bluuebunny/crossref_metadata_2025 huggingface dataset:
Collagen Induced Thrombus Formation at the Apex of Eccentric Stenoses - A Time Course Study with Non-Anticoagulated Human Blood
Authors: Barstad R Marius , Kierulf Peter , Sakariassen Kjell S
Published: July 2018

Atherosclerotic plaque rupture may trigger the formation of a mural thrombus. This thrombus formation is apparently affected by very high and complex shear conditions introduced by the luminal narrowing (stenosis) of the atheroma. To study the impact of such blood flow behaviour on thrombus formation we employed a model system where collagen-induced thrombogenesis is studied at the apex of well-defined eccentric stenoses.

Thrombus formation in non-anticoagulated human blood drawn directly from an antecubital vein over the collagen coated stenosis apex for periods of 0.5, 1, 3 or 5 min was quantified by morphometry. The stenoses reduced the cross-sectional area of the blood flow channel by 60, 80 and 89%, which corresponded to apex wall shear rates of 2600, 10,500 and 32,000 s−1, respectively. Platelet-collagen adhesion decreased by increasing shear at the stenosis apex. The corresponding adhesion rates were highest at 1 min, then they gradually decreased upon prolongation of the perfusion time. The platelet thrombus volume increased in concert with increasing shear rate up to 10,500 s−1, whereas, at 32,000 s−1, the volume was decreased. The corresponding growth rates and rates of thrombus occlusion at the apex levelled off at 3 min. Significant fibrin deposition was not observed before 3 min, and was most pronounced at 10,500 and 32,000 s−1. The plasma levels of fibrinopeptide A and P-thromboglobulin increased in concert with increasing shear and perfusion time, particularly at the two highest shear conditions.

Thus, hallmarks of thrombus formation at these stenoses with increasing shear are decreased platelet-collagen adhesion, and increased platelet-platelet interaction and fibrin deposition. A fibrin tail downstream to the collagen-attached platelet thrombus is regularly observed when thrombus occlusion exceeds 40%. However, the reduced thrombus growth at the most occlusive stenosis (89%) is presumably due to the high shear stresses which may reduce the rate of platelet incorporation into the thrombus and/or tear off thrombus fragments.
Heightened Thrombin Generation in Individuals with Resistance to Activated Protein C
Authors: Martinelli Ida , Bottasso Bianca , Duca Francesca , Faioni Elena , Mannucci Pier Mannuccio
Published: July 2018

We chose to evaluate whether or not a state of biochemical hypercoagulability was present in 74 individuals (69 heterozygotes and 5 homozygotes) resistant to activated protein C (APC) due to the Arg506 -> Gin mutation in the factor V gene. To this end, plasma levels of two markers of thrombin formation, prothrombin fragment 1+2 (F1+2) and thrombin-antithrombin complexes (TAT), were measured. High levels of F1+2 and TAT were found in 32% and 23% of APC-resistant individuals vs 4% in controls. The levels of these markers tended to be particularly elevated in three homozygous subjects. A significant positive correlation between F1+2 and TAT was present in APC-resistant individuals. No relationship between marker values and the previous occurrence of thrombotic episodes was found. Therefore, by measuring F1+2 and TAT a state of biochemical hypercoagulability has been identified in about one-third of APC-resistant individuals. This frequency is similar to that previously observed in comparable individuals with inherited deficiencies of protein C and protein S, which are usually associated with a stronger thrombotic tendency than APC-resistant individuals.
Reactivity to β2 Glycoprotein I Clearly Differentiates Anticardiolipin Antibodies from Antiphospholipid Syndrome and Syphilis
Authors: Forastiero R R , Martinuzzo M E , Kordich L C , Carreras L O
Published: July 2018

Anticardiolipin antibodies (aCL) detected by standard ELISA are found in association with autoimmune and infectious diseases. It is now recognized that P2 glycoprotein I (β2GPI) is a cofactor for aCL binding to cardiolipin (CL). To examine differences in cofactor requirements, aCL positive sera from patients with the antiphospholipid syndrome (APS) and syphilis were studied. Using an ELISA with human purified P2GPI adsorbed onto irradiated plates, we detected high binding activity in 29 out of 35 samples from APS patients and low in only 1 out of 37 aCL positive syphilis sera. Moreover, a good correlation (r = 0.79) was also observed in the former group between aCL and anti β2GPI. Whole IgG and affinity purified IgG aCL from APS patients did not bind to CL in the absence of β2GPI, but recognized β2GPI on irradiated plates in the absence of phospholipids. In contrast, IgG purified from syphilis patients only bound to CL alone. Taken together, these data indicate that performing both ELISA (aCL and anti β2GPI) it could be possible to distinguish aCL from autoimmune or infectious diseases.
Autoantibodies to Phospholipid-binding Plasma Proteins in Patients with Thrombosis and Phospholipid-reactive Antibodies
Authors: Pengo V , Biasiolo A , Brocco T , Tonetto S , Ruffatti A
Published: July 2018

