bluuebunny/crossref_metadata_2025 huggingface dataset:
Beta-2-glycoprotein I Dependent Lupus Anticoagulants Form Stable Bivalent Antibody Beta-2-Glycoprotein I Complexes on Phospholipid Surfaces
Authors: Wittevrongel C. , Vanrusselt M. , Hoylaerts M. , Vermylen J. , Arnout J.
Published: March 2018

The precise mechanism by which Beta-2-glycoprotein I (β2GPI-) dependent lupus anticoagulants lengthen phospholipid-dependent clotting reactions is still poorly understood. In order to study this, murine monoclonal antibodies (moabs) against human β2GPI were raised. Eight of the 21 anti-β2GPI moabs, obtained from 2 fusions, fulfilled the criteria for lupus anticoagulant (LA) activity as tested with a variety of sensitive screening assays and confirmatory tests. Seven moabs did not influence any clotting test. The LA positive moabs were found to compete for similar or closely spaced epitopes on immobilized β2GPI. Two moabs with potent LA activity (moabs 22 F 6 and 22 B 3) and 1 moab without LA activity (moab 16 B 3) were selected to study the interaction between antibody, β2GPI and phospholipid. Interactions were investigated by real-time biospecific interaction analysis (BIA) based on plasmon surface resonance technology on a BIA-core instrument using a sensor chip coated with phospholipid. When 22 F 6, the moab with the most pronounced LA activity, was allowed to interact with the phospholipid surface at concentrations between 0 and 400 nmol/l, no appreciable binding could be detected. Likewise, no binding could be measured when β2GPI at concentrations between 0 and 400 nmol/l was passed over the phospholipid coated sensor chip. Combinations of β2GPI and 22 F 6 resulted in significant binding. Similar results were obtained with 22 B 3, another moab with LA activity. A LA negative Moab, 16 B 3, did not cause binding of antibody-β2GPI complexes. Fab’ fragments, derived from moab 22 F 6, inhibited the binding of β2GPI-22 F 6 and β2GPI-22 B 3 in a concentration dependent way, indicating that only bivalent β2GPI-antibody complexes bind with high affinity to phospholipids. Fab’ fragments, derived from moab 22 F 6, also inhibited the LA effect of moabs 22 F 6 and 22 B 3 in diluted plasma. In summary, these experiments indicate that the β2GPI-dependent LA effect depends on the formation of bivalent β2GPI-antibody complexes on phospholipid surfaces.
Pharmacokinetics of a Slower Clearing Tissue Plasminogen Activator Variant, TNK-tPA, in Patients with Acute Myocardial Infarction
Authors: Eppler Stephen , Breed Judy , Cannon Christopher , Braunwald Eugene , Love Ted , Modi Nishit
Published: March 2018

The rapid clearance of t-PA from plasma requires administration by intravenous (IV) infusion. A slower clearing, fibrin-specific rt-PA variant may allow single intravenous bolus administration, thereby simplifying dosing. This study was designed to characterize the pharmacokinetics of the slower clearing, fibrin-specific tissue-plasminogen activator variant, TNK-tPA, in patients with acute myocardial infarction (AMI) following a single IV bolus injection. Single IV bolus doses of 5 to 50 mg of TNK-tPA were studied in an open-label, multicenter, dose escalation study. A total of 113 AMI patients were enrolled. Blood sampling for pharmacokinetics was conducted in eighty-two patients (72 men, 10 women), with 5 to 27 patients per dose. TNK-tPA was administered as an IV bolus over 5–10 s. Following IV bolus administration, there was a biphasic elimination of TNK-tPA from plasma. The initial phase had a mean half-life that ranged from 11 ± 5 to 20 ± 6 min and was followed by a terminal phase with a mean half-life that ranged from 41 ± 16 to 138 ± 84 min. Mean TNK-tPA plasma clearance was 125 ± 25 - 216 ± 98 ml/min, and the initial volume of distribution was 4.3 ± 2 - 8.4 ± 6 l. A decrease in TNK-tPA plasma clearance with increasing TNK-tPA dose was noted. In addition, women and patients with lower body weight or older age had a slower plasma clearance. In conclusion, TNK-tPA has a slower plasma clearance in patients with AMI than that reported for rt-PA, allowing administration as a single IV bolus.
Flow Cytometry Analysis of Intracellular VASP Phosphorylation for the Assessment of Activating and Inhibitory Signal Transduction Pathways in Human Platelets
Authors: Schwarz Ulrike , Geiger Jörg , Walter Ulrich , Eigenthaler Martin
Published: March 2018

Increased platelet adhesion or aggregation are key events in the pathogenesis of cardiovascular diseases. Exact determination of the platelet activation state is essential to recognize, prevent, and treat cardiovascular complications due to platelet dysfunction. Initial phases of platelet activation and inhibition are characterized by phosphorylation of specific intracellular proteins. However, methodological problems often prevent analysis of platelet protein phosphorylation under clinical conditions. A novel flow cytometry-based method using a phosphorylation-specific antibody was developed for fast and easy quantification of the phosphorylation state of a specific intracellular platelet protein. This method was used to analyze various platelet receptors and their intracellular signaling which may be impaired in genetic or acquired disorders or altered due to therapeutic interventions. In a first clinical application, the inhibitory effects of ticlopidine and clopidogrel on the platelet P2YAC ADP receptor were monitored.

