Thromboerythrocytes. In vitro studies of a potential autologous, semi-artificial alternative to platelet transfusions.

Abstract

In an attempt to overcome the limitations and drawbacks of using fresh platelets for transfusion therapy of thrombocytopenic patients, we have performed in vitro experiments on an autologous, semi-artificial alternative to platelet transfusions. Based on our previous studies of the interactions of unactivated and activated platelets with beads coated with peptides of various lengths, all of which contained the arginine-glycineaspartic acid (RGD) cell recognition sequence, the peptide Ac-CGGRGDF-NH2 was chosen for covalent coupling to erythrocytes. A heterobifunctional crosslinking reagent (N-maleimido-6-aminocaproyl ester of 1-hydroxy-2-nitrobenzene4-sulfonic acid) was used to crosslink via the peptide's free sulfhydryl group and the erythrocyte's surface amino groups. Approximately 0.5-1.5 X 106 peptide molecules bound per erythrocyte after 2 h of incubation, and most of the peptides appeared to crosslink to glycophorin A. The resulting cells, termed thromboerythrocytes, interacted selectively with activated platelets to form mixed aggregates. Studies with fluid phase RGD peptides and monoclonal antibodies indicated that the RGD peptides on the thromboerythrocytes interacted with the GPIIb/ lila receptors on activated platelets. Thromboerythrocytes could also bind to platelets adherent to collagen. There was minimal erythrocyte hemolysis during the formation of thromboerythrocytes and studies of thromboerythrocyte osmotic fragility and cellular deformability showed no significant changes from control erythrocytes. Whereas there is a 20:1 ratio of erythrocytes to platelets in the circulation of normal individuals, the erythrocytes from as little as 50 ml of blood could be transformed into the equivalent of 2 U of platelets by numbers (equivalent to 18 U of platelets by mass), and reinfused into the same individual within several hours. These data encourage us to proceed to in vivo studies to assess the hemostatic efficacy of thromboerythrocytes in thrombocytopenic animals. (J. Clin.

Studies of activated GPIIb/IIIa receptors on the luminal surface of adherent platelets. Paradoxical loss of luminal receptors when platelets adhere to high density fibrinogen.

“…Thromboerythrocytes were prepared as previously described by crosslinking the peptide AcCGGRGDF-NH2 to washed erythrocytes using the heterobifunctional cross-linking agent mal-sac-HNSA (Bachem Bioscience Inc., Philadelphia, PA) (36 To assess the ability of adherent platelets to bind a second layer of platelets as a function of the fibrinogen-and collagen-coating concentrations, the assay was modified and performed in duplicate; one well received radiolabeled platelets and thromboerythrocytes as before, and the other well received a first layer of unlabeled platelets, a subsequent 1-h incubation with platelet-poor plasma (to supply adhesive glycoproteins), and finally a second layer of radiolabeled platelets (1 h). Thus, the radioactivity in the first well reflected the binding ofthe first layer of platelets, whereas the radioactivity in the second well reflected the binding of the second layer.…”

“…As a result, we have developed an assay based on the ability of adherent platelets to bind our previously described thromboerythrocytes (36). Thromboerythrocytes are red blood cells with approximately one million peptides containing the RGDF sequence covalently coupled to their surface (36).…”

“…As a result, we have developed an assay based on the ability of adherent platelets to bind our previously described thromboerythrocytes (36). Thromboerythrocytes are red blood cells with approximately one million peptides containing the RGDF sequence covalently coupled to their surface (36). As we previously reported, the length of the RGD-containing peptide was chosen so that the thromboerythrocytes would bind to activated GPIIb/IIIa receptors on platelets, but not unactivated receptors (36,37).…”

“…Thromboerythrocytes are red blood cells with approximately one million peptides containing the RGDF sequence covalently coupled to their surface (36). As we previously reported, the length of the RGD-containing peptide was chosen so that the thromboerythrocytes would bind to activated GPIIb/IIIa receptors on platelets, but not unactivated receptors (36,37). Thus, the binding ofthromboerythrocytes to a lawn ofadherent platelets should be a direct reflection of the accessibility of activated GPIIb/IIIa receptors on the luminal surface of adherent platelets.…”

The erythrocyte viral trap: Transgenic expression of viral receptor on erythrocytes attenuates coxsackievirus B infection

“…Recombinant receptor proteins may be directly affixed to erythrocyte surfaces by using chemical crosslinkers (37) or be made to externally insert themselves into erythrocyte membranes by fusing them to a glycosyl-phosphatidylinisotol anchor (38,39). Biotinylation of erythrocytes is also readily achieved, and biotinylated erythrocytes can be safely reinfused into humans (40)(41)(42).…”