bluuebunny/crossref_metadata_2025 huggingface dataset:
Monitoring Hypocoagulant Conditions in Rat Plasma: Factors Determining the Endogenous Thrombin Potential of Tissue Factor-Activated Plasma
Authors: Nieuwenhuys Cécile M. , Feijge Marion A. , Béguin Suzette , Heemskerk Johan W.
Published: March 2018

Automated human plasma, continuous monitoring of the formation and inactivation of thrombin during the coagulation process provides an adequate way to detect hypo- and hypercoagulant conditions. Here, we describe an analogous procedure to determine the endogenous thrombin potential (ETP), i. e. the free thrombin concentration-time integral, of coagulating rat plasma. When activated with tissue factor, the ETP of plasma from Wistar rats was comparable to the ETP of human plasma, in spite of a relatively short half-life time of free thrombin in rat plasma. The ETP was highly sensitive to heparin as well as to administration of vitamin K antagonist or feeding of the animals with a vitamin K-deficient diet. In plasma that was activated under sub-optimal conditions (reduced levels of tissue factor or vitamin K-dependent coagulation factors), the ETP increased with the rate of thrombin formation in the first minutes of the coagulation process. Since both parameters are dependent of the prothrombin concentration, it appears that this level plays an important role in determining both the initial and total activity of the coagulation system. Thus, automated measurement of free thrombin during the coagulation process of rat plasma allows a detailed analysis of hypocoagulability in this animal model.
Immunoassays for the Quantitation of Porcine PAI-1 Antigen and Activity in Biological Fluid Samples
Authors: Leng Henry M. , Brouwers Els , Knockaert Isabelle , Declerck Paul
Published: March 2018

Two monoclonal antibody-based enzyme-linked immunosorbent assays (ELISAs) for the quantitation of porcine plasminogen activator inhibitor-1 (PAI-1) antigen and activity in plasma were constructed and validated. The intra-assay, interassay, and interdilution coefficients of variation were 4.3, 13, and 8%, respectively, for the antigen ELISA and 5, 16, and 11% for the activity assay. Assay recoveries, in the antigen ELISA, of either latent or active recombinant porcine PAI-1 (10 and 50 ng/ml) added to plasma were 86 ± 9% and 92 ± 22%, respectively, for the latent form and 89 ± 9% and 87 ± 7% for the active form (mean ± SD, n = 3 to 4). In the immunofunctional assay, recoveries for the same concentrations of active PAI-1 were 108 ± 16% and 92 ± 21%, respectively. In male porcine plasma the level of PAI-1 antigen was 31 ± 11 ng/ ml and the activity, 34 ± 16 ng/ml (mean ± SD, n = 10). In female plasma PAI-1 antigen levels were 20 ± 5.2 ng/ml and the PAI-1 activity 42 ± 17 ng/ml (n = 13). A linear correlation was found between PAI-1 antigen and activity levels in male (r = 0.60) and female (r = 0.70) plasma. Immunodepletion resulted in a decrease of >95% of the original PAI-1 antigen or activity levels. Incubation of plasma samples at 37° C for 16 h resulted in a significant decrease (70 to 85%) of PAI-1 activity. Under these conditions (37° C, 16 h) PAI-1 antigen levels remained unchanged in males whereas the response of the female samples in the PAI-1 antigen assay increased two-fold.

In lysed platelet-rich plasma males had 990 ± 470 ng/ml antigen and 160 ± 80 ng/ml activity and females, 920 ± 500 ng/ml antigen and 150 ± 98 ng/ml activity corresponding to 2.1 ± 0.77 fg PAI-1 antigen per platelet. Only 16% of PAI-1 released from platelets was found to be active. Linear correlations between PAI-1 antigen and activity were found for both males (r = 0.61) and females (r = 0.67).

The assays are both sensitive and specific and may, therefore, aid the elucidation of the pathophysiological role of PAI-1 in swine experimental models of atherosclerosis and other thrombotic disorders.
Long-term Clinical Follow-up in 265 Patients with Deep Venous Thrombosis Initially Treated with either Unfractionated Heparin or Dalteparin: A Retrospective Analysis
Authors: Åberg W. , Lockner D. , Paul C. , Holmström M.
Published: March 2018

The primary objective of this retrospective study was to describe the frequency of a post-thrombotic syndrome in 265 patients previously treated for deep venous thrombosis (DVT). The secondary objectives were to document the frequency of recurrent venous thromboembolism (VTE) and mortality, especially from malignant disease. The patients were evaluated 5-14 years after inclusion in three randomized trials comparing continuous intravenous (i. v.) infusion of unfractionated heparin (UFH) (n = 85) with a low molecular weight heparin (LMWH), dalteparin (n = 180). The median post-thrombotic score at follow-up was 2 (range 0-8). In a multiple step-wise regression analysis the post-thrombotic score was significantly higher among patients with initial proximal DVT (p = 0,0001) as compared with those who had distal DVT. A recurrent venous thromboembolic event was diagnosed in 29,4% of the patients treated with dalteparin and in 23,5% of the patients treated with UFH (ns). A secondary risk factor for venous thromboembolism and a longer duration of treatment with oral anticoagulants (OAC) were significantly associated with a lower risk for recurrent VTE, whereas malignant disease diagnosed during follow-up was associated with a higher risk. During follow-up a total of 40,7% of patients had died. No difference in total mortality or mortality from malignant disease was demonstrated between the two drugs. In conclusion, a severe post-thrombotic syndrome occured relatively infrequent. considering the long observation period. Proximal DVT was significantly associated with a more severe post-thrombotic syndrome. After 14 years follow-up, no significant differences were observed in overall mortality, mortality from malignant disease or recurrent VTE between UFH- and dalteparin-treated patients. Malignant disease was a risk factor for recurrent VTE, the presence of a secondary risk factor and a longer duration of treatment with OAC decreased the risk for recurrent VTE.
Comparison of Three Methods to Assess Therapeutic Quality Control of Treatment with Vitamin K Antagonists
Authors: Prins Martin , Ken Redekop W. , Tijssen Jan , Heisterkamp Siem , Büller Harry , Hutten Barbara
Published: March 2018

During treatment with vitamin K antagonists, International Normalized Ratios (INR) are determined periodically to maintain a therapeutic level of anticoagulation. We evaluated two existing methods for therapeutic quality control (linear interpolation and equidivision), with regard to their validity and reproducibility. In addition, we proposed and evaluated a (hybrid) method that takes into account potential effects of dosage modifications when INRs are far out of the target range. Validity was assessed by deleting intermediary INR results and estimating this INR based on the two surrounding INRs with each of the three methods. The estimated INRs were then compared with the observed INR.