Abbreviations: ADP: adenosine 5’-diphosphate; cAMP: cyclic adenosine-3’,5’-monophosphate; cGMP: cyclic guanosine-3’,5’-monophosphate; HUVECs: human umbilical vein endothelial cells; MAPK: mitogen-activated protein kinase; PG-E1: prostaglandin E1; PRP: platelet-rich plasma; SNP: sodium nitroprusside; VASP: vasodilator-stimulated phosphoprotein
D-glucose Increases the Synthesis of Tissue-type Plasminogen Activator (t-PA) in Human Peritoneal Mesothelial Cells
Authors: Mandl-Weber Sonja , Wörnle Markus , Haslinger Bettina , Goedde Martin , Kooistra Teake , Sitter Thomas
Published: March 2018

Physical and chemical irritation of the peritoneum through glucose-based hyperosmolar dialysis solutions results in a nonbacterial serositis with fibrinous exudation. Thereby, human peritoneal mesothelial cells (HMC) play an important role in maintaining the balance between the peritoneal generation and degradation of fibrin by expressing the fibrinolytic enzyme tissue-type plasminogen activator (t-PA) as well as the specific plasminogen activator inhibitor-1 (PAI-1). In this study, we analyzed the effect of D-glucose and metabolically inert monosaccharides on the synthesis of t-PA and PAI-1 in cultured HMC.

Incubation of HMC with D-glucose or the metabolically inert monosaccharides mannitol and L-glucose (5-90 mM) resulted in a time- and concentration-dependent increase in t-PA mRNA expression and antigen secretion without affecting PAI-1 synthesis. A similar effect was evident when HMC were first exposed sequentially to pooled spent peritoneal dialysis effluent for up to 4 hours, and subsequently incubated for 20 hours in control medium. The stimulating effect of high D-glucose on t-PA expression in HMC was prevented by treating the cells with different protein kinase C (PKC) inhibitors (Ro 31-8220, Gö 6976), but could not be mimicked by the PKC-activating phorbol ester PMA, indicating that this effect of high glucose is dependent on PKC activity, but not mediated through PKC activation. Also, using specific inhibitors (PD 98059, SB 203580) and activators (PMA, anisomycin, IL-1α) of the major routes of the mitogen-activated protein kinases (MAPKs) cascade, we found no evidence for a role of this cascade in regulating t-PA expression in HMC.

We conclude that hyperosmolarity induces t-PA (but not PAI-1) in HMC via a regulatory mechanism that requires active PKC, but that does not involve a major pathway in the MAPK cascade.
Thrombostatin Inhibits Induced Canine Coronary Thrombosis
Authors: Hasan Ahmed , Rebello Sam , Smith Edward , Srikanth Sujata , Werns Steven , Driscoll Edward , Faul Jessica , Brenner Dean , Normolle Daniel , Lucchesi Benedict , Schmaier Alvin
Published: March 2018

Thrombostatin (RPPGF), an angiotensin converting enzyme metabolite of bradykinin, is an inhibitor of α-thrombin’s ability to activate platelets. We examined the in vivo pharmacokinetics and pharmacodynamics of thrombostatin in rabbits and its ability to inhibit coronary thrombosis induced by electrolytic injury in dogs. Plasma half-life of thrombostatin had a t1/2α of 2.6 min and a t1/2β of 24 min in rabbits. Ligating the renal arteries did not prolong clearance (t1/2α = 2.4 min; t1/2β = 12 min). Thrombostatin produced a prolonged in vivo antiplatelet effect. At 30 min after a single intravenous administration in rabbits, thrombostatin’s plasma concentration was <8.7 μM (5 μg/ml). However, ex vivo 20 and 40 nM γ-thrombin-induced platelet aggregation of these rabbits’ platelets was inhibited 40% for 2.75 and 1 h, respectively. In vitro, flow cytometry studies revealed that thrombostatin specifically bound to human platelets and washed human platelets treated with thrombostatin were less responsive to γ-thrombin than control platelets. Using electrolytic injury to induce coronary artery thrombosis, dogs treated with thrombostatin, aspirin, or combined thrombostatin and aspirin occluded in 62 ± 25 (mean ± SD), 62 ± 36, or 89 ± 32 min versus untreated animals which occluded at 39 ± 27 min, (p <0.01, p <0.01 and p <0.001, respectively). These studies show that thrombostatin binds to platelets and can delay coronary occlusion in vivo.

Abbreviations: RPPGF: thrombostatin; PAR1: protease activated receptor 1, the first cloned thrombin receptor; PRP: platelet-rich plasma; PPP: plateletpoor plasma; LCX: left circumflex coronary artery; APTT: activated partial thromboplastin time; PT: prothrombin time
Efficacy of a Synthetic Pentasaccharide, a Pure Factor Xa Inhibitor, as an Antithrombotic Agent – A Pilot Study in the Setting of Coronary Angioplasty
Authors: Vuillemenot Alain , Schiele François , Meneveau Nicolas , Claudel Sophie , Donat François , Fontecave Sylvie , Cariou Roger , Samama Meyer , Bassand Jean-Pierre
Published: March 2018

Aim of the study. To assess the antithrombotic properties of SR90107/ORG31540, a sulfated pentasaccharide, which enhances specifically antithrombin III mediated inactivation of factor-Xa, in a clinical setting known to promote arterial thrombosis, i.e. coronary angioplasty.

Methods and results. Percutaneous transluminal coronary angioplasty (PTCA) was carried out with conventional balloons with a single 5 min intravenous infusion of 12 mg pentasaccharide, and 500 mg intravenous aspirin. Heparin was not allowed before, during PTCA, and within 24 h after PTCA. The primary end point was the rate of abrupt vessel closure during and within 24 h after the procedure. The sample size was set at 60 evaluable patients, in order to be able to conclude with a good level of confidence (>95%) that the abrupt vessel closure rate was less than 10%, if less than 3 abrupt vessel closures were observed. Seventy-one patients were included in the study, of whom 10 needed elective stenting, and were not considered as evaluable for efficacy. Two out of the 61 remaining evaluable patients experienced acute vessel closure during the study period [3.28%, 95% confidence interval (0.4%; 11.4%)]. No major bleeding occurred. The drug plasma concentrations reached 1.91 ± 0.39 mg/l, 10 min after pentasaccharide injection, and decreased on average to 1.18 ± 0.27 mg/l at 2 h, and to 0.36 ± 0.11 mg/l at 23 h after administration of pentasaccharide. Activated clotting time (ACT) and activated partial thromboplastin (aPTT) time remained within normal range. Thrombinantithrombin complex levels fell from 22 ± 17.1 to 4.5 ± 3.4 μg/ml, prothrombin fragment 1+2 levels decreased from 2.15 ± 1.01 to 1.73 ±
0.87, and activated factor VII levels decreased from 43.4 ± 16.8 mU/ml to 18.9 ± 7.3 mU/ml respectively from baseline to 2 h following injection of the tested drug.

