A Cluster of Basic Amino Acid Residues in the gamma370-381 Sequence of Fibrinogen Comprises a Binding Site for Platelet Integrin alpha_(IIb)beta_3 (Glycoprotein IIb/IIIa)

摘要

Adhesive interactions of platelet integrin alpha_(IIb)beta_3 with fibrinogen and fibrin are central events in hemostasis and thrombosis.However,the mechanisms by which alpha_(IIb)beta_3 binds these ligands remain incompletely understood.We have recently demonstrated that alpha_(IIb)beta_3 binds the gamma365-383 sequence in the gammaC-domain of fibrin(ogen).This sequence contains neither the AGDV nor the RGD recognition motifs,known to bind alpha_(IIb)beta_3,suggesting the different specificity of the integrin.Here,using peptide arrays,mutant fibrinogens,and recombinant mutant gammaC-domains,we have examined the mechanism whereby alpha_(IIb)beta_3 binds gamma~(365-383).The alpha_(IIb)beta_3-binding activity was localized within gamma~(370-381),with two short sequences,gamma~(370)ATWKTR~(375) and gamma~(376)WYSMKK~(381),being able to independently bind the integrin.Furthermore,recognition of alpha_(IIb)beta_3 by gamma~(370-381) depended on four basic residues,Lys~(373),Arg~(375),Lys~(380),and Lys~(381).Simultaneous replacement of these amino acids and deletion of the gamma~(408)AGDV~(411) sequence in the recombinant gammaC-domain resulted in the loss of alpha_(IIb)beta_3-mediated platelet adhesion.Confirming the critical roles of the identified residues,abnormal fibrinogen Kaiserslautern,in which gammaLys~(380) is replaced by Asn,demonstrated delayed clot retraction and impaired alpha_(IIb)beta_3 binding.Also,a mutant recombinant fibrinogen modeled after the naturally occurring variant Osaka V (gammaArg~(375) -> Gly) showed delayed clot retraction and reduced binding to purified alpha_(IIb)beta_3.These results identify the gamma~(370-381) sequence of fibrin(ogen) as the binding site for alpha_(IIb)beta_3 involved in platelet adhesion and clot retraction and define the new recognition specificity of this integrin.