On the mechanism of thrombin-induced angiogenesis: involvement of α_vβ₃-integrin

Abstract

. On the mechanism of thrombin-induced angiogenesis: involvement of ␣v␤3-integrin. Am J Physiol Cell Physiol 283: C1501-C1510, 2002. First published July 17, 2002 10.1152/ajpcell.00162.2002Thrombin has been reported to be a potent angiogenic factor both in vitro and in vivo, and many of the cellular effects of thrombin may contribute to activation of angiogenesis. In this report we show that thrombin-treatment of human endothelial cells increases mRNA and protein levels of ␣v␤3-integrin. This thrombin-mediated effect is specific, dose dependent, and requires the catalytic site of thrombin. In addition, thrombin interacts with ␣v␤3 as demonstrated by direct binding of ␣v␤3 protein to immobilized thrombin. This interaction of thrombin with ␣v␤3-integrin, which is an angiogenic marker in vascular tissue, is of functional significance. Immobilized thrombin promotes endothelial cells attachment, migration, and survival. Antibody to ␣v␤3 or a specific peptide antagonist to ␣v␤3 can abolish all these ␣v␤3-mediated effects. Furthermore, in the chick chorioallantoic membrane system, the antagonist peptide to ␣v␤3 diminishes both basal and the thrombin-induced angiogenesis. These results support the pivotal role of thrombin in activation of endothelial cells and angiogenesis and may be related to the clinical observation of neovascularization within thrombi. attachment; migration; apoptosis; reverse transcription-polymerase chain reaction THE FREQUENCY OF BLOOD COAGULATION in cancer patients, known for more than 130 years, is supported by clinical, laboratory, and histopathological evidence. This is explained at the molecular and cellular level by the thromboplastic activity of circulating tumor cells, the existence of "a cancer coagulative factor," the activation of factor X, the generation of prothrombinase by tumor cells, and the encircling of cancerous tissue by fibrin deposits (38,50). In addition, the possibility of a relation between blood clotting mechanisms and tumor progression and development of metastases was postulated as early as 1878 by Billroth (7) on the basis of the observation that cancer cells exist within thrombi. This finding was interpreted as evidence that tumor cells spread by thromboembolism. More recently, large epidemiological studies have provided evidence that the standardized incidence ratio for certain types of cancer is as high as 6.7 within a year following a thromboembolic episode (3, 41). These clinical data are in line with animal experiments where thrombin-treated B16 melanoma cells show a dramatic increase in their metastatic potential in the lung of rats (37). These observations have led to experimental use of heparin, aspirin, and warfarin for the prevention and treatment of tumors in animal models and humans (23, 50).We proposed earlier (33, 47) that the tumor-promoting effect of thrombin/thrombosis may be related to our finding that thrombin is a potent promoter of angiogenesis, a process essential for tumor growth and metastasis. The angiogenic action of thrombin was shown to b...

Thrombin Functions through Its RGD Sequence in a Non-canonical Conformation

“…Inactivation of thrombin with the S195A mutation, which replaces the active site Ser with Ala, did not abrogate this property. This is in agreement with previous studies of thrombin inactivated at the active site with diisopropylphosphofluoridate (15). The ability to promote endothelial cell attachment was also retained by mutants significantly compromised in their cleavage of fibrinogen or PARs (e.g.…”

“…Recent studies have documented that surface-absorbed thrombin promotes endothelial cell attachment and migration via interaction with ␣ v ␤ 3 integrin (15). The effect appears to be mediated by the RGD sequence, but no direct demonstration could be provided in that or any previous studies (6 -8, 16).…”

“…As a possible solution to this conundrum, Bar-Shavit et al (7,8) have suggested that thrombin functions through its RGD sequence only after denaturation or proteolytic cleavage, either by thrombin itself (7) or plasmin (8). However, recent studies have shown that surface-absorbed thrombin, or its active site inhibited form, promote attachment, migration, and survival of endothelial cells via the ␣ ␤ 3 integrin (15,16). Other studies have provided evidence that thrombin in solution can interact with ␣ ␤ 3 integrin in a way that is inhibited by RGD mimetics (17,18).…”

Thrombin and Vascular Development

“…This adhesion molecule is expressed in activated endothelial cells (eg, in tumor vasculature) and mediates signals involved in vascular remodeling. 19 Accordingly, in vitro stimulation of endothelial cells by thrombin upregulates levels of ␣v/␤3 mRNA , 20 which in turn supports the formation of new vasculature. Taken together, thrombin has proangiogenic activity in various in vitro assays involving several pathways known to be crucial in angiogenesis.…”

Thrombin physiology and pathophysiology

“…48 Upregulation of integrins is a mechanism by which thrombin promotes endothelial cell survival following detachment from basement membranes and during migration to the distal sites necessary for angiogenesis associated with tumor progression and metastasis. 30,49 Intracellular calciumsensitive kinases activated by PAR-1 cleavage include mitogen activated protein kinase and extracellular signal regulated kinase. These specific kinases are often expressed in humans with a relatively common genetic mutation of the oncogene Ras.…”