Reproducibility of time spent in an INR range was evaluated for each of the three methods by deleting at random increasing proportions of INRs and comparing these estimates with the situation without deletions. We found that estimates of time spent in INR categories obtained with equidivision were most reproducible, but least valid. The hybrid method showed slightly higher validity and reproducibility in comparison with linear interpolation. Since these differences were small, linear interpolation is preferable to the hybrid method, since the calculations involved are easier.
Allelic Discrimination of Factor V Leiden Using a 5’ Nuclease Assay
Authors: Schwaab R. , Hanfland P. , Hoernschemeyer D. , Happich D.
Published: March 2018

The G1691A (Leiden) mutation of the factor V gene is the most prevalent identified cause of venous thrombosis. Therefore, we developed a new genetic test using the TaqMan system. With this assay which combines PCR amplification and detection reaction in one closed tube, a cohort of 234 patients with a history of thrombosis was screened. In parallel, amplification products of the same patients were screened with a previously described test using endonuclease digestion of PCR products followed by gel electrophoresis. Identical results were obtained by both methods. Among cases, 122 (52%) individuals were homozygous normal, 99 (42%) were heterozygous affected and 13 (5.5%) showed homozygous pattern for the Factor V Leiden mutation. Thus, it could be demonstrated that the new TaqMan assay is a robust, rapid and automated method for high throughput application which avoids time consuming and difficult post-PCR steps.
The Biology of P-Selectin Glycoprotein Ligand-1: Its Role as a Selectin Counterreceptor in Leukocyte-Endothelial and Leukocyte-Platelet Interaction
Authors: Furie Barbara , Furie Bruce , Yang Jing
Published: March 2018

Cell-cell interactions mediating leukocyte trafficking, thrombogene-sis and inflammation are crucial for the host defense mechanism. The selectin family of integral membrane proteins includes E-selectin, L-selectin and P-selectin. Selectins mediate tethering and rolling of leukocytes to the vessel wall at the site of inflammation. The counter-receptor for P-selectin and possibly the other selectins is P-selectin glycoprotein ligand-1 (PSGL-1). This review focuses on the properties and biology of PSGL-1.
APC-resistance Is a Risk Factor for Postoperative Thromboembolism in Elective Replacement of the Hip or Knee – A Prospective Study
Authors: Lundahl Tom , Nilsson Lennart , Andersson Christer , Lindahl Tomas
Published: March 2018

Postoperative venous thromboembolic complications are commonly seen after total replacement of the hip or knee. Recently, an inherited defect with resistance to the anticoagulant activity of activated protein C (APC-resistance) has been detected. APC-resistance seems to be a common risk factor, especially in Sweden, and it increases the propensity for venous thrombosis. This study assesses the prevalence of APC-resistance in a general population and its clinical significance for patients undergoing surgery associated with a high risk of thromboembolic complications. In a prospective cohort study, we analysed for APC-resistance in 645 consecutive patients before elective replacement of the hip or knee at 3 hospitals in southern Sweden. Thromboprophylaxis with LMWH-heparin was given to all patients throughout the hospitalisation period. We recorded events of clinical thromboembolism for 3 months postoperatively. Venography, ultrasonography or pulmonary scintigraphy was requested by the clinicians according to the existing routines, i.e. only patients with symptoms of thromboembolism were examined. A thromboembolic complication was registered in 20 (3.1%) patients. Fifty per cent of the venous thrombi had a proximal location. Only 0.3% of the patients had verified pulmonary embolism. APC-resistance was found in 14.1% of the patients, of whom 9.9% had experienced postoperative thromboembolism compared with 2.0% of the patients without APC-resistance (p <0.0007). We conclude that APC-resistance is a frequent risk factor for symptomatic postoperative deep venous thrombosis with an estimated relative risk of 5.0 (95% confidence interval: from 1.9 to 12.9) in elective replacement of the hip or knee.
Crotalase, a Fibrinogen-Clotting Snake Venom Enzyme: Primary Structure and Evidence for a Fibrinogen Recognition Exosite Different from Thrombin
Authors: Henschen-Edman Agnes , Theodor Ida , Edwards Brian , Pirkle Hubert
Published: March 2018