Anti-phospholipid (aPL) antibodies are defined as antibodies detected in systems employing phospholipids (PL). This general definition is misleading as it comprises a large group of autoimmune phospholipid-reactive antibodies that are directed against specific phospholipid-binding plasma proteins, such as β2-glycoprotein I (β2GPI) and prothrombin. Definition of phospholipid-reacting antibodies according to the plasma protein against which they are directed appears more appropriate and could be useful in understanding clinical events and pathogenic mechanisms. Using ELISA systems we have studied the presence of antibodies directed against specific phospholipid-binding proteins in a series of 22 patients with thrombosis and phospholipid-reactive antibodies of the IgG isotype. High levels of anti-β2GPI IgG were detected in all 22 patients. Normal values were calculated on the basis of OD values at 405 nm (OD405) obtained for 22 age- and sex-matched healthy subjects (cut off value = 0.401). Levels of anti-β2GPI antibodies were linearly correlated with those of cardiolipin-reactive (aCL) antibodies. Eleven out of 22 patients (50%) had values of anti-prothrombin antibodies exceeding the cut-off value of 0.250. No relationship was found between the levels of anti-β2GPI and anti-prothrombin antibodies. Tests for antibodies against two natural inhibitors of blood coagulation, protein C and protein S, revealed elevated levels of anti-protein C IgG and anti-protein S IgG in 4 and 12 patients, respectively. A highly significant correlation between anti-protein C IgG and anti-protein S IgG values as well as between antibody titers against the two studied natural coagulation inhibitors and anti-prothrombin IgG was found. When comparing patients positive for aCL and presence or absence of a previous thrombotic episode (aCL+/T+ vs aCL+/T-), the positivity of anti-P2GPI IgG was found to be statistically associated with thrombosis. Conversely, among patients with previous thromboembolism with or without aCL (aCL+/T+ vs aCL-/T+) the positivity of anti-β2GPI IgG was strictly associated with the positivity of aCL, thus identifying the aPL antibody syndrome. These data demonstrate that anti-β2GPI antibodies are a marker of “autoimmune” thrombosis. Anti-prothrombin antibodies are not a marker of thrombosis and are closely associated with antibodies to protein C and protein S.
Simple and Rapid Detection of Factor V Leiden by Allele-specific PCR Amplification
Authors: Blasczyk Rainer , Ritter Markus , Thiede Christian , Wehling Jenny , Hintz Günter , Neubauer Andreas , Riess Hanno
Published: July 2018

Resistance to activated protein C is the most common hereditary cause for thrombosis and significantly linked to factor V Leiden. In this study, primers were designed to identify the factor V mutation by allele-specific PCR amplification. 126 patients with thromboembolic events were analysed using this technique, PCR-RFLP and direct sequencing. The concordance between these techniques was 100%. In 27 patients a heterozygous factor VGln506 mutation was detected, whereas one patient with recurrent thromboembolism was homozygous for the point mutation. Due to its time- and cost-saving features allele-specific amplification should be considered for screening of factor VGln506.
Increased Activation of the Haemostatic System in Men at High Risk of Fatal Coronary Heart Disease
Authors: Miller George J , Bauer Kenneth A , Barzegar Samad , Cooper Jacqueline A , Rosenberg Robert D
Published: July 2018

The haemostatic system was examined in 2951 men aged 50 to 61 years, clinically free of cardiovascular disease, who were ranked according to a risk score for fatal coronary heart disease (CHD). Risk was judged from their serum cholesterol concentration, systolic blood pressure, body mass index and smoking habit. The status of the factor VH-tissue factor pathway was estimated from the plasma levels of factor VII coagulant activity, factor VII antigen and activated factor VII. Activation of factor IX was assessed from the plasma concentration of factor IX activation peptide. Activity within the common pathway was measured as the plasma concentrations of prothrombin fragment 1+2 and fibrinopeptide A. All 6 markers of haemostatic status were positively and statistically significantly associated with risk, providing further evidence for a hypercoagulable state in men at high risk for fatal CHD. Plasma fibrinogen and serum triglyceride concentrations were also graded positively with risk.
Detection of Circulating Tissue Factor and Factor VII in a Normal Population
Authors: Albrecht Sybille , Kotzsch Matthias , Siegert Gabriele , Luther Thomas , Großmann Heinz , Großer Marianne , Müller Martin
Published: July 2018

The plasma tissue factor (TF) concentration was correlated to factor VII concentration (FVIIag) and factor VII activity (FVIIc) in 498 healthy volunteers ranging in age from 17 to 64 years. Immunoassays using monoclonal antibodies (mAbs) were developed for the determination of TF and FVIIag in plasma. The mAbs and the test systems were characterized. The mean value of the TF concentration was 172 ± 135 pg/ml. TF showed no age- and gender-related differences. For the total population, FVIIc, determined by a clotting test, was 110 ± 15% and the factor VIlag was 0.77 ± 0.19 μg/ml. FVII activity was significantly increased with age, whereas the concentration demonstrated no correlation to age in this population. FVII concentration is highly correlated with the activity as measured by clotting assay using rabbit thromboplastin. The ratio between FVIIc and FVIIag was not age-dependent, but demonstrated a significant difference between men and women. Between TF and FVII we could not detect a correlation.
Enhanced Platelet Aggregability under High Shear Stress after Treadmill Exercise in Patients with Effort Angina
Authors: Tokuue Junya , Hayashi Junichi , Hata Yoshiya , Nakahara Kazuhiko , Ikeda Yasuo
Published: July 2018

Platelet activation induced by shear forces occurring in a stenosed coronary artery is one of the mechanisms of coronary thrombosis. We evaluated the shear-induced platelet aggregation (SIPA) dynamics in patients with effort angina during treadmill exercise. SIPA was measured by a rotational cone-plate aggregometer. SIPA was markedly increased by exercise from 71.2 ± 8.9% to 81.9 ± 7.6% (p <0.01) in the patient group. Although epinephrine concentrations were elevated, its rate of increase was not correlated with that of SIPA. Yohimbine partially inhibited the exercise-induced increase in SIPA. In contrast, a significant correlation between the changing rate of plasma von Willebrand factor (vWF) larger multimers and that of SIPA (r = 0.74, p <0.05) was observed. Exercise-augmented SIPA is probably dependent on an increase in vWF larger multimers rather than platelet alpha2-receptor activation. Prevention of the interaction between vWF and its platelet receptors may play some role in decreasing the risk of coronary thrombosis during exercise.
Assessment of a Bleeding Risk Index in Two Cohorts of Patients Treated with Oral Anticoagulants
Authors: van der Meer F J M , Rosendaal F R , Vandenbroucke J P , Briët E
Published: July 2018

In two cohorts of patients on oral anticoagulant therapy, routinely treated by the Leiden Thrombosis Service, the frequency of major bleeding complications was assessed during two years (1988 and 1991). With Poisson regression analysis the influence of the risk factors age, sex, target zone, achieved INR and type of coumarin derivative used were determined. Subsequently, a bleeding risk index was calculated, combining the results of the two cohorts. For various types of patients the relative risk of major bleeding complications was assessed.

