bluuebunny/crossref_metadata_2025 huggingface dataset:
A Cross-over Pharmacokinetic Study of a Double Viral Inactivated Factor IX Concentrate (15 nm Filtration and SD) Compared to a SD Factor IX Concentrate
Authors: Peynet Jocelyne , Chambost Hervé , Négrier Claude , Briquel Marie Elisabeth , Claeyssens Ségolène , Derlon-Borel Annie , Guérois Claude , Caron Claudine , Scherrmann Jean Michel , Debray Marcel , Bridey Françoise , Goudemand Jenny
Published: March 2018

A double blind randomized cross-over multi-center study has been conducted to compare the pharmacokinetic and coagulation activation markers of high-purity factor IX concentrate subjected to both solvent/ detergent (SD) treatment and 15 nm-filtration (FIX-SD-15) with the licensed product subjected only to solvent-detergent (FIX-SD). This filtration process allows the elimination of small particles, such as non-enveloped viruses (i.e., hepatitis A and parvovirus B19). Eleven severe hemophilia B patients (FIX coagulant activity <2 IU/dl) received one infusion of 60 IU/kg of FIX-SD and one infusion of 60 IU/kg of FIX-SD-15 at least at 10 days interval. Blood samples were obtained before and at various time up to 72 h after infusion. The decay curves of factor IX (FIX:C and FIX:Ag) were evaluated by a model independent method. Bioequivalence was found between the two concentrates using the Schuirmann test. The mean FIX:C and FIX:Ag recovery of FIX-SD-15 was 1.08 and 0.89 IU/dl/IU/kg respectively with a mean half-life of 33.3 h for FIX:C and 25.6 h for FIX:Ag. Six months after initial enrollment, pharmacokinetic parameters were similar in the 7 patients tested. There was no significant variation of prothrombin fragment 1+2 and thrombin-antithrombin complexes measured up to 6 h after infusion, indicating that there was no activation process after administration of FIX. In conclusion, these data demonstrate that the introduction of a 15 nm filtration does not alter the pharmacokinetic profile of a well characterized SD FIX concentrate while providing additional viral safety.
Inhibition of Lupus Anticoagulant Activity by Hexagonal Phase Phosphatidylethanolamine in the Presence of Prothrombin
Authors: Tannenbaum Marion , Neville Carolyn , Fortin Paul , Rauch Joyce
Published: March 2018

We have previously demonstrated that lupus anticoagulant antibodies from patients with systemic lupus erythematosus (SLE) specifically recognize hexagonal (II) phase phosphatidylethanolamine (PE), but not bilayer PE (Thromb Haemost 1989; 62: 892). In those studies, the involvement of proteins in this recognition was not evaluated. To address this issue, we have isolated IgG lupus anticoagulant antibodies from the plasma of SLE patients and evaluated the inhibition of lupus anticoagulant activity by hexagonal (II) phase PE in the presence and absence of purified plasma proteins. All six of the IgG lupus anticoagulant antibodies tested were inhibited by hexagonal (II) phase PE in the presence, but not the absence, of human prothrombin. In contrast, little or no inhibition was observed with prothrombin alone or with PE in combination with either β2-glycoprotein I or annexin V. These data indicate that, for certain lupus anticoagulant antibodies, inhibition by hexagonal (II) phase PE is dependent on prothrombin, suggesting that these antibodies recognize a complex of PE and prothrombin.
Activated Platelets Induce Tissue Factor Expression on Human Umbilical Vein Endothelial Cells by Ligation of CD40
Authors: Kalbas Matthias , Willuweit Antje , Henn Volker , Kroczek Richard , Müller-Berghaus Gert , Slupsky Joseph
Published: March 2018

CD40 is a type I member of the tumour necrosis factor (TNF) receptor superfamily of proteins, and is present on a wide variety of cells including vascular endothelial cells. Ligation of this receptor on endothelial cells is known to increase expression of inflammatory adhesion molecules. We have recently demonstrated that platelets express the ligand of CD40 (CD154) within seconds of exposure to agonist, and interact with endothelial cells to participate directly in the induction of an inflammatory response. Here we show that activated platelets induce tissue factor (TF) expression on endothelial cells in a CD40/CD154-dependent manner, and that the magnitude of this response can equal that induced by TNFα. Moreover, CD40 ligation on endothelial cells downregulates the expression of thrombomodulin. We also show that CD40-mediated TF expression is less sensitive to inhibition with the oxidative radical scavenger pyrrolidine dithiocarbamate than is that mediated by TNFα, indicating that CD40 has a distinct signalling pathway. Tissue factor is a cell membrane protein which functions as the main trigger of the extrinsic pathway of blood coagulation, and its expression on endothelial cells is implicated in wound healing and angiogenesis. Since platelets are among the first cells involved in haemostasis following tissue injury, our data showing that ligation of CD40 by CD154 induces a procoagulant phenotype on vascular endothelial cells suggests that platelets may play an important role in the induction of wound healing.
Effects of Oral and Transdermal Estrogen Replacement Therapy on Markers of Coagulation, Fibrinolysis, Inflammation and Serum Lipids and Lipoproteins in Postmenopausal Women
Authors: Vehkavaara Satu , Silveira Angela , Hakala-Ala-Pietilä Tiina , Virkamäki Antti , Hovatta Outi , Hamsten Anders , Taskinen Marja-Riitta , Yki-Järvinen Hannele
Published: March 2018

