A new type of low-molecular-weight heparin (ss-LMW-H) was prepared (by controlled depolymerization and concurrent sulfation of heparin with a mixture of sulfuric and chlorosulfonic acid), to test the influence of extra-sulfate groups on biological properties of heparin fragments. The fragments had an average molecular weight ranging from 5000 to 10,000, a sulfate-to-carboxyl molar ratio of 2.8-3.1, and electrophoretic mobilities and NMR spectra distinctly different from those of the parent heparins. Depolymerization with oversulfation reduced the anticoagulant activity of heparin (ex vivo, in rats) much more than depolymerization alone, to about 10% of the original APTT and 25-30% of the original a.Xa units. By contrast, the antithrombotic activity (venous stasis model, in rats) was still comparable to that of heparin, and bleeding times were not significantly increased. The lipasemic (lipoprotein-lipase-releasing) activity of ss-LMW-H fragments was more than twice that of heparin. Results are discussed in terms of contribution of charge-density effects to different activities and to different mechanisms for the same activity of heparin.