Age and achieved INR were the most important and consistent risk factors: rate ratio (RR) for age in 1988 1.46 per 10 years increase (95% confidence interval [Cl] 1.20-1.78) and in 1991 1.57 per 10 years increase (95% Cl 1.23-2.00); RR per unit increase in achieved INR in 1988 1.42 (95% Cl 1.21-1.68) and in 1991 1.44 per unit increase in achieved INR (95% Cl 1.18-1.74). Two methods were used to combine the results of 1988 and 1991. In the first method the mean bleeding risk index was calculated: In (incidence) [natural logarithm of the incidence rate of major bleeding] = -5.64 +0.42 * age +0.26 % sex -0.29 * target range +0.36 % achieved INR -0.36 * coumarin type. In the second method only the consistent risk factors age and achieved INR were used: In (incidence) = -5.64 +0.42 % age +0.36 % achieved INR. These bleeding risk indexes can be used to assess the risk of major bleeding complications of individual patients and allow more individualized care by individual tailoring the desired anticoagulation.
Mutation in the Gene Coding for Coagulation Factor V and Resistance to Activated Protein C: Detection of the Genetic Mutation by Oligonucleotide Ligation Assay Using a Semi-automated System
Authors: Zotz Rainer B , Maruhn-Debowski Beate , Scharf Rüdiger E
Published: July 2018

Resistance of coagulation factor Va to inactivation by activated protein C (APCR) is associated with a point mutation in which adenine is substituted for guanine at nucleotide 1691 in the gene coding for factor V (FV Leiden). To date, this mutation of factor V is the most frequent genetic risk factor for venous thrombophilia. In this report, we describe the adaptation of an automatable oligonucleotide ligation assay (OLA) to detect the mutation in polymerase chain reaction-amplified DNA samples from 40 normal, 20 affected heterozygous, and 3 affected homozygous individuals. The genotypes determined by conventional allele-specific restriction enzyme site analysis were in complete concordance with the results obtained by ELISA-based oligonucleo-tide-ligation assay. The automated oligonucleotide ligation assay provides a rapid, reliable, nonisotopic method to detect the mutation responsible for APCR that can rapidly be applied to large population screening.
The International Standard for Plasminogen Activator Inhibitor-1 (PAI-1) Activity
Authors: Gaffney Patrick J , Edgell Tracey A
Published: July 2018

Since the finding that plasminogen activator inhibitor-1 (PAI-1) may influence the initiation and progression of acute myocardial infarction, the assay of PAI-1 in plasma using a variety of commercial kits has become commonplace. The need for a standard to define the activity of PAI-1 prompted an international collaborative study (ICS) to calibrate the functional potency of a lyophilised plasma PAI-1 preparation (92/654). Since PAI-1 inhibits the 2 major plasminogen activators, tissue-type plasminogen activator (t-PA) and urinary-type plasminogen activator (u-PA) in an equimolar manner it was important to establish the potency of the PAI-1 inhibitor in terms of both t-PA and u-PA neutralisation. While the ICS indicated a wide spread of data between the laboratories the mean value of 27.5 t-PA neutralisation units and 7.0 u-PA neutralisation units was confirmed by numerous assays at NIBSC using a tedious but technically reliable titration assay procedure. The plasma PAI-1 proposed standard (92/654) was stable at 20° C for 20 months.

The Fibrinolysis Subcommittee of the Scientific and Standardization Committee (SSC) of the International Society on Thrombosis and Haemostasis (ISTH), (meeting in Leuven, Belgium in September 1994) has recommended that the plasma PAI-1 (92/654) should be accepted as the International Standard for PAI-1 and should define a unitage in terms of both t-PA and u-PA neutralisation. Subsequently the Expert Committee on Biological Standardization of the World Health Organization (ECBS-WHO) meeting in Geneva, Switzerland in October 1995 approved plasma PAI-1 (92/654) as the International Standard.
Effects of NO-Donors on Thrombus Formation and Microcirculation in Cerebral Vessels of the Rat
Authors: Sasaki Yasuto , Seki Junji , Giddings John C , Yamamoto Junichiro
Published: July 2018

Sodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1), are known to liberate nitric oxide (NO). In this study the effects of SNP and SIN-1 on thrombus formation in rat cerebral arterioles and venules in vivo were assessed using a helium-neon (He-Ne) laser. SNP infused at doses from 10 Μg/kg/h significantly inhibited thrombus formation in a dose dependent manner. This inhibition of thrombus formation was suppressed by methylene blue. SIN-1 at a dose of 100 Μg/kg/h also demonstrated a significant antithrombotic effect. Moreover, treatment with SNP increased vessel diameter in a dose dependent manner and enhanced the mean red cell velocity measured with a fiber-optic laser-Doppler anemometer microscope (FLDAM). Blood flow, calculated from the mean red cell velocity and vessel diameters was increased significantly during infusion. In contrast, mean wall shear rates in the arterioles and venules were not changed by SNP infusion. The results indicated that SNP and SIN-1 possessed potent antithrombotic activities, whilst SNP increased cerebral blood flow without changing wall shear rate. The findings suggest that the NO released by SNP and SIN-1 may be beneficial for the treatment and protection of cerebral infarction
Identification of Quantifiable Hemodynamic Factors in the Assessment of Cerebral Aneurysm Behavior On behalf of the Subcommittee on Biorheology of the Scientific and Standardization Committee of the ISTH
Authors: Burleson Arrmelle C , Turitto Vincent T
Published: July 2018

