bluuebunny/crossref_metadata_2025 huggingface dataset:
A Plasma Coagulation Assay for an Activated Protein C-independent Anticoagulant Activity of Protein S
Authors: van ’t Veer Cornelis , Meijers Joost , Bertina Rogier , Wijnen Merel van , Bouma Bonno
Published: March 2018

To study the physiological importance of the activated protein C (APC)-independent anticoagulant activity of protein S, we developed an assay specific for this activity. The ability of protein S to prolong the clotting time in an APC-independent way was expressed as the ratio of the clotting time in a plasma sample divided by the clotting time in the presence of a polyclonal antibody against human protein S (both after 1:1 dilution in protein S-C4BP deficient plasma). The mean protein S-dependent anticoagulant ratio (PSdAR) was 1.53 ± 0.18 in 34 healthy controls, and was significantly lower in 16 heterozygous protein S deficient patients (PSdAR = 1.15 ± 0.09). In plasmas from patients under oral anticoagulant therapy the mean PSdAR was within the range of the control population (1.50 ± 0.18). The mean total protein S antigen level in these plasmas was 58%, suggesting a higher specific APC-independent anticoagulant activity of protein S in these patients than in normals.

This functional protein S assay can be used for the laboratory diagnosis of protein S deficiency, and to study the mechanism of the APC-independent anticoagulant activity in plasma.
Identification and Characterization of Two Thrombin-activatable Fibrinolysis Inhibitor Isoforms
Authors: Morser John , Bajzar Laszlo , Nesheim Michael , Nagashima Mariko , Zhao Lei
Published: March 2018

Thrombin-activatable fibrinolysis inhibitor (TAFI) is synthesized by the liver and is thought to circulate in plasma as a plasminogen-bound zymogen. When it is activated by the thrombin/thrombomodulin complex, activated TAFI exhibits carboxypeptidase B-like activity. To study the structure-function relationship of TAFI, we expressed recombinant human TAFI in insect cells. During the cloning of TAFI cDNA from several human liver cDNA libraries, we identified a second TAFI cDNA which differed from the published sequence at 2 positions. One of these sequences resulted in a substitution of alanine for threonine at residue 147, the other was a silent mutation. These substitutions were found in several cDNA libraries from different sources. Using Southern blot analysis, we confirmed the existence of this TAFI polymorphism in the population. In order to compare the activation and activity of TAFI isoforms, we expressed both isoforms in the baculovirus expression system, and compared the enzyme kinetics of the purified proteins. The molecular weight of recombinant TAFI is lower than plasma TAFI due to differences in glycosylation. The two recombinant TAFI iso-forms had similar activation kinetics and the activated enzymes had similar carboxypeptidase B-like activity towards small molecule substrates. Their ability to retard clot lysis was found to be similar in a plate clot lysis assay.
4G/5G Polymorphism of PAI-1 Gene Promoter and Fibrinolytic Capacity in Patients with Deep Vein Thrombosis
Authors: Sartori Maria Teresa , Wiman Björn , Vettore Silvia , Dazzi Francesco , Girolami Antonio , Patrassi Giovanni Maurizio
Published: March 2018

A deletion/insertion polymorphism (4G or 5G) in the promoter of the plasminogen activator inhibitor type 1 gene has been suggested to be involved in regulation of the synthesis of the inhibitor, the 4G allele being associated with enhanced gene expression. A relationship between 4G/5G polymorphism and PAI-1 levels was found in patients with cardiovascular and metabolic diseases, but not in healthy subjects. In the present work we studied the distribution of PAI-1 4G/5G geno-type and its relation to fibrinolytic capacity in 70 unrelated patients with deep vein thrombosis. Each patient was assayed before and after 20 min. Venous occlusion for euglobulin lysis time, t-PA antigen and activity, and PAI-1 antigen and activity. The prevalence of 5G homozygous carriers was significantly lower in patients than in controls (10% vs. 26%, p = 0.009). The 5G allele frequency was reduced, even though not significantly, in DVT patients compared to healthy subjects (0.40 vs. 0.51, respectively). In the patient group, the mean PAI-1 antigen and activity levels were significantly higher than among controls and related to the 4G/5G polymorphism. In patients with 4G/5G and 4G/4G genotype a significant correlation was found between PAI-1 levels and the global fibrinolytic activity as evaluated by euglobulin lysis time. The prevalence of a reduced fibrinolytic potential due to PAI-1 excess was 45.7% among DVT patients. Moreover, the prevalence of PAI-1 induced hypofibrinolysis was strongly related to PAI-1 polymorphism, since it was significantly lower in 5G homo-zygous patients (28.6%) than in both 4G/5G carriers (55.3%, p <0.001) and 4G homozygous patients (57.9%, p <0.001).

In conclusion, in patients with deep vein thrombosis the 4G polymorphism of PAI-1 gene promoter may influence the expression of PAI-1 and it should be taken into consideration as a facilitating condition for pathological fibrinolysis together with other environmental and genetic factors. Whether this has any significance in regard to the pathogenesis of venous thrombosis remains to be proven.
The Effects of MPL-Ligand, Interleukin-6 and Interleukin-11 on Megakaryocyte and Platelet Alpha-granule Proteins
Authors: Remmler Jill , Zucker-Franklin Dorothea , Philipp Claire
Published: March 2018

