Current concepts for a drug-induced inhibition of formation and action of thromboxane A2

Urinary and plasma metabolites of thromboxane A2 (TxA2) indicate an increased TxA2 synthesis in a number of diseases, whereby TxA2 is assumed to contribute to the underlying pathomechanisms by its profound effects on platelet aggregation and smooth muscle contraction. In some clinical situations the increment in TxA2 biosynthesis is accompanied by an increased formation of prostacyclin (PGI2) which is one of the most potent inhibitors of platelet activation and smooth muscle contraction. Therefore, drugs are being developed which suppress the formation or action of TxA2 without interfering with its functional antagonist PGI2. Low doses of acetylsalicyclic acid (ASA) preferentially inhibit cyclooxygenase activity in platelets and the synthesis of TxA2 in vivo. However, neither low doses (approximately 300 mg/day) nor very low doses spare the formation of PGI2 completely. Despite its limited selectivity, very low dose ASA (approximately 40 mg/day) provides an attractive perspective in TxA2 pharmacology. Although thromboxane synthase inhibitors selectively suppress TxA2 biosynthesis PGH2 can accumulate instead of TxA2 and substitute for TxA2 at their common TxA2/PGH2 receptors. Thromboxane synthase inhibitors can only exert platelet-inhibiting and vasodilating effects if PGH2 rapidly isomerizes to functional antagonists like PGI2 that can be formed from platelet-derived PGH2 by the vessel wall. TxA2/PGH2 receptor antagonists provide a specific and effective approach for inhibition of TxA2. These inhibitors do not interfere with the synthesis of PGI2 and other prostanoids but prevent TxA2 and PGH2 from activating platelets and inducing smooth muscle contractions. Most of the available TxA2/PGH2 receptor antagonists produce a competitive antagonism that can be overcome by high agonist concentrations. Since in certain disease states very high local TxA2 concentrations are to be antagonized, non-competitive receptor antagonists may be of particular interest. Some recent TxA2/PGH2 receptor antagonists produce such a non-competitive type of inhibition due to their low dissociation rate constant. As a consequence, agonists like TxA2 or PGH2 only reach a hemiequilibrium state at their receptors, previously occupied by those antagonists. A combination of a thromboxane synthase inhibitor with a TxA2/PGH2 receptor antagonist presents a very high inhibitory potential that utilizes the dual activities of the synthase inhibitor to increase PGI2 formation and of the receptor antagonist to antagonize PGH2 and TxA2. Such combinations or dual inhibitors, combining both moieties in one compound, prolong the skin bleeding time to a greater extent than thromboxane synthase inhibitors and even more than low dose ASA or TxA2/PGH2 receptor antagonists.     

A common binding site for primary prostanoids in vascular smooth muscles: a definitive discrimination of the binding for thromboxane A2/prostaglandin H2 receptor agonist from its antagonist

Differences in binding characteristics between agonists and antagonists for the thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor were examined in rat cultured vascular smooth muscle cells (VSMC). Scatchard analysis indicated the existence of two binding sites for the TXA2/PGH2 agonist, whereas a single class of recognition sites for the receptor antagonists were observed with approximately the same maximum binding capacity (Bmax) as a high-affinity binding site of the agonist. Weak binding inhibition by approx. 100 nM of primary prostanoids (PGE1, PGF2 alpha and PGD2) was detected only with the TXA2/PGH2 agonist, and not with the antagonist. Primary prostanoids as well as TXA2/PGH2 agonists (U46619 and STA2) suppressed the [3H]PGF2 alpha and [3H]PGE1 binding with almost the same potency, whereas TXA2/PGH2 antagonists (S-145, SQ29,548 and ONO3708) did not. The Bmax value of the binding sites was roughly identical in PGF2 alpha, PGE1 and a low-affinity binding site of U46619. These results suggest the existence of two binding sites for TXA2/PGH2 in VSMC, i.e., a high-affinity binding site corresponding to that of the TXA2/PGH2 antagonists and a low-affinity binding site in common with primary prostanoids.     

