Abstract
The activity of thrombin was revealed to remain in the clot, which was called postclotting thrombin (bound thrombin). Bound thrombin from crushed clots are complexes of α-thrombin with fibrin fragments of the N-terminal regions of α and β chains, molecular weight of which are 170 and 220 kDa. As compared with fibrinogen, exposure of cultured rabbit vascular smooth muscle (VSM) cells to bound thrombin upregulated embryonic myosin heavy chain isoform (SMemb) and plasminogen activator inhibitor-1 (PAI-1) mRNAs. To examine whether bound thrombin-induced phenotypic modulation such as cell proliferation and migration activities are prevented by knockdown of SMemb gene, an siRNA expression plasmid vector targeting open reading frame of SMemb mRNA (ORF-2/pSilencer) was constructed . Northern blot analysis revealed that treatment of the cells with ORF-2/pSilencer significantly inhibited SMemb mRNA expression by 0.51-fold. Immunofluorescent study revealed that SMemb protein expression was depressed in the ORF-2/pSilencer-treated cells. Bound thrombin significantly increased the SMemb and PAI-1 mRNA expressions by 1.4- and 2.7-folds, respectively, as compared to the treatment with PBS. Treatment with ORF-2/pSilencer abolished the bound thrombin-induced upregulations of SMemb and PAI-1 mRNA expression. Although bound thrombin had no effect on cell proliferation activity, migration activity was significantly increased by bound thrombin, which was inhibited by ORF-2/pSilencer. Thus, bound thrombin may stimulate cell migration activity through the upregulation of SMemb. [J Physiol Sci. 2007;57 Suppl:S60]
