Characterization of an ideal amphipathic peptide as a procoagulant agent

2008 ◽  
Vol 412 (3) ◽  
pp. 545-551 ◽  
Author(s):  
Jorge G. Ganopolsky ◽  
Sophie Charbonneau ◽  
Henry T. Peng ◽  
Pang N. Shek ◽  
Mark D. Blostein
Keyword(s):  
Factor Xa ◽  
Clot Lysis ◽  
Dependent Manner ◽  
Factor X ◽  
Concentration Dependent Manner ◽  
Amphipathic Peptide ◽  
Rich Domain ◽  
Factor Ixa ◽  
Direct Binding

On the basis of previous evidence that amphipathic helical peptides accelerate Factor IXa activation of Factor X [Blostein, Rigby, Furie, Furie and Gilbert (2000) Biochemistry 39, 12000–12006], the present study was designed to assess the procoagulant activity of an IAP (ideal amphipathic peptide) of Lys7Leu15 composition. The results show that IAP accelerates Factor X activation by Factor IXa in a concentration-dependent manner and accelerates thrombin generation by Factor Xa with a comparable peptide- and substrate-concentration-dependence. A scrambled helical peptide with the same amino acid composition as IAP, but with its amphipathicity abolished, eliminated most of the aforementioned effects. The Gla (γ-carboxyglutamic acid)-rich domain of Factor X is required for IAP activity, suggesting that this peptide behaves as a phospholipid membrane. This hypothesis was confirmed, using fluorescence spectroscopy, by demonstrating direct binding between IAP and the Gla-rich domain of Factor X. In addition, the catalytic efficiencies of the tenase and prothrombinase enzymatic complexes, containing cofactors Factor VIIIa and Factor Va respectively, are enhanced by IAP. Finally, we show that IAP delays clot lysis in vitro. In summary, these observations demonstrate that IAP not only enhances essential procoagulant reactions required for fibrin generation, but also inhibits fibrinolysis, suggesting a potential role for IAP as a haemostatic agent.

The Use of Ideal Amphipathic Helical Peptides To Reduce Hemorrhage In Vivo.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3152-3152
Author(s):  
Sophie Charbonneau ◽  
Jorge G. Ganopolsky ◽  
Henry T. Pang ◽  
Pang N. Shek ◽  
Mark D. Blostein
Keyword(s):  
Factor Xa ◽  
Factor X ◽  
Amphipathic Peptide ◽  
In Vivo Experiments ◽  
Factor Ixa ◽  
Carboxyglutamic Acid ◽  
Direct Binding ◽  
Gla Domain ◽  
Acid Domain

Abstract We have previously demonstrated that a 22 amino acid ideal amphipathic peptide (IAP) of K7L15 composition dramatically accelerates both factor IXa and factor Xa activity. In the present work, we investigate the activity of IAP attached to a surface in view of designing a procoagulant surface to reduce hemorrhage. Our results show that IAP maintains its catalytic enhancing properties for factor IXa and factor Xa when attached to a surface. This enhancement is dependent on the presence of the gamma-carboxyglutamic acid domain of factor X, consistent with the hypothesis that IAP behaves as a phospholipid membrane, providing a surface for the assembly of procoagulant enzymes and substrates. To further confirm this hypothesis, we demonstrate direct binding between surface-bound IAP and the Gla domain of factor X using an ELISA-based binding assay. Based on the aforementioned evidence that immobilized IAP enhances procoagulant activity, we conducted in vivo experiments using an ear-bleeding model in rabbits. We incorporated IAP into DuraSeal, a commercially available sealing agent, and found that the addition of IAP decreases the bleeding time in rabbits by 25% (p=0.0065). In conclusion, the above data provide a rationale for designing procoagulant surfaces in vivo. Further evaluation in larger animal models is warranted.

Use of Amphipathic Helical Peptides as An Anticoagulant

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4100-4100
Author(s):  
Sophie Charbonneau ◽  
Henry Peng ◽  
Pang N Shek ◽  
Mark Blostein
Keyword(s):  
Coagulation Factors ◽  
In Vitro Assays ◽  
Dependent Manner ◽  
Factor X ◽  
Concentration Dependent Manner ◽  
Free System ◽  
Active Partial Thromboplastin Time ◽  
Direct Binding ◽  
High Concentrations

