ヒト胎盤由来ecto-ATP diphosphohydrolase : その精製, 部分構造解析とずり応力惹起血小板凝集阻害作用に関する研究

Abstract

Human placental ecto-ATP diphosphohydrolase (ATPDase, a 82 kDa single-chain glycoprotein with a pI of 5.6-6.2) was highly purified by a specific murine monoclonal antibody (mAb) MK 33 (IgGi-κ) with a high yield of protein (305-451μg from one placenta) and specific activity (15-20 units/mg/min for ATP-/ADP hydrolysis), and characterized structurally and functionally. After endoglycosidase-F treatment, the 82 kDa -enzyme turned to a 62 kDa-protein with a significant loss of activity. Structurally, the N -terminal unambiguous 30 residue sequence of MKGTKDLTSQQKESNVKTFxSKNILAIL- GF was determined (where x was unidentified). The first 11-residue sequence was quite unique and not homplogous to any other protein, but the following 19-residue sequence was almost identical to that of residues 5-23 of human CD 39 lymphoid cell activation antigen deduced by cDNA sequencing. This purified enzyme immunoreacted with an anti-CD 39 mAb, indicating that these two proteins are closely related, but different molecules. Functionally, MK33 mAb-purified enzyme at a final concentration of 2μg/ml totally inhibited the secondary aggregation of platelets induced by platelet agonists. Most interest- ingly, this enzyme blocked low shear stress-induced platelet aggregation in a dose-depen- dent manner and completely at a final concentraion of O.5μg/ml. Whereas under high shear stress this protein mediated the disaggregation in the later phase of aggregation without affecting the initial aggregation. Further, the purified enzyme at a final concentraion of 1 μg/ml also totally inhibited both fibrinogen and von Willebrand factor (vWF) binding to activated platelet glycoprotein (GP) Ⅱb/Ⅲa, but did not affect vWF binding to GPIb in the presence of ristocetin. Immunohistochemical analysis of this enzyme using mAb MK 33 has localized to the syncytiotrophoblasts of placental microvilli and umbilical vein, but much less intense in umbilical arteries. These results taken together suggest that placental ecto -ATPDase, via its ADP-scavenging effect, functions as a potent inhibitor on platelet aggregation in feto-maternal circulation.