bluuebunny/crossref_metadata_2025 huggingface dataset:
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

A 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

The 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

Plasminogen 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

Like 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

The 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

Titanium 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

Inhibition 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

In 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

A 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

Patients 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

A 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

Tissue 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

The 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.
Reactivity of Soluble Fibrin Assays with Plasmic Degradation Products of Fibrin and in Patients Receiving Fibrinolytic Therapy
Authors: McCarron Bonnie , Marder Victor , Francis Charles
Published: March 2018

The ability to identify the products of thrombin and plasmin action on fibrinogen is important in patients with thrombotic and fibrinolytic disorders. New assays have been developed for “soluble fibrin” which represents soluble derivatives other than fibrinopeptides formed from fibrinogen by thrombin. These assays are either immunological, using antibodies for fibrin-specific neoepitopes, or functional and based on the cofactor activity of soluble fibrin in the tissue plasminogen activator (t-PA)-mediated conversion of plasminogen to plasmin. As plasmic derivations of fibrin share structural features with soluble fibrin, they may be reactive with assays for soluble fibrin. Therefore, we prepared plasmic digests of fibrin and determined the degree of reactivity with four soluble fibrin assays. Three assays used Mabs directed toward the fibrin-specific neoepitopes at α17-23 (A), γ312-324 (B) and α17-78 (D). A fourth (C) was based on t-PA co-factor activity. Tests A and C demonstrated marked crossreactivity with fibrin degradation products, and digests containing the largest derivatives showed greatest reactivity. Plasmic derivatives of crosslinked fibrin had greater reactivity than those of non-crosslinked fibrin. Tests B and D demonstrated minimal reactivity with plasmic derivatives of crosslinked or of non-crosslinked fibrin. Samples from patients with lower limb peripheral arterial occlusion were assayed for soluble fibrin, D-dimer and fibrinogen at presentation and eight hours after thrombolytic therapy. Variable results were seen at presentation with elevations in 13, 1, 0 and 4 of 19 patients using Tests A, B, C and D, respectively. After fibrinolytic therapy, marked increases in soluble fibrin levels were observed up to 600-fold above normal. A strong correlation between baseline levels was observed with Test B and Test D, which showed the least cross-reactivity with plasmic derivations. After thrombolytic therapy there were either weak or no correlations among the different assays. The results demonstrate that assays for soluble fibrin may react with plasmic derivatives of fibrin and this must be considered in interpreting clinical results.
Differential Regulation of Platelet Aggregation by Matrix Metalloproteinases-9 and -2
Authors: Fernandez-Patron Carlos , Martinez-Cuesta Maria , Salas Eduardo , Sawicki Grzegorz , Wozniak Mieczyslaw , Davidge Sandra , Radomski Marek
Published: March 2018

We have recently found matrix metalloproteinase-2 (MMP-2) in human platelets and reported that the release of this enzyme during platelet activation stimulates aggregation. We have now identified matrix metalloproteinase-9 (MMP-9) in human platelets and resistance-sized (~200 μm) arteries. Resting platelets released small quantities of pro-MMP-9. Maximal release of MMP-9 was detected during partial (appr. 30% maximum) aggregation with thrombin. However, maximal release of MMP-2 was associated with maximal aggregation. MMP-9 antibodies induced aggregation of resting platelets and potentiated aggregation of platelets induced by thrombin and collagen. Moreover, MMP-9 microisolated from arteries as well as recombinant human MMP-9 (0.1-30 ng/ml) inhibited thrombin and collagen-induced aggregation. We conclude that MMP-9 is an inhibitor of aggregation and in this action opposes the effects of MMP-2. The MMP-2/MMP-9 system may play an important role in the regulation of platelet-platelet and platelet-vessel wall interactions.
A Sudden Increase in Plasma Epinephrine Levels Transiently Enhances Platelet Deposition on Severely Damaged Arterial Wall
Authors: Martínez-González José , Royo Teresita , Lassila Riitta , Badimon José Juan , Badimon Lina
Published: March 2018

