bluuebunny/crossref_metadata_2025 huggingface dataset:
A Detailed Comparison of the Performance of the Standard versus the Nijmegen Modification of the Bethesda Assay in Detecting Factor VIII:C Inhibitors in the Haemophilia A Population of Canada
Authors: Verbruggen Bert , Rivard Georges , Teitel Jerome , Walker Irwin , Giles Alan ,   Association of Hemophilia Centre Directors of Canada,   Factor VIII/IX Subcommittee of Scientific and Standardization Committee of International Society on Thrombosis and Haemostasis
Published: March 2018

The Bethesda assay is widely used to monitor the development and progress of Factor VIII:C inhibitors. Factor VIII stability in the substrate plasma (normal pool) is compromised by pH shift and reduction in protein concentration. Preliminary study, by Verbruggen and colleagues (8), suggested a reduction in spuriously positive assay results may result from buffering the normal pool plasma substrate with imidazole to pH 7.4 and substituting Factor VIII deficient plasma for imidazole buffer in the control incubation mix. These laboratory findings have now been confirmed by the performance of both the standard and the modified Bethesda assays in parallel on 877 patient samples screened during the Factor VIII:C Inhibitor Surveillance Program instituted following the conversion of all Canadian haemophilia A patients to recombinant Factor VIII. Although this study does not address the question of the clinical significance of spurious positive assays, these laboratory findings do support the conclusions of Verbruggen and the modified assay has recently been endorsed by the Factor VIII/IX Subcommittee of the SSC.
Heterogeneity of Lupus Anticoagulant (LA) Antibodies: LA Activity in Dilute Russell’s Viper Venom Time and Dilute Kaolin Clotting Time Detect Different Populations of Antibodies in Patients with the “Antiphospholipid” Syndrome
Authors: Kandiah David , Krilis Steven
Published: March 2018

“Antiphospholipid” (aPL) antibodies comprise two main groups of antibodies, lupus anticoagulant (LA) antibodies and “anticardiolipin” (aCL) antibodies which can be separated by certain chromatographic techniques. In this study we analysed the plasma of 10 patients with aPL antibodies, and were able to demonstrate that in four patients with both clotting test reactivities, the dilute Russell’s Viper Venom Time (dRVVT) activity can be separated from the dilute Kaolin Clotting Time (dKCT) reactivity by using a polyacrylamide-immobilised phosphatidylserine column but not with phospholipid liposomes. The differential reactivity of the autoantibodies in this patient population is not due to binding to β2GPI, prothrombin or protein C in solid-phase immunoassays. Hence LA antibodies detected in different phospholipid-dependent clotting tests detect different populations of antibodies in some APS patients and the routine detection of LA antibodies should be performed with at least two clotting tests looking at different coagulation reactions.
The Prothrombin 20210A Allele Is the Most Prevalent Genetic Risk Factor for Venous Thromboembolism in the Spanish Population
Authors: Coll Inma , Llobet Dolors , Río Elisabeth del , Oliver Arturo , Mateo José , Borrell Montserrat , Fontcuberta Jordi , Souto Juan Carlos
Published: March 2018

We investigated the prevalence of the new recently reported mutation in the prothrombin gene (20210 A) in a sample of 116 unrelated patients with venous thromboembolism. We found 20 heterozygous carriers (17.2%, CI 95% 10.4-21.1). In comparison, we observed 13 carriers among 201 healthy unmatched controls (6.5%, CI 3.5-10.8). The 20210 A mutation seems to increase the risk of venous thrombosis 3-fold (odds ratio 3.1, 95% CI 1.4-6.6). Considering only patients with a first event (n = 62) the OR was 2.0 (p = 0.18, NS) while those with recurrent events (n = 54) showed an OR of 5.9 (95% CI 2.5-14.4). A majority of heterozygous patients (55%) presented a second thrombophilic factor and 60% of affected females had their first event before 30 years of age, while on oral contraceptive treatment. The prevalence found in this study for healthy people is the highest reported to date. The 20210 A variant appears to be the most prevalent genetic risk factor among patients with thrombosis in our geographical area.
The Effects of Standard and Low Molecular Weight Heparin on Bone Nodule Formation In Vitro
Authors: Bhandari Mohit , Hirsh Jack , Weitz Jeffrey , Young Edward , Venner Thomas , Shaughnessy Stephen
Published: March 2018

Previously, we demonstrated in a rat model of heparin-induced osteoporosis that low molecular weight heparin (LMWH) produces less bone loss than unfractionated heparin, and that only heparin increases osteoclast number and activity. In contrast, both heparin and LMWH were found to decrease osteoblast function to a similar extent, possibly because at the doses tested both agents produced maximal inhibition. To examine the relative effects of heparin and LMWH on osteoblast function more closely we used an in vitro bone nodule assay, together with measurements of alkaline phosphatase (ALP) activity. Both agents inhibited bone nodule formation and ALP activity in a concentration-dependent manner, but 6 to 8-fold higher concentrations of LMWH were required to achieve equivalent effects. The effect of heparin on osteoblast function was both chain-length and negative charge-dependent because the ability of defined heparin fragments to inhibit nodule formation correlated with their molecular weight (r = 0.98), and N-desulfated heparin was less inhibitory than heparin. In contrast, the effect of heparin on osteoblast function was pentasaccharide-independent because heparin with low affinity for antithrombin had similar activity to heparin with high antithrombin activity. These findings help to explain mounting clinical evidence that the risk of osteoporosis is lower with LMWH than with heparin.
Ristocetin- and Thrombin-induced Platelet Aggregation at Physiological Shear Rates: Differential Roles for GPIb and GPIIb-IIIa Receptor
Authors: Kasirer-Friede A. , Frojmovic M. M.
Published: March 2018