Crotalase, a fibrinogen-clotting enzyme isolated from the venom of Crotalus adamanteus, and its overlapping fragments were subjected to Edman degradation. The resulting amino acid sequence, VIGGDEC NINEHRFLVALYDYWSQLFLCGGTLINNEWVLTAAHCDRTHI LIYVGVHDRSVQFDKEQRRFPKEKYFFDCSNNFTKWDKDIM LIRLNKPVSYSEHIAPLSLPSSPPIVGSVCRAMGWGQTTSPQET LPDVPHCANINLLDYEVCRTAHPQFRLPATSRTLCAGVLEG GIDTCNRDSGGPLICNGQFQGIVFWGPDPCAQPDKPGLYTK VFDHLDWIQSIIAGEKTVNCP, is characteristic of a serine protein-ase. Comparison with thrombin, the physiological fibrinogen-clotting enzyme, showed that thrombin’s fibrinogen-recognition exosite (FRE) is poorly represented in crotalase. Hirudin, a FRE-dependent inhibitor, had no effect on crotalase. Spatial modeling of crotalase yielded a possible alternative fibrinogen-recognition site comprised of Arg 60F, Lys 85, Lys 87, and Arg 107 (underlined in the sequence above). Crotalase also lacks thrombin’s YPPW loop, as well as its functionally important ETW 146-148, and its heparin-binding site. The enzyme contains a single asparagine-linked glycosylation site, NFT, bearing neutral and amino sugars that account for 8.3% of the enzyme’s total molecular weight of 29,027. The calculated absorbance of crotalase at 280 nm, 1%, cm-1is 15.2.
Lipoprotein(a) Levels and Isoforms and Fibrinolytic Activity in Postmenopause – Influence of Hormone Replacement Therapy
Authors: Cano Antonio , Falcó Cristina , España Francisco , Gilabert Juan , Grancha Salvador , Aznar Justo , Estellés Amparo
Published: March 2018

Epidemiological studies suggest that hormone replacement therapy (HRT) decreases the risk of cardiovascular disease in postmenopausal women via several mechanisms, including modifications in the fibrinolytic system and lipoprotein(a) [Lp(a)] levels. The aim of this study was to examine the influence of the levels and isoforms of Lp(a) on fibrinolytic activity in 91 postmenopausal women in comparison with premenopause and analyze the effect of HRT on those parameters. In postmenopause, an increase in plasma Lp(a) and plasminogen activator inhibitor-1 (PAI-1) levels was found. A significant inverse correlation was observed between Lp(a) or PAI-1 levels and plasmin generation. Plasma samples with low molecular weight (MW) apo(a) isoforms showed higher plasmin inhibition than plasmas with high MW apo(a) isoforms and similar levels of total Lp(a) and PAI-1. HRT induced a significant decrease in Lp(a) and PAI-1 levels and an increase in estradiol levels, as well as an increase in fibrinolytic activity. A significant correlation was found between the percentages of variation in Lp(a) levels and in plasmin generation and between the percentages of variation in PAI-1 levels and in the euglobulin lysis time under HRT. In conclusion, the increase in fibrinolytic activity observed in women under HRT could be explained by two independent mechanisms: (a) the decrease in PAI-1 and (b) the decrease in the inhibition of plasmin generation due to the decrease in Lp(a) levels.
Clustering of Integrin αIIb-β3Differently Regulates Tyrosine Phosphorylation of pp72syk, PLCγ2 and pp125FAKin Concanavalin A-stimulated Platelets
Authors: Festetics Enrico , Bertoni Alessandra , Sinigaglia Fabiola , Balduini Cesare , Torti Mauro
Published: March 2018

Tyrosine phosphorylation of the non-receptor tyrosine kinases pp72sykand pp125FAKand of the γ2 isoform of phospholipase C (PLCγ2) in human platelets stimulated with the lectin Concanavalin A was investigated. Concanavalin A induced the rapid tyrosine phosphorylation of pp72sykand PLCγ2 with a similar kinetics, while tyrosine phosphorylation of pp125FAKoccurred in a later phase of platelet activation. When compared with other platelet agonists, Concanavalin A revealed to be at least as potent as collagen in inducing tyrosine phosphorylation of PLCγ2 and pp125FAK, while tyrosine phosphorylation of pp72sykinduced by the lectin was much stronger than that induced by thrombin or collagen. Concanavalin A-induced tyrosine phosphorylation of pp72syk, PLCγ2 and pp125FAKwas not dependent on platelet aggregation as it occurred normally even in the absence of sample stirring and when fibrinogen binding to integrin αIIb-β3was inhibited by the peptide RGDS. Tyrosine phosphorylation of pp72syk, PLCγ2 and pp125FAKrequired the binding of the lectin to the platelet surface, but was not observed in platelets treated with succinyl-Concanavalin A, a derivative of the lectin that interacts with the same receptors but does not promote clustering of membrane glycoproteins. Moreover, the aggregation-independent tyrosine phosphorylation of pp125FAKand pp72sykinduced by Concanavalin A required the expression of integrin αIIb-β3on the platelet surface as it was strongly inhibited in platelets from patients affected by Glanzmann thrombasthenia. By contrast, tyrosine phosphorylation of PLCγ2 occurred normally also in thrombasthenic platelets stimulated with Concanavalin A. These results demonstrate that, even in the absence of aggregation, the clustering of integrin αIIb-β3induced by Concanavalin A on the platelet surface directly promotes tyrosine phosphorylation of pp72sykand pp125FAKand provide further evidence that the oligomerization of the fibrinogen receptor promoted by its natural ligand during platelet aggregation may be responsible for the tyrosine phosphorylation of these proteins induced by physiological agonists.
Assay of von Willebrand Factor (vWF)-cleaving Protease Based on Decreased Collagen Binding Affinity of Degraded vWF
Authors: Gerritsen Helena , Turecek Peter , Schwarz Hans , Lämmle Bernhard , Furlan Miha
Published: March 2018