Epidemiologic evidence has shown that sympathoadrenal activation plays a triggering role in the onset of acute coronary syndromes. However, its mechanism is not yet clearly understood. The aim of this study was to assess the effect of a sudden increase in epinephrine on platelet deposition on severely damaged vessel wall at shear rate conditions modelling stenotic vessels in the porcine model. The selected epinephrine concentrations (0.5 μmol/l-1 mmol/l) alone or in combination with collagen or ADP did not affect platelet aggregation in vitro either in whole blood or in PRP, although porcine platelets express α2-adrenergic receptors as assessed by PCR. In vitro and ex vivo perfusion experiments were performed using the Badimon chamber at high shear rate conditions (1690 s-1). In vitro, epinephrine (130 nmol/l) increased platelet deposition on severely damaged vessel wall (exposing tunica media; ≈1.6-fold, p <0.05) or immobilized collagen (2.2-fold, p <0.01). Ex vivo perfusion experiments were performed from animals that received intravenous epinephrine infusion for one hour at a low (0.3 μg/kg/min; ≈17 nmol/l in plasma, at 20 min of the infusion) and a high dose (1.0 μg/kg/min; ≈106 nmol/l in plasma, at 20 min of the infusion). Only the low dose temporarily increased platelet deposition on severely damaged vessel wall during the first 30 min of infusion [2.4-fold (p <0.05) and 4.2-fold (p <0.01) at 10 and 30 min of the infusion respectively] declining afterwards. Thus, in flow conditions typical of atherosclerotic arteries, a sudden physiological release of epinephrine can temporarily enhance platelet deposition on severely damaged vessel wall while an extensive exposure leads to refractoriness.
Rapid Haemodynamic Improvement Following Saruplase in Recent Massive Pulmonary Embolism
Authors: Barnes Sarah , Hopkins Gwyn , Charbonnier Bernard , Pacouret Gérard
Published: March 2018

In a single centre pilot study, saruplase (20 mg bolus plus 60 mg infusion over 1 h) was administered to twenty patients with an angio-graphically documented recent massive pulmonary embolism: Miller index of at least 20 and mean pulmonary artery pressure of at least 20 mmHg. The lytic ability of saruplase to cause normalization of haemodynamic parameters over the first 12 h and reperfusion of pulmonary arteries at 24 h was assessed. A decrease of 25 ± 10% in total pul monary resistance was evident at 30 min. Haemodynamic parameters continued to improve with total pulmonary resistance decreasing by 29 ± 8% and 40 ± 11% at 1 and 12 h respectively. Relative improve ment in Miller index 24 ± 6 h after saruplase treatment was 38 ± 9%. Two patients suffered recurrent pulmonary embolism, two severe bleeding events were observed. One patient died following a haemorrhagic stroke.
Pharmacodynamics and Tolerance of Two Nadroparin Formulations (10,250 and 20,500 Anti Xa IU·ml-1) Delivered for 10 Days at Therapeutic Dose
Authors: Navarro C. , Cambus J. P. , Caplain H. , d’Azemar P. , Necciari J. , Duret J. P. , Gaud C. , Sié P. , Boneu B.
Published: March 2018

Venous thromboembolism may be efficiently treated by once-a-day (o.d.) administration of a high dose of low molecular weight heparin (LMWH) instead of administration of the same total dose in two injections a day (b.i. d.). To reduce the volume of the subcutaneous (s.c.) injection, a more concentrated form of the drug is advisable. This study was designed to compare the bioavailability of 2 formulations of nadroparin containing 10,250 and 20,500 anti-Xa IU·ml-1 respectively. This was an open, randomized, cross-over study where 12 healthy volunteers (age 18-35) were enrolled. They received either 90 anti-Xa IU·kg-1 b.i. d. of the 10,250 IU preparation (treatment A), or 180 anti-Xa IU·kg-1 o.d. of the 20,500 IU preparation (treatment B) for 10 days. On day 1, the subjects were sampled between 0 and 12 h (treatment A) or between 0 and 24 h (treatment B). On day 10, they were sampled between 0 and 12 h and between 12 and 24 h (treatment A) or between 0 and 24 h (treatment B). Anti-Xa and anti-IIa activities were determined by specific chromogenic assays. The main result of the study was that the bioavailability of the anti-Xa activity of the 2 nadroparin formulations was equivalent, as shown by the comparison of the AUC0-12 h plus AUC12-24 h (treatment A) and the AUC0-24 h (treatment B), calculated on day 10. This study also allowed a number of interesting observations to be made. 1) Between day 1 and day 10, there was an accumulation of the anti-Xa activity for treatment A but not for treatment B (accumulation factors: 1.6 and 1.1 respectively); 2) On day 10, the AUC0-12 h were slightly but significantly lower than the AUC12-24 h suggesting a circadian effect for anti-Xa and anti-IIa activities; 3) the clearance of the anti-Xa activity was comparable at the 2-dose regimens, while that of the anti-IIa activity was lower in treatment B than in treatment A, indicating a significant dose effect for the pharmacodynamics of the longer heparin chains; 4) On average, the clearance of the anti-IIa activity was twice as high as that of the anti-Xa activity; 5) For treatment B, significant APTT prolongations were noticed at Tmax (prolongation factor: 1.7 ± 0.25), in relation with the anti-IIa activity (0.3 ± 0.1 IU·ml–1).
4G/5G Promoter PAI-1 Gene Polymorphism Is Associated with Plasmatic PAI-1 Activity in Italians: A Model of Gene-Environment Interaction
Authors: Burzotta Francesco , Castelnuovo Augusto Di , Amore Concetta , D’Orazio Andria , Bitondo Rosa Di , Donati Maria Benedetta , Iacoviello Licia
Published: March 2018