We recently reported that washed platelets (WP) activated with ADP and expressing surface-bound vWF aggregated in flow through small tubes or in a cylindrical couette device at physiological shear rates of G = 300 s–1-1000 s–1 in the absence of exogenous ligands, with GPIb-vWF partially, and activated GPIIb-IIIa totally required for the aggregation. We have now extended these studies to aggregation of platelets “activated” with ristocetin or thrombin. Washed platelet suspensions with added soluble vWF and ristocetin (0.3-0.75 mg/ml), or activated with thrombin (0.01-0.5 U/ml) but no added ligand, were sheared in a coaxial cylinder device at uniform shear rate, G = 1000 s–1. The collision capture efficiency (αG) with which small aggregates form (= experimental/calculated initial rates of aggregation) was correlated with vWF platelet binding assessed by flow cytometry. The vWF-GPIb interaction was exclusively able to support ristocetin-mediated shear aggregation of metabolically active platelets, with very few vWF monomer equivalents bound per platelet (representing ≤10 molecules of 10 million Da) required to yield high capture efficiencies (αG = 0.38 ± .02; n = 11), suggesting rapid and stable bond formations between vWF and GPIb. However, platelet surface-expressed vWF, generated by addition of thrombin to washed platelets, was found to mediate platelet aggregation with αG = 0.08 ± .01 (n = 6), surprisingly comparable to that previously reported for WP and ADP activation. Blocking the GPIIb-IIIa receptor decreased αG by 95 ± 3% (n = 3), while a monoclonal antibody to the vWF site on GPIb caused a 49 ± 7% (n = 8) decrease in αG. The partial role for GPIb thus appears to reflect a facilitative function for increasing contact time between flowing platelets, and allowing engagement of the GPIIb-IIIa receptor to yield stable attachment.
A Heparin-coated Circuit Maintains Platelet Aggregability in Response to Shear Stress in an In Vitro Model of Cardiopulmonary Bypass
Authors: Hioki I. , Onoda K. , Shimono T. , Shimpo H. , Tanaka K. , Suzuki Y. , Shiku H. , Ikeda Y. , Yada I. , Nishikawa M.
Published: March 2018

Alterations in platelet aggregability may play a role in the pathogenesis of qualitative platelet defects associated with cardiopulmonary bypass (CPB). We circulated fresh heparinized whole blood through tubing sets coated with heparin (C group, n = 10) and through non-coated sets (N group, n = 10) as a simulated CPB circuit. Shear stress (108 dyne/cm2)-induced platelet aggregation (hSIPA), plasma von Willebrand factor (vWF) activity and platelet glycoprotein (GP) Ib expression were measured, before, during, and after this in vitro set up of circulation. In the two groups, the extent of hSIPA significantly decreased during circulation and was partially restored after circulation. Decreases in the extent of hSIPA were significantly less with use of heparin-coated circuits. There was an equivalent reduction in plasma vWF activity, in the two groups. Expression of platelet surface GP Ib decreased significantly during circulation and recovered after circulation. Reduction of surface GP Ib expression during circulation was significantly less in the C group than that in the N group. Decrease in surface GP Ib expression correlated (r = 0.88 in either group) with the magnitude of hSIPA, in the two groups. The progressive removal of surface GP Ib was mainly attributed to redistribution of GP Ib from the membrane skeleton into the cytoskeleton. Our observations suggest that use of heparin-coated circuits partly blocks the reduction of hSIPA, as a result of a lesser degree of redistribution of GP Ib.
Analysis of Platelet Glycoprotein Ia (α2 Integrin) Allele Frequencies in Three North American Populations Reveals Genetic Association between Nucleotide 807C/T and Amino Acid 505 Glu/Lys (HPA-5) Dimorphisms
Authors: Aramaki Kaelen , Teramura Gayle , Gaur Lakshmi , Reiner Alexander
Published: March 2018

Glycoprotein Ia (α2 integrin) is a subunit of the heterodimeric membrane complex (GPIa/IIa) that mediates platelet adhesion to collagen. Several nucleotide sequence variations of GPIa have been described. A nucleotide 1648 G/A dimorphism that leads to a Glu/Lys substitution at amino acid 505 is responsible for the human platelet antigen system, HPA-5. Recently, two other linked GPIa nucleotide dimorphisms involving codons Phe224 and Thr246 were identified: a C/T substitution at nucleotide 807 and a G/A substitution at nucleotide 873 (1). Using restriction enzyme digestion of amplified GPIa genomic DNA fragments (PCR-RFLP) to distinguish genotypes, we have determined the allele frequencies of the GPIa 807C/T and Glu/Lys505 dimorphisms in three North American populations, and a panel of non-human primates. Our results indicate a genetic relationship between the 807C/T and Glu/Lys505 dimorphisms that leads to an evolutionary model of GPIa isoforms.
Induction of Endothelial Cell Adhesion Molecules by Serum and Immunoglobulins G from a Patient with Vasculitis and Monoclonal Gammapathy: Potential Relevance to Vasculitis
Authors: Herbert Jean-Marc , Giraudeau Véronique , Quere Isabelle , Zini Jean-Marc , Tobelem Gérard , Dupuy Evelyne
Published: March 2018

