Quantitative description of thermodynamic and kinetic properties of the platelet factor 4/heparin bonds

Abstract

Heparin is the most important antithrombotic drug in hospitals. It binds to the endogenous tetrameric protein platelet factor 4 (PF4) forming PF4/heparin complexes which may cause a severe immune-mediated adverse drug reaction, so-called heparin-induced thrombocytopenia (HIT). Although new heparin drugs have been synthesized to reduce such a risk, detailed bond dynamics of the PF4/heparin complexes have not been clearly understood. In this study, single molecule force spectroscopy (SMFS) is utilized to characterize the interaction of PF4 with heparins of defined length (5-, 6-, 8-, 12-, and 16-mers). Analysis of the force-distance curves shows that PF4/heparin binding strength rises with increasing heparin length. In addition, two binding pathways in the PF4/short heparins (≤8-mers) and three binding pathways in the PF4/long heparins (≥8-mers) are identified. We provide a model for the PF4/heparin complexes in which short heparins bind to one PF4 tetramer, while long heparins bind to two PF4 tetramers. We propose that the interaction between long heparins and PF4s is not only due to charge differences as generally assumed, but also due to hydrophobic interaction between two PF4s which are brought close to each other by long heparin. This complicated interaction induces PF4/heparin complexes more stable than other ligand-receptor interactions. Our results also reveal that the boundary between antigenic and non-antigenic heparins is between 8- and 12-mers. These observations are particularly important to understand processes in which PF4-heparin interactions are involved and to develop new heparin-derived drugs.

Carbohydrate-Carbohydrate Interactions Mediated by Sulfate Esters and Calcium Provide the Cell Adhesion Required for the Emergence of Early Metazoans

“…Complex multiple binding events have been reported in other interactions involving carbohydrates, such as the interaction between heparin and the protein platelet factor 4, which is mediated by both electrostatic and hydrophobic bonds (52). Other example are the three different stable binding conformations formed between ricin and anti-ricin aptamer (53).…”

Not Only Heparin but Also Antibody Induces Thrombocytopenia

“…Based on special features in force-distance curves and the magnitude of PF4/H binding forces, it has been proved that long heparin bound PF4s creating additional PF4-PF4 bonds [35]. Long heparins form two types of bonds with PFs, i.e.…”

“…K A ) of PF4/H complexes is calculated. The short heparins bind to PF4s with ~10-20 times faster than long heparins [35].…”

“…Even though there is some variation in the parameters obtained by these two models, Bell-Evans model is still a powerful tool to determine the kinetics of ligand-receptor interactions [41,42]. For the PF4/H system, the PF4 tetramer is considered as one antigen or the interaction between heparin and PF4 is formed by a single bond, and therefore, applicable to the Bell-Evans model [35]. Short heparins show higher k off values than long heparins, indicating that PF4/long heparin complexes are more stable than PF4/short heparin complexes ( Table 1).…”

“…This boundary has been determined by applying multiple techniques such as atomic force microscopy-based atomic force microscopy (AFS) [35], isothermal titration calorimetry (ITC) [6], or circular dichroism (CD) spectroscopy in combination with enzyme-linked immunosorbent assay (ELISA) [7] (Figure 2). AFS shows that both numbers of specific rupture events and magnitude of rupture forces rise with an increase of heparin length, suggesting that long heparins form with PF4 more bonds than short ones [35]. A larger variation of the rupture forces for long heparins ≥8-mer compared with short heparins ≤6-mer was observed ( Figure 3A).…”