimulation, expression of TFPI mRNA and Ag was also increased in these cells. However, the increase in TFPI-Ag (6 to 8-fold) was significantly greater than the TFPI mRNA (2 to 3-fold). Notably, increased expression of TFPI persisted after the TF expression had declined. Further, increased synthesis of TFPI initially led to the saturation of heparin-releasable binding sites. TFPI-Ag was detected by Western blotting, 35S-metabolic labeling and activity assays on the conditioned media, heparin-released material from cells, and in cell lysates. TFPI-Ag was also detected by immunofluorescence staining of cells. Actinomycin D partially whereas cycloheximide completely prevented the serum-induced increased expression of TFPI synthesis by these cells, suggesting control primarily at the translational but some at the transcriptional level as well. The Mr of undegraded TFPI in all cases was ~45 kDa and was of full length. TFPI synthesized locally by fibroblasts, vascular smooth muscle cells and cardiac myocytes could play a significant role in regulating TF-initiated extravascular clotting especially since plasma TFPI that may be available at the injury site lacks a portion of the carboxyl segment and is a less efficient inhibitor.
The Fibrinogen γ Chain Dodecapeptide Inhibits Agonist-induced Aggregation of Rabbit Platelets and Fibrinogen Binding to Rabbit Glycoprotein IIb-IIIa
Authors: Packham Marian , Taylor Desirée , Yeo Erik , Gemmell Cynthia , Patil Sonali , Lam Stephen , Rand Margaret 
Published: March 2018

SummaryThe HHLGGAKQAGDV (H12) sequence at the carboxyl termini of the γ chains and the RGD sequences in the Aα chains of human fibrinogen are potential recognition sites for the binding of soluble fibrinogen to glycoprotein IIb-IIIa (GPIIb-IIIa) on activated human platelets. Thus, addition of either H12 or RGD-containing peptides inhibits aggregation of and fibrinogen binding to human platelets. In contrast, we reported previously that RGDS had relatively little inhibitory effect on these functions of rabbit platelets. In the present study, we found that H12 inhibited ADP- and thrombin-induced aggregation of rabbit platelets in a dose-dependent manner. Specificity was demonstrated by the failure of the variant HHLGGAKQAGEV peptide to inhibit ADP-induced aggregation. Furthermore, flow cytometric analyses demonstrated that H12 inhibited the binding of FITC-fibrinogen to ADP-activated rabbit platelets in a dose-dependent manner. To examine the direct interaction of H12 with rabbit GPIIb-IIIa, we performed affinity chromatography by applying an octylglucoside extract of rabbit platelet proteins onto an affinity matrix containing the fibrinogen γ chain sequence. Proteins of ∼135 kDa and ∼95 kDa were specifically eluted by soluble H12, and the 95 kDa protein band was immunoblotted by anti-LIBS1, a monoclonal antibody against human GPIIIa. In control samples, no detectable protein from rabbit platelet lysates was eluted from an RGD affinity matrix by GRGDSP. Collectively, our results demonstrated that H12 inhibits aggregation of and fibrinogen binding to rabbit platelets by directly interacting with rabbit GPIIb-IIIa. These findings suggest that rabbit platelets would serve as a suitable thrombosis model for testing the efficacy of peptide mimetics derived from H12.