bluuebunny/crossref_metadata_2025 huggingface dataset:
Common Mutation of Plasminogen Detected in Three Asian Populations by an Amplification Refractory Mutation System and Rapid Automated Capillary Electrophoresis
Authors: Ooe Asako , Kida Masafumi , Yamazaki Tomio , Park Sang-Chul , Hamaguchi Hideo , Girolami Antonio , Ichinose Akitada
Published: March 2018

Congenital deficiency and dysfunction of plasminogen (PLG) are associated with a mild thrombotic tendency. To facilitate the genetic diagnosis of dysPLGemia, we combined an amplification refractory mutation system and rapid automated capillary electrophoresis. Two different fluorescence-labeled PLG-specific primers for exon XV were designed so that each DNA amplified by PCR showed fluorescence of a different wavelength. Single peaks were detected for the normal and the mutant Ala601-Thr alleles, respectively. A study of 90 normal Caucasians revealed no individuals with the mutation, whereas its gene frequency was 0.021 in Japanese. This mutation was also detected in Korean and Chinese populations at gene frequencies of 0.016 and 0.015, respectively. All of the Korean and Chinese cases with the mutation had at least one haplotype I of the PLG gene, as did most Japanese cases. The high frequency of the Ala601-Thr mutation among these Asian populations may be due to the founder effect.
Prevention of Deep Vein Thrombosis after Hip Replacement
Authors: Samama Meyer , Lensing Anthonie , Büller Harry , Barre Jeanne , Vochelle Nicole , Beau Bertrand , Planès André
Published: March 2018

Consecutive patients undergoing total hip replacement in 43 centres were randomly assigned to receive blindly either enoxaparin (40 mg) or tinzaparin (4,500 anti-Factor IU Xa), as once daily subcutaneous injections. The first injection was administered 12 h preoperatively. Efficacy was assessed by bilateral venography performed 12-14 days postoperatively. Efficacy and safety were blindly and centrally adjudicated. Among the 499 patients included, 440 had a venogram. The total incidence of DVTs was 44 (20.1%) of the 219 patients of the enoxaparin group and 48 (21.7%) of the 221 patients of the tinzaparin group. The upper limit of the 80% confidence interval of the difference between the two treatment groups was less than 5.0%. Therefore according to the protocol’s specifications equivalence was shown. Proximal DVTs occurred in 10.5% of the enoxaparin group (23 patients) and in 9.5% (21 patients) of the tinzaparin group. No overt major bleeding was observed. One patient in the enoxaparin group developed severe thrombocytopenia and died. The LMWH tinzaparin appears clinically to be as effective and safe as enoxaparin in the prophylaxis of deep vein thrombosis after total hip replacement, at the doses used and under the conditions of this study.
Inhibitors in German Hemophilia A Patients Treated with a Double Virus Inactivated Factor VIII Concentrate Bind to the C2 Domain of FVIII Light Chain
Authors: Laub R. , Giambattista Di , Fondu P. , Brackmann H.-H. , Lenk H. , Saenko E. L. , Felch M. , Scandella D.
Published: March 2018

To reduce the risk of transmission of hepatitis A virus, an Octaphar-ma produced factor VIII (fVIII) concentrate treated with solvent detergent (FVIII-SD) was further pasteurized after purification. This product, Octavi SDPlus (FVIII-SDP), was marketed in Europe in 1993 to 1995. Inhibitors appeared from September to October, 1995, in 12 of 109 previously treated German hemophilia A patients. A study of similarly treated Belgian patients, who also developed inhibitors, had shown antibodies to the fVIII light chain (domains A3-C1-C2) only. In the present study, the epitope specificity of 8 German inhibitor plasmas was also found to be restricted to the light chain. In radioimmunoprecipitation assays to localize the light chain epitope(s), antibody binding to heavy chain (domains A1-A2-B) was 11-148 fold lower than to the C2 domain, and binding to recombinant A3-C1 was barely detectable. These results were supported by >95% neutralization of a high responder inhibitor titer by the C2 domain.
TFPIβ, a Second Product from the Mouse Tissue Factor Pathway Inhibitor (TFPI) Gene
Authors: Monroe Dougald , Oliver Julie , Roberts Harold , Chang Jen-Yea
Published: March 2018

Tissue factor pathway inhibitor (TFPI) contains three Kunitz domains separated by two connecting regions. We have cloned another naturally occurring TFPI gene product from a mouse lung cDNA library which we have called TFPI β. TFPIβ is derived from alternative splicing of the TFPI gene. Analysis of the cDNA shows that mouse TFPIβ protein is identical to TFPI from the N’-terminus through the second connecting region. However, mouse TFPIβ possesses neither a third Kunitz domain nor an Arg, Lys-rich C’-terminus but instead has a completely different C’-terminal (β-domain) sequence which is not homologous to any known protein. Northern blot analyses show that the tissues for mouse TFPIβ synthesis are heart and lung; in contrast, TFPI appears in Northern blots of heart and spleen. Both TFPIβ and TFPI messages first appear in 7-day-old mouse embryos, but only the TFPI mRNA persists until 17 days. Purified recombinant TFPIβ shows an apparent molecular weight of 38 kDa. Kinetic studies indicate that mouse TFPIβ is a slow-binding enzyme inhibitor for human factor Xa. In addition, heparin does not enhance the inhibition of factor Xa by mouse TFPIβ although it does accelerate factor Xa inhibition by TFPI.
Local Calibration of International Normalised Ratio Improves between Laboratory Agreement: Results from the UK National External Quality Assessment Scheme
Authors: Jennings I. , Woods T. A. L. , Preston F.E , Kitchen S. ,   behalf of UK NEQAS (Blood Coagulation) Steering Committee
Published: March 2018

In the present study we have performed local calibration of International Normalised Ratio (INR) measurement systems in a large series of laboratories. We assigned INRs to five lyophilised plasma calibrants, one prepared from normal plasma and four using plasma from war-farinised patients, using different International Reference Preparations for Thromboplastin. These five calibrants, and two lyophilised test plasmas were analysed by 349 centres using 60 different thromboplastin instrument combinations.

