alpha-Thrombin-induced human platelet activation results solely from formation of a specific enzyme-substrate complex.
Prior studies using the mutant thrombin, thrombin Quick I, indicate that this protease induces maximum platelet aggregation and intraplatelet [Ca2+] fluxes at agonist concentrations where dissociable, equilibrium platelet binding is undetectable and led to the conclusion that thrombin interaction with its platelet "receptor" is best described kinetically by formation of an enzyme-substrate complex. This conclusion was substantiated further in the present studies by demonstrating that both thrombin Quick I and thrombin mimicked the thrombin receptor agonist peptide in the induction of the platelet activation-dependent events required for functional Prothrombinase assembly and that a rabbit antibody raised against KATNATLDPRSFLLR, a pentadecapeptide which represents amino acids 32-46 in the platelet thrombin receptor/substrate and spans the thrombin cleavage site, inhibited both thrombin- and thrombin Quick I-induced platelet activation responses equivalently. The antipeptide antibody had a more pronounced inhibitory effect on the rate of the thrombin-induced response rather than the magnitude of the response suggesting competition for the cleavage site, consistent with the observation that pretreatment of metabolically-inhibited platelets with thrombin, which was removed by washing, eliminated specific antibody binding due to removal and/or masking of antibody epitopes. Concentrations of the antipeptide antibody that inhibited thrombin- and thrombin Quick I-induced increases in intracellular [Ca2+] flux by as much as 97% did not alter the dissociable equilibrium binding of 125-I-FPR-thrombin to platelets. These combined data indicate that the hydrolytic event initiated by thrombin or thrombin Quick I interaction with the platelet receptor/substrate for thrombin is unrelated to the dissociable equilibrium binding of thrombin to membrane sites described previously by classical receptor-ligand binding analyses.
Department of Biochemistry, University of Vermont, Burlington 05405.
K L
KL
Hayes
L
L
Leong
R A
RA
Henriksen
B A
BA
Bouchard
L
L
Ouellette
W R
WR
Church
P B
PB
Tracy
D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000201:enzymology; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D015539:Platelet Activation / Q000502:physiology*; D018179:Receptors, Thrombin / Q000819:agonists / Q000378:metabolism; D013379:Substrate Specificity; D013917:Thrombin / Q000378:metabolism / Q000494:pharmacology / Q000502:physiology*; D013925:Thromboplastin / Q000378:metabolism
D010446:Peptide Fragments; D018179:Receptors, Thrombin; C072032:thrombin receptor peptide (42-55); D013925:Thromboplastin; D013917:Thrombin; C027648:thrombin Quick