bluuebunny/crossref_metadata_2025 huggingface dataset:
Overanticoagulation Associated with Combined Use of Antibacterial Drugs and Acenocoumarol or Phenprocoumon Anticoagulants
Authors: Visser Loes , Penning-van Beest Fernie , Kasbergen A. A. , De Smet Peter , Vulto Arnold , Hofman Albert , Stricker Bruno H.
Published: March 2018

Several case reports associated combined use of coumarins and antibacterial drugs with overanticoagulation. Despite the fact that these drugs are frequently prescribed concurrently, there is little quantitative information on the risks of such complications.

To study which antibacterial drugs are associated with overanticoagulation during therapy with coumarins.

Population-based cohort study in a sample of the Rotterdam Study.

All patients who were treated with acenocoumarol or phenprocoumon in the study period from April 1, 1991 through December 31, 1998 and for whom INR data were available.

Patients were followed until an INR 2: 6.0, the end of their treatment, death or end of the study period. Proportional hazards regression analysis was used to estimate the risk of an INR 2: 6.0 in relation to concomitant use of an oral anticoagulant and antibacterial drugs after adjustment for several potentially confounding factors such as age, gender, hepatic dysfunction, malignancies, and heart failure.

Of the 1124 patients in the cohort, 351 developed an INR 2: 6.0. The incidence rate was 6.9 per 10,000 treatment days. Sulfamethoxazole combined with trimethoprim most strongly increased the risk of overanticoagulation with an adjusted relative risk of 20.1 (95% CI: 10.7-37.9). Stratification showed that the induction period of overanticoagulation varied between different antibacterial drugs.

In this study among outpatients of an anticoagulation clinic using acenocoumarol or phenprocoumon, several antibacterial drugs strongly increased the risk of overanticoagulation. Awareness of these drug interactions and more frequent monitoring of INR values during the initial stages of antibacterial drug therapy are warranted to minimize the risk of bleeding complications.
Coagulation Markers Predict Survival in Cancer Patients
Authors: Haeberli A. , Vogt A. , Woodtli K. , Henkel E. , Furrer Th. , Fey M. F. , Beer J. H.
Published: March 2018

In order to prospectively evaluate the predictive value of coagulation markers such as the fibrin Ddimer for survival of cancer patients, we analyzed their role in a prospective study at a University Hospital Institute of Medical Oncology.

268 consecutive outpatients with cancer were included, 72 in remission and 196 with active disease. All cause mortality in relation to the marker levels was measured.

99/268 patients died during the observation period of 4484 patient months (mean: 17 months). Patients with active disease had a significant, 1.5-5-fold increased marker concentration compared to patients in remission. When analyzed in quartiles, the data showed a lower than predicted death rate in the first quartile and a significantly elevated mortality in the fourth marker quartile. The odds ratio for death predicted by the fibrin monomer (FM) in the fourth vs. the first quartile was 4.1 (95% C. I.: 1.7-9.7) and p = 0.005 for the multivariate analysis of the markers.

We conclude that a single determination of coagulation markers, particularly of TAT, FM, and Ddimer is sufficient to strongly predict survival in cancer patients over the following 1-3 years.
The Role of the Factor X Activation Peptide: A Deletion Mutagenesis Approach
Authors: Mullane Michael , Porche-Sorbet Rhonda , Daust Heather , Miletich Joseph , Rudolph Amy
Published: March 2018

To understand the role of the factor X (fX) activation peptide (AP), a deletion mutagenesis approach was employed. Two single-chain, variant enzymes were generated in which 41 residues were deleted from the AP: fXdes-137-183 and fXdes-137-183;N191A, which lacks a carbohydrate moiety at Asn191 due to an alanine substitution. Deletion of the fX AP did not impact fXa catalytic activity. Activation of the variant zymogens, however, was altered. Neither mutant enzyme was activated by the fX coagulant protein from Russell’s viper venom (RVV-X1). Activation by factor VIIa (fVIIa) and fVIIa in the presence of cofactor, lipidated tissue factor (TF), occurred at an accelerated rate for both variants as compared to wild-type fX (WTfX). Similar to fVII, the mutants auto-activated in a cofactor-independent manner, which was characterized by a lag period and accelerated dose-dependently by plasma fXa (kcat/Km, 0.046 ± 0.004 µM−1s−1). Both mutants were also found to be activated by fVIIa (0.31 ± 0.03 µM−1s−1), fIXa (0.30 ± 0.03 µM−1 s−1), and thrombin (0.00078 ± 0.00015 µM−1s−1). In all cases, the rate of activation was faster for fXdes-137-183;N191A as compared to fXdes-137-183. We propose that the fX AP and Asn191 carbohydrate serve primarily as negative autoregulation mechanisms to prevent spurious activation of fX and secondarily in cofactor dependence and activator specificity.

The abbreviations used are: TF, human tissue factor; AP, activation peptide; HEPES, (N-[2-Hydroxyethyl]piperazine-N’-[2-ethanesulfonic acid]); PEG, polyethylene glycol 8000; BSA, bovine serum albumin; Tris, (Tris[hydroxymethyl] aminomethane); EDTA, (Ethylenedinitrilo)-tetraacetic acid; SDSPAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; RVV-X, X activating protein from Russell’s viper venom; gla, γ-carboxylated glutamic acid; WTfX, wild type factor X; PC:PS, phosphatidylcholine:phosphatidylserine; fXa, fIXa, f Va, fVIIa, and fVIIIa, activated coagulation factor X, IX, V, VII, and VIII; ATIII, antithrombin; TFPI, tissue factor pathway inhibitor.
Diurnal Variation in PAI-1 Activity Predominantly Confined to the 4G-allele of the PAI-1 Gene
Authors: Hoekstra Tiny , Schouten Evert , Kluft Cornelis , Geleijnse Johanna
Published: March 2018