Plasma calibrants were assigned INRs using the WHO reference thromboplastin RBT-90 or the European reference thromboplastin CRM 149R. Each participating centre determined PTs of the calibrants with their local system. These PTs were then used to construct a local calibration graph relating PT to INR. The PTs of test plasmas were converted directly into INR using the local calibration model and into INR using the conventional method. The overall medians of conventionally derived INRs of two test plasmas analysed in 349 centres were 2.50 and 3.10, compared to 2.47 and 3.04 after local calibration where RBT-90 was employed to assign INRs to calibrants. Use of CRM 149R to assign INRs to calibrants led to a significant (p <0.0001) increase in INR to 2.7 and 3.36 respectively. When results were grouped according to the thromboplastin employed, agreement between results with different reagents was improved by local calibration. There was a significant reduction (p <0.01) in the spread of results in different centres as indicated by a reduction in coefficient of variation.
A Longitudinal Study of the Relationships between Haemostatic, Lipid, and Oestradiol Changes during Normal Human Pregnancy
Authors: Greer Ian , Rumley Ann , Stewart Grace , Shepherd James , Packard Chris , Lowe Gordon , Sattar Naveed
Published: March 2018

Increased activation of both blood coagulation and fibrinolysis occurs during normal pregnancy. The responsible mechanisms are unclear, but may include increases in both oestradiol and blood lipids. We, therefore, studied the associations between fasting serum oestradiol, plasma cholesterol and triglyceride, and Factor VII activity, PAI activity, t-PA antigen, fibrin D-dimer, and vWF antigen in 10 women, each sampled on 6 occasions between 10 weeks and 35 weeks during normal pregnancy. Strong and similar individual correlations were observed between increases in FVII, PAI, t-PA and D-dimer (but not vWF) and increases in both oestradiol and triglyceride. Associations between increments in plasma cholesterol and haemostatic factors (except for FVII), were somewhat weaker. We, therefore, suggest that oestradiol-induced hypertriglyceridaemia may be a cause of elevations in plasma Factor VII activity, PAI and t-PA, and fibrin turnover (D-dimer) during normal pregnancy, but is poorly related to the increase in vWF antigen.
Effects of Heparin and Related Sulfated Polysaccharides on Tissue Factor Expression Induced by Mitogenic and Non-mitogenic Factors in Human Vascular Smooth Muscle Cells
Authors: Xuereb J. M. , Herbert J. M. , Sie P. , Boneu B. , Constans J.
Published: March 2018

Smooth muscle cells (SMCs) of the intima are generally quiescent and non proliferative. Their proliferation due to different stimulations occurs in myointimal hyperplasia and is regularly present in athero-genesis or after transluminal angioplasty leading to vascular occlusive stenosis. In the course of these pathologies, the Tissue Factor (TF) synthesis was upregulated and rapidly expressed at the membrane of the SMCs. Heparin is known to inhibit SMCs proliferation induced by FCS. We evaluated the inhibitory effect of heparin on the expression of TF induced by various mitogenic (FCS, PDGF-BB and EGF) and non-mitogenic (bacterial LPS) agents. Inhibition by heparin of SMCs proliferation induced by the same agonists was also determined.

Quiescent human vascular SMCs from normal adult arteries were treated for 1 h by heparin and related sulfated polysaccharides before stimulation by the agonists. All the agonists up-regulated the expression of TF antigen and activity. TF expression induced by the growth factors was inhibited by heparin (IC 50: 10-30 μg/ml), and other sulfated polysaccharides (IC 50: 1-5 μg/ml). SMCs proliferation, late activation of the extracellular signal-regulated kinases (ERK1/2), and PKC activity were inhibited by heparin (IC 50: 30-50 μg/ml) in SMCs stimulated by FCS but not in SMCs treated by PDGF or EGF. In contrast, heparin had no effect on LPS-induced TF expression nor on LPS-induced PKC activation. These results indicate that, besides its well known effect on SMC proliferation, heparin displays an inhibitory effect on cell mediated blood clotting processes through regulation of the TF expression.
Elevated High Molecular Weight Fibrinogen in Plasma Is Predictive of Coronary Ischemic Events after Acute Myocardial Infarction
Authors: Vila Virtudes , Ferrando Fernando , Martínez-Sales Vicenta , Fayos Laura , Ruano Miguel , Aznar Justo , Reganon Edelmiro
Published: March 2018

This study investigates the association between the concentration and function of plasma fibrinogen molecules measured at the time of hospital admission in patients with acute myocardial infarction (AMI), with reference to the risk of new coronary ischemic events during a three-day follow-up period of. Before starting fibrinolytic and anticoagulant treatment plasma fibrinogen, high molecular weight fibrinogen (HMW-fibrinogen), fibrin formation rate (FbFR) and phosphorous content in fibrinogen were determined in 90 AMI patients. During a three-day follow-up period 12 patients suffered new ischemic events. The 12 patients with coronary ischemia had higher concentrations of plasma fibrinogen (312 ± 23 vs. 270 ± 73 mg/dl, p <0.05) and HMW-fibrinogen (246 ± 35 vs. 189 ± 23 mg/dl, p <0.001) and a higher FbFR (65 ± 30 vs. 40 ± 25, p <0.001) than patients without these events. No association was found between the phosphorous content in fibrinogen and new coronary ischemic events. We conclude that after myocardial infarction an elevated plasma level of HMW-fibrinogen and a high FbFR value at the time of hospital admission are associated with new coronary ischemic events during a three-day follow-up period.
Parallel Reduction of Plasma Levels of High and Low Molecular Weight Kininogen in Patients with Cirrhosis
Authors: Scott Cheryl , Salerno Francesco , Lorenzano Elettra , Müller-Esterl Werner , Agostoni Angelo , Colman Robert , Cugno Massimo
Published: March 2018

