Cloning of Porcine Platelet Receptor GPIbα and Comparison with Human GPIbα

Abstract

Glycoprotein (GP) Ib-IX-V is a remarkable platelet adhesion receptor which initiates platelet aggregation. GPIba is the central component of GPIb-IX-V complex and it anchors the complex to the cytoskeleton and harbors the vWF-binding function. Previous studies suggested that the coagulation function of pig showed some differences with human, especially the interaction between vWF and platelet. The enhanced potential of porcine vWF to human platelet glycoprotein GPIb will trigger platelet aggregation, and contribute to coagulation dysregulation after xenotransplantation, which is proven to be an important barrier to xenograft acceptance. However, we have little knowledge about the function of porcine platelet, which is important for us to understand the molecular incompatibility between human and pig. In addition, it is also the important parameter in cardiovascular disease, thrombus and surgery researches using pig as animal model. So in this study, we focused on the differences of GPIba between pig and human. We cloned porcine GPIba sequence and analyzed its characterization. The porcine GPIba contains 1891 nucleotides with an ORF which encodes 627 amino acids. The nucleotide sequence shows 67% homology identity with human GPIba while the deduced amino acids shows 58.84% identity. The VWF binding domain shares high identity between the two species, while three amino acids difference are identified. Otherwise, the significant variations exist in the PEST domain, and there is only 35.32% homology identity. From the deduced 3-D model the porcine and human GPIba, the conformations of porcine and human vWF binding site of GPIba are generally consensus. Ristocetin-induced platelet aggregation is widely used to evaluate platelet aggregation function triggered by vWF and GPIba interaction. It is very interesting that remarkably lower aggregation has been observed in porcine platelet after adding restocetin as agonist, compared to human platelet. Normally, human platelet shows 70%-90% aggregation when the concentration of ristocetin is 0.9mg/ml. However no aggregation is observed in porcine platelet under this condition. Even when ristocetin is 2.7mg/ml, just three folds of normal concentration, only 10% aggregation is observed in porcine platelet. According to the sequence analysis and platelet aggregation tests, we proposed that there are some difference in the function of GPIba, especially the interaction of restocetin-vWF-GPIba between pig and human. This characterization of porcine GPIba will enhance our knowledge of porcine platelet and porcine coagulation system in xenotransplantation researches.