Affinity chromatography of platelets on immobilized thrombin: retention of catalytic activity by platelet-bound thrombin.
Radioactivity from I125-labeled human platelets was measured to estimate the extent of binding of platelet surface proteins to immobilized thrombin. 1-3% of the radioactivity was bound with 10-20% of this amount apparently irreversibly bound to the thrombin matrix. Site-specific chemical modification of thrombin with pyridoxal-5'-phosphate, N-bromosuccinimide or tetranitromethane resulted in a variable reduction of the amount of radiolabel bound. When thrombin modified with H-D-PheProArg-chloromethyl ketone (PPACK) was coupled to the matrix, there was no difference in the binding of platelet membrane proteins when compared to a control thrombin preparation while thrombin modified with tosyl-Lys-chloromethyl ketone (TLCK) coupled to the matrix did not bind radiolabel any more effectively than albumin which served as the control. However, when thrombin was modified with PPACK after coupling to the agarose matrix, ability to bind radiolabel was lost. Thrombin bound to platelets remained catalytically active when assayed with a peptide nitroanilide substrate. These results suggest tight binding between thrombin and platelets that is not only not dependent on active site integrity but leaves the bound thrombin catalytically competent.
Wu
H F
HF
Dental Research Center, Dental Ecology, School of Dentistry, University of North Carolina, Chapel Hill 27599-7455.
White
G C
GC
Workman
E F
EF
Jenzano
J W
JW
Lundblad
R L
RL
D000590:Amino Acid Chloromethyl Ketones; D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000378:metabolism; D002384:Catalysis; D002469:Cell Separation / Q000379:methods*; D002846:Chromatography, Affinity / Q000379:methods*; D004800:Enzymes, Immobilized; D006801:Humans; D008969:Molecular Sequence Data; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D012685:Sepharose; D013917:Thrombin / Q000378:metabolism; D014107:Tosyllysine Chloromethyl Ketone
D000590:Amino Acid Chloromethyl Ketones; D004800:Enzymes, Immobilized; D010980:Platelet Membrane Glycoproteins; D014107:Tosyllysine Chloromethyl Ketone; D012685:Sepharose; D013917:Thrombin; C040308:phenylalanyl-prolyl-arginine-chloromethyl ketone