bluuebunny/crossref_metadata_2025 huggingface dataset:
Three Different Cell Types Can Synthesize Factor XIII Subunit A in the Human Liver
Authors: Ádány Róza , Antal Miklós
Published: July 2018

The origin of human Factor XIII subunit A (FXIII A) has been a subject of intense speculation and investigation during the last decade. The major question under dispute is whether hepatocytes can produce this clotting factor. Experimental evidence obtained by FXIII A phenotype analysis in bone marrow transplant patients clearly identified hemopoietic cells (monocytes/macrophages and/or megakaryocytes/ platelets) as a source of FXIII A, and also showed that additional extra-hemopoietic site(s) of synthesis also exist. The liver has been suggested as a possible extrahemopoietic source of plasma FXIII A, but the cells responsible for synthesizing FXIII A were not identified yet. Our present study was designed to determine the cellular distribution of both FXIII A and its encoding mRNA in human liver samples by using light- and electron-microscopic immuno-morphological and in situ hybridization techniques. In paraformaldehyde/glutaraldehyde (PA/GA) fixed, araldite-embedded semithin sections that were immunostained by an ABC/DAB based postembedding immunocytochemical method, FXIII A could be detected in Kupffer cells, connective tissue histiocytes and hepatocytes. Immunoreactive hepatocytes were observed almost exclusively around the venae centrales. By using postembedding immunogold labeling, FXIII A could be electron-microscopically visualized in these hepatocytes in the immediate vicinity of the lamellae of the endoplasmic reticulum. By in situ hybridization using a mixture of five 32-40mer biotinylated oligonucleotides (ONs) to mRNA regions encoded by exons VI, XI and XV and a labeling system containing streptavidin conjugated with alkaline phosphatase/BCIP/NBT, the message for FXIII A could be detected in the same cell types. These results show that in human liver three different types of cells can synthesize FXIII A, but the extent of their contribution to the plasma FXIII A level will require further studies.
Gemfibrozil Reduces Thrombin Generation in Patients with Combined Hyperlipidaemia, without Influencing Plasma Fibrinogen, Fibrin Gel Structure or Coagulation Factor VII
Authors: Bröijersén Anders , Hamsten Anders , Silveira Angela , Fatah Kamaran , Goodall Alison H , Eriksson Sabina , Angelin Bo , Hjemdahl Paul
Published: July 2018

A double-blind, placebo-controlled, cross-over study was conducted in 21 men with combined hyperlipoproteinaemia to examine if lipid-lowering treatment with gemfibrozil (10-12 weeks) affects blood coagulation and fibrin gel structure at rest or during mental stress. Gemfibrozil lowered plasma triglycerides by 57 ± 4%, whereas high density lipoprotein (HDL) cholesterol increased by 22 ± 5%. Gemfibrozil lowered the triglyceride content of low density lipoprotein (LDL). Gemfibrozil reduced the plasma concentrations of thrombin-antithrombin complex (TAT) and prothrombin fragment F1+2 (F1+2), both at rest and during mental stress. However, there were no effects of gemfibrozil treatment on the plasma concentrations of fibrinogen, factor VII antigen, activated factor VII (Vila) or activated factor XII (XIIa), or on fibrin gel structure. Acute mental stress per se did not influence coagulation factors, reaction products or fibrin gel structure, or their responses to the study drug. Thus, gemfibrozil reduces thrombin generation in men with combined hyperlipoproteinaemia, without influencing the plasma levels of fibrinogen, Vila and XIIa, or fibrin gel structure. Attenuation of thrombin generation may contribute to the primary-preventive effects of gemfibrozil on coronary heart disease.
Coagulation Activation and Fibrinolytic imbalance in Subjects with Idiopathic Antiphospholipid Antibodies -A Crucial Role for Acquired Free Protein S Deficiency
Authors: Ames Paul R J , Tommasino Catello , Iannaccone Luigi , Brillante Massimo , Cimino Renato , Brancaccio Vincenzo
Published: July 2018

To explore the coagulation/fibrinolytic balance and its relation with free protein S (f-PS) in subjects with antiphospholipid antibodies (aPLs) outside the setting of autoimmune inflammatory disorders, we carried out a cross-sectional study on 18 thrombotic patients with primary antiphospholipid syndrome and 18 apparently healthy subjects with persistence of idiopathic aPLs. Prothrombin fragment 1+2 (F1+2), thrombin-antithrombin complex (TAT) and D-Dimer (D-D) were taken as markers of thrombin generation and fibrin turnover. Mean F1+2 levels were higher in thrombotic (p = 0.006) and non-thrombotic subjects (p = 0.0001) than in controls as were those of D-D (p <0.0001 and p = 0.003 respectively). TAT levels did not differ. Lower mean levels of f-PS were found in thrombotic (p = 0.0006) and non-thrombotic subjects (p = 0.002) than in controls. Within both groups, mean Fl+2 levels were higher in subjects who had low f-PS levels compared to those with normal f-PS levels (p = 0.01). Gender analysed data revealed blunted tPA release (venous occlusion test) in thrombotic females (from 16.80 ± 0.79 to 21.3 ± 3.9 ng/nl, NS) but not in thrombotic males (from 18.2 ± 2.0 to 33.7 ± 4.9 ng/ml, p = 0.01) nor in asymptomatic subjects of either sex. Also, in both patient groups females had higher mean PAI than males (p <0.0002) and than control females (p <0.02). Low free protein S was found in 100% of non-thrombotic and in 90% of thrombotic patients with defective fibrinolysis. These data are consistent with increased thrombin generation, accelerated fibrin turnover and fibrinolysis abnormalities also in asymptomatic carriers of aPLs and highlight a central role for acquired f-PS deficiency in the thrombotic tendency of the antiphospholipid syndrome.
Immunological Aspects of Recombinant Factor Vila (rFVIIa) in Clinical Use
Authors: Nicolaisen Else Marie , Hansen Lisbeth Lyng , Poulsen Fritz , Glazer Steven , Hedner Ulla
Published: July 2018

