Thermal Stability of Individual Domains in Platelet Glycoprotein IIbIIIa

Abstract

Thermal denaturation of platelet glycoprotein IIbIIIa (integrin aI&) was investigated by spectrofluorimetry and differential scanning calorimetry (DSC). Two forms of the protein were compared: active IIbIIIa, i.e., that fraction that binds to RGD-Sepharose, and inactive IIbIIIa, the non-binding fraction. At pH 8.5 in the presence of octyl glucoside and Ca2+ both forms exhibited a broad complex endotherm consisting of a well expressed low-temperature heat-absorption peak in the range of 40-65 "C followed by a broad peak stretching over 65-110°C. Each endotherm could be deconvoluted into at least eight transitions reflecting the melting of at least this many independently folded domains. The first two transitions in the region of the low-temperature peak had similar positions in both forms while at least some of the other transitions occurred at higher temperature in the active protein suggesting that some of the domains are more stable in the latter. When both fractions of IIbIIIa were heated in the fluorometer a sigmoidal transition was observed in the region of the first endothermic peak where the two thermolabile domains melt. This transition was destabilized by 15°C in the presence of EDTA, suggesting that these domains are formed by the 243-468 region of the IIb subunit which contains four Ca2+-binding motifs. It was further stabilized by 3°C upon addition of the G m S P K peptide in the presence of Caz+ while in EDTA the peptide had no effect. This is consistent with the involvement of Caz+-binding region in the formation of the ligand-binding site. A 66-kDa chymotryptic fragment, containing the 17-kDa NH,-terminal portion of the IIIa subunit disulfide-linked to its 50-kDa COOH-terminal portion including the cysteine-rich core, exhibited a fluorescence-detected Ca2'-independent transition in the region where the higher temperature DSC-detected transitions occur suggesting that some of the latter may be connected with the melting of the corresponding portions of IIbIIIa.Keywords : integrin ; glycoprotein IIbIIIa; fragments ; domains ; calorimetry.Platelet surface receptor glycoprotein IIbIIIa is a member of the structurally related integrin family of receptors that mediate cell-cell or cell-matrix interactions 11 -31. By mediating the fibrinogen-dependent interaction between platelets, IIbIIIa plays an essential role in platelet aggregation which is the primary mechanism of hemostatic control. All integrin receptors are composed of different variants of a and p subunits, whose combinations result in a variety of homologous non-covalently associated heterodimers [3]. Glycoprotein IIbIIIa (ar& in the integrin nomenclature) has served as a prototype to study structure/ function relationship in integrins. The IIIa v3) subunit is represented by a single chain of 762 amino acid residues while the IIb (a,,J subunit is synthesized as a single-chain polypeptide which is processed by proteolytic cleavage into a disulfidelinked heavy chain (856 residues) and light chain (149 residues) [I, 2, 41. Both IIb and IIIa subun...

Ligand Binding Promotes the Entropy-driven Oligomerization of Integrin αIIbβ3

“…In fact, this new thermostable conformer displays increased helicity, as monitored by a deeper 222-nm trough in the circular dichroic spectrum of ␣ IIb ␤ 3 , and a blue-shifted intrinsic fluorescence emission spectrum. The direction of both changes is opposite to that we and others (29,31,50) have described for guanidinium denaturation of the ␣ IIb ␤ 3 integrin. Our observations are also consistent with those of Makogoneko et al (50) who reported that ␣ IIb ␤ 3 was stable in octyl glucoside up to at least 95°C, based on fluorescence emission and scanning calorimetry measurements.…”

“…The direction of both changes is opposite to that we and others (29,31,50) have described for guanidinium denaturation of the ␣ IIb ␤ 3 integrin. Our observations are also consistent with those of Makogoneko et al (50) who reported that ␣ IIb ␤ 3 was stable in octyl glucoside up to at least 95°C, based on fluorescence emission and scanning calorimetry measurements. Combining this information with the temperature-induced changes in receptor quaternary structure observed here by dynamic light scattering, sedimentation velocity, sedimentation equilibrium, and electron microscopy, we propose that the ligand-free ␣ IIb ␤ 3 integrin undergoes conformational changes and forms aggregates at temperatures above 35°C.…”

“…Whereas the integrin-targeting sequence of each ligand is contained within chemically similar, seven-membered, cyclic rings (57,73), they exhibited different activity profiles. For example, cHArGD and eptifibatide, which share a common homoarginine residue, bound to platelet ␣ IIb ␤ 3 receptors with comparable EC 50 values, whereas cRGD bound an order of magnitude more weakly. This pattern extended to the ability of each ligand to block platelet aggregation, a fibrinogen-dependent function, as well as to their effects on platelet:fibrin adhesion.…”

“…Tryptophan fluorescence emission spectra were recorded as a function of temperature from 20 to 60°C for both free ␣ IIb ␤ 3 and the ␣ IIb ␤ 3 ⅐cHArGD complex to discern the effects of ligand binding on the thermal stability of the receptor (31,50). During heating, max shifted from 342 Ϯ 2 nm at 20°C to 337 Ϯ 2 nm at 60°C for the ligand-free integrin; in the presence of cHArGD, a shift from 340 Ϯ 3 to 338 Ϯ 2 nm was observed (n ϭ 3).…”

Decision aids can decrease decisional conflict in patients with hip or knee osteoarthritis: Randomized controlled trial

“…The second questionnaire was to determine whether patients had made their definitive decision. Health outcomes measured by the Hip Disability and Osteoarthritis Outcome Score (HOOS) and the Knee Injury and Osteoarthritis Outcome Score (KOOS)[ 12 , 13 ]. Both questionnaires include questions about patients’ symptoms, pain, activity limitations in daily life, sport, recreation and quality of life.…”