The Ca2+-mobilizing potency of alpha-thrombin and thrombin-receptor-activating peptide on human platelets -- concentration and time effects of thrombin-induced Ca2+ signaling.
In single platelets and in suspensions of platelets, alpha-thrombin evokes dose-dependent, transient increases in cytosolic Ca2+ concentration, [Ca2+]i, which are more prolonged than the [Ca2+]i transients evoked by other platelet agonists such as the thrombin-receptor-activating hexapeptide SFLLRN, thromboxane A2 analog U46619, and ADP. As a quantity taking into account both the magnitude and length of the Ca2+ response, we defined the Ca2+-mobilizing potency (CMP) of an agonist as the integrated rise in [Ca2+]i during the time of the Ca2+ signal. It was observed that: (a) the CMP increased with the agonist concentration in a saturating way, its maximal value being about four-times higher with alpha-thrombin than with SFLLRN; (b) the high CMP of alpha-thrombin was for only a small part due to endogenous production of ADP or thromboxane, and was mainly a consequence of prolonged influx of external Ca2+; (c) the CMP declined when alpha-thrombin was inactivated during the course of the Ca2+ signal; (d) CMP values increased with the agonist concentration upon sequential addition of increasing amounts of alpha-thrombin or SFLLRN; (e) when alpha-thrombin was gradually added to the platelets or formed by an in situ reconstituted prothrombinase system (with factor Xa, factor Va, and prothrombin), integrated Ca2+ responses were a function of the product of the alpha-thrombin concentration and the time of its presence. However, in these cases, the final CMP values were independent of the rate of alpha-thrombin addition or formation. We conclude that alpha-thrombin-induced Ca2+ signals in platelets rely largely upon Ca2+ influx, are not, or only slightly, subjected to homologous desensitization, and reflect the enzymatic capacity of alpha-thrombin to cleave protease-activated receptors. Thus, the high and prolonged Ca2+ signal induced by alpha-thrombin is due to continuous receptor cleavage without desensitizing effects of previously cleaved receptors.
Heemskerk
J W
JW
Department of Biochemistry, Maastricht University, The Netherlands. jwm.heemskerk@bioch.unimaas.nl
Feijge
M A
MA
Henneman
L
L
Rosing
J
J
Hemker
H C
HC
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid / Q000494:pharmacology; D000244:Adenosine Diphosphate / Q000494:pharmacology; D001081:Apyrase / Q000494:pharmacology; D001241:Aspirin / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology*; D002118:Calcium / Q000097:blood*; D003600:Cytosol / Q000378:metabolism; D004533:Egtazic Acid / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D010446:Peptide Fragments / Q000494:pharmacology*; D015539:Platelet Activation / Q000187:drug effects; D018179:Receptors, Thrombin / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D013917:Thrombin / Q000494:pharmacology*; D013997:Time Factors
D010446:Peptide Fragments; D018179:Receptors, Thrombin; C082835:thrombin receptor peptide (42-47); D004533:Egtazic Acid; D000244:Adenosine Diphosphate; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D013917:Thrombin; D001081:Apyrase; D001241:Aspirin; D002118:Calcium