Patients, receiving rFVIIa for treatment of bleeding disorders, have been followed for specific antibody formation. No antibodies against FVII were demonstrated in 170 patients, with hemophilia, or with acquired inhibitors to clotting factors. Of 6 FVII-deficient patients, one overdosed patient developed antibodies to human FVII. There was no indication of de novo formation of antibodies to potential contaminating foreign protein, which could be correlated to the rFVIIa treatment. Except for the FVII-deficient population, which may represent a risk group, rFVIIa appears to be immunologically safe for use in patient groups with bleeding disorders, including hemophilia A and B patients.
Tissue Factor Pathway Inhibitor Production by Human Mesangial Cells in Culture
Authors: Yamabe H , Osawa H , Inuma H , Kaizuka M , Tamura N , Tsunoda S , Fujita Y , Shirato K , Onodera K
Published: July 2018

Fibrin formation within the glomeruli has been observed in various forms of human and experimental glomerulonephritis and it may play an important role in progressive glomerular injury. Furthermore it has been hypothesized that glomerular fibrin deposition may occur through activation of either the intrinsic or extrinsic coagulation pathway. It has been demonstrated that a procoagulant activity (PCA) which is compatible with tissue factor is present in the glomeruli and becomes increased in human proliferative glomerulonephritis and in animal models of nephritis. Tissue factor pathway inhibitor (TFPI) regulates the extrinsic pathway of blood coagulation through its ability to inhibit tissue factor activity. TFPI is present in plasma and in platelets, and it is now thought to be produced mainly by endothelial cells. We examined whether human mesangial cells (HMC) could produce TFPI and attempted to clarify regulatory factors which affect TFPI production. Cultured HMC were used and TFPI in the cell supernatants was measured by ELISA using a specific antibody. Cultured HMC showed the production of TFPI. Immunoblot analysis revealed 40 kD protein of TFPI. The concentration of TFPI was significantly increased following the incubation with thrombin and heparin, including low molecular weight heparin, in a dose- and time-dependent manner. However, fetal calf serum, phorbol myristate acetate, lipopolysaccharide, IL-1β and tissue factor did not stimulate TFPI synthesis. Our data show that cultured HMC have the ability to produce TFPI which inhibits fibrin formation. It is possible that thrombin-induced enhancement of TFPI synthesis may be caused by the autoregulatory system of blood coagulation and that with heparin it may represent another anticoagulatory effect of heparin.
The VITA Project: Population-based Distributions of Protein C, Antithrombin III, Heparin-cofactor II and Plasminogen -Relationship with Physiological Variables and Establishment of Reference Ranges
Authors: Rodeghiero Francesco , Tosetto Alberto
Published: July 2018

A proper laboratory diagnosis of inherited thrombophilia due to defects of the coagulation system may be obtained only by a stringent definition of the diagnostic reference ranges taking into account the influence exerted by major physiological variables. To this purpose, we analyzed the data for Protein C, Antithrombin III, Heparin-Cofactor II and plasminogen coming from the first 4,000 subjects enrolled in the VITA Project. This is the first study that allows the establishment of reference ranges in a non-selected, active population using stringent standardization of clinical and laboratory measurements and multivariable regression techniques for data analysis.

Serum triglycerides and total cholesterol, together with plasma fibrinogen were found to influence the functional plasma level of the four considered proteins. Menopause increased AT-III concentration, while pill use increased Heparin-Cofactor II and plasminogen. PT and PTT ratio, gender, age, smoking, body-mass index, HDL cholesterol and blood group had minor effects. The effect of these variables should be taken into account for both clinical and epidemiologic purposes, using appropriate reference ranges or covariance analysis for adjustment.
lonomycin-stimulated Arachidonic Acid Release in Human Platelets: a Role for Protein Kinase C and Tyrosine Phosphorylation
Authors: Hargreaves Philip G , Jenner Susanne , Merritt Janet E , Sage Stewart O , Farndale Richard W
Published: July 2018

Collagen (10-90 Μg/ml) and ionomycin (1 ΜM; a calcium iono-phore) each evoked rises in intracellular free calcium, protein kinase C activity and arachidonic acid release in human platelets, and as previously demonstrated for collagen, ionomycin (1 p,M) stimulated protein tyrosine phosphorylation. However, at lower concentrations (60 and 250 nM) ionomycin selectively mobilised calcium. Ro31-8220 (a selective inhibitor of protein kinase C) inhibited (by 50%) ionomycin-stimulated arachidonic acid release. Genistein (an inhibitor of protein tyrosine kinases) also reduced by 50% ionomycin-stimulated arachidonic acid release. In combination, genistein and Ro31-8220 abolished ionomycin-stimulated arachidonic acid release. These findings show 1) that a rise in calcium is not sufficient, and 2) the activation of both protein kinase C and protein tyrosine phosphorylation is necessary, for full ionomycin-stimulated arachidonic acid release in human platelets.
A Novel Mutation Glyl672→Arg in Type 2A and a Homozygous Mutation in Type 2B von Willebrand Disease
Authors: Hagiwara Takeshi , Inaba Hiroshi , Yoshida Shinichi , Nagaizumi Keiko , Arai Morio , Hanabusa Hideji , Fukutake Katsuyuki
Published: July 2018

