bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Radiographic Contrast Agents on Thrombin Formation and Activity
Authors: Parker Andrew , Fay William
Published: March 2018

Clinical trials suggest that the risk of thrombosis during coronary angioplasty is lower with ionic contrast agents than with nonionic contrast agents. However, the molecular mechanisms underlying this effect are unknown. This study examined the effects of contrast agents on thrombin formation and its interaction with substrates, inhibitors, and ligands to define potential mechanisms by which contrast agents affect thrombus formation. Two ionic agents, diatrizoate and ioxaglate, and one nonionic agent, ioversol, were studied. Ionic agents inhibited factor X activation by the tissue factor-factor VIIa complex more potently than ioversol (53 ± 3.7, 43.0 ± 1.9, and 26.5 ± 2.4% inhibition by diatrizoate, ioxaglate, and ioversol, respectively, at concentrations of 5%). Ionic contrast agents were potent inhibitors of prothrombinase function, inhibiting thrombin formation by >75% at contrast concentrations of 0.6% (p <0.005). Ioversol inhibited prothrombinase to a significantly lesser extent than ionic agents. Clotting assays suggested that ioxaglate was the most potent inhibitor of thrombin generation in plasma despite having the least effect on fibrin polymerization. Contrast agents inhibited binding of thrombin to fibrin, with ionic agents producing a more potent effect than ioversol (p <0.02). However, contrast agents did not inhibit thrombin-mediated platelet activation, had only a minor effect on inhibition of thrombin by antithrombin III, and did not affect thrombin-hirudin interactions. In summary, these studies identify specific mechanisms by which radiographic contrast agents inhibit thrombin formation and function – i.e. inhibition of tissue factor-dependent factor Xa generation, inhibition of the prothrombinase complex, and inhibition of thrombin binding to fibrin. These findings may help to explain the reduced risk of thrombosis during coronary angioplasty associated with ionic contrast agents.
Modulation of Plasminogen Activator Inhibitor 1 by Triton X-100 – Identification of Two Consecutive Conformational Transitions
Authors: Gils Ann , Declerck Paul
Published: March 2018

Plasminogen activator inhibitor-1 (PAI-1) is a unique member of the serpin superfamily because of its conformational and functional flexibility. In the present study, we have evaluated the influence of the nonionic detergent Triton X-100 (TX-100) on the functional stability and conformational transitions of PAI-1.

At 37° C, TX-100 induced a concentration-dependent decrease of the functional half-life of PAI-1 resulting in half-lives of 177 ± 54 min (mean ± SD, n = 3), 19 ± 2 min, 1.7 ± 0.3 min and 0.53 ± 0.03 min in the presence of 0.005, 0.010, 0.020 and 0.2% TX-100, respectively, compared to a half-life of 270 ± 146 min in the absence of TX-100. Conformational analysis at various time points and at different temperatures (0° C, 25° C, 37° C) revealed that this inactivation proceeds through the formation of a substrate-like intermediate followed by the formation of the latent form. Kinetic evaluation demonstrated that this conversion fits to two consecutive first-order transitions, i.e. active substrate latent. The k1 value was strongly dependent on the concentration of TX-100 (e.g. 0.002 ± 0.0006 s -1 and 0.029 ± 0.003 s -1 for 0.01% and 0.2% TX-100 at 37° C) whereas the conversion of substrate to latent (k2) was virtually independent of the TX-100 concentration (i.e. 0.012 ± 0.002 s -1 and 0.011 ± 0.001 s -1 for 0.01 and 0.2% TX-100 at 37° C).

Experiments with a variety of other non-ionic amphiphilic compounds revealed that the amphiphilic character of the compound is, at least in part, responsible for the observed effects and strongly indicate that the currently reported mechanism of inactivation is of general importance for the conformational transitions in PAI-1.

In conclusion, TX-100 changes the initial conformation of PAI-1 resulting in altered functional properties. This observation allows us to develop a new model for the mechanism involved in the conformational flexibility of PAI-1 and may provide new insights for the development of strategies for interference with PAI-1 activity.
Purification of the Porcine Platelet GP IIb-IIIa Complex and the Propolypeptide of von Willebrand Factor
Authors: Royo Teresa , Vidal Matilde , Badimon Lina
Published: March 2018

Platelet membrane glycoproteins (GP) are involved in platelet adhesion and aggregation. The glycoprotein IIb-IIIa complex (GP IIbIIIa) is a Ca2+-dependent heterodimer that binds fibrinogen and other adhesive proteins, thereby mediating platelet aggregation and adhesion. We have purified two major glycoproteins from pig platelets by Concanavalin A-Sepharose, Heparin-Sepharose and Sephacryl S-300 HR chromatography (Fitzgerald et al. Anal Biochem, 1985): i) the GP IIb-IIIa complex, GP IIb Mr = 140,000 and GP IIIa a single chain of Mr = 95,000-100,000; and ii) a predominant glycoprotein of high molecular weight, the propolypeptide of von Willebrand factor (Mr = 80,000-100,000). Western-blot analysis of the purified GP IIb-IIIa showed that only certain monoclonal antibodies against the human receptor specifically recognize the porcine complex. Differences between the porcine and human GP IIb-IIIa glycoproteins could partially explain the decreased inhibitory effects of GP IIb/IIIa-antagonists (against the human receptor) in porcine platelets.
Characteristics of the Interaction between Thrombin Exosite1 and the Sequence 269-297 of Platelet Glycoprotein Ibα
Authors: Bouton Marie-Christine , Thurieau Christophe , Guillin Marie-Claude , Jandrot-Perrus Martine
Published: March 2018