Interactions between endothelial cell adhesion molecules and their β2 integrin adhesive receptors on leukocytes are thought to play a role in the pathogenesis of inflammatory diseases and probably vasculitis. We describe a case in whom leukocytoclastic vasculitis was associated to a monoclonal immunoglobulin G2 kappa (IgG2K). During the vasculitic crisis, the patient’s serum and the isolated IgG from this serum induced the expression of E-selectin, VCAM-1 and ICAM-1 at the HUVEC surface, but not tissue factor activity, whereas normal, control serum and patient serum at remission were without any effect.

A close relationship between the vasculitis and the serum level of the monoclonal IgG was observed. We suggest that the monoclonal IgG might induce the vasculitis by increasing the expression of E-selectin, VCAM-1 and ICAM-1 which facilitate the interaction of leukocytes with vascular endothelium.
Hemorrhagic Transformation of Cerebral Infarction – Possible Mechanisms
Authors: del Zoppo Gregory J. , von Kummer Rüdiger , Hamann Gerhard F.
Published: March 2018

To analyse the risk/benefit of cerebral thrombolysis the role of hemorrhagic transformation, either as clinically silent hemorrhagic infarction or disastreous parenchymal hemorrhage, is crucial. Thrombolysis in acute ischemic stroke increases the risk of severe, life-threatening hemorrhagic complications by up to 10 times compared to controls. In this paper, previous proposed concepts for the development of intracerebral hemorrhage and hemorrhagic transformation are presented. The role of the cerebral microvasculature will be emphasized. In experimental focal cerebral ischemia a significant loss of basal lamina components of the cerebral microvessels has been demonstrated. This loss in vessel wall integrity is associated with the development of petechial hemorrhage. The mechanisms for this microvascular damage may include plasmin-generated laminin degradation, matrix metalloproteinases activation, transmigration of leukocytes through the vessel wall, and other processes. We propose that attenuation of the microvascular integrity loss with subsequent reduction in hemorrhage is theoretically possible 1) by an improvement in the definition of an individual time window of therapy (by means of imaging techniques), 2) by a biochemical quantification of the basal lamina damage to avoid dangerous interventions, and 3) by pharmacological strategies to protect the basal lamina during thrombolysis.
Bed Rest in Deep Vein Thrombosis and the Incidence of Scintigraphic Pulmonary Embolism
Authors: Schwarz Thomas , Kropp Joachim , Prescher Yvette , Beuthien-Baumann Bettina , Daniel Werner G. , Schellong Sebastian M.
Published: March 2018

In several countries of central Europe, patients with acute proximal deep vein thrombosis (DVT) are treated not only by anticoagulation and compression therapy but additionally by strict bed rest for 6-8 days. Until now the theoretical assumption that bed rest substantially reduces the incidence of pulmonary embolism has not been subjected to empirical verification. Patients with acute proximal DVT proven by ultrasonography were randomly assigned to strict bed rest for 8 days (treatment group) or to stay mobilised (control group). In both groups, basic treatment consisted in anticoagulation by subcutaneous low molecular weight heparin/vitamin-K-antagonist and compression therapy. The incidence of pulmonary embolism was assessed by serial ventilation/perfusion SPECT on day 1 and days 8-10. Of the 309 patients with proximal DVT considered for inclusion, 180 were excluded according to the study protocol, and 3 did not give informed consent. One hundred and twenty-six patients were randomly assigned to observe bed rest (n = 62) or to keep mobilised (n = 64). Four patients refused follow-up lung scan.A new lung perfusion defect was detected in 10/59 patients in the treatment group compared to 14/63 patients in the control group (one-sided p-value = 0.25; power 0.8). Bed rest as an additional measure in the treatment of DVT is not able to substantially reduce the incidence of scintigraphically detectable pulmonary embolism. The discomfort and costs associated with the prescription of bed rest in DVT are obviously inappropriate.
Conventional Fibrinolytic Assays for the Evaluation of Patients with Venous Thrombosis: Don’t Bother
Authors: Bauer Kenneth
Published: March 2018

Hypofibrinolysis, as defined by an absent or deficient increase in blood or plasma fibrinolytic activity after an appropriate stimulus, has been known to be present in patients following an episode of deep venous thrombosis for over 30 years. The usual stimulus is 10 min of venous occlusion using a blood pressure cuff inflated to a pressure midway between systolic and diastolic pressures. The early screening fibrinolytic assays were relatively insensitive in detecting fibrinolytic system hypofunction and lacked standardization.