We examined the diurnal pattern in Plasminogen Activator Inhibitor-type 1 (PAI-1) activity and Plasminogen activator (t-PA) in relation to the 4G/5G-polymorphism in the promoter of the PAI-1 gene. The analyses were performed in the Arnhem Elderly Study, a populationbased study of 598 elderly. A single blood sample was drawn and the time of blood sampling was recorded (between 8 a. m. and 5.30 p. m.). Plasma PAI-1 activity was strongly associated with time of blood sampling, showing the highest values in the early morning. The diurnal pattern was clearly present in the 4G/4G (n = 184) and 4G/5G (n = 275) genotypes, but not in the 5G/5G-genotype (n = 139). T-PA antigen showed a weak diurnal variation, which did not differ across the variants of the 4G/5G-polymorphism. Our findings raise the hypothesis that 5G-homozygotic persons may be relatively protected from diurnal variation in the occurrence of coronary events.
Trophic Effects of Platelets on Cultured Endothelial Cells are Mediated by Platelet-associated Fibroblast Growth Factor-2 (FGF-2) and Vascular Endothelial Growth Factor (VEGF)
Authors: Pintucci Giuseppe , Froum Scott , Pinnell Jared , Mignatti Paolo , Rafii Shahin , Green David
Published: March 2018

In addition to their role in primary hemostasis, platelets serve to support and maintain the vascular endothelium. Platelets contain numerous growth factors including the potent angiogenic inducers VEGF and FGF-2. To characterize the function of these two plateletassociated growth factors, the effects of the addition of purified platelets to cultured endothelial cells were examined. The survival and proliferation of endothelial cells were markedly stimulated (2-3-fold and 5-15-fold respectively) by the addition of gel-filtered platelets. Acetylsalicylic acid-treated or lyophilized fixed-platelets were ineffective in supporting endothelial cell proliferation. In Transwell assays, the stimulatory effect of platelets on endothelial cells was preserved, consistent with an effect mediated by secreted factors. The combined inhibition of VEGF and FGF-2 by neutralizing antibodies, in contrast to inhibition of either alone, abrogated both platelet-induced endothelial cell survival and proliferation. FGF-2 isoforms were detected in platelet lysates, as well as in the releasates of agonist-stimulated platelets. Megakaryocytes generated by ex vivo expansion of hematopoietic progenitor cells with kit ligand and thrombopoietin were analyzed for expression of FGF-2. Punctate cytoplasmic staining but no nuclear staining was observed by immunocytochemistry consistent with possible localization of the growth factor to cytoplasmic granules. The addition of platelets to cultured endothelial cells activated extracellular signal-regulated kinase (ERK) in a dose and time-dependent manner. This effect was abrogated by both anti-FGF-2 and anti-VEGF antibody. Since FGF-2 and VEGF are potent angiogenic factors and known endothelial cell survival factors, their release by platelets provides a plausible mechanism for the platelet support of vascular endothelium.
Acute Thrombocytopenia in Patients Treated with the Oral Glycoprotein IIb/IIIa Inhibitors Xemilofiban and Orbofiban: Evidence for an Immune Etiology
Authors: Brassard Jacqueline , Curtis Brian , Cooper Richard , Ferguson John , Komocsar Wendy , Ehardt Maria , Kupfer Stuart , Maurath Clement , Swabb Edward , Cannon Christopher , Aster Richard
Published: March 2018

Thrombocytopenic episodes occurring in 18,845 patients treated with the GPIIb/IIIa inhibitors xemilofiban and orbofiban (“fibans”) were analyzed by a blinded review panel and 73 patients were classified as having “possible fiban-induced thrombocytopenia”. When the treatment codes were broken, a significant association between drug exposure and assignment to this group was found (p <0.001). Twenty-eight (82%) of 34 archived serum samples from these patients contained fiban-dependent antibodies specific for GPIIb/IIIa, but no such antibodies were found in 61 drug treated patients not classified as having “possible fiban-induced thrombocytopenia” (p <0.001). We conclude that fiban-dependent antibodies were the major cause of acute, severe thrombocytopenia in patients judged on the basis of clinical findings to have thrombocytopenia “possibly-induced” by xemilofiban and orbofiban. Measurement of drug-dependent antibodies may be helpful in determining the basis for acute thrombocytopenia in fiban-treated patients and possibly for identification of patients at risk to develop thrombocytopenia.
Pretreatment with Oral Anticoagulants Decreases Platelet Activation in Patients Before and After Percutaneous Coronary Intervention
Authors: Gerritsen Wim , Haas Fred , Kelder Johannes , Verheugt Freek , Plokker H. W. , Berg Jurriën M.
Published: March 2018

Platelet activation plays a major role in acute vessel closure after coronary angioplasty. In the randomized Balloon Angioplasty and Anticoagulation Study (BAAS), pretreatment with oral anticoagulants in addition to aspirin resulted in a 47% reduction of acute complications as compared with aspirin alone. This result may suggest a direct effect of oral anticoagulants on platelet activation.

Patients were randomized to aspirin alone (group A, n = 26) or to aspirin plus oral anticoagulants started one week before angioplasty (group B, n = 26). Platelet response tests were performed 1 hour before (baseline) and 1 hour after intervention and on day 1. Platelet activation was measured by flow cytometry, as the number of antibody-positive platelets per 10,000 counted. Platelet function was evaluated with use of the PFA-100® analyzer. In group B, the median number of P-selectin-positive platelets was lower before (28 vs. 54, P = 0.018) and after (13 vs. 24, P = 0.377) angioplasty than in group A. Also the median decrease in the number of P-selectin-positive platelets during angioplasty was lower in group B (Δ = 4) than in group A (Δ = 30, P = 0.022). No further significant change was observed in platelet activation on day 1 in the two groups. The ability of platelets to become stimulated as measured with the PFA-100® analyzer was not affected by oral anticoagulants.

