A number of sulfated hyaluronic acid derivatives (HyalS(2.5), HyalS(3), and HyalS(4)) were prepared by sulfation of the -OH groups present on hyaluronic acid and were generically termed HyalS(x). The anticoagulant properties of this series of compounds has previously been shown to be good in terms of their whole blood clotting inhibition and factor Xa and thrombin inactivation. The purpose of the present study was to investigate whether the use of these compounds would be beneficial to patients who would normally be given heparin, and to perform some preliminary investigations into their effects on platelets. The three compounds were thus studied by investigating their ability to inhibit von Willebrand factor-dependent platelet agglutination in comparison with unfractionated heparin. Agglutination was determined turbidometrically after the addition of ristocetin to stirred formaldehyde-fixed platelets and was demonstrated to be dependent on the presence of sulfate groups on the polysaccharide chain and correlated with the degree of HyalS(x) sulfation. Interactions possibly important in low shear environments were investigated by measuring the pharmacological action of the HyalS(x) on spontaneous platelet activation and aggregate formation by flow cytometry. The data indicate that platelet activation is not correlated with the number of sulfate or hydroxyl groups on HyalS(x), suggesting that activation occurs not via electrostatic interactions or H bonding, but via some other mechanism. A differentiation between low and high glycosaminoglycan sulfation densities is observed with respect to platelet aggregation, which is correlated with the number of sulfated groups per disaccharide unit. The ability of HyalS(x) to inhibit platelet aggregation induced by ADP and thrombin was measured by aggregometry. HyalS(4) resisted thrombin stimulation to a similar extent as heparin. All Hyal derivatives, however, were better at inhibiting ADP-induced aggregation than was heparin. We conclude, therefore, that clinical use of HyalS(x) in place of heparin may be beneficial because ristocetin-dependent agglutination, and therefore resistance to platelet aggregation in high shear environments, in addition to resistance to stimulation by ADP, has been shown to be superior to heparin. Spontaneous platelet activation and aggregation are induced at an overall low level, even at high HyalS(x) concentrations, and are comparable with that of heparin.