Molecular characterization of the variable domains of an alphaIIbbeta3-specific immunoglobulin M kappa platelet cold agglutinin in a follicular lymphoma patient with treatment refractory autoimmune thrombocytopenia: idiotypic overlap between alphaIIbbeta3 integrin antibodies.

BACKGROUND: Cold hemagglutinins are generally immunoglobulin M (IgM) kappa antibodies reactive at temperatures below 37 degrees C and if of high titer may cause hemolysis. Platelet (PLT) cold agglutinins (CAs) are rare and poorly characterized. A detailed molecular characterization of the variable domains of a pathologic, PLT-reactive, CA is presented. CASE REPORT: A 70-year-old woman was admitted with rectal bleeding accompanied by widespread petechiae, bruising, tongue and buccal mucosa bleeding, and epistaxes and proved refractory to HLA- and HPA-matched PLTs. Detailed investigation showed monoclonal heavy-chain gene rearrangement with an IgM paraprotein of 3.3 g per L and a trace of kappa Bence Jones protein in the urine, compatible with a diagnosis of secretory B-cell non-Hodgkin's lymphoma (B-NHL). PLT antibody (PAIg) investigations revealed a potent IgM kappa PLT CA. Sequencing of the rearranged variable domain genes of the malignant clone together with idiotype-specific antibodies obtained by DNA-based immunization of rabbits and matrix-assisted laser desorption/ionization-time-of-flight analysis of the PAIgM provided a irrefutable link between the thrombocytopenia, the IgM paraprotein, and the PAIgM against alphaIIbbeta3. The thrombocytopenia and bleeding were refractory to standard treatment and PLT transfusion, but treatment with rituximab resulted in a recovery of the PLT count and a complete remission of B-NHL. CONCLUSION: The IgM kappa paraprotein derived from the malignant B-cell clone was a potent and clinically significant CA against alphaIIbbeta3. The testing for PLT CAs in patients with a paraprotein and refractory to matched PLTs may aid the selection of appropriate treatment.

Production of calmodulin-tagged proteins in Drosophila Schneider S2 cells: a novel system for antigen production and phage antibody isolation.

We report the development of an expression system for the production of soluble, calmodulin (CaM)-tagged proteins in Drosophila Schneider S2 cells and the subsequent use of these proteins for the selection of phage displayed antibodies. The CaM-tag permitted the purification of recombinant protein to >90% purity in a single step at yields of >20 mg/l. Using platelet glycoprotein VI (GP6) as a model, we demonstrated that the recombinant CaM-tagged protein was post-translationally N-glycosylated and had identical ligand specificity to native protein. A novel selection strategy, exploiting the CaM tag, was then used to isolate four single chain Fv fragments (scFvs) specific for GP6 from a non-immune phage display library. In contrast to other selection methods, which can result in antibodies that do not recognise native protein, all of the scFvs we selected bound cell surface expressed GP6. In conclusion, the production of CaM-tagged proteins in Drosophila Schneider S2 cells and the selection strategy reported here offer advantages over previously published methods, including simple culture conditions, rapid protein purification, specific elution of phage antibodies and preferential selection of phage antibodies that recognise native, cell surface expressed protein.

Cutting edge: TREM-like transcript-1, a platelet immunoreceptor tyrosine-based inhibition motif encoding costimulatory immunoreceptor that enhances, rather than inhibits, calcium signaling via SHP-2.

To date, immunoreceptor tyrosine-based inhibition motifs (ITIMs) have been shown to mediate inhibitory properties. We report a novel triggering receptor expressed on myeloid cells (TREM) family member, TREM-like transcript-1 (TLT1), which differs from the activating members because its cytoplasmic tail contains two ITIMs at Y245 and Y281. A TLT1 splice variant (TLT1sp) encodes a different cytoplasmic tail lacking ITIMs. Both isoforms are expressed in resting platelet alpha-granules, which are up-regulated to the cell surface following activation. TLT1 recruited Src homology 2 domain-containing tyrosine phosphatase (SHP)-2 to the "classical" ITIM (Y281) but not the "nonclassical" ITIM (Y245). In contrast to previously characterized ITIM receptors, TLT1 enhanced, rather than inhibited, FcepsilonRI-mediated calcium signaling in rat basophilic leukemia cells, a property dependent on the SHP-2 recruiting classical Y281 ITIM. Therefore, TLT1 represents a new costimulatory ITIM immunoreceptor and is the second ITIM-bearing receptor to be identified in platelets after platelet endothelial cell adhesion molecule-1.

Identification of the primary collagen-binding surface on human glycoprotein VI by site-directed mutagenesis and by a blocking phage antibody.

Glycoprotein (GP) VI is the major receptor responsible for platelet activation by collagen, but the collagen-binding surface of GPVI is unknown. To address this issue we expressed, from insect cells, the immunoglobulin (Ig)-like ectodomains (residues 1-185) of human and murine GPVI, called hD1D2 and mD1D2, respectively. Both proteins bound specifically to collagen-related peptide (CRP), a GPVI-specific ligand, but hD1D2 bound CRP more strongly than did mD1D2. Molecular modeling and sequence comparison identified key differences between hD1D2 and mD1D2. Ten mutant hD1D2s were expressed, of which 4 had human residues replaced by their murine counterpart, and 6 had replacements by alanine. CRP binding studies with these mutants demonstrated that the exchange of lysine at position 59 for the corresponding murine glutamate substantially reduced binding to CRP. The position of lysine59 on the apical surface of GPVI suggests a mode of CRP binding analogous to that used by the related killer cell Ig-like receptors to bind HLA. This surface was confirmed as critical for collagen binding by epitope mapping of an inhibitory phage antibody against GPVI. This anti-GPVI, clone 10B12, gave dose-dependent inhibition of the hD1D2-collagen interaction. Clone 10B12 inhibited activation of platelets by CRP and collagen in aggregometry and thrombus formation by the latter in whole blood perfusion. Antibody 10B12 showed significantly reduced binding to the hD1D2-E59, and, on that basis, the GPVI:10B12 interface was modeled.