Conclusions. Administration of pentasaccharide led to the inhibition of thrombin generation without modification of aPTT and ACT. The rate of abrupt vessel closure was within range of rates reported in historical series. Thus we conclude that the anti-thrombotic activity of pentasaccharide, as shown in this pilot trial in the setting of coronary angioplasty, deserves further investigation.
Evaluation of an Adenoviral Vector Encoding Full-Length Human Factor VIII in Hemophiliac Mice
Authors: Connelly Sheila , Andrews Julie , Gallo-Penn Angela , Tagliavacca Luigina , Kaufman Randal , Kaleko Michael
Published: March 2018

Adenoviral vectors provide a promising gene therapy system for the treatment of hemophilia A. Potent vectors encoding a human factor VIII (FVIII) cDNA were developed that mediated sustained FVIII expression in normal and hemophiliac mice and complete phenotypic correction of the bleeding disorder in hemophiliac mice and dogs (Connelly and Kaleko, Haemophilia 1998; 4: 380-8). However, these studies utilized vectors encoding a truncated version of the human FVIII cDNA lacking the B-domain (BDD FVIII). In this work, an adenoviral vector encoding the human full-length (FL) FVIII cDNA was generated and characterized. While functional FL FVIII was secreted in vitro, expression of the FL protein was not detected in the plasma of vector-treated hemophiliac mice. Unexpectedly, the FL FVIII vector-treated animals demonstrated phenotypic correction of the bleeding defect as measured by a tail-clip survival study. FL FVIII protein was visualized in the mouse livers using human FVIII-specific immunohistochemical analyses. These data demonstrate that adenoviral vector-mediated in vivo expression of BDD FVIII is more efficient than that of the FL protein and that phenotypic correction can occur in the absence of detectable levels of FVIII.
Fibrinogen St. Gallen I (γ 292 Gly → Val): Evidence for Structural Alterations Causing Defective Polymerization and Fibrinogenolysis
Authors: Stucki Bettina , Schmutz Peter , Schmid Luzius , Haeberli André , Lämmle Bernhard , Furlan Miha
Published: March 2018

Fibrinogen St. Gallen I was detected in an asymptomatic Swiss woman. Routine coagulation tests revealed a prolonged thrombin and reptilase time. Functionally measured fibrinogen levels were considerably lower than those determined immunologically. Polymerization of fibrin monomers derived from purified fibrinogen was delayed in the presence of either calcium or EDTA. Normal fibrinopeptide A and B release by thrombin was established. An abnormal degradation of fibrinogen St. Gallen I by plasmin was observed. Fragment D1 of normal fibrinogen was fully protected against further proteolysis in the presence of 10 mM calcium, whereas fibrinogen St. Gallen I was partially further degraded to fragments D2 and D3. In the presence of 10 mM EDTA, the conversion of variant fragment D1 to D2 was accelerated whereas the degradation of fragment D2 to D3 was delayed in comparison to degradation of fragments D1 and D2 of normal fibrinogen. Three high-affinity calcium binding sites were found in both normal and variant fibrinogen. Mutation screening with SSCP analysis suggested a mutation in exon VIII of the → Val) as previously described in fibrinogen Baltimore I (4, 5). Dysfibrinogen St. Gallen I is characterized by delayed polymerization and abnormal fibrinogenolysis compared to normal fibrinogen. Computer modeling of the variant fibrinogen revealed structural changes that may explain its abnormal functional behaviour.γ-chain gene. Cycle sequencing of this gene portion revealed a single base substitution from G to T of the base 7527, leading to replacement of γ 292 glycine by valine. The same mutation has already been described for the fibrinogen variant Baltimore I. Molecular modeling was performed of a part of the γ-chain containing the mutation site, based on recently published X-ray crystal structures of human fibrinogen fragment D and of a 30 kD C-terminal part of the γ-chain. Significant structural alterations due to the substitution of glycine by valine at γ 292 were observed, e.g. spreading of the protein backbone, probably leading to a modified accessibility of the plasmic cleavage sites in the γ-chain at 356 Lys and 302 Lys. A shift of γ 297 Asp that is involved in interactions of fragment D with the Gly-Pro-Arg-Pro-peptide was noted by molecular modeling. The latter observation is compatible with delayed polymerization of fibrin monomers.
Profile of Recombinant Pro-urokinase Given by Intraarterial versus Intravenous Routes of Administration in a Canine Thrombosis Model
Authors: Lubbers Nathan , Nelson Richard , Wegner Craig , Cox Bryan , Burke Sandra
Published: March 2018