Little is known about the regulation of high-molecular-weight-kininogen (HK) and low-molecular-weight-kininogen (LK) or the relationship of each to the degree of liver function impairment in patients with cirrhosis. In this study, we evaluated HK and LK quantitatively by a recently described particle concentration fluorescence immunoassay (PCFIA) and qualitatively by SDS PAGE and immunoblotting analyses in plasma from 33 patients with cirrhosis presenting various degrees of impairment of liver function. Thirty-three healthy subjects served as normal controls. Patients with cirrhosis had significantly lower plasma levels of HK (median 49 μg/ml [range 22-99 μg/ml]) and LK (58 μg/ml [15-100 μg/ml]) than normal subjects (HK 83 μg/ml [65-115 μg/ml]; LK 80 μg/ml [45-120 μg/ml]) (p < 0.0001). The plasma concentrations of HK and LK were directly related to plasma levels of cholinesterase (P < 0.0001) and albumin (P < 0.0001 and P < 0.001) and inversely to the Child-Pugh score (P < 0.0001) and to prothrombin time ratio (P < 0.0001) (reflecting the clinical and laboratory abnormalities in liver disease). Similar to normal individuals, in patients with cirrhosis, plasma HK and LK levels paralleled one another, suggesting that a coordinate regulation of those proteins persists in liver disease. SDS PAGE and immunoblotting analyses of kininogens in cirrhotic plasma showed a pattern similar to that observed in normal controls for LK (a single band at 66 kDa) with some lower molecular weight forms noted in cirrhotic plasma. A slight increase of cleavage of HK (a major band at 130 kDa and a faint but increased band at 107 kDa) was evident. The increased cleavage of HK was confirmed by the lower cleaved kininogen index (CKI), as compared to normal controls. These data suggest a defect in hepatic synthesis as well as increased destructive cleavage of both kininogens in plasma from patients with cirrhosis. The decrease of important regulatory proteins like kininogens may contribute to the imbalance in coagulation and fibrinolytic systems, which frequently occurs in cirrhotic patients.
Elevated Levels of sE-selectin in Post-menopausal Females Are Decreased by Hormone Replacement Therapy to Levels Observed in Pre-menopausal Females
Authors: McLaren Margaret , Belch Jill , Seed Mary , Kennedy Gwen
Published: March 2018

Observational epidemological studies have shown that mortality from coronary heart disease is reduced in post-menopausal women by hormone replacement therapy (HRT). The aim of this study was to measure sE-selectin levels in post-menopausal females before and after HRT and to compare these with pre-menopausal females and aged matched males. Post-menopausal females (n = 70) were given HRT or no treatment to act as a control group. sE-selectin levels were significantly lower in the pre-menopausal (n = 36) when compared with the post-menopausal females (n = 70) (p = 0.027), whereas no difference between two age matched male groups was found (n = 40). Oral and transdermal HRT significantly decreased sE-selectin levels (p <0.0001 and p = 0.0005 respectively) with no change in the control group. The reduction in the levels of this marker of endothelial activation after HRT, may reflect a decrease in leucocyte/endothelial interaction which may reduce atherosclerotic risk in post-menopausal females.
An Inhibitory Anti-factor IX Antibody Effectively Reduces Thrombus Formation in a Rat Model of Venous Thrombosis
Authors: Feuerstein Giora , Toomey John , Valocik Richard , Koster Paul , Patel Arun , Blackburn Michael
Published: March 2018

An inhibitory anti-factor IX/IXa antibody (BC2) has been investigated as an anti-thrombotic agent in a rat venous thrombosis model. The treatment of rats post-injury with a single bolus dose of BC2 (3mg/kg, iv.) resulted in an ~4 fold reduction in venous thrombus mass (P = 0.043). This efficacy was matched by a minimal (<2.5 fold) prolongation of the aPTT and had no effect on the prothrombin time (PT). Heparin by comparison, given as a bolus followed by continuous infusion, at doses comparable in efficacy at reducing thrombus formation, prolonged the aPTT >50 fold. These results demonstrate that the anti-factor IX/IXa antibody (BC2), when compared to heparin, can effectively reduce venous thrombosis with less disruptive consequences on blood clotting.
Mutations in von Willebrand Factor Multimerization Domains Are not a Common Cause of Classical Type 1 von Willebrand Disease
Authors: Cumming Anthony , Hay Charles , Keeney Stephen
Published: March 2018

Type 1 von Willebrand disease (vWD) is an autosomal dominant bleeding disorder of variable penetrance. It is characterised by a mild to moderate bleeding tendency and a quantitative deficiency of von Willebrand factor (vWF) with the full range of vWF multimers. Few mutations have been described which account for the mode of inheritance in dominant vWD type 1. We screened the vWF multimerization domains (regions D1-D3 of the vWF gene) of 12 unrelated patients with dominant vWD type 1 to investigate the hypothesis that multimerization of vWF sub-units may be inhibited or reduced by a “dominant negative” mechanism. Platelet-derived RNA was reverse transcribed and the resulting vWF cDNA amplified by the polymerase chain reaction (PCR) in a series of overlapping fragments. These were subjected to a combination of single-strand conformation polymorphism (SSCP) and heteroduplex analysis. This approach identified mobility shifts on acrylamide gels that represented 12 distinct SSCP and/or heteroduplex patterns in our patient group. DNA sequencing of the region encompassing each mobility shift showed these variants to represent previously described polymorphisms within the vWF coding sequence. Examination in all 12 patients for the previously described G3389T and T3445C mutations proved negative. The molecular pathology of classical type 1 vWD remains enigmatic, mutations having been identified in only a small minority of patients. A common mechanism underlying this disease state has still to be elucidated.
Recombinant Kunitz Protease Inhibitory Domain of the Amyloid β-Protein Precursor as an Anticoagulant in Venovenous Extracorporeal Circulation in Rabbits
Authors: Annich Gail , White Tyler , Damm Deborah , Zhao Yongqiang , Mahdi Fahkri , Meinhardt Jürgen , Rebello Sam , Lucchesi Benedict , Bartlett Robert , Schmaier Alvin
Published: March 2018