Thrombopoietin (Mpl ligand), interleukin-6 (IL-6), and interleukin-11 (IL-11) differ in their effects on megakaryocyte maturation and development. In the present study, the impact of these thrombocytopoietic cytokines on biochemical and structural granule and membrane components was examined. Western blotting was performed on equivalent amounts of isolated megakaryocyte or platelet protein and the relative intensities of the enhanced chemiluminescent-visualized bands were quantitated by densitometry. Megakaryocyte growth and development factor (MGDF), a recombinant thrombopoietin-related molecule, significantly increased megakaryocyte fibronectin (72%), thrombospondin (55%), von Willebrand factor (28%) and p-selectin (CD62p) (37%) when compared to equivalent amounts of protein from saline-treated controls (p < 0.01). Megakaryocyte fibrinogen was not increased. Ultrastructurally, there was a marked increase in ribosomes and rough endoplasmic reticulum even in mature-appearing megakaryocytes. Platelets from MGDF-treated mice showed small increases in fibronectin (8%), and CD62p (18%), but did not show increases in other measured α-granule proteins. Neither IL-6 nor IL-11 increased megakaryocyte or platelet α-granule proteins over levels observed in saline controls. IL-11 treated megakaryocytes, while also exhibiting an increase in ribosomes, were characterized by prominent cytoplasmic fragmentation. The study demonstrates the impact of Mpl ligand in increasing synthesized megakaryocyte α-granule proteins and of IL-11 in promoting megakaryocyte fragmentation.
Melagatran, a Low Molecular Weight Thrombin Inhibitor, Counteracts Endotoxin-induced Haemodynamic and Renal Dysfunctions in the Pig
Authors: Larsson Anders , Saldeen Tom , Mattsson Christer , Eriksson Mats
Published: March 2018

Coagulation and fibrinolysis are crucial in septic shock and inhibition of thrombin may be beneficial in this circumstance. Since porcine endotoxaemia has been found to replicate severe septic shock, a low molecular weight thrombin inhibitor, melagatran, was infused during the first 3 out of 6 h of endotoxaemia in pigs. Plasma creatinine (p <0.01) and urinary output (p <0.05) were less affected in the melagatran + endotoxin group (n = 6) as compared to endotoxaemic controls (n = 9). The left ventricular stroke work index, systemic vascular resistance index and oxygen extraction were all less affected (p <0.05) by endotoxin during the infusion of melagatran. The plasma concentration of melagatran declined with an apparent plasma half-life of 5 h as soon as the infusion was stopped. APTT, however, continued to increase after the infusion of melagatran had stopped and reached a maximum of 113 s at 5 h (baseline 17 s). APTT in endotoxaemic control pigs reached a maximum of 22 s. Thus, melagatran may counteract some consequences of endotoxaemia.
Kenneth M. Brinkhous
Authors: Read Marjory , Nichols Timothy , White Gilbert , Wagner Robert , Roberts Harold
Published: March 2018

Kenneth M. Brinkhous, a beloved member of the faculty of the University of North Carolina, died at the age of 92 on December 11, 2000.
Modeling Human Zymogen Factor IX
Authors: Perera Lalith , Darden Thomas , Pedersen Lee
Published: March 2018

Modern theoretical techniques are employed to provide complete three dimensional structure for the zymogen and activated forms of human coagulation factors IX and IXa. These structures are fully calcium bound and equilibrated in an electrically neutral aqueous environment. The relationship of structure to mutational data is examined. We find that a substantial relative orientational change of the catalytic domain occurs on activation. Also, we find that the electrostatistically dipolar nature of the catalytic domain is substantially modified upon activation, with cleavage of the negatively charged activation peptide leaving behind a largely hydrophobic face in factor IXa. While the backbone atoms of the catalytic residues have little relative movement, nearby loops are found that do move. The presence or absence of these changes likely defines specificity.
Cell-derived Microparticles Circulate in Healthy Humans and Support Low Grade Thrombin Generation
Authors: Nieuwland Rienk , Böing Anita , Romijn Fred , Hack C. Erik , Sturk Augueste , Berckmans René
Published: March 2018

We determined the numbers, cellular origin and thrombin-generating properties of microparticles in healthy individuals (n = 15). Micro-particles, isolated from fresh blood samples and identified by flow cytometry, originated from platelets [237106/L (median; range 116-565)], erythrocytes (28106/L; 13-46), granulocytes (46106/L; 16-94) and endothelial cells (64106/L; 16-136). They bound annexin V, indicating surface exposure of phosphatidylserine, and supported coagulation in vitro. Interestingly, coagulation occurred via tissue factor (TF)-independent pathways, because antibodies against TF or factor (F)VII were ineffective. In contrast, in our in vitro experiments coagulation was partially inhibited by antibodies against FXII (12%, p = 0.006), FXI (36%, p <0.001), FIX (28%, p <0.001) or FVIII (32%, p <0.001). Both the number of annexin V-positive microparticles present in plasma and the thrombin-generating capacity inversely correlated to the plasma concentrations of thrombin-antithrombin complex (r = -0.49, p = 0.072 and r = -0.77, p = 0.001, respectively), but did not correlate to prothrombin fragment F1+2 (r = -0.002, p = 0.99). The inverse correlations between the number of microparticles and their thrombin-forming capacity and the levels of thrombin-antithrombin complex in plasma may indicate that microparticles present in the circulation of healthy individuals have an anticoagulant function by promoting the generation of low amounts of thrombin that activate protein C. We conclude that microparticles in blood from healthy individuals support thrombin generation via TF- and FVII-independent pathways, and which may have an anticoagulant function.
Prothrombin Time Ratio Is Reduced by Magnesium Contamination in Evacuated Blood Collection Tubes
Authors: Dam W. van , Sturk A. , Bertina R. M. , Besselaar A. M. H. P. van den
Published: March 2018

Magnesium ions were detected in sodium citrate solutions in several lots of evacuated blood collection tubes. The mean concentrations ranged between 1.3 and 1.6 mmol/L. Magnesium was also present in the rubber stoppers of the blood collection tubes and could be leached into the citrate solution. It was shown that magnesium added to citrated plasma shortened the prothrombin time of both coumarin and normal plasma. The effect of magnesium was relatively greater on coumarin than on normal plasma resulting in reduced prothrombin time ratio. Shortening of the prothrombin time was also observed when magnesium chloride was added to dialysed plasma, i.e., in the absence of citrate. These results indicate that magnesium contamination can interfere with accurate INR determination in the control of oral anticoagulant therapy.
Prothrombin Scranton: Substitution of an Amino Acid Residue Involved in the Binding of Na+ (LYS-556 to THR) Leads to Dysprothrombinemia
Authors: Sun William , Smirnow David , Jenkins Mallory , Degen Sandra
Published: March 2018

