Blood, 1 June 2002, Vol. 99, No. 11, pp. 3985-3992
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Isolation and characterization of an antifactor V antibody
causing activated protein C resistance from a patient with severe
thrombotic manifestations
Michael Kalafatis,
Paolo Simioni,
Daniela Tormene,
Daniel O. Beck,
Sonia Luni, and
Antonio Girolami
From the Department of Chemistry, Cleveland State
University, and the Department of Molecular Cardiology, The Lerner
Research Institute, The Cleveland Clinic Foundation, Cleveland, OH; and
Department of Medical and Surgical Sciences, Second Chair of Internal
Medicine, University of Padua, Padua, Italy.
A 44-year-old woman with a history of severe thrombotic
manifestations presented with a markedly reduced activated protein C-sensitivity ratio (APC-SR). DNA sequencing of and around the regions
encoding the APC cleavage sites in the factor Va molecule excluded the
presence of the factor VLeiden mutation and of other known genetic
mutations. No antiphospholipid antibodies were present in the
patient's plasma and both prothrombin time and activated partial
thromboplastin time were normal. The total immunoglobulin fraction was
isolated from the patient's plasma and found to induce severe APC
resistance when added to normal plasma and to factor V-deficient
plasma supplemented with increasing concentrations of factor V. Immunoblotting and immunoprecipitation experiments with the total
immunoglobulin fraction purified from the patient's plasma
demonstrated that the antibody recognizes factor V, is polyclonal, and
has conformational epitopes on the entire factor V molecule (heavy and
light chains, and B region). Thus, the immunoglobulin fraction
interferes with the anticoagulant pathway involving factor V. The
inhibitor was isolated by sequential affinity chromatography on protein
G-Sepharose and factor V-Sepharose. The isolated immunoglobulin fraction inhibited factor Va inactivation by APC because of impaired cleavage at Arg306 and Arg506 of the heavy chain of the cofactor. The
isolated immunoglobulin fraction was also found to inhibit the cofactor
effect of factor V for the inactivation of factor VIII by the
APC/protein S complex. Our data provide for the first time the
demonstration of an antifactor V antibody not related to the presence
of antiphospholipid antibodies, which is responsible for thrombotic
rather than hemorrhagic symptoms.
