Primary Recurrent Miscarriages: Anti–β2-Glycoprotein I IgG Antibodies Induce an Acquired Activated Protein C Resistance That Can Be Detected by the Modified Activated Protein C Resistance Test

To the Editor:

Using the study of affinity-purified IgG phospholipid-dependent inhibitors on the time-course of factor Va generation and inactivation, Galli et al1 demonstrated convincingly that anti–β2-glycoprotein I antibodies induce an acquired resistance to activated protein C (aPC), thus providing a new possible explanation for their thrombogenic potential.

We have studied the effect of various antiphospholipid IgG antibodies on the activated protein C-mediated factor Va proteolysis (aPC-FV) using the modified aPC-resistance test that includes predilution of patient plasma in factor V depleted plasma, according to Jorquera et al2 (Coatest APC Resistance V; Chromogenix, Mölndal, Sweden; results given as aPC-FV ratios:time obtained in the presence of aPC divided by the time obtained, using the same plasma, in absence of aPC).

All patients were nonthrombotic young women who had experienced primary unexplained recurrent miscarriages. They had been controlled to be negative for the R506Q (factor V Leiden) mutation. Lupus anticoagulant activity (LA) had been tested according to the revised criteria proposed by the Scientific Standardization Committee Subcommittee for Standardization of Lupus Anticoagulant.3 Anticardiolipin (aCLAb) and anti–β2-glycoprotein I (aβ2GP1Ab) IgG antibodies had been tested using commercially available assays (Quanta Lite [Inova Diagnostics Inc, San Diego, CA] and Varelisa β2GP1 [Elias, Freiburg, Germany]). Antiprothrombin (aFIIAb) and anti-annexinV (aAnVAb) IgG antibodies had been assayed using enzyme-linked immunosorbent assay research kits (gift from J. Amiral, Diagnostica Stago, Asnières, France). All normal values were obtained using 200 control plasmas assuming that threshold values corresponded to the 99th percentiles. All patient plasmas were tested crude and after IgG depletion using protein A-sepharose. We systematically performed mixing studies using a 1:1 mixture of patient and normal plasma. Normal plasma has been prepared pooling the 200 control plasmas and was used crude (NP) or β2-glycoprotein I depleted after immunoaffinity chromatography (NP-β2neg).

We identified 42 patients with positive antiphospholipid antibody-related markers: 10 patients with an isolated LA (7 with a KCT-type LA and 3 with a dRVVT-type LA according to Galli et al1), 10 patients with isolated aCLAb (levels ranging from 25 to 68 GPL units, normal values lower than 14.5 GPL units), 10 patients with isolated aβ2GP1Ab (levels ranging from 42 to 123 U/mL, normal values lower than 14.5 U/mL), 2 patients with isolated aFIIAb, and 10 patients with isolated aAnVAb (Table1). We observed that plasmas with isolated positive aβ2GP1Ab were the only ones to be associated with an acquired resistance to aPC, which disappeared in cases of IgG depletion. These plasmas generated resistance to aPC during mixing studies and the intensity of the inhibitory activity depended on the β2-glycoprotein I content of the normal plasma. We thereafter supplemented NP and NP-β2neg with IgG purified from plasmas with isolated positive anti–β2-glycoprotein I IgG antibodies (3:1; IgG concentration, 10 mg/mL in Tris-buffered saline). An acquired plasma resistance to aPC was induced only in the presence of plasma β2-glycoprotein I (median values and ranges of aPC-FV ratio: NP, 1.59 [1.19-1.88]; NP-β2neg, 2.22 [1.98-2.45]; P = .005; control values using IgG purified from normal plasma: NP, 2.30 [2.05-2.55], NP-β2neg, 2.28 [2.00-2.49]; nonsignificant).

Thus, the modified aPC-resistance test, together with a mixing study performed in this test, can easily allow us to identify the acquired anticoagulant activity acting as an inhibitor of the aPC-mediated factor Va proteolysis. Concerning women with recurrent miscarriages positive for antiphospholipid antibody-related markers, only aβ2GP1Ab is related with the impairment of the anti-factor V activity of aPC. This finding is concordant with data shown by Galli et al1 in their group of patients with mainly thrombotic antecedents. If acquired aPC resistance represents a pathogenic mechanism responsible for the clinical risk in a subgroup of antiphospholipid-positive patients, the modified aPC-resistance test should allow to identify plasmas positive for this peculiar anticoagulant activity.