Effect of Hirudin versus Heparin on Hemocompatibility of Blood Contacting Biomaterials: An in Vitro Study

Abstract

Hirudin serves as an alternative anticoagulant for extracorporeal blood circulation. Comparing anticoagulation with hirudin (2.5 or 5.0 μg/mL) and heparin (2.0 or 4.0 IU/mL) human blood was circulated in a modified “Chandler System” using PVC-tubes for 2 hours at 37°C. Activation of coagulation (thrombin-antithrombin III-complex, prothrombin fragment 1+2 and D-Dimer), platelet (platelet factor 4 - PF4) and complement systems was analyzed. Both heparin concentrations and 5.0 μg/dL hirudin led to as significantly less activated plasmatic coagulation as 2.5 μg/dL hirudin. Decreased levels of PF4 and anaphylatoxin C5a (P&0.05) as well as terminal complement complex demonstrated improved hemocompatibility after anticoagulation with heparin in contrast to hirudin. Because initial coagulation cascade, platelet activation and complement activation is less influenced by hirudin than by heparin, hemocompatibility is more dependent on the characteristics of the biomaterials used. This predestines hirudin as anticoagulant for in vitro studies analyzing hemocompatibility of biomaterials or surface modifications.

Hirudin versus heparin for use in whole blood in vitro biocompatibility models

“…In the presence of hirudin, there was not enough thrombin available to accomplish platelet activation, resulting in a significantly lower platelet loss than in the heparin‐containing samples. These results are similar but not identical to those reported by Kopp et al, who used the Chandler loop system to compare the behavior of hirudin and heparin in whole blood 28. There are, however, large differences in the concentrations of the tested compounds in the two studies; their study was conducted using hirudin at lower doses (2.5 and 5 μg/mL, compared to 50 μg/mL used here) and heparin at higher doses (2 and 4 IU/mL compared to 1 IU/mL used here).…”

“…We have previously studied the interaction between blood cells and artificial surfaces in a chamber model with whole blood using low‐dose heparin as the anticoagulant 15. Now we have further developed this model by replacing heparin, which affects both the coagulation and the complement systems, with the specific thrombin inhibitor hirudin, which has been reported to have minimal effect on complement activation 26, 28. The difference in anticoagulation was evaluated by performing parallel experiments with whole blood anticoagulated with low‐dose heparin (1 IU/mL) and hirudin (50 μg/mL).…”

“…Hirudin is available in recombinant form under the name lepirudin. The fact that it exclusively binds thrombin and does not affect the rest of the coagulation cascade, the complement cascade, or blood cells makes hirudin ideal for work in inflammatory research 26, 28…”

Are there sufficient standards for the in vitro hemocompatibility testing of biomaterials?

“…These assays focus on the platelet-biomaterial interactions and are well suited to assess the thrombogenic potential of a material, even though, sodium citrate is not applied as a systemic anticoagulant. For in vitro studies focusing on complement activation, anticoagulation with hirudin (commercially available as lepirudin or bivalirudine [68]) appears to mimic physiological conditions more appropriately than other anticoagulants [56,57,69].…”

Obstacles in Haemocompatibility Testing

“…Platelet activation contributes to clotting by not only exposure of activated membranes and release of clotting factors but also by release of platelet factor 4, which is neutralising heparin. Some in vitro models require other methods of anticoagulation or no anticoagulants at all [25]. Platelet studies require sometimes PPACK26, to inhibit only formed thrombin, whereas hirudin shows less aspecific inhibition than heparin [26].…”