Genetic materials from 16 unrelated Japanese patients with von Willebrand disease (vWD) were analyzed for mutations. Exon 28 of the von Willebrand factor (vWF) gene, where point mutations have been found most frequent, was screened by various restriction-enzyme analyses. Six patients were observed to have abnormal restriction patterns. By sequence analyses of the polymerase chain-reaction products, we identified a homozygous R1308C missense mutation in a patient with type 2B vWD; R1597W, R1597Q, G1609R and G1672R missense mutations in five patients with type 2A; and a G1659ter nonsense mutation in a patient with type 3 vWD. The G1672R was a novel missense mutation of the carboxyl-terminal end of the A2 domain. In addition, we detected an A/C polymorphism at nucleotide 4915 with HaeIII. There was no particular linkage disequilibrium of the A/C polymorphism, either with the G/A polymorphism at nucleotide 4391 detected with Hphl or with the C/T at 4891 detected with BstEll.
Ala244Val Is a Common, Probably Ancient Mutation Causing Factor VII Deficiency in Moroccan and Iranian Jews
Authors: Tamary Hannah , Fromovich Yonit , Shalmon Lea , Reich Ziv , Dym Orly , Lanir Naomi , Brenner Benjamin , Paz Michael , Luder Anthony S , Blau Orit , Korostishevsky Michael , Zaizov Rina , Seligsohn Uri
Published: July 2018

We investigated the molecular basis for factor VII (FVII) deficiency in Israel and found that 13 patients were homozygous and 10 heterozygous for a C to T substitution at nucleotide 10648 of the FVII gene. This predicted an Ala244Val change and was associated with decreased FVII activity and antigen level. Of the 36 Ala244Val positive alleles, 20 were observed in patients of Moroccan origin, 10 in Iranian-Jewish patients and 6 in patients of other origins. A computer model of the serine protease domain of FVII suggested that the Ala244Val substitution may cause distortion of the entire protein structure. Intragenic polymorphic sites analyses disclosed a founder effect for the Moroccan and Iranian-Jewish patients. A survey of the Ala244Val mutation revealed an allele frequency of 1:42.5 in Moroccan Jews and 1:40 in Iranian Jews. As Moroccan Jews have been separated from Iranian Jews for more than two millennia, the data suggest that the Ala244Val mutation occurred in ancient times.
Genetic Characterization of Protein C Deficiency in Japanese Subjects Using a Rapid and Nonradioactive Method for Single-Strand Conformational Polymorphism Analysis and a Model Building
Authors: Miyata Toshiyuki , Sakata Toshiyuki , Zheng Yan-Zhen , Tsukamoto Hiroaki , Umeyama Hideaki , Uchiyama Shinichiro , Ikusaka Masaomi , Yoshioka Akira , Imanaka Yasufumi , Fujimura Hironobu , Kambayashi Jun-ichi , Kato Hisao
Published: July 2018

We studied the molecular basis of protein C deficiency in 28 Japanese families including 4 asymptomatic families. Two showed a decreased level of function with a normal antigen concentration consistent with type II protein C deficiency and the remaining 26 showed type I deficiency with decreases in both function and antigen level. All the exons and intron/exon junctions of the protein C gene were studied using a strategy combining polymerase chain reaction (PCR) amplification and rapid nonradioactive single-strand conformational polymorphism (SSCP) analysis. The PCR-amplified fragments with aberrant migration on SSCP analysis were sequenced. We identified 11 missense mutations, 1 nonsense mutation, 2 neutral polymorphisms, 1 frameshift deletion, 1 inframe deletion, and 1 splice site mutation. We also identified two different rare mutations in the 5-untranslated region in the protein C gene that may be responsible for the phenotype. Of these molecular defects, ten were novel. From the results of genetic analysis of 47 Japanese families with protein C deficiency reported in this and previous studies, Phel39Val and Met364Ile substitutions and a G8857 deletion were only found in Japanese subjects and seem to be a founder effect. In contrast, Argl69Trp and Val297Met substitutions, both occurring at CG dinucleotides, were commonly observed in not only Japanese but also Western populations, indicating that these are hot spots for mutation in the protein C gene. These molecular defects were found in 22 families in total, accounting for 47% of Japanese families with protein C deficiency. The structural models of the second EGF and protease domains of activated wild-type and mutant human protein C suggest a possible substrate binding exosite on two loops; one from amino acid position 349 to 357 and the other from position 385 to 388, both of which are close to each other in the three-dimensional model.
Increased Procoagulant Activity of Red Blood Cells from Patients with Homozygous Sickle Cell Disease and β-Thalassemia
Authors: Helley Dominique , Eldor Amiram , Girot Robert , Ducrocq Rolande , Guillin Marie-Claude , Bezeaud Annie
Published: July 2018

It has recently been proved that, in vitro, red blood cells (RBCs) from patients with homozygous β-thalassemia behave as procoagulant cells. The procoagulant activity of β-thalassemia RBCs might be the result of an increased exposure of procoagulant phospholipids (i. e. phosphatidylserine) in the outer leaflet of the membrane. In order to test this hypothesis, we compared the catalytic properties of RBCs of patients with β-thalassemia and homozygous sickle cell disease (SS-RBCs) with that of controls. The catalytic parameters (Km, kcat) of prothrombin activation by factor Xa were determined both in the absence and in the presence of RBCs. The turn-over number (kcat) of the reaction was not modified by normal, SS- or (3-thalassemia RBCs. The Km was lower in the presence of normal RBCs (mean value: 9.1 µM) than in the absence of cells (26 µM). The Km measured in the presence of either SS-RBCs (mean value: 1.6 µM) or β-thalassemia RBCs (mean value: 1.5 pM) was significantly lower compared to normal RBCs (p <0.001). No significant difference was observed between SS-RBCs and p-thalassemia RBCs. Annexin V, a protein with high affinity and specificity for anionic phospholipids, inhibited the procoagulant activity of both SS-RBCs and (3-thalassemia RBCs, in a dose-dependent manner. More than 95% inhibition was achieved at nanomolar concentrations of annexin V. These results indicate that the procoagulant activity of both β-thalassemia RBCs and SS-RBCs may be fully ascribed to an abnormal exposure of phosphatidylserine at the outer surface of the red cells.
Erythromelalgia in Essential Thrombocythemia Is Characterized by Platelet Activation and Endothelial Cell Damage but not by Thrombin Generation
Authors: van Genderen Perry J J , Lucas Irene S , Strik Roel van , Vuzevski Vojislav D , Prins Fransisco J , van Vliet Huub H D M , Michiels Jan J
Published: July 2018