Supported in part by the Medical Research Service of the Department of Veterans Affairs.
Open Multicentre Study of the P
2T
Receptor Antagonist AR-C69931MX Assessing Safety, Tolerability and Activity in Patients with Acute Coronary Syndromes
Authors: Oldroyd Keith , Wilcox Robert , Storey Robert ,   the Investigators
Published: March 2018

Platelet aggregation is the central process in the pathophysiology of acute coronary syndromes. ADP contributes to thrombosis by activating platelets, and AR-C69931MX is a specific antagonist of this process acting at the P
2T
receptor. At 5 hospitals, 39 patients with unstable angina or non-Q wave myocardial infarction, who were receiving aspirin and heparin, were administered intravenous AR-C69931MX with stepped dose increments over 3 h to a plateau of either 2 μg/kg/min for 21 h (Part 1; n = 12) or up to 69 h (Part 2; n = 13) or 4 μg/kg/min for up to 69 h (Part 3: n = 14). Safety parameters, platelet aggregation (PA) induced by ADP 3 μmol/L (impedance aggregometry), bleeding time (BT) and plasma concentrations of AR-C69931XX were assessed. AR-C69931MX was well tolerated. 33 patients completed the study. There were no deaths at 30 days and no serious adverse events attributed to AR-C69931MX. Trivial bleeding (56%) was common. At 24 h, mean inhibition of PA was 96.0 ± 8.6, 94.9 ± 14.4 and 98.7 ± 2.1% and BT was 9.5 ± 8.4, 14.0 ± 9.7 and 16.0 ± 11.1 min for Parts 1, 2 and 3 respectively. At 1 h post-infusion, mean inhibition of PA was 36.2 ± 39.2, 20.7 ± 25.9 and 40.7 ± 36.7% respectively. 90% patients had a plasma half-life for AR-C69931XX of 9 min. In conclusion, AR-C69931MX is a potent, short-acting platelet ADP receptor antagonist suitable for further studies as an antithrombotic agent.
Oral Anticoagulant Therapy in Patients with Nonrheumatic Atrial Fibrillation and Risk of Bleeding
Authors: Legnani Cristina , Noventa Franco , Palareti Gualtiero , Pengo Vittorio ,   the ISCOAT
Published: March 2018

Oral anticoagulants (OA) are the drug of choice for stroke prevention in patients with non-rheumatic atrial fibrillation (NRAF). This clear benefit/risk ratio comes from several randomized clinical trials (RCT) in which highly selected patients were strictly monitored. The aim of this study was to ascertain whether the safety of OA was also obtained outside the setting of clinical trials in consecutive patients starting treatment and routinely followed at Italian anticoagulation clinics. A total of 433 patients with NRAF were enrolled in the ISCOAT study and followed up for a mean of 1.4 years. Two patients (0.3% per year) suffered from a complete non-fatal ischemic stroke, 8 patients (1.3% per year) died of thrombosis-related vascular death, and 11 patients (11 events, 1.8% per year) suffered from major bleedings (2 fatal). Major bleeding occurred more frequently in patients >75 years of age (6 events, 5.1% per year) than in younger patients (5 events, 1.0% per year).

The cumulative incidence of major bleeding in patients over 75 years of age (10.8%; 95% CI, 1.8-19.8) was significantly higher than in younger patients (2.8%; 95% CI, 0.3-5.3, p = 0.006). Major primary bleeding unrelated to organic lesions (7 patients, 1 male and 6 females) occurred in 5 elderly patients (>75 years old) with a cumulative incidence (9.6%; 95% CI 0.8-18.4) significantly higher than in younger patients (1.2%; 95% CI, 0-3.0, p = 0.0003). Univariate analysis revealed a higher frequency of major primary bleeding in females, in diabetic patients and in in those who had suffered a previous thromboembolic event. Multivariate analysis revealed that only age grater than 75 years was independently related to major primary bleedings (RR 6.6; 95% CI 1.2-37, p = 0.032). Minor bleedings (n = 27) were not more frequent in elderly patients (6% vs 4% per year, p = ns). Patients were kept at optimal intensity of treatment for 63% of the time. These data confirm the efficacy of OA but identify elderly patients as a high risk group of major bleeding.
The Amino Acid Sequence in Fibrin Responsible for High Affinity Thrombin Binding
Authors: Siebenlist Kevin , Brennan Stephen , Holyst Trudy , Mosesson Michael , Meh David
Published: March 2018

Human fibrin has a low affinity thrombin binding site in its E domain and a high affinity binding site in the carboxy-terminal region of its variant ’ chain (’408-427). Comparison of the ’ amino acid sequence (VRPEHPAETEYDSLYPEDDL) with other protein sequences known to bind to thrombin exosites such as those in GPIb , the platelet thrombin receptor, thrombomodulin, and hirudin suggests no homology or consensus sequences, but Glu and Asp enrichment are common to all. Tyrosine sulfation in these sequences enhances thrombin exosite binding, but this has not been uniformly investigated. The fibrinogen ’ chain mass determined by electrospray ionization mass spectrometry, was 50,549 Da, a value 151 Da greater than predicted from its amino acid/carbohydrate sequence. Since each sulfate group increases mass by 80 Da, this indicates that both tyrosines at 418 and 422 are sulfated. A series of overlapping ’ peptides was prepared for evaluation of their inhibition of 125I-labeled PPACK-thrombin binding to fibrin. ’414-427 was as effective an inhibitor as ’408-427 and its binding affinity was dependent on all carboxy-terminal residues. Mono Tyr-sulfated peptides were prepared by substituting non-sulfatable Phe for Tyr at ’ 418 or 422. Sulfation at either Tyr residue increased binding competition compared with non-sulfated peptides, but was less effective than doubly sulfated peptides, which had 4 to 8-fold greater affinity. The reverse ’ peptide or the forward sequence with repositioned Tyr residues did not compete well for thrombin binding, indicating that the positions of charged residues are important for thrombin binding affinity
Targeted Disruption of the Mouse Gz-alpha Gene: A Role for Gz in Platelet Function?
Authors: Matthaei Klaus , Kelleher Kim
Published: March 2018