Previous experimental and theoretical studies on the hemodynamics of saccular intracranial aneurysms have provided evidence that aneurysms tend to grow, thrombose and rupture when (1) wall shear stress and mural tension are increased compared to normal values, and (2) flow deviates from a laminar unidirectional pattern (for example flow recirculation). Aneurysm wall shear stress, however, is the only hemodynamic factor which has received special attention in terms of estimation. Additional flow-related parameters exist which could potentially bring increased insight into mechanisms for cerebral aneurysm behavior; they could also help categorize the severity of such malformations and design effective intravascular treatment techniques. The purpose of this paper is thus to present an overview of such hemodynamic factors that could assist in determining the geometries which present the greatest risks to patients. These parameters include (1) hemodynamic shear stress, (2) pressure and related stresses, (3) impingement force on the aneurysm wall, (4) inflow rate into the aneurysm, and (5) residence time of blood within the aneurysmal sac. In addition, these factors can also be currently estimated in an in vitro setting.
Two Mutations in Exon XII of the Protein Sα Gene in Four Thrombophilic Families Resulting in Premature Stop Codons and Depressed Levels of Mutated mRNA
Authors: Andersen Bente Damm , Lind Bent , Philips Malou , Hansen Annebirthe Bo , Ingerslev Jørgen , Thorsen Sixtus
Published: July 2018

Sixteen Danish unrelated thrombophilic families with plasma protein S deficiency of type I (or III) are currently under investigation in our laboratory for defects in the protein Sα gene. The present paper describes a part of this work, which deals with the identification and phenotypical presentation of two unique mutations in exon XII of the protein Sa gene in four of these families. The mutations were identified by SSCP screening followed by nucleotide sequence analysis or by direct nucleotide sequence analysis. The mutation found in one family (D) is a novel deletion of an A in either the codon for Gly448 (GGA) or Iie449 (ATT) resulting in a frameshift and a premature stop codon at position 454. The other mutation shared by three families (F, G and J) is a previously reported C → T transition within a hypermutable CG dinucleotide sequence converting Arg410 (CGA) to Stop (1GA). All affected individuals are heterozygotes for their mutation and in each family the protein S genotype, the plasma protein S phenotype (not shown for Family J) and the clinical phenotype cosegregate. The two mutations can fully explain the abnormal protein S phenotype since premature stop codons are known to disrupt gene function of the mutated allele. Analysis of protein S mRNA from platelets showed that both mutations result in a marked reduction in the amount of protein S mRNA from the mutated alleles indicating that the mutations exert their deleterious effects on gene expression at the transcriptional level. The Arg410 → Stop mutation in Families F, G and J is in all instances linked to a G at the site of a common neutral dimorphism in the codon for Pro626 (CCA/G) in exon XV. This indicates that the mutation in these families could have arisen in a common ancestor. The Arg410 (CGA) → Stop (1GA) mutation is also seen in exon XII of the normal protein Sa gene. This gives rise to the speculation as to whether the mutation in the protein Sa gene is the result of an interaction with the protein Sβ gene leading to double homologous unequal crossing-over or gene conversion of a short DNA sequence. However, this is unlikely since none of the 7 other protein S β-specific nucleotides are present in the mutated exon XII sequence of the protein Sα gene. The common Arg506 → Gin Leiden mutation in coagulation factor V is not an additional risk factor for thrombosis in any of the four families studied.
Combined Hereditary Disorders of Haemophilia B Leyden (-6 G → A) and Type 1 von Willebrand Disease
Authors: Pernod Gilles , Vinciguerra Christine , Gaucher Christine , Mazurier Claudine , Polack Benoît , Négrier Claude
Published: July 2018

Multiple coagulation disorders are unusual. We report here a combination of haemophilia B Leyden with type 1 von Willebrand disease (vWD) affecting different members of the same family. Haemophilia B Leyden was due to a -6 G → A mutation within the promoter of the factor IX gene and was responsible for a mild haemophilia in the father of the proband. The proband and her sister (age 4 and 6) exhibited a twofold lower level of factor IX activity (0.4 IU/ml) than the paternal grandmother (0.95 IU/ml). The differences in FIX levels in the three carriers of the same -6 G → A mutation suggest the implication of an age-related mechanism responsible for the increase in factor IX plasma level. Haemophilia B Leyden patient and carriers suffered also from a mild von Willebrand disease. The diagnosis of this associated type 1 vWD was performed by assaying plasma von Willebrand factor together with multimer electrophoretic studies and DDAVP test. The inheritance of this vWD was investigated by haplotype analysis of the vWF gene. Individuals affected by such an association are actually asymptomatic, but per- and post-operative bleeding risk remains to be evaluated.
Regulation of Serum Thrombopoietin Levels by Platelets and Megakaryocytes in Patients with Aplastic Anaemia and Idiopathic Thrombocytopenic Purpura
Authors: Ichikawa Naoaki , Ishida Fumihiro , Shimodaira Shigetaka , Tahara Tomoyuki , Kato Takashi , Kitano Kiyoshi
Published: July 2018