Patients with thrombotic thrombocytopenic purpura (TTP) have a deficiency of von Willebrand factor (vWF)-cleaving protease, whereas patients with hemolytic-uremic syndrome (HUS) show normal activity of this protease. Present methods for assaying vWF-cleaving protease by immunoblotting are time-intensive and cumbersome. We therefore developed a new functional assay based on the preferential binding of high-molecular-weight forms of vWF to collagen. In this assay, the diluted plasma sample to be tested is added to normal human plasma in which protease activity had been abolished. The vWF present in the protease-depleted plasma is digested by the vWF-cleaving protease in the test plasma. The proteolytic degradation leads to low-molecular-weight forms of vWF, which show impaired binding to microtiter plates coated with human collagen type III. The collagen-bound vWF is quantified using a peroxidase-conjugated rabbit antibody against human vWF. The values of vWF-cleaving protease activity in tested plasma samples are read from a calibration curve achieved by incubating the vWF-substrate with dilutions of a normal human plasma pool (NHP). Testing of plasma from patients with TTP and HUS showed that the assay can be used to distinguish between these two syndromes. The presence of an inhibitor can be detected by carrying out the test after incubation of NHP with the patient plasma sample, thus enabling differentiation of patients with familial TTP from those with non-familial TTP.
Atorvastatin Increases ecNOS Levels in Human Platelets of Hyperlipidemic Subjects
Authors: Tannous Marie , Cheung Raphael , Vignini Arianna , Mutus Bulent
Published: March 2018

Background. The purpose of this study was to probe the pleiotrophic effects of Atorvastatin on intraplatelet-nitric oxide metabolism. Methods and Results. Hyperlipidemic subjects (n = 19) were treated for 1 month (following a 3-week washout) with either Atorvastatin or placebo in a double-blinded randomized (n = 2, crossover), placebo-controlled study. Changes in the levels of intraplatelet nitric oxide synthase, nitrotyrosine were correlated with cholesterol, LDL-C, HDL-C and triglyceride levels. These studies indicate that with atrovastatin ecNOS levels increased on average by ~1.7-fold (paired t-test p = 0.009). Interestingly, levels of nitrotyrosylated platelet proteins, an indication of peroxynitrite damage, decreased as ecNOS levels increased in presence of the drug (paired t-test p = 0.33). Atorvastatin, at 10 mg per day, lowered cholesterol and LDL-C levels in all patients with the average lowering of ~21% and ~17% respectively. The effect on HDL was not significant whilst triglyceride levels were lowered by an average of ~18%. Conclusions. This study adds to the volume of evidence that statins have beneficial effects other than lipid lowering. Here, Atorvastatin is shown to significanly elevate intraplatelet ecNOS levels in hyperlipidemic subjects without affecting iNOS expression. The net result of this would be the elevation of NO production which would promote platelet deaggregation and vasodilation.
Automated Latex Agglutination and ELISA Testing Yield Equivalent D-Dimer Results in Patients with Recent Myocardial Infarction
Authors: Zareba Wojciech , Horan John , Moss Arthur , Kanouse Joel ,   for the THROMBO Research Investigators, Marder Victor
Published: March 2018

Our previous prospective study of post-infarction patients described a strong and significant association of increased plasma D-dimer concentrations in those who experienced a subsequent coronary death or non-fatal myocardial infarction. In the present study, we compare results on stored plasma obtained two months after the index myocardial infarction from 1,038 patients of this trial, using a simple automated latex agglutination (LA) assay in parallel with the standard ELISA test. Results show a somewhat higher mean value for the LA assay (702 ± 1092 vs. 638 ± 986 ng/ml, p = 0.0002), a strong linear correlation of the two assays (r = 0.86) and 88% agreement for values below 500 ng/ml by the ELISA test. D-dimer concentrations determined by each assay were highly correlated in patients with subsequent coronary artery events (p = 0.93) and quartile values for both the LA and ELISA were equally predictive of such events (p = 0.003 and p = 0.001, respectively). This is the first demonstration that a latex agglutination assay for D-dimer can be used to assess the prognostic risk of recurrent coronary thrombotic disease after myocardial infarction
Dermatan Sulfate and LMW Heparin Enhance the Anticoagulant Action of Activated Protein C
Authors: Fernández José , Petäjä Jari , Griffin John
Published: March 2018

Unfractionated heparin potentiates the anticoagulant action of activated protein C (APC) through several mechanisms, including the recently described enhancement of proteolytic inactivation of factor V. Possible anticoagulant synergism between APC and physiologic glycosaminoglycans, pharmacologic low molecular weight heparins (LMWHs), and other heparin derivatives was studied. Dermatan sulfate showed potent APC-enhancing effect. Commercial LMWHs showed differing abilities to promote APC activity, and the molecular weight of LMWHs correlated with enhancement of APC activity. Degree of sulfation of the glycosaminoglycans influenced APC enhancement. However, because dextran sulfates did not potentiate APC action, the presence of sulfate groups per se on a polysaccharide is not sufficient for APC enhancement. As previously for unfractionated heparin, APC anticoagulant activity was enhanced by glycosaminoglycans when factor V but not factor Va was the substrate. Thus, dermatan sulfate and LMWHs exhibit APC enhancing activity in vitro that could be of physiologic and pharmacologic significance.
The Effects of Angiotensin Metabolites on the Regulation of Coagulation and Fibrinolysis in Cultured Rat Aortic Endothelial Cells
Authors: Tsuji Hajime , Masuda Haruchika , Kasahara Teruhisa , Yoshizumi Masami , Sugano Tatsuya , Kimura Shinzo , Kawano Hidehiko , Kunieda Yasufumi , Yano Shingo , Nakagawa Katsumi , Kitamura Hidetsugu , Nakahara Yoshifumi , Sawada Shohei , Nakagawa Masao , Nishimura Hiromi
Published: March 2018

Not only angiotensin II (Ang II) but also other angiotensin metabolites such as angiotensin I (Ang I), angiotensin III (Ang III), angiotensin IV, or angiotensin 1-7 have recently been reported to have various activities. Few data, however, are available on the regulation of thrombus formation. In this study, we investigated the effects of angiotensin metabolites on the mRNA expression of tissue factor (TF), tissue factor pathway inhibitor (TFPI), plasminogen activator inhibitor-1 (PAI-1), and tissue type plasminogen activator (TPA) in cultured rat aortic endothelial cells. None of the used angiotensin metabolites altered TFPI or TPA mRNA expression levels. Ang I, Ang II, and Ang III made TF and PAI-1 mRNA inductions which were inhibited by an selective antagonist of angiotensin II type 1 receptors. These metabolites made TF predominant to TFPI or PAI-1 to TPA, and could render endothelial cells thrombogenic.
Differential Effects of Dietary Supplementation with Fish Oil or Soy Lecithin on Human Platelet Adhesion
Authors: Carletto Antonio , Guarini Patrizia , Galvani Simonetta , Biasi Domenico , Bellavite Paolo , Corrocher Roberto , Andrioli Giuseppe
Published: March 2018