Gz is one of nine G proteins identified in platelets and its role in these cells is unknown. Our laboratory has generated a mouse deficient in the Gz-alpha gene in the hope of determining its in vivo function. Bleeding times from the tail tip of Gzα deficient mice was significantly longer than wild type mice. Platelet aggregation and ATP secretion did not differ between wild type and Gzα deficient mice. When mice were presented with a thromboembolism challenge no differences were observed in the survival or mortality of wild type or Gzα deficient mice, however a strain difference was observed. Ignoring the genetic background of a mutant mouse might lead to a misinterpretation of results and thus it is absolutely critical to take the genetic background into account when assessing any aspect of a mutant mouse.
Factor XI Assembly and Activation on Human Umbilical Vein Endothelial Cells in Culture
Authors: Shariat-Madar Zia , Mahdi Fakhri , Schmaier Alvin
Published: March 2018

Biotin-FXI optimally bound to HUVEC in the presence of 40 nM high molecular weight kininogen (HK) and ≥ ≥7 μM Zn2+. There was little specific FXI binding in the absence of added HK and at concentrations of Zn2+ <15 μM. FXI and prekallikrein, but not prothrombin, blocked biotin-FXI binding to HUVEC in the presence of HK with an IC50 of 18 nM and 180 nM, respectively. Monoclonal antibody HKL16 and peptide SDD31 also inhibited biotin-XI binding in the presence of HK with an IC50 of 4.7 nM and 50 μM, respectively. Alternatively, peptide T249-F260 of FXI’s apple domain 3 and heparin monosulfate were weak inhibitors of FXI binding to HUVEC. FXI bound to HUVEC with an apparent K
d
of 6.9 ± 3.0 nM and B
max
of 13 ± 2.6106 sites/cell. FXI bound to HK on HUVEC, but not prothrombin, became converted to FXIa. FXI activation on HUVEC resulted from tissue culture media bovine factor XIIa. HUVEC grown in human factor XII-deficient serum did not support FXI activation. FXI binding to HUVEC in culture was mostly mediated by HK and FXI activation on HUVEC is dependent on cell-associated factor XIIa.
Cellular Consequences of the Initiation of Blood Coagulation
Authors: Camerer Eric , Røttingen John-Arne , Wiiger Merete Thune , Gjernes Elisabet , Prydz Hans
Published: March 2018

Introduction

This paper reviews some of the cell biological aspects of the consequences of blood clotting initiation. These intracellular events occur in cells carrying tissue factor (TF) when its ligand, factor VIIa, is bound to the receptor-like TF surface molecules. The intracellular signaling generated by this ligand/receptor binding and some of its consequences are described and parallel experiments with factor Xa are discussed.

The role of TF as a major player in the initiation of blood coagulation has been known since the last century1,2 and is now characterized in molecular detail. Research on TF, for a long period and for obvious reasons, concentrated on its essential role as a cofactor in this process. Its importance in the development of clinical thrombosis, be it venous or arterial, has been appreciated since it was discovered that monocytes and macrophages3 and endothelial cells,4 under certain conditions, could be induced to synthesize TF. This contributed to answering the previously unresolved question about how TF got into contact with the flowing blood in the absence of any trauma. We later demonstrated that the TF induction process, in many cases, is subject to down-regulation by cAMP5,6 and that Ca2+ influx can induce the synthesis,5,6 along with a large number of other compounds.7 We also showed that protein kinase C was a mediator in at least some of these inducing pathways.8

The purification of TF in 19739 showed that TF was an integral membrane protein. By 1977 it was clear that TF likely participated in functions other than blood clotting.10 The cloning of the gene for TF11-14 suggested that, structurally, TF was a member of the Class II cytokine receptor family.15 To fulfil the criteria for being a true receptor, it also needed a specific and high-affinity ligand, which it has in factor VII. Also, to be classified as a true receptor, ligand binding should generate an intracellular signal. In 1992, we presented the first report of such a signal in the form of Ca2+ peaks. These peaks were triggered by the addition of factor VIIa to endothelial cells carrying TF on their surface as a result of exposure to interleukin 1β. These signals were characterized further16,17 and were thought to render final proof for the function of the TF receptor.

This review discusses our findings with respect to TF/factor VIIa-induced intracellular Ca2+-signaling and concludes that there is likely a two-component receptor. The more consequential question—whether this intracellular signaling leads to altered gene expression and to other phenotypic changes—is also raised. The establishment of knockout mice18–20 and efforts to solve the three-dimensional structure of this complex by x-ray diffraction21–24 are not reviewed extensively.
Structure and Function of Fibrinogen: Insights from Dysfibrinogens
Authors: Sugo Teruko , Yoshida Nobuhiko , Terukina Shigeharu , Yamazumi Kensuke , Niwa Kazuki , Maekawa Hisato , Matsuda Michio
Published: March 2018

The structure-function relationships of dysfibrinogens and their clinical implications are discussed on the basis of the data provided from representative molecules.
Heparin-Induced Thrombocytopenia: A Clinicopathologic Syndrome
Authors: Warkentin Theodore
Published: March 2018

Introduction

The American vascular surgeons, Weismann and Tobin in 19581 and Roberts and colleagues in 1964,2 were the first to describe unusual thrombi affecting the lower limb arteries in some patients treated with heparin for 7 to 14 days. It was not until the mid-1970s however that Silver and colleagues3,4 identified the key elements of the heparin-induced thrombocytopenia (HIT) syndrome, namely thrombocytopenia associated with thrombosis beginning several days after starting heparin. Further, in vitro studies performed by these investigators suggested the presence of a heparin-dependent, platelet-aggregating factor in serum thought to be immunoglobulin G (IgG).

