Thrombin immobilized to extracellular matrix is a potent mitogen for vascular smooth muscle cells: nonenzymatic mode of action.
Esterolytically inactive diisopropyl fluorophosphate-conjugated thrombin (DIP-alpha-thrombin) stimulated 3H-thymidine incorporation and proliferation of growth-arrested vascular smooth muscle cells (SMCs), similar to native alpha-thrombin. Half-maximal mitogenic response of SMCs was obtained at 1 nM thrombin and was specifically blocked by the leech-derived, high-affinity thrombin inhibitor, hirudin. Native thrombin and a variety of thrombin species that were chemically modified to alter thrombin procoagulant or esterolytic functions were found to induce 3H-thymidine incorporation to a similar extent. Exposure of SMCs to DIP-alpha-thrombin caused a rapid and transient expression of the c-fos protooncogene, determined by Northern blot analysis. These results indicate that thrombin is a potent mitogen for SMCs through a distinct non-enzymatic domain. Binding of 125I-alpha-thrombin to SMC cultures revealed an apparent dissociation constant of 6 nM and an estimated 5.4 x 10(5) binding sites per cell. This binding was inhibited to the same extent by native thrombin and by its nonenzymatic form, DIP-alpha-thrombin. Moreover, the chemotactic fragment of thrombin (CB67-129), which failed to elicit a mitogenic response, competed for 125I-alpha-thrombin binding to SMCs. Cross-linking analysis of 125I-alpha-thrombin to SMCs revealed a specific cell-surface binding site 55 kDa in size. Finally, thrombin immobilized to a naturally produced extracellular matrix retained potent mitogenic activity toward SMCs. These observations lend support to the possibility that in vivo, subendothelial basement membranes sequester thrombin (as well as other bioactive molecules), which may stimulate localized and persistent growth of arterial SMCs. Thrombin may thus be involved directly in progression of atherosclerotic plaque formation.
Bar-Shavit
R
R
Department of Oncology, Hadassah University Hospital, Jerusalem, Israel.
Benezra
M
M
Eldor
A
A
Hy-Am
E
E
Fenton
J W
JW
Wilner
G D
GD
Vlodavsky
I
I
D000818:Animals; D002417:Cattle; D005109:Extracellular Matrix / Q000502:physiology*; D005786:Gene Expression Regulation; D006133:Growth Substances; D006801:Humans; D066298:In Vitro Techniques; D007457:Iodine Radioisotopes; D007531:Isoflurophate; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger / Q000096:biosynthesis; D011956:Receptors, Cell Surface / Q000378:metabolism; D018179:Receptors, Thrombin; D013917:Thrombin / Q000502:physiology*
D006133:Growth Substances; D007457:Iodine Radioisotopes; D011518:Proto-Oncogene Proteins; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D007531:Isoflurophate; D013917:Thrombin