Catheter-directed thrombolysis has gained increasing acceptance for the treatment of patients who present with vascular occlusion; however, intravenous injection may be preferable in selected patients. Recombinant prourokinase (r-proUK) is a recently-developed fibrin-selective thrombolytic agent with specificity for clot-bound plasminogen. To compare the effects of r-proUK on clot lysis and restoration of blood flow when injected by either intraarterial or intravenous routes of administration, we utilized a dog model of arterial thrombosis in which a radiolabelled clot is formed in the femoral artery. The r-proUK was given by intravenous infusion to one group of 18 animals in doses ranging from 10,000 IU/kg to 100,000 IU/kg; a second group of 27 dogs was treated with r-proUK administered by the intra-arterial route in a dose range from 300 IU to 10,000 IU. Clot lysis was measured by monitoring the loss of counts from the radiolabelled clot over time; blood flow was also monitored throughout the experimental period. Animals which received intravenous treatment showed dose-related clot lysis ranging from 14% to 70% at 2 h, while those which received intra-arterial infusions showed lysis ranging from 22% to 79% over the same period. For similar degrees of clot lysis attained at the highest dose levels of 100,000 IU/kg and 10,000 IU, blood flow was restored to 77% and 35% of control levels in dogs which received intravenous and intraarterial treatment, respectively. The hemostatic protein fibrinogen was not reduced in any of the treatment groups. The results indicate that 100 times more intravenous than intra-arterial r-proUK is required to produce similar clot lysis in this canine model, and that the agent can be administered at this level without induction of a systemic lytic state.
Pharmacokinetics and Pharmacodynamics of Melagatran, a Novel Synthetic LMW Thrombin Inhibitor, in Patients with Acute DVT
Authors: Eriksson H. , Frison L. , Hansson P. O. , Held P. , Holmström M. , Hägg A. , Jonsson T. , Lapidus L. , Leijd B. , Stockelberg D. , Säfwenberg U. , Taghavi A. , Thorsén M. , Eriksson U. G.
Published: March 2018

Forty-eight patients with acute proximal deep vein thrombosis (DVT) were randomised to intravenous infusions for 4 to 6 days with melagatran, a novel synthetic low molecular weight thrombin inhibitor, or unfractionated heparin adjusted by the activated partial thromboplastin time (APTT). The aim of the study was to investigate the pharmacokinetics, pharmacodynamics and the safety of melagatran therapy at three different doses. Steady-state plasma concentrations were rapidly achieved and maintained throughout the infusion period. The mean plasma concentrations in the low, medium and high dose groups were 0.17, 0.31 and 0.53 μmol/l, respectively. The prolongation of APTT was stable during the melagatran infusions and correlated to the plasma concentration. Phlebographically verified regression of thrombus size measured as decrease in Marder score was seen after 4 to 6 days in 8 of 12 patients, 6 of 12 patients and 5 of 11 patients in the low, medium and high dose groups of melagatran and in 5 of the heparin-treated patients. In the low dose group with melagatran, thrombus extension was seen in one patient. At the dose levels studied, melagatran was well tolerated with no clinically significant bleeding problems, suggesting that melagatran could safely be given to patients suffering from DVT.
Circulating Platelets Show Increased Activation in Patients with Acute Cerebral Ischemia
Authors: Tschoepe Diethelm , Kessler Christof , Zeller Joern
Published: March 2018

Platelet activation plays a central role in acute arterial stenosis as has been shown in coronary heart disease. Likewise it can be assumed to be of importance in the evolution of acute cerebral ischemia (ACI), particularly in patients with large vessel disease. Flow cytometric detection of platelet adhesion molecules as a marker of platelet activation in a group of patients with ACI and different etiologies has not been evaluated. In 72 patients with ACI and 72 controls, the exposure of activation-dependent adhesion molecules was determined using flow cytometry after immunostaining with monoclonal antibodies against CD 62, CD 63 and thrombospondin. The extent of platelet activation differed as a function of the etiology of ACI: platelets from patients with atherosclerosis of brain-supplying arteries expressed significantly more activation markers than did controls, whereas patients with cardioembolic stroke did not. By analyzing platelet adhesion molecules it is possible to describe platelet activation profiles in patients with acute cerebral ischemia. This diagnostic procedure will be useful for monitoring individualized anti-platelet therapy and may enable distinguishing different subgroups of stroke patients.
Genetic Effects for Plasma Factor VII Levels Independent of and in Common with Triglycerides
Authors: Pedersen Nancy , Egberg Nils , Faire Ulf , Hong Yuling
Published: March 2018

Background. Coagulation factor VII has been demonstrated as a potential risk factor for cardiovascular disease. Both genes and non-genetic factors are related to plasma levels of factor VII. However, the extent to which genetic effects influence variability in plasma factor VII levels is unknown. Further, increased levels of plasma factor VII are associated with serum triglycerides, yet the reason for this association is not fully understood. Methods and Results. Quantitative genetic analyses were applied to evaluate the relative importance of genetic and different environmental influences on plasma factor VII levels and to test the significance of genetic and environmental factors in common to factor VII and triglycerides in 215 pairs of middle-aged and elderly twins, of whom 104 were reared apart and 120 were women. Genetic influences were found to account for 57% of the individual differences in plasma factor VII levels, whereas shared-rearing and residual-familial environmental factors were not significant. Furthermore, a significant genetic correlation of 0.38 was found between factor VII and triglycerides, but the environmental correlation between these two measures was not significant. Genetic factors in common to factor VII and triglycerides explain about 7% of the total variance for factor VII. Conclusion. The present study suggests that there are substantial genetic influences on plasma factor VII levels. Furthermore, genetic effects explain the phenotypic association between factor VII and triglycerides.
Vitamin K Deficiency Bleeding (VKDB) in Infancy
Authors: von Kries Rüdiger , Cornelissen Marlies , McNinch Andrew , Andrew Maureen , Sutor Anton
Published: March 2018

