Assays for Phospholipid-Dependent Formation of Thrombin and Xa: A Potential Method for Quantifying Lupus Anticoagulant Activity

1991 ◽  
Vol 66 (04) ◽  
pp. 453-458 ◽  
Author(s):  
John T Brandt
Keyword(s):  
Specific Activity ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Clinical Importance ◽  
Potential Method ◽  
Quantitative Expression ◽  
Increased Risk ◽  
Apparent Association

SummaryLupus anticoagulants (LAs) are antibodies which interfere with phospholipid-dependent procoagulant reactions. Their clinical importance is due to their apparent association with an increased risk of thrombo-embolic disease. To date there have been few assays for quantifying the specific activity of these antibodies in vitro and this has hampered attempts to purify and characterize these antibodies. Methods for determining phospholipid-dependent generation of thrombin and factor Xa are described. Isolated IgG fractions from 7 of 9 patients with LAs were found to reproducibly inhibit enzyme generation in these assay systems, permitting quantitative expression of inhibitor activity. Different patterns of inhibitory activity, based on the relative inhibition of thrombin and factor Xa generation, were found, further substantiating the known heterogeneity of these antibodies. These systems may prove helpful in further purification and characterization of LAs.

A Heparin Analogue that Releases Endogenous Heparan Sulphate

1979 ◽  
Author(s):  
D.P. Thomas ◽  
T.W. Barrowcliffe ◽  
E.A. Johnson ◽  
J. Stocks ◽  
J. Dawes ◽  
...  
Keyword(s):  
Lipoprotein Lipase ◽  
Specific Activity ◽  
Anticoagulant Activity ◽  
Heparan Sulphate ◽  
Factor Xa ◽  
Fold Increase ◽  
Endothelial Lining ◽  
Drug Induced

Heparan sulphate (HS), a near-relative of heparin, but with much less anticoagulant activity in vitro, is bound to cell surfaces. We examined HS isolated from porcine intestinal mucosa, and found that although the material had low anticoagulant activity by APTT, it had a marked effect in anti-Factor Xa clotting assays, giving anti-Xa/APTT ratios of approximately 4:1 in terms of specific activity. In crossed immunoelectrophoresis experiments, HS bound to antithrombin III, but at higher concentrations than heparin. A heparin analogue (a polysulphated chondroitin), while virtually inactive in vitro, nevertheless when administered s.c. to man potentiated the effect of anti-Factor Xa to an extent comparable to that produced by low-dose heparin, but with an anti-Xa/APTT ratio of 4:1. The analogue also produced a marked release of lipoprotein lipase and a four-fold increase in the level of circulating PF4, as measured by radioimmunoassay. The anti-Xa and APTT effects of HS in vitro are very similar to those produced by the analogue in vivo, and it is suggested that the analogue releases not only lipoprotein lipase and PF4, but also HS. The drug-induced release of an endogenous glycosamino-glycan (probably from the endothelial lining of vessels) with anti-Xa activity may represent a fruitful approach to the prophylaxis of venous thrombosis.

The Anticoagulant Properties of a Modified Form of Protein S

1988 ◽  
Vol 60 (02) ◽  
pp. 298-304 ◽  
Author(s):  
C A Mitchell ◽  
S M Kelemen ◽  
H H Salem
Keyword(s):  
Monoclonal Antibody ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Factor Xa ◽  
Protein S ◽  
Human Neutrophil Elastase ◽  
Factor X ◽  
Sds Page

SummaryProtein S (PS) is a vitamin K-dependent anticoagulant that acts as a cofactor to activated protein C (APC). To date PS has not been shown to possess anticoagulant activity in the absence of APC.In this study, we have developed monoclonal antibody to protein S and used to purify the protein to homogeneity from plasma. Affinity purified protein S (PSM), although identical to the conventionally purified protein as judged by SDS-PAGE, had significant anticoagulant activity in the absence of APC when measured in a factor Xa recalcification time. Using SDS-PAGE we have demonstrated that prothrombin cleavage by factor X awas inhibited in the presence of PSM. Kinetic analysis of the reaction revealed that PSM competitively inhibited factor X amediated cleavage of prothrombin. PS preincubated with the monoclonal antibody, acquired similar anticoagulant properties. These results suggest that the interaction of the monoclonal antibody with PS results in an alteration in the protein exposing sites that mediate the observed anticoagulant effect. Support that the protein was altered was derived from the observation that PSM was eight fold more sensitive to cleavage by thrombin and human neutrophil elastase than conventionally purified protein S.These observations suggest that PS can be modified in vitro to a protein with APC-independent anticoagulant activity and raise the possibility that a similar alteration could occur in vivo through the binding protein S to a cellular or plasma protein.

