alpha-Thrombin upregulates G alpha i3 in human vascular endothelial cells.
OBJECTIVE
During thrombosis, alpha-thrombin becomes sequestered by fibrin and the subendothelial basement membrane, and it is available to interact with the vasculature over prolonged periods. In this study, we investigated the long-term effect of alpha-thrombin on G alpha i3 and G alpha s levels in human vascular endothelial cells (EC). Because obesity is associated with changes in receptor signaling in many animal models, we also explored the influence of this clinical risk factor.


METHODS
Primary cultures of human EC were exposed to alpha-thrombin for 16 hours, and immunologically detectable G alpha i3 and G alpha s levels were measured.


RESULTS
alpha-Thrombin (100 nmol/L) increased G alpha i3 levels in EC derived from the cerebral microvasculature and superficial temporal artery (4.2 +/- 1.2-fold and 2.8 +/- 0.32-fold, respectively) but had no significant effect on EC derived from omental artery (P > .6) or from the superficial temporal artery of obese (body mass index > or = 28 kg/m2) patients (P > .4). The expression of G alpha s was unchanged in all cell types (P > or = .1). Two other circulating peptides, vasoactive intestinal peptide and endothelin-1, failed to alter the expression of either G protein in EC from the cerebral microvasculature, further demonstrating the specificity of the alpha-thrombin effect. However, thrombin receptor activating protein-14 mimicked the alpha-thrombin response and increased G alpha i3 in EC derived from the cerebral microvasculature and superficial temporal artery.


CONCLUSIONS
We conclude that alpha-thrombin increases G alpha i3 expression in some EC through activation of its tethered liganded receptor. Obesity appears to suppress this action of alpha-thrombin.
Shreeve
S M
SM
Totman Laboratory for Human Cerebrovascular Research, University of Vermont, Burlington 05405, USA. sshreeve@moose.uvm.edu
Shatos
M A
MA
Thorin
E
E
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D015992:Body Mass Index; D002478:Cells, Cultured; D002536:Cerebral Arteries / Q000187:drug effects / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D005260:Female; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000096:biosynthesis* / Q000235:genetics; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000096:biosynthesis / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects*; D006629:Hirudins / Q000494:pharmacology; D006801:Humans; D008297:Male; D008875:Middle Aged; D009765:Obesity / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D009852:Omentum / Q000098:blood supply; D010446:Peptide Fragments / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D018179:Receptors, Thrombin / Q000187:drug effects / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects; D013699:Temporal Arteries / Q000187:drug effects / Q000473:pathology; D013917:Thrombin / Q000494:pharmacology*
D006629:Hirudins; D010446:Peptide Fragments; D011500:Protein Synthesis Inhibitors; D018179:Receptors, Thrombin; C072032:thrombin receptor peptide (42-55); D003513:Cycloheximide; D013917:Thrombin; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D019205:GTP-Binding Protein alpha Subunits, Gs