FXa-α2-Macroglobulin Complex Neutralizes Direct Oral Anticoagulants Targeting FXa In Vitro and In Vivo

Increasing number of patients are treated with direct oral anticoagulants (DOAC). An antidote for dabigatran inhibiting thrombin (idarucizumab) is available but no antidote is yet approved for the factor Xa (FXa) inhibitors (xabans). We hypothesized that a complex between Gla-domainless FXa and α2-macroglobulin (GDFXa-α2M) may neutralize the xabans without interfering with normal blood coagulation.Purified α2M was incubated with GDFXa to form GDFXa-α2M. Affinity of apixaban and rivaroxaban for GDFXa-α2M was only slightly decreased compared to FXa. Efficacy and harmlessness of GDFXa-α2M were tested in vitro and in vivo. Stoichiometric excess of GDFXa-α2M neutralized rivaroxaban and apixaban as attested by clot waveform assay and rotational thromboelastometry, whereas GDFXa-α2M alone had no effect on these assays. Efficacy and pro-thrombotic potential of GDFXa-α2M were also assessed in vivo. Half-life of GDFXa-α2M in C57BL6 mice was 4.9 ± 1.1 minutes, but a 0.5 mg/mouse dose resulted in uptake saturation such that 50% persistence was still observed after 170 minutes. Single administration of GDFXa-α2M significantly decreased the rivaroxaban-induced bleeding time (p < 0.001) and blood loss (p < 0.01). GDFXa-α2M did not increase D-dimer or thrombin–antithrombin complex formation, suggesting a lack of pro-thrombotic potential.GDFXa-α2M is therefore an attractive candidate for xaban neutralization neither pro- nor anticoagulant in vitro as well as in vivo.

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Heparin is more effective than apixaban in inhibiting in vitro contact activated coagulation

European Heart Journal ◽

10.1093/ehjci/ehaa946.3776 ◽

2020 ◽

Vol 41 (Supplement_2) ◽

Author(s):

M.M Engelen ◽

C Van Laer ◽

M Jacquemin ◽

C Vandenbriele ◽

K Peerlinck ◽

...

Keyword(s):

Thrombin Generation ◽

Heart Valves ◽

Thrombus Formation ◽

Oral Anticoagulants ◽

Direct Oral Anticoagulants ◽

Factor Xa ◽

Coagulation Factor ◽

Mechanical Heart Valves ◽

Contact Activation

Abstract Introduction Contact of blood with artificial surfaces such as mechanical support devices, catheters, and mechanical heart valves activates the contact activation (CA) pathway of coagulation. Furthermore, recent animal data and clinical studies suggest a more important contribution of CA in pathological thrombus formation in other cardiovascular diseases. Direct oral anticoagulants (DOACs) are recommended as first-line treatment in most patients who require long-term anticoagulation. However, because DOACs directly inhibit a single downstream coagulation factor (thrombin (fXIIa) or factor Xa (fXa)), it has been suggested that their efficacy could be reduced in the presence of strong activation of the CA pathway as compared to anticoagulants that target multiple, more upstream located coagulation factors. Purpose To compare the efficacy of a DOAC (apixaban) and heparin to suppress thrombin generation in the presence of strong CA pathway activation. Methods Pooled platelet-poor plasma was spiked with either apixaban (dissolved in DMSO and PBS) or unfractionated heparin to achieve therapeutic plasma levels. SynthASil, a commercially available mixture of phospholipids and silica, was used to stimulate the CA pathway in two different dilutions (1–80 and 5–80). Downstream coagulation was accessed by Thrombin Generation Test using Thrombinoscope by Stago and associated Thrombin Calibrator (activity 640 nM). The endogenous thrombin potential (area under the thrombin generation curve; ETP), peak thrombin generation (PTG), time to peak (ttPeak) and time to start (ttStart) were accessed. Results With decreasing concentrations of apixaban, stimulation with the lower dose SynthASil reveals an increasing ETP and PTG. As expected, ttPeak and ttStart decreased. Even supratherapeutic levels of apixaban (i.e. 1120 ng/mL) could not inhibit thrombin from being generated, in striking contrast with UFH where no thrombin was formed. Using a five times higher dose of SynthASil showed comparable ETP for all concentrations of apixaban, allocated around the control value. PTG, however, slightly increased with decreasing concentrations of apixaban. ttPeak and ttStart slightly decreased. Except for the subtherapeutic UFH concentration of 0,114 IU/mL, no thrombin was generated with UFH. Conclusion UFH is more effective in inhibiting downstream thrombin generation compared to apixaban as a response to activation of the CA pathway in vitro. These findings could help explain why direct inhibitors were not able to show non-inferiority in patients with mechanical heart valves and support the development of specific CA pathway inhibitors for patients with conditions that activate the CA pathway. Thrombin generation curves Funding Acknowledgement Type of funding source: None

