Platelet Fragmentation Requires a Specific Structural Conformation of Human Monoclonal Antibody against β3 Integrin

Abstract

We have described an autoantibody against ␤3 (GPIIIa49 -66), a region of platelet integrin ␣IIb␤3 that is unique. It induces platelet fragmentation in the absence of complement via antibody activation of platelet NADPH oxidase and 12-lipoxygenase to release reactive oxygen species, which destroy platelets. To study the mechanism of anti-GPIIIa antibody-induced platelet fragmentation, we screened a human single chain Fv antibody library with the GPIIIa49 -66 peptide. Nine monoclonal antibodies were identified that were capable of binding to GPIIIa49 -66. Surprisingly, binding avidity for GPIIIa49 -66 did not correlate with activity of induction of platelet fragmentation. We therefore investigated the requirements for platelet fragmentation. Mutations were introduced into the heavy chain complementary-determining region-3 of clones 11, 43, and 54 by site-directed mutagenesis. The capability of these clones to induce platelet fragmentation or bind to GPIIIa49 -66 subsequently changed. Molecular modeling of these clones with their mutants revealed that the ability to induce platelet fragmentation is affected by the side chain orientation of positively charged amino acids in the heavy chain of residues 99 -102. Thus, a structural change in the conformation of antiGPIIIa49 -66 antibody contributes to its binding to the ␤3 integrin and subsequent antibody-induced platelet fragmentation and aggregate dissolution.

Role of molecular mimicry of hepatitis C virus protein with platelet GPIIIa in hepatitis C–related immunologic thrombocytopenia

“…This antibody is unique in that it induces complement-independent platelet fragmentation by oxidative platelet fragmentation due to the release of reactive oxygen species through activation of 12-lipoxygenase and NADPH oxidase. [4][5][6] HIV-1 immune-related thrombocytopenia (HIV-1-ITP) is more frequent in drug abusers compared with non-drug abusers (37% vs 16% incidence, respectively), and more severe in HIV-1-seropositive drug abusers than non-drug abusers (platelet count Ͻ 10 ϫ 10 9 /L in 52% vs 9%, respectively). 7,8 A striking feature of HIV-1 infection in drug abusers is the frequent coinfection with hepatitis C virus (HCV).…”

A humanized single-chain antibody against beta 3 integrin inhibits pulmonary metastasis by preferentially fragmenting activated platelets in the tumor microenvironment

“…[18][19][20][21][22] By screening a human single-chain fragment variable region (scFv) library with the GPIIIa49-66 peptide as bait, we identified a human monoclonal scFv Ab that recognized GPIIIa49-66 (named A11), with similar functional properties to the patient autoantibody in that it preferentially binds to activated platelets and can also lyse platelet thrombus in vitro. [23][24][25] We therefore sought to determine whether A11 would be associated with any significant antimetastatic effect by clearance of functional, activated platelets in the tumor environment.…”

A Humanized Single-Chain Variable Fragment Antibody Against Beta3 Integrin in Escherichia coli

“…(5) Phage antibody is the fusion format of scFv fragment and filamentous phage coat protein 3 (g3p) (Fig. 1A), which renders it suboptimal for potential clinical use.…”

“…As illustrated by Figure 1B, all of the selected clones, including clones 11, 41, 43, 49, and 54, contain stop codon (TAG) in their heavy chain region, which limits the generation of scFv module. To circumvent this hurdle, we chose clone 11, which exhibits the highest binding activity, (5) mutated the inside stop codon (TAG) into AAG (lys) (named A11), and cloned the resultant mutant to plasmid pET-29a for expression in E. coli Rosetta. Upon induction with 1 mM IPTG, this clone produced A11 protein with a poly histidine affinity tag (His-tag) that facilitates the purification by Ni-NTA affinity column after protein refolding ( Fig.…”

“…(3,4) By screening a human phage display library with the GPIIIa49-66 peptide as bait, we developed several phagederived Abs against GPIIIa49-66 that exhibit comparable antigen-binding capacity. (5) In the present study, we have developed a scFv format (named A11) based on a previously reported phage antibody (clone 11). We demonstrate that 1.…”