Production of Soluble P-Selectin by Platelets and Endothelial Cells

A. V. Semenov^1*, Yu. A. Romanov¹, S. A. Loktionova¹, O. Yu. Tikhomirov¹, M. V. Khachikian¹, S. A. Vasil'ev², and A. V. Mazurov¹

¹Institute of Experimental Cardiology, Cardiology Research Center, Russian Ministry of Health, 3-ya Cherepkovskaya ul. 15a, Moscow, 121552 Russia; fax: (095) 414-6699

²Hematology Research Center, Russian Academy of Medical Science, Novozykovskii Proezd 4a, Moscow, 125167 Russia; fax: (095) 212-4252

^* To whom correspondence should be addressed.

Received April 5, 1999; Revision received June 3, 1999

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The distribution of a soluble form of a cell adhesion molecule, P-selectin, in human platelets and cultivated endothelial cells has been studied by enzyme-linked immunosorbent assay (ELISA). The concentration of soluble P-selectin in the blood plasma of healthy donors and patients with abnormal platelet count has also been determined. P-selectin was measured in the Triton X-100 lysate of platelets and endothelial cells (total P-selectin), in the 100,000g supernatant obtained after sedimentation of the membrane fraction from the homogenate of sonicated platelets and endothelial cells (intracellular soluble P-selectin), in the supernatant of activated and nonactivated platelets, and in the culture medium of endothelial cells. A soluble form of P-selectin which did not coprecipitate with the membrane fraction was detected in platelets and accounted for ~10% of the total P-selectin. Platelet activation by thrombin, ADP, or a thromboxane A₂ analog resulted in the secretion of 30-50% of the intracellular soluble P-selectin. Measurements of P-selectin in endothelial cell culture revealed that endothelium from aorta contained about twofold more P-selectin than endothelium from umbilical vein. Intracellular soluble P-selectin was identified in both types of endothelial cells. In endothelial cells from the umbilical vein this form made up ~10% of the total P-selectin. Soluble P-selectin was also detected in the medium of cultivated endothelial cells, where its content correlated with the total cellular P-selectin. Concentration of P-selectin in blood plasma strongly correlated with the platelet count in the blood of healthy donors and patients with thrombocytosis and thrombocytopenia. These data indicate that platelets serve as one of the main source of plasma P-selectin. However, the presence of P-selectin in the plasma of patients with severe thrombocytopenia suggests that endothelium can also be involved in plasma P-selectin production. Thus, in vitro experiments as well as measurements of plasma P-selectin have shown that both platelets and endothelial cells can produce a soluble form of the protein. Platelet-derived soluble P-selectin and plasma P-selectin were shown to react with antibodies against the cytoplasmic domain of P-selectin. These data prove that at least part of soluble P-selectin is produced by synthesis employing special mRNA which lacks the sequence encoding the transmembrane domain, but not by the proteolytic shedding of the extracellular portion of membrane P-selectin.

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KEY WORDS: P-selectin, platelets, endothelial cells, ELISA

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