Differences in αIIbβ3 Biogenesis Dynamics between Umbilical Cord Blood-Derived Human Megakaryocyte-Like Cells and Transfected HEK293 Cells as Revealed by a Mathematical Model.

Abstract Most studies of αIIbβ3 biogenesis have been conducted in transfected HEK293 and CHO cells or megakaryocyte-like cell lines, but the protein processing mechanisms in native megakaryocytes may differ. To address this issue we have studied αIIbβ3 biogenesis in human megakaryocyte-like cells derived from umbilical cord blood (UCB) and in 293 cells stably expressing αIIbβ3, and developed a mathematical model to express the kinetics. The leukocyte pool of whole UCB units was separated by Dextran and Ficoll sedimentation and enriched for CD34+ progenitor cells by negative selection using a commercial antibody panel. These cells were then cultured in the presence of 20 ng/ml thrombopoietin. By day 10 of culture approximately 90% of cells expressed αIIbβ3, 80% expressed GPIb, 45% expressed α2β1 and 55% expressed P-selectin. The cells adhered to fibrinogen and collagen, spread in a manner similar to platelets, and formed focal adhesions as judged by vinculin and phalloidin (F-actin) staining. While αIIbβ3 on the surface of 293 cells cannot undergo inside-out activation, 18% of the UCB-derived cells exhibited increased binding of PAC-1, an activation-specific monoclonal antibody, in response to TRAP, and the majority bound PAC-1 after adhesion to collagen. The ploidy of the UCB-derived cells ranged from 2 to 128. The dynamics of αIIbβ3 biosynthesis was analyzed by pulse-chase analysis with 35S-Cys/Met followed by immunoprecipitation, SDS-PAGE, and densitometry of exposed x-ray film. Using non-linear regression, curves were fitted to the observed data and a 3-compartment mathematical model (pro-αIIb, mature αIIb in complex with β3, and degraded αIIb) was used to describe the αIIb dynamics. There were important similarities and differences between the two types of cells. In both cell types, pro-αIIb decreased over time in a pattern best described by a simple exponential function, P = (a)exp(-bt), where P is the amount of pro-αIIb, a is the initial amount of pro-αIIb, and b is a rate constant. The half-lives (ln2/b) of pro-αIIb in the UCB-derived cells and the 293 cells were similar (120 and 140 min, respectively). In both cell types, mature αIIb increased over time with a pattern best described by a sigmoidal function, M = c/(1+exp(−(x−x0)/d), where M is the measured amount of mature αIIb, c is the asymptotic value of αIIb, d is the rate constant, and x0 is the time to half maximum mature αIIb. In the megakaryocyte-like cells, the time to half maximum (x0) was 120 min, while in the 293 cells it was only 55 min. The amount of pro-αIIb that has been degraded at any time point (D) can be calculated by subtracting the amounts of pro-αIIb and mature αIIb at that time point from the initial amount of pro-αIIb (D = a-P-M). In the UCB-derived cells ~ 25% of pro-αIIb is degraded without being processed to mature αIIb, while in 293 cells ~ 40% is degraded. In both cell types there is a ~90 minute lag time before the onset of net αIIb degradation. These findings suggest that while the ability to degrade αIIb may be similar between megakaryocytes and 293 cells, the folding, complex formation, and quality control mechanisms are quite different. These observations have implications for the study of integrin dynamics and may be useful in analysis of the molecular mechanisms of integrin biogenesis.