Abstract We have previously shown that an ideal amphipathic helical peptide of K7L15 composition (IAP) accelerates factor IXa-mediated factor X turnover and factor Xamediated prothrombin turnover in a phospholipid free system (Biochem J., 2008, 412:545). Under these conditions, IAP behaves as a phospholipid membrane allowing coagulation factors to bind and exert their actions. However, when IAP is used with in vitro assays that employ phospholipids such as an active partial thromboplastin time (aPTT), IAP paradoxically behaves as an anticoagulant by prolonging clotting times. We hypothesize that this anticoagulant effect occurs by blocking binding sites for coagulation factors on phospholipids membranes. To test this hypothesis, we employed three phopholipid-dependant coagulation assays, the aPTT, dilute PT and dilute RVV, with both low and high concentrations of phospholipids. We show that these coagulation times are prolonged by IAP in a concentration dependent manner and that this prolongation is abrogated by adding excess phospholipid, demonstrating phospholpid dependence for this inhibition. In purified tenase and prothrombinase assays, in the presence of phospholipids, IAP inhibits substrate turnover consistent with our hypothesis. To show direct binding between IAP and phospholipids, we conducted fluorescence spectroscopy experiments and show direct binding between IAP and phospholipid membranes. In summary, the above data demonstrate that IAP acts as an anticoagulant by blocking the interaction of coagulation factors with phospholipids membranes.

Thrombin activatable fibrinolysis inhibitor (TAFI) affects fibrinolysis in a plasminogen activator concentration-dependent manner

2004 ◽  
Vol 91 (03) ◽  
pp. 473-479 ◽  
Author(s):  
Ana Guimarães ◽  
Dingeman Rijken
Keyword(s):  
Plasminogen Activator ◽  
In Vitro Study ◽  
Inhibitory Effect ◽  
Retardation Factor ◽  
Clot Lysis ◽  
Dependent Manner ◽  
Plasma Clot ◽  
Concentration Dependent Manner ◽  
Plasmin Inhibitor

SummaryTAFIa was shown to attenuate fibrinolysis. In our in vitro study, we investigated how the inhibitory effect of TAFIa depended on the type and concentration of the plasminogen activator (PA). We measured PA-mediated lysis times of plasma clots under conditions of maximal TAFI activation by thrombin-thrombomodulin in the absence and presence of potato carboxypeptidase inhibitor. Seven different PAs were compared comprising both tPA-related (tPA, TNK-tPA, DSPA), bacterial PA-related (staphylokinase and APSAC) and urokinase-related (tcu-PA and k2tu-PA) PAs. The lysis times and the retardation factor were plotted against the PA concentration. The retardation factor plots were bell-shaped. At low PA concentrations, the retardation factor was low, probably due to the limited stability of TAFIa. At intermediate PA concentrations the retardation factor was maximal (3-6 depending on the PA), with TNK-tPA, APSAC and DSPA exhibiting the strongest effect. At high PA concentrations, the retardation factor was again low, possibly due to inactivation of TAFIa by plasmin or to a complete conversion of glu-plasminogen into lys-plasminogen. Using individual plasmas with a reduced plasmin inhibitor activity (plasmin inhibitor Enschede) the bell-shaped curve of the retardation factor shifted towards lower tPA and DSPA concentrations, but the height did not decrease. In conclusion, TAFIa delays the lysis of plasma clots mediated by all the plasminogen activators tested. This delay is dependent on the type and concentration of the plasminogen activator, but not on the fibrin specificity of the plasminogen activator. Furthermore, plasmin inhibitor does not play a significant role in the inhibition of plasma clot lysis by TAFI.

Expression and Assembly of Procoagulant Complexes by Human Pleural Mesothelial Cells

1994 ◽  
Vol 71 (05) ◽  
pp. 587-592 ◽  
Author(s):  
Anuradha Kumar ◽  
Kathleen B Koenig ◽  
Alice R Johnson ◽  
Steven Idell
Keyword(s):  
Tissue Factor ◽  
Factor Xa ◽  
Mesothelial Cells ◽  
Factor Vii ◽  
Dependent Manner ◽  
Factor X ◽  
Specific Concentration ◽  
Pleural Mesothelial Cells ◽  
Direct Binding ◽  
Thrombin Formation

SummaryMany pleural diseases involve fibrin deposition within the pleural cavity, an event that necessarily involves the mesothelium. This study of human pleural mesothelial cells (HPMC) was designed to determine how the mesothelium initiates and sustains the coagulation process. We used functional assays for activation of both factor X and prothrombin to examine expression and assembly of procoagulant activity by human pleural mesothelial cells in culture. The rates of factor Xa and thrombin formation were calcium-dependent. The rate of factor Xa formation in the presence of added factor VII increased in a concentration-dependent manner, suggesting that tissue factor is the primary procoagulant associated with HPMC. The fact that direct binding of radioiodinated factor Vila to HPMC was specific, concentration-dependent and saturable confirms that tissue factor is expressed on the cell surface. The rate of thrombin formation increased with factor Xa concentration, and the rate was 5-, 6-fold higher in presence of added factor Va indicating that HPMC support expression of prothrombinase activity. Further, direct binding of radioiodinated factor Xa to HPMC was specific, concentration-dependent and saturable, confirming that the cells support the assembly of the prothrombinase complex.