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Assessing Coagulation by Rotational Thromboelastometry (ROTEM) in Rivaroxaban-Anticoagulated Blood Using Hemostatic Agents

Prehospital and Disaster Medicine ◽

10.1017/s1049023x17006641 ◽

2017 ◽

Vol 32 (5) ◽

pp. 580-587 ◽

Cited By ~ 2

Author(s):

Jonathan Bar ◽

Alexa David ◽

Tarek Khader ◽

Mary Mulcare ◽

Christopher Tedeschi

Keyword(s):

Oral Anticoagulants ◽

Direct Oral Anticoagulants ◽

Factor Xa ◽

Hemostatic Agent ◽

Publication Type ◽

Rotational Thromboelastometry ◽

Coagulation Parameters ◽

Hemostatic Agents ◽

Clotting Cascade ◽

Data Points

AbstractIntroductionThe use of direct oral anticoagulants (DOACs) such as rivaroxaban (Xarelto) is increasingly common. However, therapies for reversing anticoagulation in the event of hemorrhage are limited. This study investigates the ability of hemostatic agents to improve the coagulation of rivaroxaban-anticoagulated blood, as measured by rotational thromboelastometry (ROTEM).Hypothesis/ProblemIf a chitosan-based hemostatic agent (Celox), which works independently of the clotting cascade, is applied to rivaroxaban-anticoagulated blood, it should improve coagulation by decreasing clotting time (CT), decreasing clot formation time (CFT), and increasing maximum clot firmness (MCF). If a kaolin-based hemostatic agent (QuikClot Combat Gauze), which works primarily by augmenting the clotting cascade upstream of factor Xa (FXa), is applied to rivaroxaban-anticoagulated blood, it will not be effective at improving coagulation.MethodsPatients (age >18 years; non-pregnant) on rivaroxaban, presenting to the emergency department (ED) at two large, university-based medical centers, were recruited. Subjects (n=8) had blood drawn and analyzed using ROTEM with and without the presence of a kaolin-based and a chitosan-based hemostatic agent. The percentage of patients whose ROTEM parameters responded to the hemostatic agent and percent changes in coagulation parameters were calculated.ResultsData points analyzed included: CT, CFT, and MCF. Of the samples treated with a kaolin-based hemostatic agent, seven (87.5%) showed reductions in CT, eight (100.0%) showed reductions in CFT, and six (75.0%) showed increases in MCF. The average percent change in CT, CFT, and MCF for all patients was 32.5% (Standard Deviation [SD]: 286; Range:-75.3 to 740.7%); -66.0% (SD:14.4; Range: -91.4 to -44.1%); and 4.70% (SD: 6.10; Range: -4.8 to 15.1%), respectively. The corresponding median percent changes were -68.1%, -64.0%, and 5.2%. Of samples treated with a chitosan-based agent, six (75.0%) showed reductions in CT, three (37.5%) showed reductions in CFT, and five (62.5%) showed increases in MCF. The average percent changes for CT, CFT, and MCF for all patients were 165.0% (SD: 629; Range:-96.9 to 1718.5%); 139.0% (SD: 174; Range: -83.3 to 348.0%); and -8.38% (SD: 32.7; Range:-88.7 to 10.4%), respectively. The corresponding median percent changes were -53.7%, 141.8%, and 3.0%.ConclusionsRotational thromboelastometry detects changes in coagulation parameters caused by hemostatics applied to rivaroxaban-anticoagulated blood. These changes trended in the direction towards improved coagulability, suggesting that kaolin-based and chitosan-based hemostatics may be effective at improving coagulation in these patients.BarJ, DavidA, KhaderT, MulcareM, TedeschiC. Assessing coagulation by rotational thromboelastometry (ROTEM) in rivaroxaban-anticoagulated blood using hemostatic agents. Prehosp Disaster Med. 2017;32(5):580–587.

