Thrombospondin-bound Integrin-associated Protein (CD47) Physically and Functionally Modifies Integrin αIIbβ3 by Its Extracellular Domain

Abstract Integrin-associated protein (IAP/CD47) is a receptor for the C-terminal cell binding domain of thrombospondin (TS). A peptide from the C-terminal cell binding domain, KRFYVVMWKK (4N1K) binds to IAP and stimulates the integrin-dependent cell functions, including platelet aggregation. We investigated the mechanism by which TS-bound IAP modulates the affinity of platelet integrin, αIIbβ3. Platelet aggregation induced by 4N1K was not completely inhibited by energy depletion with sodium azide and 2-deoxy-d-glucose, although ADP or collagen-induced platelet response was completely inhibited. The binding of ligand-mimetic antibody PAC1 to αIIbβ3 was also induced in the energy-depleted platelets. In the transfected Namalwa cells, 4N1K induced activation of the αIIbβ3 with mutated β3 (Ser-752 to Pro), which is a non-responsive form to inside-out signaling, as well as wild type αIIbβ3. The truncated form of IAP with only the extracellular immunoglobulin-like (Ig) domain was sufficient for the activation of αIIbβ3 in Chinese hamster ovary cells, although the IAP-mediated intracellular signaling was abolished, which was monitored by the absence of down-regulation of mitogen-activated protein kinase phosphorylation. Furthermore, the soluble recombinant Ig domain of IAP induced PAC1 binding to αIIbβ3 on Chinese hamster ovary cells when added with 4N1K. Physical association between the soluble recombinant Ig domain of IAP and purified αIIbβ3 was detected in the presence of 4N1K. These data indicate that the extracellular Ig domain of IAP, when bound to TS, interacts with αIIbβ3 and can change αIIbβ3 in a high affinity state without the requirement of intracellular signaling. This extracellular event would be a novel mechanism of affinity modulation of integrin.