Investigations were performed to characterize a recombinant Kunitz protease inhibitory domain of the amyloid β-protein precursor (rKPI) as anticoagulants. After a single intravenous infusion of wild type rKPI into dogs, its elimination fit a two compartment model with a t½α and t½β of 5 and 77 min, respectively. Further investigations determined if a variant form of rKPI with 178-fold more potent anti-factor Xa activity (rKPI-DD135, Ki
= 0.9 nM) could serve as an anticoagulant in a rabbit model of extracorporeal circulation using a venovenous shunt. A prospective investigation was initiated to compare standard heparin (n = 8) at 400 U/kg with different infusion concentrations of rKPI-DD135. After a single intravenous infusion of 1.89 mg/kg of rKPI-DD135 followed by a constant infusion at 0.003 (n = 3), 0.03 (n = 7), or 0.3 (n = 5) mg/kg/min, the anti-factor Xa activity of the animals’ plasma rapidly reaches a steady state for the two lower infusion concentrations of the agent. All infusions of rKPI-DD135 prolong the activated clotting time with less variation than that seen with heparin administration. rKPI-DD135 anticoagulation does not prevent a drop in the platelet counts. Fibrinogen levels decrease only slightly when the circuit is anticoagulated with rKPI-DD135. rKPI-DD135 markedly prolongs the APTT, has little effect on the PT, and reduces plasma prekallikrein and plasminogen activation. The 0.3 mg/kg/min infusion concentration of rKPI-DD135 results in reduced deposition of 111Indium-labeled platelets on the circuit when compared to heparin. Last, after a steady state level is achieved, 60% of the plasma anti-factor Xa activity of rKPI-DD135 is eliminated within 60 min after stopping the infusion. These data show the rKPI-DD135 can provide single agent anticoagulation in a rabbit extracorporeal circuit. Development of short acting factor Xa inhibitors may be useful anticoagulants for cardiopulmonary bypass.

Abbreviations: CPB: cardiopulmonary bypass; KPI: Kunitz protease inhibitor domain; rKPIWT: recombinant wild type Kunitz protease inhibitory domain of the amyloid β-protein precursor; rKPI-DD135: recombinant Kunitz protease inhibitory domain of the amyloid β-protein which is a better inhibitor of factor Xa; ECC: extracorporeal circulation; CDP: citrate phosphate dextrose anti-coagulant; PRP: platelet-rich plasma; ACT: activated clotting time; PT: prothrombin time; APTT: activated partial thromboplastin time; PPP: platelet-poor plasma.
Natriuretic Peptides Regulate the Expression of Tissue Factor and PAI-1 in Endothelial Cells
Authors: Tsuji Hajime , Nishimura Hiromi , Masuda Haruchika , Kunieda Yasushi , Kawano Hidehiko , Kimura Shinzo , Sugano Tatsuya , Kitamura Hidetsugu , Nakagawa Katsumi , Nakagawa Masao , Yoshizumi Masami
Published: March 2018

In the present study, we demonstrate that brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) interact with angiotensin II (Ang II) in regulative blood coagulation and fibrinolysis by suppressing the expressions of both tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1) induced by Ang II. The expressions of TF and PAI-1 mRNA were analyzed by northern blotting methods, and the activities of TF on the surface of rat aortic endothelial cells (RAECs) and PAI-1 in the culture media were respectively measured by chromogenic assay.

Both BNP and CNP suppressed the expressions of TF and PAI-1 mRNA induced by Ang II in a time- and concentration-dependent manner via cGMP cascade, which suppressions were accompanied by respective decrease in activities of TF and PAI-1. However, neither the expression of tissue factor pathway inhibitor (TFPI) nor tissue-type plasminogen activator (TPA) mRNA was affected by the treatment of BNP and CNP.
Expression of Plasminogen Activator Inhibitor Type I in Genotyped Human Endothelial Cell Cultures: Genotype-specific Regulation by Insulin
Authors: Benza Raymond , Li Xin-Nong , Aikens Michael , Grammer John , Brown Stephen , Booyse Francois , Grenett Hernan
Published: March 2018

Patients with non-insulin-dependent diabetes mellitus frequently have been associated with elevation in plasma levels of PAI-1. Part of the variations in individual plasma PAI-1 levels have been attributed to variations in the PAI-1 gene. In order to determine whether insulin regulates PAI-1 expression in a genotype-specific manner, individual human umbilical vein ECs (HUVECs) were genotyped using a HindIII RFLP and incubated in the absence/presence of insulin. Treatment of 1/1 PAI-1 genotype HUVECs with insulin increased secretion of PAI-1 antigen ~ 1.7 to 2.2-fold and mRNA levels were increased ~ 1.8 to 2.8-fold. Treatment of HUVECs with actinomycin D or puromycin completely abolished the induction of PAI-1 by insulin. The nuclear run-on assays indicated ~3-4 fold increase in PAI-1 transcription rates. These in vitro studies with the 1/1 PAI-1 genotyped cultured HUVECs, suggests that hyperinsulinemia may be expected to increase EC PAI-1 synthesis in those patients with the responsive 1/1 genotype.
Mutations of the Platelet Thromboxane A2 (TXA2) Receptor in Patients Characterized by the Absence of TXA2–induced Platelet Aggregation despite Normal TXA2 Binding Activity
Authors: Higuchi Wataru , Hattori Akira , Aizawa Yoshifusa , Fuse Ichiro
Published: March 2018

Previously, we reported five cases of platelet dysfunction characterized by the absence of thromboxane A2 (TXA2) – induced platelet aggregation despite normal TXA2 binding activity. In this platelet disorder, patients were divided into two groups; i.e. those whose platelets lacked or did not lack phospholipase C (PLC) activation (Group A and Group B, respectively) (Thromb Haemost 1996; 76: 1080). Furthermore, in one of the patients, we showed that a single amino acid substitution (Arg60 to Leu) in the first cytoplasmic loop of the TXA2 receptor (TXR) was responsible for this platelet disorder. However, mutational analysis of the TXR in the remaining patients has not been performed. Based on this background, we investigated the mutations of the TXR in these patients, and found that all of the patients have the same abnormality of the TXR (Arg60 → Leu), although the Group A patients were homozygous and the Group B patients were heterozygous for this mutation.

