bluuebunny/crossref_metadata_2025 huggingface dataset:
Immunoassay of Murine t-PA, u-PA and PAI-1 Using Monoclonal Antibodies Raised in Gene-inactivated Mice
Authors: Declerck Paul J , Verstreken Maria , Collen Désiré J
Published: July 2018

Three enzyme-linked immunosorbent assays for the quantitation of murine tissue-type plasminogen activator (t-PA), urokinase-type plasminogen aetivator (u-PA) and plasminogen activator inhibitor I (PAI-1), were developed using monoclonal antibodies raised against the autologous proteins in gene-inactivated mice. Dose-response was linear for t-PA and PAI-1 between 5 and 0.1 ng/ml and for u-PA between 50 and 1 ng/ml, with intra-assay, inter-assay and inter-dilution coefficients of variation of 6 to 14%. Assay recoveries of proteins (5 to 100 ng/ml) added to plasma were 73 to 95% for t-PA and PAI-1. Linear correlations (r = 0.65, r = 0.91 and r = 0.92, for t-PA, u-PA and PAI-1 respectively) were found between antigen and activity in plasma, urine and tissue extracts. Levels of t-PA and PAI-1 antigen in murine plasma were 2.5 ± 1.0 ng/ml (mean ± SD, n = 9) and 1.9 ± 0.6 ng/ml (mean ± SD, n = 8), respectively, in wild-type mice and undetectable in gene-inactivated mice. Bradykinin injection in mice provoked a 12-fold increase (p <0.0002) of t-PA and endotoxin injection an 80-fold increase (p <0.005) of PAI-1 levels. u-PA antigen levels in urine from wild-type mice ranged between 0.2 and 8.2 μ;g/ml (1.8 ± 1.9 μg/ml, mean ± SD, n = 17) and were undetectable in gene-inactivated mice.

Thus, these assays may be useful for studies on the role of these proteins in tissue remodeling, atherosclerosis, embryogenesis, etc., in established mouse models. Gene-inactivated mice may constitute a general approach for the generation of monoclonal antibodies against the deficient translation products and for the development of specific immunoassays for murine proteins.
Shear-Induced Platelet Aggregation in Normal Subjects and Stroke Patients
Authors: Konstantopoulos Konstantinos , Grotta James C , Sills Cynthia , Wu Kenneth K , David Heliums J
Published: July 2018

Elevated levels of shear stress that occur in stenotic arteries may induce platelet aggregation and initiate thrombosis. Shear-induced platelet aggregation (SIPA) was studied in groups of ischemic stroke patients and normal subjects using a viscometric-flow cytometric technique. Twenty-three patients who sustained an ischemic stroke that was not of cardiac origin were included in this study, and were classified either as atherosclerotic (n = 15) or as lacunar (n = 8) stroke patients. The results show that shear stresses at the levels which occur in arteries partially occluded by atherosclerosis or vascular spasm strongly activate and aggregate platelets, and this response is much more pronounced in non-lacunar stroke patients who had documented atherosclerotic disease of their cerebral vessels. SIPA is not affected by the time of blood drawing after the onset of stroke suggesting that these platelet abnormalities are not transient but chronic. Furthermore, the extent of platelet activation detected by an anti-P-selectin monoclonal antibody and the proportion of neutrophil-platelet aggregates circulating in vivo are significantly higher in the atherosclerotic stroke patients studied at least one month after the onset of stroke. The results indicate that the enhanced platelet responses observed in atherosclerotic stroke patients are not consequences of ischemia, and therefore both platelet activation and elevated SIPA may be considered as important risk factors for stroke. The methodology developed in this work may be useful for characterization of platelet reactivity, and may contribute to our understanding of thrombotic mechanisms.
Thrombin Receptor Activating Peptide (TRAP) Stimulates Mitogenesis, c-fos and PDGF-A Gene Expression in Human Vascular Smooth Muscle Cells
Authors: Kanthou Chryso , Benzakour Omar , Patel Geeta , Deadman John , Kakkar Vijay Vir , Lupu Florea
Published: July 2018

The synthetic peptide SFLLRNPNDKYEPF, identical in sequence to the new amino-terminus of the thrombin receptor generated following cleavage by thrombin, acts as a thrombin receptor agonist/ activating peptide (TRAP). In this study, Northern blot analysis showed that cultured human vascular smooth muscle cells (HVSMC) express a thrombin receptor transcript. TRAP, in contrast to thrombin was shown to be a weak mitogen for HVSMC. A combination of TRAP and enzymatically-inactivated thrombin (PPACK-thrombin) which provides receptor occupancy, did not potentiate TRAP-induced mitogenesis, indicating that TRAP and PPACK-thrombin do not reproduce the mitogenic effect of enzymatically-active thrombin. Both thrombin and TRAP, induced the expression of c-fos and the PDGF-A gene in a pertussis toxin (PTX)-insensitive manner. Examination of thrombin and TRAP-treated cells by immunofluorescence staining followed by computer assisted image analysis revealed that thrombin and to a lesser extent TRAP induced PDGF-AA protein expression. Antibodies to PDGF-AA partially inhibited thrombin but not TRAP-induced mitogenesis in HVSMC. This study indicates that in addition to the common signalling pathways utilised by thrombin and TRAP, enzymatically-active thrombin activates other signalling pathways and hence TRAP does not mimic fully the biological effect of thrombin on HVSMC.
Induction of Thrombosis in a Mouse Model by IgG, IgM and IgA Immunoglobulins from Patients with the Antiphospholipid Syndrome
Authors: Pierangeli Silva S , Liu Xiao Wei , Barker John H , Anderson Gary , Harris E Nigel
Published: July 2018