Several members of a family from Scranton, Pennsylvania were identified to have normal levels of prothrombin antigen while their prothrombin clotting activity was approximately 50% of normal. There has been no previous history of bleeding or other clinical manifestations in this family. The genomic DNA from the proband was amplified for all exons in the prothrombin gene and analyzed by single strand conformation polymorphism (SSCP)/heteroduplex analysis followed by DNA sequence analysis and restriction enzyme digestion. A mutation at nucleotide 20040 in exon 14 was identified and confirmed by restriction enzyme digestion. This mutation results in the substitution of Thr for Lys at amino acid 556. Amino acid 556 has been reported as one of the key residues for the binding of Na+ in the thrombin portion of the protein.
Low Levels of Thrombin Activatable Fibrinolysis Inhibitor (TAFI) in Patients with Chronic Liver Disease
Authors: George Magdalene , Fareed Jawed , Van Thiel David
Published: March 2018

Thrombin Activatable Fibrinolysis Inhibitor (TAFI) is a 60 κD glycoprotein present in plasma that regulates fibrinolysis by limiting the amount of fibrin available for fibrinolysis by tissue plasminogen activator (t-PA). Chronic liver disease is well-known to be associated with a low-grade fibrinolytic syndrome that under the appropriate stimulus proceeds to an overt disseminated intravascular coagulopathy (DIC) with demonstrable bleeding. In the present study, TAFI activity was measured in the plasma of 74 patients with advanced liver disease, and the levels of TAFI were related to those of other important coagulation and fibrinolytic factors. TAFI levels were very low and essentially undetectable in the plasma of patients with advanced hepatocellular liver disease. No relationship with the degradation products of fibrin was evident.
Characterization of Aspercetin, a Platelet Aggregating Component from the Venom of the Snake Bothrops asper which Induces Thrombocytopenia and Potentiates Metalloproteinase-induced Hemorrhage
Authors: Soto Mónica , Kamiguti Aura , Theakston David , Fox Jay , Escalante Teresa , Gutiérrez José María , Rucavado Alexandra
Published: March 2018

Thrombocytopenia occurs in a number of patients bitten by Bothrops asper, a species responsible for the majority of snakebites in Central America and southern Mexico. In this work we describe the isolation of a new platelet-aggregating protein, named aspercetin, from the venom of B. asper, which induces thrombocytopenia in mice. Isolation was carried out by a combination of ion-exchange chromatography on DEAE-Sepharose and affinity chromatography on Affi-Gel Blue. Aspercetin is a disulfide-linked heterodimer, with a pI of 4.5 and a molecular mass of 29,759 Da, detemined by MALDI-ESI mass spectrometry. N-terminal sequence shows homology with a number of venom proteins which belong to the C-type lectin family. Aspercetin has functional similarities with botrocetin, from B. jararaca venom, since it induces platelet aggregation only in the presence of plasma or purified von Willebrand factor. Aspercetin-mediated platelet aggregation results from the interaction of von Willebrand factor with platelet receptor GPIb. Aspercetin lacks anticoagulant effect and does not agglutinate erythrocytes, in contrast with other representatives of the C-type lectin family isolated from snake venoms. Moreover, aspercetin is not lethal, nor does it induce myonecrosis, hemorrhage and edema. When injected intravenously or intramuscularly in mice it induces a rapid, dose-dependent drop in platelet counts and prolongs the bleeding time, suggesting that it may play a role in the thrombocytopenia that develops in a number of B. asper envenomations. Moreover, mice injected intravenously with aspercetin and then receiving an intradermal injection of B. asper hemorrhagic metalloproteinase BaP1 develop a larger hemorrhagic lesion than mice receiving only BaP1. This suggests that aspercetin, by reducing platelet numbers, may contribute to the hemorrhagic effect characteristic of B. asper envenomations.
High Molecular Weight Kininogen Deficiency: a Patient who Underwent Cardiac Surgery
Authors: Burman J. F. , Rutherford L. C. , Keogh B. F. , Yacoub M. H. , Davidson S. J.
Published: March 2018

A 66 year old male, referred for cardiac surgery, was found to have high molecular weight kininogen deficiency (activity <1%). Apart from activated partial thromboplastin time (APTT) >300 s, tests of haemostasis were otherwise normal (factors VIII, IX, XI, XII and prekallikrein). No inhibitor of coagulation was found.

The activated coagulation time (ACT) was 800 s pre-operatively and >1000 s after heparin. Heparin levels were measured directly by an anti-Xa chromogenic assay, with values of between 2.9 and 3.2 u/ml during cardiopulmonary bypass. Thrombin-antithrombin levels rose from 2.3*g/l before surgery to a peak of 83.5*g/l at the end of cardio-pulmonary bypass. Cross linked fibrin d-dimers (XDP) levels rose from 100 ng/ml before operation to 600 ng/ml after protamine administration. The patient had no excess bleeding and no thrombotic complications from surgery.

