apyrase and glycyl-arginyl-glycyl-aspartyl-serine

In static condition, 6F1, an anti-glycoprotein Ia/IIa monoclonal antibody, almost completely prevented initial platelet adhesion to fibrillar collagen, but markedly lost its action with prolonged incubation. The platelet adhesion and spreading at the later stage were prevented by adding the peptide GRGDS, aspirin, and apyrase, suggesting that after initial recognition of platelet glycoprotein Ia/IIa with collagen the activation of glycoprotein IIb/IIIa and the release of thromboxane A2/ADP would promote platelet spreading, thus strengthening the stability of adhesion. Both initial platelet adhesion and platelet spreading were prevented by cytochalasin B, an inhibitor of actin polymerization. In contrast, BAPTA (an intracellular Ca2+ chelator) only inhibited platelet spreading. Inhibition of protein kinase C or protein tyrosine kinase by staurosporine or genistein, respectively, had only little effect on platelet adhesion. These data suggest that actin polymerization and intracellular Ca2+ mobilization are involved in the regulation of platelet spreading after initial platelet contact with collagen.

Topics: Animals; Antibodies, Monoclonal; Apyrase; Aspirin; Blood Platelets; Cattle; Collagen; Cytochalasin B; Egtazic Acid; Humans; Oligopeptides; Platelet Adhesiveness; Platelet Membrane Glycoproteins; Signal Transduction

MCF-7 cells, a metastatic human breast carcinoma line, caused dose-dependent platelet aggregation in heparinized human platelet-rich plasma (PRP). MCF-7 tumor cell-induced platelet aggregation (TCIPA) was almost blocked by apyrase (0.5 U/ml) and completely inhibited by hirudin (5 U/ml). This TCIPA was unaffected by cysteine proteinase inhibition with E-64 (10 microM), but was limited by cell pretreatment with phospholipase A2. MCF-7 cell suspension caused marked, dose-dependent decrease in plasma recalcification times using normal, Factor VIII-deficient, and Factor IX-deficient human plasma. This effect was potentiated in cell lysates but was inhibited in intact cells preincubated with sphingosine. MCF-7 cell suspension did not affect the recalcification time of Factor VII-deficient plasma. Taken together, these data suggest that MCF-7 TCIPA arises from MCF-7 tissue factor activity expression. Trigramim and rhodostomin, RGD-containing snake venom peptides which antagonized the binding of fibrinogen to platelet membrane glycoprotein IIb/IIIa, prevented MCF-7 TCIPA. Likewise, synthetic peptide GRGDS as well as monoclonal antibodies against human tissue factor, platelet membrane glycoprotein IIb/IIIa and Ib prevented MCF-7 TCIPA, which was unaffected by control peptide GRGES. On a molar basis, trigramin (IC50, 0.1 microM) and rhodostomin (IC50, 0.03 microM), were about 5,000 and 18,000 times, respectively, more potent than GRGDS (IC50, 0.54 mM).

Topics: Adenocarcinoma; Animals; Antibodies, Monoclonal; Apyrase; Breast Neoplasms; Crotalid Venoms; Cysteine Proteinase Inhibitors; Female; Hirudins; Humans; Intercellular Signaling Peptides and Proteins; Oligopeptides; Peptides; Phospholipases A; Phospholipases A2; Platelet Aggregation; Platelet Aggregation Inhibitors; Sphingosine; Tumor Cells, Cultured

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

Triflavin, an Arg-Gly-Asp (RGD)-containing peptide, purified from snake venom of Trimeresurus flavoviridis, inhibits human platelet aggregation through the blockade of fibrinogen binding to fibrinogen receptors associated with glycoprotein IIb/IIIa complex. In this report, we examined the effect of triflavin on tumor cells (human hepatoma J-5)-induced platelet aggregation (TCIPA) of heparinized platelet-rich plasma (PRP). ADP-scavenger agents, apyrase (10 U/ml) and creatine phosphate (5 mM)/creatine phosphokinase (5 U/ml) did not inhibit TCIPA while hirudin (5 U/ml) completely inhibited it. J-5 cells initially induced platelet aggregation, then blood coagulation occurred. J-5 cells concentration-dependently shortened the recalcification time of normal as well as Factor VIII, IX-deficient human plasmas, while it was inactive at shortening the recalcification time of Factor VII-deficient plasma, suggesting J-5 cells induced platelet aggregation through activation of extrinsic pathway, leading to thrombin formation as evidenced by the amidolytic activity on s-2238 by expressing tissue factor-like activity. Triflavin inhibited TCIPA in a dose-dependent manner (IC50, 0.02 microM). When compared on molar ratio, triflavin was approximately 30,000 times more potent than GRGDS (IC50, 0.58 mM). On the other hand, GRGES showed no significant effect on TCIPA, even its concentration was raised to 4 mM. Additionally, the monoclonal antibodies, raised against glycoprotein IIb/IIIa complex (i.e., 7E3 and 10 E5) inhibited J-5 TCIPA. In conclusion, we suggest the inhibitory effect of triflavin on J-5 TCIPA may be chiefly mediated by the binding of triflavin to the fibrinogen receptor associated with glycoprotein IIb/IIIa complex on platelet surface membrane.

Topics: Amino Acid Sequence; Apyrase; Binding Sites; Creatine Kinase; Crotalid Venoms; Dose-Response Relationship, Drug; Hirudins; Humans; Molecular Sequence Data; Oligopeptides; Peptides; Phosphocreatine; Platelet Aggregation; Platelet Aggregation Inhibitors; Platelet Membrane Glycoproteins; Tumor Cells, Cultured