A long-lasting, plasmin-activatable thrombin inhibitor aids clot lysis in vitro and does not promote bleeding in vivo

2009 ◽  
Vol 101 (05) ◽  
pp. 867-877 ◽  
Author(s):  
Louise Eltringham-Smith ◽  
Sharon Gataiance ◽  
Varsha Bhakta ◽  
William Sheffield
Keyword(s):  
Chimeric Protein ◽  
Fold Increase ◽  
Thrombin Inhibitor ◽  
Clot Lysis ◽  
Dependent Manner ◽  
Recombinant Hirudin ◽  
Concentration Dependent Manner ◽  
Shed Blood

SummaryThe leech protein hirudin is a potent inhibitor of thrombin, but clinical use of recombinant hirudin is restricted by haemorrhagic risks, and complicated by hirudin’s rapid clearance from the circulation. We previously employed albumin fusion to slow hirudin variant 3 (HV3) clearance. In this study, we hypothesized that reconfiguration of the chimera, appending human serum albumin (HSA) to the N-terminus of HV3, with an intervening plasmin cleavage site, would create a slowly cleared, plasmin-activatable HV3. Potential plasmin cleavage sites were screened by expression in Escherichia coli, interposed between glutathione sulfotransferase and HV3 domains. The most reactive sequence (GSGIYR-ITY) was recreated in C-terminally His-tagged albumin fusion protein HSACHV3, expressed in Pichia pastoris yeast and purified by nickel-chelate affinity chromatography. HSACHV3 showed no thrombin inhibitory activity in the absence of plasmin, but liberated active HV3 in a time- and concentration-dependent manner in its presence. In a discontinuous clot assay involving clot-bound thrombin, HSACHV3 assisted clot lysis by limiting clot extension in a tPA- and concentration-dependent manner. Similar results were obtained in plasma at higher concentrations of HSACHV3. The chimeric protein exhibited much slower clearance in mice than unfused HV3, and indistinguishable pharmacokinetics from unfused recombinant HSA. In a mouse tail transection bleeding model, doses of HSACHV3 identical to those of HV3 that elicited a four-fold increase in the volume of shed blood were without effect. Our results suggest that HSACHV3 is a fully latent, plasmin activatable, long-lasting hirudin, of potential benefit in thrombotic disorders resistant to natural or pharmacological clot lysis.

scholarly journals Inactivation of purified human alpha 2-antiplasmin and purified human C1 inhibitor by synthetic fibrinolytic agents

Blood ◽  
1981 ◽  
Vol 57 (6) ◽  
pp. 1015-1024
Author(s):  
LA Miles ◽  
JP Burnier ◽  
MS Verlander ◽  
M Goodman ◽  
AJ Kleiss ◽  
...  
Keyword(s):  
Blood Clot ◽  
Water Soluble ◽  
Flufenamic Acid ◽  
Clot Lysis ◽  
Dependent Manner ◽  
Plasmin Activity ◽  
Concentration Dependent Manner ◽  
Factor Xiia ◽  
Fibrinolytic Compounds

3-Hydroxypropyl flufenamide (Flu-HPA) is one of a series of flufenamic acid derivatives that enhances blood clot lysis in vitro. Studies of possible mechanisms of action of Flu-HPA were undertaken. The profibrinolytic activity of Flu-HPA in clot lysis assays was found to be dependent on plasminogen. The influence of Flu-HPA on the ability of purified alpha 2-antiplasmin to inhibit purified plasmin was studied. Plasmin activity was determined using 125I-fibrin plates or the spectrophotometric tripeptide substrate, Val-Leu-Lys-paranitroanilide. At Flu-HPA concentrations greater than 1 mM, the inhibitory activity of alpha 2-antiplasmin was abolished in a time-dependent and concentration- dependent manner. The influence of Flu-HPA on the ability of purified Cl inhibitor to inhibit purified plasma kallikrein and beta-Factor XIIa was also studied. Cl inhibitor activity was abolished by Flu-HPA at concentrations greater than 2 mM. Notably, Flu-HPA up to 60 mM did not affect the amidolytic activities of plasmin, kallikrein, or beta-Factor XIIa. Flu-HPA did not release enzyme activity from preformed complexes of either alpha 2-antiplasmin and plasmin of Cl inhibitor and kallikrein. A water-soluble derivative of flufenamic acid, N-flufenamyl- glutamic acid, also inactivated alpha 2-antiplasm and Cl inhibitor. This inactivation was shown to be reversible. These results indicate that synthetic fibrinolytic compounds such as flufenamic acid derivatives may promote fibrinolysis by directly inactivating alpha 2- antiplasmin and Cl inhibitor.