Soluble Thrombomodulin Purified from Human Urine Exhibits a Potent Anticoagulant Effect In Vitro and In Vivo

1995 ◽  
Vol 73 (05) ◽  
pp. 805-811 ◽  
Author(s):  
Yasuo Takahashi ◽  
Yoshitaka Hosaka ◽  
Hiromi Niina ◽  
Katsuaki Nagasawa ◽  
Masaaki Naotsuka ◽  
...  
Keyword(s):  
Human Urine ◽  
Specific Activity ◽  
Anticoagulant Activity ◽  
Rabbit Lung ◽  
Soluble Thrombomodulin ◽  
Molecular Weights ◽  
Dose Dependent Fashion ◽  
Dose Dependent

SummaryWe examined the anticoagulant activity of two major molecules of soluble thrombomodulin purified from human urine. The apparent molecular weights of these urinary thrombomodulins (UTMs) were 72,000 and 79,000, respectively. Both UTMs showed more potent cofactor activity for protein C activation [specific activity >5,000 thrombomodulin units (TMU)/mg] than human placental thrombomodulin (2,180 TMU/mg) and rabbit lung thrombomodulin (1,980 TMU/mg). The UTMs prolonged thrombin-induced fibrinogen clotting time (>1 TMU/ml), APTT (>5 TMU/ml), TT (>5 TMU/ml) and PT (>40 TMU/ml) in a dose-dependent fashion. These effects appeared in the concentration range of soluble thrombomodulins present in human plasma and urine. In the rat DIC model induced by thromboplastin, administration of UTMs by infusion (300-3,000 TMU/kg) restored the hematological abnormalities derived from DIC in a dose-dependent fashion. These results demonstrate that UTMs exhibit potent anticoagulant and antithrombotic activities, and could play a physiologically important role in microcirculation.

Effects of Heparin Oligosaccharides with High Affinity for Antithrombin III in Experimental Venous Thrombosis

1982 ◽  
Vol 47 (03) ◽  
pp. 244-248 ◽  
Author(s):  
D P Thomas ◽  
Rosemary E Merton ◽  
T W Barrowcliffe ◽  
L Thunberg ◽  
U Lindahl
Keyword(s):  
Antithrombin Iii ◽  
Specific Activity ◽  
High Specific Activity ◽  
Factor Xa ◽  
Blood Levels ◽  
Thrombin Time ◽  
High Affinity ◽  
Very High

SummaryThe in vitro and in vivo characteristics of two oligosaccharide heparin fragments have been compared to those of unfractionated mucosal heparin. A decasaccharide fragment had essentially no activity by APTT or calcium thrombin time assays in vitro, but possessed very high specific activity by anti-Factor Xa assays. When injected into rabbits at doses of up to 80 ¼g/kg, this fragment was relatively ineffective in impairing stasis thrombosis despite producing high blood levels by anti-Xa assays. A 16-18 monosaccharide fragment had even higher specific activity (almost 2000 iu/mg) by chromogenic substrate anti-Xa assay, with minimal activity by APTT. When injected in vivo, this fragment gave low blood levels by APTT, very high anti-Xa levels, and was more effective in preventing thrombosis than the decasaccharide fragment. However, in comparison with unfractionated heparin, the 16-18 monosaccharide fragment was only partially effective in preventing thrombosis, despite producing much higher blood levels by anti-Xa assays.It is concluded that the high-affinity binding of a heparin fragment to antithrombin III does not by itself impair venous thrombogenesis, and that the anti-Factor Xa activity of heparin is only a partial expression of its therapeutic potential.

