Application Of A Monoclonal Antibody (SZ-51) Against Human Platelet P-selectin

P-selectin is an integral membrane glycoprotein located in the a -granules of platelets and the Weibel-Palade bodies of endothelial cells. When these cells are activated, P-selectin rapidly redistributed to the surface membrane of these cells and became the specific marker of platelet or endothelial cell activation.To purify human platelet P-selectin we used the method of monoclonal antibody affinity chromatography. After binding monoclonal antibodies against human platelet P-selectin SZ-51-IgG to CNBr activated sepharose 4B, we got affinity chromatographies SZ-51 -sepharose 4B. Protein, which was purified from triton X-100 solubilized platelet solution through FPLC by SZ-51-sepharose 4B affinity chromatography, was characterized by SDS-PAGE and Dot blot. The binding rate of this affinitychromatography was 68% . 12 g of P-selectin protein can be purified from about 1 X 109 of platelets. Our SDS-PAGE and Dot blot indicated this 140kD protein was P-selectin . So , using SZ-51-sepharose 4B affinity chromatography to purify P-selectin was an efficient method to prepare human platelet P-selectin with high biological activity.To investigate the clinical significance in determination of the P-selectin levels in subjects with prethrombotic state or thrombosis by flow cytometry (FCM). We analyzed the P-selectin expression on platelet membrane in 42 patients with diabetes mellitus, 33 with hyperlipidemia, 23 with cerebral infarction and 20 healthy individuals, using fluorescein-labeled SZ-51 by direct FCM comparing with indirect FCM and enzyme-linked immunosorbent assay (ELISA). The level of P-selectin on platelet membrane was higher in DM (23.92 15.83%), in hyperlipidemia (18.34+9.46%) and in cerebral infarction (19.32 10.38%) than in normal subjects (3.38+1.11%) (PO.01). In addition, similar results on P-selectin were obtained by indirect FCM and ELISA in patients with DM and cerebral infarction. Therefore, FITC-labeled SZ-51-IgG can be used in FCM, and which can be a new and sensitive method to detect the platelet activation.To investigate the expression of P-selectin on the Human umbilical vein endothelial cells (FTUVECs) stimulated by H2O2 and to observe the influence of ferulic acid in this process, we cultured the HUVEC in vitro and detected the FITC-SZ-51-IgG by FCM to estimate the P-selectin expression after stimulated by H2O2. Meanwhile we observed the influence of ferulic acid to the expression of P-selectin on stimulated HUVECs by H2O2 and the role of SZ-51 to the adhesion of polymorphonuclear leukocyte (PMN) on the immobilized P-selectin.Only little P-selectin expressed on the normal culture HUVECs in vitro. But HUVECs can rapidly express the P-selectin stimulated by fairly low concentration of H2O2, in the experiments the optimal concentration of H2O2 was between 250uM and 350 uM, and the optimal stimuating time was two hours. When the concentration increasing, ferulic acid had the tendency to inhibit the expression of P-selectin stimulated by H2O2. However, SZ-51 can not inhibit the adherence of PMN to immobilized p-selectin.