Erwin Coyne

In Vitro Studies of the Interaction of Heparin, Low Molecular Weight Heparin and Heparinoids with Platelets

Heparin, low molecular weight heparins, and heparinoids were studied for their ability to inhibit the aggregation of platelets by various agonists and for their ability to adhere to collagen. Heparin was a very effective inhibitor of aggregation with collagen and with ristocetin as it was with adhesion to collagen. The heparinoids showed little effect on aggregation or adhesion. Heparan sulfate and pentosan polysulfate did show slight inhibitory activity against collagen aggregation and adhesion and both interacted with the antibody induced by heparin therapy. It is of interest that dermatan sulfate and the pentasaccharide were almost inert in these experiments, and are unlikely to induce bleeding by inhibition of platelet function. It is highly probable that interference with the interaction of von Willebrand factor with platelets and collagen is a major mechanism for bleeding in the heparinized patient.

Isolation and Characterization of Heparin From Tuna Skins

This study characterized heparin isolated from tuna skins. Glycosaminoglycans were isolated from tuna skin after digestion using anion exchange resin. Heparin was eluted from the resin by sodium chloride gradient and was further fractionated by acetone fractionation. Anticoagulant activity was determined using the activated partial thromboplastin time and Heptest assays. Potency was determined using amidolytic antifactor IIa and antifactor Xa assays. The presence of heparin in the extracted tuna skin glycosaminoglycans was confirmed using 13C-nuclear magnetic resonance. The activated partial thromboplastin time and Heptest clotting times were doubled at concentrations of about 4 and 1 µg/mL, respectively. The clotting time prolongation and antiprotease activity induced by tuna heparin was readily neutralized by 25 µg/mL protamine sulfate. These results demonstrate that biologically active heparin with properties similar to clinical grade heparin can be derived from tuna skin, a raw material with otherwise relatively little economic value.