hirudin and Osteosarcoma

Saos-2 cells, derived from a primary human osteosarcoma, caused dose-dependent platelet aggregation in heparinised human platelet-rich plasma. Saos-2 tumour cell-induced platelet aggregation (TCIPA) was completely inhibited by hirudin but unaffected by apyrase. The cell suspension shortened the plasma recalcification times of normal, factor VIII-deficient and factor IX-deficient human plasmas in a dose-dependent manner. However, the cell suspension did not affect the recalcification time of factor VII-deficient plasma. Moreover, a monoclonal antibody (MAb) against human tissue factor completely abolished TCIPA. Flow cytometric analysis using anti-integrin MAbs as the primary binding ligands demonstrated that the integrin receptors alpha v beta 3, alpha 5 beta 1 and alpha 6 beta 1 were present of Saos-2 cells, which might mediate tumour cell adhesion to extracellular matrix. Rhodostomin, an Arg-Gly-Asp (RGD)-containing snake venom peptide which antagonises the binding of fibrinogen to platelet membrane glycoprotein IIb/IIIa, prevented Saos-2 TCIPA as well as tumour cell adhesion to vitronectin, fibronectin and collagen type I. Likewise, the synthetic peptide Gly-Arg-Gly-Asp-Ser (GRGDS) showed a similar effect. On a molar basis, rhodostomin was about 18,000 and 1000 times, respectively, more potent than GRGDS in inhibiting TCIPA and tumour cell adhesion.

Topics: Amino Acid Sequence; Antibodies, Monoclonal; Apyrase; Blood Coagulation Disorders; Blood Coagulation Factors; Bone Neoplasms; Cell Adhesion; Enzyme Activation; Extracellular Matrix; Hirudins; Humans; Integrins; Molecular Sequence Data; Neoplasm Metastasis; Neoplasm Proteins; Oligopeptides; Osteosarcoma; Peptides; Platelet Aggregation; Platelet Aggregation Inhibitors; Platelet Membrane Glycoproteins; Protein Binding; Thrombin; Tumor Cells, Cultured

Some, if not all, of the cellular actions of alpha-thrombin are now believed to be mediated by proteolytic cleavage of the cell surface thrombin receptor to yield a tethered ligand that initiates signal transduction via the receptor. We have investigated the actions of alpha-thrombin on the regulation of cytosolic free Ca2+ concentration ([Ca2+]i) and intracellular pH (pHi) in human osteoblast-like Saos-2 cells. After acidification with nigericin, thrombin induced an acute increase of [Ca2+]i and a rise in pHi. The action of thrombin on pHi was dependent on activation of the Na(+)-H+ antiporter. Thrombin elicited parallel concentration-dependent increases in both [Ca2+]i and pHi, and the rise in [Ca2+]i was a prerequisite for the increase in pHi. Preincubation of thrombin with the active site proteolytic inhibitor, BOC-D-Phe-L-Pro-D,L-Lys-CF3, prevented the alkalinization response to thrombin but had little or no effect on the thrombin-induced rise in [Ca2+]i. Hirudin, a natural inhibitor of thrombin, acts by tight binding to several discrete regions on the thrombin molecule. Preincubation of thrombin with hirudin completely blocked the rise in both [Ca2+]i and pHi. These results demonstrate that the thrombin-induced rise in [Ca2+]i alone is not sufficient to cause alkalinization in Saos-2 cells. More importantly, our findings reveal that not all of the cellular actions of thrombin can be explained by proteolytic cleavage of the thrombin receptor and suggest that different domains on the thrombin molecule may be required for eliciting signals that raise [Ca2+]i and pHi in Saos-2 cells.

Topics: Alkalies; Calcium; Cytosol; Hirudins; Humans; Hydrogen-Ion Concentration; Nigericin; Osteoblasts; Osteosarcoma; Peptide Hydrolases; Rest; Thrombin; Tumor Cells, Cultured

Cultured SK-OS-10 cells (human osteogenic sarcoma metastatic to lung) shed microvesicles (dia. 300-1000 nm) that contained procoagulant and proaggregatory activities inhibitable by hirudin, by anti-tissue factor antibody and by phospholipase A2. These results show that SK-OS-10 cells belong to a group including U87MG human glioblastoma and HL-60 promyelocytic leukemia in which these activities are due to a thrombin-dependent mechanism arising from the presence of tissue factor on the surface of the tumor cells and their shed microvesicles.

Topics: Antibodies; Blood Platelets; Cell Line; Creatine Kinase; Hirudins; Humans; Inclusion Bodies; Lung Neoplasms; Osteosarcoma; Phosphocreatine; Phospholipases; Platelet Activating Factor; Platelet Aggregation; Thromboplastin