Binding of an [125I] labelled thromboxane A2/prostaglandin H2 receptor agonist to baboon platelets

To characterize the thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor on baboon platelets the binding of [125I]BOP was studied. [125I]BOP bound to washed baboon platelets in a saturable manner. Scatchard analysis of binding isotherms revealed a Kd of 1.12 +/- 0.08 nM and a binding capacity of 54 +/- 5 fmoles/10(8) platelets (326 sites/platelet). Several TXA2/PGH2 agonists and antagonists displaced [125I]BOP from its baboon platelet binding site with a rank order of potency similar to human platelets: I-BOP greater than SQ29548 greater than U46619 = I-PTA-OH greater than PTA-OH. I-BOP aggregated washed baboon platelets with an EC50 of 10 +/- 4 nM. The results indicate that [125I]BOP binds to the TXA2/PGH2 receptor on baboon platelets and that this receptor is similar to its human counterpart.     

Identification of a putative thromboxane A2/prostaglandin H2 receptor in human platelet membranes

The binding of the competitive thromboxane A2/prostaglandin H2 (TXA2/PGH2) antagonist (9,11-dimethylmethano-11, 12-methano-16-(3-aza-15 alpha beta-omega-tetranor-TXA2) ([125I]PTA-OH) to membranes prepared from human platelets was characterized. [125I]PTA-OH binding to membranes from human platelets was saturable, displaceable, and dependent on protein concentration. Scatchard analysis of equilibrium binding carried out at 30 degrees C revealed one class of binding sites with a Kd of 30 +/- 4 nM and a Bmax of 1.8 +/- 0.3 pmol/mg of protein (n = 5). Kinetic analysis of the binding of [125I]PTA-OH at 0 degrees C yielded a k1 of 1.35 X 10(6) M-1 min-1 and a k-1 of 0.032 min-1, Kd = k-1/k1 = 24 nM. The potencies of a series of TXA2/PGH2 antagonists as inhibitors of [125I]PTA-OH binding was correlated with their potencies as inhibitors of platelet aggregation induced by the TXA2/PGH2 mimetic, U46619 (1 microM) (r = 0.93, p less than 0.01). A series of TXA2/PGH2 mimetics also displaced [125I]PTA-OH from its binding site, and their potencies as inhibitors of [125I]PTA-OH binding were correlated with their potencies as stimulators of platelet aggregation (r = 0.91, p less than 0.05). The IC50 values for displacement of [125I]PTA-OH by PGF2 alpha, PGD2, and the stable PGI2 analog Iloprost were greater than 25 microM, suggesting that [125I]PTA-OH does not bind to other known platelet prostaglandin receptors. These data are consistent with the notion that this binding site may represent the platelet TXA2/PGH2 receptor.     

The affinities of prostaglandin H2 and thromboxane A2 for their receptor are similar in washed human platelets

Both thromboxane A2 (TXA2) and its precursor prostaglandin H2 (PGH2) are labile and share a common receptor. The affinities of these two compounds for their putative common receptor are unknown. We compared the potencies of TXA2 and PGH2 to aggregate human platelets and bind to the TXA2/PGH2 receptor. TXA2 was more potent than PGH2 in initiating aggregation in platelet-rich plasma, EC50 of 66 +/- 15 nM and 2.5 +/- 1.3 microM, respectively. In washed platelets, however, PGH2 was more potent than TXA2 with EC50 values of 45 +/- 2 nM and 163 +/- 21 nM, respectively. The affinity of these two compounds in washed platelets was determined in radioligand competition binding assays employing [125I]-PTA-OH. The Kd values for PGH2 and TXA2 were 43 nM and 125 nM, respectively. The results demonstrate that the affinity of PGH2 for the platelet TXA2/PGH2 receptor is greater than previously thought. The data raise the possibility that PGH2 may significantly contribute to the responses attributed to TXA2 in vivo.     

Isoelectric and mass characterization of human platelet thromboxane A2/prostaglandin H2 receptors

To further characterize the human thromboxane A2 (TXA2)/prostaglandin H2 (PGH2) receptor, preparative isoelectric focusing (IEF) was performed on solubilized platelet membranes. TXA2/PGH2 receptors, assayed by specific binding of the TXA2/PGH2 antagonist [125I]PTA-OH, were electrofocused at pH 5.6. Scatchard analysis of IEF fraction pH 5.6 revealed a 180-fold concentration of TXA2/PGH2 receptors (Bmax = 3650 +/- 228 pM/mg focused, 19 +/- 4 pM/mg unfocused) with no change in binding affinity (Kd = 47 +/- 7 nM focused, 36 +/- 14 nM unfocused). SDS-polyacrylamide gel electrophoresis of photoaffinity-labelled electrofocused receptors revealed concentration of specifically labelled proteins having molecular masses of 49,000 and 27,000 Daltons. These results suggest that the human platelet TXA2/PGH2 receptor has a pI of 5.6, molecular mass of 49,000 Daltons, and may exist as a dimer. Preparative IEF should prove useful in the eventual purification of this receptor.     

