Affinity Binding Sites For Factor Research Articles

Complement Resistance of Borrelia burgdorferi Correlates with the Expression of BbCRASP-1, a Novel Linear Plasmid-encoded Surface Protein That Interacts with Human Factor H and FHL-1 and Is Unrelated to Erp Proteins

The etiologic agent of Lyme disease, Borrelia burgdorferi, is capable of circumventing the immune defense of a variety of potential vertebrate hosts. Previous work has shown that interaction of host-derived complement regulators, factor H and factor H-like protein 1 (FHL-1), with up to five complement regulator-acquiring surface proteins (CRASPs) expressed by resistant B. burgdorferi sensu lato isolates conferred complement resistance. In addition expression of CRASP-1 is directly correlated with complement resistance of Borrelia species. This work describes the functional characterization of BbCRASP-1 as the dominant factor H and FHL-1-binding protein of B. burgdorferi. The corresponding gene, zs7.a68, is located on the linear plasmid lp54 and is different from factor H-binding Erp proteins that are encoded by genes localized on circular plasmids (cp32). Deletion mutants of BbCRASP-1 were generated, and a high affinity binding site for factor H and FHL-1 was mapped to the C terminus of BbCRASP-1. Similarly, the predominant binding site of factor H and FHL-1 was localized to the short consensus repeat 7. Factor H and FHL-1 maintain their cofactor activity for factor I-mediated C3b inactivation when bound to BbCRASP-1, and factor H is up to 6-fold more efficient in mediating C3b conversion than FHL-1. In conclusion, BbCRASP-1 (i). binds the host complement regulators factor H and FHL-1 with high affinity, (ii). is the key molecule of the complement resistance of spirochetes, and (iii). is distinct from the Erp protein family. Thus, BbCRASP-1 most likely contributes to persistence of B. burgdorferi and to pathogenesis of Lyme disease.

Collagen-bound von Willebrand Factor Has Reduced Affinity for Factor VIII

von Willebrand factor (vWf) is a multimeric adhesive glycoprotein that serves as a carrier for factor VIII in plasma. Although each vWf subunit displays a high affinity binding site for factor VIII in vitro, in plasma, only 2% of the vWf sites for factor VIII are occupied. We investigated whether interaction of plasma proteins with vWf or adhesion of vWf to collagen may alter the affinity or availability of factor VIII-binding sites on vWf. When vWf was immobilized on agarose-linked monoclonal antibody, factor VIII bound to vWf with high affinity, and neither the affinity nor binding site availability was influenced by the presence of 50% plasma. Therefore, plasma proteins do not alter the affinity or availability of factor VIII-binding sites. In contrast, when vWf was immobilized on agarose-linked collagen, its affinity for factor VIII was reduced 4-fold, with KD increasing from 0.9 to 3.8 nM. However, one factor VIII-binding site remained available on each vWf subunit. A comparable reduction in affinity for factor VIII was observed when vWf was a constituent of the subendothelial cell matrix and when it was bound to purified type VI collagen. In parallel with the decreased affinity for factor VIII, collagen-bound vWf displayed a 6-fold lower affinity for monoclonal antibody W5-6A, with an epitope composed of residues 78-96 within the factor VIII-binding motif of vWf. We conclude that collagen induces a conformational change within the factor VIII-binding motif of vWf that lowers the affinity for factor VIII.

Phosphatidylethanolamine Induces High Affinity Binding Sites for Factor VIII on Membranes Containing Phosphatidyl-L-serine

Synthetic membranes of phosphatidylcholine require inclusion of at least 5% phosphatidylserine (Ptd-L-Ser) to form binding sites for factor VIII. The relatively high requirement for Ptd-L-Ser suggests that stimulated platelets may contain another membrane constituent that enhances expression of factor VIII-binding sites. We report that phosphatidylethanolamine (PE), which is exposed in concert with Ptd-L-Ser in the course of platelet stimulation, induces high affinity binding sites for factor VIII on synthetic membranes containing 1-15% Ptd-L-Ser. The affinity of factor VIII for binding sites on membranes of Ptd-L-Ser/PE/phosphatidylcholine in a 4:20:76 ratio was 10.2 +/- 3.5 nM with 180 +/- 33 phospholipid molecules/site. PE did not induce binding sites on membranes of 4% Ptd-D-Ser, indicating that the induced binding sites require the correct stereochemistry of Ptd-L-Ser as well as PE. Egg PE and dimyristoyl-PE were equivalent for inducing factor VIII-binding sites, indicating that hexagonal phase-inducing properties of PE are not important. We conclude that PE induces high affinity factor VIII-binding sites on membranes with physiologic mole fractions of Ptd-L-Ser, possibly including those of stimulated platelets.