To investigate the possible regulating role of ω-6 and of ω-3 fatty acids on platelet adhesiveness, we randomised 60 volunteers into three groups to take 20 ml (equivalent to 0.3 g ω-6, 3.6 g ω-3; ω-6/ω-3 ratio 0.1) per day of a fish oil supplement, or to take 25 g (equivalent to 1.5 g ω-6, 0.5 g ω-3; ω-6/ω-3 ratio 3) per day of a soy lecithin supplement, or to continue on their usual diet without any supplement (control group) for a period of 15 days. Platelet adhesion on fibrinogen-coated 96-well microtitre plates was evaluated in the resting condition and after stimulation with 2 ωM ADP or 0.02 U/ml thrombin. Compared to the values before the experimental period, the fish oil group showed a significant reduction in stimulated adhesion (with ADP: from 18.8% to 15.6%, p <0.01; with thrombin: from 24.4% to 20.8%, p <0.005), whereas no difference was noted in the resting condition (from 3.6% to 3.5%, NS). In the soy lecithin group, platelet adhesion was increased in all test conditions (with ADP: from 18.7% to 23.2%, p <0.001; with thrombin: from 24.0% to 29.9%, p <0.001; resting: from 3.5% to 6.6%, p <0.001). No significant changes were observed in the control group. A good correlation was found between platelet adhesion data and the changes in the platelet fatty acid ω-6/ω-3 ratio caused by the different supplementations. Our results indicate an inhibitory effect of fish oil rich in ω-3 fatty acids on stimulated human platelet adhesiveness and a stimulatory effect of soy lecithin rich in ω-6 fatty acids on resting and stimulated adhesion. They suggest moreover that the ω-6/ω-3 ratio is a determinant of platelet adhesion.
Analysis of CD39/ATP Diphosphohydrolase (ATPDase) Expression in Endothelial Cells, Platelets and Leukocytes
Authors: Koziak Katarzyna , Sévigny Jean , Robson Simon , Siegel Jonathan , Kaczmarek Elzbieta
Published: March 2018

Purinergic signaling may influence hemostasis, inflammatory responses and apoptosis. Therefore, hydrolysis of extracellular ATP and ADP by the ATP diphosphohydrolase (ATPDase) could regulate these processes. We have previously demonstrated the identity between the vascular ATPDase and CD39. Here we show that levels of CD39 expression correlate with ATPDase activity in human endothelial cells (EC), platelets and selected monocyte, NK, and megakaryocyte cell lines. Western blotting revealed one to three isoforms of CD39/ ATPDase; mobility variations of major protein resulted from post-translational modifications. Northern blotting and primer extension indicated two major mRNA transcripts and one transcription start point, respectively. In addition, mRNAs specific for purinergic P2 receptors were detected in all of the investigated cells, suggesting that the co-expressed CD39/ATPDase may regulate purinergic signaling. Thrombotic and inflammatory responses may be modulated by the expression of CD39/ATPDase.
Novel Deletion and Insertion Mutations Cause Splicing Defects, Leading to Severe Reduction in mRNA Levels of the A Subunit in Severe Factor XIII Deficiency
Authors: Izumi Tomonori , Nagaoka Utako , Saito Tetsuo , Takamatsu Junki , Saito Hidehiko , Ichinose Akitada
Published: March 2018

In order to explore molecular mechanisms for factor XIII deficiency, a patient (Nagoya I) was examined at the DNA and RNA levels. Nucleotide sequence analysis of the patient’s DNA amplified by PCR revealed that he had a 20 bp deletion at the boundary of exon I/intron A, and an insertion of T in the invariant GT dinucleotide at the splicing donor site of exon IV/intron D. The presence of these heterozygous mutations was confirmed by restriction digestion of the amplified fragments of the proband and his parents. RT-PCR analysis demonstrated that only one kind of mRNA without exon IV was detected in Nagoya I, although its level was greatly reduced to less than 5% of normal. The other defective allele of the A subunit gene containing the 20 bp deletion was not detected. Thus, both mutations impaired normal processing of mRNA for the A subunit, resulting in his severe factor XIII deficiency.
Standardization of the APC Resistance Test. Effects of Normalization of Results by Means of Pooled Normal Plasma
Authors: Chantarangkul Veena , Negri Barbara , Mannucci Pier Mannuccio , Tripodi Armando
Published: March 2018

Results for APC resistance tests are expressed as the ratio of the clotting time with and without APC (APC ratio). Normalization by dividing the patient’s APC ratio by that of the pooled normal plasma (PNP) (n-APC ratio) was proposed to minimize between-lot reagent variability. To evaluate the merits of different expressions of the results, sets of 80 coded frozen plasmas from carriers (n = 30), non-carriers (n = 30) of the FV:Q506 mutation and 10 copies each of two control plasmas were sent to 7 expert laboratories which were asked to assess APC resistance by their methods. Results were expressed as APC ratio and nAPC ratio by the local PNP and 2 common PNP (A and B). These contained plasmas from the same (n = 20) non-carriers. In PNP A, plasma from one non-carrier was replaced with that from one heterozygous carrier. The merits of different expression of results were judged by (i) within-laboratory reproducibility; (ii) discrimination of carriers from non-carriers; (iii) between-method comparability of results. The influence of FV:Q506 plasma in the preparation of PNP was also assessed. Reproducibility, generally good even with results expressed as APC ratio (median CV 4.6% and 3.0% for normal and abnormal control), was not changed by normalization. Discrimination did not change whatever the method of result expression. Between-method comparability of results was scarcely affected by normalization with the local PNP, whereas it was considerably improved after normalization with the common PNP, but only for the non-carriers. APC ratios for PNP A by all methods were significantly lower than those for PNP B. Thus, the presence of mutant FV in a proportion as low as 2.5% may reduce the APC ratio of the PNP.
Sex Differences in the Determinants of Fibrinolytic Activity
Authors: Cooper J. A. , Howarth D. J. , Meade T. W. , Miller G. J. , MacCallum P. K.
Published: March 2018