Synthesis and biological activity of 4-methyl-3,5-dioxane derivatives as thromboxane A2 receptor antagonists

The synthesis and biological activity of novel 4-methyl-3,5-dioxane analogues are described. All compounds were produced through modification of the substituent formally corresponding to the omega-octenol side chain of thromboxane A2 (TXA2), in reference to the structure of SQ29548. Several compounds were found to be potent TXA2 receptor antagonists. Compound 8b was the most effective inhibitor of 9,11-epoxymethano PGH2 (U-46619)-induced human platelet aggregation (IC50 = 7.4 nM).     

Differential effect of pH on thromboxane A2/prostaglandin H2 receptor agonist and antagonist binding in human platelets.

The effects of changes in pH on the binding of agonists and antagonists to the human platelet thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor were determined. Competition binding studies were performed with the TXA2/PGH2 mimetic [1S-1 alpha,2 beta (5Z), 3 alpha(1E,3R*),4 alpha)]-7-[3-(3-hydroxy-4'-iodophenoxy)-1-buteny) 7-oxabicyclo-[2.2.1]-heptan-2-yl]-5-heptenoic acid ([125I]BOP). The pH optimum for binding of [125I] BOP to washed human platelets was broad with a range of pH 4-6 in contrast to that of the TXA2/PGH2 receptor antagonist 9,11-dimethyl-methano-11,12-methano-16-(3-iodo-4-hydroxyl)-13-aza-15 alpha,beta-omega-tetranorthromboxane A2 ([125I]PTA-OH) which was 7.4. Scatchard analysis of [125I]BOP binding in washed platelets at pH 7.4, 6.0, and 5.0 revealed an increase in affinity (Kd = 1.16 +/- 0.06, 0.64 +/- 0.09, and 0.48 +/- 0.05 nM, respectively) and an increase in the number of receptors (Bmax = 2807 +/- 415, 5397 +/- 636, and 7265 +/- 753 sites/platelet, respectively). The potency of I-BOP to induce shape change in washed platelets at pH 6.0 was also significantly increased from an EC50 value of 0.34 +/- 0.016 nM at pH 7.4 to 0.174 +/- 0.014 nM at pH 6.0 (n = 6, p less than 0.05). In contrast, the EC50 value for thrombin was unaffected by the change in pH. In competition binding studies with [125I]BOP, the affinity of the agonists U46619 and ONO11113 were increased at pH 6.0 compared to 7.4. In contrast, the affinity of the TXA2/PGH2 receptor antagonists I-PTA-OH, SQ29548, and L657925 were either decreased or unchanged at pH 6.0 compared to 7.4. Diethyl pyrocarbonate and N-bromosuccinimide, reagents used to modify histidine residues, reversed the increase in affinity of [125I]BOP at pH 6.0 to values equivalent to those at pH 7.4. In solubilized platelet membranes, the effects of NBS were blocked by coincubation with the TXA2/PGH2 mimetic U46619. The results suggest that agonist and antagonist binding characteristics are different for the TXA2/PGH2 receptor and that histidine residue(s) may play an important role in the binding of TXA2/PGH2 ligands to the receptor.     

Characterization of thromboxane A2/prostaglandin H2 receptors and histamine H1 receptors in cultured guinea-pig tracheal smooth-muscle cells.

We characterized thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors and histamine H1 receptors in Guinea-pig cultured tracheal smooth-muscle cells (TSMC). [3H]SQ 29,548 (a TXA2 antagonist)-binding sites were saturable and a high affinity with a dissociation constant of 6.2 +/- 0.60 nM (mean +/- S.E.) and a receptor density of 46 +/- 4.6 fmol/10(6) cells. [3H]SQ 29548 binding was completely inhibited by TXA2 mimetics or antagonists. Intracellular calcium concentration ([Ca2+]i) in TSMC was increased with U46619 stimulation and the increase was attenuated by TXA2 antagonists, the potencies of which correlated with those inhibiting the activities of the [3H]SQ 29548 binding. [3H]Mepyramine (a H1 antagonist)-binding sites were also present in TSMC. [3H]Mepyramine had a single class of low-affinity-binding sites with a dissociation constant of 2.6 +/- 0.081 microM and a receptor density of 10.6 +/- 0.11 nmol/mg protein. [3H]Mepyramine binding in TSMC membrane was inhibited by H1 antagonists, but not by H2 antagonists. The inhibition constants of mepyramine in TSMC were 910-times lower than those in tracheal membranes. In contrast, the histamine-induced increase in [Ca2+]i in TSMC was inhibited in the presence of low concentrations of H1 antagonists. All these observations provide evidence that TXA2/PGH2 receptors, mepyramine-binding sites and/or H1 receptors are expressed in cultured TSMC.     

