bluuebunny/crossref_metadata_2025 huggingface dataset:
Anti-hirudin antibodies alter pharmacokinetics and pharmacodynamics of recombinant hirudin
Authors: Hudek Renata , Piazolo Lukas , Haase Karl , Borggrefe Martin , Huhle Günter , Fischer Karl-Georg , Liebe Volker
Published: March 2018

Recombinant hirudin (r-hirudin) is a potent direct thrombin inhibitor with immunogenic properties. Anti-hirudin antibodies (aHAb) are detected in up to 74% of patients treated with r-hirudin for more than 5 days. aHAb may alter the pharmaco-kinetics and pharmacodynamics of r-hirudin.

The effects of aHAb on the pharmacokinetics of r-hirudin were investigated in rats receiving r-hirudin intravenously either without aHAb (controls), 15 min after intravenous administration of non-specific antibodies or aHAb, and after pre-incubation with aHAb. When both were compared to controls and pre-treatment with non-specific antibodies, aHAb significantly altered the pharmacokinetics of r-hirudin with similar effects in both approaches: In the presence of aHAb, the volume of distribution in a steady state and total plasma clearance were diminished, while the half-life of elimination was prolonged. Both the maximum r-hirudin plasma concentration and the area under the curve were increased. In addition, r-hirudin filtration by high-flux hemodialyzer membranes (polysulfone, AN69) was investigated 1) in the absence of aHAb, 2) in the presence of non-specific mouse antibodies, and 3) in the presence of three monoclonal aHAb. In the absence of aHAb, both hemodialyzers allowed for significant r-hirudin filtration. Non-specific mouse antibodies did not markedly affect r-hirudin filtration. By contrast, all three aHAb almost completely hindered r-hirudin filtration. aHAb varied in their capacity to neutralize r-hirudin. In conclusion, aHAb markedly alter the pharmacokinetics of r-hirudin leading to r-hirudin accumulation. In the presence of aHAb, hemofiltration does not allow for rapid reduction of r-hirudin concentration. aHAb are capable of modifying pharmacodynamics of r-hirudin. Close monitoring of aHAb-positive patients treated with r-hirudin is considered mandatory.
A new monoclonal antibody, mAb 204-11, that influences the binding of platelet GPVI to fibrous collagen
Authors: Mizuguchi Jun , Kawashima Sachiko , Nagamatsu Michiko , Miura Yoshiki , Nakagaki Tomohiro , Ito Katsuaki , Jung Stephanie , Moroi Masaaki
Published: March 2018

The newly identified platelet collagen receptor glycoprotein VI binds to fibrous collagen, inducing platelet activation. Several antibodies against GPVI have been reported, including a patient’s auto-antibodies, that activates platelets through their ability to crosslink this glycoprotein. We have developed a monoclonal antibody (mAb) against GPVI using the recombinant extracellular domain of GPVI as an antigen. This antibody, mAb 204-11, induced platelet aggregation and tyrosine phosphorylation of proteins similar to those induced by GPVI-reactive proteins, collagen and convulxin. Its interaction with GPVI was analyzed by measuring the effect of the antibody on GPVI binding to collagen using a dimeric form of recombinant GPVI, GPVI-Fc2. MAb 204-11 inhibited the binding of GPVI-Fc2 to fibrous collagen particles, but enhanced the GPVI binding to immobilized collagen, suggesting that the antibody binds to a region near the collagen binding site of GPVI. MAb 204-11 also inhibited the GPVI binding to convulxin at a low concentration, but not completely. Since mAb 204-11 reacts specifically with GPVI and is applicable for immunoblotting and immunoprecipitation, this antibody would be useful for studies on GPVI.
Regulation of the hypoxia-dependent plasminogen activator inhibitor 1 expression by MAP kinases
Authors: Jungermann Kurt , Görlach Agnes , Kietzmann Thomas
Published: March 2018

Mitogen-activated protein kinases (MAPKs) and protein kinase B (PKB) mediate growth and stress signals and have been implicated in the hypoxic response. Under hypoxic conditions, the expression of plasminogen activator inhibitor-1 (PAI-1) is mainly controlled by the hypoxia-inducible factor HIF-1. However, the role of MAPKs and PKB in HIF-1-mediated PAI-1 regulation is not clear.

Treatment with the p38 inhibitor SB203580 and the PI3K inhibitor LY294002, but not with the MEK1 inhibitor PD98059, abrogated hypoxia-dependent PAI-1 induction in HepG2 cells. Consistently, overexpression of PKB or of the p38 upstream kinases MKK6 and MKK3 and of JNK, but not of ERK, enhanced PAI-1 mRNA levels. In MKK3-,MKK6- and PKB-expressing cells luciferase (Luc) activities from a hypoxia-inducible PAI-1-Luc construct or from a HIF-dependent Luc construct and, concomitantly, HIF-1α protein levels were enhanced. These findings indicate that p38- and PKB-dependent signalling pathways contribute to enhanced PAI-1 levels in the hypoxic response.