The interaction between GPIb and thrombin promotes platelet activation elicited via the hydrolysis of the thrombin receptor and involves structures located on the segment 238-290 within the N-terminal domain of GPIbα and the positively charged exosite 1 on thrombin. We have investigated the ability of peptides derived from the 269-287 sequence of GPIbα to interact with thrombin. Three peptides were synthesized, including Ibα 269-287 and two scrambled peptides R1 and R2 which are comparable to Ibα 269-287 with regards to their content and distribution of anionic residues. However, R2 differs from both Ibα 269-287 and R1 by the shifting of one proline from a central position to the N-terminus. By chemical cross-linking, we observed the formation of a complex between 125I-Ibα 269-287 and α-thrombin that was inhibited by hirudin, the C-terminal peptide of hirudin, sodium pyrophosphate but not by heparin. The complex did not form when γ-thrombin was substituted for α-thrombin. Ibα 269-287 produced only slight changes in thrombin amidolytic activity and inhibited thrombin binding to fibrin. R1 and R2 also formed complexes with α-thrombin, modified slightly its catalytic activity and inhibited its binding to fibrin. Peptides Ibα 269-287 and R1 inhibited platelet aggregation and secretion induced by low thrombin concentrations whereas R2 was without effect. Our results indicate that Ibα 269-287 interacts with thrombin exosite 1 via mainly electrostatic interactions, which explains why the scrambled peptides also interact with exosite 1. Nevertheless, the lack of effect of R2 on thrombin-induced platelet activation suggests that proline 280 is important for thrombin interaction with GPIb.
Reconstituted High Density Lipoprotein (rHDL) Modulates Platelet Activity In Vitro and Ex Vivo
Authors: Spycher Martin , Doran Jan , Lerch Peter
Published: March 2018

A reconstituted high density lipoprotein (rHDL) prepared for clinical use was tested for its influence on platelet activity modulated by various stimuli. In a first series of in vitro experiments, rHDL was added to blood in a concentration series, and platelet rich plasma (PRP) was isolated. Platelets were stimulated with arachidonic acid, collagen, epinephrine or ADP, and platelet aggregation was assessed. rHDL mediated a dose dependent inhibition of the platelet activity. With purified platelets rHDL inhibited the release reaction induced by collagen, but not by thrombin, as measured by CD62P (P-Selectin) expression on the plasma membrane. Ex vivo experiments were performed with PRP from volunteers, previously infused with 25 mg rHDL/kg body weight and 40 mg rHDL/kg body weight, respectively. Platelet activity in PRP was assessed before, and up to 30 h after the end of the rHDL infusion. A transient inhibition of the platelet aggregation induced by arachidonic acid and collagen was observed which was more pronounced in the group receiving 40 mg rHDL/kg body weight. In both groups of experiments, in vitro and ex vivo, the inhibition of the platelet activity was also dependent on the stimulus used.
A Mutation in Plasma Platelet-activating Factor Acetylhydrolase (Val279Phe) Is a Genetic Risk Factor for Cerebral Hemorrhage but not for Hypertension
Authors: Yoshida Hidemi , Imaizumi Tadaatsu , Fujimoto Koji , Itaya Hiroyuki , Hiramoto Makoto , Yoshimizu Nobuhiro , Fukushi Kazue , Satoh Kei
Published: March 2018

Platelet-activating factor (PAF) acetylhydrolase is an enzyme that inactivates PAF. Deficiency of this enzyme is caused by a missense mutation in the gene. We previously found a higher prevalence of this mutation in patients with ischemic stroke. This fact suggests that the mutation might enhance the risk for stroke through its association with hypertension. We have addressed this hypothesis by analyzing the prevalence of the mutation in hypertension. We studied 138 patients with essential hypertension, 99 patients with brain hemorrhage, and 270 healthy controls. Genomic DNA was analyzed for the mutant allele by the polymerase-chain reaction. The prevalence of the mutation was 29.3% (27.4% heterozygotes and 1.9% homozygotes) in controls and 36.2% in hypertensives and the difference was not significant. The prevalence in patients with brain hemorrhage was significantly higher than the control: 32.6% heterozygotes and 6.1% homozygotes (p <0.05). PAF acetylhydrolase deficiency may be a genetic risk factor for vascular diseases.
Prothrombin and Its Derivatives Stimulate Motility of Melanoma Cells
Authors: Zhou Hong , Gabazza Esteban , Takeya Hiroyuki , Deguchi Hiroshi , Urano Hajime , Adachi Yukihiko , Suzuki Koji
Published: March 2018

Several studies indicated that activation of the clotting system may promote the growth and the invasive behavior of tumor cells. In the present study, we evaluated the migratory response of various melanoma cell lines to several clotting factors and prothrombin derivatives (thrombin, fragment 1, fragment 2 and kringle 1 fragment). Prothrombin, thrombin and fragment 1 stimulated chemotaxis of the murine (K-1735 M2, X21) and human A375 (SM) melanoma cell lines. Prothrombin and prothrombin fragment 1 showed their maximal chemo-tactic activity at 0.5~1 μM. Chemotaxis induced by thrombin was inhibited by hirudin, but not that induced by prothrombin or fragment 1. Other clotting proteins and the fragment 2 and kringle 1 fragment of prothrombin did not elicit chemotactic activity. Checkerboard analysis indicated that motility was directional with a significant chemokinetic component. The K-1735 M2 cells also migrated in a concentration-dependent manner to substratum-bound insoluble prothrombin, thrombin or fragment 1. Ligand binding assays showed that both prothrombin and fragment 1 bound to K-1735 M2 cells with apparent Kds of 0.5 μM. This binding was inhibited by an excess concentration of unlabeled prothrombin and fragment 1 but not by similar concentrations of other prothrombin fragments. These findings suggest that prothrombin and its fragment 1 exert chemotactic activity on melanoma cells by different mechanisms and different binding sites from that induced by thrombin.
Interaction of the A1 Subunit of Factor VIIIa and the Serine Protease Domain of Factor X Identified by Zero-length Cross-linking
Authors: Lapan Kirsty , Fay Philip
Published: March 2018