To clarify the regulatory mechanism of thrombopoietin (TPO, c-Mpl ligand) in chronic thrombocytopenic conditions, we determined TPO levels in the sera of patients with aplastic anaemia (AA; n = 26) and idiopathic thrombocytopenic purpura (ITP; n = 32) by an enzyme-linked immunosorbent assay. Despite a similarity in platelet counts, serum TPO levels in the AA group were markedly higher than those in the ITP group: 20.41 ± 9.71 f mol/ml (mean ± SD) and 1.66 ± 0.55 f mol/ml, respectively, both of which were significantly elevated compared to normal subjects (n = 41; 1.22 ± 0.37). In both groups, serum TPO level showed an inverse correlation with the platelet count. We determined the megakaryocyte volume using bone marrow clot section and found that it was markedly small in the AA group; while in the ITP group it was augmented with a correlation to serum TPO level. Our findings suggest that TPO levels may be regulated not only by platelets but also megakaryocytes in AA and ITP.
Once-daily Subcutaneous Dalteparin, a Low Molecular Weight Heparin, for the Initial Treatment of Acute Deep Vein Thrombosis
Authors: Fiessinger J N , Lopez-Fernandez M , Gatterer E , Granqvist S , Kher A , Olsson C G , Söderberg K
Published: July 2018

The aim of the study was to compare the efficacy and safety of once-daily subcutaneous injection of dalteparin, a low molecular weight heparin, with that of intravenous unfractionated heparin in the treatment of deep venous thrombosis (DVT). Patients were included if they had deep venous thrombosis distal to inguinal ligament and were randomised either before, if it was considered necessary, or after phlebographic verification of the diagnosis. There was no pre-inclusion treatment with unfractionated heparin. One hundred and twenty patients received dalteparin, administered subcutaneously once-daily at a fixed dose of 200 IU anti-factor Xa/kg, and 133 patients received a continuous intravenous infusion of unfractionated heparin (UFH). Oral anticoagulation was started on the first or second day, and initial treatment with dalteparin or UFH discontinued when the prothrombin time was in the therapeutic range (2<INR<3) on two consecutive days. Control phlebograms were taken within 4 days, thereafter. There were no significant differences between the two initial treatment groups in improvements in Marder score. Two major bleeding events occurred in the UFH group versus none in the dalteparin group. One patient in each group experienced clinically significant pulmonary embolism. During a mean follow-up period of 6.9 ± 1.5 months, recurrent DVT occurred in four patients in the dalteparin group and in two of the UFH group. These results confirm those of a previous study on dalteparin in the initial treatment of DVT, and suggest that dalteparin administered once-daily at a fixed dose of 200 UI/kg is as effective and well-tolerated as UFH in patients with DVT below the inguinal ligament. The present study also demonstrates that dalteparin can be started as soon as the diagnosis of DVT is suspected and without pre-treatment with UFH. Given that the administration of once-daily subcutaneous injections needs not require a patient to be hospitalised, studies to investigate the possibility of using dalteparin for the initial treatment of DVT in the outpatient setting are warranted.
Factor V Enhances the Cofactor Function of Protein S in the APC-Mediated Inactivation of Factor VIII: Influence of the Factor VR506Q Mutation
Authors: Váradi K , Rosing J , Tans G , Pabinger I , Keil B , Schwarz H P
Published: July 2018

Factor V and protein S are cofactors of activated protein C (APC) which accelerate APC-mediated factor VIII inactivation. The effects of factor V and protein S were quantitated in a reaction system in which plasma factor VIII was inactivated by APC and the loss of factor VIII activity was monitored in a factor X-activating system in which a chro-mogenic substrate was used to probe factor Xa formation. Factor V increased the rate of APC-mediated factor VIII inactivation in a dose-dependent manner in representative plasma samples with protein S or factor V deficiency, abnormal factor V (heterozygous or homozygous for factor VR506Q), or a combination of heterozygous protein S deficiency and heterozygous factor VR506Q. This effect was much less pronounced in the plasma samples with a decreased protein S level, but the impaired response in these plasmas was corrected by addition of protein S, indicating that both factor V and protein S are required for optimal inactivation of factor VIII by APC. The effects of factor V and protein S were also studied in a reaction system with purified proteins. APC-catalysed factor VIII inactivation was enhanced 3.7-fold in the presence of 1.1 nM factor V and 1.5-fold in the presence of 2.4 nM protein S. When both 1.1 nM factor V and 2.4 nM protein were present the rate enhancement was 11-fold. Factor V is a more potent cofactor than protein S, as can be concluded from the fact that 0.04 nM factor V gave the same stimulation as 2.4 nM protein S. Protein S lost its cofactor function after complexation with C4b binding protein, which indicates that it is free protein S that acts as a cofactor. To investigate the effect of the R506Q mutation in factor V on APC-mediated factor VIII inactivation, factor V was purified from the plasma of patients homozygous for factor VR506Q. In the absence of protein S, factor VR506Q did not enhance factor VIII inactivation by APC, but in the presence of 2.4 nM protein S a slight enhancement was observed. The APC cofactor activity of factor V was lost when factor V was activated with thrombin or with the factor V activator from Russell’s viper venom. These data indicate that optimal inactivation of factor VIII by APC requires the presence of an intact factor V molecule and free protein S.
Probenecid Inhibits Platelet Responses to Aggregating Agents in Vitro and Has a Synergistic Inhibitory Effect with Penicillin G
Authors: Packham M A , Rand M L , Perry D W , Ruben D H , Kinlough-Rathbone R L
Published: July 2018

