Introduction
The anticoagulant glycosaminoglycan heparin binds to the plasma protein antithrombin, potentiating its activity and so inhibiting the coagulation cascade. Among other therapeutic applications, it is used to prevent and treat deep vein thrombosis. As heparin binding is an attribute shared by many proteins other than antithrombin, sequestration by plasma proteins could potentially reduce the available concentration of therapeutically administered heparin, and thus modulates its biological activity. Although the heparin-binding ability of some abundant plasma proteins is well characterized, the binding potential of the vast majority of plasma constituents is unknown. We have therefore initiated a systematic study of the heparin-binding plasma proteome, using affinity capture on immobilized heparin, followed by salt elution and protein identification by mass spectrometry.
Materials and methods
Five platelet-depleted human plasma pools were fractionated by affinity chromatography on HiTrap heparin columns. Bound material was eluted stepwise with 0.5 m, 0.7 m, 1.2 m and 2.2 m NaCl. The approximate protein concentration of each plasma pool and fraction was determined by optical density reading at 280 nm. The fractions were separated by SDS-PAGE and protein bands were visualized by staining with Coomassie blue or silver nitrate. Stained bands were excised, digested in gel with trypsin, and the liberated peptides were analysed by tandem electrospray mass spectrometry using a Micromass Q-Tof. Proteins were identified by correlation of the uninterpreted tandem mass spectra to entries in the UniProt database.
Results
Over 50 proteins were identified in whole plasma. No systematic differences in protein composition were observed between the different plasma pools. Heparin-binding proteins recovered in salt washes of 0.7 m or above included fibronectin, histidine-rich glycoprotein, apolipoproteins, complement factors C3 and C4b, and antithrombin III. Antithrombin was the major protein eluting at 1.2 m NaCl or above.
Discussion
The rationale of this study was to investigate plasma for the presence of proteins potentially able to compete with antithrombin for therapeutically administered heparin. Of over 50 different plasma proteins identified in this preliminary study, only antithrombin remained bound to heparin above 1.2 m NaCl. We anticipate that use of larger volumes of albumin- and immunoglobulin-depleted plasma will reveal low concentrations of additional high-affinity binding proteins, but these are unlikely to bind significant amounts of heparin. However, in the present study, we detected several abundant proteins, which bound to immobilized heparin at 0.7 m NaCl, and these may reduce the bioavailability of heparin by nonspecific binding.