We have previously used a solid phase binding assay to localize a Factor X (FX) interactive site to the acidic C-terminus of the A1 subunit of FVIIIa (Lapan KA, Fay PJ. J Biol Chem 1997; 272: 2082-2088). The complex of FVIII-FX was made covalent following reaction with the zero-length cross-linking reagent 1-ethyl-3-(3-dimethylaminopropyl-)carbodiimide hydrochloride (EDC). Western blotting of the thrombin-cleaved complex showed that the A1 subunit of FVIIIa associated with FX heavy chain. The FX-A1 product was also detected following cross-linking to the A1/A3-C1-C2 dimer, but not the activated protein C-cleaved A1336/A3-C1-C2 form, indicating that a residue(s) in the region spanning Met337-Arg372 contributed to the intermolecular ion pair(s). A synthetic peptide to this acidic region (FVIII337-372) cross-linked to FX and the product was alkaline resistant indicating that amide linkage(s) were formed. Sequence analysis of the FX-FVIII337-372 adduct suggested that the first 12 NH2-terminal residues of the FX and peptide do not participate in cross-link formation. Conversion of the cross-linked product to FXa by RVV-X showed that the peptide was associated with the serine protease-forming domain of the heavy chain. These results indicate that the association of FVIIIa and FX occurs from a salt linkage(s) formed between residues of the A1 acidic C-terminus of the cofactor (within residues 349-372) and the serine protease-forming domain of the substrate.
Platelet Associated Fibrinogen and ICAM-2 Induce Firm Adhesion of Neutrophils under Flow Conditions
Authors: Kuijper P. H. M. , Torres H. I. Gallardo , Lammers J.-W. J. , Sixma J. J. , Koenderman L. , Zwaginga J. J.
Published: March 2018

Surface-bound platelets support selectin-mediated rolling and β2-integrin-mediated firm adhesion of neutrophils (PMN) under flow conditions. We examined which ligands on platelets mediate this firm adhesion. Surface-bound platelets express ICAM-2 and GPIIbIIIa-bound fibrinogen, which are ligands for LFA-1 and MAC-1. In a well defined model for vessel wall injury, blood from an afibrinogenemic patient was perfused over ECM-coated coverslips to obtain fibrinogen-free platelet surfaces. At high shear rates, PMN-adhesion to fibrinogen-free platelet surfaces decreased compared to fibrinogen-containing controls. Under these conditions, firm adhesion and not rolling was blocked demonstrating the importance of fibrinogen in this process. In addition, MAC-1 and LFA-1 on PMN and ICAM-2 on platelets played a role in firm adhesion; the effect of blocking antibodies was most evident at high shear. The effects of fibrinogen depletion and ICAM-2 blocking were additive. In conclusion, multiple redundant ligands, like ICAM-2 and fibrinogen, induce firm and shear resistant PMN adhesion to platelets under flow conditions. Individually these ligands become critical at higher shear. Blocking of two or more interactions also interferes with low shear adhesion.
Two Repressor Elements Inhibit Expression of the von Willebrand Factor Gene Promoter In Vitro
Authors: Ardekani Ali , Greenberger Joel , Jahroudi Nadia
Published: March 2018

A fragment of the human von Willebrand factor (VWF) gene promoter corresponding to sequences –487 to +247 bp functions as an endothelial specific promoter in cell culture. We have previously reported that a GATA transcription factor functions as an activator and an NF1 like protein functions as a repressor of this promoter fragment. We have now identified a second negative regulatory element in the VWF promoter that interacts with nuclear factor(s) (designated R) in both bovine aortic endothelial and smooth muscle cells. Inhibition of either the NF1 or the R repressor alone is not sufficient to activate the VWF promoter in smooth muscle cells. The present studies reveal that simultaneous inhibition of both repressors activates the VWF promoter in smooth muscle cells. The data support a model of selective derepression to explain the endothelial cell specific activity of the –487 to +247 fragment of the VWF promoter in vitro.
Purification and Characterization of Kaouthiagin, a von Willebrand Factor-Binding and -Cleaving Metalloproteinase from Naja kaouthia Cobra Venom
Authors: Hamako Jiharu , Nishida Sachiyo , Nomura Shosaku , Fujimura Yoshihiro , Ito Masayuki , Ozeki Yasuhiro , Titani Koiti , Matsui Taei
Published: March 2018

