Compositional Differences in Commercially Available Prothrombin Complex Concentrates

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4391-4391 ◽  
Author(s):  
Nasir Sadeghi ◽  
Daniel Kahn ◽  
Josephine Cunanan ◽  
Arthur Only ◽  
Debra Hoppensteadt ◽  
...  
Keyword(s):  
Protein Content ◽  
Tissue Factor ◽  
Oral Anticoagulant ◽  
Factor Xa ◽  
Factor Ix ◽  
Thrombin Inhibitors ◽  
Prothrombin Complex Concentrates ◽  
Thrombin Activity ◽  
Sds Page ◽  
Anticoagulant Drugs

Abstract Abstract 4391 Prothrombin complex concentrates (PCCs) are used in the management of bleeding complications with conventional oral anticoagulant drugs such as warfarin. These concentrates are also used in supportive therapy for hemostatic disorders. More recently these agents have been investigated for neutralization of the newer oral anticoagulant drugs such as the direct factor Xa and thrombin inhibitors. Since the activation of these complexes results in the generation of factor Xa and IIa, these agents may potentially neutralize both Xa and thrombin inhibitors. However, the potency of these agents is defined in units which represent the level of factor IX, other factors including factor II, VII, and X are also present, and in unspecified amounts. Moreover, other vitamin K-dependent proteins such as protein C, protein S, and protein Z may also be present. Varying amounts of albumin and other agent such as heparin and antithrombin may also be present as an additive. The purpose of this study is to compare the compositions of the currently available PCCs such as Profilnine®, Beriplex®, Cofact®, Octaplex®, Prothromplex®, and the older agents such as Konyne®, Preconetiv®, and Feiba®. Materials and Methods: Commercially available PCCs were obtained from various suppliers. Each of the individual vials of these concentrates was diluted with saline to obtain a 10 U/ml factor IX solution. Protein content was measured using Lowry's method. Sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) electrophoresis was carried out by dilution of each concentrates to 2 U/ml. Western blot analysis was performed to determine presence of prothrombin, prothrombin-1, and thrombin using anti-human recombinant thrombin antibody capable recognizing these proteins. Tissue factor activation profiles of each PCCs was also studied using Innovin®. The protein composition of native and activated prothrombin complexes was also investigated using surface-enhanced laser desorption/ionization (SELDI) mass spectrometry utilizing the gold chip array (BioRad). Tissue factor mediated thrombin generation by each of the prothrombin complex was studied using a fluorometric method (Technoclone, Vienna, Austria). Results: The total protein content of these PCCs ranged from 18–106 mg/100U. Some of the products were found to contain varying amounts of albumin, antithrombin and heparin as evident in both SDS-PAGE and SELDI analysis. The SDS-PAGE profile of these complexes showed multiple protein bands ranging from 15 to 250 kDa. Beriplex and Profilnine showed fewer bands; Profilnine® mainly exhibited 250, 110, 75 kDa bands and Beriplex® mainly 75 and 66 kDa bands. The other products contain additional bands in the range of 15 to 66 kDa representing albumin and other products. In the SELDI analysis multiple peaks consistent with the SDS-PAGE profile were noted. The immunoblotting showed a major band 70–75 kDa (prothrombin) along with a 50 kDa band representing prethrombin-1. Prior to activation, Feiba® exhibited a distinct additional 37 kDa dense band, (thrombin). SELDI analysis also indicated variable amounts of prothrombin in the products. Upon activation all PCC's were capable of generating thrombin as measured by SELDI and immunoblotting. The prothrombin band completely disappears from all PCCs except Preconetiv®, the prevalent products being prethombin-1 and thrombin. The amount of the prethrombin-1 band varied widely among products; and nearly disappears from all as it is converted to thrombin with time of incubation. The amount of thrombin activity generated from each prothrombin complex was concentration dependent and ranged from 30–1044nm/1.25 units/ml. Octaplex and Cofact produced the strongest thrombin activity whereas Beriplex and Prothromplex produced the least thrombin activity. Conclusions: This study shows that despite standardization in factor IX units, at equivalent IX unit potency these agents widely vary in their composition. Beriplex® and Profilnine® represent purer preparations. Upon activation of prothrombin initiated by tissue factor each complex is capable of generating varying amounts of thrombin. Because of these wide variations in protease generation the relative neutralization potential of each of these PCCs may also differ widely. Thus each of these products should thus be considered as a distinct agent and their efficacies individually determined for a given indication. Disclosures: No relevant conflicts of interest to declare.