This mutation is the only abnormality which has been found in this platelet disorder, and in patients heterozygous for this mutation, the mutant type TXR suppresses wild-type receptor-mediated platelet aggregation by a mechanism independent of PLC activation.
Comparative Study of Vanadate- and Phorbol Ester-induced Cyclo-oxygenase-2 Expression in Human Endothelial Cells
Authors: Hirai Kenzo , Ezumi Yasuharu , Nishida Eisuke , Uchiyama Takashi , Takayama Hiroshi
Published: March 2018

Our previous study showed that vanadate, an inhibitor of protein tyrosine phosphatases, induced the expression of cyclo-oxygenase (COX)-2 in a protein-tyrosine-kinase (PTK)-dependent manner in human umbilical vein endothelial cells (HUVEC). Here, we further compared the actions of vanadate and phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), on induction of COX-2 with special reference to mitogen-activated protein kinases (MAPKs) in HUVEC. Vanadate induced activation of three families of MAPKs, extracellular signal-regulated kinases 1 and 2 (ERK1/2), p38, and c-Jun amino-terminal kinase (JNK) 1, while activation of ERK1/2 alone was induced by PMA. The former activation by vanadate and the latter one by PMA were inhibited by tyrphostin-47, an inhibitor of PTKs, and by Ro31-8220, a PKC inhibitor, respectively. Either tyrphostin-47, PD98059, a specific inhibitor of the upstream kinase toward ERK1/2, or SB203580, a specific inhibitor of p38, completely suppressed vanadate-induction of COX-2 mRNA and protein. On the other hand, PMA-induction of COX-2 mRNA and protein was abolished by Ro31-8220 or PD98059 but not by SB203580. These data indicate that PMA-induced and PKC-dependent expression of COX-2 requires mainly activation of ERK1/2 among MAPKs, while activation of both ERK1/2 and p38 or possibly of all three families of MAPKs is necessary for vanadate-induced and PTK-dependent expression of COX-2.
Factor X Nagoya 1 and Nagoya 2: a CRM– Factor X Deficiency and a Dysfunctional CRM+ Factor X Deficiency Characterized by Substitution of Arg306 by Cys and of Gly366 by Ser, respectively
Authors: Kojima Tetsuhito , Suzuki Kyoko , Umeyama Hideaki , Yamazaki Tomio , Kamiya Tadashi , Toyoda Hideki , Kato Hisao , Miyata Toshiyuki
Published: March 2018

We have identified, in two unrelated patients, factor X deficiency that we have designated factor X Nagoya 1 and Nagoya 2, respectively. The proband with factor X Nagoya 1 showed factor X activity level of 3% and factor X antigen level <10% of the normal control value. All the exons and intron/exon junctions of the factor X gene were studied using a strategy combining polymerase chain reaction (PCR) amplification and nonradioactive single-strand conformational polymorphism (SSCP) analysis. Exon 8 containing DNA fragment of the proband with factor X Nagoya 1 showed aberrant migration on SSCP analysis. All exon-containing DNA fragments amplified by PCR were sequenced, and we identified a C-to-T substitution in exon 8 in the human factor X gene of the proband, which results in the replacement of Arg306 by Cys. This genetic defect has been transmitted from her father, and her sister also carried the same mutation; both showed almost half the normal levels of both factor X activity and antigen. The coordinates of human factor Xa indicated that Arg306 in the catalytic domain is positioned at the beginning of the alpha-helix near the second EFG-like domain. The substitution for Arg of Cys has been supposed to cause the destruction of local alpha-helix formation, possibly leading to the secretion problem. The proband with dysfunctional factor X Nagoya 2 was characterized by factor X activity level of 34% with normal factor X antigen level of 80%. We identified one substitution of G for A in exon 8 in the human factor X gene of the proband, which results in the replacement of Gly366 by Ser. As the Gly366 is positioned at the primary substrate binding pocket, the replacement of Gly with Ser would cause a defect of substrate binding, leading to the loss of enzymatic activity.
Elevated Tissue Factor and Tissue Factor Pathway Inhibitor Circulating Levels in Ischaemic Heart Disease Patients
Authors: Gori Anna Maria , Fedi Sandra , Chiarugi Ludia , Simonetti Ignazio , Dabizzi Roberto Piero , Prisco Domenico , Pepe Guglielmina , Abbate Rosanna , Gensini Gian Franco , Serneri Gian Gastone Neri , Falciani Michela
Published: March 2018

Several studies have shown that thrombosis and inflammation play an important role in the pathogenesis of Ischaemic Heart Disease (IHD). In particular, Tissue Factor (TF) is responsible for the thrombogenicity of the atherosclerotic plaque and plays a key role in triggering thrombin generation. The aim of this study was to evaluate the TF/Tissue Factor Pathway Inhibitor (TFPI) system in patients with IHD.

We have studied 55 patients with IHD and not on heparin [18 with unstable angina (UA), 24 with effort angina (EA) and 13 with previous myocardial infarction (MI)] and 48 sex- and age-matched healthy volunteers, by measuring plasma levels of TF, TFPI, Prothrombin Fragment 1-2 (F1+2), and Thrombin Antithrombin Complexes (TAT).