A von Willebrand factor (vWF)-binding and -cleaving metalloproteinase, termed “kaouthiagin”, was purified from the venom of cobra snake Naja kaouthia. Kaouthiagin is a monomer with a molecular mass of about 46 kDa and 51 kDa under non-reducing and reducing conditions, respectively, and the N-terminal amino acid sequence is homologous to high molecular mass snake venom metalloproteinases. Kaouthiagin bound to vWF in a divalent ion-independent manner, but the reduced kaouthiagin failed to interact with vWF, suggesting that the protein conformation maintained by intrachaindisulfide linkages of the molecule is essential for the binding to vWF. Neither botrocetin nor bitiscetin, vWF-binding modulators from another snake venom, interfered with the binding between kaouthiagin and vWF, but a monoclonal antibody VW92-3 specific to the N-terminal region of vWF (residues 1-910) inhibited the binding. Without affecting platelet GPIb/IX and GPIIb/IIIa, kaouthiagin specifically cleaved vWF between residues Pro-708 and Asp-709 in a divalent ion-dependent manner to diminish the multimeric structure of vWF in plasma, resulting in the loss of ristocetin-induced platelet aggregability and the collagen-binding activity of vWF. These results indicate that kaouthiagin is a unique metalloproteinase which specifically binds to and cleaves vWF at a specific site and that it will be a useful tool for functional dissection of vWF.
Plaque Rupture and Plaque Erosion
Authors: Burke Allen P. , Farb Andrew , Virmani Renu
Published: March 2018

There are multiple substrates for coronary thrombosis overlying an atherosclerotic plaque. The most common, plaque rupture, consists of an interruption of a thin fibrous cap overlying a lipid rich core. Plaque rupture is a result of macrophage infiltration and matrix degradation, is often seen in calcified plaques, and is highly associated with hypercholesterolemia. A less common substrate, plaque erosion, is not associated with elevated cholesterol and is the prime cause of coronary thrombosis in premenopausal women. The characteristic histologic features are abundant surface smooth muscle cells and proteoglycans, and a small or absent lipid rich core. The mechanisms of plaque erosion are unclear, and there are no consistent risk factors, although patients are often smokers.
TNK-tPA for Acute Myocardial Infarction: The Clinical Experience
Authors: Pislaru Sorin V. , Van de Werf Frans
Published: March 2018

Thrombolytic therapy has become the mainstay of treatment for acute transmural myocardial infarction. Present fibrinolytic regimens have a number of shortcomings, including the failure to induce early and sustained reperfusion in as many as 40-50% of the patients, and to prevent reocclusion in another 10-20% of the patients. The efforts for improving thrombolysis are focused on the development of new agents (fibrinolytics, anticoagulants, and antiplatelet agents). TNK-tPA is a triple combination mutant of wild-type tissue plasminogen activator that exhibits a longer plasma half-life, an enhanced fibrin-specificity, and an increased resistance to the plasminogen activator inhibitor-1. This paper summarizes the results of clinical trials with TNK-tPA in acute myocardial infarction.
Heparin-induced Thrombocytopenia – Pathogenesis and Treatment
Authors: Greinacher A.
Published: March 2018

Heparin-induced thrombocytopenia (HIT) is now recognized as the most frequent immune-mediated adverse drug reaction. During the last decade, fundamental aspects of the pathogenesis of HIT have been resolved. The understanding of some the mechanisms underlying the development of new, paradox thromboembolic complications in HIT led to the concept that thrombin generation plays a key-role in clinically manifest HIT. Consequently new therapeutic concepts imply the use of drugs with either indirect of direct anti-thrombin activity such as donaparoid-sodium and the recombinant hirudin lepirudin. During the last years results of first prospective studies assessing various treatment regimens in HIT became available. Although data of randomized trials are still missing some treatment recommendations can already be drawn from these studies. This review summarizes key aspects of the pathogenesis of HIT and provides an overview of current treatment strategies.
Treatment of Carotid Artery Stenosis by Elective Stent Placement Instead of Carotid Endarterectomy in Patients with Severe Coronary Artery Disease
Authors: Krämer Jochen , Vogel Peter , Friedrich Ingrid , Michael Gross C. , Uhlich Frank , Tamaschke Christian , Dietz Rainer , Waigand Jürgen
Published: March 2018

Patients with concomitant cardiac and cerebrovascular disease undergoing revascularization procedures are at high risk of both, cardiac and cerebrovascular complications. The purpose of our study was to evaluate the feasibility of prior elective carotid artery stenting as an alternative treatment procedure to carotid endarterectomy (CEA) in patients with concomitant coronary artery disease (CAD), who clearly needed coronary revascularization. We offered extracranial internal carotid stenting to 85 patients with 89 significant carotid stenoses. Out of these, 19 patients were symptomatic. The quantitative mean reduction in diameter was 77 ± 11%. Stent implantation was successful in 88 lesions. Two disabling major and 3 reversible minor strokes occurred periprocedurally. Three patients showed asymptomatic restenosis and stent deformation was detected in 2 patients. Based on this experience, carotid stenting in high risk patients with severe coronary artery disease is feasible and safe and might be indicated as an alternative procedure for combined surgery.
Impact of Apolipoprotein(a) Isoform Size Heterogeneity on the Lysine Binding Function of Lipoprotein(a) in Early Onset Coronary Artery Disease
Authors: Simó Josep , Vilella Elisabet , Ribas Montserrat , Pujana Miguel , Sundaram Indirana , Hammel Jeffrey , Hoover-Plow Jane , Joven Jorge
Published: March 2018