Antiphospholipid syndrome is a disorder of recurrent thrombosis and pregnancy losses associated with production of anticardiolipin antibodies and lupus anticoagulant positivity. Recently, we have adapted a mouse model of induced venous thrombosis to study the role of autoantibodies in thrombus formation. To determine whether immunoglobulins from patients with the antiphospholipid syndrome play a role in thrombosis, we injected groups of CDI mice either with immunoglobulins purified from seven patients with the antiphospholipid syndrome (nine preparations studied: four IgG, three IgM and two IgA) or with immunoglobulins of the same isotype from healthy controls. Seventy- two h after injection, a non-occlusive thrombus was induced in the femoral veins of experimental mice by a pinch injury; the thrombus areas as well as times of formation and disappearance of the thrombi were measured. Eight of the nine antiphospholipid syndrome immunoglobulin preparations caused a significant increase in mean thrombus area and a significant delay in mean thrombus disappearance time as compared with normal controls. To determine whether anticardiolipin antibodies might be involved, separate groups of mice were injected with affinity-purified IgG (n = 2) or IgM (n = 2) anticardiolipin antibodies or with normal immunoglobulins of the same isotype, and the effects on thrombus formation compared. Mean thrombus area and mean disappearance times were again significantly increased in all four groups injected with affinity-purified antibodies. This is the first study to show that anticardiolipin antibodies of IgG, IgM and IgA isotypes may play a role in thrombosis in vivo.
Molecular Epidemiology of Factor IX Germline Mutations in Mexican Hispanics: Pattern of Mutation and Potential Founder Effects
Authors: Thorland Erik C , Weinshenker Brian G , Liu Jing-zhong , Ketterling Rhett P , Vielhaber Erica L , Kasper Carol K , Ambriz Raul , Paredes Rogelio , Sommer Steve S
Published: July 2018

Germline mutations in patients with hemophilia B generally have arisen within the past 150 years. Evidence suggests that these germline mutations generally result from endogenous processes. However, a unique pattern would be expected if a population were exposed to a physiologically important germline mutagen since mutagens generally produce characteristic patterns, or “fingerprints”, of mutation. To determine the pattern of mutation in Mexican Hispanics, the regions of likely functional significance in the factor IX gene were screened by di-deoxy fingerprinting (ddF) in 31 families with hemophilia B. Mutations were found in 30 of these families. Haplotype analysis was performed on individuals with identical mutations to help distinguish independent, recurrent mutations from founder effects. Analysis of these 30 mutations, along with 7 mutations reported previously in Mexican Hispanic families, reveals a pattern of independent mutation that is similar to the pattern of mutation observed in 127 U. S. Caucasian families (p = 0.89). These results may reflect either an underlying pattern of germline mutation due to endogenous processes or the presence of an ubiquitous mutagen. Further analyses of the recurrent mutations revealed that two mutations, T296M and R248Q, accounted for 19% of the mutations found in the Mexicans. Haplotype data suggest that the multiple occurrences of T296M and R248Q are associated with founder effects and that screening for these mutations may allow rapid mutation detection and carrier diagnosis in a significant minority of Mexican families with hemophilia B. These two mutations also are associated with founder effects in the U. S. Caucasian population. However, the haplotypes are different in these two populations, indicating independent origins. The occurrence of identical founder mutations in distinct populations provides evidence for the previous hypothesis that the number of different mutations giving rise to mild or borderline mild/moderate hemophilia B is small compared to deleterious mutations causing more severe disease.
Subcutaneous Low-Molecular-Weight Heparin Fragmin Versus Intravenous Unfractionated Heparin in the Treatment of Acute Non Massive Pulmonary Embolism: An Open Randomized Pilot Study
Authors: Meyer G , Brenot F , Pacouret G , Simonneau G , Juvin K Gillet , Charbonnier B , Sors H
Published: July 2018

Low-molecular-weight heparins have been extensively investigated in the treatment of deep venous thrombosis but limited data are available concerning their use in pulmonary embolism. In an open, pilot, randomized study, we compare the safety and efficacy of Fragmin, a low-molecular-weight heparin with those of unfractionated heparin in 60 patients with non massive pulmonary embolism (Miller Index < 20). Thirty one patients received unfractionated heparin intravenously and 29 received a fixed dose of 120 Anti-Xa IU/kg of Fragmin administered subcutaneously twice a day for 10 days. There was no pulmonary embolism recurrence nor major bleeding in either group during the treatment period. The decrease in pulmonary vascular obstruction on perfusion lung scan between day 0 and day 10 was 17 ± 13% in the Fragmin group and 16 ± 13% in the heparin group (NS). These results indicate that Fragmin may be a safe and effective treatment of submassive pulmonary embolism.
Gelatin Use Impairs Platelet Adhesion during Cardiac Surgery
Authors: Tabuchi Norlyuki , de Haan Jacob , Gallandat Huet Rolf C G , Boonstra Plet W , van Oeveren Willem
Published: July 2018

Artificial colloids based on gelatin are used as plasma expander to replace donor blood products. In laboratory experiments, gelatin reduced both the velocity and extend of platelet agglutination by ristocetin, and only the agglutination velocity by polybrene (p <0.05). Furthermore, gelatin delayed the in-vitro platelet plug formation under shear-stress in the absence of ADP (p <0.05), whereas gelatin induced no delay in the presence of ADP. Thus, after induction of vWF release from platelets by polybrene or ADP, platelet function was normal. These results indicate that gelatin affects in particular the functionality of plasma-vWF and partly inhibits platelet adhesion.