Erythromelalgia, a characteristic aspirin-responsive microvascular thrombotic complication in essential thrombocythemia (ET), may develop despite oral anticoagulant treatment or treatment with heparin, suggesting that the generation of thrombin is not a prerequisite for its development. To study this, a cross-sectional comparison of the plasma levels of thrombomodulin (TM), platelet factor 4 (PF4), β-thrombo-globulin (β-TG), prothrombin fragment 1+2 (F1+2) and total degradation products of fibrin(ogen) (TDP) was made between 5 ET patients suffering from erythromelalgia, 16 asymptomatic ET patients and 20 control subjects, and after treatment with aspirin, respectively. Furthermore, 2 ET patients with a history of erythromelalgia were studied at regular time intervals after discontinuation of aspirin until erythromelalgia recurred. As compared with asymptomatic ET patients and control subjects erythromelalgia was characterized by significantly higher β-TG and TM levels but no significant differences were detected in either F1+2 or TDP levels. Treatment of erythromelalgia with aspirin resulted in disappearance of erythromelalgic signs and symptoms, which was paralleled by a significant decrease of β-TG and TM levels. Histopathologic and immunohistochemical analysis of biopsies derived from erythromelalgic skin areas of 2ET patients showed that erythromelalgic thrombi stained positively for von Wille-brand factor opposed to only a weak fibrin staining. Our data suggest that erythromelalgia is caused by the intravascular activation and aggregation of platelets with subsequent sludging or occlusion of the acral arterial microvasculature. The generation of thrombin appears not to be essential for the formation of these platelet thrombi, thereby giving a plausible explanantion for the inefficacy of coumadin derivatives and heparin in the prevention and treatment of erythromelalgia in essential thrombocythemia.
Conjunctive Administration of Intravenous Heparin Attenuates Cross-linked Fibrin Degradation in Patients Treated with Streptokinase
Authors: Galvani Marcello , Abendschein Dana R , Ferrini Donatella , Ottani Filippo , Rusticali Franco , Eisenberg Paul R
Published: July 2018

Increases in thrombin activity occur in patients treated with streptokinase, but conjunctive therapy with intravenous heparin does not appear to improve either the rate of early infarct artery patency or survival in patients with acute myocardial infarction. In a recent study we found that concentrations of fibrinopeptide A, a marker of thrombin-mediated fibrin formation, were lower in the first 3 h in patients treated with intravenous heparin (5000 U bolus followed by a fixed-dose 1000 U/h infusion, n = 14) compared with subcutaneous (12,500 U every 12 h, started 4 h after streptokinase, n = 14) administration, but were increased in both groups of patients, consistent with persistent thrombin activity. To determine whether the differential effects of the intensity of heparinization on thrombin formation were reflected in differences in fibrin degradation, we measured cross-linked fibrin degradation products (XL-FDP) before and 1,2,3,8,12, and 24 h after streptokinase in the same cohort of patients, with a new ELISA with a D-dimer-specific capture antibody (MAb 3B6) and a fibrin-specific tag antibody (MAb 1D2, Agen, Brisbane, Australia). The incidence of early coronary recanalization assessed by creatine-kinase MM isoforms (increase in activity ≥0.18%/min), was similar in both groups (79 vs 86%). Concentrations of XL-FDP were similar in patients with and without recanalization, but were lower in patients treated with intravenous compared with subcutaneous heparin at 8 h, but the results did not reach statistical significance (627 ±151 ng/ml versus 1007 ± 157 ng/ ml, p = 0.06), and were significantly lower at 12 h (327 ± 72 versus 781 ± 162 ng/ml, p = 0.03 at 12 h) (mean ± SEM). Concentrations of cross-linked fibrin degradation products were also lower in patients in whom the activated partial thromboplastin time was greater than two times the control, compared with those with inadequate anticoagulation (498 ± 105 versus 1084 ± 179 ng/ml; p = 0.02) (mean ± SEM). Thus, more effective inhibition of thrombin with conjunctive intravenous heparin therapy results in less cross-linked fibrin turnover in the first 12 h after thrombolysis with streptokinase. This probably reflects decreased fibrin formation with therapeutic anticoagulation.
Cyclosporine Therapy for Acquired Factor VIII Inhibitor in a Patient with Systemic Lupus Erythematosus
Authors: Schulman S , Langevitz P , Livneh A , Martinowitz U , Seligsohn U , Varon D
Published: July 2018

The case of a 27-year-old woman with systemic lupus erythematosus and development of an autoantibody against factor VIII during an exacerbation of her underlying disorder is described. Attempts to eliminate the antibody with high dose gammaglobulin and repeated courses of cyclophosphamide failed, whereafter she received cyclosporine in increasing doses. When therapeutic serum levels of cyclosporine were achieved (150-350 ng/ml) the inhibitor rapidly decreased and disappeared with a concomitant normalization of the factor VIII levels. Treatment with cyclosporine was subsequently reduced and discontinued after one year, and at present no inhibitor is detectable. In view of the successful results with cyclosporine treatment in 4 of 6 previous cases and in all 3 previous cases with autoimmune disorders, this regimen should be evaluated in a systematic manner as a potential first line drug in patients with acquired hemophilia and an underlying autoimmune disorder.
Phosphatidylethanolamine Induces Specific Conformational Changes in the Kininogens Recognizable by Antiphosphatidylethanolamine Antibodies
Authors: Sugi Toshitaka , McIntyre John A
Published: July 2018

