| Summary: | 碩士 === 國立臺灣大學 === 藥理學研究所 === 88 === 英文摘要
a- and b-crotroxin are members of disintegrin family derived from the venom of the Crotalus atrox. The purification procedure consisted of ion-exchanger chromatography, gel filtration and C18 reverse-phase HPLC. a- and b-crotroxin antagonized the binding of fibrinogen to platelet membrane glycoprotein IIb/IIIa . The purified a- and b-crotroxin were homogenous as judged by SDS-PAGE and NH2-terminal sequence analysis. a-crotroxin was shown as a long-chain disintegrin with a molecular weight of 12,336 daltons, and b-crotroxin, a medium disintegrin of 7,543 daltons. In human platelet suspension, a-crotroxin inhibited thrombin- and collagen-induced human platelet aggregation in a dose-dependent manner with IC50 values of 2.8 ´ 10-7 M and 3.6 ´ 10-7 M, respectively. b-crotroxin dose-dependently inhibited the platelet aggregation triggered by thrombin and collagen. The IC50 values were estimated to be 4.9 ´ 10-7 M and 5.3 ´ 10-7 M, respectively. However, a- and b-crotroxin apparently did not affect the shape change caused by these agonists.
Using flow cytometry, we found that a- and b-crotroxin inhibited the binding of monoclonal antibodies raised against a2 , a5 , b1 , b3 and 7E3 (which recognizes integrin aIIbb3 and aVb3) to HUVEC and GF. Regarding the cell adhesion to extracellular matrices (ECMs), a-crotroxin inhibited HUVEC adhesion to immobilized fibrinogen, fibronectin and vitronectin in a dose-dependent manner. The IC50 values were estimated to be 4.3 ´ 10-7 M, 1.39 ´ 10-6 M and 1.7 ´ 10-7 M, respectively. b-crotroxin also had the similar effect and its IC50 values were estimated to be 1.17 ´ 10-6 M, 2.21 ´ 10-6 M and 6.5 ´ 10-7 M, respectively. In addition, a-crotroxin also concentration-dependently inhibited GF adhesion to immobilized fibrinogen, fibronectin, collagen and vitronectin with IC50 values of 4.7 ´ 10-7 M, 5.3 ´ 10-7 M, 2.3 ´ 10-7 M and 4.2 ´ 10-7 M, and b-crotroxin also inhibited adhesion reaction with IC50 values of 7.1 ´ 10-7 M, 9.2 ´ 10-7 M, 4.4 ´ 10-7 M and 1.24 ´ 10-6 M, respectively. These results indicate that a- and b-crotroxin inhibited HUVEC and GF adhesion to extracellular matrices mainly through the blockade of integrin a2b1, a5b1 and aVb3 expressed on these cells.
The effects of a- and b-crotroxin on HUVEC and GF proliferation were evaluated by using MTT assays. a- and b-crotroxin dose-dependently inhibited the proliferation of HUVECs. The IC50 values were estimated to be 6.4 ´ 10-7 M and 1.43 ´ 10-6 M, respectively. In GF, a- and b-crotroxin also concentration-dependently inhibited the cell proliferation with IC50 values of 4.4 ´ 10-7 M and 9.8 ´ 10-7 M, respectively. In another proliferation assay, a- and b-crotroxin dose-dependently inhibited BrdU incorporation. In HUVECs, the IC50 values were estimated to be 8.5 ´ 10-7 M and 2.71 ´ 10-6 M, respectively. In GFs, the IC50 values were estimated to be 2.9 ´ 10-7 M and 1.01 ´ 10-6 M, respectively. Regarding integrin-mediated signal transduction, a-crotroxin almost completely inhibited phosphorylation of focal adhesion kinase (FAK) triggered by GF adhesion to ECMs (fibrinogen, fibronectin and collagen), while b-crotroxin showed a less pronounced effect. GF adhesion to immobilized a- or b-crotroxin also induced FAK phosphorylation. PKC inhibitor (i.e. calphostin C) inhibited a- or b-crotroxin-induced FAK activation. In addition, we found that GF adhesion to immobilized of a- and b-crotroxin also caused F-actin formation during cytoskeleton rearrangement. This effect was also inhibited by PKC inhibitor, calphostin C, and monoclonal antibodies raised against b1and b3 .
In conclusion, a- and b-crotroxin modulate the functional behaviors of endothelial cells, fibroblasts and platelets by interacting with integrins (a2 , a5 , b1 , b3) expressed on these cells. The working mechanisms of their anti-platelet aggregation, anti-adhesive, anti-proliferative activities and induction of cytoskeleton reorganization may be related to blockade of outside-in signaling. a- and b-crotroxin blocked focal adhesion kinase activation caused by ligation of GF with extracellular matrices. GF adhesion to immobilized a- or b-crotroxin also induced protein kinase-dependent phosphorylation of FAK. However, the mechanism of the anti-proliferation activity of a- and b-crotroxin on HUVECs and GFs still needs to be investigated.
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