Theme paper: Part of this paper was originally presented at the joint meetings of the 16th International Congress of the International Society of Fibrinolysis and Proteolysis (ISFP) and the 17th International Fibrinogen Workshop of the International Fibrinogen Research Society (IFRS) held in Munich, Germany, September, 2002.
Role of Gender and Genetic Variance in Plasminogen Activator Inhibitor-1 Secretion from Human Adipose Tissue
Authors: Wahrenberg Hans , Eriksson Per , Arner Peter , Van Harmelen Vanessa
Published: March 2018

Gender and the 4G/5G polymorphism in the plasminogen activator inhibitor 1 (PAI-1) gene are believed to play a role in the regulation of plasma PAI-1 activity. Adipose tissue has been found to be an important source of PAI-1. The possible influence of gender and the 4G/5G polymorphism in the PAI-1 gene on PAI-1 secretion from abdominal subcutaneous adipose tissue was investigated in 59 women and 32 men. The subjects were apparantly healthy, although they differed markedly inter-individually in body mass index (21-53 kg/m2). The 4G/5G polymorphism did not influence the adipose secretion rate of PAI-1 or plasma PAI-1 activity. There was no gender difference in the adipose secretion of PAI-1. In multiple regression, including body mass index (BMI), waist-to-hip ratio (WHR), plasma insulin and plasma triglycerides as the independent and adipose PAI-1 secretion as the dependent variable, only BMI and plasma triglycerides correlated independently with adipose PAI-1 secretion (r = 0.54, p <0.05; r = 0.51, p <0.05, respectively). Men had a two times higher plasma PAI-1 activity than women (p <0.05). This gender difference was mainly due to gender differences in WHR. In multiple regression analysis, BMI and WHR were identified to be independently correlated with plasma PAI-1 activity (r = 0.60, p <0.05; r = 0.52, p = 0.01, respectively). In conclusion, neither gender nor the 4G/5G polymorphism in the PAI-1 gene are associated with secretion of PAI-1 from abdominal subcutaneous adipose tissue.
Effects of Fluvastatin and Bezafibrate Combination on Plasma Fibrinogen, t-plasminogen Activator Inhibitor and C Reactive Protein Levels in Coronary Artery Disease Patients with Mixed Hyperlipidaemia (FACT Study)
Authors: Cofrancesco E. , Boschetti C. , Cortellaro F. , Mancini M. , Mariani M. , Paoletti R. , Cortellaro M. ,   the FACT Study Centers
Published: March 2018

We studied the effects of fluvastatin and bezafibrate in monotherapy and in combination on plasma fibrinogen, t-plasminogen activator inhibitor (PAI-1) and C reactive protein (CRP) in patients with coronary artery disease (CAD) and mixed hyperlipidaemia

In this randomised, double blind, multicentre trial 333 patients with stable angina pectoris or previous myocardial infarction or coronary revascularisation and mixed hyperlipidaemia (LDL-cholesterol 135-250 mg/dl and triglycerides (TG) 180-400 mg/dl) were randomised to fluvastatin 40 mg, bezafibrate 400 mg, fluvastatin 20 mg + bezafibrate 400 mg or fluvastatin 40 mg + bezafibrate 400 mg treatments for 24 weeks.

Plasma fibrinogen significantly decreased after treatment with the combinations fluvastatin+bezafibrate (−14 and −16%) and with bezafibrate monotherapy (−9%). No significant reduction was observed after fluvastatin monotherapy (−4%). No significant changes were observed in PAI-1 and CRP plasma levels. Combination therapy significantly decreased both LDL-C and TG, and significantly increased HDL-C.

The combined effects on fibrinogen and plasma lipids achieved by fluvastatin and bezafibrate combination treatment might be more useful than the simple reduction of cholesterol in preventing ischaemic cardiovascular disease.
The Role of Vitamin B12 in Fasting Hyperhomocysteinemia and Its Interaction with the Homozygous C677T Mutation of the Methylenetetrahydrofolate Reductase (MTHFR) Gene
Authors: Coppola Antonio , Madonna Pasquale , Fermo Isabella , Pagano Anna , Mazzola Giuseppina , Galli Laura , Cerbone Anna , D’Angelo Armando
Published: March 2018

Total fasting plasma homocysteine (tHcy), homozygosity for the C677T mutation of the methylenetetrahydrofolate reductase (MTHFR) gene and for the A2756G mutation of the methionine synthase (MS) gene, vitamin B12 and folate plasma levels were evaluated in 170 consecutive patients (89 M, 81 F; mean age 41 ± 12 yrs) with documented early-onset thrombosis (89 venous, 69 arterial, 12 both; mean age at first episode 36 ± 11 yrs), and in 182 age- and sex-matched healthy control subjects. Moderate hyperhomocysteinemia (HHcy, tHcy >19.5 µM in men and >15 µM in women) was detected in 45 patients (26.5%) and in 18 controls (9.9%, Mantel-Haenszel OR and 95% C.I. after stratification for arterial or venous thrombosis: 3.25, 1.78–5.91). The 677TT MTHFR genotype was not significantly more prevalent in patients (27.6%) than in controls (21.4%, RR = 1.42; 0.84–2.41), and markedly contributed to HHcy (Mantel-Haenszel RR after stratification for case/control status: 8.29, 4.61–14.9). The 2756GG MS genotype, observed in 4 patients (2.4%) and 8 controls (4.4%), was not associated to HHcy. tHcy was negatively correlated to folate and vitamin B12 levels, with better correlation found in subjects with the 677TT mutation (r = −0.42 and −0.25) than with the 677CC or CT MTHFR genotype (r = −0.37 and −0.11). However, folate was similar in patients and controls and vitamin B12 was higher in patients (460 ± 206 vs. 408 ± 185 pg/ml, p = 0.011). In a generalized linear model, 44% of the variation in tHcy levels was explained by folate and vitamin B12 levels, the MTHFR genotype, gender, and by the interaction of the MTHFR genotype with folate (p ≤0.028); the interactions of vitamin B12 with the MTHFR genotype, gender and patient/control status also significantly contributed to the variation in tHcy levels (p ≤0.028). A 4-week administration of 5-methyltetrahydrofolate (15 mg/day) markedly lowered plasma tHcy in 24 patients with MTHFR 677TT genotype, but the response to treatment correlated with vitamin B12 levels (p = 0.023). Subjects carrying the MTHFR 677TT genotype have higher folate and vitamin B12 requirements irrespective of the A2756G polymorphism of the MS gene. Yet unidentified abnormalities of MS or of any of the enzymes participating in the synthesis of methylated vitamin B12 may play an important role in the phenotypic expression of moderate hyperhomocysteinemia.
Proteolytic Modulation of Thrombopoietin Activity: Comparison of Thrombin, Plasmin, and Urokinase
Authors: Aramaki Kaelen , Reiner Alexander
Published: March 2018