We compared the effects of oral estradiol (2 mg), transdermal estradiol (50 g), and placebo on measures of coagulation, fibrinolysis, inflammation and serum lipids and lipoproteins in 27 postmenopausal women at baseline and after 2 and 12 weeks of treatment. Oral and transdermal estradiol induced similar increases in serum free estradiol concentrations. Oral therapy increased the plasma concentrations of factor VII antigen (FVIIag) and activated factor VII (FVIIa), and the plasma concentration of the prothrombin activation marker prothrombin fragment 1+2 (F1+2). Oral but not transdermal estradiol therapy significantly lowered plasma plasminogen activator inhibitor-1 (PAI-1) antigen and tissue-type plasminogen activator (tPA) antigen concentrations and PAI-1 activity, and increased D-dimer concentrations, suggesting increased fibrinolysis. The concentration of soluble Eselectin decreased and serum C-reactive protein (CRP) increased significantly in the oral but not in the transdermal or placebo groups. In the oral but not in the transdermal or placebo estradiol groups low-density-lipoprotein (LDL) cholesterol, apolipoprotein B and lipoprotein (a) concentrations decreased while high-density-lipoprotein (HDL) cholesterol, apolipoprotein AI and apolipoprotein AII concentrations increased significantly. LDL particle size remained unchanged. In summary, oral estradiol increased markers of fibrinolytic activity, decreased serum soluble E-selectin levels and induced potentially antiatherogenic changes in lipids and lipoproteins. In contrast to these beneficial effects, oral estradiol changed markers of coagulation towards hypercoagulability, and increased serum CRP concentrations. Transdermal estradiol or placebo had no effects on any of these parameters. These data demonstrate that oral estradiol does not have uniformly beneficial effects on cardiovascular risk markers and that the oral route of estradiol administration rather than the circulating free estradiol concentration is critical for any changes to be observed.
Platelet Glycoprotein Receptor IIIa Polymorphism PlA1/PlA2
and Coronary Risk: a Meta-Analysis
Authors: Castelnuovo Augusto Di , Gaetano Giovanni de , Donati Maria Benedetta , Iacoviello Licia
Published: March 2018

Membrane glycoprotein IIb/IIIa plays a major role in platelet function. The gene encoding the glycoprotein IIIa shows a common polymorphism PlA1/PlA2
that was variably associated with vascular disease. To clarify the role of PlA1/PlA2
polymorphism in coronary risk, a meta-analysis of published data was conducted. Studies were identified both by MEDLINE searches, and hand searching of journals and abstract books.

A total of 34 studies for coronary artery disease (CAD), and 6 for restenosis after revascularization were identified, for a total of 9,095 cases and 12,508 controls. In CAD, the overall odds ratio for carriers of the PlA2
allele was 1.10 (95% CI: 1.03 to 1.18), and it was 1.21 (95% CI: 1.05 to 1.38) in subjects younger than 60. Overall odds ratio was 1.31 (95% CI: 1.10 to 1.56) after revascularization procedures.

The association of PlA2
status with overall cardiovascular disease in the general population is significant but weak; higher risk has been identified in less heterogeneous subgroups as in the younger cohorts and in the restenosis subset with stents.
Protein Z Circulates in Plasma in a Complex with Protein Z-Dependent Protease Inhibitor
Authors: Tabatabai Amir , Fiehler Ryan , Broze George
Published: March 2018

Protein Z (PZ) is a vitamin K-dependent plasma protein that forms a Ca++-dependent complex with factor Xa at phospholipid surfaces. This interaction between PZ and factor Xa enhances by >1000-fold the inhibition of factor Xa by the serpin called protein Z-dependent protease inhibitor (ZPI). These experiments show that PZ also binds ZPI in a process that does not require Ca++ or phospholipids. In pooled normal plasma, which contains excess ZPI relative to PZ, all the PZ appears to be bound in a complex with ZPI. The binding of PZ to ZPI reduces the rate and extent of factor XIa inhibition produced by ZPI. During the course of these studies, it was noted that a PZ purification procedure, that included NaSCN (2.0 M) elution of PZ from an immunoaffinity column, produced aggregated, inactive forms of PZ.
Studies on the Binding of 3H-SR121566, an Inhibitor of Gp IIb-IIIa Activation
Authors: Savi P. , Zamboni G. , Rescanières O. , Herbert J. M.
Published: March 2018

SR121566 is a new synthetic agent which inhibits the binding of fibrinogen to activated platelets, and platelet aggregation. 3H-SR121566 bound with nanomolar affinity (KD ranging from 45 to 72 nM) to Gp IIb-IIIa expressing cells only. On activated human platelets, this ligand allowed the detection of a maximal number of 100-140,000 binding sites. The binding of SR121566 to platelets, was displaced by several agents including RGD-containing peptides and synthetic RGD mimetics, but not by ReoPro®, a humanised monoclonal antibody which inhibits the binding of fibrinogen to the Gp IIb-IIIa complex. Neither the fibrinogen dodecapeptide nor fibrinogen itself were able to compete with SR121566 whether platelets were activated or not.

Flow cytometry studies indicated that SR121566 which did not activate Gp IIb-IIIa by itself, dose-dependently prevented the detection of activation-induced binding sites on TRAP-stimulated platelets in the presence or absence of exogenous fibrinogen, indicating a direct effect on the activation state of the Gp IIb-IIIa complex. Moreover, SR121566 was able to reverse the activation of Gp IIb-IIIa and to displace the binding of fibrinogen when added up to 5 min after TRAP stimulation of platelets. When added at later times (15 to 30 min), SR121566 failed to displace fibrinogen binding, even if SR121566 binding sites were still accessible and the Gp IIb-IIIa complex not activated.

In conclusion, our study is in accordance with the finding that fibrinogen is recognised by the activated Gp IIb-IIIa complex through the dodecapeptide sequence present on its gamma chain, and that this interaction is inhibited by SR121566 by preventing and reversing the activated conformation of Gp IIb-IIIa and not by direct competition with fibrinogen.
Evidence from Site-directed Mutagenesis that the Cytoplasmic Domain of the β3 Subunit Influences the Conformational State of the αvβ3 Integrin Ectodomain
Authors: Schaffner-Reckinger Elisabeth , Brons Nicolaas , Kieffer Nelly
Published: March 2018

