Evidence that activation of human T cells by porcine endothelium involves direct recognition of porcine SLA and costimulation by porcine ligands for LFA-1 and CD2.

In this study we present a comprehensive evaluation of the molecular interactions between human T cells and porcine aortic endothelial cells (PAEC) that contribute to human T cell activation. Binding assays demonstrated that porcine erythrocytes (E) and PAEC express ligand(s) for the human T cell glycoprotein CD2. Prior incubation of human T cells with a blocking monoclonal antibody directed against CD2 (alpha CD2-BL) completely inhibited T cell/E and T cell/PAEC interaction.

Protection of porcine aortic endothelial cells from complement-mediated cell lysis and activation by recombinant human CD59.

Discordant xenogeneic organ transplantation is a potential solution to the critical shortage of suitable donor organs. However, clinical application of xenotransplantation with physiologically suitable organs such as those from the pig, is currently limited by the lack of agents to prevent antibody and complement-mediated hyperacute rejection of the transplanted organ. We have used retrovirus-mediated gene transfer to express the terminal complement inhibitor protein, human CD59, in neonatal porcine aortic endothelial cells (nPAEC).

2-[3-[2-(4,5-diphenyl-2-oxazolyl) ethyl] phenoxy] acetic acid (BMY 42393): 2). Oral activity and efficacy in animal models of arterial thrombosis.

The oral activity and antithrombotic efficacy of BMY 42393 was examined in ex vivo platelet aggregation studies and arterial thrombosis animal models. In a heterologous ex vivo platelet aggregation assay, ADP-induced human platelet aggregation was inhibited when washed human platelets were combined with rat platelet-poor plasma, taken from rats previously orally-dosed with BMY 42393. The IC50 for platelet aggregation inhibition was approximately 10 mg/kg.

2-[3-[2-(4,5-Diphenyl-2-oxazolyl) ethyl] phenoxy] acetic acid (BMY 42393): a new, structurally-novel prostacyclin partial agonist: 1). Inhibition of platelet aggregation and mechanism of action.

BMY 42393, (2-[3-[2-(4,5-diphenyl-2-oxazolyl)ethyl]phenoxy]acetic acid), is a new prostacyclin partial agonist that inhibited ADP, collagen and thrombin-induced platelet aggregation (IC50 range 0.3 - 2.0 microM).

Nonprostanoid prostacyclin mimetics. 4. Derivatives of 2-[3-[2-(4,5-diphenyl-2-oxazolyl)ethyl]phenoxy]acetic acid substituted alpha to the oxazole ring.

The 4,5-diphenyloxazole derivatives 2-4 were previously identified as nonprostanoid prostacyclin (PGI2) mimetics. A series of derivatives of 2-4 bearing substitutents at the carbon atom alpha to the oxazole ring were synthesized and evaluated as inhibitors of ADP-induced aggregation of human platelets in vitro. In the unsaturated series, the alpha-carbethoxy derivative 10a, evaluated as an equal mixture of geometrical isomers, inhibited platelet aggregation with an IC50 of 0.

Inhibitors of blood platelet cAMP phosphodiesterase. 4. Structural variation of the side-chain terminus of water-soluble 1,3-dihydro-2H-imidazo[4,5-b]quinolin-2-one derivatives.

1-(Cyclohexylmethyl)-4-[4-[(2,3-dihydro-2-oxo-1H-imidazo[4,5-b] quinolin-7-yl)oxy]-1-oxobutyl]piperazine (2) was previously identified as a potent, water-soluble inhibitor of human blood platelet cAMP phosphodiesterase and of induced aggregation in vitro that demonstrated effective antithrombotic activity in animal models of thrombosis. Although 2 exhibited 25% oral bioavailability in rats, pharmacokinetic studies conducted in monkeys revealed that the parent compound was less than 5% bioavailable, the result of extensive first-pass biotransformation in the liver. In an effort to identify potent platelet aggregation inhibitors with enhanced metabolic stability, the side-chain amide moiety of 2 was replaced with chemically more stable urea (6a-s), sulfonamide (13a-m), sulfone (19a-r), and tetrazole (23a-s) moieties.

Nonprostanoid prostacyclin mimetics. 2. 4,5-Diphenyloxazole derivatives.

4,5-Diphenyl-2-oxazolenonanoic acid (18b) was synthesized and found to inhibit ADP-induced aggregation of human platelets with an IC50 of 2.5 microM. Acid 18b displaced [3H]iloprost from human platelet membranes in a concentration-dependent fashion, consistent with 18b inhibiting platelet function by acting as a prostacyclin mimetic.

Calcium/calmodulin transduces thrombin-stimulated secretion: studies in intact and minimally permeabilized human umbilical vein endothelial cells.

Thrombin stimulates cultured endothelial cells (EC) to secrete stored von Willebrand factor (vWF), but the signal transduction pathways are poorly defined. Thrombin is known to elevate the concentration of intracellular calcium ([Ca2+]i) and to activate protein kinase C (PKC) in EC. Since both calcium ionophores and phorbol esters release vWF, both second messenger pathways have been postulated to participate in vWF secretion in response to naturally occurring agonists.

Multiple pathways of thrombin-induced platelet activation differentiated by desensitization and a thrombin exosite inhibitor.

Recently a thrombin receptor with a unique mechanism of activation was cloned from a megakaryocyte-like cell line (Vu et al., Cell 64:1057-1068, 1991). Thrombin cleaves a portion of this receptor creating a new N-terminus that acts as a "tethered-ligand" to activate the receptor.