Oral Anti-Factor Xa and Factor IIa Agent Mediated Inhibition Of Tissue-Factor Mediated Generation Of Thrombin In Prothrombin Complex Concentrates

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4810-4810
Author(s):  
Daneyal Syed ◽  
Debra Hoppensteadt ◽  
Daniel Kahn ◽  
Job Harenberg ◽  
Jawed Fareed
Keyword(s):  
Tissue Factor ◽  
Oral Anticoagulant ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Factor Xa ◽  
Onset Time ◽  
Thrombin Inhibitors ◽  
Bleeding Complications ◽  
Prothrombin Complex Concentrates ◽  
Inhibition Of Thrombin

Introduction Several oral anti-factor IIa and factor Xa agents have recently been developed. These include the thrombin inhibitors Ximelagatran/Melagatran (M) and Dabigatran Etexilate/Dabigatran (D), which require endogenous conversion to the active agents D and M. The factor Xa inhibitors, Rivaroxaban (R) and Apixaban (A), are anti-Xa agents that do not require any endogenous activation. Ximelagatran was withdrawn from the market due to adverse reactions. Dabigatran, Rivaroxaban, and Apixaban are approved for various clinical indications. Antagonism of the anticoagulant effect may be required in bleeding complications. Contradictory results were reported for the efficacy of various prothrombin complex concentrates (PCCs) with these new oral anticoagulants (NOACs). The purpose of this study was to determine the differences in the thrombin generation inhibitory profiles of the newer oral anticoagulant agents. Methods Commercially available PCCs namely Octaplex and Beriplex, were used as a source of Factors II, VII, IX and X. To investigate the effect of each of these agents, a working solution of 1U/ml of both PCCs were supplemented in a graded concentration of 0-1250ng/ml with M, D, R and A. Thrombin generation studies were carried out using a thromboplastin activator (RC High, Technoclone Vienna, Austria). Total thrombin generated was measured in terms of nM’s. The IC-50 for each agent was calculated individually. The time course of thrombin generation was also measured following the kinetic profiles and AUC. Results Dabigatran and Melagatran produced relatively weaker inhibition of thrombin generation with the IC-50 values ranging from 410-110ng/ml in Beriplex and 350-1120ng/ml in Octaplex. Both Rivaroxaban and Apixaban produced strong inhibition of thrombin generation, with the IC-50 ranging from 58-62ng/ml in Octaplex; whereas, in Beriplex these values ranged from 48-50ng/ml. The onset time for thrombin generation and total thrombin formation was concentration dependent. The kinetics of thrombin generation with A and R were distinct from D and M. At concentrations below 310ng/ml the total amount of thrombin generated was comparable to the control; however, its formation was delayed. In both systems, D exhibited the weakest thrombin generation inhibitory potential. While the onset time of thrombin generation was delayed at concentrations below 310ng/ml the levels were comparable to or higher than the control. Discussion This data suggests that PCC’s such as Octaplex and Beriplex can be used to generate thrombin and it’s inhibition by new oral anticoagulant drugs. Octaplex generates much higher amount of thrombin than Beriplex at equivalent units. These results also show that in comparison to the oral anti-Xa agents, the oral anti-IIa agents are relatively weaker inhibitors of thrombin generation. These studies also suggest that the differential inhibition of the generation of thrombin through tissue factor by the anti-Xa and IIa agents may contribute to the potential neutralization profile of PCC’s for these drugs. Disclosures: No relevant conflicts of interest to declare.

