Incompatibility between pig thrombomodulin (TM) and primate thrombin is thought to be an important factor in the development of microvascular thrombosis in rejecting pig-to-primate xenografts. To examine this interaction at the molecular level, we cloned pig TM and measured its ability to bind human thrombin and act as a cofactor for the activation of human protein C and TAFI. The 579-residue pig TM protein showed approximately 69% sequence identity to human TM. Within the EGF domains necessary for binding of thrombin (EGF56), protein C (EGF4) and TAFI (EGF3), all of the amino acids previously identified as critical for the function of human TM, with the exception of Glu-408 in EGF5, were conserved in pig TM. Comparison of transfected cells expressing pig or human TM demonstrated that both proteins bound human thrombin and inhibited its procoagulant activity. However, pig TM was a poor cofactor for the activation of human protein C and TAFI, with domain swapping showing that EGF5 was the most important determinant of compatibility. Thus, while pig TM may be capable of binding thrombin generated in the vicinity of xenograft endothelium, its failure to promote the activation of human protein C remains a significant problem.
Background
The two common sialic acid (Sia) in mammals are N-Acetylneuraminic acid (Neu5Ac) and its hydroxylated form N-Glycolylneuraminic acid (Neu5Gc). Unlike most mammals, humans cannot synthesize Neu5Gc that is considered foreign and recognized by circulating antibodies. Thus, Neu5Gc is a potential xenogenic carbohydrate antigen in bioprosthetic heart valves (BHV) that tend to deteriorate in time within human patients.
Methods
We investigated Neu5Gc expression in non-engineered animal-derived cardiac tissues and in clinically used commercial BHV, and evaluated Neu5Gc immunogenicity on BHV through recognition by human anti-Neu5Gc IgG.
Results
Neu5Gc was detected by immunohistochemistry in porcine aortic valves and in porcine and bovine pericardium. Qualitative analysis of Sia-linkages revealed Siaα2–3>Siaα2–6 on porcine/bovine pericardium while the opposite in porcine aortic/pulmonary valve cusps. Similarly, six commercial BHV containing either porcine aortic valve or porcine/bovine/equine pericardium revealed Siaα2–3>Siaα2–6 expression. Quantitative analysis of Sia by HPLC showed porcine/bovine pericardium express four-fold higher Neu5Gc levels compared to the porcine aortic/pulmonary valves, with Neu5Ac at six-fold over Neu5Gc. Likewise, Neu5Gc was expressed on commercial BHV (186.3±16.9 pmol Sia/μg protein), with Neu5Ac at eight-fold over Neu5Gc. Affinity-purified human anti-Neu5Gc IgG showing high specificity towards Neu5Gc-glycans (with no binding to Neu5Ac-glycans) on a glycan microarray, strongly bound to all tested commercial BHV, demonstrating Neu5Gc immune recognition in cardiac xenografts.
Conclusions
We conclusively demonstrated Neu5Gc expression in native cardiac tissues, as well as in six commercial BHV. These Neu5Gc xeno-antigens were recognized by human anti-Neu5Gc IgG, supporting their immunogenicity. Altogether, these findings suggest BHV-Neu5Gc/anti-Neu5Gc may play a role in valve deterioration warranting further investigation.
FLNA-MVD is a developmental and degenerative disease with complex phenotypic expression which can influence patient management. FLNA-MVD has unique features with both MVP and paradoxical restricted motion in diastole, sub-valvular mitral apparatus impairment and polyvalvular lesions in males. FLNA-MVD conveys a substantial lifetime risk of valve surgery in men.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.