Analysis of GTP-binding proteins, phosphoproteins, and cytosolic calcium in functional heterogeneous human blood platelet subpopulations.
The biochemical basis for the functional heterogeneity of human blood platelets was investigated in terms of protein phosphorylation, cytoplasmic calcium ([Ca2+]i), the ratio of 46 and 50 kDa vasodilator-stimulated protein (VASP), and GTP-binding proteins (G-proteins). Platelets were fractionated by density. Comparing resting low-density platelets (LDP) to high-density platelets (HDP) revealed higher phosphorylation of proteins in the 47, 31, and 24 kDa ranges. A higher phosphorylation of the 20 kDa protein in LDP compared to HDP was related to an enhanced [Ca2+]i, an increased ADP-ribosylation of the inhibitory G-protein (G(i alpha1-3)) and rhoA, and a decreased ADP-ribosylation of the stimulatory G-protein (G(s alpha)). The differences in the ribosylation patterns of the subpopulations were not influenced by thrombin stimulation or exposure to prostaglandin E1 (PGE1). An 18 kDa phosphoprotein was more highly phosphorylated in resting HDP than in LDP. Thrombin exposure caused dephosphorylation of the 18 kDa phosphoprotein in the HDP, but generally increased phosphorylation of both HDP and LDP in the 47, 31, 24, and 20 kDa bands. Preincubation with prostaglandin E1 or sodium nitroprusside diminished the subsequent thrombin-induced increase in phosphorylation, particularly in HDP. In unstimulated HDP, the 50 kDa VASP phospho form was enhanced, whereas in unstimulated LDP the 46 kDa VASP dephospho form was increased. Our findings suggest that the functional heterogeneity of platelets is partly derived from differences in signal transduction mechanisms reflected in varying phosphoprotein patterns and G-protein properties of platelet stimulatory and inhibitory pathways.
Opper
C
C
Institute of Physiological Chemistry, University of Marburg, Germany. opper@mailer.uni-marburg.de
Schuessler
G
G
Kuschel
M
M
Clement
H W
HW
Gear
A R
AR
Hinsch
E
E
Hinsch
K
K
Wesemann
W
W
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000328:Adult; D000527:Alprostadil / Q000494:pharmacology; D001792:Blood Platelets / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000032:analysis*; D006801:Humans; D008297:Male; D009599:Nitroprusside / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D015398:Signal Transduction
D010750:Phosphoproteins; D009599:Nitroprusside; D000246:Adenosine Diphosphate Ribose; D019204:GTP-Binding Proteins; D000527:Alprostadil; D002118:Calcium