Pretreatment with oral anticoagulants resulted in less activated platelets before and after coronary angioplasty, which is in agreement with its clinical effect of reducing procedural complications. Platelet function was not affected by oral anticoagulants.
Coagulation Factor VII Activity, Arg/Gln353 Polymorphism and Features of Insulin Resistance in First-Degree-Relatives of South Asian Patients with Stroke
Authors: Catto Andrew , Young John , Bamford John , Bavington John , Grant Peter , Kain Kirti
Published: March 2018

The aim of the study was to determine associations of factor VII:C levels in 140 South Asian stroke subjects and 143 first-degree relatives versus age-sex matched 146 control subjects without a personal or a family history of stroke subjects living in UK. There were no significant differences in Factor VII:C levels or FVII Msp I gene polymorphism (Arg-Gln 353) R353Q genotype frequency between the groups. R353Q genotype determined Factor VII:C levels in all the three groups. Factor VII:C levels correlated with triglycerides (patients, r = 0.23; relatives r = 27; control subjects, r = 0.24) and plasminogen activator inhibitor activity (patients, r = 0.30; relatives r = 0.22; control subjects r = 0.20) in all the three groups, but with insulin only in patients (p = 0.19). Circulating levels of Factor VII:C are determined by R353Q genotype and cluster with other risk factors associated with insulin resistance in South Asian ischaemic stroke patients, first-degree relatives and control subjects but are not related to stroke or a family history of stroke.
Targeted Inactivation of the Mouse Locus Encoding Coagulation Factor XIII-A: Hemostatic Abnormalities in Mutant Mice and Characterization of the Coagulation Deficit
Authors: Lauer Peter , Metzner Hubert , Zettlmeißl Gerd , Li Meng , Smith Austin , Lathe Richard , Dickneite Gerhard
Published: March 2018

Blood coagulation factor XIII (FXIII) promotes cross-linking of fibrin during blood coagulation; impaired clot stabilization in human genetic deficiency is associated with marked pathologies of major clinical impact, including bleeding symptoms and deficient wound healing. To investigate the role of FXIII we employed homologous recombination to generate a targeted deletion of the inferred exon 7 of the FXIII-A gene. FXIII transglutaminase activity in plasma was reduced to about 50% in mice heterozygous for the mutant allele, and was abolished in homozygous null mice. Plasma fibrin γ-dimerization was also indetectable in the homozygous deficient animals, confirming the absence of activatable FXIII. Homozygous mutant mice were fertile, although reproduction was impaired. Bleeding episodes, hematothorax, hematoperitoneum and subcutaneous hemorrhage in mutant mice were associated with reduced survival. Arrest of tail-tip bleeding in FXIII-A deficient mice was markedly and significantly delayed; replacement of mutant mice with human plasma FXIII (Fibrogammin® P) restored bleeding time to within the normal range. Thrombelastography (TEG) experiments demonstrated impaired clot stabilization in FXIII-A mutant mice, replacement with human FXIII led to dose-dependent TEG normalization. The mutant mice thus reiterate some key features of the human genetic disorder: they will be valuable in assessing the role of FXIII in other associated pathologies and the development of new therapies.
Localization and Identification of Thrombin and Plasminogen Activator Activities in Model Human Thrombi by in situ Zymography
Authors: Robbie Linda , Berry Susan , Bennett Bruce , Mutch Nicola , Moir Elaine , Booth Nuala
Published: March 2018

Human thrombi vary in their susceptibility to lysis and this is clinically important. Several potential contributory factors were examined in this study by using model thrombi, created under flow; these provide a robust, reproducible and easily-manipulated system. Here we identify the plasminogen activators (PA) active in model thrombi of known age and define the cellular and plasma contribution to activity in different areas. The cell-rich head of model thrombi had strong thrombin and PA activity, with coagulant activity also at the tail. Thrombin activity decreased as model thrombi were aged. PA activity in the thrombus head also decreased on ageing of thrombi but activity emerged around the thrombi, including the tail. Activity in the head of fresh model thrombi was primarily due to uPA, with some contribution from tPA. Experiments with thrombi prepared from platelet-rich plasma and added leucocytes showed that uPA activity at the head of fresh thrombi was derived from PMN. Older thrombi had tPA activity around the tail of the thrombus; this activity occurred in the absence of cells. This study highlights the importance of PMN-derived uPA activity in the lysis of fresh thrombi, with activity originating in the leucocyte-rich head. It also shows that thrombi are dynamic structures in which fibrin can be repeatedly laid down and lysed, observations that are relevant to therapeutic lysis and potential rethrombosis.
Thrombin Activatable Fibrinolysis Inhibitor (TAFI) Levels in Patients with Coronary Artery Disease Investigated by Angiography
Authors: Schroeder Verena , Chatterjeel Tushar , Mehta Haresh , Windecker Stephan , Pham Trinh , Devantay Nicolas , Meier Bernhard , Kohler Hans
Published: March 2018