The Factor VIII Bypassing Activity of Prothrombin Complex Concentrates: The Roles of Factor VIla and of Endothelial Cell Tissue Factor

1991 ◽  
Vol 66 (05) ◽  
pp. 559-564 ◽  
Author(s):  
Jerome M Teitel
Keyword(s):  
Factor Viii ◽  
Tissue Factor ◽  
Factor Viia ◽  
Factor Xa ◽  
Procoagulant Activity ◽  
Tissue Factor Expression ◽  
Prothrombin Complex Concentrates ◽  
Cell Tissue ◽  
Factor Expression ◽  
Necrosis Factor

SummaryAn experimental model incorporating cultured endothelial cells (EC) was used to study the "factor VIII bypassing" activity of prothrombin complex concentrates (PCC), a property exploited in the treatment of hemophiliacs with alloantibodies to factor VIII. Two PCC preparations were ineffective as stimuli of tissue factor expression by EC. However, incubation with a combination of PCC plus endotoxin (lipopolysaccharide, LPS) or tumor necrosis factor (TNF) induced much greater tissue factor expression than was seen in response to either substance alone. PCC expressed an additional direct procoagulant activity at the EC surface, which could not be attributed to either thrombin or factor Xa, and which was diminished by an anti-tissue factor antibody. Therefore factor VIIa, which was detectable in both PCC preparations, likely provided this additional direct procoagulant activity at the EC surface. We also excluded the possibility that coagulation proteases contained in or generated in the presence of PCC are protected from inactivation by AT III. Therefore, PCC can indirectly bypass factor VIII by enhancing induced endothelial tissue factor expression, and also possess direct procoagulant activity, probably mediated by factor VIIa.

scholarly journals Studies of a mechanism inhibiting the initiation of the extrinsic pathway of coagulation

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 645-651 ◽  
Author(s):  
LV Rao ◽  
SI Rapaport
Keyword(s):  
Tissue Factor ◽  
Factor Viia ◽  
Factor Xa ◽  
Factor Ix ◽  
Factor Vii ◽  
Factor X ◽  
Extrinsic Pathway ◽  
Mechanism Of Inhibition ◽  
Peptide Release ◽  
Release Assay

Abstract We have extended earlier studies (Blood 66:204, 1985) of a mechanism of inhibition of factor VIIa/tissue factor activity that requires a plasma component (called herein extrinsic pathway inhibitor or EPI) and factor Xa. An activated peptide release assay using 3H-factor IX as a substrate was used to evaluate inhibition. Increasing the tissue factor concentration from 20% to 40% (vol/vol) overcame the inhibitory mechanism in normal plasma but not in factor VII-deficient plasma supplemented with a low concentration of factor VII. A second wave of factor IX activation obtained by a second addition of tissue factor to plasma with a normal factor VII concentration was almost abolished by supplementing the reaction mixture with additional EPI and factor X. Factor Xa's active site was necessary for factor Xa's contribution to inhibition, but preliminary incubation of factor Xa with EPI in the absence of factor VIIa/tissue factor complex or of factor VIIa/tissue factor complex in the absence of EPI did not replace the need for the simultaneous presence of factor Xa, factor VIIa/tissue factor, calcium, and EPI in an inhibitory reaction mixture. Inhibition of factor VIIa/tissue factor was reversible; both tissue factor and factor VIIa activity could be recovered from a dissociated, inhibited factor VIIa/tissue factor complex. EPI appeared to bind to a factor VIIa/tissue factor complex formed in the presence of factor Xa but not to a factor VIIa/tissue factor complex formed in the absence of factor Xa.

New anticoagulants – towards the development of an "ideal" anticoagulant

VASA ◽  
2009 ◽  
Vol 38 (1) ◽  
pp. 13-29 ◽  
Author(s):  
Haas
Keyword(s):  
Unfractionated Heparin ◽  
Vitamin K Antagonists ◽  
Dabigatran Etexilate ◽  
Factor Xa ◽  
Rapid Onset ◽  
Factor Ix ◽  
Thrombin Inhibitors ◽  
Therapeutic Monitoring ◽  
Direct Thrombin Inhibitors ◽  
Onset Of Action

