Activated Protein C-Resistance Induced by a Low Molecular Weight Inhibitor.

Activated protein C (APC) inactivates factor Va (fVa) following three cleavages in the heavy chain at R⁵⁰⁶, R³⁰⁶ and R⁶⁷⁹. Cleavage at R⁵⁰⁶ precedes cleavage at R³⁰⁶. Cleavage at Arg³⁰⁶ is strictly lipid-dependent and results in total inactivation of the factor Va molecule with dissociation of fragments from the A2 domain from the rest of the molecule. Factor V^Leiden is associated with an R→Q substitution at position 506 and is present at approximately 8% of the Caucasian population. The heterozygous presentation of factor V^Leiden results in delayed inactivation of factor Va and “APC-resistance” with attendant increased risk of venous thrombosis. However, not all cases of “APC-resistance” are explained by factor V^Leiden. We observed “APC-resistance” in a patient displaying heterozygous factor V^Leiden, Waldenstrom’s macroglobulinenemia, systemic lupus erythrematosus (anticoagulant) and a history of coronary artery disease. The patient’s plasma resistance to APC inactivation was not repaired by immunodepletion of his factor V^Leiden and replacement by normal plasma factor V. Conversely when the patient’s fVa was returned to factor V immunodepleted normal plasma it did not display APC-resistance. Cleavage of the patient’s plasma fVa at R³⁰⁶ was not detected following prolonged incubation of his clotted plasma at 37°C even when 2 nM APC was added following clotting. These data suggested that the APC-resistance observed in the patient was not due to the presence of factor V^Leiden, but due to some property which inhibited the lipid dependent cleavage at Arg³⁰⁶. The patient’s plasma was depleted of IgG/IgM and the purified immunoglobulin fraction assessed for inhibition of APC cleavage and inactivation of fVa in a system using purified reagents. The data were compared with the inhibition of fVa inactivation by APC by an IgG/IgM fraction obtained from normal plasma under similar experimental conditions. No inhibition of APC cleavage and inactivation of fVa by the IgM/IgG fraction obtained from either plasma were observed. In addition, the fVa molecule contained in the IgM/IgG-depleted patient plasma was still resistant to cleavage and inactivation by APC. Following dialysis the patient’s plasma lost its ability to inhibit fVa cleavage and inactivation by APC. Overall these studies indicate that inhibition of fVa cleavage and inactivation by APC in the patient’s plasma is caused by a hitherto undescribed metabolite of low molecular weight. The mechanism of action of this metabolite is not yet known, but the evidence suggests that the metabolite interferes with the lipid-dependent cleavage and inactivation of fVa by APC at R³⁰⁶. These data demonstrate the existence of an as yet unknown APC inhibitor of low molecular weight in the plasma of a patient with lupus anticoagulant and severe thrombotic symptoms.