In order to explore the mechanisms leading to conformational changes of the vitronectin receptor αvβ3 following ligand or divalent cation binding, we have investigated the expression of epitopes known as ligand-induced binding sites (LIBS) on 3 cytoplasmic tail mutants expressed in CHO cells. Truncation of the entire 3 cytoplasmic domain induced constitutive LIBS exposure on αvβ3 and IIb β3. Deletion of the C-terminal NITY759 sequence or disruption of the NPLY747 motif by a Y747A substitution impaired extracellular conformational changes on αvβ3 following RGDS, echistatin or Mn2+ binding, whereas the substitutions Y747F, Y759A or Y759F allowed normal LIBS exposure. Furthermore, metabolic energy depletion totally prevented Mn2+-dependent LIBS exposure, but had only a minor effect on RGDS-induced conformational changes. Our results demonstrate that the structural integrity of the NPLY747 motif in the β3 cytoplasmic domain, rather than potential phosphorylation of Tyr747 or Tyr759, is a prerequisite for conformational changes within the αvβ3 ectodo-main, and suggest that two different mechanisms are responsible for RGDS- and Mn2+-dependent conformational changes.
Influence of Contact System Deficiencies during Cardiopulmonary Bypass
Authors: Cugno Massimo , Agostini Angelo
Published: March 2018

As a result of the expanding indications for cardiopulmonary bypass (CPB) there is a growing number of patients undergoing this procedure who are at high surgical risk, and increasing efforts to investigate the mechanisms involved in CPB complications are warranted. Blood contact with foreign surfaces triggers systemic inflammatory response and may induce coagulation (1), this is believed to be responsible of most of the unwanted effects associated with CPB. During this procedure, contact phase activation occurs (2), involving factor XII (FXII), prekallikrein (PK) and high molecular weight kininogen (HK). FXII binds to negatively charged surfaces undergoing autoactivation with cleavage of Arg353-Val354 to form a two-chain molecule of 80 kD, i.e. activated FXII (FXIIa, also known as FXIIa). FXIIa can cleave PK to kallikrein which in turn cleaves FXII to produce FXIIa and a factor XII fragment of 28-30 kD (FXIIf, also known as FXIIa) and cleaves HK to HKa generating the nonapeptide bradykinin. FXIIa can cleave both FXI and PK to active enzymes, while FXIIf (which cannot bind to surfaces) cleaves PK (2) and the first component of complement (C1) (3). These mechanisms of activation differ from those initiating the coagulation cascade during in vivo haemostasis through the tissue factor pathway as a result of tissue injury (4).
Plasma Tumor Necrosis Factor α Levels and the –238* A Promoter Polymorphism in Patients with Antiphospholipid Syndrome
Authors: Bertolaccini M. L. , Lanchbury J. S. , Caliz A. R. , Katsumata K. , Vaughan R. W. , Kondeatis E. , Khamashta M. A. , Koike T. , Hughes G. R. V. , Atsumi T.
Published: March 2018

Objectives. To explore the possible involvement of the proinflammatory and prothrombotic cytokine TNFα in APS by determining the plasma levels in patients and to test for association of TNFA promoter polymorphisms and HLA class II genotypes with both plasma TNF and disease. Patients and Method. We studied 83 Caucasoid patients with APS and two groups of healthy controls. TNFα levels were determined in plasma from 35 patients’ and 21 controls using a highly sensitive sandwich ELISA. The full patient group was genotyped together with 95 ethnically matched healthy controls. -308 and -238 TNFA promoter polymorphisms were assessed by ARMS-PCR. HLA-DQB1, DQA1 and DRB1 genotypes were determined by PCR using sequence specific primers. Results. TNFα levels were significantly higher in patients with APS than healthy controls (median 2.95 pg/ml [range 0.51-10.75] vs. 0.95 pg/ml [0.51-1.6], respectively; p = 0.0001). Frequencies of TNFA-308*2 genotype did not differ between patients and controls. In contrast, TNFA-238*A positive genotype was more frequent in APS patients with arterial thrombosis and pregnancy loss than in controls (OR 3.7 [95% CI 1.37-10.1], p = 0.007 and OR 3.95 [95% CI 1.3-11.7], p = 0.01; respectively). DQB1*0303-DRB1*0701 haplotype was associated with TNFA-238*A in the control group (OR 96.0 [95% CI 9.6-959], p 0.0001) as well as in APS patient’s group (OR 54.2 [95% CI 9.6-306.5], p 0.0001). Conclusions. Raised plasma TNFα levels were found in patients with APS. As a prothrombotic and proinflammatory cytokine, TNFα may be involved in the development of clinical features of APS. The lack of correlation between the TNFA-238 polymorphism and plasma levels associated with disease suggests that the TNFα genetic marker may only indirectly relate to protein levels by virtue of allelic association with a functional marker which may reside in the HLA class II region.
An Update of the Grützbalgϕ Hypothesis: The Role of Thrombosis and Coagulation in Atherosclerotic Progression
Authors: Yee Karen , Ikari Yuji , Schwartz Stephen
Published: March 2018

While a great deal has been learned about the initiation of atherosclerosis, much less is known about the factors that ultimately determine progression and clinical manifestations. In this review we will focus on the possible role of coagulation, especially intramural fibrin in lesion progression.
Time and Dose Dependent Augmentation of Inhibitory Effects of Abciximab by Aspirin
Authors: Baumann Patricia , Holmes Michael , Taatjes Douglas , Sobel Burton , Schneider David
Published: March 2018

