Thrombospondin in essential thrombocythemia

Abstract

Essential thrombocythemia is a myeloproliferative disorder characterized by frequent bleeding and thrombotic complications. On a molecular level, two abnormalities of platelet thrombospondin have been identified: abnormal glycosylation of the intact 185,000-dalton chain has been detected and a shortened form of the thrombospondin chain is present. We have used two monoclonal antibodies and Lens culinaris lectin to probe the structure of thrombospondin in the platelets from three patients with essential thrombocythemia; one patient with polycythemia vera and two patients with secondary thrombocytosis. The presence of abnormal thrombospondin fragments with molecular weights of 160,000 and 30,000 was detected in the intact platelets and in the supernatant from thrombin-treated platelets, in all of the individuals except one of the secondary thrombocytosis patients. Monoclonal antibody binding studies indicate that both fragments are produced by proteolysis at a single site, which results in the removal of a 30,000- dalton fragment from the NH2-terminal. Lens culinaris lectin-binding studies revealed that some of the carbohydrate moieties of thrombospondin are near this cleavage site. The results are consistent with the hypothesis that the abnormal thrombospondin fragments observed under conditions of increased platelet production are due to increased susceptibility to proteolysis which, in turn, may be due to defective glycosylation.

Studies of platelet volume, chemistry and function in patients with essential thrombocythaemia treated with Anagrelide

“…In one patient before therapy and in two patients after therapy it was accompanied by a similar increase in platelet membrane CD36, as measured by flow cytometry by Thibert et al (1995). In parallel, the abnormal migration of TSP on immunoblot, that we and others already have described (Booth et al , 1984; Lawler et al , 1987; Legrand et al , 1991a), was present before and during Anagrelide therapy for the three patients studied.…”

The structure of human thrombospondin, an adhesive glycoprotein with multiple calcium-binding sites and homologies with several different proteins.

“…At higher enzyme-to-substrate ratios the 30,000-D fragment is converted to a 25,000-D fragment which no longer binds the lectin. Since the NH2-terminal sequence of the 25,000-D fragment is identical to that of the intact molecule, it can be concluded that the carbohydrate is near the COOH terminal of the 30,000-D fragment (Galvin et al, 1985;Lawler et al, 1986a) exactly as predicted by the sequence.…”

“…The glycosylation site is eleven residues from the COOH-terminus of this fragment. Chymotryptic digestion at low enzyme-to-substrate ratios results in the production of a 30,000-D fragment which binds the Lens culinaris lectin (Lawler et al, 1986a). At higher enzyme-to-substrate ratios the 30,000-D fragment is converted to a 25,000-D fragment which no longer binds the lectin.…”