The PAI-1 gene promoter 4G/5G polymorphism was found to be associated with plasma PAI-1 activity in Northern and Central Europe populations, but no data are available on the association between this polymorphism and PAI-1 levels in Southern Europe countries (such as Italy) where the incidence of ischemic disorders is lower. This study shows that among populations with different incidence of atherothrombotic disorders the 4G/5G PAI-1 gene promoter polymorphism has the same importance in the regulation of plasma PAI-1 activity.

Moreover, we have analysed some gene-environmental interactions: the correlation between PAI-1 and cholesterol in non dyslipidemic subjects and the correlation between PAI-1 activity and tryglicerides in dyslipidemic subjects differed according to the 4G/5G genotype class. Thus, our findings suggest that, among subjects with or without metabolic disorders such as dyslipidemia, completely different gene-environment interactions may occur.
Thrombomodulin in Human Plasma Contributes to Inhibit Fibrinolysis through Acceleration of Thrombin-dependent Activation of Plasma Procarboxypeptidase B
Authors: Hosaka Yoshitaka , Takahashi Yasuo , Ishii Hidemi
Published: March 2018

Thrombomodulin (TM) expressed on endothelial cells binds thrombin and initiates anticoagulant pathways. Soluble functional proteolytic fragments of TM are also present in circulating plasma. Recently, it was reported that TM accelerated thrombin-dependent plasma procarboxypeptidase B (pro-pCPB) activation in a purified system and suggested that TM may inhibit fibrinolysis in crude plasma. The aim of present study was to evaluate any functional role of soluble TM fragments in plasma or purified TM added into plasma to the regulation of coagulation and fibrinolysis. Addition of rabbit TM (1-200 ng/ml) to plasma resulted in a concentration-dependent prolongation of urokinase (UK)- or tissue plasminogen activator (t-PA)-induced clot lysis time. The concentration of TM required for the inhibition of fibrinolysis was lower than that required for the inhibition of coagulation. Addition of anti-rabbit TM IgG or anti-human TM IgG into plasma reduced UK- or t-PA-induced clot lysis time without affecting clotting times, indicating that exogenous TM or soluble TM fragments in normal human plasma participated in regulation of fibrinolysis. Moreover, the TM-dependent inhibition of fibrinolysis was observed only in the presence of thrombin and blocked by addition of carboxypeptidase B inhibitors, but not mediated by protein C activation or direct inhibition of UK, t-PA or plasmin. Analysis of various substrates and inhibitors indicated that TM accelerated thrombin-dependent pro-pCPB activation in plasma. The present results indicate that TM, including soluble TM fragments in plasma, inhibit fibrinolysis via activation of pro-pCPB in plasma.
Anti-thrombotic Effects of CX-397, a Recombinant Hirudin Analog, in a Canine Model of Coronary Artery Thrombosis
Authors: Hori Toshimitsu , Moriike Mie , Asano Taiji , Hayashi Hideya , Iwade Kazunori , Hosoda Saichi , Ohyama Tadashi
Published: March 2018

