PROTEOGLYCANS AND SULFATED PROTEINS OF PLATELETS: CHARACTERIZATION AND RESPONSE TO THROMBIN AND ADP

Abstract

The proteoglycans (PG) and sulfated proteins of guinea pig platelets were labeled in vivo by intraperitoneal injection of (35S)sulfate. At 3 days after injection, platelets contained 3 distinct populations of chondroitin-6-sulfate proteoglycans which together constitute about 65% of the cellular (35S) label. Most PG elute from a DEAE-Sephacel column with 4M Gdn HC1 (PG-1, 87%), and elute at Kav 0.12 on Sepharose CL-6B. The PG-1 can be resolved by SDS-PAGE into two fractions. The remainder (PG-2, 13%) elutes from the DEAE-Sephacel column with 4M Gdn HCl/2% Triton X-100 or 2% CHAPS, and has a Kav of 0.07 on Sepharose CL-6B. About 20-25% of the cell (35S) label elutes from DEAE-Sephacel in the wash-through or with 0.23M NaCl, and can be resolved by SDS-PAGE into at least 8 distinct bands which we have tentatively characterized as sulfated glycoproteins. The remainder of the (35S) is in low molecular weight (LMW) material which does not adhere to DEAE-Sephacel and has not been further characterized.Platelets were treated with either thrombin or ADP, and the cells were then separated from the supernatant by centrifugation. The radiolabeled molecules in the supernatant and the cells were analyzed by DEAE-Sephacel and Sepharose CL-6B column chromatography. About 65% of the total cell (35S) was released from the cells by thrombin. Most of this radiolabel adhered to the DEAE-Sephacel column, and was found to be PG-1. The remainder of the released (35S) was about half the LMW material. In contrast, only 10-15% of the (35S)labeled material retained by the cells adhered to DEAE-Sephacel and was found to be PG-2. The remainder of the (35S)-labeled material retained by the platelets was the sulfated proteins and the LMW material. ADP caused release of about 15% of the (35S), and this was found to be in part PG-1 and in part the LMW material, but not PG-2. None of the (35S)-labeled molecules appeared to be degraded during platelet activation. We suggest that the PG-1 represent the a-granule and PG-2 the membrane proteoglycans. The sulfated proteins have not been described previously. Their role is not known, but we hypothesize that they may form part of the negative charge of the glycocalix and thus be part of the reactive surface of the platelet.