Due to its role in the balance between coagulation and fibrinolysis, thrombin activatable fibrinolysis inhibitor (TAFI) may be involved in the development of cardiovascular diseases. We studied 362 patients with coronary artery disease (CAD) and 134 control subjects free of CAD, both groups investigated by angiography. TAFI antigen levels were determined in venous and intracoronary plasma samples and were related to metabolic and hemostatic risk factors and extent of coronary atherosclerosis. Venous TAFI levels tended to be higher in CAD patients compared to controls, whereas this difference was significant in intracoronary samples. A subgroup of patients who had not experienced acute myocardial infarction or undergone previous cardiac interventions showed significantly higher TAFI levels in both venous and intracoronary plasma samples. TAFI levels correlated with acute phase reactants indicating a role for TAFI in inflammation. However, TAFI levels did not correlate with extent of coronary atherosclerosis and among the classical cardiovascular risk factors TAFI levels only correlated with total cholesterol and fibrinogen concentration. Our results suggest that TAFI might be a risk factor for the development of CAD.
Flow Conditions Modulate Homocysteine Induced Changes in the Expression of Endothelial Cell Genes Associated with Cell-Cell Interaction and Cytoskeletal Rearrangement
Authors: Savion Naphtali , Gal Nurit , Varon David , Dardik Rima
Published: March 2018

In the present study we analyzed changes in the pattern of gene expression in endothelial cells (HUVEC) exposed to elevated homocysteine levels under flow conditions. RNA samples of untreated and homocysteine treated EC were analyzed using a human cDNA array containing 588 cDNAs relevant to the cardiovascular system. Exposure to homocysteine under flow resulted in altered expression of 8 genes, the alterations of 3 of which were further confirmed by RT-PCR analysis: upregulation of MCP-1 and of profilin-I, and downregulation of alpha-catenin. The increased expression of profilin-I and the decreased expression of alpha-catenin were also confirmed at the protein level by Western blot analysis. Furthermore, immunostaining for alpha-catenin demonstrated complete loss of the protein from intercellular junctions following exposure to homocysteine under flow. All these changes were not observed in cells exposed to homocysteine under static conditions. We show that under flow conditions, homocysteine modulates the expression of genes/proteins involved in actin cytoskeleton reorganization, as well as in the formation and/or maintenance of cellcell junction, thus contributing to the morphological changes (e.g. cell retraction) reflecting endothelial cell injury.
A single complete ultrasound investigation of the venous network for the diagnostic management of patients with a clinically suspected first episode of deep venous thrombosis of the lower limbs
Authors: Mallard Luc , Elias Marie , Alquier Catherine , Guidolin François , Gauthier Bruno , Viard Alain , Mahouin Pierre , Vinel Anne , Boccalon Henri , Elias Antoine
Published: March 2018

In patients clinically suspected of deep-vein thrombosis (DVT) of the lower limbs, it is safe to withhold anticoagulant therapy after a negative ultrasound (US) limited to the popliteal and the femoral veins, provided that this can either be repeated or combined with other diagnostic procedures. To assess the safety of withholding anticoagulants after a single negative complete US, we performed a multicenter, prospective, cohort study including consecutive ambulatory outpatients from institutional and private practice settings, with a clinically suspected first episode of DVT. Patients fulfilling the inclusion criteria were enrolled after careful clinical assessment. A complete US examination of the proximal and the distal veins was performed according to a standardized and detailed protocol. Anticoagulant therapy was administered in patients with proximal or isolated distal DVT and withheld in those with negative results. The main outcome measure was the occurrence of objectively documented clinical thromboembolic events during a three-month follow-up after a negative US.

Out of 623 patients,401 (64.4%) had a baseline negative US, were not anticoagulated and could be followed-up for three months. Two patients presented a calf DVT within three months. The incidence of venous thromboembolic events, including distal DVT, was 0.5% [95% confidence interval: 0.1-1.8]. No proximal DVT, or non-fatal or fatal pulmonary embolism occurred (incidence: 0.0% [95% confidence interval: 0.0-0.9]).

In conclusion, it is safe to withhold anticoagulant therapy in patients with clinically suspected DVT after a single, negative, complete US. Integrating this method within diagnostic strategies for DVT could improve management and be more acceptable for patients and physicians.
Complete compression ultrasonography of the leg veins as a single test for the diagnosis of deep vein thrombosis
Authors: Schwarz Thomas , Halbritter Kai , Beyer Jan , Siegert Gabriele , Oettler Wolfram , Schmidt Benjamin , Schroeder Hans , Schellong Sebastian
Published: March 2018

Noninvasive diagnosis of deep vein thrombosis (DVT) is based on ultrasound examination of the leg veins, usually restricted to only compression of the proximal veins (CUS). Patients with negative CUS findings require a second examination or a combination with other tests, which impairs clinical efficiency. In this prospective outcome study, 1646 consecutive patients with clinically suspected DVT were examined once by a standardized protocol of complete compression ultrasound comprising all proximal and distal veins (CCUS) as the only diagnostic test. The examination was equivocal in 15 patients (1% technical failure rate). Another 366 patients (22%) were tested positive for proximal DVT, distal DVT, muscle vein thrombosis, or phlebitis. Of 1265 patients in whom CCUS findings were negative, 242 met exclusion criteria for follow-up (age <18, life expectancy <3 months, other reasons for anticoagulation, postthrombotic lesions of the leg veins, or lack of informed consent). During the 3 months of follow-up, three of 1023 patients with negative CCUS findings experienced a symptomatic venous thromboembolic event (0.3% [95% CI 0.1%-0.8%]). We conclude that the CCUS protocol has a low technical failure rate and is safe with respect to excluding DVT, thereby reducing the diagnostic workup of patients with suspected DVT to a single ultrasound examination.
Impaired prothrombinase activity of factor X Gly381Asp results in severe familial CRM+ FX deficiency
Authors: Pinotti Mirko , Camire Rodney , Baroni Marcello , Rajab Anna , Marchetti Giovanna , Bernardi Francesco
Published: March 2018