Aspirin and abciximab independently decrease the incidence of cardiac events. To identify potential interactions, antiplatelet effects of abciximab were characterized in blood from healthy subjects given aspirin. Platelet activation was determined in whole blood with and without abciximab (2 μg/ml) added in vitro. Flow cytometry was used to quantify fibrinogen binding (glycoprotein IIb-IIIa activation). Binding of fluorochrome-labeled and 125I-labeled abciximab was determined before and after exposure to aspirin. In blood from subjects given aspirin for 5 days, abciximab-induced inhibition of the capacity to bind fibrinogen in response to 1 μM ADP was greater when the daily dose had been 325 mg compared with 81 mg (% inhibition: no aspirin 53 ± 6; 81 mg daily 62 ± 5; 325 mg daily 69 ± 6). The effect of 5 daily doses of aspirin was greater than that of one. Larger single doses elicited larger effects (% inhibition 2 h after 325 mg 59 ± 6; 2 h after 650 mg 78 ± 5). Neither salicylsalicylic acid nor naproxen sodium potentiated the effect of abciximab. Exposure of platelets to 14Cacetylsalicylic acid led to acetylation of glycoprotein IIb and IIIa. Binding of 125I-abciximab to platelets was increased after 30 and 60 min. Acetylation of glycoprotein IIb-IIIa by aspirin augments inhibitory effects of abciximab in a dose- and time-dependent manner by increasing binding of abciximab to platelets.
F X Nottingham and F X Taunton Two Novel Mutations in Factor X Resulting in Loss of Functional Activity and an Interpretation Using Molecular Modelling
Authors: Srinivasan N. , Westby J. , Horn E.H. , Dolan G. , Deam S.
Published: March 2018

We report two novel mutations in the Factor X gene which result in a bleeding tendency in two unrelated Caucasian families. Although the mutations occur at adjacent codons in exon 8 and result in reduced functional activity with normal antigen levels, the patterns of inheritance appear to be quite distinct. Factor X Nottingham (alanine 404 threonine) appears to be associated with an autosomal recessive pattern of inheritance. In contrast, Factor X Taunton (arginine 405 glycine) results in a mode of inheritance consistent with an autosomal dominant pattern, all five of the heterozygotes in this family being clinically affected. Molecular modelling studies suggest that, in the case of Factor X Nottingham, a drastic conformational change causes major unfolding of the protein. For Factor X Taunton, less extreme conformational changes occur causing loss of functional activity such that substrate binding sites might be maintained. It is proposed that competition with wild type for substrate binding could occur leading to a dominant negative effect.
Factor V Arg306 → Gly Mutation Is not Associated with Activated Protein C Resistance and Is rare in Taiwanese Chinese
Authors: Lin Jen-Shiou , Tsay Woei , Shen Ming-Ching
Published: March 2018

Polymerase chain reaction amplification followed by BstOI enzyme digestion and DNA sequencing was employed to detect the mutation of factor V gene. The subjects consisted of 105 venous thrombophilic patients and 183 healthy controls. Only one patient was found to have factor V Arg306 → Gly mutation, his elder son also had an identical mutation. None of the healthy subjects studied had Arg306 → Thr mutation. The rare event of factor V Arg306 → Gly mutation in patients and controls suggest that this mutation is not associated with increased risk of venous thrombosis. Conventional, modified and extended activated protein C (APC) resistance assays in this patient and his family members clearly showed that factor V Arg306 → Gly mutation is not associated with APC resistance (APC sensitivity ratio <2). In conclusion, factor V Arg306 → Gly mutation is rare in Taiwanese Chinese and not associated with APC resistance, it is possibly not a risk factor for venous thrombophilic thrombosis.
Lack of Association between the Platelet Glycoprotein Ia C807T Gene Polymorphism and Myocardial Infarction in Japanese
Authors: Kurihara Hiroki , Imai Yasushi , Sugiyama Takao , Hamada Chikuma , Sakai Eiichi , Mori Mitsuko , Nagai Ryozo , Morita Hiroyuki
Published: March 2018

The platelet-collagen receptor, glycoprotein Ia/IIa (integrin α2β1) plays a fundamental role in the adhesion of platelets to fibrillar collagen, an event leading to platelet activation and thrombus formation and contributing to the pathogenesis of thrombotic disease. Further, glyco-protein Ia/IIa receptor density and function may be associated with two linked and silent polymorphisms (807C/T and 873G/A) within the glyco-protein Ia gene. We tested the extent to which these polymorphisms serve as genetic markers of myocardial infarction in a Japanese population. A case-control study was carried out using 210 Japanese myocar-dial infarction patients and 420 age- and sex-matched controls. Geno-typing was accomplished using PCR followed by melting curve analysis with specific fluorescent hybridization probes. The 807CC, CT, TT genotypes linked perfectly to the 873GG, GA, AA genotypes, respectively. Allele frequencies of the 807T (873A) variant were similar in the control and patient groups (0.373 vs. 0.352). The 807T and 873A variants of platelet glycoprotein Ia gene are common and in a perfect linkage in the Japanese population, but it appears unlikely that the 807T (873A) variant represents a useful marker of increased risk for myocardial infarction.
Prothrombotic Changes in Hemostatic Parameters and C-reactive Protein in the Elderly with Winter Acute Respiratory Tract Infections
Authors: Horan John , Francis Charles , Falsey Ann , Kolassa John , Smith Brian , Hall William
Published: March 2018

Mortality rates attributable to cerebrovascular and ischemic heart disease increase among older adults during the winter. Prothrombotic changes in the hemostatic system related to seasonal factors, such as ambient temperature changes, and winter acute respiratory tract infections, may contribute to this excess seasonal mortality. A prospective nested case-control study was conducted to assess the impact of winter acute respiratory tract infections on fibrinogen, factor VII, factor VIIa, D-dimer, prothrombin fragment 1.2, PAI-1, soluble P-selectin and C-reactive protein (CRP) in older adults. The change in laboratory parameters from baseline (fall) to the time of infection in both middle-aged and elderly individuals was compared with matched non-infected controls. In older adult participants with winter acute respiratory tract infections, significant increases occurred in fibrinogen and C-reactive protein, but not in any other markers. The mean fibrinogen increased 1.52 g/L (38%) and the mean CRP increased 37 mg/L (370%) over baseline (both p < 0.001). In a multivariate analysis, both infection and season were associated with the increase in fibrinogen, but only infection was associated with the CRP increase. Old age magnified the increase in CRP but not in fibrinogen. Winter acute respiratory tract infections induce an exaggerated inflammatory response in older adults. The associated increase in fibrinogen, an independent risk factor for ischemic heart disease, may be partly responsible for the excess winter vascular mortality.
Whole Blood Tissue Factor Procoagulant Activity Remains Detectable during Severe Aplasia following Bone Marrow and Peripheral Blood Stem Cell Transplantation
Authors: Ozcan Muhit , Morton Colleen , Solovey Anna , Dandelet Luke , Bach Ronald , Hebbel Robert , Slungaard Arne , Key Nigel
Published: March 2018

