Thrombin-stimulated growth, clustering, and collagen lattice contraction of human gingival fibroblasts is associated with its protease activity.
BACKGROUND
Thrombin is a serine protease produced following gingival tissue injury or inflammation. It regulates the functional behavior of injury-neighboring cells via the activation of specific protease-activated receptors (PAR). Thrombin's role in gingival tissue healing and inflammatory response processes is not yet well understood.


METHODS
We investigated the effects of thrombin on gingival fibroblast (GF) growth [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay], collagen lattice contraction, and associated morphological changes.


RESULTS
Thrombin (>1 U/ml), but not thrombin receptor (PAR-1) agonist peptide (SFLLRN, single letter amino acid code, abbreviated as TRAP, 1 to 50 microg/ml), stimulated the growth and clustering of cultured human GF in vitro. Growth-stimulatory effects of thrombin were inhibited by D-Phe-Pro-ArgCH2Cl (PPACK), a serine protease inhibitor. By contrast, trypsin (>10 microg/ml), a PAR-2 activator, suppressed the growth of GF. Thrombin (>0.2 U/ml) and TRAP (10 to 25 microg/ml), but not trypsin, prostaglandin E2 (0.01 to 0.5 microg/ml), or bovine serum albumin (BSA) (1 to 80 microg/ml), induced the GF-populated collagen lattice contraction within 30 to 60 minutes of exposure. The thrombin-induced collagen lattice contraction was inhibited by PPACK (20 microg/ml) and an actin filament polymerization inhibitor, cytochalasin B (1 microg/ml). The collagen lattice contraction induced by TRAP was also inhibited by cytochalasin B, but not by PPACK. Using a reverse-transcriptase polymerase chain reaction (RT-PCR), the expression of PAR-1, and to a lesser extent PAR-3, was observed for human GF, although little PAR-2 and PAR-4 expression was noted.


CONCLUSIONS
These results indicate that thrombin is important in periodontal wound healing and inflammatory processes by promoting the growth and contraction of GF. The stimulatory effects of thrombin are associated with its protease activation of thrombin receptors.
Chang
M C
MC
Team of Biomedical Science, Chang-Gung Institute of Nursing, Taoyuan, Taiwan.
Chan
C P
CP
Wu
H L
HL
Chen
R S
RS
Lan
W H
WH
Chen
Y J
YJ
Jeng
J H
JH
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000991:Antithrombins / Q000494:pharmacology; D002449:Cell Aggregation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D003094:Collagen / Q000187:drug effects*; D004396:Coloring Agents; D003460:Crystallization; D003571:Cytochalasin B / Q000494:pharmacology; D015232:Dinoprostone / Q000494:pharmacology; D004789:Enzyme Activation; D005347:Fibroblasts / Q000187:drug effects* / Q000201:enzymology; D015870:Gene Expression; D005881:Gingiva / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D006801:Humans; D010446:Peptide Fragments / Q000494:pharmacology; D044463:Receptor, PAR-1; D044464:Receptor, PAR-2; D018179:Receptors, Thrombin / Q000187:drug effects* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D013223:Statistics as Topic; D013778:Tetrazolium Salts; D013844:Thiazoles; D013917:Thrombin / Q000819:agonists / Q000494:pharmacology*; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology
D000590:Amino Acid Chloromethyl Ketones; D000991:Antithrombins; D004396:Coloring Agents; D010446:Peptide Fragments; D044463:Receptor, PAR-1; D044464:Receptor, PAR-2; D018179:Receptors, Thrombin; D015842:Serine Proteinase Inhibitors; D013778:Tetrazolium Salts; D013844:Thiazoles; C082835:thrombin receptor peptide (42-47); D012710:Serum Albumin, Bovine; D003571:Cytochalasin B; D003094:Collagen; D014357:Trypsin; D013917:Thrombin; C022616:thiazolyl blue; D015232:Dinoprostone; C040308:phenylalanyl-prolyl-arginine-chloromethyl ketone