TF plasma levels in IHD patients (median 215.4 pg/ml; range 72.6 to 834.3 pg/ml) were significantly (p<0.001) higher than those found in control subjects (median 142.5 pg/ml; range 28.0-255.3 pg/ml).

Similarly, TFPI plasma levels in IHD patients were significantly higher (median 129.0 ng/ml; range 30.3-316.8 ng/ml; p <0.001) than those found in control subjects (median 60.4 ng/ml; range 20.8-151.3 ng/ml). UA patients showed higher amounts of TF and TFPI plasma levels (TF median 255.6 pg/ml; range 148.8-834.3 pg/ml; TFPI median 137.7 ng/ml; range 38.3-316.8 ng/ml) than patients with EA (TF median 182.0 pg/ml; range 72.6-380.0 pg/ml; TFPI median 115.2 ng/ml; range 47.0-196.8 ng/ml) and MI (TF median 213.9 pg/ml; range 125.0 to 341.9 pg/ml; TFPI median 130.5 ng/ml; range 94.0-207.8 ng/ml). Similar levels of TF and TFPI were found in patients with mono- or bivasal coronary lesions. A positive correlation was observed between TF and TFPI plasma levels (r = 0.57, p <0.001). Excess thrombin formation in patients with IHD was documented by TAT (median 5.2 μg/l; range 1.7-21.0 μg/l) and F1+2 levels (median 1.4 nmol/l; range 0.6 to 6.2 nmol/l) both significantly higher (p <0.001) than those found in control subjects (TAT median 2.3 μg/l; range 1.4-4.2 μg/l; F1+2 median 0.7 nmol/l; range 0.3-1.3 nmol/l).

As in other conditions associated with cell-mediated clotting activation (cancer and DIC), also in IHD high levels of circulating TF are present. Endothelial cells and monocytes are the possible common source of TF and TFPI. The blood clotting activation observed in these patients may be related to elevated TF circulating levels not sufficiently inhibited by the elevated TFPI plasma levels present.
Regulation of Monocyte Tissue Factor Activity by Allogeneic and Xenogeneic Endothelial Cells
Authors: Kopp Christoph , Siegel Jonathan , Anrather Josef , Winkler Hans , Grey Shane , Kaczmarek Elzbieta , Bach Fritz , Geczy Carolyn , Robson Simon
Published: March 2018

The regulation of tissue factor (TF) activity by the cell associated tissue factor pathway inhibitor (TFPI) during monocyte (Mo) and endothelial cell (EC) interactions is not fully understood. This report describes co-ordinate induction of TF antigen (TF-Ag) and membrane-associated TFPI-Ag on human Mo following coculture with human aortic (HAEC) or porcine aortic EC (PAEC) or after stimulation with TNFα. We show that both allo- and xenogeneic EC induce human Mo-TF antigen in short-term culture. However, the TF activity of TNFα-primed Mo is suppressed when these cells are cocultured with HAEC [by 40.3 ± 6.3% (p <0.02)] or PAEC [by 50.5 ± 10.6% (p <0.001)]. Antibody (Ab) blocking studies confirm that TFPI is the principal anticoagulant associated with this suppression of TF-activity. Our data indicate that anti-TF activity originates, at least in part, from the activated human Mo in the coculture; additionally, specific generation of TFPI by Mo is observed under the xenogeneic culture conditions. As Mo associated TF, induced by allo- or xenogeneic EC interactions, is regulated by cell-associated TFPI, we propose that infiltrating Mo may modulate the thrombotic process at sites of vascular injury in association with both allo- and xenograft rejection.

Aspects of the work were presented at the American Society for Hematology meeting in Orlando, FL, December 1996.
Biochemical and Pharmacological Characterization of YM-60828, a Newly Synthesized and Orally Active Inhibitor of Human Factor Xa
Authors: Sakai Yumiko , Hisamichi Nami , Kayama Makoto , Mano Yuji , Sato Kazuo , Hirayama Fukushi , Koshio Hiroyuki , Matsumoto Yuzo , Kawasaki Tomihisa , Taniuchi Yuta
Published: March 2018

YM-60828 was found to potently inhibit human factor Xa following oral administration. YM-60828 showed high affinity for factor Xa (Ki = 1.3 μM), but did not affect thrombin (Ki > 100 μM). YM-60828 doubled factor Xa clotting time, prothrombin time (PT) and activated partial thromboplastin time (APTT) at 0.10, 0.21, 0.24 μM, respectively. Importantly, it did not prolong thrombin time at 100 μM. YM-60828 also inhibited factor Xa in the prothrombinase complex with an IC50 value of 7.7 nM. In addition to its anticoagulant activity, YM-60828 inhibited platelet aggregation induced by various agonists (IC50 = 3 to 23 μM). Squirrel monkeys were used to study the ex vivo anticoagulant activity and pharmacokinetic properties of YM-60828. One hour after oral administration at 3 mg/kg, YM-60828 strongly prolonged PT and APTT by 4.8- and 1.9-fold, respectively, and plasma concentration reached 788 ± 167 ng/ml. Bioavailability was calculated to be 20.3%. These results strongly suggest that YM-60828 will be a valuable orally active and potent anticoagulant agent showing potential antithrombotic activity.
Activation of the Coagulant Pathway in Cigarette Smokers
Authors: Bauer K. A. , Cooper J. A. , Rosenberg R. D. , Miller G. J.
Published: March 2018

The effects of chronic cigarette smoking on the coagulation system were examined in 2964 men aged 50 to 61 years and clinically free of cardiovascular disease. Factor VII activity (VIIc), factor VII antigen (VIIag), prothrombin fragment 1+2 (F1.2), fibrinopeptide A (FPA) and fibrinogen were measured in all participants, and activated factor VII (VIIa), factor IX activation peptide (IX pep) and factor X activation peptide (X pep) in a large sub-sample. The levels of all indices except FPA differed significantly between non-smokers, ex-smokers and current smokers. After adjustment for other conventional cardiovascular risk factors, mean VIIc was raised slightly by 3% in ex-smokers and current smokers as compared with non-smokers, owing to increases in VIIa and VIIag. Plasma IX pep, X pep, F1.2 and fibrinogen concentration were highest in current smokers, intermediate in ex-smokers and lowest in non-smokers. These findings accord with the increased risk of arterial thrombosis in smokers.
Thrombomodulin Levels during Normal Pregnancy, at Delivery and in the Postpartum: Comparison with Tissue-type Plasminogen Activator and Plasminogen Activator Inhibitor-1
Authors: Mermillod N. , Amiral J. , Reber G. , Moerloose de
Published: March 2018

Some studies suggest that soluble thrombomodulin (TM) could be used as a marker of preeclampsia or eclampsia. However little is known about the sequential changes of TM during the course of normal pregnancy.