Probenecid is an anion channel blocker and uricosuric agent, originally developed to slow the rate of excretion of penicillin. It is now also administered with many other drugs to reduce their required dosages. Recently, probenecid (2.5 mM) has been used to prevent leakage of fura-2 or fluo-3 when these indicators of cytosolic Ca2+ levels have been introduced into cells. However, we found that probenecid markedly inhibited the increases in cytosolic Ca2+ caused by ADP, thrombin, the thrombin receptor-activating peptide (SFLLRN, TRAP), ADP, sodium arachidonate, the thromboxane A2 (TXA2) mimetic U46619, and platelet-activating factor (PAF). This finding precluded the use of probenecid with platelets in measurements of cytosolic Ca2+ with indicators such as fura-2. We then investigated the effects of probenecid on aggregation and release of 14C-serotonin from prelabeled platelets. Responses to all the agonists were inhibited by 2.5 mM probenecid, but concentrations as low as 0.25-0.5 mM inhibited responses to agonists that act largely via TXA2 (collagen, sodium arachidonate and U46619). Collagen-induced TXA2 formation was inhibited in a dose-dependent manner. Responses of aspirin-pretreated platelets to thrombin, SFLLRN, U46619 and PAF were also inhibited by probenecid, indicating that prevention of TXA2 formation does not account for all the inhibitory effects. The combination of probenecid with penicillin G produced additive or synergistic inhibition of platelet responses; responses dependent on TXA2 were synergistically inhibited by concentrations of the drugs that are reached in vivo. The synergistic inhibitory effect of probenecid on platelet functions could further impair hemostasis if it has already been partially compromised by the administration of other drugs.
Elevated Circulating P-Selectin in Insulin Dependent Diabetes Mellitus
Authors: Jilma Bernd , Fasching Peter , Ruthner Christine , Rumplmayr Anna , Ruzicka Sabine , Kapiotis Stylianos , Wagner Oswald F. , Eichler Hans-Georg
Published: July 2018

Based on findings that showed increased P-selectin expression on platelets and on choroidal microvessels of patients with insulin dependent diabetes mellitus (IDDM), we hypothesized that also plasma concentrations of circulating (c)P-selectin would be increased in these patients.

The aim of this study was to compare the plasma levels of cP-selec-tin between non-smoking patients with IDDM, treated with an intensified insulin therapy, and healthy controls. The study design was prospective, cross-sectional and analyst-blinded. Subjects were matched individually for sex, age and body mass index. Plasma levels of cP-selectin and of von Willebrand antigen (vWF-Ag) were determined by enzyme linked immunoassays.

Forty-two pairs were available for intergroup comparison. Median plasma concentrations of cP-selectin in patients with IDDM (285 ng/ml; interquartile range: 233-372) were on average 21% higher than those of controls (236 ng/ml; interquartile range: 175-296; p = 0.004). Also, median plasma levels of vWF-Ag were 10% higher in patients (96 U/dl; interquartile range: 82-127) than controls (87 U/dl; interquartile range: 70-104; p = 0.025). There was no correlation between plasma concentrations of cP-selectin and vWF-Ag levels in either group (p ώ0.05).

In conclusion, our results of increased cP-selectin levels are in line with increased P-selectin expression on platelets and on choroidal microvessels found in patients with IDDM. In view of the currently developed small molecule inhibitors of cell adhesion molecules, these independent observations together may provide a sound rationale to select P-selectin as a target for treating or preventing IDDM-associated micro- or macrovascular complications.
Effect of Heparin on the Activation of Factor XI by Fibrin-bound Thrombin
Authors: von dem Borne Peter A Kr , Meijers Joost C M , Bouma Bonno N
Published: July 2018

Fibrin-bound thrombin is protected from inactivation by antithrombin III, while its coagulant potential is retained. In the presence of heparin, ternary complexes between thrombin, fibrin and heparin are formed. In these complexes the coagulant activity of thrombin is retained, whereas the anticoagulant activity of fibrin-bound heparin is neutralized. The limited effectiveness of heparin in the prevention of both venous thrombosis and coronary reocclusion is probably related to the protective effect of fibrin on the inactivation of thrombin by antithrombin III. Recently, it has been shown that factor XI can be activated by thrombin, resulting in the generation of additional thrombin via the intrinsic pathway. This additional thrombin is capable of stabilizing the clot by protecting it from fibrinolysis. We studied the effect of heparin on the activation of factor XI by fibrin-bound thrombin. First, we used fibrin monomers coupled to Sepharose to which thrombin and unfractionated heparin (UFH) were bound. Factor XI activation by thrombin was the same in the presence of fibrin-Sepharose or control-Sepha-rose. The addition of heparin (0.1 U/ml) resulted in a 91 and 15-fold enhancement in the presence of control-Sepharose and fibrin-Sepharose, respectively. Next, we added complexes of heparin, thrombin and fibrin monomer to factor XII and XI double-deficient plasma in the presence or absence of a reconstituting amount of factor XI. In the presence of factor XI, additional fibrin formation was observed indicating that factor XI activation by thrombin in complex with fibrin and heparin can take place in plasma. We then studied the effect of other heparin-like anticoagulants on the thrombin-mediated factor XI activation. UFH enhanced thrombin-mediated factor XI activation 68-fold, LMWH (low molecular weight heparin, Fragmin) 12-fold, danaparoid (Orgaran) 3-fold, while the pentasaccharide ORG 31540 did not result in an enhancement. Binding studies of these anticoagulants to fibrin-Sepharose showed that LMWH bound with approximately the same affinity as UFH, while danaparoid and the pentasaccharide did not bind to fibrin.