scholarly journals Contribution of the NH2-terminal EGF-domain of factor IXa to the specificity of intrinsic tenase

2012 ◽  
Vol 108 (12) ◽  
pp. 1154-1164 ◽  
Author(s):  
Shabir Qureshi ◽  
Likui Yang ◽  
Alireza Rezaie
Keyword(s):  
Active Site ◽  
Membrane Surface ◽  
Factor Xa ◽  
Resonance Energy ◽  
Catalytic Efficiency ◽  
Normal Activity ◽  
Factor X ◽  
Factor Ixa ◽  
Direct Binding ◽  
Egf Domain

SummaryFactor IXa (FIXa) is a vitamin K-dependent coagulation serine protease which binds to factor VIIIa (FVIIIa) on negatively charged phospholipid vesicles (PCPS) to catalyse the activation of factor X (FX) to factor Xa (FXa) in the intrinsic pathway. Fluorescence resonance energy transfer (FRET) studies have indicated that the Gla-domain-dependent interaction of FIXa and FX with PCPS in the presence of FVIIIa positions the active-site of the protease at an appropriate height above the membrane surface to optimise the catalytic reaction. In this study, we investigated the contribution of the NH2-terminal EGF-domain (EGF1) of FIXa to the recognition specificity of intrinsic tenase by constructing an EGF1 deletion mutant of FIXa (FIXa-desEGF1) and characterising the properties of the mutant in kinetic, direct binding and FRET assays. The results of direct binding and kinetic studies demonstrated that the binding affinity of the mutant for interaction with FVIIIa on PCPS has been impaired greater than 10-fold and the catalytic efficiency of the mutant protease FVIIIa-PCPS complex in the activation of FX has been decreased 100-fold. By contrast, the mutant protease exhibited a normal activity toward FX in the absence of the protein cofactor. FRET measurements revealed that the distance of the active-site of the mutant FIXa relative to PCPS vesicles has been decreased 10 Åfrom 75 ±2 Åfor FIXa to 65 ±2 Åfor FIXa-desEGF1 independent of FVIIIa. These results suggest that the NH2-terminal EGF-domain of FIXa provides a binding-site for FVIIIa and plays an essential spacer function in the intrinsic tenase complex.

Characterization of an Ideal Amphipathic Peptide as a Potential Hemostatic Agent.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1618-1618 ◽  
Author(s):  
Jorge G. Ganopolsky ◽  
Sophie Charbonneau ◽  
Henry Peng ◽  
Mark Blostein
Keyword(s):  
Thrombin Generation ◽  
Coagulation Cascade ◽  
Hemostatic Agent ◽  
Dependent Manner ◽  
Factor X ◽  
Amphipathic Peptide ◽  
Helical Peptides ◽  
Factor Ixa ◽  
Gla Domain ◽  
Factor X Activation

Abstract We have previously demonstrated that amphipathic helical peptides can accelerate the turnover of the substrate factor X by the enzyme, factor IXa in a gamma-carboxyglutamic acid (Gla) domain dependent manner (Biochemistry,39:12000Biochemistry,39:12000). Such improvement was due to a remarkable decrease in KM and a mild increase in kcat, mimicking the presence of phospholipid membranes. It is hypothesized that amphipathic helical peptides could exert similar activities in other reactions of the blood coagulation cascade, such as thrombin generation and in whole blood clotting. In the present work, we analyze the activity of a 22-amino acid ideal amphipathic helical peptide (IAP) of K7L15 composition, in factor X activation and thrombin generation. The addition of IAP accelerates factor X activation by factor IXa in a concentration dependent manner. IAP also accelerates thrombin generation by factor Xa with a comparable peptide and substrate concentration dependence. Further, the Gla domain is also required for peptide activity confirming the hypothesis this peptide behaves as a membrane mimetic. Using fluorescence spectroscopy and an ELISA-based binding assay, we demonstrate direct binding between IAP and the Gla domain of factor X. Additionally, when IAP is immobilized to a reaction surface, factor X activation and thrombin generation are dramatically enhanced, as compared to the corresponding reactions on untreated surfaces. Finally, activated partial thromboplastin times (APTTs) of pooled plasma are significantly shortened on surfaces treated with IAP, in comparison with clotting times measured on untreated surfaces. The above results suggest that immobilized IAP may serve as a potential hemostatic agent, as demonstrated in isolated critical reactions of the coagulation cascade and in whole plasma.