A Comparison of Anticoagulation Activity of a Potent Heparin Preparation in Different Test

1979 ◽  
Author(s):  
A.S. Bhargava ◽  
J. Heinick ◽  
Chr. Schöbel ◽  
P. Günzel
Keyword(s):  
Blood Clotting ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Thrombin Time ◽  
Beagle Dogs ◽  
Clotting Time ◽  
Relative Potencies ◽  
Heparin Preparation

The anticoagulant effect of a new potent heparin preparation was compared with a commercially available heparin in vivo after intravenous application in beagle dogs. The anticoagulant activity was determined using thrombin time, activated partial thromboplastin time and whole blood clotting time after 5, 10 and 30 minutes of application. The relative potency of the new heparin preparation (Scherinq) was found to be 1.62 to 2.52 times higher than heparin used for comparison (150 USP units/mg, Dio-synth). The anticoagulant properties of both preparations were also studied in vitro using dog and human plasma. The relative potencies in vitro correlated well with those obtained in vivo. Further characterization with amidolytic method using chromogenic substrate for factor Xa and thrombin (S-2222 and S-2238 from KABI, Stockholm) showed that heparin (Schering) contains 243 to 378 USP units/raq depending upon the test systems used to assay the anticoagulation activity and in addition, proves the validity of the amidolytic method.

The Synthetic Pentasaccharide, Fondaparinux, Reduces Inflammation and Neutrophil Accumulation in Kidney Ischemia-Reperfusion Injury.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 217-217 ◽  
Author(s):  
Gernot Schabbauer ◽  
Rolf D. Frank ◽  
Todd Holscher ◽  
Yuichiro Sato ◽  
Michael Tencati ◽  
...  
Keyword(s):  
Ischemia Reperfusion Injury ◽  
Inflammatory Diseases ◽  
Anticoagulant Activity ◽  
Ischemia Reperfusion ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Inflammatory Cells ◽  
Neutrophil Accumulation ◽  
Kidney Ischemia

Abstract Acute inflammatory diseases are often accompanied by coagulation activation leading to local thrombotic complications and disseminated intravascular coagulation. Recent studies support the concept of crosstalk between coagulation and inflammation. The synthetic pentasaccharide, fondaparinux, is a selective antithrombin-dependent inhibitor of coagulation factor Xa. In this study, we investigated the effect of fondaparinux in a lethal murine model of kidney ischemia-reperfusion (I/R) injury that is associated with coagulation and inflammation. Fondaparinux treatment of I/R-injured mice significantly reduced serum creatinine levels and increased survival from 0 to 44% compared with saline treated control mice. In contrast, depletion of fibrinogen with ancrod was not protective, suggesting that fondaparinux may have additional properties beyond its anticoagulant activity. Indeed, fondaparinux significantly reduced IL-6 and MIP-2 expression but did not reduce MCP-1 expression. Furthermore, fondaparinux significantly decreased neutrophil accumulation in the injured kidneys. Finally, we showed that fondaparinux reduced recruitment of neutrophils into the peritoneum in a model of acute peritonitis and inhibited the binding of U937 cells to P-selectin in vitro. Our data indicate that fondaparinux has both anticoagulant and anti-inflammatory activity reducing fibrin deposition and blocking the binding of inflammatory cells to activated endothelium. Fondaparinux may be useful in the treatment of acute inflammatory diseases.

In Vitro Evaluation of Apixaban, a Novel, Potent, Selective and Orally Bioavailable Factor Xa Inhibitor.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4130-4130 ◽  
Author(s):  
Joseph M. Luettgen ◽  
Tracy A. Bozarth ◽  
Jeffrey M. Bozarth ◽  
Frank A. Barbera ◽  
Patrick Y. Lam ◽  
...  
Keyword(s):  
Human Plasma ◽  
Serine Proteases ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Competitive Inhibitor ◽  
Rat Plasma ◽  
Reversible Inhibitor ◽  
Antithrombotic Agent