In 1992, Amiral and colleagues5 identified the antigen target of HIT antibodies. The unusual nature of the HIT antigen, a multimolecular complex of heparin and platelet factor 4 (PF4),5,6 together with the remarkable, perhaps unparalleled, platelet- and endothelium-activating properties of the pathogenic HIT antibodies,7-10 are central to the thrombotic events that occur in these patients. From its obscure beginnings, HIT is now accepted as a common and distinct immunohematologic syndrome with broad implications ranging from public health prevention issues to fundamental questions of pathogenesis, which are leading to new insights into the molecular pathogenesis of thrombosis.
Treatment of Heparin-Induced Thrombocytopenia
Authors: Greinacher Andreas
Published: March 2018

Introduction

Unfractionated heparin (UFH) and low molecular weight heparin (LMWH) are the most widely used anticoagulants when parenteral anticoagulation with a short half-life is required. Both can be administered subcutaneously and intravenously, and both have been shown to be effective in a variety of clinical settings.1 UFH has several limitations. One is its poor bioavailability after subcutaneous injection and the marked variability in its anticoagulant response in patients with an acute thromboembolic complication.2,3 Another major issue associated with UFH is the induction of heparin-induced thrombocytopenia (HIT). Both limitations are closely linked,4 as the underlying cause is the high density of negative charges on the heparin molecule and its molecular weight. Both are responsible for the binding of heparin to plasma proteins other than antithrombin (AT), such as platelet factor 4 (PF4) and to several cell types. This leads to heparin-platelet interaction, the formation of HIT antigen (i.e., PF4/heparin complexes), and inhibition of the anticoagulant effect of heparin (aPTT-nonresponder).
Development of High-Capacity Adenoviral Vectors for Gene Therapy
Authors: Kochanek Stefan
Published: March 2018

Introduction

Articles about mutations that cause genetic disorders frequently include a suggestion that gene therapy will likely provide a cure for the disease in the future. In fact, ongoing efforts to sequence the human genome, to understand the roles of genes during development, and to determine the molecular pathogenesis of disease will almost “naturally” lead to the use of nucleic acids for the treatment of diseases in the future.

Somatic gene therapy can be defined as the treatment of inherited or acquired diseases by the introduction and expression of genetic information in somatic cells. Genetic disorders that are caused by loss-of-function mutations might be treated by introducing a functional copy of the mutated gene into the appropriate tissue. Good candidate disorders for somatic gene therapy include the many inborn errors of metabolism, such as hemophilia A or B, which are caused by either a low amount or the complete absence of a single functional gene product. These disorders have been characterized at the DNA level, and the disease-causing mutations can be relatively easily identified in individual patients.

Many acquired diseases, such as different types of cancer, are less well-defined at the molecular level. However, because of their frequency and their impact on public health, interest in the development of gene therapy for these disorders is particularly high. In fact, most of the clinical gene therapy trials that have been conducted so far relate to the treatment of malignant tumors. Despite the many ongoing clinical trials, as yet, there have been no clear examples of the successful treatment of any human disease by gene transfer.

Possibly the single most important reason preventing a broad application of gene transfer in the treatment of diseases, whether inherited or acquired, is the lack of both safe and effective gene transfer technologies. What are the requirements for the use of a vector as a vehicle for gene transfer in patients with inborn errors of metabolism? The vector should efficiently transport and deliver foreign genes to different cell types, both in vitro and in vivo. Following gene transfer, these genes should be permanently expressed at physiological levels. In many cases, expression will need to be tissue-specific and regulated. The vector should be safe, stable, and easily manufactured to high yields.

While several nonviral and viral gene transfer vectors have been tested in different systems, currently, there is no vector system available that meets all of these requirements. With the realization that efficient delivery of genes into tissues is at the heart of successful somatic gene therapy, many research groups are concentrating their efforts on refining the different aspects of the multi-step process of gene transfer.
Effects of Hormone Replacement Therapy on Coagulation, Fibrinolysis, and Thrombosis Risk in Postmenopausal Women
Authors: Koh Kwang Kon , Horne McDonald , Cannon Richard
Published: March 2018

Introduction

In the United States and other developed countries, more women die from cardiovascular disease than from any other disease. The Nurses’ Health Study, among others, has suggested that postmenopausal women who take estrogen therapy have fewer cardiovascular events over time compared with untreated women.1,2 The favorable effects of orally-administered estrogen on lipoprotein levels, namely reductions in low-density lipoprotein (LDL) cholesterol and lipoprotein(a) levels, and increases in high-density lipoprotein (HDL) cholesterol levels, likely account for much of this apparent benefit. These effects have not yet, however, been proven in randomized clinical trials. Because estrogen has a multitude of biological effects, many other potential mechanisms by which estrogen provides protection against atherosclerosis and its consequences are currently being studied.

