The Design of Competitive, Small-molecule Inhibitors of Coagulation Factor Xa

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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Isolation and Structural Charaderization of a Potent Inhibitor of Coagulation Factor Xa from the Leech Haementeria ghilianii

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646610 ◽

1989 ◽

Vol 61 (03) ◽

pp. 437-441 ◽

Cited By ~ 28

Author(s):

Cindra Condra ◽

Elka Nutt ◽

Christopher J Petroski ◽

Ellen Simpson ◽

P A Friedman ◽

...

Keyword(s):

Molecular Weight ◽

Salivary Gland ◽

Amino Acid ◽

Amino Acid Sequence ◽

Western Blot ◽

Potent Inhibitor ◽

Factor Xa ◽

Coagulation Factor ◽

Sds Page ◽

Bovine Factor

SummaryThe present work reports the discovery and charactenzation of an anticoagulant protein in the salivary gland of the giant bloodsucking leech, H. ghilianii, which is a specific and potent inhibitor of coagulation factor Xa. The inhibitor, purified to homogeneity, displayed subnanomolar inhibition of bovine factor Xa and had a molecular weight of approximately 15,000 as deduced by denaturing SDS-PAGE. The amino acid sequence of the first 43 residues of the H. ghilianii derived inhibitor displayed a striking homology to antistasin, the recently described subnanomolar inhibitor of factor Xa isolated from the Mexican leech, H. officinalis. Antisera prepared to antistasin cross-reacted with the H. ghilianii protein in Western Blot analysis. These data indicate that the giant Amazonian leech, H. ghilianii, and the smaller Mexican leech, H. officinalrs, have similar proteins which disrupt the normal hemostatic clotting mechanisms in their mammalian host’s blood.

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Activation of Human Coagulation Factor VIII by Activated Factor X, the Common Product of the Intrinsic and the Extrinsic Pathway of Blood Coagulation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657137 ◽

1982 ◽

Vol 47 (02) ◽

pp. 096-100 ◽

Cited By ~ 22

Author(s):

K Mertens ◽

R M Bertina

Keyword(s):

Factor Viii ◽

Human Factor ◽

Intrinsic Factor ◽

Factor Xa ◽

Coagulation Factor ◽

Factor X ◽

Extrinsic Factor ◽

Extrinsic Pathway ◽

Factor Ixa ◽

The Common

SummaryThe intrinsic activation of human factor X has been studied in a system consisting of purified factors and in plasma. In both these systems factor Xa stimulated the activation of factor X by factor IXa plus factor VIII This is due to the activation of factor VIII by factor Xa. When this factor Xa is formed via the extrinsic pathway, the extrinsic factor X activator functions as a stimulator of the intrinsic factor X activator.

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