Several observations suggest that limited proteolysis of full-length 70 kD human thrombopoietin (Tpo) may be important for Tpo biology. Recently, it was reported that thrombin cleaves full-length recombinant human Tpo (rhTpo) sequentially at two sites, Arg195 within the glycan domain followed by Arg117 within the cytokine domain, and that these cleavages modulate Tpo activity in vitro. We demonstrate that urokinase and plasmin also cleave rhTpo in a time-dependent manner. Urokinase cleavage is confined to the glycan domain, and generates a 35 kD N-terminal fragment that contains the intact cytokine domain, and is associated with increased Tpo activity. In contrast, plasmin cleaves Tpo sequentially at two specific sites (Arg205 within the glycan domain followed by Lys52 within the cytokine domain), and is associated with a marked decrease in Tpo activity. These proteolytic events have potential implications for regulation of Tpo activity in vivo.
A Case of Congenital Afibrinogenemia: Fibrinogen Hakata, a Novel Nonsense Mutation of the Fibrinogen γ-Chain Gene
Authors: Iida Hiroko , Ishii Eiichi , Nakahara Mutsuko , Urata Michiyo , Wakiyama Machiko , Kurihara Masako , Watanabe Kumiko , Kai Takeshi , Ihara Kenji , Kinoshita Sachiko , Hamasaki Naotaka
Published: March 2018

Congenital afibrinogenemia due to a novel homozygous nonsense mutation of the fibrinogen γ-chain gene, fibrinogen Hakata, was found in an 18-year-old Japanese girl who had received supplemental fibrinogen therapy since she was 4 months old. The plasma fibrinogen concentrations of the proband were measured as less than 10 mg/dl by a functional method and less than 17 mg/dl by an immunological method. Fibrinogen concentrations of her family were in the range of 94-164 mg/dl. The proband and her family had no other clinical symptoms.

Genomic DNA of the proband and her family was isolated from leukocytes, and all exons of fibrinogen subunits and their intron/exon boundaries were analyzed. A genetic mutation, a guanine-to-thymine (G-to-T) transversion at the nucleotide position of 5860, was identified on exon 7 of the γ-chain gene. This mutation changed the codon for the 231st residue of the γ chain from GAG (Glu) to TAG (stop). No other mutation was observed. Aα, Bβ and γ chains were observed in plasma of the heterozygous family members. However, only a trace amount of Aα chain and no γ chain was detected in the plasma of the proband.
Monoclonal Antibodies against Platelet Membrane Glycoproteins IIb/IIIa and Ibα Inhibit Platelet Dependent Thrombin Generation by Different Mechanisms
Authors: Metcalfe Paul , Goodall Alison , Gray Elaine , Thomas Stephen
Published: March 2018

The antithrombotic effect of antiplatelet agents is principally due to their anti-aggregatory action, but these agents may also interfere with coagulation. We have investigated the effect of monoclonal antibodies (MAb) to platelet membrane glycoproteins (GP) IIb/IIIa and Iba on thrombin generation.

Antibodies to platelet membrane glycoprotein IIb/IIIa (RFGP56 and c7E3) were shown to inhibit platelet-mediated thrombin generation stimulated by both intrinsic and extrinsic methods. An antibody to GP Ibα (RFGP37) also inhibited thrombin generation in these systems.

FITC-annexin V was used to determine the effect of these antibodies on the exposure of procoagulant phospholipids on the platelet membrane, and it was found that the anti-IIb/IIIa antibodies reduced this, whereas the anti-Ibα antibody caused an increase.

We conclude that our monoclonal antibodies against platelet membrane glycoproteins IIb/IIIa and Ibα inhibit platelet dependent thrombin generation by different mechanisms.
Tissue Factor Is Rapidly Elevated in Plasma Collected from the Pericardial Cavity during Cardiopulmonary Bypass
Authors: Philippou Helen , Adami Antonella , Davidson Simon , Pepper John , Burman John , Lane David
Published: March 2018

There is growing evidence that the tissue factor/factor VIIa pathway of coagulation is enhanced during cardiopulmonary bypass. Hitherto, available evidence has suggested that upregulated monocyte bound tissue factor is made available, either in the blood collected from the site of surgery or on circulating cells. However, cellular upregulation is slow, while generation of factor VIIa in blood collected from the pericardial cavity is rapid. We have therefore investigated the possibility of an alternative source of tissue factor, plasma (as opposed to cellular) tissue factor in blood samples taken from the central vein catheter (systemic circulation) and collected from the pericardial cavity during cardiopulmonary bypass. Six patients undergoing first time cardiopulmonary bypass grafting were studied. Tissue factor antigen was found to be rapidly elevated (by 15 min) in the pericardial plasma, ∼5-fold above systemic levels (p <0.004). Similar elevations were found in markers of coagulation activation, factor VIIa antigen (p = 0.066), prothrombin fragment F1+2 (p <0.003) and thrombin-antithrombin complex (p <0.03). To explore whether plasma tissue factor was (or had been) functionally active, factor VIIa was measured also with the soluble tissue factor functional assay after removal of heparin. Functional factor VIIa activity fell significantly in the systemic circulation, probably due to the heparin-induced increase (∼15-fold) in tissue factor pathway inhibitor (TFPI), but was elevated in pericardial blood compared with that taken from the central line catheter (p <0.006). These results demonstrate that both components of the activation complex for the extrinsic pathway of coagulation are rapidly generated in pericardial blood during bypass.
Plasma Concentration of von Willebrand Factor in Acute Myocardial Infarction
Authors: Kim Jae-Young , Aoki Naoto , Abe Sumihisa , Ichikawa Noriko , Yoshida Minako , Nagaoka Yuta , Handa Shunnosuke , Sakai Hiroyuki , Goto Shinya
Published: March 2018