We conclude that fibrin-bound thrombin is capable of factor XI activation. Furthermore, heparin bound in a complex with fibrin can act as a cofactor for this activation. This factor XI activation capacity may play a role in the limited effectiveness of heparin. Provided that thrombin-mediated factor XI activation plays an important role in vivo, danaparoid and especially the pentasaccharide may be better anticoagulants than UFH and LMWH.
Long-term Treatment with Growth Hormone Decreases Plasminogen Activator Inhibitor-1 and Tissue Plasminogen Activator in Growth Hormone-deficient Adults
Authors: Johansson Jan-Ove , Landin Kerstin , Johannsson Gudmundur , Tengborn Lilian , Bengtsson Bengt-Åke
Published: July 2018

The syndrome of growth hormone deficiency (GHD) in adults is associated with premature atherosclerosis, increased cardiovascular mortality, abnormal lipoprotein patterns and abnormal body composition. We have previously shown that GH-deficient adults have increased concentrations of fibrinogen and plasminogen activator inhibitor (PAI-1) activity.

The aim of the present investigation was to study coagulation and fibrinolysis in 17 patients with adult-onset GHD during two years of treatment with recombinant human GH (12 μg/kg body weight/day). The impact of the contemporary changes in metabolic variables and body composition on coagulation and fibrinolysis was studied. The patients received conventional thyroid, adrenal and gonadal hormone replacement therapy.

PAI-1 activity, PAI-1 antigen and tissue plasminogen activator (t-PA) antigen levels decreased during the GH treatment period (p <0.05). The decrease was more pronounced in patients with high pre-treatment levels of the different variables. a2-antiplasmin decreased (p <0.05), while plasminogen was unchanged during two years of GH treatment.

Fibrinogen concentrations tended to decrease after two years of GH treatment (p = 0.06), while the coagulation factors VII and VIII were unchanged, von Willebrand factor demonstrated a transient decrease after 18 months of GH treatment. The coagulation inhibitor, protein C, decreased (p <0.05), while antithrombin was unchanged.

Fasting plasma insulin increased (p <0.01), but blood glucose did not differ after two years of GH treatment. Serum high-density lipoprotein cholesterol, total cholesterol and triglycerides were unaltered. Body fat decreased during the initial GH treatment but was unaltered after two years, while lean body mass increased (p <0.001) and the waist over hip circumference ratio tended to decrease (p = 0.06).

In conclusion, PAI-1 activity, PAI-1 antigen and t-PA antigen decreased during long-term GH treatment. These changes may be a direct effect of GH itself or may be secondary to the favourable changes in body composition. It remains to be seen whether changes in these fibrinolytic variables during rhGH treatment reduces the cardiovascular risk in patients with GHD. The present results suggest that GH plays a role in the regulation of fibrinolysis.
Ca2+-dependent Activation of the 33-kDa Protein Kinase Transmits Thrombin Receptor Signals in Human Platelets
Authors: Ido Masaru , Kato Shinya , Ogawa Hiroyuki , Hayashi Kenji , Komada Yoshihiro , Zhang Xao Li , Sakurai Minoru , Suzuki Koji
Published: July 2018

Thrombin stimulation induces a dramatic increase in the activity of the 33-kDa serine/threonine kinase (PK33) in human platelets (10). The Arg-Gly-Asp (RGD) peptide, an inhibitor of the thrombin-mediated aggregation of platelets, did not affect the PK33 activation induced by thrombin suggesting that the activation of this kinase occurs independently from platelet aggregation. To identify a potential role of Ca2+ and calmodulin in the regulation of PK33, the effect of several Ca2+/calmodulin inhibitors on the thrombin-induced activation of PK33 was assessed using denaturation/renaturation method. Pretreatment of platelets with EGTA decreased the maximum PK33 activity induced by thrombin. The chelation of both the extra- and the intracellular Ca2+ by EGTA and by acetoxymethyl ester of 5,5′ -dimethyl-bis-(<9-aminophen-oxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA-AM) decreased further the PK33 activation by thrombin. Preincubation of platelets with the anticalmodulin agent, N-(4-aminobutyl)-5-chloro-2-naphtha-lenesulfonamide (W13), inhibited markedly the activation of PK33 by thrombin, whereas the inactive structural analog N-(4-aminobutyl)-2-naphthalenesulfonamide (W12) and the myosin light chain kinase inhibitor 1 -(5-chloronaphthalene-1 -sulfonyl)-1 H-hexahydro-1,4-diaze-pine (ML9) showed very weak inhibitory effects. Treatment of resting platelets with the calcium ionophore, A23187, activated PK33 in a dose-dependent manner; phorbol 12-myristate 13-acetate enhanced this effect. However, the two foregoing agents did not induce similar degree of PK33 activities as thrombin. These results indicate that the activation of PK33 is independent of the formation of the GPIIb/IIIa-fibrinogen complex and that it might be regulated by a Ca2+-dependent pathway.
Triglyceride Dependence of Factor VII Coagulant Activity in Deep Venous Thrombosis
Authors: Mukherjee Manjari , Dawson Gloria , Sembhi Kuldip , Kakkar Vijay V
Published: July 2018