Terminology. Replace the term “Hemorrhagic Disease of the Newborn” (HDN) by “Vitamin K Deficiency Bleeding” (VKDB), as neonatal bleeding is often not due to VK-deficiency and VKDB may occur after the 4-week neonatal period. Definition. VKDB is bleeding due to inadequate activity of VK-dependent coagulation factors (II, VII, IX, X), correctable by VK replacement. Diagnosis. In a bleeding infant a prolonged PT together with a normal fibrinogen level and platelet count is almost diagnostic of VKDB; rapid correction of the PT and/or cessation of bleeding after VK administration are confirmative. Warning signs. The incidence of intracranial VKDB can be reduced by early recognition of the signs of predisposing conditions (prolonged jaundice, failure to thrive) and by prompt investigation of “warning bleeds”. Classification. VKDB can be classified by age of onset into early (<24 h), classical (days 1-7) and late (>1 week <6 months), and by etiology into idiopathic and secondary. In secondary VKDB, in addition to breast feeding, other predisposing factors are apparent, such as poor in-take or absorption of VK. VK-Prophylaxis: Benefits. Oral and intramuscular VK (one dose of 1 mg) protect equally well against classical VKDB but intramuscular VK is more effective in preventing late VKDB. The efficacy of oral prophylaxis is increased with a triple rather than single dose and by using doses of 2 mg vitamin K rather than 1 mg. Protection from oral doses repeated daily or weekly may be as high as from i.m. VK. VK-Prophylaxis: Risks. VK is involved in carboxylation of both the coagulation proteins and a variety of other proteins. Because of potential risks associated with extremely high levels of VK and the possibility of injection injury, intramuscular VK has been questioned as the routine prophylaxis of choice. Protection against bleeding should be achievable with lower peak VK levels by using repeated (daily or weekly) small oral doses rather than by using one i.m. dose. Breast feeding mothers taking coumarins. Breast feeding should not be denied. Supervision by pediatrician is prudent. Weekly oral supplement of 1 mg VK to the infant and occasional monitoring of PT are advisable. Conclusion. VKDB as defined is a rare but serious bleeding disorder (high incidence of intracranial bleeding) which can be prevented by either one i.m. or multiple oral VK doses.
Factor XIII Val34Leu Is a Genetic Factor Involved in the Aetiology of Venous Thrombosis
Authors: Reitsma P. H. , Lourenço D. , Maffei F. H. , Morelli V. , Tavella M. H. , Araújo A. G. , Piccinato C. E. , Zago M. A. , Franco R F.
Published: March 2018

A mutation in the factor XIII gene (FXIII Val34Leu) gene was recently reported to confer protection against myocardial infarction, but its relationship with venous thrombosis is unknown. In addition, a mutation in the 5’-untranslated region of the FXII gene (46 C→T) was identified which is associated with low plasma levels of the protein. Its prevalence in patients with venous thrombosis is also unknown. We investigated the frequency of the FXIII Val34Leu and FXII 46 C→T mutations in 189 patients with deep venous thrombosis and in 187 age-, gender- and race-matched controls. FXIII Val34Leu was detected in 38.6% of the patients and in 41.2% of the controls. Interestingly, homozygosity for the FXIII mutation was found in 1.6% of the patients and in 9.6% of the controls, yielding an odds ratio (OR) for venous thrombosis of 0.16 (95% CI: 0.05-0.5). The OR for hetero-zygotes was 1.1 (95% CI: 0.7-1.7). The FXII 46 C→T mutation was detected in 46.0% of the patients and in 48.6% of the controls. The OR for heterozygotes was 0.9 (95% CI: 0.6-1.4) and for homozygotes the OR was 0.8 (95% CI: 0.3-1.9). Our data indicate that the FXII 46 C→T mutation is unlikely to be a major risk factor for venous thrombotic disease. In contrast, the homozygous state for FXIII Val34Leu is a strong protective factor against venous thrombosis, which emerges as a novel genetic factor involved in the aetiology of thrombophilia.
The Relationship of Mutations in the MTHFR, Prothrombin, and PAI-1 Genes to Plasma Levels of Homocysteine, Prothrombin, and PAI-1 in Children and Adults
Authors: Balasa Vinod , Glueck Charles , Stroop Davis , Becker Ann , Pillow Ann , Wang Ping , Gruppo Ralph
Published: March 2018

Studies in adults have demonstrated that the genetic mutations C677T methylenetetrahydrofolate reductase (MTHFR), prothrombin 20210A, and the 4G polymorphism of the plasminogen activator inhibitor-1 (PAI-1) gene are associated with elevated plasma levels of homocysteine, prothrombin and PAI-1, respectively and with an increased risk of thrombosis. No similar data is available in children. Therefore, we assessed the relationship of plasma levels of homocysteine, prothrombin and PAI-1 with their respective mutations in 197 normal children, compared to 40 adults. By stepwise multiple regression, homocysteine was positively associated with age, PAI-1 activity was negatively associated with age, while PAI-1 antigen and prothrombin levels were associated with gender, being higher in girls than boys. When the genotypes were added to the regression model as additional explanatory variables, the MTHFR genotype accounted for 2.9% of the variance of homocysteine (p = 0.024), and the PAI-1 gene accounted for 2.7% of the variance of PAI-1 antigen levels (p = 0.023). Of children homozygous for the MTHFR mutation, 35% had homocysteine levels ≥ the age-specific 95th percentile, compared to 2% hetero-zygotes and 5% wild type normals (p = 0.0001). The mean homocysteine level was higher in children homozygous for the MTHFR gene (8.4 μmol/l) than in heterozygotes (5.5 μmol/l), p <0.05. Of children homozygous for the 4G polymorphism of the PAI-1 gene, 19% had PAI-1 activity levels ≥ the age-specific 95th percentile, compared to 2% of heterozygotes and 3% of wild type normals (p = 0.003). Studies of the incidence of the MTHFR, prothrombin, and PAI-1 4G/5G genotypes in children with thrombosis, when compared to these healthy normals, will provide evidence as to which of these genes are associated with thrombophilia.