Recent investigations have revealed the crucial role of von Willebrand factor (vWF) in platelet thrombus formation under flow conditions. The plasma concentrations of vWF were measured together with various hemodynamic and hemostatic parameters in 51 cases of acute myocardial infarction. In 10 randomly selected cases, the plasma concentrations and distribution of multimers vWF were serially determined after reperfusion therapy by percutaneous transluminal coronary angioplasty (PTCA). The vWF concentration at the onset of the acute myocardial infarction was significantly higher than in an age-matched control group (vWF AG: 18.7 ± 1.2 µg/ml vs. 10.3 ± 0.5 µg/ml, p = 8.43×10− (12), mean ± SE). Simultaneous determination of hemodynamic and hemostatic parameters revealed that the only two parameters that were significantly correlated with the patients` plasma vWF concentrations were their pulmonary capillary wedge pressure (PCWP) and heart rate, suggesting a relationship between hemodynamic changes induced by the onset of myocardial infarction and the vWF plasma concentrations. Serial determinations revealed that the vWF concentrations had not changed 1 h after reperfusion therapy, but that they significantly increased by 24 to 72 h. The distribution of the larger multimers of vWF also increased in the acute and subacute phase. The vWF concentration and multimer distribution normalized 14 days after the onset of the myocardial infarction. Our findings suggest that the vWF concentration increased in acute myocardial infarction patients, possibly in association with the hemodynamic deterioration that occurs in acute myocardial infarction.
The Second Laminin G-type Domain of Protein S Is Indispensable for Expression of Full Cofactor Activity in Activated Protein C-catalysed Inactivation of Factor Va and Factor VIIIa
Authors: Evenäs Petra , García de Frutos Pablo , Nicolaes Gerry , Dahlbäck Björn
Published: March 2018

Vitamin K-dependent protein S is a cofactor to the anticoagulant serine protease activated protein C (APC) in the proteolytic inactivation of the procoagulant, activated factor V (FVa) and factor VIII (FVIIIa). In the FVa degradation, protein S selectively accelerates the cleavage at Arg306, having no effect on the Arg506 cleavage. In the FVIIIa inactivation, the APC-cofactor activity of protein S is synergistically potentiated by FV, which thus has the capacity to function both as a pro- and an anticoagulant protein. The SHBG-like region of protein S, containing two laminin G-type domains, is required for the combined action of protein S and FV. To elucidate whether both G domains in protein S are needed for expression of APC-cofactor activities, chimeras of human protein S were created in which the individual G domains were replaced by the corresponding domain of the homologous Gas6, which in itself has no anticoagulant activity. In a plasmabased assay, chimera I (G1 from Gas6) was as efficient as wild-type recombinant protein S, whereas chimera II (G2 from Gas6) was less effective. The synergistic cofactor activity with FV in the inactivation of FVIIIa was lost by the replacement of the G2 domain in protein S (chimera II). However, chimera I did not exert full APC-cofactor activity in the FVIIIa degradation, indicating involvement of both G domains or the entire SHBG-like region in this reaction. Chimera I was fully active in the degradation of FVa in contrast to chimera II, which exhibited reduced cofactor activity compared to protein S. In conclusion, by using protein S-Gas6 chimeric proteins, we have identified the G2 domain of protein S to be indispensable for an efficient inactivation of both FVIIIa and FVa, whereas the G1 domain was found not to be of direct importance in the FVa-inactivation experiments.
Anticoagulant Activity and Pharmacokinetic Properties of a Sub-cutaneously Administered Mixed Micellar Formulation of Argatroban in Experimental Animals
Authors: Lunven Catherine , Lecoffre Catherine , Lainée Pierre , O’Connor Stephen , André Frédéric , Roger Bénédicte , Garrigou-Gadenne Danielle , Rouchouse Alain , Roome Nigel , Vivan Nathalie , Berry Christopher
Published: March 2018

We have studied the anticoagulant properties of a novel mixed micellar formulation containing 14 mg/ml argatroban administered by the sub-cutaneous (s. c.) route to rats, rabbits, dogs and primates. Blood samples were taken at various times post-treatment for the determination of the thrombin time (TT), Ecarin clotting time (ECT) and the activated partial thromboplastin time (aPTT). Plasma levels of argatroban were determined in the dog and primate.

Mixed micelles alone (0.15 M sodium glycocholate and 0.15 M egg lecithin) were without effect on the clotting parameters. The mixed micellar formulation of argatroban dose-dependently increased all three clotting parameters in the rat (1–4 mg/kg), the rabbit (1 and 2 mg/kg), the dog (1 and 2 mg/kg) and the primate (0.25 and 0.5 mg/kg). In each case the TT was the most sensitive parameter, followed by the ECT and the aPTT. The duration of action of argatroban in each species was dose dependent and varied from 3 h in the rat to 6 h in the dog. In the latter, the mixed micelle formulation had a significantly increased plasma half-life and mean residence time without affecting the overall area under the curve. The increases in the clotting time were strongly correlated with the plasma levels of argatroban and were linear across the range of concentrations obtained in the dog and the primate, although the aPTT plasma concentration response curve was very flat. Species differences were noted between the increase in clotting time for a given plasma concentration, with the primate being more sensitive than the dog (e. g. 4.7 times more so in terms of the ECT).