Autoantibodies to the zwitterionic phospholipid (PL), phosphatidylethanolamine (PE) have been described in patients with thrombotic diseases. Recently, we reported that many antiphosphatidylethanolamine antibodies (aPE) are not specific for PE per se, but are directed to plasma proteins which can bind PE, for example, high molecular weight kininogen (HK), low molecular weight kininogen (LK) and/or proteins in complex with HK, factor XI or prekallikrein. In the ELISA, we now show that intact HK binds to all PL tested. By using a monoclonal antibody (mAb) to the HK light chain, binding to cardiolipin (CL) was highest followed by phosphatidylserine (PS), PE and phosphatidylcholine (PC). By contrast, LK bound best to PE, with lesser amounts binding to PC and CL. Many aPE recognize only a kininogen-PE complex and neither PE nor kininogen when they are tested independently. We now report that these aPE are specific for the kininogen-PE complex as they do not recognize the kininogens when the latter are presented on other PL substrates. This indicates that PE induces unique antigenic conformational changes in the kininogens which are not induced when the kininogens bind to other PL. The kininogens bind to platelets and endothelial cells through an as yet undefined receptor. Since PE is a normal constituent of the outer layer of cell and platelet plasma membranes, it is available for the kininogens to bind. In vitro studies showed that purified IgG which bound kininogen-PE complexes in ELISA also caused platelets to undergo irreversible aggregation when stimulated by subthreshold concentrations of bovine thrombin.
Two New Frequent Dimorphisms in the Protein S (PROS1) Gene*
Authors: Mustafa Stefan , Pabinger Ingrid , Mannhalter Christine
Published: July 2018

Two new polymorphisms were identified in the proteinS gene (PR0S1): an intronic T/A-dimorphism (PIPS1) in intron K, and an exonic C/A-dimorphism (PEPS2), located in the 3'untranslated trailer of exon 15. Allelic frequencies of 24% (PIPS 1-A) and 17% (PEPS2-A) respectively, were determined in the normal population. The identification of an intronic and an exonic PR0S1 dimorphism, in addition to the known BstXI dimorphism, enlarges the molecular tool box for gene analysis and transcript quantification in hereditary protein S deficiency. Haplotype analysis showed that variability of both new polymorphisms occurred almost exclusively in the A-allele of the known intragenic BstXI dimorphism. Therefore, PEPS2 and PIPS1 are especially valuable in individuals homozygous for the BstXI A-variant.
Plasminogen Activator Inhibitor 1 Is Elevated in the Children of Men with Premature Myocardial Infarction
Authors: Rallidis Loukianos S , Megalou Ageliki A , Papageorgakis Nikos H , Trikas Athanasios G , Chatzidimitriou Grammatiki I , Tsitouris George K
Published: July 2018

To assess whether plasminogen activator inhibitor 1 (PAI-1) activity is elevated in the progeny of young coronary men, 193 young subjects were recruited and divided into two groups. Group A consisted of 104 children whose fathers had suffered a myocardial infarction before the age of 55 (“cases”). Eighty-nine young subjects matched for age, sex, body mass index (BMI) and smoking habits without familial history of coronary artery disease (CAD) served as controls (group B). Children with a family history of diabetes mellitus or hypertension were excluded from both groups. We measured PAI-1 activity, tissue-type plasminogen activator (t-PA) antigen, a2-antiplasmin, fibrinogen, lipids and apolipoproteins in both groups. PAI-1 activity levels were also determined in the men who suffered a premature myocardial infarction 4 months after their discharge. PAI-1 activity levels were higher in cases compared to controls (3.13 ± 1.9 vs 2.17 ± 1.9 U/ml, p = 0.0014). t-PA antigen and a2-antiplasmin did not differ significantly between the two groups, while fibrinogen, total cholesterol, low-den-sity lipoprotein cholesterol, apolipoprotein B and lipoprotein(a) were significantly higher in group A. PAI-1 was positively correlated with triglycerides (r = 0.22, p = 0.024), apolipoprotein B (r = 0.21, p = 0.039) and fibrinogen (r = 0.22, p = 0.029 ) in cases and with BMI in both cases (r = 0.37, p = 0.0003) and controls (r = 0.23, p = 0.044). In stepwise multiple regression analysis, only apolipoprotein B (p = 0.008) and BMI (p = 0.0014) were significant determinants of PAI-1 activity in cases. There was also a positive correlation between PAI-1 activity levels of the affected fathers and their children (r = 0.30, p = 0.01). The present data support the hypothesis that elevated PAI-1 levels in the offspring of men with premature myocardial infarction impair their fibrinolytic capacity contributing to their familial predisposition to CAD.
Characterization of Recombinant von Willebrand Factors Mutated on Cysteine 509 or 695
Authors: Siguret Virginie , Ribba Anne-Sophie , Christophe Olivier , Chérel Ghislaine , Obert Bernadette , Rouault Christine , Nishikubo Toshiya , Meyer Dominique , Girma Jean-Pierre , Piétu Geneviève
Published: July 2018

