Molecular characterization of a human monoclonal antibody that interacts with a sulfated tyrosine-containing epitope of the GPIb receptor and inhibits platelet functions.

Modification of tyrosine residues in extracellular proteins by a sulfate moity plays an important role in many ligand/receptors interactions. In the present work, we describe a unique human monoclonal antibody, termed Y1-scFv, that is specific for a sulfated epitope in the platelat receptor GPIb. The Y1-scFv single chain antibody (scFv) competes with von Willebrand factor (vWF) for binding to human platelets and thus effectively inhibits platelet aggregation. Limited proteolysis of GPIb molecule, using the endoproteases, mocarhagin and cathepsin G, revealed that a seven amino-acid epitope, Tyr-276 to Glu-282, contains the recognition site for Y1-scFv. This GPIb region contains three sulfated tyrosine residues. Binding studies of Y1-scFv to cells and to synthetic peptides in vitro indicated that of the seven residues comprising the epitope only sulfo-Tyr-276 and adjacent Asp-277 are critical for the interaction. To identify the reciprocal sequences in the antibody that recognize the sulfated epitope, we introduced mutations within the complementary-determining region of the heavy chain (CDR3H) of Y1-scFv (MRAPVI). Arginine residue in the second position was critical for the binding. Moreover, a mutant, containing two sequential arginine residues, in the second and third positions of the CDR3H (MRRPVI), showed a nine-fold increased binding to GPIb. This antibody mutant also demonstrated a significant increase in inhibition of vWF-dependent platelet aggregation and adhesion under flow. In conclusion, this unique antibody and mutants, that recognize a sulfated epitope in GP1b receptor, efficiently inhibited platelet adhesion and aggregation, making it a candidate for a new anti-thrombotic agent.

Impaired platelet aggregation in melanoma patients treated with interferon-alpha-2b adjuvant therapy.

BACKGROUND: High-dose interferon (INF)- alpha-2b is the only Food and Drug Administration-approved adjuvant treatment for patients with melanoma who are at high risk of recurrence. Although circumstantial evidence points to a potentially harmful effect of INF-alpha-2b on platelet function, to the authors' knowledge this has never been studied in humans. METHODS: The study group was comprised of patients who had undergone surgery for melanoma and were free of disease but at a high risk of recurrence. All patients were candidates for adjuvant INF treatment (high-dose) and were undergoing routine evaluation to which platelet aggregation was added. Aggregation was triggered in standard fashion with adenosine diphosphate, epinephrine, collagen, thrombin, arachidonic acid, and ristocetin. Blood samples were drawn immediately before treatment, during the intravenous loading phase, during the subcutaneous maintenance phase, and 3-6 weeks after cessation of treatment. Patients receiving low-dose, long-standing INF-alpha-2b treatment also were tested. All results at each phase were compared with those of normal controls. RESULTS: In those patients receiving high-dose INF-alpha-2b, ristocetin-induced aggregation did not appear to be affected. However, the response to > or = 1 of the other agonists was impaired in 5 of 6 samples during loading, 14 of 15 samples during the maintenance phase, and 8 of 13 samples after treatment, compared with only 1 of 8 samples before treatment (P = 0.025, P = 0.002, and P = 0.067, respectively). During treatment with low-dose INF, platelet function was affected to a lesser extent. CONCLUSIONS: INF treatment in melanoma patients appears to be associated with severe impairment of platelet aggregation, which appears to be dose-dependent and cumulative-dose-dependent. This is not detectable by the standard coagulation profile. This effect has significant implications in the event of accidental injury or elective surgery. The antiaggregation activity may be the mechanism by which INF delays, reduces, or prevents the formation of melanoma metastases.