This patient shows that high molecular weight kininogen is not required for thrombin formation or fibrinolysis during cardiac surgery and illustrates the need to measure heparin directly in patients with such contact factor deficiencies.
Multicenter Study of Homocysteine Measurement – Performance Characteristics of Different Methods, Influence of Standards on Interlaboratory Agreement of Results
Authors: Chantarangkul Veena , Lombardi Rossana , Lecchi Anna , Mannucci Pier Mannuccio , Cattaneo Marco , Tripodi Armando ,   for the ad hoc Study Group
Published: March 2018

After the demonstration that moderate hyperhomocysteinemia is associated with thrombosis, many hematological labs are becoming interested in total homocysteine (tHcy) measurement. This prompted us to organize a collaborative study to investigate the performance of methods used in this setting and to assess the between-lab comparability of results. Two pairs of pooled plasma (A1-A2 and B1-B2) were prepared at the coordinating Center. tHcy levels were normal in A1-A2 and moderately high in B1-B2. Within each pair tHcy levels were similar but not identical. Aliquots were taken from each pool to prepare sets of 100 samples (coded from 1 to 100). Each set consisted of 25 replicates for each pool. Samples were frozen and shipped in dry ice to 16 labs with a common frozen aqueous standard. Labs were asked to measure (in blind) tHcy with their methods and standards. Results were sent to the coordinating Center both as raw readings and as tHcy levels. The following methods were used: High Pressure Liquid Chromatography (HPLC) in 12 labs (home-made in 10 and commercial in 2); Enzyme Immuno Assays (EIA) in 2; Fluorescence Polarization Immunoassay (FPIA) in 2 and Capillary Electrophoresis (CE) in one. Results for paired pools (A1-A2 and B1-B2) were analyzed by the Student t test to assess for the ability to discriminate between similar but not identical tHcy levels. Results for each pool were used to assess within-lab reproducibility and between-lab comparability. Within-lab reproducibility expressed as median CV ranged from 12.6 to 13.9% (home-made HPLC); from 9.2 to 11.4% (commercial HPLC); from 21.8 to 24.2% (EIA); from 2.7 to 3.3% (FPIA) and from 11.2 to 22.0% (CE). All labs, except one using CE and 2 using home-made HPLC, were able to discriminate between similar tHcy levels in the normal range (pools A1-A2). Ten labs (4 using home-made HPLC, 2 commercial HPLC, 2 FPIA, one EIA and one CE) were able to discriminate between similar moderately high tHcy levels (pools B1-B2). Between-lab comparability expressed as CV was 14.0% 13.9%, 15.6% and 14.5% for pools A1, A2, B1, and B2. These values were considerably lower (CV values < 5.2%) when a common plasma standard was used for calculation of tHcy levels, while the use of a common aqueous standard failed to achieve the necessary harmonization. In conclusion, performance characteristics of the FPIA method compare favorably with the well-established HPLC methods. It is simpler and more suitable to be used by general hematological labs. Between-lab comparability of results is still a problem. The establishment of an international plasma standard would be of help to harmonize tHcy measurement across laboratories.
β2-glycoprotein I Binding to Platelet Microparticle Membrane Specifically Reduces Immunoreactivity of Glycoproteins IIb/IIIa
Authors: Vallar Laurent , Regnault Véronique , Latger-Cannard Véronique , Lecompte Thomas
Published: March 2018

We have investigated β2-glycoprotein I (β2GPI) binding to platelet-derived microparticles (PMP) and its effect on GPIIb/IIIa. PMP were isolated from washed human platelets after stimulation with A23187, and analyzed by surface plasmon resonance spectroscopy. β2GPI as well as activated protein C (APC) or annexin V bound to PMP-coated sensorchips, demonstrating exposure of anionic phospholipids on immobilized PMP. β2GPI binding was impaired by calcium and occurred in a concentration-dependent manner with apparent kon = 2.6104 M-1.s-1 and koff = 4.410-3 s-1, corresponding to a KD value of 1.710-7 M. When analyzed by flow cytometry, the binding of certain mAbs specific for GPIIb and/or GPIIIa was reduced in the presence of β2GPI but not of APC or annexin V, whereas the binding of anti-GPIb or anti-P-selectin mAbs, or of soluble fibrinogen remained unchanged. These results suggest a broad but specific influence of β2GPI on GPIIb/IIIa immunoreactivity, and indicate that β2GPI may act as a modulator of GPIIb/IIIa-dependent functions of PMP.
Development and Assessment of Enzyme Immunoassay for Platelet-derived Microparticles
Authors: Osumi Kazuoki , Ozeki Yasushi , Saito Sonoko , Nagamura Yoshie , Ito Hideki , Kimura Yukio , Ogura Hiroshi , Nomura Shosaku
Published: March 2018

Platelet-derived microparticles (PMPs) are released from platelets through the platelet activation by high shear stress, collagen, or calcium ionophore (A23187). PMPs are observed in patients with acute myocardial infarction, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome, heparin-induced thrombocytopenia and other thrombotic disorders, but the importance of circulating PMPs in the pathogenesis of these diseases is still debated. Numbers of PMPs are usually determined by flowcytometry (FCM), but easier and reproducible PMP assay systems are needed. To develop a better ELISA for PMPs, we used antibodies against the platelet antigens anti-GPIb (NNKY5-5), anti-GPIIb/IIIa (NNKY2-11, anti-CD41), anti-GPIX (KMP-9), and anti-CD9 (NNKY1-19). PMPs were detected with all combinations of these antibodies, but the ELISA having the highest and most specific absorbance was obtained with a combination of KMP-9 (capture antibody) and NNKY5-5 (detecting antibody). PMPs in blood samples were measured by ELISA and FCM. ELISA correlated with PMPs quantitated by FCM. By shaking ELISA plates during incubation, nonspecific binding of platelets was eliminated. The level of PMPs was not increased in diabetes mellitus, thrombotic thrombocytopenic purpura, anti-phospholipid syndrome, or sepsis. The concentration of PMP was elevated in hemolytic uremic syndrome. Activated PMPs were absorbed to 0.8 m filter, but circulating PMPs were not absorbed. These results suggest that activated PMPs are likely to adhere to leukocytes or endothelial cells at the activation site and that the circulating form of PMPs are likely to be a residue of activated PMPs. To detect only the activated form of PMPs, a new ELISA needs to be developed, and it will likely use a combination of antibodies that detect platelet activation markers such as P-selectin (CD62P) or activated GPIIb/IIIa.
Potentiation of Thromboxane A2-induced Platelet Secretion by Gi Signaling through the Phosphoinositide-3 Kinase Pathway
Authors: Dangelmaier Carol , Jin Jianguo , Smith Bryan , Kunapuli Satya
Published: March 2018

