Glycocalicin: A new assay - The normal plasma levels and its potential usefulness in selected diseases

Abstract

Platelet glycocalicin (GC) is the extramembranous portion of GPIbα that can be rapidly cleaved by enzymes such as calpain, plasmin, trypsin, elastase, etc. Quantitative cleavage will ultimately result in an acquired Bernard-Soulier-like bleeding disorder, and circulating GC may act as a potential inhibitor of platelet adhesion. We have developed and standardized a new enzyme-linked immunosorbent assay (ELISA), which uses two monoclonal antibodies (mAbs), both of which bind to the amino-terminal 45-kD fragment of GC and inhibit platelet-von Willebrand interactions and the streptavidin- biotin system. First, the methodology was evaluated and standardized with special emphasis on the anticoagulant and the inhibitors (EDTA, prostaglandin E1 [PGE1] aprotinin, N-ethyl-maleimide), the mode of high-speed centrifugation (to avoid platelet microparticles), and the standards used (purified GPIb and GC). This assay was then used to analyze the GC levels of healthy subjects (2.04 ± 0.46 μg/mL) and of patients with selected diseases. The results of patients with aplastic anemia and thrombocytosis confirmed that GC levels are clearly dependent on the platelet count, which was the basis for the introduction of the GC index, the standardization of GC for a platelet count of 250 x 109/L. The GC index discriminates reliably patients with active immune thrombocytopenic purpura from those in remission. GC levels are elevated in patients on hemodialysis (3.62 ± 0.75 μg/mL, P < .001). The high GC index (6.93 ± 4.21, P