Thus, a mixed micellar formulation of argatroban, which markedly enhances its solubility, could be useful as a potential anticoagulant for sub-cutaneous administration.
Tissue Factor Encryption/de-encryption Is not Altered in the Absence of the Cytoplasmic Domain
Authors: Bromberg Michael , Carson Steven
Published: March 2018

Since the cytoplasmic domain of tissue factor (TF) appears to have a role in TF function beyond coagulation, experiments were conducted to determine whether the cytoplasmic domain also has a role in regulating procoagulant activity of TF present in the cell membrane. TF encryption was quantitated in human YU-SIT1, U87-MG, and mouse 3T3 cells which were transfected for expression of human tissue factor or a construct lacking the cytoplasmic domain (TFCD). Comparison of intact cells (encrypted) with fully disrupted cells (de-encrypted) showed that TF and TFCD were equally encrypted with respect to function in fX activation. Moreover, cells expressing TF and TFCD were indistinguishable in their procoagulant responses to A23187-calcium and varied concentrations of nonionic detergents. TF in membrane vesicles spontaneously shed by U87-MG cells was largely, but incompletely, de-encrypted, and the degree of de-encryption was independent of the cytoplasmic domain. We conclude that the predominant mechanism(s) for encrypting TF procoagulant activity is independent of the cytoplasmic domain.
Role of α5β1 and αvβ3 Integrins on Smooth Muscle Cell Spreading and Migration in Fibrin Gels
Authors: Yee Karen , Schwartz Stephen , Ikari Yuji
Published: March 2018

Fibrin is found at sites of vascular injury and is one of the major matrix ligands for β3 integrins. Blocking the β3 integrin on smooth muscle cell is hypothesized as a potential target to prevent restenosis because it could inhibit cell attachment and migration into fibrin provisional matrix. Human aortic smooth muscle cells (HNB18E6E7) spread stably in plasma gels within 24 h. Cell spreading was dramatically blocked by simultaneous use of α5β1 and αvβ3 integrin antibodies (P <0.0001), however, blocking of either integrin alone failed to inhibit spreading. GPenGRGDSPCA, which has been considered a specific αvβ3 antagonist, inhibited spreading at 500 µM, suggesting that the peptide blocked both α5β1 and αvβ3. Similarly, invasive migration into fibrin gels was blocked by simultaneous use of both α5β1 and αvβ3 antibodies, however, blocking of either integrin alone failed to effect cell migration. Another migration assay using transwell indicated similar results. In conclusion, both α5β1 and αvβ3 integrins are responsible for smooth muscle cell spreading and migration into fibrin gels. These data suggest that blocking β3 integrin alone would not affect smooth muscle cell interaction with fibrin.
Analysis of the 677 C→T Mutation of the Methylenetetrahydrofolate Reductase Gene in Different Ethnic Groups
Authors: Franco R. F. , Araújo A. G. , Guerreiro J. F. , Elion J. , Zago M. A.
Published: March 2018

A recently described mutation in the methylenetetrahydrofolate reductase (MTHFR) gene (a C to T transition at nucleotide 677) is associated with a thermolabile phenotype and decreased enzyme activity. In homozygotes, the mutation is also related to hyperhomocysteinemia and increased risk for atherosclerotic disease and (apparently) venous thrombosis. The prevalence of this mutation in different human populations is unknown. We have investigated the frequency of the 677 C→T mutation in the MTHFR gene in 337 individuals (674 chromosomes) belonging to four ethnic groups: Whites, African and Brazilian Blacks, Asians and Amerindians. The frequencies of the positive allele among Whites and Asians were similar to those previously reported for Caucasian populations. The positive allele seems to be slightly rarer among the Amerindians (frequency 24.0%) in comparison to Whites and Asians, with a heterogeneous distribution among the five Indian tribes analysed. In contrast, the mutation has a very low prevalence in Blacks, especially among the African Blacks, for whom the mutation was absent in homozygosity. Our data indicate that the 677 C→T MTHFR mutation has a significantly heterogeneous distribution among different ethnic groups, a fact that may contribute to explain geographical or racial differences in the risk for vascular disease.
Vitamin K Intake and Sensitivity to Warfarin in Patients Consuming Regular Diets
Authors: Lubetsky Aharon , Dekel-Stern Edit , Chetrit Angela , Lubin Flora , Halkin Hillel
Published: March 2018

The effect of dietary vitamin K intake on warfarin sensitivity is known only from case reports and few small clinical studies. We followed 50 patients commencing warfarin and consuming their regular diets (for 8 weeks) to study this relationship. A one-week recall dietary questionnaire was completed at weeks 2 and 8. Daily intake of nutrients and vitamin K was calculated from standard tables. Warfarin sensitivity index (WSI) was defined as final INR/final warfarin dose (mg/day/m2 of body surface area) (week 8).

Vitamin K intake was 17-974 (median: 179) μg/day. Median WSI was 0.82 (0.31-4.47). A WSI value of 1.1 significantly separated excess (≥250 μg/day) from normal (<250 μg/day) vitamin K consumers (16/18 vs. 15/32, respectively, p <0.01). The former had lower day 5 INR (median: 1.9 vs. 3.0, p <0.001), needed more warfarin to achieve INR ≥2.0 (32.0 ± 9.2 mg vs. 25.4 ± 6.4 mg, p = 0.009) and required a higher maintenance steady state warfarin dose (5.7 ± 1.7 mg/day vs. 3.5 ± 1.0 mg/day, p <0.001).