Platelet activation results in shape change, aggregation, generation of thromboxane A2, and release of granule contents. We have recently demonstrated that secreted ADP is essential for thromboxane A2-induced platelet aggregation (J. Biol. Chem. 274: 29108-29114, 1999). The aim of this study was to investigate the role of secreted ADP interacting at P2 receptor subtypes in platelet secretion. Platelet secretion induced by the thromboxane A2 mimetic U46619 was unaffected by adenosine-3’phosphate-5’-phosphate, a P2Y1 receptor selective antagonist. However, AR-C66096, a selective antagonist of the P2T
AC
receptor, inhibited U46619-induced platelet secretion, indicating an important role for Gi signaling in platelet secretion. Selective activation of either the P2T
AC
receptor or the α2A adrenergic receptor did not cause platelet secretion, but potentiated U46619-induced platelet secretion. SC57101, a fibrinogen receptor antagonist, failed to inhibit platelet secretion, demonstrating that outside-in signaling was not required for platelet secretion. Since Gi signaling results in reduction of basal cAMP levels through inhibition of adenylyl cyclase, we investigated whether this is the signaling event that potentiates platelet secretion. SQ22536 or dideoxyadenosine, inhibitors of adenylyl cyclase, failed to potentiate U46619-induced primary platelet secretion, indicating that reduction in cAMP levels does not directly contribute to platelet secretion. Wortmannin, a selective inhibitor of PI-3 kinase, minimally inhibited U46619-induced platelet secretion when it was solely mediated by Gq, but dramatically ablated the potentiation of Gi signaling. We conclude that signaling through the P2TAC receptor by secreted ADP causes positive feedback on platelet secretion through a PI-3 kinase pathway.
Expression and Anticoagulant Function of the Endothelial Cell Protein C Receptor (EPCR) in Cancer Cell Lines
Authors: Tsuneyoshi Naoko , Horiguchi Shin-ichiro , Ye Xiaofen , Matsuzaki Miwako , Toi Masakazu , Suzuki Koji , Kimoto Masao , Fukudome Kenji
Published: March 2018

Induction of procoagulant factors in malignant cells is considered to be the major cause of coagulation disorders in cancer. Thrombomodulin (TM), a negative regulator of coagulation was also found to be expressed in cancer cells. We report here evidence for another anticoagulant, the endothelial cell protein C receptor (EPCR), in cancer cells. EPCR was detected in several cell lines derived from various types of cancer. Significant levels of protein C (PC) activation were detected only with cell lines expressed both EPCR and TM. Anti-EPCR monoclonal antibodies (mAbs) specifically inhibited the activation. Thus, EPCR function appears to be important for PC activation by cancer cells. In addition, we detected EPCR expression in tumor cells from breast cancer patients, with an extremely high frequency. EPCR function may contribute to progression or pathogenesis of some types of cancer, and may explain the complexity of coagulopathy in cancer patients.
Anticoagulant Dysfunction of Human Arg352Trp-Activated Protein C Caused by Defective Factor Va Inactivation
Authors: Rintelen Claudia , Yegneswaran Subramanian , Griffin John
Published: March 2018

The dysfunctional mutant R352W-protein C was found in two patients with venous thrombosis. The mutant R352A-protein C was constructed to define the contribution of charge/size of the residue at 352 on protein C (chymotrypsin numbering 187). Compared with wild type-protein C, R352W-protein C showed no difference in activation by thrombin·thrombomodulin or α-thrombin. However, R352W-activated protein C (APC) anticoagulant activity (aPTT assay) was reduced to ~65%. Although the catalytic efficiency of R352W-APC towards the oligopeptide substrate S-2366 was unperturbed, factor Va and R506Q-factor Va were not efficiently inactivated by R352W-APC compared with wild type-APC. R352A-APC showed reduced anticoagulant activity and reduced efficiency in factor Va inactivation and in factor VIIIa-inactivation in the presence of protein S. These observations suggest that the dysfunction of R352W-APC in factor Va inactivation may be one of the mechanisms leading to venous thrombosis in affected patients and that R352 plays an important role in the physiological functioning of APC.
Factor VIII Arg2304 → His Binds to Phosphatidylserine and Is a Functional Cofactor in the Factor X-ase Complex
Authors: Lewis Deborah , Moore Karen , Ortel Thomas
Published: March 2018

Four factor VIII light chain constructs containing hemophilia A mutations at R2304 and R2307 were prepared and expressed in mammalian cells. These mutations are located in a putative phosphatidylserine binding site identified by peptide studies (spanning amino acids 2303-2332). The levels of all four mutants in conditioned medium were significantly less than wild type by immunoprecipitation and ELISA. R2304H and wild type factor VIII light chains were concentrated by cation exchange chromatography from medium. R2304H and wild type factor VIII light chains bound immobilized phosphatidylserine similarly. The reconstituted cofactor activity of R2304H factor VIII light chain was slightly greater than wild type factor VIII light chain. These results are consistent with the recently reported crystal structure of factor VIII C2 domain that suggests R2304H is not directly involved in phospholipid binding. The observed clinical phenotype is probably due to decreased circulating levels of a functional protein.
Flow Cytometry Detection of Platelet Procoagulant Activity and Microparticles in Patients with Unstable Angina Treated by Percutaneous Coronary Angioplasty and Stent Implantation
Authors: Vidal Catherine , Spaulding Christian , Picard Françoise , Schaison Frédéric , Melle Josiane , Weber Simon , Fontenay-Roupie Michaëla
Published: March 2018