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Synthesis and Thrombin, Factor Xa and U46619 Inhibitory Effects of Non-amidino and Amidino N2-Thiophenecarbonyl- and N2-Tosylanthranilamides

10.20944/preprints201702.0009.v1 ◽

2017 ◽

Author(s):

Soo Hyun Lee ◽

Wonhwa Lee ◽

Nguyen Thi Ha ◽

Il Soo Um ◽

Jong-Sup Bae ◽

...

Keyword(s):

Platelet Aggregation ◽

Bleeding Time ◽

Ex Vivo ◽

Factor Xa ◽

Inhibitory Effects ◽

Human Endothelial Cells ◽

Antithrombotic Activity ◽

Pharmacological Targets

Thrombin (factor IIa) and factor Xa (FXa) are key enzymes at the junction of the intrinsic and extrinsic coagulation pathways and are the most attractive pharmacological targets for the development of novel anticoagulants. Twenty non-amidino N2-thiophencarbonyl- and N2-tosyl anthranilamides 1-20 and six amidino N2-thiophencarbonyl- and N2-tosylanthranilamides 21-26 were synthesized and evaluated prothrombin time (PT) and activated partial thromboplastin time (aPTT) using human plasma at concentration 30 μg/mL in vitro. From these results, compounds 5, 9, and 21-23 were selected to study the further antithrombotic activity. The anticoagulant properties of 5, 9, and 21-23 significantly exhibited a concentration-dependent prolongation of in vitro PT and aPTT, in vivo bleeding time, and ex vivo clotting time. These compounds concentration-dependently inhibited the activities of thrombin and FXa and inhibited the generation of thrombin and FXa in human endothelial cells. In addition, data showed that 5, 9, and 21-23 significantly inhibited thrombin catalyzed fibrin polymerization and mouse platelet aggregation and inhibited platelet aggregation induced U46619 in vitro and ex vivo. N-(3'-Amidinophenyl)-2-((thiophen-2''-yl)carbonyl amino)benzamide (21) was most active.

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Direct oral anticoagulants; a review for the non-specialist

Hematology Reports ◽

10.4081/hr.2021.9239 ◽

2021 ◽

Vol 13 (4) ◽

Author(s):

Thilina Gunawardena

Keyword(s):

Oral Anticoagulants ◽

Direct Oral Anticoagulants ◽

Factor Xa ◽

Thrombin Inhibitors ◽

Onset Of Action ◽

Novel Anticoagulants ◽

Number Of Patients ◽

Direct Factor Xa Inhibitors ◽

Pharmacokinetic Properties ◽

Clinical Overview

Thrombin inhibitors and direct factor Xa inhibitors represent a major breakthrough in the field of anticoagulation pharmacotherapy. These novel agents have replaced warfarin as the oral anticoagulant of choice in certain indications, as they possess equal or superior efficacy and better safety profiles. They have a quick onset of action, predictable pharmacokinetic properties and minimal drug and food interactions. So they do not require frequent blood monitoring and dose adjustments as with warfarin. Considering all the advantages, there seems to be a rapid increase in the number of patients who are started on these novel anticoagulants. In this review, we highlight the pharmacology of these direct oral anticoagulants and the evidence-based indications for their use. We aim to provide a clinical overview for the non-specialist who may be called upon to manage a patient who is currently on one of these novel anticoagulants.

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Impact of Idarucizumab and Andexanet Alfa on DOAC Plasma Concentration and ClotPro® Clotting Time: An Ex Vivo Spiking Study in A Cohort of Trauma Patients

Journal of Clinical Medicine ◽

10.3390/jcm10163476 ◽

2021 ◽

Vol 10 (16) ◽

pp. 3476

Author(s):

Daniel Oberladstätter ◽

Christoph J. Schlimp ◽

Johannes Zipperle ◽

Marcin F. Osuchowski ◽

Wolfgang Voelckel ◽

...