Synthesis and biological activity of 1-phenylsulfonyl-4-phenylsulfonylaminopyrrolidine derivatives as thromboxane a(2) receptor antagonists

The synthesis and biological activity of novel 1-phenylsulfonyl-4- phenylsulfonylaminopyrrolidine analogues are described. All compounds were produced through modification of the substituent formally corresponding to the 1,3-dioxane ring system and the omega-octenol side chain of thromboxane A(2) (TXA(2)), in reference to the structure of Daltroban. Several compounds were found to be potent TXA(2) receptor antagonists. Compound 51a was the most effective inhibitor of 9,11-epoxymethano PGH(2) (U-46619)-induced rat aortic strip contraction (IC(50)=0.48 nM).     

Substrate inactivation of lung thromboxane synthase preferentially decreases thromboxane A2 production

Bovine lung thromboxane synthase was immobilized on phenyl-Sepharose beads by adsorption. The immobilized enzyme was catalytically active and synthesized both TXA2 and HHT. The production of both products was inhibited by 1-benzylimidazole and furegrelate. Multiple additions of PGH2 dramatically reduced the ability of the enzyme to synthesize TXA2, but did not effect the synthesis of HHT. In addition, 1-benzylimidazole did not protect thromboxane synthase from inactivation with multiple additions of PGH2. When the enzyme was incubated with PGH2 in the presence of 1-benzylimidazole, the synthesis of TXA2 was inhibited. When the inhibitor was removed the enzyme had still been inactivated by PGH2 in the presence of 1-benzylimidazole. Thus the substrate inactivation of the enzyme does not require the production of TXA2. Our data suggests that the synthesis of TXA2 and HHT can be differentially inactivated and may occur at different sites on the enzyme.     

On the mechanism of prostacyclin and thromboxane A2 biosynthesis

The present research describes studies which address the mechanism of prostacyclin (PGI2) and thromboxane A2 (TXA2) biosynthesis. In addition to prostaglandin H1 (PGH1), PGG2, PGH2, and PGH3, also 8-iso-PGH2, 13(S)-hydroxy-PGH2, and 15-keto-PGH2 were applied to determine the substrate specificities and kinetics of prostacyclin and thromboxane synthase in more detail. Human platelet thromboxane synthase converted PGH1, 8-iso-PGH2, 13(S)-hydroxy-PGH2 and 15-keto-PGH2 into the corresponding heptadecanoic acid (C17) plus malondialdehyde, whereas the thromboxane derivative was formed only from PGG2, PGH2, and PGH3 together with the corresponding C17 metabolite and malondialdehyde in a 1:1:1 ratio. In contrast, PGG2, PGH2, 13(S)-hydroxy-PGH2, 15-keto-PGH2 and PGH3 were almost completely isomerized to the corresponding prostacyclin derivative by bovine aortic prostacyclin synthase, whereas PGH1 and 8-iso-PGH2 only produced the corresponding C17 hydroxy acid plus malondialdehyde. Isotope-labeling experiments with [5,6,8,9,11,12,14,15-2H]PGH2 revealed complete retention of label and no isotope effect in the course of thromboxane biosynthesis, but the loss of one 2H atom at C-6 with an isotope effect of 1.20 during PGI2 formation. Prostacyclin and thromboxane synthase bind both 9,11-epoxymethano-PGF2 alpha and 11,9-epoxymethano-PGF2 alpha at the heme iron, but according to their difference spectra in opposite ways with respect to the 9- and 11-position. In agreement with published model studies, a cage radical mechanism is proposed for both enzymes according to which the initial radical process is terminated through oxidation of carbon-centered radicals by the iron-sulfur catalytic site, followed by ionic rearrangement to PGI2 or TXA2. Various Fe(III) model compounds as well as liver microsomes or cytochrome P-450CAM can also form small amounts of PGI2 and TXA2, but mainly yield 12(S)-hydroxy-5,8,10-heptadecatrienoic acid plus malondialdehyde probably by a radical fragmentation pathway.     

