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In Vitro LA Mechanism

From Kathy Doig, PhD, Michigan State University (Go Spartans!). George – what is the current thinking on how LA interferes with our assays?  Does the antibody physically block the proteins so they can’t react properly even though they embed in the phospholipids (PLs)?  Does it prevent them from embedding in the PLs as they should?  Or is it something else entirely?

Dr. Doig gets these penetrating questions from her graduate students, sending us back to the books for answers. Here is George’s effort at a reply:

Kathy, I found no information describing the in vitro sequence of events, though numerous references theorize that the in vivo pathophysiology starts with LA binding plasma β-2 glyprotein I (β-2GPI, also annexin or prothrombin) generating an allosteric change that causes β-2GPI to bind endothelial cell phospholipids (platelets, in the case of prothrombin). The endothelial response then seems to cause thrombosis. One theory considered a reduction in endothelial nitric oxide. Here is a 2013 review: Willis R, Pierangeli SS. Anti-β2-glycoprotein I antibodies. Ann N Y Acad Sci. 2013;1285:44-58 that is a gateway to several primary research articles.

The apparent lack of data allows me to make up a parallel in vitro sequence. In the assay the LA-modified β-2GPI binds reagent PL and limits its availability so that both the VIIIa-IXa and the Xa-Va complex formation are reduced and the PTT is prolonged. The theory assumes that native β-2GPI does not bind PL.

The PTT reagents we employ for routine screening and heparin monitoring, like HemosIL aPTT-SP contain moderate to large PL concentrations so there is plenty of PL left over to support coagulation, but LA-sensitive reagents like Siemens Actin FSL are formulated with low PL concentrates designed to be “used up” when LA is around. Please see our article, Fritsma GA, Dembitzer FR, Randhawa A, et al. Recommendations for appropriate activated partial thromboplastin time reagent selection and utilization. Am J Clin Pathol 2012;137:904–8.

When you perform the enzyme immunoassay for anti-cardiolipin antibody, there is β-2GPI in the reaction mix, so that is what you are really detecting, and that is why the anti-β-2GPI test kit is more sensitive and specific for the antibody.

Realizing that my in vitro mechanism is theoretical, I welcome comments and updates from our many well-informed participants.

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