STUDIES ON PLATELET PLASMA-MEMBRANES .2. CHARACTERIZATION OF SURFACE PROTEINS OF RABBIT PLATELETS INVITRO AND DURING CIRCULATION INVIVO USING DIAZOTIZED (DIIODOSULFANILIC-I-125 ACID AS A LABEL
Diazotized (125I)-diiodosulfanilic acid (DD125ISA) binds specifically to the exposed proteins on the surface of the rabbit platelet plasma membrane. This was demonstrated by the following observations with the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole platelets and the isolated plasma membrane fraction: the specific activity of isolated membrane protein was 7-fold that of whole platelet protein; no proteins of intact platelets were labeled which were not represented in the isolated plasma membrane; DD125ISA-labeled proteins were altered by trypsin treatment of intact, labeled platelets and the pattern of labeling produced by reaction of isolated membranes with DD125ISA differed from that produced by the labeling of intact platelets. Reaction with DD125ISA with intact platelets produced labeling of only the 3 membrane glycoproteins (MW: 180,000, 125,000 and 92,000 daltons) with greatest labeling of the largest glycoprotein and least labeling of the smallest glycoprotein. When rabbit platelets were labeled simultaneously with DD125ISA and 51Cr, the 2 isotopes were similarly distributed in various density populations of platelets. Some DD125ISA was solubilized from labeled and washed platelets by sonication, but all platelet DD125ISA was recovered in the plasma membrane fraction after 30 min. circulation in vivo. In vivo 51Cr recovery and survival were not altered by simultaneous labeling of platelets with DD125ISA. The disappearance of DD125ISA from circulating platelets (T1/2 = 17 h) was more rapid than 51Cr (T1/2 = 30 h) and appeared exponential in contrast to the linear 51Cr disappearance. DD125ISA did not disappear from platelets faster than 51Cr when doubly labeled platelets were harvested after 3 h circulation and incubated in autologous plasma (T1/2 of DD125ISA elution = 43 h, 51Cr = 33 h). SDS-PAGE analysis of DD125ISA-labeled platelets after 14-20 h circulation in vivo demonstrated the same pattern of DD125ISA distribution on membrane glycoproteins as on the platelets prior to infusion. This symmetrical loss of the membrane label was interpreted to indicate symmetrical loss of membrane proteins, suggesting that the platelet may lose pieces of membrane and not specific surface proteins during circulation. This could occur during reversible adhesion encounters during the process of hemostasis and cause the smaller size and decreased effectiveness of older platelets.