Levels of TM were determined in 100 women with uneventful pregnancies. Samples (n = 394) were divided into five study intervals, three during pregnancy, one at delivery and one three days postpartum.

As compared with TM levels (median 34.3 ng/ml, range 17.6-61) of a control group of 60 healthy non-pregnant women, TM levels were shown to increase throughout pregnancy, median (and range) values being respectively 38.5 (17.6-72.7) from 11 to 20 weeks, 45.2 (22.6-75.2) from 21 to 30 weeks and 54.3 (25.1-114.5) ng/ml from 31st week to delivery. One hour after delivery TM levels were still elevated and dropped three days postpartum to 40.5 (20.9-79.4) ng/ml. The increase of TM levels was correlated with those of tissue-type plasminogen activator and plasminogen activator inhibitor-1 antigens. The large overlap in TM levels between the study periods seems to preclude a clinical use of TM based on reference values from a control group. Our data suggest that it would be more appropriate to take into account TM baseline values in a given woman to examine her TM increase during pregnancy.
Enhancement of Fibrinolysis by Plactins: Structure-activity Relationship and Effects in Human U937 Cells and in Mice
Authors: Inoue Toshiki , Hasumi Keiji , Sugimoto Maki , Endo Akira
Published: March 2018

Plactin D, a cyclic pentapeptide [
cyclo
(-D-Val-L-Leu-D-Leu-LPhe-D-Arg-)] produced by a fungal strain, enhances fibrinolytic activity (6). The present study deals with the structure-activity relationship of plactins and their effects in U937 cells and mice. The results obtained from 50 plactin D analogues with a single amino acid substitution demonstrated that the following substitutions were detrimental: the enantiomer for each of the five residues; a polar, an acidic or a basic residue for D-Val, L-Leu, D-Leu or L-Phe; a polar, a hydrophobic or an acidic residue for D-Arg. On the other hand, a compound with L-Leu or L-Val in place of L-Phe was seven times as active as plactin D. These results suggest an essential role of a sterically restricted arrangement of four hydrophobic residues and the adjacent basic residue. The enhancement of fibrinolysis was dependent on plasma, ranging from 2- to 3-fold when U937 cells were incubated with 15-30 μM plactin D in the presence of 6-50% plasma, while no elevation was observed when cells were incubated in the absence of plasma. Plasminogen alone could not substitute for plasma. The plactin D effect was totally abolished by anti-urokinase IgG but not by anti-tissue plasminogen activator IgG. Plactin D caused a plasma-dependent, transient increase in the cellular urokinase activity. This urokinase activation may have accounted for the increased fibrinolytic activity of plactin D-treated U937 cells. Homogenates of the lung obtained from mice 0.5 to 2 h after intravenous plactin D (5 mg/kg) showed 2- to 3-fold increased levels of fibrinolytic activity, while activities of the brain, heart, liver, spleen, kidney and aorta were not significantly affected. In conclusion, plactin D enhances fibrinolysis both in cultured mammalian cells and in experimental animals.
Platelets and Endothelial Cells Act in Concert to Delay Thrombolysis – Evidence from an In Vitro Model of the Human Occlusive Thrombus
Authors: Jerome W. G. , Handt S. , Hantgan R. R.
Published: March 2018

The molecular and cellular mechanisms that over a period of hours render a human thrombus progressively resistant to fibrinolysis have been probed with a novel in vitro model. The kinetics of clot formation and fibrinolysis were monitored by laser light scattering with platelet-rich model thrombi contained in cylindrical flow chambers. In selected experiments, human umbilical vein endothelial cells were also cultured to confluence on the inner walls of these “glass blood vessels”. Following an “aging” period (0.5, 2 or 4 h), each thrombus was gently perfused with a bolus of plasminogen/recombinant tissue plasminogen activator to induce fibrinolysis. Platelets delayed lysis of 2 h-aged thrombi by ~70% and (non-stimulated) endothelial cells by ~30%, compared to cell-free control clots. However, even greater lytic delays (~260%) resulted when both vascular cells were present in the same 2 h-aged thrombus. In contrast, rapid lysis was consistently achieved with R298E,R299E t-PA, a genetically engineered plasminogen activator that is insensitive to inhibition by plasminogen activator inhibitor type 1. These observations suggest platelets and endothelial cells act in concert to enrich the fibrin scaffold of an aging human thrombus in plasminogen activator inhibitor. We propose that the presence of both platelets and endothelial cells may contribute to progressive thrombolytic resistance.
Suppression of Intimal Hyperplasia by a 5-lipoxygenase Inhibitor, MK-886: Studies with a Photochemical Model of Endothelial Injury
Authors: Umemura Kazuo , Ohmura Tomohiro , Hashimoto Hisakuni , Nakashima Mitsuyoshi , Kondo Kazunao
Published: March 2018