This work was carried out following an institutional research committee approved protocol with signed informed consent.
Effects of Statins in Thrombosis and Aortic Lesion Development in a Dyslipemic Rabbit Model
Authors: Alfon J. , Pueyo Palazon C. , Royo T. , Badimon L.
Published: March 2018

HMG-CoA reductase inhibitors (statins) are effective in primary and secondary prevention of coronary heart disease. The mechanism of action is mainly attributed to their plasma cholesterol lowering activity, although additional effects have been suggested. Our objective was to study whether atorvastatin and simvastatin exhibited an inhibitory effect on platelet deposition onto a triggering damaged vessel wall in addition to an antiatherosclerotic effect in the dyslipemic rabbit model. Statins were administered at identical doses of 2.5 mg/kg/day with a hyperlipidemic diet during 10 weeks. Both drugs similarly lowered total cholesterol and, moderately, triglycerides. Mural platelet deposition on damaged vessel wall placed in an ex-vivo flow perfusion system was reduced in atorvastatin treated animals (39.7 ± 6.2 × 106 PLT/cm2) vs. controls (94.8 ± 15.9 × 106 PLT/cm2, p <0.02). Simvastatin reduced aortic fatty streak surface coverage (31,7 ± 5.3%) vs. controls (47.9 ± 4.1%, p <0.005) and intimal thickening in thoracic aorta (0.15 ± 0.05 intima to total area ratio in simvastatin treated animals vs. 0.36 ± 0.03 in control animals, p <0.05). Atherosclerotic fatty streak coverage correlated positively with total cholesterol, tryglicerides and LDL-cholesterol levels in all groups. HMG-CoA reductase inhibitors similarly lowered plasma lipids but exhibited significantly different effects in the modulation of atherosclerotic development and platelet response at the tested dose. Therefore, the effect of statins on the progression and manifestation of cardiovascular disease might be also mediated by regulating platelet response to vessel injury.
The Prothrombin 20210A Allele and Its Association with Myocardial Infarction
Authors: Daly M. E. , Steeds R. P. , Channer K. S. , Samani N. J. , Hampton K. K. , Croft S. A.
Published: March 2018

The relationship between the prothrombin (PT) 20210A allele and arterial disease is controversial. We conducted a case-control study to assess its contribution to risk of myocardial infarction (MI). Five hundred and thirty-nine acute MI patients and 498 control subjects aged <75 years were studied. Two percent of cases carried the PT20210A allele compared to 2.8% of controls. The odds ratio for MI was 0.72 (95% CI 0.32-1.60) indicating that the PT20210A allele confers no increased risk for MI. Subgroup analysis showed no association between the PT20210A allele and either premature MI or MI in females. We conclude the PT20210A allele is not a risk factor for MI and suggest that discrepancies in studies relating the PT20210A allele to MI may be due to difficulties in estimating its low allelic frequency in the general population and thus random differences in the observed frequencies in the control populations studied.
Free and Total Platelet Glycoprotein IIb/IIIa Measurement in Whole Blood by Quantitative Flow Cytometry during and after Infusion of c7E3 Fab in Patients Undergoing PTCA
Authors: Metz Damien , Nazeyrollas Pierre , Nguyen Philippe , Simon Gérard , Daliphard Sylvie , Droullé Chantal , Elaerts Jacques , Potron Gérard , Hézard Nathalie
Published: March 2018

A quantitative flow cytometry assay was used to evaluate the ex vivo kinetics of c7E3 Fab platelet effect in 16 patients undergoing PTCA treated with abciximab and compared with aggregometry assay. Immunolabeling of platelets was directly assessed on whole blood, using in parallel two monoclonal antibodies (Mabs) raised against GPIIIa, Mab1, the binding of which is inhibited by c7E3 Fab, and Mab2, the binding of which is not affected by c7E3 Fab. We found a severe and sustained inhibition of both GPIIb/IIIa receptors and platelet functions. The inter-individual variation in response to abciximab was low. A significant transient increase at H24 and H48 in the binding of Mab2 was found as an unexpected result, and confirmed in vitro. Results demonstrate that flow cytometry is a reliable method in agreement with aggregation. In addition, our results show that it is a standardized tool and a time-saving technique.
Lupus Anticoagulant Testing in Europe: An Analysis of Results from the First European Concerted Action on Thrombophilia (ECAT) Survey Using Plasmas Spiked with Monoclonal Antibodies against Human β2-Glycoprotein I
Authors: Meijer P. , Vermylen J. , Arnout J.
Published: March 2018

Lupus anticoagulants (LA) are immunoglobulins directed to either prothrombin or Beta-2-glycoprotein I (β2GPI) bound to phospholipids. Most patients with LA have both β2GPI- and prothrombin-dependent antibodies. Several recent reports have shown that LA is more strongly associated with thrombosis than anticardiolipin antibodies (aCL). Therefore, an accurate detection of LA is of utmost importance in patients suspected of an antiphospholipid syndrome. We recently raised a series of murine monoclonal antibodies against human Beta-2-glycoprotein I (β2GPI) with LA activity similar to affinity purified human β2GPI-dependent LAs. A normal plasma pool, and the same pool spiked with LA positive anti-β2GPI antibodies at two potency levels, were used as materials in an external quality assessment scheme organised by the European Concerted Action on Thrombosis (ECAT). Fifty nine laboratories participating in this trial were asked to test for the presence of a LA in the 3 samples submitted. The majority (82%) of the participants found the high potency LA sample to be positive. Only 37% of the laboratories considered the weak potency LA sample to be positive. The submission of a normal sample, a weakly positive sample and a clearly positive sample enabled us to compare the relative LA responsiveness of the different screening assays used. Clotting time ratios varied from 0.81 to 3.28 for sample B and from 0.66 to 5.32 for sample D. In general, the highest clotting time ratios were found with the dilute prothrombin time (dPT), the dilute Russell Viper Venom time (dRVVT) and the Kaolin Clotting time. The most frequently used screening tests were the aPTT and the dRVVT. With the various assay systems, LA responsiveness varied largely according to the reagents used. For the β2GPI-dependent LA used in this study, PTT LA clearly showed the highest responsiveness among the aPTT reagents and Innovin among the dPT reagents. The present study also shows that many laboratories still rely on poorly responsive screening assays for their LA tests. Other laboratories rely on sensitive and more specific integrated test systems based on a sensitive screening assay with a low phospholipid content and a confirmatory test employing high phospholipid concentrations. The most used integrated system was dRVVT based. However, also here the LA responsiveness was largely reagent dependent.