Currently available anticoagulants, such as unfractionated heparin, low molecular weight heparins and vitamin K antagonists, have proved effective in the prevention and treatment of thromboembolic disorders. However, these drugs have some drawbacks, such as unpredictability (in the case of unfractionated heparin), non-specificity and parenteral mode of administration, which limit their use in the clinical setting. There is a need for new agents with efficacy similar to that of these classes of anticoagulants and none of their associated drawbacks. Advances are being made in the development of more convenient and more specific drugs, with the aim to improve substantially the prevention and management of thromboembolic disorders. This review will emphasize how the development of an ideal anticoagulant, with potential benefits including high efficacy, safety, low levels of bleeding, fixed dosing, rapid onset of action, ability to bind clot-bound coagulation factors and no requirement for therapeutic monitoring, is a considerable challenge. This review will present the most relevant preclinical data, as well as the clinical studies performed to date, for several drug classes. Direct thrombin inhibitors, such as dabigatran etexilate, will be reviewed, as well as indirect (fondaparinux and idraparinux) and direct (rivaroxaban, apixaban, among others) Factor Xa inhibitors, Factor IXa inhibitors and monoclonal antibodies against Factor IX/IXa.

Tissue Factor Mediated Activation of Prothrombin Complex Concentrates (PCCs) Is Differently Inhibited by Dabigatran, Rivaroxaban and Apixaban. Potential Clinical Implications

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3410-3410
Author(s):  
Jawed Fareed ◽  
Nasir Sadeghi ◽  
Daniel Kahn ◽  
Josephine Cunanan ◽  
Kimberly Bartosiak ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Oral Anticoagulants ◽  
Protein G ◽  
Prothrombin Complex Concentrates ◽  
Sds Page ◽  
Activation Products ◽  
Tissue Factors

Abstract Abstract 3410 The newly developed oral anticoagulants represent specific antithrombin (dabigatran, Boehinger, Ingelheim) and antifactor Xa agents (rivaroxaban, Bayer Health Care/Jhonsen) and apixaban, Bristol Myers Squibb/Pfizer). Prothrombin Complex Concentrates (PCCs) such as profilnine® and beriplex® are reported to partially neutralize the anticoagulant effects of these agents. Since these PCCs are capable of generating factor Xa and thrombin, the newer anticoagulants may be neutralized differentially by the proteases generated by PCCs. Coagulation and thrombosis are activated substantially by tissue factor in vivo. The purpose of this study is to compare the inhibitory effects of dabigatran, rivaroxaban and apixaban in tissue factor mediated thrombin generation using profilnine, by utilizing various approaches to characterize activation products including thrombin. Materials and Methods Dabigatran, rivaroxaban, and apixaban were synthesized and/or commercially obtained. Profilnine (Grifols Biologicals Inc.) was also commercially obtained. One commercial lot of a recombinant thrombin preparation Recothrom® was obtained from ZymoGenetics Inc for the development of polyclonal antibodies. To generate specific antisera, individual groups of rabbits (n = 3–6) were challenged repeatedly with human recombinant thrombin, over a 9-month period. At the end of this time the antisera from each rabbit was collected and pooled. Immunglobulin (IgGs) were isolated using a protein G column (HiTrap Protein G HP – GE Helathcare Bio-Science Crop). Buffered profilnine (2.5 u/ml) was activated with routinely used tissue factor reagents by adding commercially available PT reagents such as thromboplastin C, neoplastinPlus, and simplastin at a 1:4 ratio and incubated for 30 minutes. The activation of profilnine was measured by using thrombin generation utilizing a fluorogenic substrate method (Technoclone) and the protease generation profile was evaluated using mass spectrometry method (SELDI), SDS-PAGE analysis and immunoblotting using a specific antithrombin (Recothrombin) antibody to profile the activation products. Similar studies were carried out in profilnine supplemented with graded amount of various oral anticoagulants in the concentration range of 0–2.5ug/ml. Results All tissue factors produce varying degrees of time dependent activation of profilnine as measured by consumption of prothrombin peak at 71 KDa and generation of thrombin peaks at 3l–37 KDa as observed in the SELDI. Varying amounts of prothrombin generation at 52 KDa was also evident. Distinct immunoblot for thrombin in western blotting analysis was consistent with SDS-PAGE and SELDI analysis showing the generation of thrombin. The anti-Xa agents blocked the generation of thrombin whereas dabigatran failed to produce this effect. This phenomenon was also observed in all three methods used to study generation of the thrombin when using other PCCs such as octaplex and thromboplex activated by various tissue factors. In the fluorometric thrombin generation assays both apixaban and rivaroxaban produced a relatively stronger inhibition of thrombin generation (IC50= 20–200ng/ml) wheras > 500ng/ml for dabigatran in various PCCs. Conclusion These results suggest that in contrast to dabigatran both rivaroxaban and apixaban produce a much stronger inhibition of tissue factor mediated generation of the thrombin in PCCs. Inhibition of the functional generation of thrombin was weaker with dabigatran in contrast to apixiban and rivaroxiban. The observed ex-vivo neutralization profile of these agents by PCCs may be due to the differential interactions with the protease generated during their activation. These differences along with the compositional variations in the PCCs should be taken into account while considering prothrombin complexes for the neutralization of new oral anticoagulants. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Factor XI contributes to thrombin generation in the absence of factor XII