Platelet activation is known to participate to the pathogenesis of acute coronary syndromes. Aminophospholipid exposure and micro-particles shedding are hallmarks of full platelet activation and may account for the dissemination of prothrombotic seats. Using flow cytometry analysis of annexin V binding to externalized aminophospholipids, we followed platelet procoagulant activity (PPA) and platelet microparticles (PMP) shedding in venous and coronary whole blood samples from 30 patients with unstable angina before and after percutaneous coronary angioplasty (PTCA) and stent implantation. Baseline values of PPA and PMP were significantly more elevated in patients than in control subjects (p <0.005). PMP percentage was significantly higher in coronary than in venous blood, and in coronary blood of patients with proximal instead of mid/distal lesions of coronary arteries. No enhancement of platelet reactivity to TRAP and collagen was induced by procedure. Whereas activated GpIIb-IIIa and P-selectin expression decreased 24 h and 48 h after procedure, PPA and PMP remained as elevated as before. Thus, flow cytometry is a reliable method for detection of fully activated platelets in whole blood samples. Annexin V binding analysis demonstrates the persistance of in vivo platelet activation, despite the use of antiaggregating agents.
Risk of Pregnancy-related Venous Thrombosis in Carriers of Severe Inherited Thrombophilia
Authors: Legnani Cristina , Bucciarelli Paolo , Grandone Elvira , De Stefano Valerio , Mannucci Pier Mannuccio , Martinelli Ida
Published: March 2018

Homozygous carriers of factor V Leiden have an approximately 80-fold increased risk of venous thrombosis. Also double heterozygous carriers of both the factor V Leiden and the prothrombin gene mutations are at high thrombotic risk. The magnitude of the risk of venous thrombosis in pregnant women with the two severe thrombophilic conditions has not been estimated so far. We performed a multicenter retrospective family study in women with homozygous factor V Leiden, double heterozygous factor V Leiden and the prothrombin gene mutation, and women with normal coagulation. Only relatives of index patients with thrombosis formed the study cohort. Fifteen homozygous and 39 double heterozygous women were compared to 182 women with normal coagulation. Venous thrombosis occurred in 3 of 19, 2 of 50 and 1 of 221 pregnancies, respectively. One thrombotic episode occurred in the third trimester, the remaining 5 in the postpartum. The prevalence of venous thrombosis was 15.8% (95% CI 3.4-39.6) for homozygotes, 4.0% (95% CI 0.5-13.7) for double heterozygotes and 0.5% for women with normal coagulation. The relative risk of pregnancy-related venous thrombosis was 41.3 (95% CI 4.1-419.7) for homozygous and 9.2 (95% CI 0.8-103.2) for double heterozygous carriers. In conclusion, homozygous carriers of factor V Leiden and, to a lesser extent, double heterozygous carriers of factor V Leiden and of the prothrombin mutation have an increased risk of venous thrombosis during pregnancy, particularly high during the postpartum period. On the basis of these findings we recommend that these women receive anticoagulant prophylaxis at least in the postpartum, that should perhaps be extended to the whole pregnancy in homozygous carriers.
Assembly of High Molecular Weight Kininogen and Activation of Prekallikrein on Cell Matrix
Authors: Shariat-Madar Z. , Mahdi F. , Sampaio C. A. M. , Schmaier A. H. , Motta G.
Published: March 2018

Investigations determined if extracellular matrix of endothelial cells (EC) is a platform for HK assembly and PK activation. In buffers containing bovine serum albumin, biotin-HK binding to ECV304 cells or their matrix requires ≥ 50 µM added Zn 2+. Ortho-phenanthroline or a HK domain 5 peptide blocks HK binding. Binding to umbilical vein EC or matrix, but not ECV304 cells or matrix, is mediated by cytokeratin 1. Biotin-HK binds to ECV304 cells or matrix with a Kd
of 15.8 or 9.0 nM and a Bmax
of 2.6 107 or 2.4 107 sites/cell, respectively. PK activation on ECV304 cells or matrix is blocked by antipain or SBTI and corn trypsin inhibitor partially inhibits kallikrein formation. PK activation occurs on ECV304 cells or matrix prepared without serum or in human factor XII deficient serum, indicating that the PK activator is not factor XIIa. EC matrix promotes plasminogen activation after the assembly of HK, PK and pro-urokinase. These studies indicate that matrix of various EC has the ability to assemble HK allowing for PK activation and subsequent activities.

Abbreviations: EC: endothelial cells, FXII: factor XII, HK: high molecular weight kininogen, HKa: bradykinin-free HK, PK: plasma prekallikrein, Pro-UK: pro-urokinase, uPAR: urokinase plasminogen activator receptor, tcuPA: twochain urokinase, CK1: cytokeratin 1, SBTI: soybean trypsin inhibitor, HUVEC: human umbilical vein endothelial cell, SDS-PAGE: sodium dodecyl sulfatepolyacrylamide gel electrophoresis, CTI: corn trypsin inhibitor, p-APMSF: p-amidinophenylmethylsulfonylfluoride, EBSS: Earle’s Balanced Salt Solution
Plasmin Proteolysis of Endothelial Cell and Vessel Wall Associated Tissue Factor Pathway Inhibitor
Authors: Stalboerger Paul , Panetta Carmelo , Simari Robert , Caplice Noel
Published: March 2018

