Initiation of proliferative events by human α‐thrombin requires both receptor binding and enzymic activity

Abstract

To determine the role of thrombin high-affinity receptor occupancy and enzymic activity in thrombin initiation of cell proliferation, we have utilized thrombin derivatives which separate these functions. We previously showed that enzymically active gamma-thrombin stimulates ion fluxes without binding to high-affinity sites, whereas proteolytically inhibited DIP-alpha-thrombin which binds to high-affinity receptors does not. Since neither derivative initiates DNA synthesis by itself, this suggested that two separate sequences of events might be necessary for a complete initiation signal. We now report that the combination of DIP-alpha-thrombin and gamma-thrombin initiate DNA synthesis and cell proliferation to levels approaching the maximal initiation by native alpha-thrombin. This combinatory effect is dose-dependent for both gamma-thrombin and DIP-alpha-thrombin in the same concentration range as alpha-thrombin alone. Thus, these same concentrations of alpha-thrombin alone may be required to initiate each sequence of events. The combinatory stimulation could be achieved even if the derivatives were added individually up to 8 hr apart. Moreover, preincubation with either derivative shortened the lag period for initiation of DNA synthesis by native alpha-thrombin. These results indicate that both receptor occupancy and enzymic activity are necessary for thrombin initiation of cell proliferation and that each action initiates a sequence of early events which moves the cell forward toward entry into a proliferative cycle.

Thrombin Inhibitor, Argatroban, Prevents Tumor Cell Migration and Bone Metastasis

“…Thrombin is a multifunctional protein involved in a variety of biologic functions, including blood coagulation, platelet adhesion, platelet aggregation, mitogenesis of fibroblasts and smooth muscle cells [25, 26, 27, 28, 29, 30], as well as tumor metastasis [31, 32, 33]. It has been well documented that thrombin stimulates further tumor cell adhesion to platelets [31, 32], endothelial cells and extracellular matrix proteins [33], as well as tumor cell mitogenesis [13].…”

Role of thrombin as a tumor growth factor

“…10,12 Indeed, there are several reports that relatively high concentrations of thrombin can act as a mitogenic agent for mesenchymal tissue such as fibroblasts, endothelial cells and smooth muscle cells in the absence of serum. [13][14][15][16][17][18] Neither is there any direct proof that thrombin enhances primary tumor growth in vivo because this is dependent upon tumor implantation and angiogenesis. Indeed, there is a large body of evidence pointing to the stimulatory role of thrombin on the induction of angiogenesis in vivo.…”

Thrombin Primes Responsiveness of Selective Chemoattractant Receptors at a Site Distal to G Protein Activation

“…Diisopropylphosphofluoridate-inactivated ␣-thrombin did not stimulate Ca 2ϩ mobilization in RBL-2H3 cells and had no effect on the fMLP response (Table II). ␥-Thrombin retains its catalytic activity, but its binding site for the tethered ligand thrombin receptor is disrupted (26). ␥-Thrombin itself caused only a small increase in Ca 2ϩ mobilization but primed the response to fMLP by about 3-fold.…”

“…Hirudin completely blocked both Ca 2ϩ mobilization and priming of the fMLP response by thrombin but had no effect on the fMLP response itself (Table II). Thrombin inactivated by diisopropylphosphofluoridate has no catalytic activity but still binds to the seven transmembrane domain thrombin receptor via the anion binding exosite (26). Diisopropylphosphofluoridate-inactivated ␣-thrombin did not stimulate Ca 2ϩ mobilization in RBL-2H3 cells and had no effect on the fMLP response (Table II).…”