Elevated plasma Lp(a) is an independent risk factor for cardiovascular disease. Unique to Lp(a) is the apoprotein, apo(a) which can vary from 250 to 800 kDa in molecular weight. Small isoforms are also associated with the risk of cardiovascular disease. The purpose of this study was to examine the association of Lp(a) concentration, apo(a) size, and Lp(a) lysine-binding site(s) (LBS) function in patients with early onset heart disease, and age-matched controls. Mean values of Lp(a) were significantly higher in the patients than for the age-matched group. The smallest molecular weight isoform for each subject had significantly fewer kringles for the patients than the age-matched controls. There was a significant correlation between LBS activity and kringle number in the single-banded phenotypes of the patients, but not the controls. LBS activity was significantly higher in patients with small isoforms (≤18 kringles) compared to controls. The odds ratio for coronary artery disease for high LBS activity and high Lp(a) concentration was 4.4 (p = 0.002) and for high LBS activity and small isoforms was 10.1 (p = 0.002). In the patients, Lp(a) concentration was higher, apo(a) size was smaller, and LBS activity higher in the small isoforms compared to the controls. This study suggests an association of high LBS activity in small isoforms of Lp(a) with disease in humans.
Genetic Predisposition to Bleeding during Oral Anticoagulant Therapy: Evidence for Common Founder Mutations (FIXVal-10 and FIXThr-10) and an Independent CpG Hotspot Mutation (FIXThr-10)
Authors: Kriz K. , Wuillemin W. A. , Maly F. E. , von Felten A. , Siegemund A. , Keeling D. M. , Baker P. , Chu K. , Konkle B. A. , Lämmle B. , Albert T. , Oldenburg J. ,   the Study Group on Hereditary Warfarin Sensitivity
Published: March 2018

The recent discovery of five patients with coumarin sensitive FIX-variants due to a missense mutation in the FIX propeptide, either Ala-10Val or Ala-10Thr, has highlighted a novel type of genetic predisposition to bleeding during oral anticoagulant therapy (OAT). In the present study, we report six additional patients with such FIX variants. Haplotype analysis of FIX polymorphisms revealed a founder effect in the five German and Swiss patients with the Val-10 variant. Also, four Thr-10 variants detected in Germany, Switzerland and Great Britain derived from a common founder. Two Thr-10 variants from USA showed an independent de novo origin at a CpG dinucleotide that in general represents a mutation hotspot. These findings implicate the existence of additional subjects with corresponding variants in the populations of various countries. Even though the rare occurrence of these variants does not justify a general aPTT screening during OAT, it is recommended to monitor each bleeding event during OAT in males in order to exclude a genetic predisposition to bleeding by means of the following testing strategy: a) aPTT-testing in each bleeding complication of male patients during OAT, b) if aPTT is disproportionately prolonged, determination of FIX:C, and c) if FIX:C is disproportionately decreased as compared to FII:C, FVII:C and FX:C, sequencing of exon 2 of the FIX gene. This strategy will provide a cost-effective and safe procedure to identify patients that carry the FIX variants. Moreover, such a strategy accumulates data about the prevalence of these FIX mutations in a given population.
Elevated Plasma Levels of Crosslinked Fibrinogen Gamma-chain Dimer Indicate Cancer-related Fibrin Deposition and Fibrinolysis
Authors: Steinkellner Werner , Holzmann Klaus , Gsur Andrea , Grimm Rudolf , Ensinger Christian , Obrist Peter , Sauermann Georg , Gerner Christopher
Published: March 2018

Cancer-related fibrin deposition and fibrinolysis were investigated by two-dimensional gel electrophoresis of human solid tumor and effusion specimen in addition to plasma samples. Fibrinogen gamma-chain dimer indicating fibrin deposition and plasmin-generated fibrinogen beta-chain fragments were identified in various solid tumor types by amino acid sequencing, mass spectrometry analysis and Western blotting. In tumor-associated effusions, these techniques allowed to observe plasmin-generated fragments of fibrinogen alpha, beta and gamma-chains in addition to elevated levels of acute-phase proteins. Similar observations were made in case of inflammation-associated effusions. No fibrin degradation product was observed in plasma samples, however, high amounts of fibrinogen gamma-chain dimer crosslinked by transglutaminase were detected in plasma from tumor patients, but not in plasma from controls and patients suffering acute infections and/or inflammations. This finding demonstrated that high transglutaminase activity may be associated with cancer. The presented data indicate that the amount of crosslinked fibrinogen gamma-chain dimer in plasma may correlate with tumor-associated fibrin deposition. The tumor-biological relevance of this potential marker protein is discussed.
Antiplatelet and Antithrombotic Activity of SL65.0472, a Mixed 5-HT1B/5-HT2A Receptor Antagonist
Authors: Lorrain Janine , Lochot Sylvette , Delahaye Monique , Lalé Alain , Savi Pierre , Lechaire Irène , Ferrari Patrice , Bernat André , Schaeffer Paul , Janiak Philippe , Duval Nicole , Grosset Alain , Herbert Jean-Marc , O’Connor Stephen , Berry Christopher
Published: March 2018

The antiplatelet and antithrombotic activity of SL65.0472 (7-fluoro-2-oxo-4-[2-[4-(thieno [3,2-c]pyrin-4-yl) piperazin-1-yl]ethyl]-1,2-dihydroquinoline-acetamide), a mixed 5-HT1B/5-HT2A receptor antagonist was investigated on 5HT-induced human platelet activation in vitro, and in rat, rabbit and canine platelet dependent thrombosis models. SL65.0472 inhibited 5-HT-induced platelet shape change in the presence of EDTA (IC50 values = 35, 69 and 225 nM in rabbit, rat and human platelet rich plasma (PRP)), and also inhibited aggregation induced in human PRP by 3-5 μM 5-HT + threshold concentrations of ADP (0.5-1 M) or collagen (0.3 g/ml) with mean IC50 values of 49 ± 13 and 48 ± 6 nM respectively. SL65.0472 inhibited thrombus formation when given both intravenously 5 min and orally 2 h prior to assembly of an arterio-venous (A-V) shunt in rats as from 0.1 and 0.3 mg/kg respectively. It was active in a rabbit A-V shunt model with significant decreases in thrombus weight as from 0.1 mg/kg i. v. and at 10 mg/kg p. o. The delay to occlusion in an electric current-induced rabbit femoral artery thrombosis model was increased by 251% (p <0.05) after 20 mg/kg p. o. SL65.0472 (30 μg/kg i. v.) virtually abolished coronary cyclic flow variations (7.2 ± 1.0/h to 0.6 ± 0.6/h, p <0.05) and increased minimum coronary blood flow (1.2 ± 0.8 ml/ min to 31.8 ± 8.4 ml/min, p <0.05) in a coronary artery thrombosis model in the anaesthetised dog. Finally, SL65.0472 significantly increased the amount of blood lost after rat tail transection at 3 mg/kg p. o. Thus the anti-5-HT2A component of SL65.0472 is reflected by its ability to inhibit 5-HT-induced platelet activation, and platelet-rich thrombus formation.
Effect of Ca2+ Chelation on the Platelet Inhibitory Ability of the GPIIb/IIIa Antagonists Abciximab, Eptifibatide and Tirofiban
Authors: Marciniak Stanley , Jordan Robert , Mascelli Mary
Published: March 2018