Atherosclerosis is associated with chronic inflammation in response to injury and with a number of risk factors, such as elevated serum cholesterol, smoking, diabetes, and hypertension.3 Local activation of platelets and thrombus formation adjacent to atheromatous plaques, especially where ruptured or eroded, is now recognized to be of pathophysiological importance in the acute and chronic clinical expression of this disease.4 Thus, the favorable effects of estrogen on hemostasis may be relevant to the progression of atherosclerosis and associated clinical manifestations.

In this regard, several groups have reported on the favorable effects of hormone replacement therapy on fibrinolysis in postmenopausal women. These results might be interpreted as protection by estrogen against the propagation of thrombi that could result in myocardial ischemia and infarction. On the other hand, coagulation activation and increased risk of thromboemboli and myocardial infarction have also been reported with hormone replacement therapy in postmenopausal women. In an effort to gain a better understanding of the complex effects of estrogen on hemostasis, this chapter will review a number of observational trials and treatment studies conducted in postmenopausal women receiving estrogen therapy.
Disseminated Intravascular Coagulation: Clinical Spectrum and Established as well as New Diagnostic Approaches
Authors: ten Cate Hugo , Levi Marcel , Müller-Berghaus Gert
Published: March 2018

Introduction

Although disseminated intravascular coagulation (DIC) is an “intermediary mechanism of disease”1 that has been intensively studied during the last three decades,2-4 several aspects relating to the clinical problem of DIC are still under debate. For example, a standardized definition of disseminated intravascular coagulation has not yet been agreed upon. A diagnosis can be accurately made using the facilities of a specialized laboratory, but time constraints make the diagnosis of DIC by the general laboratory difficult. Since DIC can be prevented if the right therapy is initiated early, the ability to quickly and accurately diagnose DIC and to monitor the involved dynamic processes are essential prerequisites for the effective management patients with DIC.

Due to the problems associated with defining DIC, making an early diagnosis, and effectively treating the condition following diagnosis, the clinical management of DIC can be difficult. The transition from an activated hemostatic system to a well-defined state of DIC is indistinct, and the borderlines cannot easily be distinguished. In addition, different underlying diseases mediating DIC induce different clinical symptoms associated with DIC and demand different therapeutic approaches.
The Pathophysiology of Disseminated Intravascular Coagulation
Authors: Timmerman Janneke , Levi Marcel , ten Cate Hugo
Published: March 2018

Introduction

Disseminated intravascular coagulation (DIC) can be defined as “an acquired syndrome characterized by the activation of intravascular coagulation up to intravascular fibrin formation. The process may be accompanied by secondary fibrinolysis or inhibited fibrinolysis.”1 Being an acquired disorder, DIC occurs in a wide range of underlying disease states, including sepsis (both by Gram positive and Gram negative bacteria), burns, preeclampsia, malignancy, and polytrauma.1 In the majority of conditions, the pathogenesis of DIC remains only partly understood, an exception is Gram negative septicemia. In the following chapter, we will discuss the presumed mechanism by which DIC is elicited in different pathological states. The initiation of DIC follows the presentation of the glycoprotein tissue factor (TF) at a cellular surface, being either an intact or perturbed cell, or a cell membrane remnant.2 The extracellular TF molecules may interact with circulating factor VIIa to form a catalytic complex. The complex binds the coagulation zymogen factors IX and X. This leads to proteolytic cleavage of these molecules, yielding enzymatically active factors IXa and Xa and promoting the activation of prothrombin to thrombin (Fig. 1).
Hemostatic Gene Expression and Vascular Disease in Obesity: Insights from Studies of Genetically Obese Mice
Authors: Samad Fahumiya , Loskutoff David
Published: March 2018

Introduction

Obesity is a major public health problem in Western societies with substantial economic consequences. In the United States alone, over 30% of the population is defined as clinically obese, and thereby, are subject to increased risk for obesity-associated disorders, including cardiovascular disease (CVD), hypertension, insulin resistance, and non-insulin dependent diabetes mellitus (NIDDM).1,2 In spite of the magnitude of this problem, the molecular changes in obesity that promote these conditions are far from resolved. The fact that human obesity is a polygenic disorder with complex environmental and behavioral characteristics has made obesity research one of the more difficult areas of investigation in the medical sciences.1,3 However, recent studies of genetically obese mice and rats have produced breakthroughs in several areas of obesity-related research, primarily because obesity in these animals appears to be monogenic (i.e., to result from mutations in single genes). In this regard, five different genes, all mapped to different chromosomal locations, have been shown to cause distinct syndromes of spontaneous obesity with severe insulin resistance in mice.3 These include the obese (ob), diabetes (db), tubby (tub), lethal yellow (Ay) and fat (fat) mutations. Genes encoding intercellular adhesion molecule 1 (ICAM-1) and the leukocyte integrin αmβ2 (MAC-1) also have been implicated in the regulation of adipose tissue mass.4 These animal studies, together with studies of cultured adipocytes, have provided fundamental new information about the factors and cells that may be responsible for the altered hemostatic balance leading to increased cardiovascular risk in obesity/NIDDM.