In-vitro evaluation of anti-factor IXa aptamer on thrombin generation, clotting time, and viscoelastometry

2009 ◽  
Vol 101 (05) ◽  
pp. 827-833 ◽  
Author(s):  
Kenichi Tanaka ◽  
Fania Szlam ◽  
Christopher Rusconi ◽  
Jerrold Levy
Keyword(s):  
Thrombin Generation ◽  
Lag Time ◽  
Clot Formation ◽  
Dependent Manner ◽  
Plasma Samples ◽  
Clotting Time ◽  
Concentration Dependent Manner ◽  
Factor Ixa ◽  
Prolonged Aptt

SummaryThe REG1 system consists of factor IXa inhibitor, RB006, an ap-tamer-based anticoagulant and its antidote, RB007. The optimal use of RB006 can be facilitated by understanding its effect on the formation of thrombin and fibrin, and other standard tests of coagulation. Blood from consented volunteers was drawn into 3.2% citrate (9:1 v/v) and either used immediately or centrifuged to obtain platelet-poor plasma. Increasing concentrations of ap-tamer (6–24 μg/ml) alone or in combination with heparin (0.1 U/ml) or lepirudin (0.2 μg/ml) were added to blood and plasma samples. Activated clotting times (ACT+, low range-ACT), thrombelastometry (ROTEM™) or thrombelastography (TEG®) were performed in recalcified whole blood samples. Thrombin generation, prothrombin time (PT) and activated partial thromboplastin time (aPTT) were performed in plasma samples. To some samples the antidote RB007 was added to neutralise the anticoagulation activity of RB006. In all experiment the ratio of RB006 to RB007 was kept 1:2. RB006 dose-dependently prolonged aPTT and low range-ACT, but, as expected, had no effect on PT. RB006 prolonged the lag time and decreased the peak of Actin-triggered thrombin generation. Thrombin-activated TEG demonstrated that RB006 decreases the rate of clot formation. These effects were potentiated when RB006 was combined with heparin or lepirudin. In all experiments RB007 reversed the effects of RB006 back to baseline. In conclusion, RB006 inhibits thrombin generation and clot formation in a concentration-dependent manner. It is feasible to monitor RB006 and its reversal with RB007 using aPTT, low range-ACT, and thrombin-activated TEG.

Lupus Anticoagulants Form Immune Complexes With Prothrombin and Phospholipid That Can Augment Thrombin Production in Flow

Blood ◽  
1999 ◽  
Vol 94 (10) ◽  
pp. 3421-3431 ◽  
Author(s):  
Susan L. Field ◽  
Philip J. Hogg ◽  
Elise B. Daly ◽  
Yan-Ping Dai ◽  
Barbara Murray ◽  
...  
Keyword(s):  
Calcium Ions ◽  
Flow Reactor ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Plasma Phospholipid ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Lupus Anticoagulants

Lupus anticoagulants (LA) are a family of autoantibodies that are associated with in vitro anticoagulant activity but a strong predisposition to in vivo thrombosis. They are directed against plasma phospholipid binding proteins, including prothrombin. We found that a murine monoclonal antiprothrombin antibody and 7 of 7 LA IgGs tested enhanced binding of prothrombin to 25:75 phosphatidyl serine:phosphatidyl choline vesicles in a concentration-dependent manner. We hypothesized that enhanced binding of prothrombin to phospholipid in the presence of LA IgG might result in increased thrombin production when reactions are performed in flow. Thrombin production by purified prothrombinase components was measured in a phospholipid-coated flow reactor. The flow reactor was incubated with prothrombin, calcium ions, and the IgGs and then perfused with prothrombin, calcium ions, the IgGs, factor Va, and factor Xa. A murine monoclonal antiprothrombin antibody and 4 of 6 LA IgGs from patients with a history of thrombosis increased thrombin production up to 100% over control in the first 15 minutes. In summary, LA IgGs concentrate prothrombin on a phospholipid surface that can augment thrombin production by prothrombinase in flow. These observations suggest that LA might propagate coagulation in flowing blood by facilitating prothrombin interaction with the damaged blood vessel wall.

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