Impaired whole blood fibrinolytic activity (FA), measured by the dilute clot lysis time (DCLT), is associated with first episodes of ischaemic heart disease (IHD) in the Northwick Park Heart Study in men, especially under 55 years, and in women. In a community-based study to investigate possible determinants of the DCLT, and therefore to assess which fibrinolytic components might be predictors of first IHD events, we measured fibrinolytic variables in a sub-sample of 150 healthy adults (73 males, 77 females) randomly selected from a single general practice.

Most of the variance in DCLT (68% in men, 63% in women) was explained by tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor type-1 (PAI-1) activities. In multiple regression analysis there was a significant difference in the strength of the association of t-PA activity with DCLT in men compared to women (test for interaction p = 0.05), the association of t-PA activity with DCLT being significant in males but not in females. Plasma PAI-1 activity was strongly associated with DCLT in both sexes. There was no independent association of DCLT with plasma fibrinogen, t-PA antigen, other fibrinolytic inhibitors, body mass index, serum lipids or C-reactive protein.

Plasma PAI-1 activity in females and both t-PA and PAI-1 activities in males are the main determinants of whole blood FA measured by DCLT. It is therefore likely that these modulators of the plasma fibrinolytic system are associated with the onset of first clinical episodes of IHD. Elevated levels of t-PA antigen were positively associated with DCLT after adjustment for age and sex and therefore indicate impaired rather than enhanced FA. Further studies of the association of FA with risk of IHD should include not only “global” measures but also assessment of t-PA and PAI-1 activities, particularly as our results suggest that their associations with IHD may differ in men and women.
Atrial Natriuretic Peptide Inhibits the Expression of Tissue Factor and Plasminogen Activator Inhibitor 1 Induced by Angiotensin II in Cultured Rat Aortic Endothelial Cells
Authors: Tsuji Hajime , Nishimura Hiromi , Kasahara Teruhisa , Sugano Tatsuya , Masuda Haruchika , Nakagawa Katsumi , Nakahara Yoshifumi , Kitamura Hidetsugu , Yamada Keizou , Yoneda Mitsuru , Sawada Shohei , Nakagawa Masao , Yoshizumi Masami
Published: March 2018

The pharmacological characteristics of atrial natriuretic peptide (ANP), such as natriuresis, vasodilation, or suppression of smooth muscle cell proliferation, are well investigated. However, this is the first study to report its role on blood coagulation and fibrinolysis mediated by vascular endothelial cells. In this study, the effects of ANP on the enhanced expression of tissue factor (TF) and plasminogen activator inhibitor 1 (PAI-1) by angiotensin II (Ang II) in cultured rat aortic endothelial cells (RAECs) were examined. The expressions of TF and PAI-1 mRNA were detected by northern blotting methods. The activities of TF on the surface of RAECs and PAI-1 in the culture media were measured by chromogenic assay. ANP suppressed mRNA expressions of TF and PAI-1 induced by Ang II in a concentration-dependent manner. This suppression was accompanied by the decreased activities of TF and PAI-1.
Role of Integrin αvβ3 in Vascular Biology
Authors: Byzova Tatiana , Rabbani Ramin , D’Souza Stanley , Plow Edward
Published: March 2018

Introduction

A defining characteristic of vascular cells is their adhesive status. The predominant cells of the blood vessel, endothelial cells (EC) and smooth muscle cells (SMC), are normally adherent but can be induced to migrate in response to vascular injury and angiogenic stimuli. The circulating blood cells are ordinarily nonadhesive but can rapidly acquire an adhesive phenotype in response to physiologic and pathophysiologic stimuli. As prime examples, platelets become adherent to the subendothelial matrix and to one another during thrombus formation, and leukocytes first adhere to EC and then transmigrate during the inflammatory response. At a molecular level, the adhesive properties of the vascular cells are determined by the adhesion receptors on their cell-surface and the functional state of these receptors. To match the variety of requisite cellular adhesive reactions, the repertoire of adhesion receptors expressed by vascular cells is broad. Multiple representatives of the immunoglobulin-like, the selectin, the cadherin and the integrin families of adhesion receptors are present on and have been implicated in the functions of the vascular cells. The importance of these adhesion receptors in vascular cell function is underscored by the severe pathogenetic consequences of their congenital deficiencies, such as in Glanzmann’s thrombasthenia, LAD (Leucocyte Adhesion deficiency) I and LAD II (1-3).