CX-397, a recombinant hirudin analog, is a potent and specific inhibitor of human α-thrombin. We conducted a comparative study of CX-397 and heparin in a canine model of left circumflex (LCX) coronary artery thrombosis to evaluate the anti-thrombotic efficacy of CX-397. Administration of drugs (i. v.; bolus + infusion) was commenced 10 min prior to the initiation of LCX coronary artery electrolytic injury (100 μA for 300 min). All saline-treated control animals (7/7) developed thrombotic occlusion during the experimental period, leaving a residual thrombus mass of 15.4 ± 3.8 mg. Treatment with CX-397 at three incremental dose levels reduced the incidence of occlusion (4/7, 2/5 and 0/7) and decreased thrombus weight (12.6 ± 2.5 mg, 6.3 ± 3.0 mg and 2.1 ± 1.3 mg, respectively) in a dose-dependent manner. At the in termediate dose (15,000 ATU/kg + 15,000 ATU/kg/h) or higher, CX-397 showed significant anti-thrombotic effects (p <0.05 and p <0.01) and suppressed increases in thrombin-antithrombin III complex (TAT) levels (p <0.01 and p <0.001). In the heparin (80 U/kg + 60 U/kg/h)-treated group, the incidence of occlusion (5/7) and thrombus weight (14.1 ± 6.2 mg) did not differ significantly from the control group. Plas ma TAT levels in the heparin group decreased compared with the control group (p <0.01), but was less potent than the intermediate dose CX-397 (p <0.01). The anti-coagulation (activated partial thromboplastin time and activated clotting time) and template bleeding time prolongation effects of heparin were more potent than those of the intermediate dose CX-397 which showed significant anti-thrombotic effects. In conclusion, CX-397 dose-dependently suppressed thrombus formation by inhibition of thrombin activity in a canine coronary artery injury model. The anti-thrombotic efficacy of CX-397 was more potent than that of heparin at equivalent anti-coagulation dosage.
Nucleotide Structure and Characterization of the Murine Gene Encoding Anticoagulant Protein C
Authors: Jalbert Louise , Rosen Elliot , Lissens Ann , Carmeliet Peter , Collen Désiré , Castellino Francis
Published: March 2018

The 15,160 bp murine gene encoding anticoagulation protein C (PC) was cloned and sequenced, including 414 bp upstream of exon 1 and 80 bp downstream of the translation stop codon. Nine exons and eight introns were identified. The first exon was untranslated and contained the major transcriptional start site, the surrounding nucleotide sequence of which matched reasonably well with the consensus eukaryotic Cap element sequence. The translational initiator methio-nine residue was located in exon 2. The other introns were positioned as splices between the major domain units of the protein. The 5’ untranslated region contained two possible CCAAT sequences and GC boxes, but no TATA box was obvious within the optimal range of distances from the transcription start site. The 3’-flanking nucleotides included a probable polyadenylation site (ATTAAA), beginning 80 nucleotides downstream of the translation stop codon, and a downstream consensus sequence (AGTGTTTC) required for the efficient formation of a 3’ terminus of mRNA. Several high probability transcription factor recognition sequences, including proteins that are enriched in, or specific to, the liver, such as C/EBP, C/EBP, HNF1, and HNF3, have been located in the 5’ region of the gene. These results indicate that all elements are present for liver-based transcription of the gene for murine PC.
A Novel Mutation in Intron K of the PROS1 Gene Causes Aberrant RNA Splicing and Is a Common Cause of Protein S Deficiency in a UK Thrombophilia Cohort
Authors: Daly M. E. , Makris M. , Preston F. E. , Peake I. R. , Beauchamp N. J.
Published: March 2018

In the course of investigating the molecular basis of protein S deficiency in 31 index cases with thrombophilia, we identified seven kindred where the underlying defect was a novel A to G transition 9 bp upstream of exon 12 in intron K of the PROS1 gene. In all but one case, the mutation caused type I deficiency. One individual had type III deficiency. While ectopic transcript analysis using the BstXI dimorphism in exon 15 failed to detect a transcript from the mutated allele, analysis of transcripts spanning exons 11 and 12 revealed a minor mRNA species. Sequencing confirmed the mutation created a new RNA acceptor site introducing 8 nucleotides of intronic sequence into the mature mRNA. Haplotype analysis of the defective PROS1 alleles in six families revealed the same haplotype in all affected individuals suggesting the presence of a common ancestor. Six of the fourteen relatives with the mutation experienced at least one venous thrombotic event strongly supporting the association of the mutation with venous thrombosis.
Low-dose Oral Vitamin K Reliably Reverses Over-anticoagulation due to Warfarin
Authors: Donovan D. , Harrison L. , McGinnis J. , Ginsberg J. , Crowther M. A.
Published: March 2018

