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

Abstract

Heparin has traditionally been a widely used anticoagulant in blood research, but has been shown to be inappropriate for work with the complement system because of its complement-interacting properties. In this work, we have compared the effects of heparin with those of the specific thrombin inhibitor hirudin on complement and blood cells in vitro. Whole blood collected in the presence of hirudin (50 microg/mL) or heparin (1 IU/mL) was incubated in the slide chamber model. The plasma was analyzed for complement activation markers C3a and sC5b-9, and the polyvinylchloride test slides were stained for adhering cells. The integrity of the complement system was tested by incubating serum and hirudin-treated plasma in the presence of various activating agents. In contrast to heparin, the addition of hirudin generally preserved the complement reactivity, and complement activation in hirudin plasma closely resembled that in normal serum. Importantly, immunochemical staining of surface-bound cells demonstrated the inducible expression of tissue factor on bound monocytes from hirudin-treated blood, an effect that was completely abolished in heparin-treated blood. Our results indicate that hirudin as an anticoagulant produces more physiological conditions than heparin, making hirudin well-suited for in vitro studies, especially those addressing the regulation of cellular processes.

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

“…Most commonly used anticoagulants are heparin for whole blood studies and sodium citrate for studies that focus on platelet-biomaterial interactions in vitro [52,55]. More recently, hirudin appears in an increasing number of hemocompatibility studies and clinically applied routine test systems [56,57]. The decision for a specific anticoagulant should be driven by the clinical background, the choice of tested blood (e.g.…”

“…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].…”

Human Endothelial Cell Activation by Escherichia coli and Staphylococcus aureus Is Mediated by TNF and IL-1β Secondarily to Activation of C5 and CD14 in Whole Blood

“…As a necessary limitation of the model, coagulation has to be prevented. However, using the thrombin-specific agent lepirudin, only the end stage of the coagulation process is inhibited, providing a more physiological environment compared with the more commonly used heparin (23). HUVEC do not express ABO Ags, and consequently ABO matching between EC and whole blood donors is not necessary (24).…”

Biocompatibility and Biotolerability Assessment of Microspheres Using a Whole Blood Model

“…In addition, initial coagulation activation up-stream thrombin, can still be measured, including thrombin cleavage products [76]. Tissue factor, the main inducer of the extrinsic pathway, is still inducible on monocytes using lepirudin as anti-coagulant [45] while not by soluble heparin [77]. Thrombin might have some impacts on leukocyte performance, as monocytes chemotactic responses [78] and IL-8 secretion from PBMCs [79].…”