Sustained integrin ligation involves extracellular free sulfhydryls and enzymatically catalyzed disulfide exchange

Abstract

Studies have suggested a pivotal role for free sulfhydryls in platelet integrin function, and enzyme-mediated reduction of disulfide bonds on platelets has been implicated. The platelet fibrinogen receptor ␣ IIb ␤ 3 is the best-studied platelet integrin and serves as a model system for studying the structure-function relation in this family of adhesion receptors. The demonstration of free sulfhydryls on the exofacial domain of purified ␣ IIb ␤ 3 , specifically in its activated conformation, prompted us to explore the potential for activation-dependent, enzymatically catalyzed thiol expression on intact platelets and the possible role of surface-associated protein disulfide isomerase (PDI) in ␣ IIb ␤ 3 ligation. Using the membrane-impermeant sulfhydryl blocker para-chloromercuriphenyl sulfonate, the inhibitor of disulfide exchange bacitracin, and the monoclonal anti-PDI antibody RL90, we examined fibrinogen binding to ␣ IIb ␤ 3 as well as ligation-induced allosteric changes in the conformation of ␣ IIb ␤ 3 . We sought to distinguish the possible involvement of disulfide exchange in agonist-induced platelet stimulation from its role in integrin ligation. Analysis of the role of free thiols in platelet aggregation suggested a thiolindependent initial ligation followed by a thiol-dependent stabilization of binding. IntroductionThe affinity of integrins for their ligands is tightly regulated, both by cellular events and subsequent to ligand binding. [1][2][3][4][5] Such cellular control over the interaction of extracellular proteins implies the existence of a mechanism to propagate information back and forth between the extracellular head and the cytoplasmic tail of the integrin receptor. In recent years, the posited mechanism has been the allosteric conformational changes occurring in the integrin receptor, both in response to cellular events that switch the receptor from low to high affinity (inside-out signaling) and external events that relay information about occupancy of the receptor (outside-in signaling). [6][7][8][9] Although information about the nature of the changes in conformation is slowly emerging (for review, see Woodside et al 10 ) and consequent changes in intracellular interactions are widely documented, 10-13 the molecular mechanism regulating such changes, particularly on the exofacial domain, has not been elucidated. We previously reported that extracellular sulfhydryls participate in conformational changes triggered by interaction of the platelet integrin ␣ 2 ␤ 1 with its natural ligand collagen. 14 We also found that disulfide exchange is required for platelet adhesion mediated by integrins ␣ 5 ␤ 1 and ␣ IIb ␤ 3 as well as ␣ 2 ␤ 1 and that surface-expressed protein disulfide isomerase (PDI) is involved in this exchange. 15 Furthermore, involvement of disulfide exchange in receptor function is a feature specific to the integrin-receptor family (J. L. et al, submitted manuscript, 2002). We therefore proposed that conformational changes subsequent to ligand interaction with the integrin lead to ...

Factor XIII improves platelet adhesion to fibrinogen by protein disulfide isomerase-mediated activity

“…These data also confirm our previous observation that FXIII can bind directly to platelets [23]. However, in another study, we showed that PDI-dependent platelet adhesion is specific to platelet integrin-mediated adhesion [12,13]. Thus, the addition of FXIII following established platelet integrin-fibrinogen interaction, as in the present work, leaves FXIIIA PDI activity free to enhance this interaction.…”

“…In the present study, on the basis of our earlier findings that PDI plays a mediatory role in platelet adhesion [11][12][13] and that FXIIIA exerts PDI activity [10], we sought to examine the relative contributions of FXIII PDI or transglutaminase activity to platelet adhesion to fibrinogen.…”

“…Cellsurface-localized, membrane-bound PDI is essential for sustaining the binding of fibrinogen, fibronectin, and collagen to platelet integrins αIIbβ3, α5β1, and α2β1, respectively [11,12], thereby regulating platelet adhesion [11,12] and aggregation [13]. PDI also regulates the activity of L-selectin, a cell-adhesion molecule found on leukocytes [14].…”

Interactions between cell surface protein disulphide isomerase and S-nitrosoglutathione during nitric oxide delivery

“…Free thiols become available on the platelet surface upon activation [10] and those present on integrin α IIb β 3 provide a direct target for redox modification [33]. Thiol-disulphide exchange within α IIb β 3 , catalysed by an endogenous isomerase activity [34], by PDI [11,35], or by ERP5 [36] is necessary for transition to its active conformation. Thus the thiol redox state of α IIb β 3 influences platelet adhesive function and any agent that modifies this redox state may interfere with platelet aggregation.…”

Conformational Regulation of α4β1-Integrin Affinity by Reducing Agents

“…Another model suggests that integrin conformation is regulated by reduction of the disulfide bonds and possibly involves protein-disulfide isomerase (PDI) (17)(18)(19). PDI regulates disulfide exchange and conformationally induced shedding of Lselectin (20).…”