Changes in cytoplasmic pH in platelets activated through the high- and moderate-affinity receptor pathways by highly purified human alpha-thrombin.
Platelets in plasma were loaded with the probe BCECF/AM, and changes in cytoplasmic pH levels induced by highly purified human alpha-thrombin (2900 NIH U/mg) were studied in washed platelets having high- and moderate-affinity receptors and in platelets from which the high-affinity alpha-thrombin receptor had been removed by treatment with Serratia marcescens protease. In intact platelets, cytoplasmic acidification reached a maximum within 2 minutes of -0.072 +/- 0.009 pH units at 0.3 nmol/L alpha-thrombin concentration (0.03 U/ml). Cytoplasmic pH values were higher at both lower and higher alpha-thrombin concentrations and were significantly (p = 0.018) higher at 2 nmol/L alpha-thrombin, which induced -0.037 +/- 0.013 pH units of acidification. Five nanomoles of alpha-thrombin, however, induced cytoplasmic alkalinization of +0.027 +/- 0.033 pH units. In platelets lacking the high-affinity receptor where there is a 10 to 20-fold reduction in sensitivity to alpha-thrombin, acidification reached a maximum of -0.175 +/- 0.033 pH units at 2 nmol/L alpha-thrombin, but alkalinization was observed at 5 nmol/L (+0.038 +/- 0.025) and 10 nmol/L (+0.042 +/- 0.007) alpha-thrombin. These results show that the transition from acidification to alkalinization occurs in the same range of alpha-thrombin concentrations (2 to 5 nmol/L) in both preparations, despite the rightward shift in sensitivity caused by the absence of the high-affinity receptor. However, the maximum acidification reached in control platelets (-0.037 pH units at 2 nmol/L) was much less than the value obtained in platelets lacking the high-affinity receptor (-0.175 pH units at 2 nmol/L alpha-thrombin).(ABSTRACT TRUNCATED AT 250 WORDS)
Cell Biology Department, American Red Cross, Rockville, MD 20855.
G D
GD
Jones
T E
TE
Tenner
N J
NJ
Greco
S
S
Kitagawa
N N
NN
Tandon
G A
GA
Jamieson
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015539:Platelet Activation; D011956:Receptors, Cell Surface / Q000032:analysis / Q000502:physiology*; D018179:Receptors, Thrombin; D013917:Thrombin / Q000494:pharmacology*
D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D000584:Amiloride; D013917:Thrombin; C039614:ethylisopropylamiloride