Subsecond calcium dynamics in ADP- and thrombin-stimulated platelets: a continuous-flow approach using indo-1.
The regulation and kinetics (less than 5 seconds) of cytosolic calcium changes ([Ca2+]i) in stimulated blood platelets have been investigated under physiological blood flow conditions. Using a newly-developed continuous-flow approach with indo-1-loaded human platelets, adenosine diphosphate (ADP, 10 mumol/L) and thrombin (5 U/mL) were equally effective in significantly increasing [Ca2+]i by 0.5 seconds. ADP induced a transient [Ca2+]i peak of 1 to 2 mumol/L near 2 seconds, whereas thrombin caused a sustained and larger response. The first phase (less than 2 seconds) was not influenced by a lack of extracellular Ca2+, in contrast to the subsequent [Ca2+]i increase that only reached about 0.7 mumol/L for either ADP or thrombin. The shear rates used in our continuous-flow apparatus were physiological (less than 1,258 sec-1) and only slightly increased the basal [Ca2+]i of 0.1 mumol/L. Platelet aggregation (less than 5 seconds), assessed by single-particle counting, was not altered in platelets loaded with indo-1/AM (2.5 mumol/L).
Jones
G D
GD
Department of Biochemistry, University of Virginia, Charlottesville 22908.
Gear
A R
AR
D000244:Adenosine Diphosphate / Q000494:pharmacology*; D001792:Blood Platelets / Q000378:metabolism*; D002118:Calcium / Q000097:blood*; D003593:Cytoplasm / Q000502:physiology; D006801:Humans; D066298:In Vitro Techniques; D007211:Indoles; D007700:Kinetics; D010974:Platelet Aggregation; D013917:Thrombin / Q000494:pharmacology*
D007211:Indoles; D000244:Adenosine Diphosphate; D013917:Thrombin; C048960:indo-1; D002118:Calcium