Direct fibrinolytic agents: biochemical attributes, preclinical foundation and clinical potential

Abstract

Summary. Direct fibrinolytics are proteolytic enzymes that degrade fibrin without requiring an intermediate step of plasminogen activation. This review summarizes the current information available for five such agents, namely, plasmin (the prototypical form), three derivatives of plasmin (mini‐plasmin, micro‐plasmin, and delta‐plasmin), and alfimeprase, a recombinant variant of a snake venom α‐fibrinogenase, fibrolase. Biochemical attributes of molecular size, fibrin binding and inhibitor neutralization are compared. Preclinical investigations that assess the potential for thrombolytic efficacy in vitro and in animal models of vascular occlusion and for hemostatic safety in animal models of bleeding are detailed. Clinical potential has been assessed in patients with peripheral arterial and graft occlusion, acute ischemic stroke, and access catheter and hemodialysis shunt occlusions. The direct fibrinolytic agents have impressive biochemical and preclinical foundations for ultimate clinical application. However, clinical trial results for micro‐plasmin and alfimeprase have not measured up to their anticipated benefit. Plasmin has thus far shown encouraging hemostatic safety, but efficacy data await completion of clinical trials. Whether direct fibrinolytics will provide clinical superiority in major thrombotic disorders over currently utilized indirect fibrinolytics such as tissue plasminogen activator remains to be determined.

The evolution of recombinant thrombolytics: Current status and future directions

“…Thus, direct acting thrombolytics have potential for improved thrombolysis and enhanced hemostatic safety. 16,139 In a porcine model, plasmin was found to possess superior characteristics than t-PA in dissolving arteriovenous graft thrombi. 140 Several studies conducted in animal models indicate the effectiveness and safety of plasmin administration for thrombolytic therapy.…”

“…Kringle 1 is also involved in plasmin binding to the a2-antiplasmin inhibitor, one of the fastest protein-protein interactions (rate of the inhibition reaction is »10 7 M ¡1 s ¡1 ). 16 Consequently, plasmin bound to fibrin impedes inactivation by this inhibitor. The C-terminal light chain carries the serine protease domain with His 603 , Asp 646 and Ser 741 as the catalytic triad.…”

“…On the other hand, catheter-based (local) administration of plasminogen activators helps in achieving thrombolysis faster, but bleeding complications may still result since the local concentration of the plasminogen activator may exceed the PAI-1 inhibitory capacity. 16 In such a scenario, the attributes of direct-acting thrombolytics can be exploited. Direct thrombolytic agents are proteolytic enzymes that do not require the intermediate step of plasminogen activation and degrade fibrin directly.…”

“…The C-terminal light chain carries the serine protease domain with His 603 , Asp 646 and Ser 741 as the catalytic triad. 16 The heavy chain also accommodates a 77 residues N-terminal peptide region, which is cleaved during in vitro activation of plasminogen by autolysis. The Glu 1 -Pm and Lys 78 -Pm can catalyze the hydrolysis of the N-terminus 77 amino acids of Glu 1 -Pg or Glu 1 -Pm, converting Glu 1 -Pg to Lys 78 -Pg and Glu 1 -Pm to Lys 78 -Pm.…”

“…The molecule is composed of the catalytic protease domain and the kringle 5 domain of plasminogen. 16 Mini-plasminogen has been expressed in E. coli as inclusion bodies (500-600 mg/L) using the T7 expression system. The inclusion bodies were refolded, purified and activated to mini-plasmin using urokinase.…”