We conclude that in 32% (16/50) of anticoagulated patients under usual dietary conditions sensitivity to warfarin is decreased by vitamin K intake ≥250 μg/day.
Solvent Effects on Activity and Conformation of Plasminogen Activator Inhibitor-1
Authors: Egelund R. , Jensen S. , Rodenburg K. W. , Andreasen P. A.
Published: March 2018

We have studied effects of the solvent composition on the activity and the conformation of human plasminogen activator inhibitor-1 (PAI-1) from HT-1080 fibrosarcoma cells. Non-ionic detergents, including Triton X-100, reduced the inhibitory activity of PAI-1 more than 20-fold at 0° C, but less than 2-fold at 37° C, while glycerol partly prevented the detergent-induced activity-loss at 0° C. The activity-loss was associated with an increase in PAI-1 substrate behaviour. Evaluating the PAI-1 conformation by proteolytic susceptibility of specific peptide bonds, we found that the V8-proteinase susceptibility of the Glu332-Ser333 (P17-P16) bond, part of the hinge between the reactive centre loop (RCL) and β-strand 5A, and the endoproteinase Asp-N susceptibility of several bonds in the β-strand 2A-α-helix E region were increased by detergents at both 0 and 37° C. The susceptibility of the Gln321-Ala322 and the Lys325-Val326 bonds in β-strand 5A to papain and trypsin, respectively, was increased by detergents at 0° C, but not at 37° C, showing a strict correlation between proteinase susceptibility of β-strand 5A and activity-loss at 0° C. Since the β-strand 2A-α-helix E region also showed differential susceptibility to endoproteinase Asp-N in latent, active, and reactive centre-cleaved PAI-1, we propose that a detergent-induced conformational change of the β-strand 2A-α-helix E region influences the movements of β-sheet A, resulting in a cold-induced conformational change of β-strand 5A and thereby an increased substrate behaviour at low temperatures. These results provide new information about the structural basis for serpin substrate behaviour.
The Activation of Plasminogen Activator Inhibitor-1 Expression by IL-1β Is Attenuated by Estrogen in Hepatoblastoma HepG2 Cells Expressing Estrogen Receptor α
Authors: Scicchitano Marshall , Kilbourne Edward
Published: March 2018

A low estrogen status in postmenopausal women is associated with elevated plasma levels of plasminogen activator inhibitor-1 (PAI-1). In this study, the ability of estrogen compounds to regulate PAI-1 expression was determined in a hepatocyte HepG2 cell line made to stably express estrogen receptor α (ERα). In both the wild type and ER expressing HepG2 cells, estrogen had no effect on basal PAI-1 expression. However, in the ER expressing cells the ability of IL-1β to increase PAI-1 mRNA and protein levels was attenuated by 17β-estradiol, tamoxifen and twelve estrogen components of Premarin. In contrast, the mixed agonist/antagonist raloxifene had weak agonist activity and like the pure antagonist ICI 182780, it dose dependently blocked the effect of 17β-estradiol on IL-1β stimulated PAI-1 levels. These results suggest that estrogen agonists may lower PAI-1 levels in vivo by inhibiting cytokine activated PAI-1 expression by an ER dependent mechanism.
Function of Glycoprotein VI and Integrin α2β1 in the Procoagulant Response of Single, Collagen-Adherent Platelets
Authors: Siljander Pia , Vuist Wim , Breikers Githa , Reutelingsperger Chris , Barnes Michael , Graham Knight C. , Lassila Riitta , Farndale Richard , Heemskerk Johan
Published: March 2018

Various collagen-based materials were used to assess the structural requirements of collagen for inducing the procoagulant response of adhering platelets, as well as the collagen receptors involved. Cross-linked or monomeric collagen-related peptide (CRP), Gly-Cys-Hyp-(Gly-Pro-Hyp)10-Gly-Cys-Hyp-Gly was highly adhesive for platelets in a glycoprotein VI- (GpVI-)dependent manner. Adhesion was followed by a prolonged increase in cytosolic [Ca2+]i, formation of membrane blebs, exposure of phosphatidylserine (PS) and generation of prothrombinase-stimulating activity. Fibrils of type-I collagen were less adhesive but, once adhered, many of the platelets presented a full procoagulant response. Monomeric type-I collagen was unable to support adhesion, unless Mg2+-dependent integrin α2β1 interactions were facilitated by omission of Ca2+ ions. With all surfaces, however, post-addition of CaCl2 to adhering platelets resulted in a potent Ca2+-influx signal, followed by PS exposure and bleb formation. The procoagulant response elicited by binding to CRP was inhibited by anti-GpVI Fab fragments, but not by impeding integrin α2β1-mediated events. With fibrillar collagen, it was inhibited by blocking either the GpVI- or integrin α2β1- mediated interactions. This suggests that the triple-helical Gly-Pro-Hyp repeat in CRP and analogous sequences in fibrillar collagen stimulate the procoagulant response of adherent platelets by acting as ligands for GpVI. Influx of Ca2+ is required for this response, and adhesion via integrin α2β1 serves to potentiate the signaling effects of GpVI.
Eicosanoids and Iso-Eicosanoids: Constitutive, Inducible and Transcellular Biosynthesis in Vascular Disease
Authors: Folco Giancarlo , Patrono Carlo , Maclouf Jacques
Published: March 2018

Introuduction

Current understanding of the mechanisms underlying plaque development (1-3) assigns a key role to oxidative modifications of specific phospholipids that are carried into the subendothelial space with low-density lipoproteins (LDL). These variably oxidized lipids in turn trigger a chronic inflammatory response, largely orchestrated by monocytes/macrophages (1-3). Thrombotic occlusion of a major coronary or cerebral vessel can complicate the sudden fissuring or rupture of a plaque and lead to myocardial infarction or ischemic stroke, respectively. The determinants of lipid oxidation, plaque fragility and of the hemostatic response to plaque rupture, as well as the hemodynamic factors influencing the multifactorial process of atherothrombosis represent targets of pharmacological interventions aimed at reducing the risk of cardiovascular and cerebrovascular complications.

The oxidative modifications of the arachidonic acid backbone, that lead to the formation of enzymatic (eicosanoids) and non-enzymatic (iso-eicosanoids) derivatives (Fig. 1), can provide autacoid mechanisms modulating activation of the major cellular players of atherothrombosis, including platelets, endothelial cells, neutrophils and monocytes/macrophages (4-7). The remarkable clinical effects of low-dose aspirin in reducing the risk of stroke and myocardial infarction by 20 to 50% in various clinical settings (8) provide perhaps the most convincing evidence for the pathophysiologic importance of eicosanoid mechanisms in modulating the thrombotic outcome of plaque fissuring. That interference with a single mechanism of amplification of platelet activation, such as that provided by thromboxane (TX) A2-thromboxane receptor (TP) interactions on the platelet membrane, can lead to clinically detectable consequences is perhaps surprising, but certainly encouraging in the search of novel targets for pharmacological intervention.