Abstract Apixaban, previously known as BMS-562247, is a high affinity, highly selective, orally-active, reversible inhibitor of coagulation factor Xa (fXa), in clinical studies as a therapeutic agent for prevention and treatment of thromboembolic diseases. The in vitro characteristics of apixaban were evaluated in purified systems and in human blood from healthy volunteers. Detailed kinetic analysis of apixaban inhibition of human fXa showed that it is a readily reversible, potent and competitive inhibitor versus a synthetic tripeptide substrate with a Ki of 0.08 nM, an association rate of 2 × 107 M−1s−1and a dissociation half life of 3.4 min. Weak affinity (Ki ~3 μM) is observed for thrombin, plasma kallikrein, and chymotrypsin. Affinity for trypsin and all other serine proteases tested is negligible with Ki > 15 μM. Apixaban is an effective inhibitor of free fXa and of prothrombinase, in buffer, platelet poor plasma, and whole blood. The anticoagulant activity of apixaban was determined in platelet-poor human plasma. Apixaban causes concentration dependent prolongation of the fXa mediated clotting assays. The human plasma concentration required to produce a doubling of the clotting time is 3.6 μM for prothrombin time, 7.4 μM for activated partial thromboplastin time and 0.4 μM for HepTest. To support preclinical efficacy and safety studies purified fXa from rabbit, dog and rat plasma was also found to be inhibited by apixaban (0.17, 2.6, and 1.3 nM, respectively). In summary the in vitro properties of apixaban show that it is a highly selective and potentially potent antithrombotic agent for venous and arterial thrombotic diseases.

scholarly journals Characterization of the Highly Active Polyhydroxyalkanoate Synthase of Chromobacterium sp. Strain USM2

2011 ◽  
Vol 77 (9) ◽  
pp. 2926-2933 ◽  
Author(s):  
Kesaven Bhubalan ◽  
Jo-Ann Chuah ◽  
Fumi Shozui ◽  
Christopher J. Brigham ◽  
Seiichi Taguchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Weight Percent ◽  
Pha Synthase ◽  
Content Type ◽  
Highly Active ◽  
Key Enzyme ◽  
Polyhydroxyalkanoate Synthase

ABSTRACTThe synthesis of bacterial polyhydroxyalkanoates (PHA) is very much dependent on the expression and activity of a key enzyme, PHA synthase (PhaC). Many efforts are being pursued to enhance the activity and broaden the substrate specificity of PhaC. Here, we report the identification of a highly active wild-type PhaC belonging to the recently isolatedChromobacteriumsp. USM2 (PhaCCs). PhaCCsshowed the ability to utilize 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) monomers in PHA biosynthesis. Anin vitroassay of recombinant PhaCCsexpressed inEscherichia colishowed that its polymerization of 3-hydroxybutyryl-coenzyme A activity was nearly 8-fold higher (2,462 ± 80 U/g) than that of the synthase from the model strainC. necator(307 ± 24 U/g). Specific activity using a Strep2-tagged, purified PhaCCswas 238 ± 98 U/mg, almost 5-fold higher than findings of previous studies using purified PhaC fromC. necator. Efficient poly(3-hydroxybutyrate) [P(3HB)] accumulation inEscherichia coliexpressing PhaCCsof up to 76 ± 2 weight percent was observed within 24 h of cultivation. To date, this is the highest activity reported for a purified PHA synthase. PhaCCsis a naturally occurring, highly active PHA synthase with superior polymerizing ability.

scholarly journals Immunological Characterization of HIV and SARS-CoV-2 Coinfected Young Individuals

Cells ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 3187
Author(s):  
Claudia Vanetti ◽  
Daria Trabattoni ◽  
Marta Stracuzzi ◽  
Antonella Amendola ◽  
Clara Fappani ◽  
...  
Keyword(s):  
Immune Response ◽  
General Population ◽  
Crucial Role ◽  
Rna Virus ◽  
Hiv Positive ◽  
Specific Antibodies ◽  
Increased Risk ◽  
Set Up

While the risk of SARS-CoV-2 infection and/or COVID-19 disease progression in the general population has been largely assessed, its impact on HIV-positive individuals remains unclear. We present clinical and immunological data collected in a cohort of HIV-infected young individuals during the first wave of COVID-19 pandemic. SARS-CoV-2 RNA, virus-specific antibodies, as well as the expression of factors involved in the anti-viral immune response were analyzed. Moreover, we set up an in vitro coinfection assay to study the mechanisms correlated to the coinfection process. Our results did not show any increased risk of severe COVID-19 in HIV-positive young individuals. In those subjects who contracted SARS-CoV-2 infection, an increase in IL-10 expression and production was observed. Furthermore, in the in vitro coinfection assay, we revealed a reduction in SARS-CoV-2 replication associated to an upregulation of IL-10. We speculate that IL-10 could play a crucial role in the course of SARS-CoV-2 infection in HIV-positive individuals. These results might help defining clinical management of HIV/SARS-CoV-2 co-infected young individuals, or putative indications for vaccination schedules in this population.

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