Plasmin is an important protease that mediates clot fibrinolysis and vessel wall extracellular matrix proteolysis. Recently, in vitro studies have suggested that plasmin can cleave and inactivate recombinant TFPI, a major inhibitor of TF-mediated coagulation. We hypothesized that such an interaction may occur in vascular cells expressing TFPI, or in the vessel wall, with implications for thrombolysis. In a series of experiments, we examined the effects of plasmin on cell surface and extracellular matrix (ECM) associated TFPI in endothelial cells (EC) in culture and on EC and smooth muscle cells (SMC) in the vessel wall. Plasmin (0.2 μM) decreased cell surface and matrix associated TFPI activity in cultured endothelial cells by 77 ± 5 % and 69 ± 6% respectively (p < 0.01). Plasminogen, the proenzyme form of plasmin had no such effect on cell surface TFPI or matrix TFPI. Cell surface TFPI antigen measured by fluorescence activated cell sorter (FACS) was also significantly reduced by plasmin. Proteolysis of conditioned medium TFPI was suggested by loss of a ~45kD TFPI on Western Blot analysis following plasmin treatment. Plasmin also proteolysed a ~45kD TFPI protein in the intact ECM of EC, an effect which was inhibited by preincubation with aprotinin, a plasmin inhibitor. Incubation of similar concentrations of plasmin, with homogenates of normal vessel decreased a ~45kD TFPI immunoreactive band on Western blot analysis. Plasmin also decreased surface TFPI activity on frozen sections of normal vessel as measured by an amidolytic assay. Finally, plasmin treatment of atherosclerotic plaque sections caused complete removal of TFPI immunoreactivity associated with luminal EC and intimal SMC, when compared to control treated plaque (n = 3). Together these data suggest that plasmin proteolyses the majority of EC-associated (surface and matrix) TFPI and may remove TFPI from the luminal surface and intima of the vessel wall. TFPI proteolysis in cultured EC was associated with significant reduction in TFPI anticoagulant activity. These data provide evidence that plasmin degradation of TFPI occurs in vascular cells and in the vessel wall and may have implications for rethrombosis following thrombolysis in vivo.
Transfer, Transition und kontinuierliche Erwachsenenbetreuung von Patienten mit Phenylketonurie (PKU)
Authors: Mütze U. , Rohde C. , Arelin M. , Kirmse S. , Ceglarek U. , Baerwald C. , Beblo S. , Thiele A. G.
Published: March 2018

Durch moderne Screeningmethoden, verbesserte Diagnostik und neue Therapieoptionen steigt die Zahl der langfristig zu betreuenden Patienten mit seltenen angeborenen Stoffwechselerkrankungen stetig an. Im Jahr 2005 wurde am Universitätsklinikum Leipzig eine der ersten deutschen Spezialambulanzen für diese Patienten gegründet und ein Transitionsprogramm etabliert. Vier und zehn Jahre später wurde retrospektiv die Langzeitbetreuung am Beispiel von Patienten mit Phenylketonurie (PKU) im Hinblick auf die metabolische Einstellung, Transitions-, Therapie- und Lebenszufriedenheit und den sozioökonomischen Status untersucht. 96 PKU-Patienten (56 w/40 m) wurden bis 2015 transferiert. 81 % wurden zuletzt noch betreut. Die metabolische Einstellung lag stets im angestrebten Bereich. Über 80 % der Patienten waren mit der Transition und Erwachsenenbetreuung zufrieden. Bei Lebensqualität und gesamtsozioökonomischen Daten gab es keine Unterschiede zur Kontrollpopulation. Unter enger multidisziplinärer Zusammenarbeit der erfahrenen Mitarbeiter aus Pädiatrie und Erwachsenenmedizin gelang eine erfolgreiche Transition bei den meisten Patienten. Zur Vermeidung eines Therapieabbruchs ist die weitere Etablierung spezialisierter Ambulanzen für angeborene Stoffwechselerkrankungen für Kinder und insbesondere Erwachsene dringend notwendig.
Franz Anton Mesmer (1734–1815) als Pionier der Psychotherapie
Authors: Boerner R. J.
Published: March 2018

Mesmer bzw. der „Mesmerismus“ haben über Jahrzehnte wichtige Denkanstöße in Medizin, Wissenschaft und Kultur gegeben, die bis heute nachwirken. Dessen Bedeutung für die Psychotherapie wurde jedoch nur partiell zur Kenntnis genommen. Es wird gezeigt, dass durch den „Mesmerismus“ mit dessen geisteswissenschaftlicher Position in der Dialektik von Natur und Geist, mit der Hervorhebung des besonderen Stellenwerts der Persönlichkeit des Therapeuten, der Therapiemethoden mit Suggestion, Meditation und hypnoseähnlichen Interventionen zentrale Themen und Richtungen in der heutigen Psychotherapie (-debatte) angestoßen wurden.
Suchtbehandlung bei komorbid psychischen Krankheiten
Authors: Soyka M. , Aichmüller C.
Published: March 2018

Das Auftreten von komorbiden psychischen Störungen bei Suchterkrankungen ist häufig, dabei sind die Verläufe im Vergleich zu Patienten, die nur eine Diagnose aufweisen, schlechter. Eine effektive Suchttherapie ist nur im Rahmen der Mitbehandlung der psychischen Komorbidität möglich. Integrierte Behandlungskonzepte scheinen dabei von Vorteil zu sein. Die dafür erforderlichen pharmakologischen und psychosozialen Interventionen sind bei den jeweiligen psychischen und Suchterkrankungen teilweise gut untersucht. Die Datenlage bei komorbiden Suchterkrankungen ist kaum evidenzbasiert. Dies liegt unter anderem an der Heterogenität der Studien, aber auch jedoch an der Vielzahl möglicher Komorbiditätsformen. Anhand einer Kasuistik wird das therapeutische Procedere und das Behandlungsangebot der Spezialstation für Abhängigkeitserkrankungen mit komorbiden Störungen der Privatklinik Meiringen dargestellt.
Fluorescence and Cerenkov luminescence imaging
Authors: Schwenck J. , Fuchs K. , Eilenberger S. H. L. , Rolle A.-M. , Castaneda Vega S. , Thaiss W. M. , Maier F. C.
Published: March 2018

This review addresses small animal optical imaging (OI) applications in diverse fields of basic research. In the past, OI has proven to be cost- and time-effective, allows real-time imaging as well as high-throughput analysis and does not imply the usage of ionizing radiation (with the exception of Cerenkov imaging applications). Therefore, this technique is widely spread – not only geographically, but also among very different fields of basic research – and is represented by a large body of publications. Originally used in oncology research, OI is nowadays emerging in further areas like inflammation and infectious disease as well as neurology. Besides fluorescent probe-based contrast, the feasibility of Cerenkov luminescence imaging (CLI) has been recently shown in small animals and thus represents a new route for future applications.