We investigated three members of a large Omani family affected by severe factor X (FX) deficiency (coagulant activity <1%) and showing marked differences in the onset of severe hemorrhagic symptoms. All patients were homozygous for a novel FX mutation (Gly381Asp) in the structurally conserved region of the serine protease active site. Expression levels of recombinant 381D-FX were similar to those of wt-FX, indicating the presence of a severe CRM+ FX deficiency, a poorly investigated condition. The 381D-FX was normally activated and did not show a detectable amidolytic activity. Instead, we observed a residual activity in a prothrombin-time based assay (1%) and in prothrombinase assays both in plasma (1%) and in purified systems (3%). Comparison with FX variants characterized by reduced activation suggests that mutations affecting FX activity might result in a more pronounced impairment of coagulation and thus in severe hemorrhagic phenotype. In addition, this study indicates that the hemorrhagic heterogeneity observed in FX deficiencies is only partially explained by molecular analysis of FX gene.
Risk factors and coagulation parameters in relationship to phlebographic response and clinical outcome in the treatment of acute deep vein thrombosis
Authors: Kadziola Zbigniew , Scully Mike , Nakov Roumen , Misselwitz Frank , Kakkar Vijay , Breddin Hans
Published: March 2018

Possible correlation of the effects of pharmacotherapy on the inhibition of the in-vivo generation of thrombin and on the prevention of thrombus extension in patients with deep vein thrombosis (DVT) could help to define patients at higher risk.

Patients with symptomatic deep vein thrombosis confirmed by phlebography were randomised to intravenous unfractionated heparin (UFH), or a subcutaneous low-molecular-weight heparin (reviparin) twice daily for one week, or a subcutaneous reviparin once daily for four weeks. The patients were treated with oral anticoagulants for at least 3 months. Main endpoints were regression of thrombus on phlebography on Day 21 and recurrent symptomatic venous thromboembolism up to 3 months. Coagulation parameters, markers of in-vivo thrombin generation, and TFPI-release were determined at randomisation, weeks 1 and 3.

Four hundred sixty six responders (reduction of at least 30 per cent in Marder score) and 419 non-responders (Marder score unchanged or changed less than ±30%) showed no significantly different baseline characteristics. The non-responder group had a higher median Marder score at baseline and after one and three weeks of treatment, and had significantly higher fibrinogen levels, TAT complexes and F1+2 values than responders. There were no significant differences in coagulation parameters between non-responders and patients with asymptomatic + symptomatic VTE with the exception of higher TAT complexes at baseline.

Significant differences in Marder score and coagulation parameters at baseline were found between responders and non-responders. Non-responders have a higher risk tosuffer recurrent VTE and may need intensified treatment.
Activation of human platelets by 2-arachidonoylglycerol is enhanced by serotonin
Authors: Bari Monica , Del Principe Domenico , Finazzi-Agrò Alessandro , Maccarrone Mauro
Published: March 2018

The endocannabinoid 2-arachidonoylglycerol (2-AG) has been shown to activate human platelets in platelet-rich plasma, by binding to a “platelet-type” cannabinoid receptor (CBPT). Here, washed human platelets were used to characterize the binding of [3H]2-AG to CBPT, showing a dissociation constant (Kd) of 140 ± 31 nM and a maximum binding (Bmax) of 122 ± 10 pmol.mg protein-1. Selective antagonists of both CB1 and CB2 cannabinoid receptors inhibited this binding, which was enhanced up to ~230% over the controls by 1 µM serotonin (5-hydroxytryptamine, 5-HT). Human platelets were also able to bind [3H]5-HT (Kd = 79 ± 17 nM, Bmax = 14.6 ± 1.3 pmol.mg protein-1), and 1 µM 2-AG enhanced this binding up to ~150%. Moreover, they were able to take up [3H]5-HT through a high affinity transporter (Michaelis-Menten constant = 22 ± 2 nM, maximum velocity = 344 ± 15 pmol.min-1.mg protein-1), which was not affected by 2-AG. Interestingly, 5-HT did not affect the activity of the 2-AG transporter of human platelets. Treatment of washed platelets with 1 µM 2-AG led to increased intracellular inositol-1,4,5-trisphosphate (up to ~300%) and decreased cyclic AMP (down to ~50%). Furthermore, treatment of pre-loaded platelets with 1 µM 2-AG induced a ~300% increase in [3H]2-AG release, according to a CBPT-dependent mechanism. Also, 1 µM 5-HT enhanced the effect of 2-AG on inositol-1,4,5-trisphosphate (~500% of the controls), cyclic AMP (~20%) and [3H]2-AG release (~570%), and the latter process was shown to be partly (~50%) involved in the 5-HT-dependent platelet activation. Taken together, reported findings represent the first demonstration that 2-AG and 5-HT can mutually reinforce their receptor binding on platelet surface, which might have therapeutic implications.
Fibrinolytic capacity increases with age in healthy humans, while endothelium-dependent vasodilation is unaffected
Authors: Hrafnkelsdóttir Thórdís , Wall Ulrika , Swedberg Karl , Jern Sverker , Gudnason Thorarinn
Published: March 2018

The capacity for stimulated endothelial release of tissue-type plasminogen activator (tPA) and endothelium-dependent vasodilation is diminished in patients with cardiovascular risk factors. We examined the effect of age on desmopressin-stimulated tPA release and endothelium-dependent vasodilation, using the perfused-forearm model. Thirty-two healthy subjects were divided into quartiles by age (mean age 24, 36, 54, and 72 years, respectively). Baseline fibrinolytic parameters, baseline forearm blood flow (FBF), and increase in stimulated FBF were all similar across age. By contrast, the capacity for desmopressin-stimulated release of tPA increased linearly by age. For tPA antigen, the total amount released (area-under-the-curve) was 1015, 1282, 2139, and 2845 ng/L tissue (p = 0.011) and the peak release rates were 62, 80, 113, and 163 ng/min/L tissue (p = 0.008) in the age-quartiles, respectively. A similar significant age-related response was observed for tPA activity. We conclude that in healthy individuals there is an up-regulation of the fibrinolytic response by age.
Human/chicken urokinase chimeras demonstrate sequences outside the serine protease domain that dictate autoactivation
Authors: Aimes Ronald , Regazzoni Karine , Quigley James
Published: March 2018

