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.
Andersen:Aalborg University: Patents & Royalties. Nielsen:Aalborg University: Patents & Royalties.
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