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...
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