The effect of incubation of plasma with lipoprotein lipase on factor VII coagulant (FVII: C) activity was examined in 40 patients, 22 male and 18 female, aged 28 to 77 years, with history of venograph-ically proven deep venous thrombosis (DVT). While the mean (± SEM) FVII: C activity of the 40 patients was 100.9 ± 4.1%, 19 patients had FVII: C activity less than 100%, 11 had 100 to 120% activity and 10 patients had greater than 120% FVII:C activity. The mean triglyceride level of all the patients was 84.0 ± 6.5 mg/dl. The FVII: C activity correlated significantly with triglyceride (r = 0.36; n = 40; p = 0.021). There was about 30% average loss of FVII: C activity upon incubation of plasma with lipoprotein lipase. The mean activity loss increased from 23.8% to 31.5% and 42.6% in patients whose FVII:C activity levels were less than 100%, between 100 and 120% and more than 120% respectively, the variation in the means being statistically significant (p <0.001). While according to current opinion, FVII:C activity represents the total FVII mass (FVII plus FVIIa) and activity state, the present findings demonstrate a lipid dependence of FVII: C activity, and raises the possibility of a therapeutic option of controlling FVII: C by controlling triglyceride levels.
A Heparin Cofactor II Mutation (HCII Rimini) Combined with Factor V Leiden or Type I Protein C Deficiency in Two Unrelated Thrombophilic Subjects
Authors: Bernardi F , Legnani C , Micheletti F , Lunghi B , Ferraresi P , Palareti G , Biagi R , Marchetti G
Published: July 2018

305 patients with juvenile thromboembolic episodes were screened for the presence of heparin cofactor II deficiency. The heterozygous deletion of two bases was found in the exon 5 of the heparin cofactor II gene in two unrelated patients, very likely due to a founder effect. This molecular lesion, causing a frameshift and elongated translation, affects the core of the molecule and should cause the complete unfolding of the protein, which is in accordance with the observed type I deficiency. The corresponding region of antithrombin III gene is affected by a cluster of frameshift mutations suggesting that heparin cofactor II and antithrombin III could share similar mutational patterns.

The heparin cofactor II gene alteration was associated with, in one patient, the factor V Leiden mutation and, in the other, type I protein C deficiency. The tracing of the single defects in several family members indicated that the mutations became clinically manifest only when present in the doubly heterozygous condition. This study provides two examples, based on molecular findings, of the interplay of risk factors which is potentially useful to define a role for heparin cofactor II deficiency in inherited thrombophilia.
Risk Factor Profiles in Patients with Different Clinical Manifestations of Venous Thromboembolism: A Focus on the Factor V Leiden Mutation
Authors: Manten Bert , Westendorp Rudi G J , Koster Ted , Reitsma Pieter H , Rosendaal Frits R
Published: July 2018

Background. Patients with venous thromboembolic disease may present with different clinical manifestations. Factor V Leiden mutation leading to resistance to activated protein C is associated with a sevenfold increased risk for presenting with deep-vein thrombosis. It is not yet established whether carriers of the mutation have a similarly increased risk for manifesting with pulmonary embolism.

Methods. From an Anticoagulation Clinic monitoring coumarin therapy, a consecutive series of patients with a first thromboembolic event (objectively proven by current radiological methods) were enrolled. All patients were interviewed and blood was drawn for geno-typing. From the hospital charts and the personal interview, information was obtained on acquired risk factors and the signs and symptoms on hospital admission.

Results. 45 patients presented with symptoms of pulmonary embolism only, 211 had only symptoms of deep-vein thrombosis whereas 23 had clinical features of both. In about half of the patients acquired risk factors for venous thromboembolism were present which did not differ between the three groups of patients. Recent surgery had been performed more often in patients presenting with pulmonary embolism than in other patients (33.3% vs. 18.5%, p <0,05). Factor V Leiden was present in 9% of the patients presenting with pulmonary embolism (relative risk: 3.3 95% Cl: 1.0-10.6) and 17% of the patients presenting with deep-vein thrombosis (relative risk: 6.9 95% Cl: 3.6-12.8). The prevalence of factor V Leiden was intermediate in patients with both clinical characteristics.

Conclusion. These data suggest that patients with venous thromboembolism have different clinical presentation depending on the risk factor profile. Factor V Leiden may preferentially lead to manifest deep-vein thrombosis. Differences in structure of venous thrombi could underlie differences in embolic tendency.
Plasmin Is a Specific Stimulus of the 5-Lipoxygenase Pathway of Human Peripheral Monocytes
Authors: Weide Ingo , Tippler Bettina , Syrovets Tatjana , Simmet Thomas
Published: July 2018

The objective of this study was to characterize the plasmin-induced stimulation of leukotriene (LT) B4 biosynthesis in human peripheral monocytes (PM). Plasmin up to 175 × 10-3 CTA U/ml triggers a concentration-dependent release of 5-lipoxygenase-derived LTB4 while release of the cyclooxygenase products thromboxane (TX) B2 and prostaglandin (PG) E2 remained unaffected. The stimulatory effect appeared to be specific in as much as 1) it was found in PM, but not in polymorphonuclear neutrophils (PMN), 2) it requires the lysine binding sites of plasmin molecule since it was inhibited by the lysine analogues 6-aminohexanoic acid (6-AHA) and trans-4(aminometh-yl)cyclohexane-l-carboxylic acid (t-AMCA), 3) the intact catalytic center of plasmin is required since neither plasminogen nor catalytic center-blocked plasmin share the stimulatory effect of active plasmin, 4) other serine proteases such as a-chymotrypsin, human neutrophil elastase and cathepsin G did not stimulate release of detectable amounts of LTB4 from PM. In addition, catalytic center-blocked plasmin antagonized the stimulatory effect of active plasmin. Plasmin-mediated monocyte activation apparently proceeds via a pertussis toxin-sensitive G protein. Plasmin did not increase inositol (1,4,5) trisphosphate levels, but a time- and concentration-dependent stimulation of cyclic GMP formation was observed. The data show that plasmin is a specific stimulus for human peripheral monocytes. Plasmin may be an important link between the coagulation cascade and inflammatory reactions.