Keyword(s):

Plasma Concentration ◽

Healthy Volunteers ◽

Oral Anticoagulants ◽

Direct Oral Anticoagulants ◽

Factor Xa ◽

Factor Xa Inhibitors ◽

Clotting Time ◽

Andexanet Alfa ◽

The Impact

Specific antagonists have been developed for the reversal of direct oral anticoagulants (DOAC). We investigated the impact of these reversal agents on the plasma concentration and visco-elastic test results of dabigatran and factor Xa inhibitors. After baseline measurements of dabigatran, the plasma concentration, and the visco-elastic ClotPro® ecarin clotting time (ECA-CT), we added the reversal agent Idarucizumab in vitro and these two analyses were repeated. Likewise, the baseline plasma concentration of apixaban, edoxaban, and rivaroxaban as well as ClotPro® Russell’s viper venom test clotting time (RVV-CT) were measured and reanalyzed following Andexanet alfa spiking. We analyzed fifty blood samples from 37 patients and 10 healthy volunteers. Idarucizumab decreased the measured dabigatran plasma concentration from 323.9 ± 185.4 ng/mL to 5.9 ± 2.3 ng/mL and ECA-CT from 706.2 ± 344.6 s to 70.6 ± 20.2 s, (all, p < 0.001). Andexanet alfa decreased the apixaban concentration from 165.1 ± 65.5 ng/mL to 9.8 ± 8.1 ng/mL, edoxaban from 152.4 ± 79.0 ng/mL to 36.4 ± 19.2 ng/mL, and rivaroxaban from 153.2 ± 111.8 ng/mL to 18.1 ± 9.1 ng/mL (all p < 0.001). Andexanet alfa shortened the RVV-CT of patients with apixaban from 239.2 ± 71.7 s to 151.1 ± 30.2 s, edoxaban from 288.2 ± 65.0 s to 122.7 ± 37.1 s, and rivaroxaban from 225.9 ± 49.3 s to 103.7 ± 12.1 s (all p < 0.001). In vitro spiking of dabigatran-containing blood with Idarucizumab substantially reduced the plasma concentration and ecarin-test clotting time. Andexanet alfa lowered the concentration of the investigated factor Xa-inhibitors but did not normalize the RVV-CT. In healthy volunteers’ blood, Idarucizumab spiking had no impact on ECA-CT. Andexanet alfa spiking of non-anticoagulated blood prolonged RVV-CT (p = 0.001), potentially as a consequence of a competitive antagonism with human factor Xa.

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Protein Inhibitor of Coagulation Factor Xa (PifXa): A New Protein-Based Specific Factor Xa Inhibitor Is An Effective, Reversible Inhibitor of Blood Coagulation.

Blood ◽

10.1182/blood.v116.21.1095.1095 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1095-1095

Author(s):

Mette Sondrup Andersen ◽

Aage Kristian Olsen Alstrup ◽

Julie Kirstine Andersen ◽

Søren Risom Kristensen ◽

Kåre Lehmann Nielsen

Keyword(s):

Blood Coagulation ◽

Bleeding Time ◽

Factor Xa ◽

Coagulation Factor ◽

Coagulation Cascade ◽

Specific Factor ◽

Low Molecular Weight ◽

University Patents

Abstract Abstract 1095 Heparin was discovered in 1916 and put into clinical trials in 1935. Despite advent of several anticoagulants during the last 75 years, heparin still remains the most widely used anticoagulant. None the less, several drawbacks of heparin exist i) it is difficult to determine the correct dosage, ii) heparins binds many different targets in humans, iii) side effects such as Heparin Induced Thrombocytopenia (HIT) is known (Hirsh J et al, Chest 2001). Consequently, intense emphasis have been put on finding new and improved inhibitory agents towards specific factors in the blood coagulation. Especially factor Xa (fXa) is considered an interesting target for inhibitors due to its central place in the coagulation cascade (Gross PL and Weitz, JI, Clinical Pharmacology and therapeutics 2009). Here we present a novel direct specific inhibitor of fXa, PifXa (protein inhibitor of coagulation factor Xa), which has been isolated from potato tubers. The inhibitor of the legume Kunitz type protein family was able to inhibit the activity of fXa using a mixed mode of inhibition with an apparent Ki of 2.5 nM, as determined using a low molecular weight substrate. Noteworthy, no inhibition of thrombin could be detected. Furthermore, the effect of the inhibitor could be detected using the activated partial thromboplastin time (aPTT) assay, which suggests that PifXa is not only capable of inhibiting free fXa but also complex/clot-bound fXa. Other known specific fXa inhibitors such as the pentasaccharide fondaparinux (Arixtra, GlaxoSmithKline) and low molecular weight heparin (LMWH) give rise to little or no effect in the aPTT assay. This observation has been attributed to the fact that these inhibitors only inhibit free fXa (Hirsh J et al, Chest 2001). PifXa was capable of significantly prolonging the tail bleeding time, but did not increase the bleeding amount significantly compared to the control in in vivo experiments conducted in rats. Hence, PifXa is highly specific towards the blood coagulation cascade, but do not interfere with the platelet plug formation in contrast to heparin, that can interfere with the thrombin induced platelet activation (Day, J et al, J of Cardiothoracic and Vascular Anesthesia 2004). Indeed, inhibition of activation of the platelets by PifXa could not be detected in in vitro experiments using platelet aggreometry. Furthermore, PifXa given in combination with the anti-platelet drug acetylsalicylic acid increased both the bleeding time and amount in the in vivo rat experiment significantly, demonstrating an additive effect of PifXa and the antiplatelet drug. The combined effect exceeded that of both heparin and fondaparinux. In contrast to other specific factor Xa inhibitors, the effect of PifXa, being a protein, can be fully reversed by addition of a specific polyclonal antibody. That this is in fact possible was demonstrated in vitro. The specificity of the inhibitor combined with the possibility to reverse the effect makes PifXa an interesting candidate drug during cardio pulmonary bypass where the general inconvenient requirement for IV administration of protein drugs is tolerable, a large dose of anticoagulants in a limited period of time is necessary, and thus administration of an antidote to reverse the effect at the end of the procedure is desired. Disclosures: Andersen: Aalborg University: Patents & Royalties. Nielsen:Aalborg University: Patents & Royalties.