Using a novel whole blood assay, we recently demonstrated that tissue factor procoagulant activity (TF PCA) is present in normal individuals. Preliminary experiments suggested that this activity is localized in the mononuclear cell fraction. Postulating that whole blood TF PCA would therefore be undetectable when monocytes and neutrophils are absent from peripheral blood, we assayed TF PCA during the peri-transplant period in 15 consecutive patients undergoing allogeneic (n = 12) or autologous (n = 3) bone marrow transplantation (BMT) or peripheral blood stem cell transplantation (PBSCT). Baseline (pre-transplant) mean TF PCA was higher in patients compared to normal controls (P <0.005). Unexpectedly, although TF PCA during the period of profound aplasia was significantly reduced compared to baseline (p <0.05), fully 55% of the initial activity remained detectable. During the engraftment phase, TF PCA returned to pre-transplant levels, with a linear correlation between monocyte counts and TF PCA (r = 0.63). In contrast to normal whole blood, incubation of aplastic samples with E. Coli lipopolysaccharide ex vivo failed to induce TF PCA. Throughout the period of study – but especially during the aplastic phase – the absolute number of circulating endothelial cells (CECs) that were TF antigen-positive was increased compared to normals (P <0.001). However, removal of these cells from whole blood samples failed to significantly diminish total TF PCA indicating that CECs alone could not account for the detectable TF PCA during aplasia. We conclude that neither circulating mature myelo-monocytic cells nor endothelial cells can account for all the functionally intact TF in peripheral blood. Further studies are needed to identify the other source(s) of TF PCA.
The TF:VIIa Complex: Clinical Significance, Structure-function Relationships and Its Role in Signaling and Metastasis
Authors: Konigsberg William , Kirchhofer Daniel , Riederer Markus , Nemerson Yale
Published: March 2018

In healthy humans, blood vessel-tissue factor is found primarily in the adventitia and thus physically separated from coagulation factors, which mainly circulate in an inactive form. Following injury, TF is exposed to blood and initiates the coagulation cascade. The resulting fibrin formation is an essential part for the initial repair of vessel damage to minimize blood-loss (reviewed by (1-4). Therefore, TF may be considered to form a hemostatic sheath around blood vessels essential for hemostasis and appears to be essential for life inasmuch as no TF deficiency has been reported and TF knockout mice do not survive beyond the perinatal period.
Increased Platelet Glycoprotein V Levels in Patients with Coronary and Peripheral Atherosclerosis
Authors: Lanza F. , Galajda P. , Gurney D. , Moog S. , Cazenave J. P. , Lip G. Y. H. , Blann A. D.
Published: March 2018

As platelet hyperactivity is important in atherosclerosis and smoking, we hypothesized higher levels of soluble platelet membrane glycoprotein V (gpV) in 95 patients with peripheral artery disease (PAD) and 92 with coronary artery disease (CAD) compared to 99 healthy controls, and examined the effects of aspirin and of smoking two cigarettes on soluble gpV and platelet function. Soluble gpV (ELISA) was significantly raised in, but not between, both PAD and CAD patients, compared to controls (p <0.05). In multivariate analysis, systolic blood pressure, smoking and atherosclerosis (all p <0.01) were significant influences on soluble gpV in the whole study cohort. There was a weak correlation between soluble gpV and another platelet marker, soluble P selectin (p = 0.048). Acute smoking in 14 subjects increased platelet aggregability and beta-thromboglobulin, but not soluble gpV: there were no changes in 11 non-smokers. Five days consumption of aspirin (325 mg daily) by 14 subjects did not influence levels of soluble gpV. Our data indicate that soluble gpV may be a useful new marker of platelet activation in atherosclerosis, but may be influenced by smoking status and blood pressure.
Combined Effect of Factor V Leiden and Prothrombin 20210A on the Risk of Venous Thromboembolism
Authors: Rosendaal Frits , Cattaneo Marco , Margaglione Maurizio , De Stefano Valerio , Cumming Tony , Arruda Valder , Hillarp Andreas , Reny Jean-Luc , Emmerich Joseph ,   the study group for pooled-analysis in venous thromboembolism
Published: March 2018

Factor V Leiden and factor II G20210A mutations are two frequent genetic risk factors involved in venous thromboembolism (VTE). The goal of this pooled analysis of 8 case-control studies, comprising a total of 2310 cases and 3204 controls, was to precisely estimate the risk of VTE in patients bearing both mutations (double heterozygotes). Odds ratios for VTE were 4.9 (95% CI; 4.1-5.9) for the factor V Leiden and 3.8 (3.0-4.9) for the factor II G20210A mutation. Fifty-one cases (2.2%) and none of the controls were double heterozygotes. The odds ratio for venous thrombosis in double heterozygotes was 20.0 (11.1-36.1). Twelve percent of patients heterozygous for factor V Leiden were also heterozygous for factor II G20210A and conversely 23% of patients heterozygous for factor II G20210A were also heterozygous for factor V Leiden. Furthermore, in this large population we analyzed the effect of oral contraceptive (OC) in women carrying one of these mutations. Odds ratio for VTE associated with OC was 2.29 (1.72-3.04). In factor V Leiden carriers using OC, the odds ratio for VTE was 10.25 (5.69-18.45). The odds ratio of the association of factor II mutation and OC use was 7.14 (3.39-15.04). Finally, we also confirmed that the frequency of factor V Leiden was lower in patients with pulmonary embolism than in patients with deep vein thrombosis without PE (odds ratio 0.69). Conversely, factor II G20210A mutation was equally balanced in both patient groups.
Tissue Distribution of Factor VIII Gene Expression In Vivo – A Closer Look
Authors: Hollestelle Martine , Thinnes Terri , Crain Karen , Stiko Ann , Kruijt Johan , van Berkel Theo , Loskutoff David , van Mourik Jan
Published: March 2018

