Inhibition of Heparinase I by Defibrotide with Potential Clinical Implications.

Defibrotide represents a poly-deoxyribonucleotide derived antischemic and antithrombotic agent. Currently this agent is used for the management of transplantation associated with vascular complications. Defibrotide is a polyanionic electrolyte capable of releasing endogenous antithrombotic mediators such as (tissue factor pathway inhibitor (TFPI) and heparans. Co-administration of defibrotide with heparin has been shown to produce an augmentation of the effect of heparin and an increase in the biologic half-life. This may be due to the drug interactions or inhibition of endogenous heparin digesting enzyme by defibrotide. To test the hypothesis that defibrotide may inhibit heparin digesting enzymes such as heparinases (I–III), the effects of defibrotide were investigated on the digestion of unfractionated heparin. Unfractionated heparin was subjected to bacterial heparinase I (flavobacterion heparinicum) in an isolated biochemical systems where unfractionated heparin was used as a substrate at a fixed concentration of 750 μg/ml. Graded amounts of defibrotide in the range of 6.25–250 μg/ml were supplemented to this mixture. The degree of heparinase digestion of unfractionated heparin was determined by using high performance liquid chromatographic methods and computation of oligosaccharide profiles. Heparinase I was found to digest heparin (MW=15.2kDa) converting it to low molecular mass heparins (MW=4.1kDa). At concentrations of >125 μg/ml defibrotide produced an almost complete inhibition of heparinase I digestion. This inhibition of heparinase digestion was dependent on defibrotide concentration and the IC₅₀ was found to be 12.5 μg/ml. These results suggest that polyelectrolytes such as defibrotide are capable of inhibiting heparin/heparan digestion enzymes. The observed potentiation of anticoagulant effect of heparin in patients treated simultaneously with defibrotide may partially be due to the inhibition of endogenous heparin digesting enzymes. Subsequent studies in primates have revealed that the anticoagulant effects of both unfractionated heparin and low-molecular mass heparins are potentiated by defibrotide as determined by AUC measurements for the circulating level of these agents. Thus, these interactions should be taken into account to optimize anticoagulant management where these drugs are used in a combination regimen.