Thus, this review will focus on examples for OI applications in inflammation, infectious disease, cell tracking as well as neurology, and provides an overview over CLI.
Optical imaging probes and their potential contribution to radiotracer development
Authors: Hermann S. , Schäfers M. , Höltke C. , Faust A.
Published: March 2018

Optical imaging has long been considered a method for histological or microscopic investigations. Over the last 15 years, however, this method was applied for preclinical molecular imaging and, just recently, was also able to show its principal potential for clinical applications (e.g. fluorescence-guided surgery). Reviewing the development and preclinical evaluation of new fluorescent dyes and target-specific dye conjugates, these often show characteristic patterns of their routes of excretion and biodistribution, which could also be interesting for the development and optimization of radiopharmaceuticals. Especially ionic charges show a great influence on biodistribution and netcharge and charge-distribution on a conjugate often determines unspecific binding or background signals in liver, kidney or intestine, and other organs.

Learning from fluorescent probe behaviour in vivo and translating this knowledge to radio-pharmaceuticals might be useful to further optimize emerging and existing radiopharmaceuticals with respect to their biodistribution and thereby availability for binding to their targets.
Endothelial Perturbation in Cystic Fibrosis
Authors: Romano Mario , Collura Mirella , Iapichino Luciana , Pardo Francesca , Falco Angela , Chiesa Pierluigi Lelli , Caimi Gregorio , Davì Giovanni
Published: March 2018

Cystic fibrosis (CF) is characterized by a persistent inflammatory state, which can be secondary to chronic pulmonary infection and may affect vascular endothelium. We measured circulating levels of von Willebrand factor (vWF), tissue-plasminogen activator (t-PA), and P-selectin in 20 CF patients and 20 healthy subjects. vWF, t-PA and P-selectin levels were significantly higher in CF patients. Endothelial perturbation (> 2 SD increase in both vWF and t-PA) was present in 65% of CF patients. These patients displayed lower FEV1 values compared to individuals without endothelial perturbation and an inverse correlation between FEV1 and P-selectin levels was observed. Tumor necrosis factor- (TNF-) and interleukin (IL)-6 levels were also increased in CF patients and significant direct correlations were found between TNF- and vWF, t-PA or P-selectin levels. These results indicate that CF patients exhibit signs of endothelial dysfunction/perturbation, which are likely to be related to a persistent inflammatory state due to chronic pulmonary infection, and may play a role in the progression of this disease.
Compound Heterozygosity of the GPIbα Gene Associated with Bernard-Soulier Syndrome
Authors: Larrucea Susana , Pastor Angela , Butta Nora , Arias-Salgado Elena , Ayuso Matilde , Parrilla Roberto , González-Manchón Consuelo
Published: March 2018

We report the molecular genetic analysis of the Bernard-Soulier syndrome (BSS) phenotype in two related patients showing absence of glycoprotein (GP) Ibα and detectable amounts of GPIX on the platelet surface, and a truncated form of GPIbα in solubilized platelets and plasma. They both were compound heterozygotes for the GPIbα gene: a maternal allele with a T insertion at position 1418 causing a translational frameshift and premature polypeptide termination, and a paternal allele with a T715A substitution changing Cys209 to Ser. Heterozygotes for either one of these mutations were asymptomatic. Transient transfection of cells coexpressing GPIbβ and GPIX failed to detect surface expression of the GPIbα mutants. Cells transfected with [1418insT]GPIbα-cDNA showed a truncated protein of the predicted size in both cell lysate and conditioned medium, indicating the inability of the mutant protein to anchor the plasma membrane. In contrast, transfection of [T715A]GPIbα-cDNA yield a mutated protein barely detectable in the cell lysate and absent in the medium, indicating that the loss of Cys209 renders GPIbα more vulnerable to proteolysis and unable to undergo the normal secretory pathway. Our findings indicate that the additive effects of both mutations are responsible for the BSS phenotype of the patients.
Characterization and Structural Impact of Five Novel PROS1 Mutations in Eleven Protein S-deficient Families
Authors: Andersen Bente Damm , Bisgaard Marie Luise , Lind Bent , Philips Malou , Villoutreix Bruno , Thorsen Sixtus
Published: March 2018

Heterozygozity for four novel missense mutations (W108C, W342R, E349K and L485S) and one novel 4 bp deletion (ACdelAAAG affecting codons 632-633) was identified in PROS1 of unrelated thrombosis prone Danish families with protein S type I or III deficiency. The 4 bp deletion results in a frameshift leading to replacement of the coding sequence for the 3 C-terminal amino acids by an abnormal extended sequence that codes for 9 amino acids. The E349K substitution was found in 7 families. Haplotype analysis using 7 microsatellite markers flanking PROS1 was consistent with a common founder for this mutation. The mutations reported here are most likely the cause of the protein S deficiency. Firstly, the four missense mutations cosegre-gate with the abnormal plasma protein S phenotype and lead to the loss of highly conserved amino acids. Secondly, computer analysis of structural models of protein S predicts that the substitutions could affect proper protein folding and/or stability. Analysis of platelet mRNA from subjects with the W108C, E349K, L485S mutation or the 4 bp deletion showed that mutated mRNA was expressed in significant amounts suggesting that mutated molecules are synthesized. Our results are compatible with defective protein folding/unstable molecules, impaired secretion and intracellular degradation of mutated protein, which appear to be the major molecular disease mechanisms for missense mutations and certain other mutations found in genetic disorders.