Previous studies have shown that factor VIII (FVIII) is expressed by multiple tissues. However, little is known about its cellular origin or its level of expression in different organs. In the present study, we examined FVIII gene expression in different tissues on a quantitative basis. Most of the tissues, especially liver and kidney, expressed high levels of FVIII mRNA compared to their level of expression of other hemostatic proteins, including von Willebrand factor (VWF). This was unexpected since FVIII is a trace protein. In situ hybridization analysis confirmed that liver and kidney were rich in FVIII mRNA. In the liver, a clear hybridization signal was detected in cells lining the sinusoids. FVIII mRNA analysis of purified liver cells confirmed the expression of FVIII mRNA by sinusoidal endothelial cells and Kupffer cells. Low but significant levels of FVIII mRNA were also detected in the hepatocytes. VWF mRNA was not detectable in these cells. Similarly, immunohistochemical staining of liver tissue revealed that FVIII protein is primarily associated with sinusoidal cells. VWF protein was predominantly located in the endothelium of larger vessels. In the kidney, FVIII synthesis was localized to the glomeruli and to tubular epithelial cells. Taken together, these results suggest that besides hepatocytes, non-parenchymal cells (e.g. sinusoidal endothelial cells) contribute to FVIII synthesis. VWF synthesis is primarily confined to extra-hepatic tissues.
Hemostatic/Fibrinolytic Protein Changes in C3H/HeN Mice Infected with Rickettsia conorii
Authors: Srikanth Sujata , Elghetany Tarek , Normolle Daniel , Gokhale Sumita , Feng Hui-Min , Walker David , Schmaier Alvin
Published: March 2018

Changes in plasma hemostatic and fibrinolytic proteins were determined during courses of a murine model of fatal and non-fatal Rocky Mountain spotted fever. C3H/HeN mice were infected with Rickettsia conorii and coagulation and histopathologic studies were performed at prescribed periods of time. A significant decrease in plasma factor VIII activity and rise in plasma factor V procoagulant activity correlated with a fatal infection. Factor VII levels were unchanged; factor XI levels dropped early in the course in the lethally infected animals, but returned to normal. Factor XII, high molecular weight kininogen, and prekallikrein levels were unchanged by the sublethal infection. Prekallikrein levels fell during the lethal infection. Antithrombin concentrations were decreased significantly in all animals, but plasma plasminogen levels did not change in either group of animals. Nonocclusive thrombi were microscopically observed rarely and only in animals surviving a sublethal infection. A fall in tissue plasminogen activator activity and a rise in plasminogen activator inhibitor activity highly correlated with a lethal outcome. Lethal infection with R. conorii is associated with primary endothelial cell injury resulting in decreased tissue plasminogen activator and increased plasminogen activator inhibitor.
Impaired Platelet Function among Platelet Donors
Authors: Jilma-Stohlawetz Petra , Hergovich Nicole , Homoncik Monika , Dzirlo Larisa , Horvath Michaela , Janisiw Michael , Panzer Simon , Jilma Bernd
Published: March 2018

Background: Platelet transfusions are effective for the prevention and treatment of bleeding in patients with disorders of platelet number and/or function. In recent years plateletpheresis concentrates have outnumbered pooled platelet concentrates, albeit with significant differences between nations. Thus, the platelet quality of individual donors has become increasingly important. The aim of this study was to gain an estimate for the prevalence of impaired platelet function among platelet donors. Study design and methods: We determined the inter-donor variability in platelet plug formation with a PFA-100 analyzer, the prevalence of impaired thromboxane formation, and effects of the density in alpha2 integrin polymorphism and density. Results: (i) Collagen-epinephrine induced closure time (CEPI-CT) showed a great inter-subject variability in platelet donors and was higher than in healthy controls (p = 0.008). One-fifth of donors had abnormal CEPI-CT values and 11% exceeded >300 s (max measurable value). (ii) Decreased serum thromboxane B2 levels were found in 9% of all donors, compatible with surreptitious intake of cyclooxygenase inhibitors or with an aspirin-like defect. CEPI-CT correlated inversely with TxB2-levels in donors and controls. (iii) The density of the alpha2-integrin correlated negatively with CEPI-CT and CADP-CT values in controls, but was not responsible for the observed impaired platelet function in donors. (iv) Finally, the ABO blood group system modulates closure times. Conclusion: In sum, a large number of platelet donors present with prolonged closure times. Decreased thromboxane formation and frequent platelet donation partly account for this observation.

Abbreviations: 807 CC/CT/TT polymorphisms of the alpha2-integrin gene, CD36… GPIIIb, an alternative collagen receptor, CD42b… GpIb, the von Willebrand receptor, CD49b… alpha2-integrin, subunit of the main collagen receptor, CADP-CT… collagen adenosine diphosphate induced closure time, CEPI-CT… collagen/epinephrine induced closure time, PFA-100… platelet function analyzer, vWF-Ag… von Willebrand factor antigen, TXB2… thromboxane B2
Distinct Domains of αIIbβ3 Support Different Aspects of Outside-In Signal Transduction and Platelet Activation Induced by LSARLAF, an αIIbβ3 Interacting Peptide
Authors: Derrick Jerry , Shattil Sanford , Poncz Mortimer , Gruppo Ralph , Gartner Kent
Published: March 2018