Blood ◽  
2009 ◽  
Vol 114 (2) ◽  
pp. 452-458 ◽  
Author(s):  
Dmitri V. Kravtsov ◽  
Anton Matafonov ◽  
Erik I. Tucker ◽  
Mao-fu Sun ◽  
Peter N. Walsh ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Factor Ix ◽  
Factor Xii ◽  
Factor Xi ◽  
Factor Xii Deficiency ◽  
Low Concentrations

Abstract During surface-initiated blood coagulation in vitro, activated factor XII (fXIIa) converts factor XI (fXI) to fXIa. Whereas fXI deficiency is associated with a hemorrhagic disorder, factor XII deficiency is not, suggesting that fXI can be activated by other mechanisms in vivo. Thrombin activates fXI, and several studies suggest that fXI promotes coagulation independent of fXII. However, a recent study failed to find evidence for fXII-independent activation of fXI in plasma. Using plasma in which fXII is either inhibited or absent, we show that fXI contributes to plasma thrombin generation when coagulation is initiated with low concentrations of tissue factor, factor Xa, or α-thrombin. The results could not be accounted for by fXIa contamination of the plasma systems. Replacing fXI with recombinant fXI that activates factor IX poorly, or fXI that is activated poorly by thrombin, reduced thrombin generation. An antibody that blocks fXIa activation of factor IX reduced thrombin generation; however, an antibody that specifically interferes with fXI activation by fXIIa did not. The results support a model in which fXI is activated by thrombin or another protease generated early in coagulation, with the resulting fXIa contributing to sustained thrombin generation through activation of factor IX.

scholarly journals Studies of the activation of factor VII bound to tissue factor [see comments]

Blood ◽  
1996 ◽  
Vol 87 (9) ◽  
pp. 3738-3748 ◽  
Author(s):  
LV Rao ◽  
T Williams ◽  
SI Rapaport
Keyword(s):  
Tissue Factor ◽  
Factor Viia ◽  
Tissue Injury ◽  
Factor Xa ◽  
Inhibitory Effect ◽  
Factor Ix ◽  
Factor Vii ◽  
Activate Factor ◽  
Factor X ◽  
Ovarian Carcinoma Cell Line

Experiments were performed to evaluate activation of factor VII bound to relipidated tissue factor (TF) in suspension and to TF constitutively expressed on the surface of an ovarian carcinoma cell line (OC-2008). Activation was assessed by measuring cleavage of 125I- factor VII and by the ability of unlabeled factor VII to catalyze activation of a variant factor IX molecule that, after activation, cannot back-activate factor VII. Factor Xa was found to effectively activate factor VII bound to TF relipidated in either acidic or neutral phospholipid vesicles. Autoactivation of factor VII bound to TF in suspension was dependent on the preparation of TF apoprotein used and the technique of its relipidation. This highlights the need for caution in extrapolating data from TF in suspension to the activation of factor VII bound to cell surfaces during hemostasis. A relatively slow activation of factor VII bound to OC-2008 monolayers in the absence of added protease was observed consistently. Antithrombin in the presence or absence of heparin prevented this basal activation, whereas TF pathway inhibitor (TFPI/factor Xa complexes had only a limited inhibitory effect. Adding a substrate concentration of factor X markedly enhanced basal activation of factor VII, but both TFPI/factor Xa and antithrombin/heparin abolished this enhancement. Overall, our data are compatible with the hypothesis that not all factor VII/TF complexes formed at a site of tissue injury are readily activated to factor VIIa (VIIa)/TF complexes during hemostasis. The clinical significance of this is discussed.

