Tsopanoglou Nikos E, Papaconstantinou Matthew E, Flordellis Christodoulos S, Maragoudakis Michael E
Department of Pharmacology, Medical School, University of Patras, Rio Patras, Greece.
Thromb Haemost. 2004 Oct;92(4):846-57. doi: 10.1160/TH04-04-0208.
In a previous report we have presented evidence that thrombin interacts with alpha(v)beta(3) integrin in endothelial cells at the molecular and cellular level. This interaction was shown to be of functional significance in vitro and in vivo and contributed to activation of angiogenesis by thrombin. In the present study, we have used a synthetic thrombin peptide, TP508, which represents residues 183 to 200 of human thrombin. This peptide lacks the catalytic site of thrombin but contains the thrombin RGD sequence. Immobilized (surface-coated) TP508 peptide, like thrombin, supported alpha(v)beta(3) integrin-dependent endothelial cell attachment and haptotactic migration. These effects were specific (a scrambled TP508 peptide was without effect), and dosedependent. The RGD sequence was essential since a modified TP508 peptide, which contained RAD sequence instead of RGD, was inactive. Immobilized TP508 peptide stimulated phosphorylation of mitogen-activated protein kinases and focal adhesion kinase, the signal transduction pathways characteristic for integrin activation. On the other hand, TP508 peptide, when in solution, did not mimic other thrombin-promoted angiogenic effects, such as that of activation gelatinase A, upregulation of expression of vascular endothelial growth factor receptor mRNA or prostacyclin PGI(2) release in endothelial cells. On the contrary, soluble TP508 acted as an antagonist for the aforementioned effects of thrombin. TP508 peptide inhibited these thrombin-induced effects through a RGD and alpha(v)beta(3)-related mechanism. The antagonism with thrombin or thrombin receptor activating peptide was specific and involved at least in part mitogen-activated protein kinases activation. These results point to the importance of RGD sequence of thrombin in mediating effects on endothelial cells and angiogenesis.
在之前的一份报告中，我们已提供证据表明，凝血酶在分子和细胞水平上与内皮细胞中的α(v)β(3)整合素相互作用。这种相互作用在体外和体内均显示出功能意义，并有助于凝血酶激活血管生成。在本研究中，我们使用了一种合成的凝血酶肽TP508，它代表人类凝血酶的183至200位残基。该肽缺乏凝血酶的催化位点，但含有凝血酶RGD序列。固定化（表面包被）的TP508肽与凝血酶一样，支持α(v)β(3)整合素依赖性内皮细胞黏附和趋触性迁移。这些效应具有特异性（无序的TP508肽无作用）且呈剂量依赖性。RGD序列至关重要，因为含有RAD序列而非RGD的修饰TP508肽无活性。固定化的TP508肽刺激丝裂原活化蛋白激酶和黏着斑激酶的磷酸化，这是整合素激活的特征性信号转导途径。另一方面，TP508肽在溶液中时，不会模拟凝血酶促进的其他血管生成效应，如激活明胶酶A、上调血管内皮生长因子受体mRNA的表达或内皮细胞中前列环素PGI(2)的释放。相反，可溶性TP508作为凝血酶上述效应的拮抗剂。TP508肽通过RGD和α(v)β(3)相关机制抑制这些凝血酶诱导的效应。与凝血酶或凝血酶受体激活肽的拮抗作用具有特异性，且至少部分涉及丝裂原活化蛋白激酶的激活。这些结果表明凝血酶的RGD序列在介导对内皮细胞和血管生成的作用中具有重要性。