Upregulation of thromboxane synthase in human colorectal carcinoma and the cancer cell proliferation by thromboxane A2

Tumor growth of colorectal cancers accompanies upregulation of cyclooxygenase-2, which catalyzes a conversion step from arachidonic acid to prostaglandin H(2) (PGH(2)). Here, we compared the expression levels of thromboxane synthase (TXS), which catalyzes the conversion of PGH(2) to thromboxane A(2) (TXA(2)), between human colorectal cancer tissue and its accompanying normal mucosa. It was found that TXS protein was consistently upregulated in the cancer tissues from different patients. TXS was also highly expressed in human colonic cancer cell lines. Depletion of TXS protein by the antisense oligonucleotide inhibited proliferation of the cancer cells. This inhibition was rescued by the direct addition of a stable analogue of TXA(2). The present results suggest that overexpression of TXS and subsequent excess production of TXA(2) in the cancer cells may be involved in the tumor growth of human colorectum.     

A photoaffinity label for the thromboxane A2/prostaglandin H2 receptor in human blood platelets

A photoactive iodoarylazide derivative (I-APA-PhN3) of the competitive thromboxane A2/prostaglandin H2 (TXA2/PGH2) antagonist 13-azaprostanoic acid is evaluated. Upon photoactivation, the compound was found to inhibit specifically and irreversibly human platelet aggregation induced by the TXA2/PGH2 mimetic U46619. In receptor-binding studies using [3H]U46619, I-APA-PhN3 exhibited an IC50 of 300 nM for inhibition of U46619 binding. Photoactivation of I-APA-PhN3 resulted in an irreversible 58% reduction in specific binding of U46619. This compound and its corresponding ratio-iodinated form will prove to be useful tools for the isolation and purification of the TXA2/PGH2-binding protein in human platelets.     

A possible role of thromboxane A2 (TXA2) and prostacyclin (PGI2) in circulation.

Recently two local hormones, thromboxane A2 (TXA2) and prostacyclin (PGI2) have been discovered. These hormones are labile metabolites of arachidonic acid. TXA2 is generated by blood platelets, while PGI2 is produced by vascular endothelium. TXA2 is a potent vasoconstrictor. It also initiates the release reaction, followed by platelet aggregation. PGI2 is a vasodilator, especially potent in coronary circulation. It also inhibits platelet aggregation by virtue of stimulation of platelet adenyl cyclase. Common precursors for both hormones are cyclic endoperoxides PGG2 and PGH2, being formed by cyclooxygenation of arachidonic acid. This last enzymic reaction is more efficient in platelets than in vascular endothelium, and therefore the generation of PGI2 by vasuclar wall is accelerated by an interaction between platelets and endothelial cells. During this interaction platelets supply the endothelial PGI2 synthetase with their cyclic endoperoxides. The newly formed PGI2 repels the platelets from the intima. When PGI2 synthetase is irreversibly inactivated by low concentration of lipid peroxides, then the platelets are not rejected but stick to the endothelium, generate TXA2 and mature thrombi are formed. A balance between formation and release of PGI2, TXA2 and/or cyclic endoperoxides in circulation is of utmost importance for the control of intra-arterial thrombi formation and possibly plays a role in the pathogenesis of atherosclerosis.     

Mechanism for antiplatelet effect of onion: AA release inhibition, thromboxane A(2)synthase inhibition and TXA(2)/PGH(2)receptor blockade

Antiplatelet actions of aqueous extract of onion were investigated in rat and human platelet. IC(50)values of onion extract for collagen-, thrombin-, arachidonic acid (AA)-induced aggregations and collagen-induced thromboxane A(2)(TXA(2)) formation were 0.17 +/- 0. 01, 0.23 + 0.03, 0.34 +/- 0.02 and 0.12 +/- 0.01 g/ml, respectively. [(3)H]-AA release induced by collagen (10 microg/ml) in rat platelet was decreased by onion compared to control (22.1 +/- 2.13 and 5.2 +/- 0.82% of total [(3)H]-AA incorporated, respectively). In fura-2 loaded platelets, the elevation of intracellular Ca(2+)concentration stimulated by collagen was inhibited by onion. Onion had no cytotoxic effect in platelet. Onion significantly inhibited TXA(2)synthase activity without influence on COX activity. Platelet aggregation induced by U46619, a stable TXA(2)mimetic, was inhibited by onion, indicating its antagonism for TXA(2)/PGH(2)receptor. These results suggest that the mechanism for antiplatelet effect of onion may, at least partly, involve AA release diminution, TXA(2)synthase inhibition and TXA(2)/PGH(2)receptor blockade.     