scholarly journals Coagulation-dependent inhibition of fibrinolysis: role of carboxypeptidase-U and the premature lysis of clots from hemophilic plasma

Blood ◽  
1996 ◽  
Vol 88 (10) ◽  
pp. 3815-3823 ◽  
Author(s):  
GJ Jr Broze ◽  
DA Higuchi
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Feedback Inhibition ◽  
Factor Viia ◽  
Factor Xa ◽  
Factor Ix ◽  
Factor X ◽  
Dependent Inhibition ◽  
Fibrinolysis Inhibitor ◽  
Normal Hemostasis

Coagulation is initiated by the binding of factor VIIa to tissue factor, with resultant limited factor IX and X activation and thrombin production. Owing to the feedback inhibition of the factor VIIa/tissue factor complex by tissue factor pathway inhibitor (TFPI), additional factor X activation and thrombin generation must proceed through a pathway involving factors VIII, IX, and XI. Experiments designed to elucidate the requirement for amplified factor Xa and thrombin generation in normal hemostasis show that the resistance of plasma clots to tissue plasminogen activator (tPA)- and urokinase-induced fibrinolysis is related to the extent of thrombin generation. Inhibition of fibrinolysis is mediated in part by plasma carboxypeptidase-U ([CPU] carboxypeptidase-R, procarboxypeptidase-B, thrombin-activatable fibrinolysis inhibitor), a proenzyme that is proteolytically activated by thrombin in a process enhanced dramatically by the cofactor thrombomodulin. A clot induced in factor IX-deficient plasma with limited amounts of tissue factor in the presence of urokinase (100 U/mL) lyses prematurely, and this defect is corrected by supplementation of the deficient plasma with factor IX (5 micrograms/mL) or thrombomodulin (20 ng/mL). These additions enhance the rate and extent of CPU activation: in the case of factor IX, presumably by permitting amplified generation of factor Xa and thrombin, and in the case of thrombomodulin, presumably by increasing the degree of CPU activation produced by the low levels of thrombin generated in the absence of factor IX. Pretreatment of the factor IX-deficient plasma with specific anti-CPU antibodies prevents the increased resistance to fibrinolysis produced by addition of factor IX and thrombomodulin. Likewise, when coagulation is induced by thrombin (2 U/mL) in the presence of tPA (60 U/mL), clots formed from plasmas deficient in factors VIII, IX, X, or XI lyse prematurely unless the missing factor is replaced or thrombomodulin (20 ng/mL) is added.

Aptamer ARC19499 mediates a procoagulant hemostatic effect by inhibiting tissue factor pathway inhibitor

Blood ◽  
2011 ◽  
Vol 117 (20) ◽  
pp. 5514-5522 ◽  
Author(s):  
Emily K. Waters ◽  
Ryan M. Genga ◽  
Michael C. Schwartz ◽  
Jennifer A. Nelson ◽  
Robert G. Schaub ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Tissue Factor Pathway Inhibitor ◽  
Hemophilia A ◽  
Factor Viia ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Intrinsic Pathway ◽  
Extrinsic Pathway ◽  
Tissue Factor Pathway

Abstract Hemophilia A and B are caused by deficiencies in coagulation factor VIII (FVIII) and factor IX, respectively, resulting in deficient blood coagulation via the intrinsic pathway. The extrinsic coagulation pathway, mediated by factor VIIa and tissue factor (TF), remains intact but is negatively regulated by tissue factor pathway inhibitor (TFPI), which inhibits both factor VIIa and its product, factor Xa. This inhibition limits clot initiation via the extrinsic pathway, whereas factor deficiency in hemophilia limits clot propagation via the intrinsic pathway. ARC19499 is an aptamer that inhibits TFPI, thereby enabling clot initiation and propagation via the extrinsic pathway. The core aptamer binds tightly and specifically to TFPI. ARC19499 blocks TFPI inhibition of both factor Xa and the TF/factor VIIa complex. ARC19499 corrects thrombin generation in hemophilia A and B plasma and restores clotting in FVIII-neutralized whole blood. In the present study, using a monkey model of hemophilia, FVIII neutralization resulted in prolonged clotting times as measured by thromboelastography and prolonged saphenous-vein bleeding times, which are consistent with FVIII deficiency. ARC19499 restored thromboelastography clotting times to baseline levels and corrected bleeding times. These results demonstrate that ARC19499 inhibition of TFPI may be an effective alternative to current treatments of bleeding associated with hemophilia.

