Neutralase ™ Reverses the Anti-coagulant but Not the Anti-thrombotic Activity of Heparin in a Rabbit Model of Venous Thrombosis

Abstract

Neutralase™ (heparinase I; E.C. 4.2.2.7) is a heparin-degrading enzyme undergoing clinical evaluation as an alternative to protamine for reversing the anticoagulant effects of heparin in coronary bypass surgery. The objective of this study was to assess the relative effects of Neutralase and protamine on reversal of heparin-dependent elevations in coagulation parameters and inhibition of clot formation in a rabbit vena caval stasis model. Rabbits were treated with saline or heparin (300 U/kg) for 10 minutes, followed by saline, protamine (2.6 mg/kg), or Neutralase (10 or 30 μg/kg, representing 1.23 IU/kg and 3.69 IU/kg, respectively). Twenty minutes later, venous stasis was induced, and vena caval clots were excised, weighed, and characterized. Coagulation parameters [activated partial thromboplastin time (aPTT) and thrombin clotting time (TCT)] and antiFactor IIa and Xa levels were measured throughout the protocol. Both protamine and Neutralase reversed heparin-mediated increases in aPTT (>300 seconds to 26–35 seconds) and TCT (>300 seconds to 29–56 seconds) to values that were not different from saline-treated, nonheparinized animals. Thrombus weight in the nonheparinized saline group was 62±7 mg; heparin-treated animals had no detectable clots. Protamine reversal of heparin was associated with clot formation (89±20 mg) while Neutralase reversal was not (no clots). Heparin-induced increases in antiFactor IIa activity were reversed similarly by protamine and Neutralase (from 4.3–8.8 U/ml to 0.2–0.3 U/ml) while antiFactor Xa activity was differentially reversed (from 3.9–5.9 U/ml to 0.7–1.3 U/ml Neutralase; 5.5 U/ml to 0.02 U/ml protamine). These results are consistent with a hypothesis that Neutralase cleaves heparin into fragments, which are devoid of antiFactor IIa activity that retain modest antiFactor Xa activity, resulting in reversal of anticoagulant, but not antithrombotic, heparin activity. This property of Neutralase may be beneficial in reducing post-surgical thrombotic events after reversal of heparin.