In conclusion, many laboratories still rely on poorly responsive screening assays by which weakly positive LA samples are misdiagnosed. LA positive anti-β2GPI moabs have a potential for the unlimited production of LA control specimens, that may help hemostasis laboratories choose more LA responsive assay systems and to assess intra-laboratory precision of their LA testing.
Precise Carrier Diagnosis in Families with Haemophilia A: Use of Conformation Sensitive Gel Electrophoresis for Mutation Screening and Polymorphism Analysis
Authors: Williams I. J. , Abuzenadah A. , Winship P. R. , Preston F. E. , Dolan G. , Wright J. , Peake I. R. , Goodeve A. C.
Published: March 2018

Causative mutations in the factor VIII gene of seven unrelated patients with severe haemophilia A were identified using the mutation screening procedure conformation sensitive gel electrophoresis (1) and characterised by direct sequencing. Female family members of all patients had requested either carrier status determination or prenatal diagnosis. However, lack of the factor VIII gene inversion, a prior family history or informative polymorphisms prevented diagnosis in these families. Identification of a mutation in each family enabled female carrier status to be determined in all cases. Six mutations were previously unreported. One Afro-Caribbean patient had two sequence changes; A670 2G and A6769G. The latter, resulting in Met2238Val and previously reported as a FVIII mutation, was shown to be polymorphic with a 42% heterozygosity rate in an Afro-Caribbean population. Conformation sensitive gel electrophoresis was found to be technically simple and efficient at locating previously unknown FVIII gene mutations.
Superficial Vein Thrombosis: Incidence in Association with Pregnancy and Prevalence of Thrombophilic Defects
Authors: Ramsay J. E. , Tait R. C. , Walker I. D. , McCall F. , Conkie J. A. , Carty M. J. , Greer I. A. , McColl M. D.
Published: March 2018

Superficial venous thrombotic (SVT) events are a feature of thrombophilic abnormalities, particularly those involving the protein C pathway. We have determined the incidence of SVT associated with pregnancy and the early postpartum period in a retrospective study involving 72 000 deliveries. Fourty-nine cases occurring in 47 individuals were recorded, with an overall incidence of 0.68/1000 deliveries (95% CI 0.48-0.88). None had a previous history of deep vein thrombosis or pulmonary embolism. Most events occurred in the early post-partum period (0.54/1000 deliveries). Twenty-four/fourty-seven were screened for established thrombophilic abnormalities, with only 1 abnormality detected (FVLeiden heterozygote). Thrombophilia may play a minor role in the aetiology of SVT associated with pregnancy, although a larger study is required to confirm this.
The Contribution of Anti-prothrombin-antibodies to Lupus Anticoagulant Activity
Authors: Oort Erica van , Derksen Ronald , de Groot Philip , Horbach Daniëlle
Published: March 2018

The presence of lupus anticoagulant (LAC) is strongly correlated with a history of thrombosis in patients with SLE. LAC activity can be caused by anti-prothrombin (FII)- and/or anti-β2glycoprotein I (β2GPI)-antibodies.

In the present study, the contribution of anti-FII-antibodies to LAC activity was measured in 28 LAC positive plasmas. Plasmas were incubated with prothrombin or BSA, immobilized on CNBr-activated Sepharose, to absorb all anti-FII-antibodies. In 4 out of the 28 plasmas LAC activity was completely dependent on anti-FII-antibodies. In 7 out of the 28 plasmas, anti-FII-antibodies did not contribute to LAC activity. These anti-FII-antibodies can be regarded as non-functional antibodies. In the majority (17/28) of the samples, LAC activity within a single plasma was caused by a combination of antibodies with different specificities. Both dRVVT and KCT showed comparable sensitivity for the detection of functional anti-FII-antibodies.

In conclusion, in most samples LAC activity is not caused by anti-FII-antibodies alone but by a combination of different types of antibodies. The presence of LAC activity and anti-FII-antibodies in one plasma does not automatically implicate that these antibodies are responsible for the LAC activity.
Effects of Skin Mast Cells on Bleeding Time and Coagulation Activation at the Site of Platelet Plug Formation
Authors: Kauhanen Petteri , Kovanen Petri , Reunala Timo , Lassila Riitta
Published: March 2018

We studied the effects of stimulated skin mast cells on bleeding time and thrombin generation which was measured using prothrombin fragment F 1+2 (F 1+2) and thrombin-antithrombin-III-complex (TAT). In 10 patients with urticaria pigmentosa (chronic cutaneous mast cell accumulation) the mean bleeding time was significantly prolonged in wounds made on urticaria pigmentosa lesions vs. normal skin (460 ± 34 vs. 342 ± 27 s, p = 0.005). In 10 atopic subjects skin incisions were made on prick-tested sites 30, 60, 120 and 240 min after administration of an allergen (acute mast cell stimulation), histamine or vehicle. The mean bleeding time was significantly prolonged at all time points, being maximal at 120 min (60% prolonged) in wounds made on allergen-stimulated skin areas (p <0.01) compared with histamine or vehicle sites. Administration of allergen or histamine lowered the TAT concentration in the bleeding-time blood. Furthermore, TAT and F 1+2 levels in the bleeding-time blood were lower at 60, 120 and 240 min after allergen or histamine application in comparison with samples collected at 30 min. We conclude that skin mast cells can regulate primary hemostasis by prolonging bleeding time and by inhibiting thrombin generation.
Superficial Vein Thrombosis of Lower Limbs: Influence of Factor V Leiden, Factor II G20210A and Overweight
Authors: Wutschert Robert , Heinzmann Monica , Perneger Thomas , Reber Guido , Bounameaux Henri , Moerloose Philippe de
Published: March 2018

