Characterization of native human thrombopoietin in the blood of normal individuals and of patients with haematologic disorders

Thrombopoietin (TPO) isolated from thrombocytopenic plasma of various anima l species has previously been shown to comprise only truncated forms of the molecule? presumably generated by proteolysis. Native TPO has now been par tially purified from normal human plasma by immunoaffinity chromatography a nd was confirmed to be biologically active. Gel filtration in the presence of SDS revealed that TPO eluted in two peaks: a major peak corresponding to the elution position of fully glycosylated recombinant human TPO (rhTPO) c onsisting of 332 amino acid residues, and a minor peak corresponding to a s maller molecular size. Immunoblot analysis also revealed that most plasma-d erived TPO migrated at the same position as fully glycosylated rhTPO, corre sponding to a molecular size of similar to 80 to 100 kDa. Furthermore, the size distribution of circulating TPO in patients with various haematologic disorders did not differ markedly from that of plasma-derived TPO from heal thy individuals. These results indicate that the truncation of circulating TPO is not related to disease pathophysiology, and that the predominant for m of TPO in brood is a biologically active similar to 80- to 100-kDa specie s. The size distribution of TPO extracted from normal platelets was similar to that of TPO in plasma; the proportion of truncated TPO was decreased by prior incubation of platelets with hirudin, indicating that the endogenous truncated TPO, at least in platelet extract, was generated by thrombin-med iated cleavage.