All thrombolytic agents in current clinical usage are plasminogen activators. Although effective, plasminogen activators uniformly increase the risk of bleeding complications, especially intracranial hemorrhage, and no laboratory test is applicable to avoid such bleeding. We report results of a randomized, blinded, dose-ranging comparison of tissue-type plasminogen activator (TPA) with a directacting thrombolytic agent, plasmin, in an animal model of fibrinolytic hemorrhage. This study focuses on the role of plasma coagulation factors in hemostatic competence. Plasmin at 4-fold, 6-fold, and 8-fold the thrombolytic dose (1 mg/kg) induced a dose-dependent effect on coagulation, depleting antiplasmin activity completely, then degrading fibrinogen and factor VIII. However, even with complete consumption of antiplasmin and decreases in fibrinogen and factor VIII to 20% of initial activity, excessive bleeding did not occur. Bleeding occurred only at 8-fold the thrombolytic dose, on complete depletion of fibrinogen and factor VIII, manifest as prolonged primary bleeding, but with minimal effect on stable hemostatic sites. Although TPA had minimal effect on coagulation, hemostasis was disrupted in a dose-dependent manner, even at 25% of the thrombolytic dose (1 mg/kg), manifest as rebleeding from hemostatically stable ear puncture sites. Plasmin degrades plasma fibrinogen and factor VIII in a dose-dependent manner, but it does not disrupt hemostasis until clotting factors are completely depleted, at an 8-fold higher dose than is needed for thrombolysis. Plasmin has a 6-fold margin of safety, in contrast with TPA, which causes hemorrhage at thrombolytic dosages. (Blood.

Summary. While protamine sulfate reverses the anticoagulant effect of standard heparin, there currently is no effective antidote for low molecular weight heparin (LMWH)-induced bleeding. Recently, recombinant activated factor VII (rFVIIa) was approved by the FDA for use in hemophilia patients with factor (F)VIII or FIX inhibitors. However, this new pro-hemostatic agent has potential utility in other clinical scenarios. In this study, we utilized a well-characterized rabbit ear puncture model to test the ef®cacy of rFVIIa to reverse LMWH-induced prolonged bleeding. Animals were ®rst treated with bolus intravenous LMWH (1800 anti-FXa U kg À1 ) which increased the primary bleeding time approximately fourfold and raised the plasma anti-FXa activity immediately and continuously throughout the 90-min experiment. In a randomized and blinded fashion, animals then received either rFVIIa (400 mg kg À1 ) or placebo by bolus intravenous injection, following which the ear puncture bleeding times were measured, along with blood levels of heparin (anti-FXa activity) and FVII. FVII activity increased 5.3-fold over baseline in treated animals, decreasing by only 24% over the full observation period. The rFVIIatreated animals showed a slight decrease in bleeding time immediately after injection, but there was no statistically signi®cant difference in bleeding after rFVIIa or placebo administration. In this study using a rabbit ear bleeding model, rFVIIa was not an effective antidote to LMWH-induced bleeding. However, the bolus injection of LMWH produced a very high blood anti-FXa level, which may have precluded rFVIIa effectiveness.

Summary. Plasmin is a direct thrombolytic which has been shown to have a strikingly favorable bene®t to risk pro®le in comparison with plasminogen activators, notably tissue plasminogen activator (t-PA). As heparin is known to increase the risk of hemorrhage when co-administered with a plasminogen activator, we asked whether adjunct antithrombotic agents such as aspirin and heparin would affect the safety of plasmin. Three groups of rabbits were administered plasmin at a dose (4 mg kg À1 ) designed to induce signi®cant decreases in antiplasmin, ®brinogen and factor (F)VIII, to about 25, 40 and 40%, respectively, of baseline values, but not cause prolongation of the ear puncture bleeding time. In a blinded and randomized trial, the results show that an intravenous aspirin bolus plus heparin administered as a bolus followed by a maintenance continuous infusion did not signi®cantly prolong the bleeding time during plasmin infusion. These data indicate that in the rabbit, concomitant use of aspirin plus heparin does not affect the safety of a therapeutic dose of plasmin.Keywords: aspirin, heparin, plasmin, safety.Locally delivered thrombolytic therapy is effective for hastening the lysis of deep vein thromboses [1,2] and thrombosed catheters [3] and for improving the clinical outcome of peripheral arterial graft occlusion [4] or ischemic stroke [5]. All agents that are currently approved and used widely for clinical thrombolysis are plasminogen activators (PA) [6], but their use is uniformly associated with an increased risk of hemorrhagic complications, even when delivered regionally to a thrombus [7,8]. We recently reported studies of the direct-acting proteolytic enzyme plasmin in animal models of thrombolysis and bleeding, and show a dramatic superiority in the safety of plasmin over TPA [9,10], without sacri®ce of thrombolytic ef®cacy [11].The combined use of heparin or hirudin anticoagulant therapy with PA increases the risk of bleeding [12±15]. Thus, the rate of intracranial hemorrhage in patients with peripheral arterial occlusion treated with regional recombinant urokinase was 0.5% (1/210), compared with a rate of 4.8% (3/62) when heparin was co-administered with the PA [15]. Since our prior studies of plasmin were performed without concomitant anticoagulant administration, the experimental conditions may not have re¯ected actual clinical conditions in which heparin, aspirin and/or other antithrombotic therapies are utilized with PA. Thus, the study reported here tests whether the hemostatic safety of plasmin [9,10] is disrupted by the concomitant administration of aspirin or by aspirin plus heparin. MethodsHuman plasmin (BAY 57±9602, Lot TPM03 bulk) was puri®ed by SK activation of human blood plasma-derived plasminogen. The starting material for plasminogen puri®cation was the sodium caprylate precipitate from an intravenous immunoglobulin process [16]. Plasminogen was puri®ed by adsorption on lysine-Sepharose FF (Amersham Pharmacia, Uppsala, Sweden) and eluted with epsilon-aminocaproic acid. The resulti...