Superficial vein thrombosis (SVT) has been reported in patients with thrombophilia. In the present unmatched case-control study, the two most common thrombophilic abnormalities (factor V Leiden and factor II G20210A) were searched for in 112 consecutive patients with SVT of lower limbs and in 180 healthy donors. FV Leiden was present in 16/112 (14.3%) SVT patients and 11/180 (6.1%) controls (odds ratio 2.51, 95% CI 1.04-6.24) and FII G20210A in 4/112 (3.6%) patients and 2/180 (1.1%) controls (OR 3.28, 95% CI 0.46-36.84). In addition, body mass index (BMI) ≥28 kg/m2 was also associated with SVT (OR 2.81, 95% CI 1.60-5.00). After adjustement for BMI ≥28 kg/m2, the association between FV Leiden and SVT remained strong though no longer statistically significant. Among patients with SVT, the presence of FV Leiden was independently associated with the absence of varicose veins (OR 4.62, 95% CI 1.25-18.0) and with a BMI ≥28 kg/m2 (OR 3.74, 95% CI 1.05-15.1). In conclusion, both FV Leiden and overweight seem to predispose to SVT, a finding that should be confirmed in larger studies.
Increased Fraction of Circulating Activated Platelets in Acute and Previous Cerebrovascular Ischemia
Authors: Ruf Andreas , Vogt Axel , Lichy Christoph , Buggle Florian , Patscheke Heinrich , Hacke Werner , Grau Armin
Published: March 2018

Determination of circulating activated platelets may be helpful to estimate the prognosis and to stratify therapies in arterial vascular disorders including stroke. We used flow cytometry and phase contrast microscopy to study whether the fraction of platelets expressing p-selectin and CD63 and the fraction of platelets with shape change are increased in patients with acute and previous cerebrovascular ischemia.

The proportion of platelets expressing activation dependent antigens was higher in patients with acute (n = 24; p-selectin: 8.23 ± 4.21%; CD63: 3.53 ± 2.53%) and with previous cerebrovascular ischemia (n = 46; 3.86 ± 1.98%; 2.80 ± 1.79%) as compared to age- and sex-matched control subjects (n = 35; 2.17 ± 0.96%; 1.79 ± 0.75%; p ≤0.005, respectively). In patients with previous ischemia, there was no difference between treatment with aspirin (n = 25) or phenprocoumon (n = 21). Hypertension, diabetes mellitus and smoking were not associated with increased antigen expression (analysis of variance). The fraction of discoid platelets and platelet counts were not significantly different between groups.

Our results indicate increased expression of platelet neoantigens in acute and to a less degree in previous cerebrovascular ischemia. Ongoing platelet activation after cerebrovascular ischemia despite therapy with aspirin or phenprocoumon indicates that new anti-platelet drugs may be of benefit for these patients. Flow cytometry appears to be a useful tool to assess platelet function in cerebrovascular ischemia.
Coronary Artery Disease and Fibrinolysis: From the Blood to the Vessel Wall
Authors: Sobel Burton E.
Published: March 2018

This review addresses a continuum of the role of derangements in the fibrinolytic system from the blood to the vessel wall in the pathogenesis of acute coronary events and the underlying vasculopathy. Pharmacologic modification of fibrinolysis has become a primary therapy for acute myocardial infarction caused by thrombotic occlusion of infarct-related coronary arteries. Patients with type 2 diabetes and other insulin resistant states exhibit impaired fibrinolysis in blood, implicated in the pathogenesis of macroangiopathy. An altered balance of activity of proteins involved in the fibrinolytic system within vessel walls (the proteo[fibrino]lytic system) has been recognized as well and appears likely to contribute to the acceleration of macroangiopathy. Accordingly, normalization of the proteo(fibrino)lytic system in blood and in vessel walls is a particularly attractive target for retardation of the progression of macrovascular disease associated with insulin resistant states.
Oxidative Stress and Atherosclerosis: Its Relationship to Growth Factors, Thrombus Formation and Therapeutic Approaches
Authors: Peter Karlheinz , Kübler Wolfgang , Ruef Johannes , Nordt Thomas K. , Runge Marschall S. , Bode Christoph
Published: March 2018

The initiating event of atherogenesis is thought to be an injury to the vessel wall resulting in endothelial dysfunction. This is followed by key features of atherosclerotic plaque formation such as inflammatory responses, cell proliferation and remodeling of the vasculature, finally leading to vascular lesion formation, plaque rupture, thrombosis and tissue infarction. A causative relationship exists between these events and oxidative stress in the vessel wall. Besides leukocytes, vascular cells are a potent source of oxygen-derived free radicals. Oxidants exert mitogenic effects that are partially mediated through generation of growth factors. Mitogens, on the other hand, are potent stimulators of oxidant generation, indicating a putative self-perpetuating mechanism of atherogenesis. Oxidants influence the balance of the coagulation system towards platelet aggregation and thrombus formation. Therapeutic approaches by means of antioxidants are promising in both experimental and clinical designs. However, additional clinical trials are necessary to assess the role of antioxidants in cardiovascular disease.