Binding of a thromboxane A2/prostaglandin H2 agonist [3H]U46619 to washed human platelets

The binding characteristics of [3H]U46619 to washed human platelets were studied. [3H]U46619 binding to washed human platelets was saturable and displaceable. Kinetic studies yielded a Kd of 11 +/- 4 nM (n = 4). Scatchard analysis of equilibrium binding studies revealed one class of high affinity binding sites with a Kd of 20 +/- 7 nM and a Bmax of 9.1 +/- 2.3 fmole/10(7) platelets (550 +/- 141 binding sites per platelet) (n = 4). A number of compounds that act as either agonists or antagonists of the TXA2/PGH2 receptor were tested for their ability to inhibit the binding of [3H]U46619 to washed human platelets. The Kds of the agonists and antagonists were similar to their potencies to induce or inhibit platelet aggregation. These data provide some evidence that [3H]U46619 binds to the putative human platelet TXA2/PGH2 receptor.     

Regulation of microvascular prostacyclin and thromboxane with inhibitors of arachidonic acid metabolism

The levels of the stable degradation products of prostacyclin (PGI2) and thromboxane A2 (TXA2): 6-oxo-prostaglandin E1 alpha (6-oxo-PGE1 alpha) and thromboxane B2 (TXB2) respectively were determined in the effluent of the rabbit epigastric skin flap after infusion of exogenous arachidonic acid. The blood to the flap passes through the microcirculation and thus the changes in eicosanoid biosynthesis in this part of the vasculature were recorded. The aim was to use inhibitors of arachidonic acid metabolism to increase the PGI2/TXA2 ratio. This may be potentially beneficial to ischaemic skin flaps by reducing platelet aggregation associated with damaged microvascular endothelium, overcoming vasospasm and increasing microvascular blood flow. Increased PGI2/TXA2 ratios (up to 5-fold) were best achieved using TXA2 synthetase inhibitors such as dazoxiben hydrochloride. These were significantly more potent than the phosphodiesterase inhibitor dipyridamole, and the lipoxygenase inhibitor Bay g6575. No increase in blood flow was achieved. The cyclooxygenase inhibitor indomethacin did slow the blood flow at high concentrations (above 10(-5) M), and inhibited both PGI2 and TXA2 synthesis. Approximately 2-fold higher concentrations of dazoxiben hydrochloride and dipyridamole were required to produce the same TXA2 synthetase inhibition in the flap microvasculature in vivo compared with platelets in vitro.     

Activation of cellular invasion by trefoil peptides and src is mediated by cyclooxygenase- and thromboxane A2 receptor-dependent signaling pathways.

We have investigated the possible functional relationships between cellular invasion pathways induced by trefoil factors (TFFs), src, and the cyclooxygenases COX-1 and COX-2. Pharmacological inhibitors of the Rho small GTPase (C3 exoenzyme), phospholipase C (U-73122), cyclooxygenases (SC-560, NS-398), and the thromboxane A2 receptor (TXA2-R) antagonist SQ-295 completely abolished invasion induced by intestinal trefoil factor, pS2, and src in kidney and colonic epithelial cells MDCKts.src and PCmsrc. In contrast, invasion was induced by the TXA2-R mimetic U-46619, constitutively activated forms of the heterotrimeric G-proteins Galphaq (AGalphaq), Galpha12, Galpha13 (AGalpha12/13), which are signaling elements downstream of TXA2-R. Ectopic overexpression of pS2 cDNA and protein in MDCKts.src-pS2 cells and human colorectal cancer cells HCT8/S11-pS2 initiate distinct invasion signals that are Rho independent and COX and TXA2-R dependent. We detected a marked induction of COX-2 protein and accumulation of the stable PGH2/TXA2 metabolite TXB2 in the conditioned medium from cells transformed by src. This led to activation of the TXA2-R-dependent invasion pathway, which is monitored via a Rho- and Galpha12/Galpha13-independent mechanism using the Galphaq/PKC signaling cascade. These findings identify a new intracrine/paracrine loop that can be monitored by TFFs and src in inflammatory diseases and progression of colorectal cancers.