Background: Patients receiving long-term warfarin frequently develop asymptomatic excessive prolongation of their international normalized ratio (INR) results. The most appropriate management strategy in these patients is unknown. This prospective cohort study was designed to address whether 1 mg of oral vitamin K effectively reduces the INR value of such patients. Methods: A prospective cohort study was performed in two tertiary care teaching hospitals, in which 62 patients receiving warfarin who had INR values between 4.5 and 10.0 received 1 mg of oral vitamin K. All patients had daily INR values and clinical assessments performed. Results: The mean INR value at presentation was 5.79 (95% confidence interval (CI) 5.48 to 6.09, range 4.5 to 9.5). Sixteen hours after receiving the 1 mg of oral vitamin K, the mean INR was 2.86 (95% CI 2.50 to 3.23). On the second and third days after vitamin K, the mean INR values were 2.20 (1.93 to 2.47) and 2.14 (1.85 to 2.44), respectively. No adverse events or bleeding complications were observed. In three patients (6%) the INR value rose between the time of vitamin K administration and the next INR determination; two patients received a further 2 mg dose of subcutaneous vitamin K. Conclusions: In patients receiving warfarin who have asymptomatic excessive prolongations in their INR results, 1 mg of oral vitamin K reliably reduces the INR to the therapeutic range within 24 h. This therapy is more convenient, less expensive, and might be safer than parenteral vitamin K. Thus, it should be considered in all non-bleeding patients receiving warfarin, who present with INR results of 4.5 to 9.5.

Dr. Crowther is the recipient of a Medical Research Council of Canada Research Fellowship.

Dr. Ginsberg is a Career Investigator of the Heart and Stroke Foundation of Ontario.
Activated Protein C Sensitivity, Protein C, Protein S and Coagulation in Normal Pregnancy
Authors: Brennand J. , Conkie J. A. , McCall F. , Greer I. A. , Walker Isobel , Clark P.
Published: March 2018

A prospective study of activated protein C sensitivity, protein C, protein S, and other coagulation factors in 239 women during normal pregnancy was carried out. Protein C activity appeared unaffected by gestation, although an elevation of protein C activity was observed in the early puerperium. A fall in total and free protein S with increasing gestation was observed. Activated protein C sensitivity ratio (APC:SR) showed a progressive fall through pregnancy. This fall correlated with changes in factor VIIIc, factor Vc and protein S. 38% of subjects, with no evidence of Factor V Leiden or anticardiolipin antibodies, showed a low APC:SR (APC:SR <2.6) in the third trimester of pregnancy. Aside from a significant reduction in birth weight, no difference in pregnancy outcome was observed between these subjects and those with a normal APC:SR. Activated protein C sensitivity ratio, modified by pre-dilution of patient samples with factor V depleted plasma, showed no consistent trend with gestation.
Difference of [Ca2+]i Movements in Platelets Stimulated by Thrombin and TRAP: the Involvement of αIIbβ3-Mediated TXA2 Synthesis
Authors: Tomiyama Yoshiaki , Honda Shigenori , Senzaki Kayoko , Tanaka Akito , Okubo Mitsuru , Takahashi Fumie , Takasugi Hisashi , Seki Jiro , Aoki Toshiaki
Published: March 2018

