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Antithrombin is a blood plasma serpin that inhibits coagulation serine proteases, including factor Xa. Its activity is enhanced by binding an appropriate sequence in heparin. The inhibitory protein and its activating carbohydrate partner therefore play different molecular roles.^[ref]

In a 1983 experiment, investigators synthesized related four- and five-sugar fragments and checked their structures with nuclear magnetic resonance. The tested five-sugar fragment bound antithrombin and enhanced inhibition of factor Xa; the tested four-sugar fragment did neither in that comparison.^[ref]

A 1997 structural study connected binding to changes in the main beta sheet and exposure of the reactive-center loop. This supplied a structural explanation for activation rather than merely another measurement of binding.^[ref] The sequence-specific comparison matters: counting five sugars does not guarantee that an arbitrary five-sugar chain has the same activity.

Heparin is a strongly sulfated glycosaminoglycan containing alternating glucosamine-derived and uronic acid residues, including iduronic acid and glucuronic acid. Chains vary in length and chemical substitution, so a preparation is not adequately described by one small-molecule molecular formula. Sulfation and carboxylate groups contribute substantial negative electric charge.^[ref]

A defined fragment can test which structural details matter. In 1983, J. Choay and collaborators synthesized related four- and five-sugar fragments and verified their structures with nuclear magnetic resonance. The five-sugar fragment bound antithrombin and enhanced its inhibition of factor Xa, while the tested four-sugar fragment did not.^[ref]

The experiment shows why counting sulfate groups or measuring total charge cannot by itself specify biological activity: a particular arrangement of residues also matters. This is a molecular structure–function example, distinct from selecting a clinical dose or predicting every property of an unfractionated preparation.

J. Choay is the first author of a 1983 study testing which defined fragments of heparin support binding to antithrombin. The team synthesized related four- and five-sugar structures, checked their chemical structures by nuclear magnetic resonance, and measured binding and enhancement of factor Xa inhibition.^[ref]

The tested five-sugar fragment formed an equimolar complex with antithrombin and enhanced its inhibitory activity; the four-sugar fragment did not show the same behavior. Comparing chemically defined fragments helped separate a particular residue arrangement from the heterogeneous mixture of chains in a heparin preparation. Structure confirmation and activity measurement answered different questions, and both were needed for the argument.^[ref]