A large-scale procedure for the isolation of integrin GPIIb/IIIa, the human platelet fibrinogen receptor.
The heterodimer GPIIb/IIIa, formed by the Ca(2+)-dependent association of glycoproteins IIb (GPIIb) and IIIa (GPIIIa), is the major integrin at the platelet surface, where it serves as the receptor for fibrinogen and other adhesive proteins and plays a central role in platelet aggregation and in platelet adhesion to the subendothelium. Here we describe a procedure for the isolation of GPIIb/IIIa using as starting material either the whole particulate fraction, obtained by differential centrifugation after hypoosmotic lysis of glycerol-loaded platelets, or any of the fractions obtained by density gradient centrifugation of the whole particulate fraction. The procedure consists simply of differential extraction with Triton X-100 of the starting particulate fraction, anion-exchange chromatography of the 4% Triton X-100 supernatant, and size-exclusion chromatography of the GPIIb/IIIa-rich fraction retained in the ion-exchange column. The use of particulate fractions instead of whole platelets as the starting material for extraction together with differential extraction with Triton X-100 (two steps that are simple and inexpensive to perform) results in the early removal of many unwanted proteins, which otherwise would have to be removed at later stages at the expense of severely impairing the final yield of GPIIb/IIIa. Pure GPIIb/IIIa is obtained with a yield of about 48%, the highest so far reported, calculated with respect to the GPIIb and GPIIIa content in the starting particulate fraction. The final product can be stored in freeze-dried form without apparent changes in its physical and chemical properties.(ABSTRACT TRUNCATED AT 250 WORDS)
Rivas
G A
GA
Instituto de Química Física, CSIC, Madrid, Spain.
Calvete
J J
JJ
González-Rodríguez
J
J
D001709:Biotechnology; D001792:Blood Platelets / Q000737:chemistry; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006801:Humans; D017830:Octoxynol; D010980:Platelet Membrane Glycoproteins / Q000302:isolation & purification*; D011092:Polyethylene Glycols
D010980:Platelet Membrane Glycoproteins; D011092:Polyethylene Glycols; D017830:Octoxynol