This study investigated the difference of [Ca2+]i movement in platelets in response to thrombin and TRAP. The involvement of αIIbβ3 in this signaling was also studied. Stimulation of platelets with thrombin at 0.03 U/ml caused platelet aggregation and a two-peak increase in [Ca2+]i. The second peak of [Ca2+]i, but not the first peak was abolished by the inhibition of platelet aggregation with αIIbβ3 antagonists or by scavenging endogenous ADP with apyrase. A cyclooxygenase inhibitor, aspirin, and a TXA2 receptor antagonist, BM13505, also abolished the second peak of [Ca2+]i but not the first peak, although these regents did not inhibit aggregation. Under the same assay conditions, measurement of TXB2 demonstrated that αIIbβ3 antagonists and aspirin almost completely inhibited the production of TXB2. In contrast to thrombin-stimulation, TRAP caused only a single peak of [Ca2+]i even in the presence of platelet aggregation, and a high level of [Ca2+]i increase was needed for the induction of platelet aggregation. The inhibition of aggregation with αIIbβ3 antagonists had no effect on [Ca2+]i change and TXB2 production induced by TRAP. Inhibition studies using anti-GPIb antibodies suggested that GPIb may be involved in the thrombin response, but not in the TRAP. Our findings suggest that low dose thrombin causes a different [Ca2+]i response and TXA2 producing signal from TRAP. Endogenous ADP release and fibrinogen binding to αIIbβ3 are responsible for the synthesis of TXA2 which results in the induction of the second peak of [Ca2+]i in low thrombin- but not TRAP-stimulated platelets.
Recombinant Tumor Necrosis Factor Receptor p75 Fusion Protein (TNFR:Fc) Alters Endotoxin-Induced Activation of the Kinin, Fibrinolytic, and Coagulation Systems in Normal Humans
Authors: Majluf-Cruz Abraham , Stadnicki Antoni , Tropea Margaret , Reda Debra , Agosti Jan , Colman Robert , Suffredini Anthony , DeLa Cadena Raul
Published: March 2018

The effects of inhibition of tumor necrosis factor (TNF) on cell and protease activation were evaluated in 18 normal volunteers given endotoxin (4 ng/kg, iv) after an infusion of low (10 mg/m2 iv, n = 6) or high dose (60 mg/m2 iv, n = 6) recombinant human dimeric TNF receptor protein (TNFR:Fc) or its vehicle (placebo n = 6). Activation of the coagulation system occurred by 2 h in the TNFR:Fc vehicle-placebo group manifested by decreased prekallikrein functional levels and increased levels of prothrombin F1+2 fragments (p <0.0001). High or low dose TNFR:Fc delayed the fall in prekallikrein functional levels by 1 h and 4 h, respectively (p <0.0002), but did not inhibit the increase in circulating levels of prothrombin F1+2 fragments. In contrast, endothelium activation, characterized by increased levels of tissue plasminogen activator, plasminogen activator inhibitor-1, and von Willebrand Factor antigen was blunted by both low and high dose TNFR:Fc (p <0.001). While the endotoxin-associated decrease in platelet number was not altered, platelet-derived β-thromboglobulin peak levels were blunted and delayed by TNFR:Fc (p <0.02). Increased levels of neutrophil elastase were attenuated by low and high dose TNFR:Fc (p <0.001). These results suggest that although TNF is functionally linked to the activation of endothelium, neutrophils, coagulation, and fibrinolysis, alternative pathways are present in vivo that result in activation of the kallikrein-kinin system after endotoxin-induced TNF release. These alternative pathways may limit some of the anti-inflammatory effects of TNFR:Fc.
Turnover of Thrombomodulin at the Cell Surface Occurs at a Similar Rate to Receptors that Are Not Actively Internalized
Authors: Chu Michael , Bird Catherina , Teasdale Melvena , Bird Phillip
Published: March 2018

Loss of thrombomodulin (TM) from the endothelial cell surface is thought to contribute to thrombosis encountered in malignant and inflammatory disease. Internalization or endocytosis of TM from the cell surface has been proposed to be one mechanism by which TM levels are reduced. Previous work has led to a view that TM is rapidly internalized using a non-conventional pathway, under the direction of a signal motif in its extracellular domain. This is contrary to the clathrin-dependent route taken by most rapidly internalized receptors that possess signals in their cytoplasmic domains. However the internalization rate of TM has never been directly compared to known internalizing or non-internalizing molecules. Here we show that the rate and amount of uptake of TM (5-10% per hour) is indistinguishable from the non-internalizing influenza virus haemagglutinin (HA), and is considerably less than the actively internalizing low density lipoprotein receptor, which reaches a steady state of approximately 50% internalized in 15 min. The low level rate of TM and HA internalization observed is comparable to the rate of normal plasma membrane turnover. Furthermore, this uptake of TM does not require a signal in its extracellular domain.