Background/Purpose:

Antiphospholipid Syndrome (APS) is an autoimmune rheumatic
disorder in which antiphospholipid antibodies (aPL)
cause clinical events including vascular thrombosis (VT) and pregnancy
morbidity (PM). In clinical practice aPL are detected
by three tests, the anti-cardiolipin and
anti-beta-2-glycoprotein I (beta2GPI) ELISAs and the lupus anticoagulant (LA)
test. The LA test measures prolongation of clotting time caused by aPL present in the serum of patients. LA positivity is strongly
associated with thrombosis and is the strongest predictor of PM in patients
with APS. In testing potential efficacy of new therapeutic agents for APS it
would therefore be very useful to be able to assay their ability to inhibit the
LA effect of polyclonal IgG aPL
in samples from patients. Here we describe a novel method for carrying out such
tests and its application to testing PEGylated human recombinant domain I (DI) of beta2GPI. Recombinant DI has previously been shown to block
binding and pro-thrombotic properties of purified IgG antibodies from patients
with APS. PEGylation (chemical addition of polyethylene glycol) can increase
half-life and reduce immunogenicity of small molecules, enhancing their
potential for development as therapeutic agents. 

Methods:

We used a Sysmex-CA coagulometer with a dRVVT Plus
kit. The principle of the assay is that the sample to be tested
 is allowed to clot either in the presence or absence of a reagent
containing excess phospholipid (two step test). aPL
that cause an LA effect prolong clotting time in the LA sensitive  test
(LS) but not in the LA resistant test (LR) containing excess phospholipid. Thus
the ratio of LS to LR clotting time is a measure of LA effect. We purified IgG
from serum of patients with APS (n=4, all fulfilling Sydney criteria and known
to be LA-positive) and added it to normal human plasma at a concentration of
500mcg/ml. These samples were then tested in the coagulometer
assay either with or without pre-incubation with DI alone or DI PEGylated with
three different sizes of PEG (20kDa, 30kDa and 40kDa).

Results:

The figure shows results for all four patients individually.
In each case, purified IgG in the absence of inhibitor gave LS/LR ratio >
1.1, showing an LA effect. For all patients, addition of PEGylated DI reduced
the LA effect on LS/LR ratio and the effect of PEGylated DI was greater than
the effect of non-PEGylated DI in 3 out of 4 cases. This finding is
particularly interesting because PEGylation generally reduces biological
effects of small molecules. PEG alone has minimal effect.

Conclusion:

Using this assay, we demonstrated that it is possible to
test ability of a putative therapeutic agent for APS to inhibit the LA effect.
PEGylation of DI did not reduce its ability to inhibit the LA effect of IgG
purified from patients with APS but actually increased inhibition. This finding
may be beneficial in development of PEG-DI as a therapeutic.