Activity of Maleimide Functionalized Unfractionated and Low Molecular Weight Heparins

The development of modern anti-coagulant high performance surface coatings can require the immobilization of heparin in a modular orthogonal fashion. The precision of current commercial technologies as the Carmeda® BioActive Surface coating for this application is limited. Here we present the use of click chemistry as a potential approach for the immobilization of maleimide functionalized heparin molecules to thiol decorated surfaces. Thereby the influence of maleimide functionalization on the anti-coagulant activity was investigated and compared for different heparins of varying molecular size.

Three different LMWH (enoxaparin, certoparin and tinzaparin) as well as two UFH (Merck, ratiopharm) were functionalized with N-(2-aminoethyl)maleimide TFA to a final degree of one maleimide group/heparin molecule. Functionalization degree was analyzed by HPSEC and NMR analysis. To determine reactivity of the maleimide moieties binding of TNB^2- as well as an RGDSP peptide was measured by substrate consumption. Inhibitory activities of the unmodified and functionalized heparin molecules for FIIa and FXa were measured as chromogenic assay in a buffer system. Besides heparins were incubated with whole blood together with coagulation stimulating or non-stimulating surfaces and analyzed for their inhibitory potential and hemocompatible properties.

For all five tested heparin molecules, a functionalization degree of one reactive maleimide per heparin molecule was achieved. The dependence of anti-FIIa activity on the heparin molecule size was confirmed, but anti-FXa and anti-FIIa activity were not affected by the maleimide functionalization. Upon exposure of blood heparinized with comparable anti-FXa units under coagulant or non-coagulant conditions, a heparin size dependent clot formation was detected. Maleimide functionalized compounds did not induce increased cell activation or inflammatory response. However, the maleimide moiety triggered an immediate loss of heparin activity upon its addition to whole blood and an increased clot formation.

Heparin molecule size dependent clot formation of heparinized whole blood under coagulant conditions

The functionalization of heparins with a reactive maleimide moiety offers a potential strategy for surface immobilization of both UFH and LMWH. The approach does not affect the heparins potential to inhibit the coagulation factors FIIa and FXa, but an immediate loss of activity was detected for the functionalized compounds in whole blood.