CHANGES IN CYTOPLASMIC PH IN PLATELETS ACTIVATED THROUGH THE HIGH-AFFINITY AND MODERATE-AFFINITY RECEPTOR PATHWAYS BY HIGHLY PURIFIED HUMANALPHA-THROMBIN

Platelets in plasma were loaded with the probe BCECF/AM, and changes i n cytoplasmic pH levels induced by highly purified human alpha-thrombi n (2900 NIH U/mg) were studied in washed platelets having high- and mo derate-affinity receptors and in platelets from which the high-affinit y alpha-thrombin receptor had been removed by treatment with Serratia morcescens protease, In intact platelets, cytoplasmic acidification re ached a maximum within 2 minutes of 0.072 +/- 0.009 pH units at 0.3 nm ol/L alpha-thrombin concentration (0.03 U/ml). Cytoplasmic pH values w ere higher at both lower and higher alpha-thrombin concentrations and were significantly (p = 0.018) higher at 2 nmol/L alpha-thrombin, whic h induced -0.037 +/- 0.013 pH units of acidification. Five nanomoles o f alpha-thrombin, however, induced cytoplasmic alkalinization of + 0.0 27 +/-0.033 pH units. In platelets lacking the high-affinity receptor where there is a 10 to 20-fold reduction in sensitivity to alpha-throm bin, 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. Th ese results show that the transition from acidification to alkalinizat ion occurs in the same range of alpha-thrombin concentrations (2 to 5 nmol/L) in both preparations, despite the rightward shift in sensitivi ty caused by the absence of the high-affinity receptor. However, the m aximum acidification reached in control platelets (-0.037 pH units at 2 nmol/L) was much less than the value obtained in platelets lacking t he high-affinity receptor (-0.175 pH units at 2 nmol/L alpha-thrombin) . These studies suggest that at relatively low concentrations (< 2 nmo l/L), alpha-thrombin induces cytoplasmic acidification in both platele t preparations, whereas higher doses ( > 5 nmol/L) induce cytoplasmic alkalinization. The significance of these cytoplasmic pH changes remai ns to be elucidated.