These negative effects of gelatin on hemostasis were demonstrated in two clinical studies during cardiac surgery. In a randomized study of sixty patients undergoing cardiac surgery, gelatin as prime in the heart-lung machine appeared to result in diminished efficacy of aprotinin on hemostasis, whereas it did not affect hemostasis in non-aprotinin patients. An additional retrospective clinical study showed that only high dose of gelatin affected hemostasis. This suggests a limited role of plasma-vWF and a strong back-up mechanism of platelet-vWF in achieving hemostasis.
Pharmacokinetics and Tolerance of the Natural Pentasaccharide (SR90107/ORG31540) with High Affinity to Antithrombin III in Man
Authors: Boneu B , Necciari J , Cariou R , Sié P , Gabaig A M , Kieffer G , Dickinson J , Lamond G , Moelker H , Mant T , Magnani H
Published: July 2018

This paper reports the results of the first administration of the synthetic natural pentasaccharide with high affinity to antithrombin III (NP) in man. The study was mainly focused upon the pharmacokinetic properties and general tolerance of the compound. Subcutaneous injections of doses <1.43 mg (1000 anti Xa IU) did not generate measurable anti-Xa activities. After subcutaneous injection of increasing doses from 1.43 to 22.9 mg (1000 to 16000 anti-Xa IU) to young healthy volunteers, it was found that the maximal concentration (Cmax) and the area under curve (AUC) were linearly correlated to the dose, that the total plasma clearances (Cl) were constant and almost 3 times lower than those of the current low molecular weight heparins. Cmax were reached between 1 h and 3 h after the injection and the half-lives (t1/2) were remarkably constant (13.1 h to 13.9 h). During the first 24 h following the injection, around 50% of the total administered dose was recovered in the urine in an active form, indicating that kidney plays a major role in the elimination of NP. Consistent with these results, when NP was administered to healthy elderly volunteers having a lower creatinine clearance, the half-life of the compound was longer and the clearance lower. At doses exceeding 22.9 mg, Cmax, and AUC were slightly lower than expected, the percentage of the dose recovered in the urine and the total apparent plasma clearance increased, suggesting that the excess of NP unbound to antithrombin III was excreted faster. NP was also administered at various dosages once or twice a day for 7 days to 20 elderly volunteers. Due to the long half-life of the compound the “steady state” was obtained 2 to 3 days after the first injection at which the mean Cmax was increased 1.5 to 2 times. The general tolerance of the compound was excellent. No relevant prolongations of the prothrombin time, of the activated partial thromboplastin time or of the bleeding time were observed. A re-bleeding phenomenon of the bleeding time incision, probably related to friability of the haemostatic plug, occurred in 3 subjects treated with the highest dose regimens: single injection of 26.6 mg (20000 anti-Xa IU) (young volunteers) and repeated injections of 11.4 mg (8000 anti-XaIU) once a day for 7 days (elderly volunteers). At these times, plasma NP concentrations were between 2.9 and 3.6 μg ⋅ ml-1 (2 and 2.5 anti-Xa IU ⋅ ml-1).
Modulation of Platelet Activation In Vitro by Thrombopoietin
Authors: Kojima Hiroshi , Hamazaki Yoko , Nagata Yuka , Todokoro Kazuo , Nagasawa Toshiro , Abe Tsukasa
Published: July 2018

Effect of human recombinant thrombopoietin (TPO) on platelet activation in vitro was studied. Although TPO by itself did not cause platelet aggregation, it upregulated ADP-induced aggregation, especially the second wave of aggregation. This effect was dose-dependent for up to 5 ng/ml of TPO. When platelets were activated by epinephrine, collagen, or α-thrombin, similar effect was observed. However, TPO did not affect A23187- or PMA-induced aggregation, suggesting that TPO may have modulated the signal transduction pathway upstream of inositol 1,4,5-trisphosphate and diacylglycerol production. TPO also upregulated thrombin-induced α-granule secretion. To clarify the involvement of protein tyrosine phosphorylation, platelets were activated by TPO and/or suboptimal concentration of ADP, then tyrosine phosphorylation was detected by immunoblot analysis, using anti-phosphotyrosine monoclonal antibody. TPO by itself caused significant tyrosine phosphorylation of 146,130,122,108, 97,94, and 88 kDa proteins. Further, by using antibodies against signal transduction molecules for immunoprecipitation, we observed the significant tyrosine phosphorylation in Jak2 and Tyk2 molecules after TPO-stimulation. The results of the present experiment clearly indicate that TPO directly activated platelets and modulated intracellular signal transduction pathway.
On the Role of Platelet FcγRIIa Phenotype in Heparin-Induced Thrombocytopenia
Authors: Brandt John T , Isenhart Craig E , Osborne Jeanne M , Ahmed Alaa , Anderson Clark L
Published: July 2018

Heparin induced thrombocytopenia (HIT) is characterized by the formation of antibodies that activate normal donor platelets in vitro in the presence of heparin. We asked whether the commonly observed donor-specific variation in the platelet aggregation response to HIT antibodies is influenced by the density of FcγRII on platelets or by the Arg/His 131 allelic polymorphism of platelet FcγRII. We found that platelets with the His/Hisl31 FcγRII phenotype were unresponsive to HIT antibody (0/9) whereas platelets with the Arg/Argl31 phenotype responded well (7/9). His/Hisl31 platelets were largely unresponsive also to a murine IgG1 antiplatelet monoclonal antibody (UR1) known to activate platelets by FcγRII clustering. We then determined the frequency distribution of FcγRIIa Arg/His 131 phenotypes on a series of 200 patients evaluated for HIT and 100 non-thrombocytopenic hospitalized patients. The frequency of the His/Hisl31 phenotype was significantly increased (34.4%) in the 96 thrombocytopenic patients with HIT antibody compared to the 104 thrombocytopenic patients without HIT antibody and the 100 non-thrombocytopenic patients (∼19%). Thus, the FcγRII phenotype regulates the in vitro activation response of normal platelets to HIT antibody and is a risk factor for the thrombocytopenia of HIT
Laboratory Identification of Lupus Anticoagulants: Results of the Second International Workshop for Identification of Lupus Anticoagulants
Authors: Brandt John T , Barna Linda K , Triplett Douglas A
Published: July 2018