SummaryThe direct fibrinolytic enzyme, plasmin, was compared with tissue plasminogen activator (TPA) in rabbit models of local thrombolysis and fibrinolytic hemorrhage. Plasmin was produced by solid-phase urokinase activation of plasminogen and purified on benzamidine Sepharose. Applied as an intra-arterial infusion into the thrombosed abdominal aorta under conditions of unimpeded blood flow, plasmin (4 mg/kg) and TPA (2 mg/kg) achieved equivalent clot dissolution and flow restoration. Using the model of restricted blood flow into the thrombosed aorta, which limits local plasminogen supply, plasmin was superior to TPA in clot lysis and vascular reperfusion. Using similar dosages of plasmin (2 or 4 mg/kg) and TPA (1 or 2 mg/kg) in the earpuncture rebleed model. TPA induced rebleeding in a dose-dependent manner from prior puncture sites in 9 of 10 animals, while none of the 10 animals exposed to plasmin rebled from these sites.These results suggest that plasmin is an effective, unique thrombolytic agent, distinguished from the plasminogen activators in current usage by its striking safety profile.

Plasmin is a direct-acting thrombolytic agent with an impressive safety margin in experimental models, as it is tolerated without bleeding at a 6-fold greater dose than needed for thrombolysis. At higher plasmin dosages, above a reproducible dose threshold, alpha-2-antiplasmin (AP) is consumed, accompanied by complete depletion of fibrinogen and factor VIII. As a result of clotting factor exhaustion, the primary bleeding time is prolonged. We assessed whether supplemental AP could prevent bleeding induced by a purposefully toxic dosage of plasmin, and whether AP could arrest such bleeding once it occurs. Using the rabbit ear puncture model, plasmin (8 mg/kg) alone was compared with plasmin plus supplemental AP (5 mg/kg), injected either prior to or after plasmin infusion. We dosed plasmin 8-fold higher than that required for thrombolytic efficacy, based upon our previous observations of its capacity to prolong primary bleeding in greater than 80% of treated animals. Parameters for comparison between treatment groups were: primary bleeding time at timepoints prior to, during, and after experimental infusion, incidence of rebleeding at previously stable hemostatic sites, and quantification of AP, fibrinogen, and factor VIII. Pre-treatment AP doubled the plasma concentration, preserved circulating fibrinogen and factor VIII, and prevented bleeding. Post-plasmin AP corrected the plasmin-associated depletion of AP, but fibrinogen and factor VIII concentrations remained very low, and prolonged bleeding was uncorrected. To test whether AP could be supplemented with coagulation factors in order to arrest bleeding following high dose plasmin, human cryoprecipitate was co-administered. While the fibrinogen and factor VIII levels increased to 200% and 50% of baseline values, respectively, prolonged bleeding persisted. Unexpectedly, administration of human cryoprecipitate led to excess bleeding in association with acute thrombocytopenia and gross hemolysis, presumably mediated by xenographic antibody present in the product. Thus, we find that supplemental AP administered prior to a toxic dose of plasmin prevents coagulation factor consumption and preserves normal hemostasis. Once coagulation factor depletion occurs, AP alone is insufficient to correct the hemorrhagic state that ensues. Clinically, plasmin may be useful as a regional thombolytic agent, and AP supplementation may permit higher dosing as well as protect against accidental overdose. Further studies are needed to determine whether combined AP and coagulation factor repletion may correct the prolonged bleeding induced by massive plasmin infusion.