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Andexanet Alfa, the Possible Alternative to Protamine for Reversal of Unfractionated Heparin

Annals of Pharmacotherapy ◽

10.1177/1060028020943160 ◽

2020 ◽

Vol 55 (2) ◽

pp. 261-264

Author(s):

John N. Maneno ◽

Genevieve Lynn Ness

Keyword(s):

Unfractionated Heparin ◽

Vitamin K Antagonists ◽

Oral Anticoagulants ◽

Direct Oral Anticoagulants ◽

Hospital Setting ◽

Factor Xa ◽

Thrombin Inhibitors ◽

Thrombin Time ◽

Reversal Agent

The recent shortage of protamine prompted an investigation of alternatives for reversal of unfractionated heparin. Heparin is an anticoagulant utilized in the hospital setting. Available options for anticoagulation include direct oral anticoagulants, vitamin K antagonists, thrombin inhibitors, low-molecular-weight heparins, and heparin. Protamine is the approved reversal agent for heparin with few alternatives under investigation. Although andexanet was designed as an antidote for apixaban and rivaroxaban, in vitro studies show that in a dose-dependent technique, andexanet had near full reversal of heparin, reversed anti–factor Xa activity, and neutralized anticoagulant effects of activated partial thromboplastin time and thrombin time induced by heparin.

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Relative Interfering Effects of In Vivo Direct Oral Anticoagulants on Routine Coagulation Tests

Blood ◽

10.1182/blood-2021-152088 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4238-4238

Author(s):

Jinju Kim ◽

Yejin Song ◽

Hyun-Jeong Kim ◽

Mi-Sook Yang ◽

Jaewoo Song

Keyword(s):

Conflicts Of Interest ◽

Oral Anticoagulants ◽

Direct Oral Anticoagulants ◽

Regression Line ◽

Anticoagulation Therapy ◽

Blood Samples ◽

Coagulation Tests ◽

Anticoagulated Patients

Abstract Background: The interfering effects of DOACs on the screening coagulation tests, such as prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen assay, have been shown mainly by in vitro spiking experiments. However, the effects of DOACs on coagulation tests in real-world samples from anticoagulated patients are unknown because of the difficulty in selectively eliminating DOAC from blood samples already containing DOACs. Method: Citrated blood samples were drawn from patients on anticoagulation therapy (rivaroxaban and edoxaban). In addition, blood samples from patients not on anticoagulation were collected. PT INR and APTT were measured from those samples by coagulometers from two manufacturers (Roche t711, Swiss and ACL-TOP, USA). We also measured DOAC levels from the same samples by anti-FXa activity (Hyphen Biomed, France). Then, we compared the test results in relation to the DOAC levels. Results: The PT INR, APTT, and fibrinogen assay results from non-anticoagulated patients measured by the two coagulometers were comparable (PT INR: y = -3.353 + 1.029 x; APTT: y = -6.276 + 1.101x; fibrinogen: y = -3.353 + 1.029 x; Passing Bablok). We included blood samples from 61 patients on rivaroxaban and 75 patients on edoxaban. From the rivaroxaban samples we observed the regression line change for PT INR (y = 0.6303 + 0.3712x) and for APTT (y = -10.71+1.358x). The comparability of fibrinogen assay was not affected significantly (y = -17.39+1.01x). From the edoxaban samples we also observed the similar change of the regression line (PT INR: y = 0.4728 + 0.5661x; APTT: y = -133.07+2.014x). Fibrinogen levels were comparable (y = -28.95+1.082x). Conclusion: The susceptibility of screening coagulation tests to the interfering effects of in vivo DOAC is dependent on the reagents and coagulometers. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Stroke-prevenció időskorban: az edoxabán hatékonysága és biztonságossága idős betegekben az ENGAGE AF vizsgálat eredményei alapján