The integrins are the largest and most broadly distributed of the families of cellular adhesion receptors. Of the integrins, αvβ3, originally identified as the vitronectin receptor, is particularly widely distributed. It is expressed at variable density on many types of vascular cells. Obviously, the adhesive properties of a cell are determined by its full repertoire of adhesion receptors. As an example, the adhesion of EC to fibrinogen/fibrin is mediated by no fewer than five receptors. Nevertheless, it is possible to dissect out the contributions of individual adhesion receptors, and αvβ3 has been implicated in many functional responses of vascular cells. This review focusses upon the role of αvβ3 in vascular cell biology. Other contributions of this multifunctional receptor, such as its role in neoplastic growth and invasion and in osteoclast-mediated bone resorption, are beyond the scope of this article and have been reviewed elsewhere (4, 5).
Factor V Leiden and Prothrombin Gene G 20210 A Variant in Children with Ischemic Stroke
Authors: Bodó Z. , Plotho Jutta , Streif W. , Male Ch. , Bernert G. , Rauter L. , Ebetsberger G. , Kaltenbrunner K. , Kurnik P. , Lischka A. , Paky F. , Ploier R. , Höfler G. , Mannhalter Christine , Muntean W. , Zenz W.
Published: March 2018

Objective: To investigate if the factor V Leiden mutation (F-V-LM) and/or the prothrombin gene G 20210 A variant (P-G20210A-V) are risk factors for acute stroke in Austrian children. Patients: 33 children with acute ischemic stroke documented by computer tomography and/or magnetic resonance imaging of the brain were enrolled in an open multicenter survey. Results: 6/33 children had F-V-LM (5 heterozygous, 1 homozygous). This represents 18% (95% CI: 6.7-39.9%) of our pediatric stroke population and thus exceeds the expected prevalence in the Austrian population of 4,6% (Fischer’s exact test, p = 0.01). F-V-LM was not found in 11 children with neonatal stroke but in 6/22 children with stroke after the neonatal period. 5/6 children with F-V-LM had an underlying disorder that is a risk factor for stroke in children. The P-G20210A-V was detected in 1/26 (3.85%; 95% CI: 0.1-21.4%) patients. Comparison of the prevalence of P-G20210A-V in our study with that in the general population of Austria of 1% revealed no statistical significance (Fischer’s exact test, p = 0.38). Conclusion: Our data suggest that the F-V-LM is a risk factor for acute stroke in Austrian children beyond the neonatal period. The P-G20210A-V apparently does not represent a risk factor for stroke in Austrian children.
Isolation of Frog and Chicken cDNAs Encoding Heparin Cofactor II
Authors: Colwell Niall , Tollefsen Douglas
Published: March 2018

Heparin cofactor II (HCII) is a serpin that inhibits thrombin rapidly in the presence of heparin or dermatan sulfate. HCII activity has been detected in human, rabbit, and mouse plasma, and cDNA clones for HCII have been isolated previously from human, rabbit, rat, and mouse liver libraries, suggesting a conserved physiologic role for HCII among mammals. In this report, we show that both frog and chicken plasma contain a dermatan sulfate-dependent inhibitor that forms a 118-kDa complex with human 125I-thrombin. Screening of frog and chicken liver cDNA libraries in bacteriophage λ with a human HCII cDNA probe yielded nearly full-length clones with inserts of 1.8 and 1.7 kb, respectively. The amino acid sequences deduced from the frog and chicken HCII cDNAs are ~60% identical to one another and to each of the mammalian sequences. In particular, the N-terminal acidic domain, the glycosaminoglycan-binding site, and the reactive site sequences are highly conserved. Our results indicate that HCII is widely distributed among vertebrates and may have a common function in birds, amphibians, and mammals.
Platelet Aggregation Induced In Vitro by Rabbit Plasma Clot-associated Thrombin, and Its Inhibition by Thrombin Inhibitors
Authors: Gandossi Eric , Lunven Catherine , Gauffeny Christiane , Roome Nigel , Berry Christopher
Published: March 2018

The activation of rabbit platelets by rabbit plasma clots, and the inhibition of clot-associated thrombin by heparin:antithrombin III, recombinant hirudin (rHV2Lys47) and argatroban, a low molecular weight thrombin inhibitor, was studied.

Plasma clots caused the aggregation of platelets suspended in a plasma-free medium as assessed by single platelet counting, and by scanning electron microscopy (platelet aggregates present on the clot surface). Platelet aggregation, induced by clot-associated thrombin, was inhibited by argatroban with an IC50 of 14 ± 3 nM compared to an IC50 of 12 ± 2 nM when human thrombin in solution titrated to give the same decrease in the platelet count as plasma clots was used. rHV2Lys47 also inhibited aggregation induced by clot-associated thrombin with an IC50 of 1.6 ± 0.4 nM compared to 1.6 ± 0.5 nM with thrombin in solution. Heparin was less active against clot-associated thrombin (IC50 = 69 ± 9 mU/ml) than against thrombin in solution (IC50 = 15 ± 5 mU/ml).

This study shows that plasma clot-bound thrombin activates platelets and that direct-acting thrombin inhibitors such as argatroban and rHV2Lys47 are more effective than heparin:antithrombin III in inhibiting this phenomenon.
The Effects of Hormone Replacement Therapy (HRT) on Hemostatic Variables in Women with Previous Venous Thromboembolism – Results from a Randomized, Double-Blind, Clinical Trial
Authors: Qvigstad Erik , Andersen Trine Opstad , Mowinckel Marie-Christine , Sandset Per Morten , Høibraaten Else
Published: March 2018

In a recent randomized, double-blind, placebo-controlled trial of women with a history of venous thromboembolism (VTE), we found that hormone replacement therapy (HRT) was associated with an early excess risk of recurrent thrombosis. The aims of the present study were to characterize the effects of HRT on coagulation in these women to elucidate the mechanism(s) by which HRT increases the risk of thrombosis. The study comprised 140 women who were randomized to receive continuous treatment for 24 months with once daily 2 mg 17- -estradiol plus 1 mg norethisterone acetate (n = 71) or placebo (n = 69). HRT caused significant increases in prothrombin fragments 1+2, thrombin-antithrombin complex, and D-Dimer after 3 months, but these changes were less pronounced on prolonged treatment. The increases in markers of activated coagulation was higher in those women who subsequently developed recurrent thrombosis, but was similar in carriers and non-carriers of the factor V Leiden mutation. HRT had no effects on fibrinogen and factor VIII. Activated factor VII, but not factor VII antigen, decreased significantly on HRT as compared with placebo. The coagulation inhibitors antithrombin, protein C, and TFPI, but not protein S, all showed significant sustained decreases in the HRT group as compared with placebo. Antithrombin and protein C decreased by 8-12% on HRT, whereas TFPI activity decreased by 12-17% and TFPI free antigen by 29-30%. In multivariate analysis, only TFPI activity was a significant predictor for the increased activation of coagulation. We conclude that HRT was associated with early activation of coagulation, which corroborates the finding of an early risk of recurrent VTE. This activation may in part be explained by reduction in circulating anticoagulants.
Clonal Restriction of Platelet-associated Anti-GPIIb/IIIa Autoantibodies in Patients with Chronic ITP
Authors: Lopez-Dee Jennifer , Bowditch Ronald , McMillan Robert
Published: March 2018