Considerable progress has been made in the eicosanoid field during the last ten years, well beyond the thromboxane/prostacyclin balance hypothesis (9), with the elucidation of at least three distinct modalities of eicosanoid biosynthesis, i.e., constitutive, inducible and transcellular. In this review, we shall discuss the molecular, cellular and pharmacological aspects of eicosanoid and iso-eicosanoid biosynthesis as they relate to the multifactorial process of atherothrombosis, with the aim of suggesting novel pathophysiologic mechanisms as well as potential therapeutic targets.
Sex Differences in Coagulation and Fibrinolysis in Subjects with Coronary Artery Disease
Authors: Ossei-Gerning Nicholas , Wilson Ian , Grant Peter
Published: March 2018

Women with coronary artery disease (CAD) have a prognosis at least as bad and possibly worse than men. Differences in classical risk factors do not fully account for these findings and there is evidence that circulating levels of haemostatic factors may predict CAD risk. In this study sex differences in haemostatic risk factors were examined in relation to coronary stenosis.

609 (420 men, 69%) subjects admitted for coronary angiography for suspected CAD were recruited. Levels of Factor VII:C (FVII:C), fibrinogen, plasminogen activator inhibitor-1 (PAI-1) and von Willebrand factor (vWF) were estimated in 296 subjects from one centre. Of these, women (n = 107) had higher levels of FVII:C (134% vs 117%, p <0.0005), and fibrinogen (3.4 g/l vs 3.2 g/l p = 0.01) than men (n = 189) and these differences remained after adjusting for other covariates. In subjects with angiographically significant atheroma these differences in haemostatic factors (n = 50 for women vs n = 147 for men) were exaggerated, (FVII:C 139% vs 117, p <0.0001, fibrinogen 3.7 g/l vs 3.3 g/l p = 0.003), PAI-1 (26.2 ng/ml vs 19.7 ng/ml, p = 0.02) with a trend towards higher levels of vWF in the women. Women with significant atheroma at angiography (n = 50) had higher levels of PAI-1 (25.0 ng/ml vs 13.4 ng/ml p <0.0001) and vWF (1.25 IU/ml vs 1.06 IU/ml, p = 0.02) and a trend towards higher levels of both fibrinogen and FVII:C than women with normal or in significant coronary vessel disease (n = 57).

Elevated circulating levels of PAI-1, vWF, fibrinogen and FVII:C in women with angiographically proven CAD may contribute to an adverse cardiovascular risk factor profile and the poorer prognosis in females than male patients with proven coronary artery disease.
High Sensitivity Detection of Activated Factor IX: Application to the Analysis of Different Therapeutical Factor IX Concentrates and Prothrombin Complexes
Authors: Kusch Manuela , Seitz Rainer , König Herbert
Published: March 2018

A very sensitive and highly reliable test system for the detection of activated coagulation factor IX (FIXa) has been established. This assay system is based on the cleavage of a fluorogenic substrate by activated factor X (FXa) which is generated by FIXa. This assay can be used to process a large number of samples at a time and, being based on the convenient microtiter plate format, can easily be adapted to automated processing for routine screening of large sample numbers.

With this assay at hand we determined the FIXa content of different commercially available therapeutic FIX sources, such as high purity FIX (HPFIX) and prothrombin complex concentrates (PCC). Here we demonstrate that PCC from several suppliers do not contain significantly higher levels of FIXa as compared to HPFIX from the same supplier. In fact, there is a tendency for HPFIX to contain more FIXa than PCC. Moreover, HPFIX from certain manufacturers who do not produce PCC are characterized by an exceptionally high content of FIXa. Therefore, the higher thrombogenic potential of PCC which is well documented clinically cannot be explained solely – if at all – by an increased content of FIXa. Rather, it will be necessary to identify other components responsible for this phenomenon.
Influence of Lipoprotein (a) Levels and Isoforms on Fibrinolytic Activity – Study in Families with High Lipoprotein (a) Levels
Authors: Falcó Cristina , Dalmau Jaime , España Francisco , Aznar Justo , Estellés Amparo
Published: March 2018

Increased levels of lipoprotein (a) [Lp(a)] have been considered an independent risk factor for cardiovascular disease, but the mechanism behind this relationship is not completely understood. A high concentration of Lp(a) may interfere with fibrinolysis because of the structural similarity between apo(a) and plasminogen. The aim of the present study was to examine the influence of apo(a) levels and isoforms on fibrinolytic activity in 82 subjects from 24 families in which the Lp(a) levels were ≥30 mg/dl in at least one child and one parent. Several fibrinolytic parameters, including plasmin generation by fibrin-bound tissue plasminogen activator, the lipid profile and apo(a) isoforms were studied. Subjects with high circulating Lp(a) levels (n = 44) had significantly reduced plasmin generation compared with their relatives with normal Lp(a) levels (n = 38). A significant inverse correlation between Lp(a) levels and plasmin generation was observed. The individuals with a combination of high levels of plasma Lp(a) and a major apo(a) isoform ≤580 kD molecular weight show the lowest fibrinolytic activity. A high correlation was found between the levels of apo(a) isoforms in children and the levels of the corresponding parental apo(a) iso-forms. We conclude that the antifibrinolytic effect of Lp(a) in subjects with two apo(a) isoforms may depend not only on the total plasma level of Lp(a) but also on the relative concentration of the small apo(a) isoform.
Measurement of Platelet Microparticles during Cardiopulmonary Bypass by Means of Captured ELISA for GPIIb/IIIa
Authors: Miyamoto Shinji , Marcinkiewicz Cezary , Edmunds L. Henry , Niewiarowski Stefan
Published: March 2018