In this chapter, we summarize our studies on plasminogen activator inhibitor 1 (PAI-1), tissue factor, and transforming growth factor (TGF)-β expression in obesity, using genetically obese mice as a model. These studies emphasize the key role played by the adipocyte, a cell whose numbers, size, and metabolic activity are grossly altered in obesity/NIDDM. They also implicate multiple cytokines, hormones, and growth factors in the abnormal expression of these and perhaps other hemostatic genes by adipocytes in obesity/NIDDM (Fig. 1). These studies emphasize the key role played by tumor necrosis factor (TNF)-α in the expression of hemostatic genes in this disorder.
Why Atherosclerotic Vessels Narrow: The Fibrin Hypothesis
Authors: Yee Karen , Schwartz Stephen
Published: March 2018

Introduction

Conventional wisdom holds that atherosclerosis narrows vessels simply by growth of the plaque.1 We now know, however, that this is incorrect. Morphological studies from the last 10 years have shown that there is no correlation between lesion size and lumen diameter. This lack of correlation is also true in the lesions found in the apolipoprotein E (apoE)-deficient mouse model.2 Pathologic studies in the mouse model and in humans suggest that the media remodels as the plaque enlarges to maintain normal blood flow through the vessel.3,4 This normal ability of the vessel to adapt to changes in lumen diameter has been called either “adaptive remodeling” or “compensatory enlargement.” We will use the former term since “remodeling” has long been used by physiologists to describe adaptive changes of vessels to pressure or flow.

The mechanism for adaptive remodeling is not known. Critical processes may include shear stress caused by the lesion, signaling in response to medial attenuation by proteases, or poorly defined apoptotic processes.3-5 This chapter will review the evidence for adaptive remodeling and propose a mechanism for the ultimate failure of remodeling and subsequent narrowing of the affected vessel. We will suggest that the critical processes in vascular narrowing are the same processes seen in wound healing, with fibrin playing a role as a provisional matrix.
Gene-Environment Interaction in the Determination of Levels of Plasma Fibrinogen
Authors: Luong Le-Anh , Montgomery Hugh , Day Ian , Mohamed-Ali Vidya , Yudkin John , Humphries Steve
Published: March 2018

Introduction

This review will focus on the inflammatory risk factors that may influence changes in plasma fibrinogen levels and that may influence an individual’s risk of ischemic heart disease (IHD). These inflammatory risk factors occur to a different extent in individuals as their environment changes. Although a specific genotype may be associated, in healthy subjects, with modest differences in levels of a risk factor for thrombosis, this effect may be larger or smaller in subgroups of subjects. Documenting such gene-environment interactions is important if genotype information is ever to be used in a clinical or diagnostic setting. Understanding the molecular mechanisms of such interactions is vital to the development of novel therapeutic approaches to reduce risk of myocardial infarction (MI).

We review some of the gene-environment interactions detected to date for the G-455A β-fibrinogen gene promoter polymorphism. Carriers of the A allele, representing roughly 20% of the population, consistently have, on average, 7% to 10% higher fibrinogen levels than those with the genotype GG. Data will be presented to demonstrate interaction between situations of inflammatory stimulation (e.g., smoking habit, presence of ischemic disease, and level of physical exercise) in the determination of the magnitude of the effect of the A allele on plasma fibrinogen levels.

The cytokine interleukin-6 (IL-6) is the likely link between inflammatory processes and IHD. Recently we have identified a functional G/C polymorphism at -174bp in the IL-6 promoter, with the G allele being a 2 to 4 times stronger promoter upon stimulation with interleukin-1 (IL-1) or lipopolysaccharide (LPS). In a small study of healthy subjects, the C allele was associated with significantly lower mean plasma levels of IL-6, an effect which may be protect against the development of IHD. Finally, we describe a rapid throughput genotyping method that is useful for large-scale genetic epidemiology studies.
Regulation of Fibrinolysis in the Development of Atherothrombosis: Role of Adipose Tissue
Authors: Alessi M.C. , Juhan-Vague I.
Published: March 2018

Introduction

Among the list of coronary risk factors, it has recently been proposed that fibrinolytic system impairment, due to increased plasminogen activator inhibitor-1 (PAI-1) levels in plasma, could predict complications of atherosclerosis. The fibrinolytic system1 is regulated by a balance between activators and inhibitors. PAI-1, which inhibits t-PA and u-PA, is the main inhibitor of plasminogen activation. An increased PAI-1 concentration induces a reduction in plasmin formation and leads to fibrin accumulation.2-5 PAI-1 also induces changes in vessel wall remodeling through the activation of metalloproteinases and growth factors and the degradation of the extracellular matrix.4 In addition to its antiprotease activity, PAI-1 also participates in the cellular adhesion and migration processes. Because it binds to vitronectin in the same part of the molecule as vitronectin receptor or u-PA receptor, it mediates the release of cells from their substrate.6,7 All of these properties indicate that PAI-1 can be involved in tissue remodeling and thrombotic processes and, therefore, participates in the development of atherothrombosis.

In plasma, an elevated PAI-1 concentration is presently considered a risk factor for coronary vascular events. It appears that PAI-1 is an inducible target controlled for in insulin resistance and obesity, which are situations that favor the development of atherothrombosis. It has been postulated that PAI-1 could contribute to an increased susceptibility to atherothrombosis in insulin-resistant patients. The production of PAI-1 by adipose tissue has recently been demonstrated. The elucidation of the regulation of PAI-1 synthesis and the identification of factors responsible for increased plasma PAI-1 concentration in the insulin-resistant state might lead to therapeutic concepts for the prevention of atherothrombosis.