Lupus anticoagulants (LAs) are antibodies that interfere with phospholipid dependent coagulation reactions in vitro. This workshop was designed to provide the participants with an experience in identification of LAs, to evaluate different criteria for mixing studies, to assess the performance of recently introduced confirmatory studies and to assess the performance of two potential surrogate LA control plasmas. The results demonstrate that there continues to be significant variation in the sensitivity and responsiveness of APTT reagents to the presence of LAs, confirming the need for more than one screening assay before the presence of a LA can be ruled out. In this workshop, the best distinction between factor deficiency and inhibitors was obtained using a 1:1 mix of normal plasma with patient plasma and the criterion defining correction as shortening of the APTT to within 5 s of the APTT of pooled normal plasma. A 4:1 mix of patient to normal plasma did not work well in distinguishing factor deficiency from inhibitors. The platelet neutralization procedure, DVVconfirm® and StaClot LA® all gave positive results with the LA samples. False positive platelet neutralization procedures were seen with the samples from patients on oral anticoagulants and a factor V inhibitor. False positive StaClot LA results were obtained with high titer factor VIII inhibitors. Both of the potential surrogate plasmas gave variable results with multiple assays; they can not be recommended for routine use at present.
The 1691 G → A Mutation in the Factor V Gene: Relationship to Activated Protein C (APC) Resistance and Thrombosis in 65 Patients
Authors: Leroy-Matheron Catherine , Levent Martine , Pignon Jean-Michel , Mendonça Caio , Gouault-Heilmann Michèle
Published: July 2018

Four hundred fifty subjects were screened for the 1691 G → A mutation in the factor V gene. Two hundred ninety-seven patients were referred to us for unexplained thrombosis, 133 were family members of these patients and 20 were normal subjects. We studied the relationships between the mutation, resistance to APC and thrombosis. Among the 450 subjects tested, 65 belonging to 42 families were found to have the 1691 G → A mutation in one (n = 61) or both alleles (n = 4). The prevalence of the mutation in the thrombotic patients was 13%. Resistance to APC was tested for in 247 subjects not on anticoagulant treatment (4 homozygous and 44 heterozygous for the mutation, and 199 individuals without the mutation). Incomplete cosegregation of heterozygosity for the 1691 G → A mutation with APC resistance (APC-SR <2.4 or n-APC-SR <0.75) was observed, showing that the functional assay alone is insufficient for a firm diagnosis. In patients carrying the mutation, elevated levels of prothrombin fragment 1+2 and D-dimers pointed to increased thrombin generation in vivo. Clinical manifestations in the heterozygous subjects were very similar to those reported in heterozygous PC or PS deficiencies, but the first thrombotic event occurred later than in PC- or PS-deficient patients. Homozygosity for the factor V gene mutation appears to be a far more benign thrombotic disorder than homozygous PC and PS deficiencies.
Safety and Efficacy of Monoclonal Antibody Purified Factor IX Concentrate in Previously Untreated Patients with Hemophilia B
Authors: Shapiro Amy D , Ragni Margaret V , Lusher Jeanne M , Culbert Steven , Koerper Marion A , Bergman Garrett E , Hannan Margaret M
Published: July 2018

The safety and efficacy of a monoclonal antibody purified factor IX concentrate were evaluated in two continuing trials of 32 previously untreated patients with mild, moderate, or severe hemophilia B. Patients were evaluated every 2 weeks for 24 weeks and every 3 months thereafter for at least 1 year. No patients became positive for human immunodeficiency virus antibody or hepatitis C virus antibody during the trial. Two patients developed a false-positive hepatitis B core antibody, one transiently, but neither had elevated levels of alanine aminotransferase (ALT). None of the 25 patients evaluable for non-A, non-B, non-C hepatitis by strict International Society of Thrombosis and Hemostasis criteria developed elevated levels of ALT indicative of posttransfusion infection. Anaphylaxis occurred in one subject who also developed an inhibitor to factor IX (19.3 Bethesda units). Five of the eight adverse events reported (63%) were mild in severity, and the relationship of three of these to therapy was considered remote. Hemostasis with monoclonal antibody purified factor IX concentrate was excellent in all patients.
Effect of Heterologous Factor V Heavy Chain Sequences on the Secretion of Recombinant Human Factor VIII
Authors: Ortel Thomas L , Moore Karen D , Ezban Mirella , Kane William H
Published: July 2018

Factor VIII and factor V share a repetitive domain structure of A1-A2-B-A3-C1-C2. To define the region(s) within the factor VIII heavy chain that result in inefficient expression of the recombinant protein, we expressed a series of factor VIH/factor V chimeras that contained heterologous sequences from the A1 and/or A2 domains. Substitution of the factor VIIIA1 domain dramatically reduced secretion of factor V ~ 500-fold, whereas substitution of the factor VIII A2 domain had minimal effect on secretion. Conversely, substitution of the factor V A1 domain increased secretion of factor VIII ~3-fold, whereas substitution of the factor V A2 domain actually reduced secretion ~4-fold. Pulse chase experiments confirmed that reduced expression levels were due to decreased secretion rather than instability of secreted protein. Smaller substitutions did not further localize within the A1 domain the regions responsible for inefficient secretion.
Mutations which Introduce Free Cysteine Residues in the Gla-Domain of Vitamin K Dependent Proteins Result in the Formation of Complexes with α1-Microglobulin
Authors: Wojcik E G C , Simioni P , Berg M v d , Girolami A , Bertina R M
Published: July 2018