Intimal thickening is a major complication following percutaneous transluminal coronary angioplasty, which leads to restenosis and requires reoperation. We have investigated the effect of a 5-lipoxygenase inhibitor, MK-886, a leukotriene B4 (LTB4) receptor antagonist, ONO-4057 or a LTC4 and LTD4 receptor antagonist, ONO-1078, on intimal thickening. Photochemical reaction between green light and systemically administered Rose Bengal produced intimal thickening in the rat femoral artery. Each drug was administered orally, once a day for 7 days, starting just after the endothelial injury. Both MK-886 administration, 10 mg/kg, and ONO-4057 administration, 100 mg/kg, suppressed intimal thickening level examined three weeks after endothelial injury, while similarly administered ONO-1078 did not. In cultured rat-derived smooth muscle cells, LTB4, an active metabolite of 5-lipoxygenase whose biosynthesis in air pouch exudate was suppressed by MK-886, stimulated cell migration. Based on these observations, the 5-lipoxygenase may have a key role in intimal thickening via its metabolites such as LTB4.
Tissue Factor Pathway Inhibitor in Tetracycline-induced Pleuritis in Rabbits
Authors: Pendurthi Usha , Pueblitz Siegfried , Koenig Kathleen , Williams Todd , Rao Vijay Mohan , Idell Steven
Published: March 2018

Pleural fibrin deposition that promotes loculation and fibrosis after pleural injury is initiated by tissue factor (TF). In this study, we sought to determine if tissue factor pathway inhibitor (TFPI), an inhibitor of the TF-factor VIIa complex, was likewise expressed in tetracycline (TCN)-induced pleural injury and, if so, whether TFPI was locally elaborated. Pleural fluid TFPI activity approximated that of plasma by 24 h and doubled by 3 days after intrapleural TCN. By contrast, pleural fluid coagulation factors VII and V remained below plasma concentrations at these intervals. Immunohistochemical studies demonstrated TF, TFPI and fibrin localized in pleural and subpleural tissues and within intrapleural adhesions. TFPI activity and mRNA were also elaborated by rabbit pleural mesothelial cells and lung fibroblasts. TFPI is locally expressed and pleural fluid TFPI exceeds plasma levels during TCN-induced pleural injury. Resident cells as well as extravasation likely contribute to intrapleural TFPI. TFPI expression temporally and anatomically approximates that of TF and may limit TF-induced fibrin deposition in evolving TCN-induced pleuritis.
Adhesion of Leukocytes to Growing Arterial Thrombi
Authors: Roald Helge Einar , Lyberg Torstein , Hagberg Inger Anne
Published: March 2018

Since the role of leukocytes found present in thrombi and haemo-static plugs is not clearly understood, we have investigated the interaction between leukocytes and growing thrombi in a human ex vivo model of arterial thrombogenesis. At a wall shear rate characteristic of moderately stenosed arteries (2600 s–1), granulocytes selectively accumulated at the luminal surface of platelet thrombi. The leukocyte adhesion seemed independent of fibrin formation and was clearly correlated to thrombus growth and platelet activation. In contrast, flow cytometry revealed that the expression of adhesion molecules (CD11a, CD11b, CD11c, CD3, CD14, CD62L, HLA-DR and binding of fibrinogen) on the surface of circulating leukocytes passing the thrombi was, on short term conditions (15 min), independent of thrombus growth. The adhered granulocytes probably play a pivotal role in limiting the size of the evolving thrombi, as suggested by our electron micrographs of the arterial thrombi showing lysed and phagocytosed platelets. Thus, granulocytes might play an active role in the acute/semiacute phase of local thromboregulation.
C4b-binding Protein Inhibits the Factor V-dependent but not the Factor V-independent Cofactor Activity of Protein S in the Activated Protein C-mediated Inactivation of Factor VIIIa
Authors: van de Poel Robbert , Meijers Joost , Bouma Bonno
Published: March 2018

Activated protein C (APC) is an important inactivator of coagulation factors Va and VIIIa. In the inactivation of factors Va and VIIIa, protein S serves as a cofactor to APC. Protein S can bind to C4b-binding protein (C4BP), and thereby loses its cofactor activity to APC. By modulating free protein S levels, C4BP is an important regulator of protein S cofactor activity. In the factor VIIIa inactivation, protein S and factor V act as synergistic cofactors to APC. We investigated the effect of C4BP on both the factor V-independent and factor V-dependent cofactor activity of protein S in the factor VIIIa inactivation using a purified system. Protein S increased the APC-mediated inactivation of factor VIIIa to 60% and in synergy with protein S, factor V at equi-molar concentrations increased this effect further to 90%. The protein S/factor V synergistic effect was inhibited by preincubation of protein S and factor V with a four-fold molar excess of C4BP. However, C4BP did not inhibit the factor V-independent protein S cofactor activity in the purified system whereas it inhibited the cofactor activity in plasma. We conclude that C4BP-bound protein S retains its cofactor activity to APC in the factor VIIIa inactivation.
Coagulation Factor VII in Middle-aged Women with and without Coronary Heart Disease
Authors: Silveira Angela , Orth-Gomér Kristina , Hamsten Anders , Schenck-Gustafsson Karin , Eriksson-Berg Margita
Published: March 2018

Epidemiological studies of coagulation factor VII as a risk factor for coronary heart disease (CHD), mainly conducted in men, have shown discrepant results. We examined the associations of coagulation factor VII antigen (VIIag) and activated factor VII (VIIa) with manifest CHD in a community-based case-control study of women aged ≤65 years. Mean plasma concentrations of VIIag and VIIa in patients and controls were 443 ± 108 and 418 ± 89 ng/L (p <0.01) and 5.26 ± 2.21 and 4.90 ± 1.65 ng/L (NS), respectively. The odds ratio (OR) for CHD for the highest versus the lowest quartile of VIIag was 1.75 (95% CI, 1.05 to 2.92). The adjusted OR was 0.76 (95% CI, 0.28-1.98) after controlling for other cardiovascular risk factors. The corresponding ORs for VIIa were non-significant. In conclusion, the plasma concentration of VIIa was not significantly increased in a large group of women with precocious CHD, and VIIag levels, although elevated, were not independently associated with manifest disease.