A captured enzyme-linked immunosorbent assay (ELISA) using the disintegrin, kistrin, is described and used to measure platelet microparticles (PMP) generated during open heart surgery. This ELISA detects 75 ng/ml of glycoprotein IIb/IIIa (GPIIa/IIIa) in solution and is more sensitive and less variable than flow cytometry and radioimmunoassay. By ELISA, mean values of GPIIb/IIIa in PMP are 14.2 ± 7.9 μg/ml for outdated platelets and 0.28 ± 0.1 μg/ml in fresh blood from healthy donors. Normal washed platelets (108) contain 8.8 μg of GPIIb/IIIa.

In 12 cardiac surgical patients, PMP measured by ELISA significantly increased (p = 0.039) to 0.58 ± 0.3 μg/ml at the end of cardio-pulmonary bypass, but the increase measured by flow cytometry (1207 to 1447 events in PMP gate) was not significant. Neither heparin nor protamine alter PMP. After cardiopulmonary bypass, PMP concentrations return to baseline values before protamine is given. Concentrations of PMP in pericardial blood are greater than in simultaneous perfusate. This ELISA is more sensitive and accurate than alternate methods for measuring PMP and shows the PMP production and rapid clearance during open cardiac surgery.

Present address: Dr. Shinji Miyamoto, Department of Cardiovascular Surgery, Oita Medical University, 1-1 Idaigaoka, Oita, Japan
Heparin-induced Thrombocytopenia: IgG Binding to PF4-Heparin Complexes in the Fluid Phase and Cross-reactivity with Low Molecular Weight Heparin and Heparinoid
Authors: Newman Peter , Swanson Rebecca , Chong Beng
Published: March 2018

Early diagnosis of heparin-induced thrombocytopenia (HIT) is essential to reduce morbidity and mortality. We report an enzyme immunoassay which detects the binding of HIT IgG to PF4-heparin in the fluid phase. Our fluid phase assay produces consistently low background and can detect low levels of anti-PF4-heparin. It is suited to testing alternative anticoagulants because, unlike in an ELISA, a clearly defined amount of antigen is available for antibody binding. We were able to detect anti-PF4-heparin IgG in 26/28 (93%) HIT patients. We investigated cross-reactivity of anti-PF4-heparin antibodies with PF4 complexed to alternative heparin-like anticoagulants. Low molecular weight heparins cross-reacted with 23/26 (88%) of the sera from HIT patients while half of the HIT sera weakly cross-reacted with PF4-danaparoid (Orgaran). The thrombocytopenia and thrombosis of most of these patients resolved during danaparoid therapy, indicating that detection of low affinity antibodies to PF4-danaparoid by immunoassay may not be an absolute contraindication for danaparoid administration.
Prevention and Therapy of Experimental Venous Thrombosis in Rabbits by Desmin 370
Authors: Rossiello Maria Rosaria , Barbanti Miriam , Calanni Fiorella , Semeraro Nicola , Colucci Mario
Published: March 2018

Desmin 370 (D370), a low molecular weight dermatan sulfate, has been shown to reduce the size of preformed thrombi in rats, via a mechanism largely independent of its anticoagulant activity. In the present study we investigated the therapeutic efficacy of D370 in rabbits with experimental jugular vein thrombosis. Experiments performed to evaluate the antithrombotic dosages in rabbits indicated that D370 prevented the formation of venous thrombi (Wessler model) in a dose-dependent manner with complete inhibition at 20 mg/kg. When injected to rabbits bearing a 30 min aged thrombus, D370 caused a time- and dose-dependent reduction in thrombus weight. Thrombi harvested 2 h after injection of 50 mg/kg of D370 were 71% smaller than thrombi from saline-treated rabbits and 50% smaller than pretreatment thrombi, suggesting a double effect of the drug: inhibition of thrombus accretion and reduction of the existing thrombus. Interestingly, pretreatment with the fibrinolytic inhibitor EACA (1 g/kg), significantly attenuated the therapeutic efficacy of D370, suggesting a possible involvement of the fibrinolytic system. Heparin (50 and 200 U/kg) was less active as therapeutic agent, the maximal decrease in thrombus weight, as compared to untreated rabbits, amounting to 38%. Heparin, moreover, caused a more pronounced prolongation of APTT than comparable antithrombotic dosages of D370. Our present data extend previous results on the therapeutic efficacy of D370 and underscore its potential as an alternative antithrombotic drug.
Thromboembolic Complications Associated with the Use of Prothrombin Complex and Factor IX Concentrates
Authors: Hellstern Peter , Lechler Eckhard , Überfuhr Peter , Müller-Berghaus Gert , Köhler Michael
Published: March 2018

In 1994, shortly after a heat-treated prothrombin complex concentrate (PCC) had been withdrawn from the German market due to transmission of hepatitis B, the license of another brand was withdrawn, due to 3 acute fatalities associated with the use of this product. We report on the clinical data of altogether 5 patients, who died during a 3 month period in Germany after having received this brand of PCC. All patients had surgery, acquired deficiencies of coagulation factors, and underlying diseases predisposing for thrombosis or disseminated intravascular coagulation. PCC was administered for the prevention of bleeding. In three patients, a drug interaction of PCC with aprotinin may also have played a role. Several points, however, are suspicious of a major causative effect of the respective product, (a) the close temporal correlation between administration of the drug and the subsequent clinical as well as laboratory deterioration, (b) the accumulation of these adverse events in a short period of time, when the use and market share of this brand increased due to the shortage of other products, and (c) laboratory abnormalities of this brand which have been consistently observed in several in vitro studies.