We have previously described a genetic factor IX variant (Cys18→Arg) for which we demonstrated that it had formed a heterodimer with armicroglobulin through formation of a disulphide bond with the remaining free cysteine residue of the disrupted disulphide bond in the Gla-domain of factor IX. Recently, we observed a similar high molecular weight complex for a genetic protein C variant (Arg-1→Cys). Both the factor IX and the protein C variants have a defect in the calcium induced conformation. In this study we show that the aminoterminus of this protein C variant is prolonged with one amino acid, cysteine. This protein C variant, as well as protein C variants with Arg9→Cys and Ser12→Cys mutations which also carry a free cysteine residue, are shown to be present in plasma as a complex with α1-microglobulin. A prothrombin variant with a Tyr44→Cys mutation, had not formed such a complex. Furthermore, complexes between normal vitamin K-dependent clotting factors and α1-microglobulin were shown to be present in plasma at low concentrations. The data suggest that the presence of an unpaired cysteine residue in the propeptide or the N-terminal half of the Gla-domain has strongly promoted the formation of a complex with α1-microglobulin in the variants.
Nafamostat Mesilate, a Broad Spectrum Protease Inhibitor, Modulates Platelet, Neutrophil and Contact Activation in Simulated Extracorporeal Circulation
Authors: Sundaram Sumuk , Gikakis Nicolas , Erik Hack C , Niewiarowski Stefan , Edmunds L H , Koneti Rao A , Sun Ling , Cooper S L , Colman Robert W
Published: July 2018

Activation of humoral and cellular participants in inflammation enhances the risk of postoperative bleeding and multiple organ damage in cardiopulmonary bypass (CPB). We now compare the effects of heparin alone in combination with nafamostat mesilate (NM), a protease inhibitor with specificity of trypsin-like enzymes, in an extracorporeal circuit which simulates CPB. NM significantly inhibits the release of platelet (β-thromboglobulin (βTG) at 60 and 120 min. Platelet counts do not differ. ADP-induced aggregation decreases in circuits with NM, which is due to a direct effect of NM on platelet function. NM prevents any significant release of neutrophil elastase; at 120 min, plasma elastase-α1antitrypsin complex is 0.16 μg/ml in the NM group and 1.24 μg/ml in the control group. NM completely inhibits formation of complexes of C1 inhibitor with kallikrein and FXIIa. NM does not alter markers of complement activation (C1-C1-inhibitor complex and C5b-9), or indicators of thrombin formation (F1.2). However, at 120 min, thrombin activity as measured by release of fibrinopeptide A is significantly decreased. The data indicate that complement activation during CPB correlates poorly with neutrophil activation and that either kallikrein or FXIIa or both may be more important agonists. The ability of NM to inhibit two important contact system proteins and platelet and neutrophil release raises the possibility of suppressing the inflammatory response during clinical CPB.
The Importance of Platelets in the Expression of Monocyte Tissue Factor Antigen Measured by a New Whole Blood Flow Cytometric Assay
Authors: Amirkhosravi A , Alexander M , May K , Francis D A , Warnes G , Biggerstaff J , Francis J L
Published: July 2018

Previous methods for the determination of monocyte tissue factor (TF) have been technically complex, difficult to standardize, prone to spuriously elevated results and difficult to implement in a clinical laboratory environment. We report the development of a two-color whole blood cytometric technique that overcomes many of these disadvantages. The assay uses small volumes of citrated blood (1.0 ml), can be performed in under one hour (if endotoxin stimulation is not performed), is reproducible (CV = 5%) and uses methodology commonly available in clinical laboratories. Baseline (mean ± SD) expression of monocyte TF in normal subjects was very low (1.1 ± 0.95%, Mean Fluorescence [Mean FL] 0.20 ± 0.01) making relatively small increases easy to detect. Monocyte TF expression following endotoxin (LPS) stimulation for 1 h was 34.6 ± 11.2% (Mean FL 0.32 ± 0.04). LPS-stimulated activity varied between subjects (21-68%) but was remarkably consistent for individual subjects (CV = 5.4%). Stimulated monocyte TF expression was directly proportional to the platelet count and was reduced by platelet protective anticoagulants and by ingestion of aspirin. Non LPS-stimulated monocyte TF was markedly increased, in a dose-dependent manner, by adding collagen to whole blood. This was apparently associated with platelet-monocyte binding and could be abolished by anti-P-Selectin. We conclude that the whole blood flow cytometric assay of monocyte TF may be a valuable tool for clinical use and a useful model system for evaluating the humoral and cellular factors governing monocyte TF expression in a natural environment.
Promotion of the Crosslinking of Fibrin and α2-antiplasmin by Platelets
Authors: Hevessy Zsuzsa , Haramura Gizella , Boda Zoltán , Udvardy Miklós , Muszbek László
Published: July 2018

Factor XIII (FXIII) is of high importance in the regulation of fibrinolysis. It crosslinks α2-antiplasmin (α2AP) and fibrin and by this way protects fibrin from the prompt elimination by plasmin. Although FXIII of platelets has been implicated in this protective mechanism, the role of platelets and platelet FXIII in the crosslinking process is far from being elucidated. As demonstrated by SDS PAGE and by immunoblotting for α2AP, intact normal platelets resuspended in FXIII-free plasma or FXIII-free fibrinogen solution catalyzed the crosslinking of fibrin chains and also the crosslinking of α2AP to fibrin α-chains. With FXIII-deficient platelets no crosslinking reaction could be observed indicating that the crosslinking with normal platelets was, indeed, due to platelet FXIII and not to another, putative platelet transglutaminase. However, the crosslinking of α2AP to fibrin induced by the FXIII of intact platelets resuspended in FXIII-free plasma was considerably less extensive than the crosslinking carried out by the FXIII of normal plasma in the presence of FXIII-free platelets. Furthermore, the replacement of FXIII-free platelets by normal platelets in normal FXIII-containing plasma resulted in little, if any, difference in the crosslinking process. When crosslinking was induced by highly purified plasma FXIII the presence of intact FXIII-free platelets significantly accelerated the formation of α-chain polymers as well as the incorporation of α2AP-fibrin α-chain hetero-dimer into these polymers. The results indicate that, in physiological conditions, platelet FXIII plays only a minor role in the crosslinking of α2AP and fibrin; however, platelets, independently of their FXIII content, promote the crosslinking reaction by providing a catalytic surface on which the formation of highly crosslinked fibrin polymers is accelerated.
Epinephrine Augments Platelet Recruitment to Immobilized Collagen in Flowing Blood - Evidence for a von Willebrand Factor-mediated Mechanism
Authors: Mustonen Pirjo , Lassila Riitta
Published: July 2018

