Asymmetric distribution of sites with high affinity for antithrombin III in rat skin heparin proteoglycans.

The distribution of sites with high affinity for antithrombin III in [35S]heparin proteoglycans from rat skin was studied by affinity chromatography of the intact proteoglycans (Mr congruent to 1 x 10(6)) and degradation products. Unfractionated proteoglycan and proteoglycan fractions with low affinity and high affinity separated on antithrombin III-agarose were treated with alkali, releasing heparin chains (Mr congruent to 1 x 10(5)). Each chain preparation was fractionated on antithrombin III-agarose into fractions with low affinity and high affinity. Unfractionated proteoglycan and proteoglycan fractions with low affinity and high affinity were incubated with rat serum at pH 6.0, which gave products of similar size to commercial heparins (Mr congruent to 1 x 10(4)) termed heparin fragments. Each fragment preparation was fractionated on antithrombin III-agarose, yielding fractions with no affinity, low affinity, and high affinity, 40% of the proteoglycan preparation had low affinity, containing 4% high affinity chains and 7% high affinity fragments. The high affinity proteoglycan fraction yielded 40% high affinity chains and 22% high affinity fragments. The data show that the distribution of binding sites with high affinity for antithrombin III in heparin proteoglycans is highly asymmetric. Therefore, the concept that polymer modification reactions occurring during heparin biosynthesis, which must be involved in the formation of high affinity binding sites, occur in a random way must be reappraised.