Modulation of red blood cell aggregation and blood viscosity by the covalent attachment of Pluronic copolymers

Despite many years of research, the physiologic or possible pathologic sign ificance of RBC aggregation remains to be clearly determined. As a new appr oach to address an old question, we have recently developed a technique to vary the aggregation tendency of RBCs in a predictable and reproducible fas hion by the covalent attachment of nonionic polymers to the RBC membrane. A reactive derivative of each polymer of interest is prepared by substitutio n of the terminal hydroxyl group with a reactive moiety, dichlorotriazine ( DT), which covalently bonds the polymer molecule to membrane proteins. Plur onics are block copolymers of particular interest as these copolymers can e nhance or inhibit RBC aggregation. Pluronics exhibit a critical micellizati on temperature (CMT): a phase transition from predominantly single, fully h ydrated copolymer chains to micelle-like structures. The CMT is a function of both copolymer molecular mass and concentration. This micellization prop erty of Pluronics has been utilized to enhance or inhibit RBC aggregation a nd hence to vary low-shear blood viscosity. Pluronic coated RBCs were prepa red using reactive DT derivatives of a range of Pluronics (F68, F88, F98 an d F108) and resuspended in autologous plasma at 40% hematocrit. Blood visco sity was measured at a range of sheer rates (0.1-94.5 s(-1)) and at 25 and 37 degreesC using a Contraves LS-30 couette low shear viscometer. RBC aggre gation and whole blood viscosity was modified in a predictable manner depen ding upon the CMT of the attached Pluronic and the measurement temperature: below the CMT, RBC aggregation was diminished; above the CMT it was enhanc ed. This technique provides a novel tool to probe some basic research quest ions. While certainly of value for in vitro mechanistic studies, perhaps th e most interesting application may be for in vivo studies: typically, intra vital experiments designed to examine the role of RBC aggregation in microv ascular flow require perturbation of the suspending plasma to promote or re duce aggregation (e.g., by the addition of dextran). By binding specific Pl uronics to the surface, we can produce RBCs that intrinsically have any des ired degree of increased or decreased aggregation when suspended in normal plasma, thereby eliminating many potential artifacts for in vivo studies. T he copolymer coating technique is simple and reproducible, and we believe i t will prove to be a useful tool to help address some of the longstanding q uestions in the field of hemorheology.