Protein-Bound Heparin/Heparan Sulfates in Human Adult and Umbilical Cord Plasma

Abstract

We used thrombin times and a competitive radiometric assay to identify, quantitate and characterize endogenous heparin-like molecules in umbilical cord (n = 58) and normal adult (n = 25) plasma. Thrombin times for cord plasma (29.6 ± 3.6 s) were significantly longer (p ≤ 0.0005) than those for adult plasma (18.9 ± 2.3 s), suggesting increased endogenous heparins. A radiometric assay based on the displacement of ¹²⁵I-heparin from protamine-Sepharose revealed that protease-digested plasma contained heparin/heparan sulfate, and plasma that was not digested with protease appeared not to contain heparin/heparan sulfate. More heparin/heparan sulfate was identified in cord than in adult plasma (p ≤ 0.05), but heparinase digestion produced significantly (p ≤ 0.001) reduced concentrations of heparin/heparan sulfate in only 39% of the samples. The lack of heparinase sensitivity in 61% of the protease-digested samples apparently was due to low molecular weight (LMW) heparins, for control heparin fragments of 5 kD that did not extend thrombin times were also less affected by heparinase, but the same LMW heparins were detected by radiometric assay. Despite normal thrombin times in all samples, the amounts of endogenous heparin/heparan sulfate identified in protease-digested samples by radiometric assay were of sufficient concentrations to produce inordinately prolonged thrombin times when compared with the same concentrations of unfractionated heparin. Collectively, these findings suggest the presence of a plasma reservoir of endogenous heparin/heparan sulfates in normal cord and adult plasma. These endogenous heparin/heparan sulfates are bound to plasma proteins, and an as yet undetermined proportion of these bound heparin/heparans are most likely LMW molecules.