Small Molecule Compounds for Heparin Reversal and Their Use in Heparin Quantitation

Background: Heparin is a class of naturally occurring anticoagulants frequently used to prevent blood clots. Heparins are rapidly reversed by protamine, but this approach has limitations, including protamine’s short half-life and hemorrhagic risk if used in excess, emphasizing the need for alternative reversal agents. Here, we screened a library of 262 glycan-interacting small molecules of diverse chemical structures, to identify those capable of binding heparin and blocking its anticoagulant activity.

Methods: 262 compounds were screened for their ability to inhibit heparin anticoagulant activity in plasma-based assays. Two of the compounds possessed fluorescent properties and were further tested for their ability to measure heparin concentration in plasma.

Results: Initial screens were performed using a thrombin-initiated fibrin formation assay, which identified 7 compounds capable of reversing the anticoagulant activity of unfractionated heparin. Subsequent studies, utilizing fibrin formation and tissue factor- or kaolin-initiated thrombin generation, sought to assess their efficacy as protamine alternatives. Compounds GTC-3155 and GTC-3302 were most potent, completely reversing unfractionated heparin in all systems, but exhibited some heparin-independent anticoagulant activity at high concentrations (100 μM). GTC-3062 and GTC-3308 restored fibrin formation, partially restored thrombin generation, and did not have any apparent heparin-independent activity. These compounds were next evaluated against low molecular weight heparin. While all four compounds substantially reversed unfractionated heparin, only GTC-3155 and GTC-3062 were effective with low molecular weight heparin. Based on these results, GTC-3062, a quinazoline derivative shows the most promise for therapeutic use. GTC-3062 has acceptable metabolic stability in vitro, a plasma half-life of ~35min in C57BL/6N mice, and no apparent acute toxicity when injected up to 300 mg/kg. Direct binding of GTC-3062 to heparin was revealed as a hypochromic effect in UV absorbance and CD studies.

GTC-3155, a carbazole derivative, also possesses fluorescent properties, exciting at 350nm and emitting at 450nm, suggesting potential use for heparin detection and quantification. GTC-3155 binds directly to heparin, as revealed in fluorescence studies. Heparin binding, either in buffer or human plasma, reduced the emission in a dose-dependent manner (0.2-2U/mL), allowing for quantitation within this range. Thus, GTC-3155 may be useful for monitoring heparin dose, and for measurement of non-anticoagulant heparins, which cannot be monitored by anti-Xa activity.

Conclusions: Our data support the hypothesis that compounds which target the glycan moieties in heparin may be safer than protamine for reversing heparin anticoagulant activity. Notably, GTC-3062 partially reversed anticoagulation, restored fibrin formation, was effective against both unfractionated and low molecular weight heparins, and did not exhibit heparin-independent activity. The therapeutic potential of these compounds will next be evaluated using in vivo hemostatic and thrombotic mouse models.