The peptide LSARLAF causes αIIbβ3-dependent platelet activation exemplified by secretion, aggregation, spreading and adhesion on fibrinogen, and tyrosine phosphorylation. αIIbβ3-dependent outside-in signal transduction induced by LSARLAF was investigated in variant thrombasthenic platelets which lack most of the cytoplasmic domain of the integrin β3 subunit (αIIbβ3 Δ724). These studies revealed that only certain aspects of this αIIbβ3-dependent outside-in signaling were affected by the β3 truncation. Specifically, αIIbβ3 724 supported LSARLAF-induced platelet aggregation, agglutination and secretion, but failed to trigger cytoskeletal reorganization and platelet spreading on fibrinogen, despite the fact that PMA-induced non αIIbβ3 mediated signaling caused spreading of these platelets on fibrinogen. Thus, distinct domains of αIIbβ3 are required to support different aspects of LSARLAF-induced platelet activation. Furthermore, these studies suggest that not all αIIbβ3-dependent platelet responses require an intact β3 cytoplasmic tail.
The 33-kDa Platelet α-granule Membrane Protein (GMP-33) Is an N-terminal Proteolytic Fragment of Thrombospondin
Authors: Damas Conchi , Vink Tom , Nieuwenhuis Karel , Sixma Jan
Published: March 2018

GMP-33 is a platelet membrane associated protein that is recognised by RUU-SP 1.77, an antibody raised against activated platelets. GMP-33 is predominantly associated with the membrane of platelet α-granules and it is translocated to the plasma membrane upon platelet activation (Metzelaar et al. Blood 1992; 79: 372-9). In this study we have isolated the protein by immunoaffinity chromatography. The N-terminus was sequenced and was identical to the N-terminal sequence of human thrombospondin. The protein was N-glycosylated and bound to heparin as would be expected of the N-terminal part of thrombospondin. RUU-SP 1.77 reacted only with reduced thrombospondin. Plasmin and trypsin digestion of thrombospondin yielded fragments of approximately the same size as GMP 33 that reacted with RUU-SP 1.77 after reduction. No evidence for alternative splicing was found. We postulate that GMP 33 is an N-terminal proteolytic fragment of thrombospondin that is membrane associated.
Segmentale Überwuchssyndrome
Authors: Rössler J.
Published: March 2018

In den letzten Jahren wurden einige segmentale Überwuchssyndrome genetisch weiter aufgeklärt. Es handelt sich um sehr seltene Erkrankungen, deren Phänotypen über viele Jahre nicht eindeutig voneinander abgegrenzt werden konnten. Vom Überwuchs betroffen können das Skelettsystem, das Fett- und Bindegewebe, aber auch ganze Extremitäten und Organe sein. Beim Proteus-Syndrom sind somatische, aktivierende Mutationen im AKT1-Gen gefunden worden. Bei den PROS „PI3K related overgrowth spectrum“-Syndromen wie dem CLOVE-, dem Fibroadipösen Hyperplasie-Syndrom und dem Klippel-Trénaunay-Syndrom wurden Mutationen in der Kinase PI3KCA beschrieben. Auch hier ist die Mutation somatisch, d. h. im betroffenen Gewebe zu finden. Mutationen in der Keimbahn von PTEN führen zu den PTEN-Hamartom-Tumor-Syndromen, wie dem Cowden-, dem Bannayan-Riley-Ruvalcaba(BRR) und dem SOLAMEN-Syndrom. Letztendlich muss die Überprüfung der Mutationen aus dem mTOR-Signalweg eine abschließende Diagnose erbringen. Die Erkenntnisse über die Genetik der segmentalen Überwuchssyndrome bringen Grundlagen für neue therapeutische Ansätze mit mTOR-Inhibitoren, wie z. B. Sirolimus. Aktuell wird eine klinische Studie (SIPA-SOS) in Deutschland hierzu vorbereitet.
Erstversorgung und Reanimation des Neugeborenen
Authors: Dzietko M. , Deindl P. , Sallmon H. , Stein A. , Hansmann G.
Published: March 2018

Hintergrund: Die Wiederbelebung des Neugeborenen ist ein seltenes Ereignis. Deswegen kann es gerade in solchen stressbeladenen Situationen zu Fehleinschätzungen der betreuenden Personen kommen.

Methode: Es erfolgte eine selektive Literaturrecherche in Pubmed und Medline unter Bezug auf nationale und internationale Leitlinien und systematische Übersichtsarbeiten.

Ergebnisse: Es werden der seit 2015 geltende Algorithmus für die kardiopulmonale Reanimation des Neugeborenen und die Veränderungen im Vergleich zu 2010 beschrieben. Darüber hinaus wird dargestellt, welchen Bedarf an Hilfe ein Neugeborenes nach der Geburt hat, welches Equipment zur Versorgung mindestens notwendig ist und wie die Wiederbelebung des Neugeborenen im Detail inklusive Gefäßzugängen und Medikamenten durchgeführt werden sollte.

Schlussfolgerungen: Reanimationen beim Neugeborenen treten sehr selten auf. Aus diesem Grunde sollten der Algorithmus nach aktuellsten Richtlinien wiederholt und praktisches Training in regelmäßigen Abständen durchgeführt werden.
ADHS und Sucht im Erwachsenenalter
Authors: Ridinger M.
Published: March 2018

Gegenstand und Ziel: Übersichtsartikel zur Komorbidität von ADHS und Sucht im Erwachsenenalter. Die Prävalenz des kindlichen ADHS beträgt 5% und ist mit einem erhöhten Suchtrisiko verbunden, was zu ADHS-Prävalenzraten bis zu 23,1% bei erwachsenen Substanzabhängigen führt. ADHS führt ab der Kindheit zu Beeinträchtigungen im Entwicklungsverlauf. Als Haupthypothese für die Suchtentwicklung wird eine fehlgeleitete Selbstmedikation mit psychotropen Substanzen zur Linderung der ADHS-Symptome angenommen. Behandlungsansätze: Bei ADHS sind Stimulanzien Medikamente der ersten Wahl. Bei komorbider Sucht, muss eine individuelle Risiko-Nutzen-Abwägung unter Berücksichtigung von Haltequoten und Wechselwirkungen zwischen ADHS-Medikamenten und Suchtmitteln erfolgen. Achtsamkeitsbasierte Verfahren und Neurofeedbackmethoden gelten als ergänzende Behandlungsformen. Schlussfolgerungen: Eine konsequente Behandlung des ADHS ab der Kindheit gilt als präventiv für die Suchtentwicklung. Die Behandlung der Komorbidität von ADHS und Sucht sollte integrativ unter Einbezug von Pharmakotherapie, Psychoedukation und Verhaltenstrainings erfolgen.
Thrombin-mediated In Vitro Processing of Pro-von Willebrand Factor
Authors: Váradi Katalin , Turecek Peter , Mitterer Artur , Dorner Friedrich , Schwarz Hans Peter
Published: March 2018

