Integrins are cysteine-rich heterodimeric cell-surface adhesion molecules that alter their affinity for ligands in response to cellular activation. The molecular mechanisms involved in this activation of integrins are not understood. Treatment with the thiol-reducing agent, dithiothreitol, can induce an activation-like state in many integrins suggesting that cysteine-cysteine dithiol bonds are important for the receptor's tertiary structure and may be involved in activation-induced conformational changes. Here we demonstrate that the platelet-specific integrin, ␣ IIb  3 , contains an endogenous thiol isomerase activity, predicted from the presence of the tetrapeptide motif, CXXC, in each of the cysteinerich repeats of the  3 polypeptide. This motif comprises the active site in enzymes involved in disulfide exchange reactions, including protein-disulfide isomerase (EC 5.3.4.1) and thioredoxin. Intrinsic thiol isomerase activity is also observed in the related integrin, ␣ v  3 , which shares a common -subunit. Thiol isomerase activity within ␣ IIb  3 is time-dependent and saturable, and is inhibited by the protein-disulfide isomerase inhibitor, bacitracin. Furthermore, this activity is calcium-sensitive and is regulated in the EDTA-stabilized conformation of the integrin. This novel demonstration of an enzymatic activity associated with an integrin subunit suggests that altered thiol bonding within the integrin or its substrates may be locally modified during ␣ IIb  3 activation.Integrins are cell-surface, calcium-dependent, heterodimeric adhesion molecules that play a critical role in cell-cell and cell-substrate adhesion. In cells at rest, integrins are present in a latent or resting conformation. Following cellular activation, they undergo conformational changes to become high affinity receptors for their specific ligand(s). The "switch" mechanism whereby integrins are converted from their resting conformation is critically important to their cellular function. However, the mechanisms underlying these conformational changes have not yet been deduced.The conformational changes in the platelet-specific integrin, ␣ IIb  3 , are the composite result of at least two processes. First, intracellular signals converge on the cytoplasmic tails of the integrin conveying the intention to activate. Second, the extracellular domains, which constitute Ͼ95% of the molecules, respond with an increased affinity for ligand and an altered display of antibody epitopes suggestive of altered protein folding. We have shown that the conserved ␣-subunit cytoplasmic sequence, KVGFFKR, is critical for the intracellular-mediated activation of the platelet integrin (1). The precise role played by this peptide sequence remains uncharacterized. However, Vinogradova et al. (2), have recently proposed a structural basis for this effect which proposes a protein-protein interaction with the integrin cytoplasmic tails. Deletion or mutation of this cytoplasmic sequence from the ␣ IIb subunit were found to increase the ligand binding affinity...
Bernard-Soulier syndrome is a rare bleeding disorder caused by a quantitative or qualitative defect in the platelet glycoprotein (GP) Ib-IX-V complex. The complex, which serves as a platelet receptor for von Willebrand factor, is composed of 4 subunits: GPIb, GPIbβ, GPIX, and GPV. We here describe the molecular basis of a novel form of Bernard-Soulier syndrome in a patient in whom the components of the GPIb-IX-V complex were undetectable on the platelet surface. Although confocal imaging confirmed that GPIb was not present on the platelet surface, GPIb was readily detectable in the patient's platelets. Moreover, immunoprecipitation of plasma with specific monoclonal antibodies identified circulating, soluble GPIb. DNA-sequence analysis revealed normal sequences for GPIb and GPIX. There was a G to A substitution at position 159 of the gene encoding GPIbβ, resulting in a premature termination of translation at amino acid 21. Studies of transient coexpression of this mutant, W21stop-GPIbβ, together with wild-type GPIb and GPIX, demonstrated a failure of GPIX expression on the surface of HEK 293T cells. Similar results were obtained with Chinese hamster ovary IX cells, a stable cell line expressing GPIb that retains the capacity to re-express GPIX. Thus, we found that GPIbβ affects the surface expression of the GPIb-IX complex by failing to support the insertion of GPIb and GPIX into the platelet membrane.
Mab1 and Mab2 bind to GPIIb/IIIa and are differentially displaced by abciximab and small molecular weight antagonists. These antibodies may be used to monitor receptor number and occupancy during administration of a GPIIb/IIIa antagonist.
Bernard-Soulier syndrome is a rare bleeding disorder caused by a quantitative or qualitative defect in the platelet glycoprotein (GP) Ib-IX-V complex. The complex, which serves as a platelet receptor for von Willebrand factor, is composed of 4 subunits: GPIb, GPIbβ, GPIX, and GPV. We here describe the molecular basis of a novel form of Bernard-Soulier syndrome in a patient in whom the components of the GPIb-IX-V complex were undetectable on the platelet surface. Although confocal imaging confirmed that GPIb was not present on the platelet surface, GPIb was readily detectable in the patient's platelets. Moreover, immunoprecipitation of plasma with specific monoclonal antibodies identified circulating, soluble GPIb. DNA-sequence analysis revealed normal sequences for GPIb and GPIX. There was a G to A substitution at position 159 of the gene encoding GPIbβ, resulting in a premature termination of translation at amino acid 21. Studies of transient coexpression of this mutant, W21stop-GPIbβ, together with wild-type GPIb and GPIX, demonstrated a failure of GPIX expression on the surface of HEK 293T cells. Similar results were obtained with Chinese hamster ovary IX cells, a stable cell line expressing GPIb that retains the capacity to re-express GPIX. Thus, we found that GPIbβ affects the surface expression of the GPIb-IX complex by failing to support the insertion of GPIb and GPIX into the platelet membrane.
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