Mammalian urokinase-type plasminogen activator (uPA) is produced as a stable single polypeptide chain zymogen and requires a distinct proteolytic cleavage to become an active, two-chain enzyme. In contrast, chicken uPA, both native and recombinant, is found predominantly as a two-chain, active enzyme even in the absence of plasmin, a physiological activator. Here we show that the proclivity to autoactivate is not a unique property of the chicken uPA catalytic domain but requires sequences distinct from and independent of the serine protease domain. Human/chicken chimeric uPA molecules and point mutants were used to determine the structural requirements for uPA autoactivation versus zymogen stability. The amino terminal fragment of chicken uPA engineered onto the human uPA molecule can induce the autoactivation of the human uPA. In fact, the first twenty residues of the chicken uPA are necessary and sufficient to induce the autoactivation of chicken and human uPA. These results indicate that sequence motifs, distal to the active site, control the substrate specificity and catalytic efficiency of uPA activity in autolytic activation.
Contributions of Asn2198, Met2199, and Phe2200 in the factor VIII C2 domain to cofactor activity, phospholipid-binding, and von Willebrand factor-binding
Authors: Lewis Deborah , Pound Mary , Ortel Thomas
Published: March 2018

The crystal structure of the factor VIII C2 domain consists of a β-sandwich core from which β-hairpins and loops extend to form a hydrophobic surface. The hydrophobic surface includes M2199 and F2200 at the tip of the 1st β-hairpin. To determine the individual contributions of residues N2198, M2199, and F2200 to phospholipid and von Willebrand factor (vWF) binding properties of factor VIII, we prepared mutant proteins with single alanine substitutions. We found that single mutations at N2198 and M2199 had relatively little impact on cofactor activity, or phospholipid and vWF binding. However the F2200A mutant had slightly lower cofactor activity at subsaturating phospholipid concentrations. Competitive ELISAs suggested that F2200 plays a more important role in both phospholipid-binding and vWF-binding than N2198 and M2199. All mutant proteins were still recognized by a monoclonal antibody and two factor VIII inhibitors that neutralized cofactor activity and blocked factor VIII binding to phospholipids.

Presented in part at the XVIII Congress of the International Society on Thrombosis and Haemostasis, Paris, France, 6-12 July 2001, and the 43rd Annual Meeting of the American Society of Hematology, Orlando, Florida, 7-11 December 2001
Effects of recombinant factor VIIa on platelet function and clot structure in blood with deficient prothrombin conversion
Authors: Martin Erika , Kuhn Jan , Seremetis Stephanie , Carr Marcus
Published: March 2018

While recombinant factor VIIa (rFVIIa) shows promise as a broad-spectrum hemostatic agent, questions remain regarding the most appropriate dose and the best way to monitor its effects. In this study we tested the sensitivity of a thrombin dependent platelet assay, platelet contractile force, to the effects of rFVIIa in normal, factor-deficient, and inhibitor-containing blood samples. Dose dependent effects of rFVIIa on platelet contractile force (PCF) and clot elastic modulus (CEM) were measured in all blood samples.

rFVIIa minimally affected PCF and CEM in normal blood clotted with thrombin or batroxobin. While rFVIIa minimally altered PCF and CEM in factor VIII (FVIII) deficient blood clotted with thrombin, rFVIIa increased PCF and CEM and shortened the lag phase in a dose dependent manner in batroxobin-induced clots. The effects of rFVIIa in factor IX (FIX) deficient blood mirrored the effects seen in FVIII deficient samples. Whether clotted with thrombin or batroxobin, baseline PCF and CEM were abnormally low in FVIII deficient samples containing FVIII inhibitors. In such samples, rFVIIa caused dose dependent improvement of PCF, CEM, and lag phases. In one patient with a spontaneous inhibitor, rFVIIa caused dose dependent increases in PCF and CEM in blood clotted with either enzyme.

rFVIIa corrects the deficient thrombin generation seen in FVIII and FIX deficiency, and in blood containing FVIII inhibitors. As a consequence, platelet function is improved and clot structure is enhanced. Platelet contractile force and clot elastic modulus measurements are sensitive to the dose dependent effects of rFVIIa.
Green tea epigallocatechin-3-gallate inhibits platelet signalling pathways triggered by both proteolytic and non-proteolytic agonists
Authors: Deana Renzo , Turetta Loris , Donà Massimo , Brunati Anna , Michiel Lucia , Garbisa Spiridione , Donella-Deana Arianna
Published: March 2018

Epigallocatechin-3-gallate (EGCG), a component of green tea, inhibits human platelet aggregation and cytosolic [Ca2+]cincreases more strongly when these processes are induced by thrombin than by the non-proteolytic thrombin receptor activating peptide (TRAP), thromboxane mimetic U46619, or fluoroaluminate. In line with the previously demonstrated EGCG anti-proteolytic activity, a marked inhibition on aggregation is obtained by pre-incubation of thrombin with EGCG prior to addition to cellular suspension. The catechin also reduces cellular Ca2+ influx following thapsigargin-induced calcium emptying of endoplasmic reticulum, and the agonist-promoted cellular protein tyrosine phosphorylation. Both tyrosine kinases Syk and Lyn, immuno-precipitated from stimulated platelets, are greatly inhibited upon cellular pre-incubation with EGCG, which also inhibits the in vitro auto-phosphorylation and exogenous activity of these two enzymes purified from rat spleen. Both thrombin-induced aggregation and [Ca2+]c increase are reduced in platelets from rats that drank green tea solutions. It is concluded that EGCG inhibits platelet activation, by hindering the thrombin proteolytic activity, and by reducing the agonist-induced [Ca2+]cincrease through inhibition of Syk and Lyn activities.
Anandamide induces apoptosis in human endothelial cells: its regulation system and clinical implications
Authors: Yamaji Kazuyo , Sarker Krishna , Kawahara Koichi , Iino Satoshi , Yamakuchi Munekazu , Abeyama Kazuhiro , Hashiguchi Teruto , Maruyama Ikuro
Published: March 2018