The interacting domain of vWF with platelet GPIb has been shown to overlap the large A1 loop formed by the intra-chain disulfide bond linking Cys 509 to Cys 695. In order to further investigate the role of the conformation of this region, we have expressed in COS-7 cells three mutated full-length recombinant vWFs (rvWFs) in which the substitutions Cys509Gly, Cys509Arg or Cys695Gly have been introduced by site-directed mutagenesis. SDS-agarose gel electrophoresis demonstrated an impaired multimerization of the mutants with undetectable high molecular weight multimers and a decrease of the relative amounts of the intermediate sized multimers. Binding analysis showed that rvWFC509G and rvWFC509R did not interact with botrocetin but spontaneously interacted with GPIb; the latter binding remained unchanged in the presence of ristocetin. This indicates that the substitution of Cys509 by Gly or Arg creates a conformation of vWF that increases its binding to GPIb. In contrast, rvWFC695G which did not react with botrocetin was also unable to interact with GPIb even in the presence of ristocetin, indicating that sequences interacting with GPIb are masked and/or disrupted. In conclusion, the substitution of each of the Cys509 and 695 results in mutant proteins which may be “locked” into active or inactive conformations in regard to the binding to platelet GPIb receptor.
Evaluation of the Abnormal Platelet Function in von Willebrand Disease by the Blood Filtration Test
Authors: Pareti Francesco I , Cattaneo Marco , Carpinelli Luca , Zighetti Maddalena L , Bressi Caterina , Mannuccio Mannucci Pier , Ruggeri Zaverio M
Published: July 2018

We have evaluated platelet function in different subtypes of von Willebrand disease (vWD) by pushing blood through the capillarysized channels of a glass filter. Patients, including those with type IIB vWD, showed lower than normal platelet retention and increased cumulative number of blood drops passing through the filter as a function of time. In contrast, shear-induced platelet aggregation, measured in the cone-and-plate viscometer, was paradoxically increased in type IIB patients. Treatment with l-desamino-8-D-arginine vasopressin (DDAVP) tended to normalize the filter test in patients with type I-platelet normal and type I-platelet low vWD, but infusion of a factor VUI/von Willebrand factor (vWF) concentrate lacking the largest vWF multimers was without effect in type 3 patients. Experiments with specific monoclonal antibodies demonstrated that the A1 and A3 domains of vWF, as well as the glycoproteins Ibα and Ilb-IIIa on platelets, are required for platelet retention in the filter. Thus, the test may reflect vWF function with regard to both platelet adhesion and aggregation under high shear stress, and provide relevant information on mechanisms involved in primary hemostasis.
Haemostatic Studies in Carbohydrate-deficient Glycoprotein Syndrome Type I
Authors: Fiumara A , Barone R , Buttitta P , Musso R , Pavone L , Nigro F , Jaeken J
Published: July 2018

CDG syndrome (CDGS) type I is the most frequent form of a group of metabolic disorders characterised by a defect of the carbohydrate moiety of glycoproteins. A large number of plasma glycoproteins, including clotting factors and inhibitors, are decreased and stroke-like episodes have been described in about half of the reported patients. We studied blood coagulation factors, inhibitors and D-dimer plasma levels in four subjects, aged 12-23 years, with CDGS type I. Factors VIII, XI, antithrombin III activity, antigen plasma levels of antithrombin III, free protein S and protein C were decreased whereas protein C as activity was normal. In addition two patients had reduction of factors II, V, VII, IX, X reflecting the phenotypic heterogeneity associated with CDGS type I. D-dimer plasma concentrations were elevated in all subjects. The hypercoagulable state as consequence of the combined deficiencies of coagulation inhibitors could contribute to the stroke-like phenomena in CDGS type I.
Characterization of Cellular Binding Sites and Interactive Regions within Reactants Required for Enhancement of Plasminogen Activation by tPA on the Surface of Leukocytic Cells
Authors: Félez Jordi , Miles Lindsey A , Fabregas Pere , Jardi Merce , Plow Edward F , Lijnen Roger H
Published: July 2018

Plasminogen and tPA bind to a common set of binding sites on nucleated cells. To assess the functional consequences of cellular binding, we have measured the kinetic changes induced by plasminogen activation by tPA on cell surfaces. These studies were carried out with U937 and THP-1 monocytoid cells, with Raji, Nalm6 and Molt4 lymphoid cells and with peripheral blood monocytes and neutrophils. The interactions of plasminogen and tPA with cells induced an increase in the rate of plasmin generation which depended upon the cell concentration. With saturating amounts of U937 monocytoid cells (1.25 × 105/ml) the rate of plasmin generation was 0.39 nM.s-1 versus 0.07 and 0.09 nM.s-1 without cells or without tPA, respectively. The catalytic efficiency of Glu- or Lys-plasminogen activation by tPA increased by 7.2- and 24.2-fold, respectively. These changes were induced by a 72-242-fold reduction in the Km of these interactions which was in the range of 0.3-0.9 µM. These values are below the plasminogen concentration in plasma (1-2 µM). Moreover, we provide new data indicating that 1) only a specific subset of plasminogen binding sites, i.e. molecules exposing carboxyl terminal lysines on the cell surface, promotes plasminogen activation on cells; 2) the first four kringles of plasminogen and the finger of tPA are critical for enhanced plasmin generation on cell surfaces; 3) the simultaneous co-localization of tPA with plasminogen on cell surfaces is required for enhanced plasminogen activation; 4) modulation of plasminogen/tPA receptor expression induces concomitant modulation of the stimulatory effects of cells on plasminogen activation and 5) in a direct comparison, the mechanism by which cells and fibrin fragments accelerate plasminogen activation are similar but not identical. These data suggest that modulation of plasminogen/tPA binding sites permits local and efficient generation of plasmin on cell surfaces.
Recombinant Full-length Tissue Factor Pathway Inhibitor (TFPI) Prevents Thrombus Formation and Rethrombosis after Lysis in a Rabbit Model of Jugular Vein Thrombosis
Authors: Kaiser Brigitte , Fareed Jawed
Published: July 2018

In the jugular vein of rabbits thrombus formation was induced by vessel wall damage using a balloon catheter and following reduction of blood flow by 80-90% for 60 min (partial stasis). The blood flow in the vein was measured continuously and the incidence of primary thrombus formation, the time until lysis with recombinant tissue-type plasminogen activator (rt-PA) as well as the incidence of rethrombosis after lysis were determined. At the end of the experiment the wet weight of the thrombus formed inside the vessel was measured. For the determination of haemostaseological parameters blood was drawn from the cannulated femoral artery.