Objective. Enhanced GPIIb/IIIa binding and inhibition of platelet aggregation of eptifibatide by the reduction of ionized plasma calcium concentrations have been reported. The present study compared the importance of Ca2+ chelation on the in vitro platelet inhibitory profiles of the GPIIb/IIIa antagonists abciximab, eptifibatide and tirofiban. Methods and Results. Turbidimetric platelet aggregation dose response curves of the various GPIIb/IIIa antagonists were performed using platelet rich plasma (PRP) anticoagulated with either trisodium citrate, or the non-chelating anticoagulant, PPACK. The concentrations of antagonist that resulted in 50% inhibition of TRAP-induced (10 M) platelet aggregation (IC50) were measured in the presence of either citrate or PPACK. In addition, the influence of Ca2+ chelation on the binding properties (relative affinity, on- and off-rates) of abciximab for the GPIIb/IIIa receptor on platelets was measured. For all three agonists, the IC50 concentrations were lower for platelets treated with citrate than PPACK, but the degree of difference varied among the agents. The mean TRAP IC50 values for citrate and PPACK were 88.2 ± 12.2 nM and 126.1 ± 28.4 nM for abciximab (1.4 fold enhancement; p = 0.0007), 75.9 ± 13.3 nM and 142.6 ± 32.6 nM for tirofiban (1.9-fold enhancement; p = 0.001), and 260.2 ± 62.5 nM and 810.3 ± 182.5 nM for eptifibatide (3.1-fold enhancement; p = 0.001). A similar shift in effective inhibitor concentrations for abciximab was observed with ADP (10 M). The relative affinities (EC50), on- and off-rates of abciximab for the platelet GPIIb/IIIa receptor in the presence of trisodium citrate and PPACK were equivalent. Conclusions. These data confirm previous observations that Ca2+ chelation afforded by citrate decreases the effective inhibitor concentrations of GPIIb/IIIa antagonists, as assessed by turbidimetric platelet aggregation. However, the extent of decrease was less for abciximab and tirofiban, compared to eptifibatide.
The Subendothelium of the HMEC-1 Cell Line Supports Thrombus Formation in the Absence of von Willebrand Factor and Collagen Types I, III and VI
Authors: Bonnefoy Arnaud , Harsfalvi Jolan , Pfliegler George , Fauvel-Lafève Françoise , Legrand Chantal
Published: March 2018

The macromolecular composition of the extracellular matrix (ECM) produced by the human microvascular endothelial cell line (HMEC-1) was determined by ELISA and its thrombogenicity was studied in blood perfusion assays. Results were compared with those obtained with the ECM produced by human umbilical vein endothelial cells (HUVEC). The HMEC-1’s ECM contains collagen type IV, fibronectin, laminin and thrombospondin, but no detectable levels of collagen types I, III and VI, or von Willebrand factor (vWF), whereas all these components were found in the ECM synthesized by HUVEC. HMEC-1’s ECM was perfused with low-molecular-weight heparin-anticoagulated blood at two wall shear rates (650/s and 2600/s), representative of moderate and high arterial wall shear rates, in parallel plate flow chambers for 5 min. This resulted in the formation of large platelet aggregates, compared to essentially a monolayer of adherent platelets on HUVEC’s ECM. Interestingly, large thrombi were formed at 2600/s when HMEC-1’s ECM was perfused with the blood of a patient with severe type III von Willebrand disease lacking both plasma and platelet vWF, indicating that vWF was not absolutely required for thrombus formation on this matrix. Thrombin generated on the HMEC-1’s ECM contributed importantly to the large platelet thrombi formed, shown by performing blood perfusion experiments in the presence of thrombin inhibitors. Our results indicate that 1) platelet adhesion and aggregate formation on a subendothelium may occur at a high shear rate (2600/s) without the participation of collagen types I, III and VI, and vWF; and 2) the HMEC-1 cell line may prove useful for in vitro studies of the thrombogenic properties of microvascular subendothelium which in most cases does not contain fibrillar collagens and vWF.
P-Selectin Antagonism Causes Dose-dependent Venous Thrombosis Inhibition
Authors: Schaub Robert , Wrobleski Shirley , Londy Frank , Fex Beverly , Chapman Amy , Greenfield Lazar , Wakefield Thomas , Myers Daniel
Published: March 2018