Although elevated plasma epinephrine (epi) levels are associated with clinical atherothrombosis, the role of epi in platelet-vessel wall interaction has not been established. Our aim was to study the effect of high physiological epi (10 nM) in an experimental model which tests the interaction between platelets and immobilized collagen in whole blood. Shear forces and anticoagulation were modulated. Epi significantly enhanced platelet deposition, but only at high shear rate (1,600 s-1)- In PPACK- or LMWH-anticoagulated blood, the increase in platelet deposition was 32 to 85 % (p <0.02-0.05). Furthermore, platelet aggregation was cotriggered with subthreshold concentrations of epi and ristocetin, and monoclonal antibodies against glycoprotein (GP) lb (AN 51 and SZ 2) attenuated epi-induced aggregation. We conclude that epi is capable of augmenting platelet functions, which are dependent on the interaction of vWF with GP lb and GP IIb/IIIa. Via this mechanism epi may promote arterial thrombosis in vivo.
The Antithrombotic Effect of Aurin Tricarboxylic Acid in the Guinea Pig Is not Solely due to Its Interaction with the von Willebrand Factor-GPIb Axis
Authors: Azzam K , Cissé-Thiam M , Drouet L
Published: July 2018

Commercial aurin tricarboxylic acid (ATA) has been reported to interfere specifically with von Willebrand factor-glycoprotein lb (vWF-GPIb) axis. This study was designed to explore the antithrombotic effects of AT A by examining its effects on guinea pig platelet function in vitro, in vivo and ex vivo. In vitro, addition of various concentrations of ATA to platelet-rich guinea pig plasma totally inhibited ristocetin-induced platelet aggregation, as expected. Unexpectedly, however, ATA similarly inhibited the aggregation induced by ADP, PAF, collagen, I-BOP (a thromboxane A2/prostaglandin H2 analogue) and arachidonic acid.

In vivo, the antithrombotic action of ATA was assessed in a model of acute platelet-dependent guinea pig mesenteric artery thrombosis triggered by laser-induced intimal injury. As the thrombotic response of arteries to such injury is a spontaneous cyclic recurrent process, 5 arteries in each animal were consecutively studied for 15 min each after i.v. bolus injection of 5, 7.5 or 10 mg/kg of ATA, which reduced the number of recurrent thrombi per artery in a dose-dependent manner. The highest dose of 10 mg/kg induced maximal inhibition of thrombus formation (72%, p <0.001) 5 min after injection.

Ex vivo, platelet aggregation was assessed in blood samples taken before and after i.v. bolus injection of 10 or 15 mg/kg ATA. Ten mg/kg significantly inhibited collagen-induced aggregation, and 15 mg/kg, the aggregation induced by ristocetin, ADP, PAF, collagen, I-BOP and arachidonic acid.

The results of the in vivo studies confirmed that ATA is an effective antithrombotic agent. In the in vitro and ex vivo studies, ristocetin-induced platelet aggregation confirmed that ATA interacts with the vWF-GPIb axis, and suggests that the final common pathway of the aggregation induced by other agents tested consists of fibrinogen binding to the platelet GPIIb/IIIa receptor. We conclude that ATA interferes with vWF binding to GPIb, that it may interact with fibrinogen binding to GPIIb/IIIa, and that it might possess potent antithrombotic properties in platelet-mediated thrombosis.
Increased Cyclin Dependent Kinase in Aortic Tissue of Rats Fed Homocysteine
Authors: Lubec Barbara , Arbeiter Klaus , Hoeger Harald , Lubec Gert
Published: July 2018

Background. Hyperhomocyst(e)inemia is strongly associated with occlusive arterial disease. Several mechanisms for the development of vascular lesions have been described. A direct effect of homocysteine on proliferation of smooth muscle cells and collagen expression was proposed recently. These observations led us to examine the effect of homocysteine on cyclin dependent kinase, the starter of mitosis and reflecting proliferation.

Methods and results. Thirty Him: OF A rats were divided into three groups. Ten animals were fed for a period of six weeks 50 mg/kg body wt per day homocysteine, ten the same dose of homocysteic acid and ten remained untreated controls. At the end of the experiment we determined aortic cyclin dependent kinase, phosphokinases A and C, aortic homocyst(e)ine and aortic hydroxyproline. Aortic cyclin dependent kinase was significantly (p = 0.0001) elevated in the homocysteine treated group (mean 120 ± 15) compared with the homocysteic acid treated group (mean 71 ± 11) or the untreated group (mean 72 ± 10 fmol/mg aortic tissue). Aortic homocyst(e)ine was significantly higher in homocysteine treated animals (p = 0.0002) strongly correlating with cyclin dependent kinase (r squared = 0.85, p = 0.0001) and with aortic hydroxyproline (r squared = 0.66, p = 0.0001), which in turn was significantly (p = 0.0001) increased in the homocysteine treated group. Phosphokinases A and C determined to rule out nonspecific effects on kinases were not increased by administered homocysteine.