Chronic immune thrombocytopenic purpura is due to platelet destruction induced by autoantibodies against platelet surface antigens. Prior studies show that some serum autoantibodies are light-chain restricted, suggesting a clonal origin. Since plasma and platelet-associated antibody from the same patient may bind to different epitopes, it is important to evaluate the clonality of platelet-associated antibody. Platelet-associated autoantibodies from 28 ITP patients were studied. Of 23 platelet-associated antibodies tested directly, 16 showed significant light chain restriction (7 complete and 9 partial) when compared to plasma IgG light chain distribution. Similarly, 9 of 12 platelet-associated antibody eluates were light chain restricted, 5 complete and 4 partial. In all cases where platelet-associated antibody and antibody eluate from the same patient were studied, the results were concordant. We conclude that a significant proportion of platelet-associated antibodies from ITP patients show apparent clonality, as evaluated by light chain restriction. These results are consistent with other studies in ITP suggesting a limited antigenic repertoire.
Evidence for a Role for Phospholipase C, but not Phospholipase A2, in Platelet Activation in Response to Low Concentrations of Collagen
Authors: Lockhart Leslie , Pampolina Caroline , Nickolaychuk Brent , McNicol Archibald
Published: March 2018

The release of arachidonic acid is a key component in platelet activation in response to low concentrations (1-20 g/ml) of collagen. The precise mechanism remains elusive although a variety of pathways have been implicated. In the present study the effects of inhibitors of several potentially key enzymes in these pathways have been examined. Collagen (1-10 g/ml) caused maximal platelet aggregation which was accompanied by the release of arachidonic acid, the synthesis of thromboxane A2, and p38MAPK phosphorylation. Preincubation with the dual cyclooxygenase/lipoxygenase inhibitor BW755C inhibited aggregation and thromboxane production, and reduced p38MAPK phosphorylation. A phospholipase C inhibitor, U73122, blocked collagen-induced aggregation and reduced arachidonic acid release, thromboxane synthesis and p38MAPK phosphorylation. Pretreatment with a cytosolic phospholipase A2 inhibitor, AACOCF3, blocked collagen-induced aggregation, reduced the levels of thromboxane formation and p38MAPK phosphorylation but had no significant effect on arachidonic acid release. In contrast inhibition of PKC by Rö31-8220 inhibited collagen-induced aggregation, did not affect p38MAPK phosphorylation but significantly potentiated arachidonic acid release and thromboxane formation. Collagen caused the tyrosine phosphorylation of phospholipase C 2 which was inhibited by pretreatment with U73122, unaffected by AACOCF3 and enhanced by Rö31-8220. These results suggest that cytosolic phospholipase A2 plays no role in the arachidonic acid release in response to collagen. In contrast, the data are consistent with phospholipase C 2 playing a role in an intricately controlled pathway, or multiple pathways, mediating the release of arachidonic acid in collagen-stimulated platelets.
Human Endothelial Cells Maintain Anti-Aggregatory Activity for Platelets during Apoptosis
Authors: Favaloro Emmanuel , Medbury Heather , Zoellner Hans , Xu Wei
Published: March 2018

Despite evidence of elevated levels of tissue factor and platelet binding by apoptotic endothelial cells, microthrombi do not appear to be associated with apoptotic endothelium and this suggests maintained anti-aggregatory activity for platelets. We report that anti-aggregatory activity is maintained by apoptotic endothelium obtained by serum and or matrix deprivation, which we propose as models for apoptotic endothelial cells released during microvascular remodelling and traumatic detachment respectively. Both apoptotic and non-apoptotic endothelium had strong anti-aggregatory activity for platelets stimulated with either ADP or thrombin. Inhibition experiments using L-NAME and indo-methacin indicated a role for nitric oxide and prostacyclin in this activity. Experiments with latex beads further confirmed that inhibited platelet aggregation by endothelium was not merely a non-specific phenomenon. These data support the idea that EC maintain active anti-thrombotic activity during apoptosis, consistent with maintained urokinase levels and canalicular fragmentation reported elsewhere.
A Standard Nomenclature for von Willebrand Factor Gene Mutations and Polymorphisms
Authors: Eikenboom J. C. J. , Ginsburg D. , Hilbert L. , Mazurier C. , Peake I. R. , Sadler J. E. , Rodeghiero F. , Goodeve A. C.
Published: March 2018

Examination of the entire von Willebrand factor (VWF) gene for mutations, particularly in types 1 and 3 von Willebrand disease (VWD) is becoming more widely practised. The sequence of the entire VWF gene will soon be compiled as a single sequence. For these reasons, a clearly defined nomenclature to use for numbering the VWF nucleotide and amino acid sequence is required.

The following recommendations are made for VWF numbering. VWF cDNA nucleotide sequence should be numbered from the A of the initiator ATG as the +1 position. Genomic DNA should be prefixed with a “g” and also numbered from this position. Amino acid (aa) numbering should be from the initiator methionine as the +1 position with sequential numbering of aa throughout VWF. To avoid confusion with previously used numbering schemes for mature VWF, which started from serine 764 of pre-pro VWF, the use of the single letter amino acid code is recommended.