Orvosi Hetilap ◽

10.1556/650.2018.31100 ◽

2018 ◽

Vol 159 (20) ◽

pp. 798-802

Author(s):

László Márk

Keyword(s):

Atrial Fibrillation ◽

Vitamin K Antagonists ◽

Oral Anticoagulants ◽

Direct Oral Anticoagulants ◽

Factor Xa ◽

The Elderly ◽

Thromboembolic Events ◽

Systemic Embolization ◽

Number Of Patients ◽

Net Clinical Benefit

Abstract: Atrial fibrillation is the most common clinically relevant arrhythmia frequently causing systemic thromboembolic events. Traditionally vitamin K antagonists had been used for decades to prevent these events. The emerging of the new direct anticoagulants has revolutionized this treatment and a gradual growth and extensive spread of usage is expected. The latest one approved in Hungary, edoxaban, is a factor Xa inhibitor. Once-daily administration and favourable safety profile are major benefits of this drug. In a large clinical study with a high number of patients it proved to be at least as effective as warfarin in the prevention of stroke and systemic embolization while causing significantly less major bleedings. As the incidence of atrial fibrillation increases with age, the observation that, compared with the other direct oral anticoagulants, the administration of edoxaban in the elderly has a favourable net clinical benefit (in the rate of prevented thromboembolic events and the number of caused bleedings) may have a great importance. Orv Hetil. 2018; 159(20): 798–802.

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Ancylostoma caninum Anticoagulant Peptide Blocks Metastasis In Vivo and Inhibits Factor Xa Binding to Melanoma Cells In Vitro

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615117 ◽

1998 ◽

Vol 79 (05) ◽

pp. 1041-1047 ◽

Cited By ~ 20

Author(s):

Kathleen M. Donnelly ◽

Michael E. Bromberg ◽

Aaron Milstone ◽

Jennifer Madison McNiff ◽

Gordon Terwilliger ◽

...

Keyword(s):

Active Site ◽

Thrombin Generation ◽

Factor Xa ◽

Coagulation Factor ◽

Melanoma Cells ◽

Factor V ◽

Ancylostoma Caninum ◽

Metastatic Activity

SummaryWe evaluated the in vivo anti-metastatic activity of recombinant Ancylostoma caninum Anticoagulant Peptide (rAcAP), a potent (Ki = 265 pM) and specific active site inhibitor of human coagulation factor Xa originally isolated from bloodfeeding hookworms. Subcutaneous injection of SCID mice with rAcAP (0.01-0.2 mg/mouse) prior to tail vein injection of LOX human melanoma cells resulted in a dose dependent reduction in pulmonary metastases. In order to elucidate potential mechanisms of rAcAP’s anti-metastatic activity, experiments were carried out to identify specific interactions between factor Xa and LOX. Binding of biotinylated factor Xa to LOX monolayers was both specific and saturable (Kd = 15 nM). Competition experiments using antibodies to previously identified factor Xa binding proteins, including factor V/Va, effector cell protease receptor-1, and tissue factor pathway inhibitor failed to implicate any of these molecules as significant binding sites for Factor Xa. Functional prothrombinase activity was also supported by LOX, with a half maximal rate of thrombin generation detected at a factor Xa concentration of 2.4 nM. Additional competition experiments using an excess of either rAcAP or active site blocked factor Xa (EGR-Xa) revealed that most of the total factor Xa binding to LOX is mediated via interaction with the enzyme’s active site, predicting that the vast majority of cell-associated factor Xa does not participate directly in thrombin generation. In addition to establishing two distinct mechanisms of factor Xa binding to melanoma, these data raise the possibility that rAcAP’s antimetastatic effect in vivo might involve novel non-coagulant pathways, perhaps via inhibition of active-site mediated interactions between factor Xa and tumor cells.

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