Inhibition of P-selectin by antibody or selectin antagonist decreases inflammation and thrombosis. This study evaluates the dose-response relationship using a selectin receptor antagonist. Eight male baboons (Papio anubis) underwent inferior vena caval thrombosis using a 6 h balloon occlusion model. Three animals received 500 μg/kg P-selectin antagonist (rPSGL-Ig) and five 1 mg/kg rPSGL-Ig with or without a non-anticoagulant dose of Dalteparin. These animals were compared to our published results in this model with 4 saline controls and 8 animals that received 4 mg/kg rPSGL-Ig. A statistically significant dose-response relationship existed between rPSGL-Ig dose and thrombosis (p < 0.01), and between rPSGL-Ig dose and spontaneous recanalization (p < 0.05). Inflammatory assessment revealed decreased gadolinium enhancement in all rPSGL-Ig groups compared to previously reported control, despite no significant differences in inflammatory cell extra-vasation. No dose of rPSGL-Ig caused anticoagulation. Selectin antagonism results in a dose-dependent decrease in thrombosis and increase in spontaneous recanalization.
Pulmonary Fibrosis Is Increased in Mice Carrying the Factor V Leiden Mutation following Bleomycin Injury
Authors: Xu Zuojun , Westrick Randal , Shen YueChun , Eitzman Daniel
Published: March 2018

Increased fibrin deposition following inflammatory lung injury has been proposed to facilitate the development of pulmonary fibrosis. Therefore, factors predisposing to thrombosis may affect the fibrotic response to injury. Activated protein C (aPC) resistance due to the factor V Leiden mutation (FνL) is a common genetic risk factor for vascular thrombosis. To examine the relationship between aPC resistance and the development of pulmonary fibrosis, lung inflammation was induced by bleomycin in mice carrying the FvL mutation. Three weeks following the instillation of 0.0375 U of bleomycin, the lungs of mice homozygous and heterozygous for FvL contained significantly more hydroxyproline (35 ± 4 and 36 ± 7 ug hydroxyproline/ mg total protein, respectively) than wild-type mice (26 ± 6 ug/mg protein, p <0.01 for both comparisons). These data demonstrate a strong relationship between aPC resistance and the pulmonary fibrosis that occurs following inflammatory lung injury.
The Glycoprotein Ib-IX-V Complex in Platelet Adhesion and Signaling
Authors: Andrews Robert , Shen Yang , Gardiner Elizabeth , Dong Jing-fei , López José , Berndt Michael
Published: March 2018

Introduction

Thrombosis can result in unstable angina, acute myocardial infarction, or stroke, all major causes of death in the Western world. Circulating platelets become adherent and form an occlusive thrombus, either by exposure to sclerotic lesions following plaque rupture or in response to pathological shear stress in obstructed coronary arteries. This process parallels normal haemostasis, where platelets adhere to the subendothelium at sites of vascular injury, become activated, and recruit additional platelets to the developing thrombus. At high shear, thrombus formation is initiated by a specific platelet membrane adhesion receptor, the glycoprotein (GP) Ib-IX-V complex, which binds the adhesive glycoprotein, von Willebrand factor (vWF), in the vessel wall or plasma. Recent evidence also suggests that platelet adhesion to endothelial cells and leukocytes may be involved in atherogenesis, thrombosis, and inflammation. Preliminary findings indicate that GP Ib-IX-V specifically recognizes P-selectin, a member of the selectin superfamily, an interaction that may, at least partially, regulate platelet-endothelial cell adhesion. This review focuses on recent advances in understanding structure-activity relationships of GP Ib-IX-V.
The Role of Genetics in Inhibitor Formation
Authors: Gill Joan Cox
Published: March 2018

Introduction

Treatment of hemorrhages in patients with inherited severe hemophilia A is dependent on transfusion replacement therapy with either plasma-derived or recombinant factor VIII concentrates.1 Hemorrhages become very difficult to treat after inhibitors develop, resulting in significant decreases in qualityof- life and life expectancy.2-6 Strategies to treat hemorrhages in the face of an inhibitor include the use of “bypass” therapy with activated and nonactivated prothrombin complex concentrates, high-dose factor VIII, porcine factor VIII, and recombinant factor VIIa. None of these is as effective as treatment of the noninhibitor patient with factor VIII.2,6 Therefore, several strategies to induce tolerance to factor VIII administration have been attempted, including administration of daily high-dose intravenous infusions of factor VIII concentrate alone, or in combination with, immune-modulating therapy.2,6 These strategies are extraordinarily difficult and expensive and are frequently unsuccessful, particularly when the inhibitor is well established. Thus, the ability to predict which patients are at risk for inhibitor development and to identify factors that predispose to inhibitor development would allow investigation of therapies targeted to prevent the establishment of the inhibitor response or to provide the ability to initiate early and, thus, more effective, immune tolerance induction for those at risk.
Recombinant Clotting Factors in the Treatment of Hemophilia
Authors: Lee Christine
Published: March 2018

Introduction

The field of hemophilia has always been one of rapid change with improvements in clinical practice rapidly following the advances in basic science and clinical research. The major achievements of the last decade have reflected the application of molecular biology, and this has been particularly true for the development of recombinant clotting factors. The interest in developing recombinant factors VIII and IX was driven by the desire for a “safe” clotting factor following the terrible epidemics of transfusion-transmitted disease in the 1970s and 1980s.

