Different pathways for control of Na+/H+ exchange via activation of the thrombin receptor.
The aim of the present study was to clarify the control of Na+/H+ exchange in platelets activated via the thrombin receptor. When human BCECF-loaded platelets were stimulated with the thrombin-receptor-activating peptide (TRAP; amino acid sequence SFLLRN), which activates the receptor independently of proteolysis, the cytosolic pH (pHi) rose from 7.13 +/- 0.04 (n = 6) to 7.27 +/- 0.04 (n = 5), followed by a rapid decrease to resting values. Trypsin, which cleaves the receptor, induced a rapid and irreversible rise in pHi to 7.31 +/- 0.06 (n = 5). gamma-Thrombin, which cleaves the receptor but is unable to bind to the hirudin-like domain, induced a slow and irreversible rise in pHi to 7.31 +/- 0.04 (n = 14). alpha-Thrombin, which cleaves the receptor and binds to its hirudin-like domain, induced a rapid and irreversible rise in pHi to 7.31 +/- 0.04 (n = 22). Changes in pHi induced by TRAP, trypsin, gamma- and alpha-thrombin were accompanied by similar changes in cytosolic Ca2+ concentration ([Ca2+]i) and 32P-pleckstrin, a substrate of protein kinase C (PKC). The separate chelation of Ca2+i (30 microM BAPTA-AM) or inhibition of PKC (1 microM staurosporine) induced about 50% inhibition of the pHi responses triggered by TRAP, trypsin, gamma- and alpha-thrombin, but the combination induced complete inhibition. Thus the different types of activation of the thrombin receptor control Na+/H+ exchange via the same mechanism. Binding of thrombin to the hirudin-like domain accelerates exchange activation, whereas proteolysis of the receptor is essential for a sustained increase in pHi.
Nieuwland
R
R
Department of Haematology, University Hospital Utrecht, The Netherlands.
van Willigen
G
G
Akkerman
J W
JW
D000470:Alkaloids / Q000494:pharmacology; D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D001798:Blood Proteins / Q000378:metabolism; D002118:Calcium / Q000097:blood; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005452:Fluoresceins / Q000494:pharmacology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000494:pharmacology; D010750:Phosphoproteins; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D018179:Receptors, Thrombin / Q000187:drug effects / Q000502:physiology*; D017923:Sodium-Hydrogen Exchangers / Q000097:blood*; D019311:Staurosporine; D013917:Thrombin / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology
D000470:Alkaloids; D001798:Blood Proteins; D005452:Fluoresceins; D010446:Peptide Fragments; D010750:Phosphoproteins; D018179:Receptors, Thrombin; D017923:Sodium-Hydrogen Exchangers; C039077:platelet protein P47; C072032:thrombin receptor peptide (42-55); D004533:Egtazic Acid; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D011493:Protein Kinase C; D014357:Trypsin; D013917:Thrombin; D019311:Staurosporine; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium