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Heparinases and their heparin-related applications

Heparin, a highly sulfated glycosaminoglycan with the highest negative charge density of any known biological molecule, is located in the extracellular matrix and plays important physiological roles in anticoagulation and angiogenesis. The acidic polysaccharide of heparin consists of a heterogeneous disaccharide repeating unit of hexosamine and uronic acid (L-iduronic or D-glucuronic acid) connected through 1-4 linkages and modified with various functional groups. Conserved among a number of widely different species, heparin is usually produced by basophils and mast cells and released only into the vasculature at sites of tissue injury. In addition to its well-known usage as an injectable anticoagulant, heparin can also be used to form an inner anticoagulant surface on various experimental and medical devices such as test tubes and renal dialysis machines. Whether the right dose of heparin is being used to a patient can be monitored by the activated partial thromboplastin time (aPTT), a blood test measuring the time that it takes the blood plasma to clot.

The three heparinase enzymes (heparinase I, heparinase II, and heparinase III) produced by Flavobacterium heparinum specifically recognize and cleave at different sequences of heparin. Heparinase I enzyme (EC cleaves heparin at the linkages between hexosamines and O-sulfated iduronic acids, yielding oligoaccharides (mainly disaccharides). In addition, Heparinase I cleaves heparin at the antithrombin III binding pentasaccharide domain. Heparinase II enzyme cleaves at the 1-4 linkages between hexosamines and uronic acid residues (both glucuronic and iduronic) of heparin, yielding oligoaccharides (mainly disaccharides). Heparinase III enzyme (EC does not cleaves heparin.

Heparin can bind to RNA polymerase and interfere with DNA transcription by blocking RNA polymerase from binding to the promoter region. Heparin can also bind to DNA and interfere with PCR in diagnostic procedures if the sample is from the patient treated with heparin. Heparinase I and II enzymes can be used to treat the samples and eliminate the interference caused by heparin.

The activated clotting time (ACT) is the most commonly used method for monitoring heparin during cardiopulmonary bypass surgery. Heparinase I can be used to treat the specimen in ACT to monitor non-heparin-related alterations in coagulation function.

Heparin contamination in specimens is a common cause of the unexpected PTT prolongation. By treating the plasma with heparinase I, one can determine if the PTT prolongation is due to heparin.

Thromboelastography (TEG) during cardiopulmonary bypass (CPB) to diagnose excessive postoperative hemorrhage can be interfered by even small amount of heparin, producing a nondiagnostic tracing. Heparinase can be used to treat TEG blood samples and remove the interference caused by heparin.

In addition to neutralization of heparin, the single and mixture of heparinases can be used for detection and determination of plasma heparin, quality control in heparin manufacturing, and preparation of low molecular weight heparins, disaccharide or oligoaccharide from unfractionated heparin.