Although the goals for treatment for hemophilia, as stated by the World Federation of Hemophilia, are “to minimize disability and prolong life, to facilitate general social and physical wellbeing, and to help each patient achieve full potential, while causing no harm,”1 the cost of recombinant clotting factors are comparatively high. As a result, in many instances, the provision has been rationed, even though such therapy is considered state-of-the-art treatment for all patients requiring replacement therapy.
In Vivo Gene Delivery by Lentiviral Vectors
Authors: Naldini Luigi
Published: March 2018

Introduction

Successful gene therapy requires the efficient delivery and sustained expression of a therapeutic gene into the tissues of a human body. Most of the candidate tissues for therapeutic gene transfer are made of quiescent cells, such as from the brain, liver, and muscle. Thus, the optimal vector should infect nondividing cells, become stably associated with the genome of target cells, and support a high, steady-state level of transcription.1,2 Like all vectors derived from retroviruses, lentiviral vectors integrate into the chromatin of target cells and do not transfer any viral genes. Both of these features are important for achieving sustained expression of the transgene. Moreover, lentiviral vectors infect nondividing cells, a feature sharply distinguishing them from simple or onco-retroviral vectors.3 Upon infection, retroviruses deliver a nucleoprotein complex that reverse transcribes the viral RNA and integrates the newly made DNA into the chromatin. The nucleoprotein complexes of onco-retroviruses are excluded by the nucleus, and they reach the chromatin only when the nuclear membrane is fragmented during mitosis. This explains the dependence of a productive onco-retroviral infection on cell division occurring shortly after viral entry.4,5 In contrast, the nucleoprotein complexes of lentiviruses contain nuclear localization signals that mediate their active transport through the nucleopores during interphase. This explains the capacity of lentiviruses to infect macrophages, a nondividing cell type.6-9
Animal Testing of Retroviral-Mediated Gene Therapy for Factor VIII Deficiency
Authors: Jolly Douglas , Greengard Judith
Published: March 2018

Introduction

Hemophilia A results from the plasma deficiency of factor VIII, a gene carried on the X chromosome. Bleeding results from a lack of coagulation factor VIII, a large and complex protein that circulates in complex with its carrier, von Willebrand factor (vWF).1 Severe hemophilia A (<1% of normal circulating levels) is associated with a high degree of mortality, due to spontaneous and trauma-induced, life-threatening and crippling bleeding episodes.2 Current treatment in the United States consists of infusion of plasma-derived or recombinant factor VIII in response to bleeding episodes.3 Such treatment fails to prevent cumulative joint damage, a major cause of hemophilia-associated morbidity.4 Availability of prophylactic treatment, which would reduce the number and severity of bleeding episodes and, consequently, would limit such joint damage, is limited by cost and the problems associated with repeated venous access. Other problems are associated with frequent replacement treatment, including the dangers of transmission of blood-borne infections derived from plasma used as a source of factor VIII or tissue culture or formulation components. These dangers are reduced, but not eliminated, by current manufacturing techniques. Furthermore, approximately 1 in 5 patients with severe hemophilia treated with recombinant or plasma-derived factor VIII develop inhibitory humoral immune responses. In some cases, new inhibitors have developed, apparently in response to unnatural modifications introduced during manufacture or purification.5

Gene therapy could circumvent most of these difficulties. In theory, a single injection of a vector encoding the factor VIII gene could provide constant plasma levels of factor in the long term. However, long-term expression after gene transfer of a systemically expressed protein in higher mammals has seldom been described. In some cases, a vector that appeared promising in a rodent model has not worked well in larger animals, for example, due to a massive immune response not seen in the rodent.6 An excellent review of early efforts at factor VIII gene therapy appeared in an earlier volume of this series.7 A summary of results from various in vivo experiments is shown in Table 1. This chapter will focus on results pertaining to studies using vectors based on murine retroviruses, including our own work.
Gene Therapy for Hemophilia A and B: Patient Selection and Follow-up, Requirements for a Cure
Authors: Lusher Jeanne
Published: March 2018

Introduction

The treatment of hemophilia A and B has improved considerably in recent years. The availability of hepatitis A and B vaccines, safer clotting factor concentrates (particularly recombinant factor VIII and recombinant factor IX concentrates), and synthetic agents, such as desmopressin,1 has resulted in earlier, more aggressive treatment and prophylactic regimens aimed at preventing chronic, debilitating joint disease.2-8There have been no new cases of human immunodeficiency virus (HIV) disease attributable to clotting factor in North America since 1987, and documented instances of hepatitis transmission by clotting factor concentrates have been rare in the 1990s. Concerns remain that certain nonenveloped viruses, such as human parvovirus B19 and hepatitis A virus, can still be transmitted by some plasma-derived clotting factor concentrates,9and questions linger as to whether the agents causing Creutzfeld-Jacob disease (CJD) and new variant CJD might also be transmitted. Overall, however, the products available to treat hemophilia today are safer than ever before.

An increasing number of persons with hemophilia are receiving exclusively recombinant (r) products, and manufacturers are now producing new, second-generation r-factor VIII products that are stabilized with sugars, rather than albumin, or are smaller, truncated molecules.10 Scientists are now designing specific changes into the factor VIII genes in an attempt to derive unique and improved forms of r-factor VIII.11 The next logical areas of focus are to bring to fruition the promise of an “unlimited supply” of r-factor VIII and r-factor IX products, to meet the needs of persons with hemophilia, not only in developed countries, but throughout the world, and to be able to cure hemophilia through gene therapy.

As gene therapy trials begin in humans with hemophilia, the scientists involved, the United States Food and Drug Administration (FDA), and perhaps most importantly, members of the hemophilia community must decide which categories of affected individuals should be entered in these trials, particularly the earliest, Phase I trials. Who is most likely to benefit if gene therapy proves to be both effective and safe? Who should be the first patients to be enrolled in each new trial? Who is at greatest risk if something unexpected happens? What would be considered a good outcome? Clearly, some of these questions are more difficult to answer than others.