Recombinant full-length tissue factor pathway inhibitor (TFPI) was studied with regard to its effect both on primary thrombus formation and on rethrombosis after lysis. In control animals damage of the vessel wall combined with partial stasis led to the formation of occlusive venous thrombi. In vitro bolus injection of TFPI (5,10,20 Μg/kg) at the time of thrombus induction prevented the formation of venous thrombi during the 1 h period of partial stasis. In the subsequent observation period a dose-dependent inhibition of later occurring partial or complete thrombotic occlusion was found. At the dose of 20 Μg/kg i.v. in all animals TFPI prevented a complete thrombotic occlusion of the vein up to 3 h after stasis. To study the effectiveness of TFPI on rethrombosis after lysis TFPI was injected i.v. after lysis of the thrombus with rt-PA (600 Μg/kg i.v. bolus + 600 Μg/kg i.v. infusion over 60 min). In saline treated control rabbits a reocclusion of the vessel was seen in 8 of 10 animals. TFPI (10, 20, 40 Μg/kg i.v.) injected at the end of rt-PA administration caused a dose-dependent inhibition of thrombotic reocclusion after lysis. At 40 Μg/kg i.v. the formation of occlusive thrombi was prevented up to 3 h after lysis. TFPI at the doses used caused modest anticoagulant effects in global clotting assays; activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) were only slightly prolonged. A clear and dose-dependent prolongation of clotting times was only seen in the Heptest® assay.

The results show that the physiologic coagulation inhibitor TFPI acts as a strong antithrombotic agent in an experimental model of venous thrombus formation and thrombotic reocclusion after lysis in rabbits.
An Evaluation and Improvement Program for Inpatient Anticoagulant Control
Authors: Phillips W S , Smith J , Greaves M , Preston F E , Channer K S
Published: July 2018

To improve the management and therapeutic control of inpatients on anticoagulant drugs, combined prescription and monitoring charts have been developed for both heparin and warfarin which incorporate clinical guidelines. These have been introduced throughout a 700-bedded acute teaching hospital via a structured program of change management.

We have demonstrated improvements in the quality of anticoagulant control (assessed with a custom-written computer program), adherence to clinical guidelines and quality of monitoring and prescribing of anticoagulants in inpatients.

The percentage time spent under-anticoagulated with heparin (activated partial thromboplastin time ratio <1.5) fell from 32.7% to 18.5% (p<0.0001), whereas there was no change in percentage time over-anticoagulated (5.1% vs. 5.8%; p = ns). The percentage time spent under-anticoagulated with warfarin was unaltered (26.3% vs. 29.8%; p = ns) but the percentage time spent over-anticoagulated (International Normalised Ratio >4.5) was halved from 5.4% to 2.7% (p<0.001).

We conclude that the introduction of the charts led to significant improvements in anticoagulant control.
Plasma Macrophage Colony-Stimulating Factor and P-Selectin Levels in Malaria-Associated Thrombocytopenia
Authors: Lee S H , Looareesuwan S , Chan J , Wilairatana P , Vanijanonta S , Chong S M , Chong B H
Published: July 2018

Thrombocytopenia is a common finding in malaria. In clinical trials, recombinant macrophage colony-stimulating factor (M-CSF) causes a reversible, dose-dependent thrombocytopenia, and high M-CSF has been reported in autoimmune thrombocytopenias. P-selectin, which is secreted into the plasma following platelet/endothelial activation or damage, is elevated in certain consumptive thrombocytopenic disorders. The relationships between thrombocytopenia, M-CSF and P-selectin were analysed in 63 patients with severe (n = 13) or uncomplicated (n = 26) P. falciparum (PF) or P. vivax (PV) malaria (n = 24). On admission, 69% of PF patients and 75% of PV patients were thrombocytopenic (platelets < 150 X 109/1). M-CSF was elevated in PF (3021 ± 1844 pg/ml) and PV (2602 ± 1668 pg/ml) patients, compared to controls (589 ± 200 pg/ml). The platelet count was inversely correlated with M-CSF in PF (r = -0.681), and in PV malaria (r = -0.548). Elevated P-selectin was found in severe PF malaria, but not in PV malaria. Severe PF malaria was associated with marked thrombocytopenia, very high M-CSF, elevated P-selectin and compelling evidence of disseminated intravascular coagulopathy (DIC). Platelet counts, M-CSF and P-selectin returned to control values in 7-14 days. These data suggest that elevated M-CSF in malaria, by enhancing macrophage activity, may result in increased macrophage-mediated platelet destruction. Further, platelet/endothelial activation or damage, as measured by P-selectin, or DIC could intensify thrombocytopenia in severe PF malaria, but does not appear to contribute to thrombocytopenia in uncomplicated PF or PV malaria.
Differential Effects of Reconstituted High-density Lipoprotein on Coagulation, Fibrinolysis and Platelet Activation during Human Endotoxemia
Authors: Pajkrt Dasja , Lerch Peter G , van der Poll Tom , Levi Marcel , Illi Marlies , Doran Jan E , Arnet Beat , van den Ende Abraham , ten Cate Jan Wouter , van Deventer Sander J H
Published: July 2018

