Antithrombin III Independent Anticoagulants Benedict R. Lucchesi, M.D., Ph.D. Department of Pharmacology University of Michigan Medical School

Antithrombin III Independent Anticoagulants Hirudin – From the medicinal leech – Synthesized by recombinant DNA techniques – Direct inhibitor of thrombin Lepirudin (Refludin™) – desulfohirudin - a recombinant hirudin* derived from yeast cells. Hirugen (bivalirudin, Angiomax™) – Synthetic dodecapeptide derived from hirudin. Argatroban – Arginine based compound – Weak competitive inhibitor of thrombin. These compounds are used in those patients who have developed thrombocytopenia during treatment with heparin.

Lepirudin (Refludin™)

Lepirudin [rDNA] (Refludan™) highly specific direct inhibitor of thrombin - [Leu1-Thr2]-63- desulfohirudin - a recombinant hirudin* derived from yeast cells. polypeptide - 65 amino acids MW= identical to natural hirudin except for the substitution of leucine for isoleucine at the N-terminal end of the molecule and the absence of a sulfate group on tyrosine 63. action independent of ATIII and not inhibited by platelet factor 4 one molecule of lepirudin binds one molecule of thrombin - all thrombin-dependent coagulation pathways are affected approved for clinical use in the treatment of heparin-induced thrombocytopenia type II. * Hirudin derived from the leech Hirudo medicinalis

Bivalirudin (Hirulog, Angiomax ™ )

Synthetic 20-amino acid peptide analog of naturally occurring hirudin Bivalirudin is a specific and reversible direct thrombin inhibitor that binds to the catalytic site and the anion- binding exosite of circulating and clot-bound thrombin. Inhibition of thrombin prevents activation of factors V, VIII, and XIII; conversion of fibrinogen to fibrin; platelet activation and aggregation).

Bivalirudin (Hirulog, Angiomax™) The effects of bivalirudin are reversed as thrombin slowly cleaves the bilvalirudin-Arg3-Pro4 bond, resulting in recovery of thrombin active site function. The onset of anticoagulant effect is immediate after direct IV injection of bivalirudin. Bivalirudin therapy prolongs several coagulation assays: – activated clotting time (ACT), – activated partial thromboplastin time (aPTT), – thrombin time (TT), – prothrombin time (PT) – Coagulation times return to the normal range approximately 1—2 hours after discontinuance of the drug.

(Gly) 4 D-Phe-Pro-Arg-Pro (active-site-binding moiety) C-terminal dodecapeptide (Exosite 1-binding portion) (Gly) 4 D-Phe-Pro-Arg-Pro (active-site-binding moiety) 2 1 Thrombin Bivalirudin binds bivalently to thrombin’s active site and exosite 1. Bivalirudin is a synthetic molecule, designed as a bivalent direct thrombin inhibitor. Molecule consists of an N-terminal Gly-Pro-Arg-Pro sequence that binds to the active site of thrombin, linked via a four glycine residue spacer to a dodecapeptide analog of the C- terminal of hirudin (an anticoagulant protein isolated from the medicinal leech). Binds specifically to thrombin and directly to both the active catalytic site and the anion- binding exosite 1 with high affinity. Bivalirudin inhibits both circulating and fibrin-bound thrombin.

Bivalirudin can displace fibrin bound to thrombin. 2 1 Thrombin A 2 1 Bivalirudin is cleaved by thrombin at the active site. B 2 1 Thrombin The bond between thrombin and the remaining portion of bivalirudin is weaker. Thrombin can resume hemostatic functions. C

Fibrin 2 1 Thrombin Bivalirudin inhibits both fibrin-bound and circulating thrombin.

(A) Structure of bivalirudin and (B) bivalirudin / hirudin complexes Bivalirudin consists of an active site-directed moiety linked by a poly-glycine spacer to a dodeca- peptide analogue of the carboxy terminal of hirudin. Once bivalirudin complexes thrombin, it is converted from a noncompetitive inhibitor that interacts with both the active site and exosite 1 on thrombin to a competitive inhibitor that only binds to exosite 1.

Potential for enhanced protein C activation by bivalirudin. Fluid- phase thrombin is complexed and inhibited by bivalirudin. Upon arrival to the microcirculation where thrombomodulin is concen- trated, the amino-terminal domain of bivalirudin is released, leaving only the carboxy-terminal domain bound to exosite 1 on thrombin. The affinity of thrombomodulin for thrombin is higher than that of the carboxy-terminal dodecapeptide of bivalirudin, thus thrombin binds to thrombomodulin, where it activates protein C.

110% Bivalirudin concentration (ng/mL) Percent aggregation response to activators ADP Collagen Thrombin Therapeutic range Weitz J, Maraganore J. TCT 2001 Bivalirudin blocks thrombin-mediated platelet aggregation.

Argatroban

Argatroban Argatroban, a synthetic piperidine carboxylic acid derivative of l-arginine, is an anticoagulant. Commercially available argatroban is a racemic mixture of the R- and S-diastereoisomers in a ratio of approximately 65 to 35, with the S-isomer having about twice the thrombin-inhibitory potency of the R-isomer. Argatroban, a highly selective, reversible, small-molecule direct thrombin inhibitor that binds rapidly to the catalytic site/a polar region of both circulating (free) and clot-bound thrombin.

Argatroban Inhibition of thrombin prevents various steps in the coagulation process (e.g., activation of factors V, VIII, and XIII and of protein C; conversion of fibrinogen to fibrin; platelet activation and aggregation). At infusion rates up to 40 mcg/kg per minute, argatroban produces dose-dependent increases inactivated partial thromboplastin time (aPTT) and several other coagulation assays (activated clotting time [ACT], prothrombin time [PT], and thrombin time [TT]). Metabolized principally by the liver via hydroxylation and aromatization. Does not induce antibody formation to itself nor does it interact with heparin-induced antibodies.

Administered by continuous IV infusion. Before administering argatroban, all parenteral anticoagulants must be discontinued and a baseline activated partial thromboplastin time (aPTT) obtained. Supplied, as a concentrated drug (100 mg/ml), which must be diluted 100-fold prior to infusion. Should not be mixed with other drugs prior to dilution in a suitable intravenous fluid. Argatroban