Conclusions. Our findings indicate that homocysteine stimulates aortic cyclin dependent kinase with the possible consequence of proliferation of aortic cells. Aortic collagen accumulation could be explained by either the homocysteine-effect on collagen synthesis described in literature, or secondarily, by increased proliferation of collagen producing aortic cells.
Mutation Spectrum in Patients with Wiskott-Aldrich Syndrome and X-linked Thrombocytopenia: Identification of Twelve Different Mutations in the WASP Gene
Authors: Schwartz Marianne , Békássy Albert , Donnér Mikael , Hertel Thomas , Hreidarson Stefan , Kerndrup Gitte , Stormorken Helge , Stokland Tore , Tranebjcerg Lisbeth , Ørstavik Karen Helene , Skovby Flemming
Published: July 2018

Twelve different mutations in the WASP gene were found in twelve unrelated families with Wiskott-Aldrich syndrome (WAS) or X-linked thrombocytopenia (XLT). Four frameshift, one splice, one nonsense mutation, and one 18-base-pair deletion were detected in seven patients with WAS. Only missense mutations were found in five patients diagnosed as having XLT. One of the nucleotide substitutions in exon 2 (codon 86) results in an Arg to Cys replacement. Two other nucleotide substitutions in this codon, R86L and R86H, have been reported previously, both giving rise to typical WAS symptoms, indicating a mutational hot spot in this codon. The finding of mutations in the WASP gene in both WAS and XLT gives further evidence of these syndromes being allelic. The relatively small size of the WASP gene facilitates the detection of mutations and a reliable diagnosis of both carriers and affected fetuses in families with WAS or XLT.
Anticoagulant Response to Agkistrodon Contortrix Venom (ACV test): a New Global Test to Screen for Defects in the Anticoagulant Protein C Pathway
Authors: Robert A , Eschwège V , Hameg H , Drouet L , Aillaud M-F
Published: July 2018

As specific assays used to identify defects in the protein C (PC) anticoagulant pathway are laborious and expensive, we describe here a global test to screen for these defects. This assay is expressed as the ratio of two activated partial thomboplastin times, one in the absence and one in the presence of 0,125 U/ml of the PC activator of Agkistrodon contortrix venom (ACV). Eight of the 168 healthy volunteers of the control group exhibited an ACV ratio below the lower normal limit of 3.37 [6 subjects with the mutation Arg 506 to Gin in their factor V gene (FV R506Q) and one with PS deficiency]. 128 patients who have had at least one episode of deep-vein thrombosis were retrospectively studied. All patients carrying FV Q506R (n = 48), PC deficiency (n = 14) or combined defects, i. e. FV Q506R and PC deficiency (n = 4) or FV Q506R and PS deficiency (n = 3), had ACV ratios < 3.37. PS deficient plasmas (n = 20) exhibited ACV ratios which overlapped normal range. ACV ratios of one out of seven patients with antithrombin deficiency, and 10% of patients without identified defect in the PC anticoagulant pathway (n = 30) were < 3.37. An ACV ratio raised to 3.70 could lead to a test identifying all patients with a defect in the PC anticoagulant pathway.
Low Plasma Levels of Factor Vile and Antigen Are More Strongly Associated with the 10 Base Pair Promoter (-323) Insertion than the Glutamine 353 Variant
Authors: Humphries Steve , Temple Anne , Lane Anne , Green Fiona , Cooper Jackie , Miller George
Published: July 2018

In a group of 705 healthy middle-aged men, we have examined the relationship between plasma levels of Factor VII (FVII) c and Factor VII antigen (FVIIag) and two polymorphisms in the FVII gene. One polymorphism alters arginine at position 353 to glutamine (R/Q), and the other is the result of a 10 base pair (bp) insertion in the promoter region at position -323 from the start of translation (0/1 Obp). The frequency for the Q allele was 0.105 (95% Cl 0.09-0.12) and for the lObp allele was 0.117 (95% Cl 0.10-0.13). Men who were carriers of either of the rare alleles had levels of FVIIc and FVIIag that were approximately 20% lower than non-carriers, and both of these effects were highly statistically significant (p < 0.0001). Strong allelic association was observed between the two polymorphisms, with R and 0 bp being on the same chromosome in 96% of cases, and Q and 10 bp being on the same chromosome in 90% of cases where this could be determined unambiguously (Delta = 0.92, X2 = 1206, p < 0.0001). This strong allelic association created three major genotype groups which were found to have significantly different levels of FVIIc (p < 0.001). In the 547 men homozygous for both common alleles (R/R & 0/0), mean (SD) FVIIc was 101 (29) as compared with 85 (30) in the 20 men with the genotype R/R & 0/10 and 81(23) in the 126 men with the genotype R/Q & 0/10, suggesting a larger lowering effect associated with the 10 bp allele (16%) compared to the Q allele (an additional 4%). The lowering effect on FVII associated with the 10 bp allele remained statistically significant after adjusting for the effect of the Q allele (p = 0.004 for FVIIc and p = 0.06 for FVIIag), but the effect associated with the Q allele was no longer significant after adjusting for the 10 bp allele, suggesting that the strongest effect on levels of FVIIc was associated with the 0/10 bp genotype. In the sample overall, plasma FVIIc was associated positively with serum triglyceride concentration and the slope of this relationship was significantly greater in those with the genotype R/R compared to the other groups combined (0.12 versus 0.02, p = 0.008) with differences of similar size seen in the 0/0 compared to 0/10+10/10 groups. However, using the combined genotypes, the slope of this relationship in the R/R & 0/10 group was 0.38 and was significantly steeper (p = 0 .0 1 ) than in the other two groups who did not differ in this respect (slopes 0.11 and 0.08). This effect was seen on four subsequent annual examinations, and was also evident in the relationship between FVIIag and triglyceride concentration (p = 0.003 for difference between groups measured at baseline only). These data suggest that part of the previously described effects on FVIIc levels associated with the R/Q polymorphism may be explained by genetic variation in the promoter region of the FVII gene.
Experimental Haemorrhagic Effect of Two-Domain Non-Glycosylated Tissue Factor Pathway Inhibitor Compared to Low Molecular Weight Heparin
Authors: Holst Jan , Lindblad Bengt , Matthíasson Stefan E , Stjernquist Ulf , Ezban Mirella , Østergaard Per B , Hedner Ulla
Published: July 2018

The glycosylated multivalent three-domain Kunitz inhibitor TFPI is a natural inhibitor of tissue factor-FVIIa complex in the presence of FXa. TFPI has an experimental antithrombotic capacity indistinguishable from LMWH in a prophylactic dose, regardless of glycosylation and of the third domain. An inherited equilibrium between antithrombosis and haemorrhage exists. The aim of the study was to evaluate whether a two-domain non-glycosylated TFPI (117QTFPI1−161) has a bleeding potential in a rat gastric mucosa model. Groups; placebo, LMWH (tinzaparin) 60 and 250 anti-Xa IU/kg and 117QTFPIM61−161 1.0 and 10.0 mg/kg, given i.v. (bolus injection), randomised double dummy design.