Anandamide (AEA), an endogenous cannabinoid, is generated by macrophages during shock conditions, and is thought to be a causative mediator of septic shock. Thus, we hypothesized that AEA plays a crucial role in endothelial cell (EC) injury. Here, we demonstrate that AEA induces apoptosis in a time-and dose-dependent manner in human umbilical vein endothelial cells (HUVECs). AEA triggered phosphorylation of c-Jun NH2-terminal kinase (JNK) and p38 mitogen activated protein kinase. AEA also showed a marked increase of interleukin 1β–converting enzyme (ICE)CED-3 family protease (caspase-3) activity. AEA-induced EC death was inhibited by a selective vanilloid receptor 1 (VR1) antagonist, capsazepine, and was enhanced by a VR1 agonist, capsaicin, indicating that AEA induces apoptosis in ECs via VR1. In conclusion, we propose that AEA may play a crucial role in EC injury under conditions of shock, and that the use of inhibitors of the AEA regulation system may have a therapeutic effect under these conditions.
Reactive oxygen species modulate HIF-1 mediated PAI-1 expression: involvement of the GTPase Rac1
Authors: Berchner-Pfannschmidt Utta , Wotzlaw Christoph , Cool Robbert , Fandrey Joachim , Acker Helmut , Jungermann Kurt , Görlach Agnes , Kietzmann Thomas
Published: March 2018

The hypoxia-inducible transcription factor HIF-1 mediates upregulation of plasminogen activator inhibitor-1 (PAI-1) expression under hypoxia. Reactive oxygen species (ROS) have also been implicated in PAI-1 gene expression. However, the role of ROS in HIF-1-mediated regulation of PAI-1 is not clear. We therefore investigated the role of the GTPase Rac1 which modulates ROS production in the pathway leading to HIF-1 and PAI-1 induction.

Overexpression of constitutively activated (RacG12V) or dominant-negative (RacT17N) Rac1 increased or decreased, respectively, ROS production. In RacG12V-expressing cells, PAI-1 mRNA levels as well as HIF-1α nuclear presence were reduced under normoxia and hypoxia whereas expression of RacT17N resulted in opposite effects. Treatment with the antioxidant pyrrolidinedithiocarbamate or coexpression of the redox factor-1 restored HIF-1 and PAI-1 promoter activity in RacG12V-cells. In contrast, NFκB activation was enhanced in RacG12V-cells, but abolished by RacT17N. Thus, these findings suggest a mechanism explaining modified fibrinolysis and tissue remodeling in an oxidized environment.
Molecular characterization of coagulation factor XII deficiency in a Japanese family
Authors: Nishioka Junji , Kasai Yasunari , Kato-Nakazawa Keiko , Abe Yasunori , Morishita Yoshitaka , Nakatani Kaname , Nobori Tsutomu , Wada Hideo
Published: March 2018

We report the identification in a Japanese family of a novel homozygous W486C mutation in the protease domain of coagulation factor XII (FXII), which was associated with the reduction of plasma FXII activity and antigen level to less than 5% of normal. Sequences of each exon for FXII gene was analysed in family members by polymerase chain reaction (PCR) amplification followed by a direct sequencing method. Sequence analysis showed a homozygous substitution of G to C at nucleotide position 10587 (cDNA position 1458) in proband’s FXII gene, resulting in a Trp to Cys substitution in the catalytic domain of FXII. PCR-fragment length polymorphism analysis of 55 healthy volunteers showed no such mutation. Transient expression of FXII in HK-293T cells and analysis of FXII antigen in culture media and cell lysates showed reduced secretion of mutant protein by more than 84% relative to that of wild type protein although the intracellular contents were similar. Our results suggest that the reduced secretion of FXII protein was due to incorrect folding caused by the introduction of Cys486. We designated this mutation as FXII Mie-1.
Effect of hemostatic risk factors on the individual probability of thrombosis during pregnancy and the puerperium
Authors: Gerhardt Andrea , Zotz Rainer , Scharf Rüdiger
Published: March 2018

In a retrospective study of 190 women with a first history of venous thromboembolism during pregnancy and the puerperium and 190 age-matched women with at least one prior pregnancy and no history of venous thromboembolism, the individual probability of thrombosis was determined. Assuming an overall risk of 1 in 1500 pregnancies, the probability of pregnancy-related thrombosis in carriers of homozygous factor V Leiden was 1 in 80 (odds ratio 20.6, p=0.005) and among carriers of combined heterozygous factor V Leiden and heterozygous G20210A mutation in the prothrombin gene 1 in 20 (odds ratio 88, p<0.001). The probability of thrombosis per pregnancy among women with elevated levels of factor VIII:C (>172 % activity) was 1 in 385 (odds ratio 4.5, p<0.001) and among those with increased levels of von Willebrand factor antigen (>190 %) 1 in 435 (odds ratio 4.0, p=0.002), independent of elevated factor VIII:C levels. The high prevalence of combined and homozygous defects of hemostatic components (21.6%) in patients as compared with normal women (0.86%) supports the concept that venous thromboembolism is a multicausal disorder.
Thromboembolic risk in patients with high titre anticardiolipin and multiple antiphospholipid antibodies
Authors: Neville Carolyn , Rauch Joyce , Kassis Jeannine , Chang Erika , Joseph Lawrence , Le Comte Martine , Fortin Paul
Published: March 2018

Asymptomatic antiphospholipid antibody (aPL) carriers with high risk for thrombosis may benefit from preventive anticoagulation.