High-density lipoproteins (HDL) can bind and neutralize lipopoly- saccharides (LPS) in vitro and in vivo. HDL can also affect fibrinolytic activity and can directly influence platelet function by reducing platelet aggregation. In this study, the effects of reconstituted HDL (rHDL) on LPS-induced coagulation, fibrinolysis and platelet activation in humans were investigated. In a double-blind, randomized, placebo-controlled, cross-over study, eight healthy male volunteers were injected with LPS (4 ng/kg) on two occasions, once in conjunction with rHDL (40 mg/kg, given as a 4 h infusion starting 3.5 h prior to LPS injection), and once in conjunction with placebo. rHDL significantly reduced LPS-induced activation of coagulation (plasma levels of prothrombin fragment F1+2) and fibrinolysis (plasma levels of tissue type plasminogen activator antigen, t-PA). No effect was observed on LPS-induced inhibition of the fibrinolytic pathway (PAI-1) or on the transient thrombocytopenia elicited by LPS. Furthermore, rHDL treatment significantly enhanced the inhibition of collagen-stimulated inhibition of platelet aggregation during endotoxemia, but had no such effect on arachido- nate-stimulated platelet aggregation. rHDL treatment per se also reduced collagen-induced platelet aggregation. These results indicate that rHDL modifies the procoagulant state associated with endotoxemia.
The Effect of Factor Xa/Phospholipid Infusion on the Acute Phase Response in Baboons
Authors: Kruithof Egbert K O , Agay Diane , Mestries Jean Claude , Gascon Marie-Paule , Ythier Arnaud
Published: July 2018

Disseminated intravascular coagulation (DIC) is a frequent complication of septicemia or tissue injury and may be accompanied by elevations of interleukin-6, a mediator of the acute phase response. It is not known whether thrombin or fibrin deposition may directly induce an acute phase response. To study this, we employed a baboon model of in vivo thrombin generation, induced by the administration of purified bovine Factor Xa and phospholipid vesicles. Two Xa/phospholipid dosages were used, a low dosage (2 animals) leading to a rapid 49% decrease in fibrinogen and a high dosage (two injections at 5h interval; 3 animals) leading to complete fibrinogen depletion. Thereafter, fibrinogen levels increased in both treatment groups, reached a maximum of 2.52 ± 0.23 g/1 (mean ± SE, n = 5; p <0.01 with respect to basal levels) at day 2, and returned to normal by day seven. In five control (injection of 0.15% NaCl) baboons no significant changes of fibrinogen were observed (maximal values: 1.88 ± 0.12 g/1). Serum concentrations of C-reactive protein, an acute phase protein, increased from 3.7 ± 0.4 mg/1 to a maximum of 33.0 ± 7.3 at day one, which was five-fold higher (p <0.01) than in control animals at day one (6.2 ± 0.5 mg/1). Transient increases were observed within 6 h for interleukin-6 from basal values of 6.2 ± 1.7 ng/1 to peak plasma levels of 42.9 ±21.4 ng/1, a value threefold higher (p = 0.07) than in control animals (14.8 ± 4.0 ng/1).

The preliminary results of this observational study suggest that factor Xa/phospholipid infusion is followed by an acute phase response, leading after one day to significant increases of fibrinogen and of C-reactive protein.
Evaluation of Sensitivity and Specificity of a Standardized Procedure Using Different Reagents for the Detection of Lupus Anticoagulants
Authors: Goudemand J , Caron C , De Prost D , Derlon A , Borg J Y , Sampol J , Sié P ,   for the Working Group on Hemostasis of the Société Française de Biologie Clinique and for the Groupe d’Etudes sur I’Hémostase et la Thrombose
Published: July 2018

This study was designed to test the sensitivity and specificity of a combination of 3 phospholipid-dependent assays performed with various reagents, for the detection of lupus anticoagulant (LA). Plasmas containing an LA (n = 56) or displaying various confounding pathologies [58 intrinsic pathway factor deficiencies, 9 factor VIII inhibitors, 28 plasmas from patients treated with an oral anticoagulant (OAC)] were selected. In a first step, the efficiency of each assay and reagent was assessed using the Receiving Operating Characteristic (ROC) method. Optimal cut-offs providing both sensitivity and specificity ≥80% were determined. The APTT assay and most of the phospholipid neutralization assays failed to discriminate factor VIII inhibitors from LA. In a second step, using the optimal cut-offs determined above, the results of all the possible combinations of the 3 assays performed with 4 different reagents were analyzed. Thirteen combinations of reagents allowed ≥ 80% of plasmas of each category (LA, factor deficiency or OAC) to be correctly classified (3/3 positive test results in LA-containing plasmas and 0/3 positive results in LA-negative samples).
PAI-1 Concentrations in First-degree Relatives of Patients with Non-insulin-dependent Diabetes: Metabolic and Genetic Associations
Authors: Mansfield Michael W , Stickland Max H , Grant Peter J
Published: July 2018

Aim: To identify whether levels of PAI-1 are elevated in relatives of NIDDM patients and the extent to which they relate to features of insulin resistance and to genotype at a common PAI-1 promoter polymorphism.

Results: In 132 first-degree relatives of NIDDM patients and 151 controls PAI-1 activity was higher in relatives 14.4 U/ml than controls U/ml (p <0.0005) with higher body mass index 27.3 v. 24.7 kg/m2 p <0.0005, fasting insulin 9.3 v. 7.6 mU/l p <0.005 and triglyceride v. 1.2 mmol/1 p <0.05. PAI-1 activity levels were higher in men and in smokers and showed a trend to being higher with increasing number of 4G alleles at the PAI-1 promoter polymorphism. After adjustment for age, sex, smoking, BMI and triglyceride levels, PAI-1 levels remained 26% higher in the NIDDM relatives (p = 0.01).

Conclusion: In relatives of NIDDM patients PAI-1 levels are elevated, presenting an additional mechanism for their increased cardiovascular risk.