Glycocalicin: a new assay--the normal plasma levels and its potential usefulness in selected diseases

Abstract

Platelet glycocalicin (GC) is the extramembranous portion of GPIb alpha 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 micrograms/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 10(9)/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 micrograms/mL, P < .001). The high GC index (6.93 +/- 4.21, P < .001) in cirrhosis patients suggests an increased platelet turnover and/or abnormal proteolysis. In contrast to other groups, we have not found that recombinant tissue plasminogen activator (rtPA) treatment of patients with myocardial infarction increases GC levels. However, concentrations are elevated in leukemia and the highest levels found are approximately 40 micrograms/mL. These studies suggest that GC is a useful platelet marker in certain diseases, which directly reflects platelet damage and possibly platelet dysfunction.

The role of thrombopoietin in post‐operative thrombocytosis

“…Although platelet counts decreased immediately after surgery and gradually increased over time, no change in the GC concentration was observed. Previously, GC has been shown to be a marker for platelet turnover and is thought to be enzymatically cleaved from the platelet during destruction (Coller et al, 1984;Steinberg et al, 1987;Beer et al, 1994). It is probable that the follow-up of the patients in this study was too short to detect an increment in GC concentration because the newly produced platelets were still viable at the end of the follow-up.…”

High levels of reticulated platelets and thrombopoietin characterize fetal thrombopoiesis

“…If the observed correlation between GC levels in the fetuses and mothers was not merely by chance, it may indicate a placental transfer of GC. However, this is rather unlikely for a protein with a MW of 135 kDa (Beer et al, 1994) unless there is an active transport system, as has been observed for IgG.…”

Platelet activation mechanisms and markers in arterial thrombosis

“…It is unclear, however, whether these larger platelets are responsible for platelet hyperreactivity. Another marker, plasma glycocalicin, the extramembranous portion of GPIb, has recently been investigated by an enzymeimmunoassay [69]. It may have the potential for use as a marker of platelet damage [69].…”