All actively treated groups significantly prolonged both the bleeding volume (493-984 Μl) and the bleeding time (10-20 min) compared to placebo (41 Μl, 2 min). It was not possible to distinguish a difference between the lower dose of LMWH and 117QTFPI1−161 in either parameter (p = 0.23-0.71). The two doses of 117QTFPI1−161 caused elevation of plasma-TFPI, 18 and 150 times baseline value. Both LMWH doses (0.6-3.2 anti-Xa IU/ml) and both 117QTFPI1−161 doses (0.2-2.7 anti-Xa IU/ml), caused significant effect in the anti-Xa assay, however 117QTFPI1−161 significantly less. Only the largest dose of 117QTFPI1−161 caused significant prolongation in the APTT assay (34 s). Both doses of LMWH caused significant prolongation (60-300 s). LMWH was the only substance to prolong the dilute-PT assay.

Non-glycosylated two-domain 1.0 mg/kg TFPI, yielding supra-physiological plasma concentration, has an experimental haemorrhagic potential indistinguishable from LMWH in a prophylactic dose. The effect mediated by this type of TFPI could primarily be due to an inhibition of FXa.
Prothrombin Time Sensitivity and Specificity to Mild Clotting Factor Deficiencies of the Extrinsic Pathway: Evaluation of Eight Commercial Thromboplastins
Authors: Massignon D , Moulsma M , Bondon P , Debize G , Abidi H , Buttin T , Bon C , Pillonchery G , Coeur P
Published: July 2018

Prothrombin-time (PT) sensitivity and specificity to mild clotting factor II, V, VII and X deficiencies have rarely been studied. We therefore carried out a prospective study, in 350 patients, of eight commercial thromboplastins (CTs) in their ability to detect mild clotting factor deficiencies, notably in factor VII. In each patient the factor II, V, VII and X clotting activities and PT performed with each CT were determined. For each CT, PT sensitivity and specificity in detecting factor deficiencies below 0.5 U/ml or below 0.4 U/ml were determined at various PTs, and then Receiver Operator Characteristic curves constructed. At optimum PT threshold level (sensitivity = specificity), exactitude varied from 0.64 to 0.74 (p < 0.01) and from 0.67 to 0.81 (p < 0.0001) in detecting deficiencies below 0.5 and 0.4 U/ml respectively. In conclusion, this study shows the limits of the PT test as performed with 8 CTs in patients with mild clotting factor deficiencies. The impact of such differences in sensitivity and specificity on monitoring certain patients subject to decrease in coagulation factor, and, in particular, of those under low-dose oral anticoagulant, remains to be determined.
Antiplatelet and Antithrombotic Effects of YM337, the Fab Fragment of a Humanized Anti-GPIIb/IIIa Monoclonal Antibody in Monkeys
Authors: Kaku Seiji , Kawasaki Tomihisa , Hisamichi Nami , Sakai Yumiko , Taniuchi Yuta , Inagaki Osamu , Yano Shinya , Suzuki Ken-ichi , Terazaki Chinami , Masuho Yasuhiko , Satoh Noboru , Takenaka Toichi , Yanagi Kennichi , Ohshima Norio
Published: July 2018

The antiplatelet and antithrombotic effects of the Fab fragment of the humanized antiplatelet glycoprotein (GP) IIb/IIIa monoclonal antibody C4G1 (YM337) were investigated in monkeys. First, the relationship between the inhibition of platelet aggregation and the prolongation of bleeding time was studied in rhesus monkeys. YM337 dose-dependently inhibited ex vivo platelet aggregation, with complete inhibition at doses higher than 0.25 mg/kg intravenous injection or 1.5 μg/kg/min infusion. At 0.25 mg/kg bolus injection followed by 1.5 μg/kg/min infusion, YM337 immediately and continuously inhibited platelet aggregation during the 6-h infusion period with platelet aggregation rapidly returning to over 50% of baseline within 1 h after the cessation of infusion. Template-bleeding time was significantly prolonged during the period of complete inhibition of platelet aggregation.

Second, the antithrombotic effects of YM337 were investigated in a photochemically-induced thrombosis model in squirrel monkeys. YM337 at a dose of 1 mg/kg intravenous injection followed by 6 μg/kg/min infusion for 60 min prevented occlusive thrombus formation in all 4 monkeys. In contrast, time to occlusive thrombus formation did not change on intravenous bolus injection of aspirin 17 mg/kg (11.3 ± 5.2 min) or sodium ozagrel (9.4 ± 3.0 min) compared with saline (13.3 ± 4.0 min). YM337 but not aspirin or sodium ozagrel significantly inhibited ex vivo ADP-induced platelet aggregation, while all drugs completely inhibited arachidonic acid-induced platelet aggregation. However, while aspirin and sodium ozagrel inhibited the thromboxane B2 generation accompanying arachidonic acid-induced platelet aggregation, YM337 had no effect on this variable. Platelet counts and bleeding time showed no significant change in any group in this squirrel monkey model.

These results indicate that YM337, with a short half-life, may be a useful therapeutic agent in patients with thrombotic disorders.