It was our objective to test whether the risk of thrombosis increases with: 1) increasing titres of anticardiolipin antibodies (aCL) after adjustment for other cardiovascular risk factors and 2) the number of aPL detected.

In a cross-sectional study, blood was collected from clinics in two teaching hospitals. The study included 208 individuals suspected of having an aPL and 208 age- and sex-matched controls having blood drawn for a complete blood count.

Clinical variables included history of previous arterial (ATE) or venous (VTE) thrombotic events, traditional risk factors for cardiovascular disease, and systemic lupus erythematosus (SLE). Laboratory variables included IgG/IgM aCL, lupus anticoagulant, and IgG/IgM anti-β2-glycoprotein I.

Mean age was 46.5 years and 83% were female. Seventy-five of the 416 participants had > 1 aPL, and 69 had confirmed > 1 ATE or VTE. Family history was positive in 48% of participants, smoking in 28%, hypertension in 16%, diabetes in 6%, and SLE in 20%. A 10-unit increase in aCL IgG titre was associated with an odds ratio (OR) [95% CI] of 1.07 [1.01-1.13] for ATE and 1.06 [1.02 - 1.11] for VTE. The odds of a previous thrombosis increased with each additional aPL detected: 1.5 [0.93-2.3] for ATE and 1.7 [1.1-2.5] for VTE.

These results indicate that increased titres of aCL and multiple aPL were associated with an increased risk of a previous thrombotic event.
Unexpected persistence of platelet hyporeactivity beyond the neonatal period: a flow cytometric study in neonates, infants and older children
Authors: Hézard Nathalie , Potron Gérard , Schlegel Nicole , Amory Catherine , Leroux Bernard , Nguyen Philippe
Published: March 2018

Previous studies, using flow cytometry, have reported a lower platelet reactivity in neonates compared to adults. Only few studies were carried out in older children, and results were controversial in terms of age to reach adult platelet function. We studied a total of 125 healthy neonates, infants and older children, and 15 adults. αIIbβ3 expression on resting and activated platelets was lower in all children, with an impaired capability of αIIbβ3 activation (PAC1 and bound fibrinogen). This defect was observed until the age of fifteen with a gradual recovery with age. In neonates, we observed a defect of GPIbα internalization, and demonstrated that this defect persisted in older children as well. In contrast with αIIbβ3 integrin activation, we did not observe a gradual age-dependent recovery. These unexpected results point out the need for reference values in childhood.
Differential effects of oral and transdermal postmenopausal estrogen replacement therapies on C-reactive protein
Authors: Oger Emmanuel , Le Gal Grégoire , Blouch Marie-Thérèse , Abgrall Jean-François , Kerlan Véronique , Scarabin Pierre-Yves , Mottier Dominique , Lacut Karine ,   and the SARAH investigators
Published: March 2018

C-reactive protein (CRP) is one of the main independent predictors of cardiovascular events. Oral post-menopausal estro-gen replacement therapy (ERT) increases CRP levels, but the effect of transdermal ERT is not well documented.

CRP, interleukine-6 (IL-6) and tumor necrosis factor-α (TNF-α) levels were evaluated in a randomised study of 196 healthy postmenopausal women, who were allocated to receive continuous oral estradiol-17β, (n=63) or transdermal estradiol-17β, (n=68) both combined with micronised progesterone, or placebo (n=65). Oral estrogen increased CRP levels compared with both placebo (p=0.010) and transdermal estrogen (p=0.004) at 6 months. There was no significant effect of transdermal estro-gen on CRP levels compared with placebo (p=0.997). No significant difference was found in the median changes for IL-6 and TNF-α between the three treatment groups.

In conclusion, transdermal estrogen has no significant effect on CRP levels at 6 months, but CRP concentrations increased significantly with oral estrogen although no changes in cytokine levels were detected. The clinical relevance of these effects remains to be determined.
Haemostatic and Lipid Determinants of Prothrombin Fragment F1.2 and D-dimer in Plasma
Authors: Cooper Jackie , Martin Janet , Howarth David , Meade Thomas , Miller George , MacCalum Peter
Published: March 2018

The determinants of plasma levels of prothrombin fragment F1.2 (F1.2) and D-dimer in different populations are unclear and this may complicate their interpretation as predictors of thrombotic risk, particularly in the case of D-dimer. We therefore measured F1.2 and D-dimer levels together with a number of other haemostatic and lipid variables in a cross-sectional community-based study of 150 healthy adults (73 male, 77 female), age range 23-80 years, identified from the list of a general practice by stratified random sampling within sex and decade of age. Plasma F1.2 was significantly higher in females than males and was independently and positively associated with age, factor VII activity (FVIIc) and C1 inhibitor, and inversely associated with high density lipoprotein (HDL) cholesterol. Plasma D-dimer showed a quadratic association with age (p <0.0001). In those ≤55 years D-dimer was inversely associated with dilute clot lysis time (DCLT) and activated protein C (APC) ratio. In those >55 years it was significantly higher in females than males and associated positively with age, fibrinogen and, in males, activated factor XII (FXIIa). In a multiple-linear model which combined both age groups, F1.2 and D-dimer were independently associated with each other (r = 0.22, p = 0.03). Thus, thrombin generation and fibrin turnover/fibrinolysis are associated in healthy subjects. HDL cholesterol (inversely) and FVIIc are associated with basal thrombin generation (i.e. F1.2). Determinants of D-dimer differ according to age and interpretation of the biological significance of D-dimer levels in epidemiological studies may therefore not be straightforward.