Effects of Dabigatran, a Direct Thrombin Inhibitor, as Compared to the Direct Factor Xa Inhibitors, Rivaroxaban and Apixaban, on Tissue Factor-Induced Human Platelet Aggregation in Platelet Rich Plasma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1884-1884 ◽  
Author(s):  
Joanne van Ryn ◽  
Monika Kink-Eiband ◽  
Norbert Hauel ◽  
Henning Priepke ◽  
Wolfgang Wienen
Keyword(s):  
Platelet Aggregation ◽  
Tissue Factor ◽  
Potent Inhibitor ◽  
Platelet Rich Plasma ◽  
Factor Xa ◽  
Thrombin Inhibitors ◽  
Factor Xa Inhibitors ◽  
Direct Thrombin Inhibitors ◽  
Direct Factor ◽  
Direct Factor Xa Inhibitors

Abstract Direct thrombin inhibitors (DTIs) have been shown to be very potent inhibitors of platelet function when platelets are activated with thrombin. This action does not occur by direct binding of the DTI to the platelet PAR-1/-4 receptor, but indirectly, by reducing thrombin concentrations and thereby reducing the interactions of thrombin with its receptor on the platelet. It was hypothesized that both thrombin and factor Xa inhibitors could inhibit platelet aggregation, if the stimulus to initiate aggregation was higher in the cascade than factor Xa, such as tissue factor. Thus, dabigatran, a DTI, and the direct factor Xa inhibitors, rivaroxaban and apixaban were tested. Free flowing whole blood (60 ml) was obtained from an antecubital vein using an 18 gauge needle from healthy human volunteers. Blood was collected in tubes containing 3.13% sodium citrate (1 in 10 dilution with whole blood). Blood was centrifuged at 200x g to obtain platelet rich plasma (PRP). Samples (300 μL PRP) were placed in a 6-channel aggregometer, equilibrated for 5 min at 37°C and calibrated with PPP from same individual (0–1 Volts). Photometric tracings were continuously digitally recorded over 5 min following the addition of tissue factor and curves were evaluated as AUC over this time interval. Each PRP sample was incubated with 2 mg/ml Pefabloc®FG (Gly-Pro-Arg-Pro) to prevent fibrin polymerisation, 5 mM CaCl2 and increasing concentrations of dabigatran or factor Xa inhibitor. Tissue factor stimulus (range, 5–27 μl of 10 ml Innovin solution) was tailored for each individual, so that the minimum concentration that resulted in maximum aggregation was used. As positive controls, aggregation was also performed after stimulating with ADP (10 μM), collagen (2 μg/ml), TRAP (20 μM) or ecarin (0.1 U/ml). All substances inhibited tissue factor-induced platelet aggregation in a concentration-dependent manner. Dabigatran was the most potent inhibitor of platelet aggregation among the substances tested, with an IC50 of 35 nM, rivaroxaban and then apixaban followed, with IC50s of 312 and 817 nM, respectively. All substances had no effect on platelet aggregation induced by ADP, collagen and TRAP. Dabigatran was a potent inhibitor of ecarin-induced platelet aggregation, while the factor Xa inhibitors had no effect, as expected from their mechanism of action. Thus, these studies demonstrate that both direct thrombin inhibitors (by inhibiting thrombin) and direct factor Xa inhibitors (by preventing thrombin generation) indirectly inhibit platelet aggregation, though dabigatran was more potent than rivaroxaban and apixaban under these experimental conditions. Thus, these substances may not only be effective in venous/stasis thrombotic episodes where fibrin formation plays an important role, but may also be effective in more platelet dominant, arterial thrombosis settings.

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