Von Willebrand factor (vWF) is synthesized in endothelial cells as pre-provWF and processed intracellularly to propeptide (vWFpp) and mature vWF. Building on previous studies indicating that recombinant provWF when infused into animals can also be processed extracellularly in vivo, we investigated the processing of provWF in vitro. Incubation of a recombinant provWF (rpvWF) preparation with canine and human vWF-deficient plasma induced a time-dependent decrease in provWF antigen and an increase in vWFpp antigen without changing total vWF antigen or collagen-binding activity. Multimer analysis showed the gradual transformation of the provWF multimers to mature vWF multimers and cleaved vWFpp was visualized on autoradiograms of SDS-polyacrylamide electrophoresis gels using
125
I-labeled provWF. Processing was facilitated by CaCl2, but prevented by a thrombin inhibitor and did not occur in prothrombin-depleted plasma. When recombinant provWF was incubated with increasing amounts of purified thrombin, the extent of provWF processing was dose-dependent. The specific cleavage of vWFpp was confirmed by immunoblots using an anti-vWFpp antibody and by amino terminal amino-acid analysis. Binding of provWF to collagen decreased the thrombin concentration necessary for propeptide removal to a concentration in the range of that found during blood clotting. Meizothrombin, an intermediate of prothrombin activation, was also able to induce dose-dependent removal of the propeptide from rpvWF. Hirudin preconditioning of vWF-deficient mice attenuated processing of infused rpvWF suggesting that thrombin plays a part in the processing events in vivo.
Effect of Cardiolipin Oxidation on Solid-Phase Immunoassay for Antiphospholipid Antibodies
Authors: Schlame Michael , Haller Ivan , Sammaritano Lisa , Blanck Thomas
Published: March 2018

Diagnostic assays for antiphospholipid antibodies are routinely performed on microtitre plates coated with cardiolipin. Here we show that contact between cardiolipin and NUNC-Immuno® plates leads to extensive oxidation, generating a series of peroxy-cardiolipins which were identified by electrospray ionization mass spectrometry. To investigate the impact of oxidation on the antibody assay, cardiolipin was resolved into 12 molecular species, including oxidized species and non-oxidized species with different degrees of unsaturation. All 12 species reacted under anaerobic conditions with serum from patients with primary antiphospholipid syndrome. Immune reactivity was similar for tetralinoleoyl-cardiolipin, trilinoleoyl-oleoyl-cardiolipin, and peroxycardiolipins, but somewhat lower for tristearoyl-oleoyl-cardiolipin. Oxidative treatment of cardiolipin with air, cytochrome c, or Cu2+/tert-butylhydroperoxide, either before or during the assay, did not enhance immune reactivity. Similar results were obtained with a monoclonal IgM from lupus-prone mice, that binds cardiolipin in the absence of protein cofactors. We conclude that the solid-phase assay for antiphospholipid antibodies can be supported by various oxidized and nonoxididized molecular species of cardiolipin.
Vollblutaggregometrie zur Kontrolle der Wirksamkeit von Azetylsalizylsäure bei Patienten mit koronarer Herzkrankheit
Authors: Dzijan-Horn M. , Bohner J. , Spannagl M. , Weisser H. , Calatzis A. , von Pape K.-W.
Published: March 2018

In dieser Studie wurden zwei Methoden evaluiert und verglichen, welche die Wirkung von Azetylsalizylsäure (ASS) auf die Thrombozytenaggregation im Vollblut erfassen. Die In-vitro-Blutungszeit wurde im PFA-100 (Dade Behring, Marburg) mit der Kollagen/Adrenalin-Messzelle im Zitratblut bestimmt. Die Vollblutaggregation wurde am Multiplate- System (Dynabyte medical, München) mit Hirudinblut (25 μg/ml) bestimmt. Zur Aggregation wurden die Induktoren Arachidonsäure (ASPItest), Kollagen (COLtest) sowie TRAP-6 (thrombin receptor activating peptide, TRAPtest) eingesetzt. Nach Aufklärung und Einwilligung wurden zur Analyse der In-vitro-Blutungszeit sowie der Vollblutaggregometrie Zitratblut und Hirudinblut von 76 Patienten mit koronarer Herzkrankheit, die unter langfristiger Azetylsalizylsäure- Behandlung standen (ASS-Patienten), entnommen. Zusätzlich wurde von 57 gesunden Blutspendern Hirudinblut für die Vollblutaggregometrie entnommen.

Im PFA-100 betrugen die Verschlusszeiten der ASS-Patienten 273 ± 49 s. Basierend auf dem Grenzwert (cut-off) von 170 s wurde bei 5 von 76 Patienten kein Ansprechen des Tests auf die ASS-Therapie nachgewiesen (Aspirin-Non-Response). Die Vollblutaggregometrie war bei den ASS-Patienten im Vergleich (vs. AUC-Werte) zu den Blutspendern in TRAPtest vergleichbar, hingegen im COLtest und ASPItest signifikant vermindert (p <0,05). Von den fünf Patienten, die im PFA-100 normwertige Verschlusszeiten zeigten, hatte nur einer eine normwertige Aggregation im ASPItest, und ebenfalls nur eine normwertige Aggregation im COLtest.

Die in der Studie gefundenen hohen Ansprechraten des PFA-100 sowie ASPItest auf die Azetylsalizylsäuretherapie kann durch die erwartete gute Compliance im untersuchten Kollektiv erklärt werden. In den verschiedenen Tests wurden unterschiedliche Patienten als Non-Responder zugeordnet. Dies verdeutlicht die Bedeutung der Testbedingungen für die Diagnose einer Azetylsalizylsäure-Resistenz oder Aspirin- non-Response.