THERMAL-STABILITY OF INDIVIDUAL DOMAINS IN PLATELET GLYCOPROTEIN IIBIIIA

Thermal denaturation of platelet glycoprotein IIbIIIa (integrin alpha( IIB)/beta(3)) was investigated by spectrofluorimetry and differential scanning calorimetry (DSC). Two forms of the protein were compared: ac tive IIbIIIa, i.e., that fraction that binds to RGD-Sepharose, and ina ctive IIbIIIa, the non-binding fraction. At pH 8.5 in the presence of octyl glucoside and Ca2+ both forms exhibited a broad complex endother m consisting of a well expressed low-temperature heat-absorption peak in the range of 40-65 degrees C followed by a broad peak stretching ov er 65-110 degrees C. Each endotherm could be deconvoluted into at leas t eight transitions reflecting the melting of at least this many indep endently folded domains. The first two transitions in the region of th e low-temperature peak had similar positions in both forms while at le ast some of the other transitions occurred at higher temperature in th e active protein suggesting that some of the domains are more stable i n the latter. When both fractions of IIbIIIa were heated in the fluoro meter a sigmoidal transition was observed in the region of the first e ndothermic peak where the two thermolabile domains melt. This transiti on was destabilized by 15 degrees C in the presence of EDTA, suggestin g that these domains are formed by the 243-468 region of the IIb subun it which contains four Ca2+-binding motifs. It was further stabilized by 3 degrees C upon addition of the GRGDSPK peptide in the presence of Ca2+ while in EDTA the peptide had no effect. This is consistent with the involvement of Ca2+-binding region in the formation of the ligand -binding site. A 66-kDa chymotryptic fragment, containing the 17-kDa N H2-terminal portion of the ma subunit disulfide-linked to its 50-kDa C OOH-terminal portion including the cysteine-rich core, exhibited a flu orescence-detected Ca2+-independent transition in the region where the higher temperature DSC-detected transitions occur suggesting that som e of the latter may be connected with the melting of the corresponding portions of IIbIIIa.