Regulation of the phosphoinositide cycle by Na+/H+ exchange and intracellular pH in human platelets.

We have found that thrombin-induced activation of protein kinase C (PKC) in platelets, measured by phosphorylation of the 47 kDa protein, is synergistically enhanced by the amiloride analogue ethylisopropylamiloride (EIA), a specific inhibitor of Na+/H+ exchange. This EIA effect was further synergistically enhanced by lowering intracellular pH (pHi) with either nigericin or sodium propionate, and reversed by raising pHi with monensin or ammonium chloride. The synergistic enhancement of thrombin-activated PKC by EIA plus nigericin was not observed when PKC was directly activated by phorbol esters.

Enhancement of thrombin- and ionomycin-stimulated prostacyclin and platelet-activating factor production in cultured endothelial cells by a tumor-promoting phorbol ester.

Tumor-promoting phorbol esters such as 4 beta-phorbol 12-myristate 13-acetate (PMA) have been shown to act synergistically with Ca2+ ionophores in cell activation, including stimulation of arachidonic acid metabolism. The effects of PMA on unstimulated and Ca2+ ionophore- or thrombin-stimulated PGI2 and platelet-activating factor (PAF) production in cultured bovine aortic endothelial cells (BAEC) and human umbilical vein endothelial cells (HUVEC) were investigated. Incubation of BAEC or HUVEC for 5-10 min with 100 nM PMA alone slightly increased basal PGI2 production.

Synergistic release of arachidonic acid from platelets by activators of protein kinase C and Ca2+ ionophores. Evidence for the role of protein phosphorylation in the activation of phospholipase A2 and independence from the Na+/H+ exchanger.

The protein kinase C activators phorbol myristate acetate (PMA), mezerein, oleoylacetylglycerol, and (-)-indolactam V, although without direct effect on arachidonic acid release, greatly enhance the release of platelet arachidonic acid caused by the Ca2+ ionophores A23187 and ionomycin. In contrast, 4 alpha-phorbol 12,13-didecanoate and (+)-indolactam V, which lack the ability to activate kinase C, do not potentiate arachidonate release. Release of arachidonic acid occurs without activation of phospholipase C and is therefore mediated by phospholipase A2.

Second messenger function of phosphatidic acid in platelet activation.

Phosphatidic acid (PA) is synthesized as the result of the receptor-mediated response of platelets to physiologic agonists. The role of PA in platelet signal transduction, however, is largely unknown. We have examined the responses of platelets to 1-stearoyl-2-arachidonoyl phosphatidic acid (SAPA), the predominant molecular species of human platelet PA.

IL-1 and related cytokines enhance thrombin-stimulated PGI2 production in cultured endothelial cells without affecting thrombin-stimulated von Willebrand factor secretion or platelet-activating factor biosynthesis.

We examined the effects of various cytokines on alpha-thrombin-stimulated prostaglandin (PG) I2 production, von Willebrand factor (vWF) secretion, and platelet-activating factor (PAF) synthesis in cultured human umbilical vein endothelial cells (HUVEC). A 24-h pretreatment with IL-1 beta doubled the low level of constitutive PGI2 production. In contrast, alpha-thrombin increased PGI2 production fivefold in untreated HUVEC.

Release of Ca2+ by inositol 1,4,5-trisphosphate in platelet membrane vesicles is not dependent on cyclic AMP-dependent protein kinase.

In contrast with previous reports, it was found that membrane-protein phosphorylation by the catalytic subunit (CS) of cyclic AMP-dependent protein kinase had no effect on Ca2+ uptake into platelet membrane vesicles or on subsequent Ca2+ release by inositol 1,4,5-trisphosphate (IP3). Furthermore, IP-20, a highly potent synthetic peptide inhibitor of CS, which totally abolished membrane protein phosphorylation by endogenous or exogenous CS, also had no effect on either Ca2+ uptake or release by IP3. Commercial preparations of protein kinase inhibitor protein (PKI) usually had no effect, but one preparation partially inhibited Ca2+ uptake, which is attributable to the gross impurity of the commercial PKI preparation.

Ca2+ mobilization can occur independent of acceleration of Na+/H+ exchange in thrombin-stimulated human platelets.

Intracellular free Ca2+ [( Ca2+]i) and pH (pHi) were measured simultaneously by dual wavelength excitation in thrombin-stimulated human platelets double-labeled with the fluorescent probes fura2 and 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein to determine the relationship between changes in [Ca2+]i and pHi, respectively. At 37 degrees C, thrombin (0.5 or 0.

Control of platelet protein kinase C activation by cyclic AMP.

Experiments were performed to elucidate the role of adenosine 3': 5'-cyclic monophosphate (cAMP) in the control of platelet protein kinase C (PKC) activation. Platelet aggregation and secretion in response to 4 beta-phorbol 12-myristate 13-acetate (PMA) or 1-oleoyl-2-acetylglycerol (OAG) were inhibited by dibutyryl cAMP in a dose-dependent manner. Inhibition of these functional activities paralleled a decrease in the PMA-induced phosphorylation of the Mr 47,000 substrate (p47) of PKC by pre-incubation of platelets with dibutyryl cAMP.

Synergistic inhibition of platelet activation by plasmin and prostaglandin I2.

Endothelial cell prostacyclin (PGI2) inhibits platelet activation by raising platelet cyclic AMP. Previously, platelet activation was also shown to be blocked by plasmin formed by endothelium-derived tissue plasminogen activator (TPA). We have now studied interactions between PGI2 and plasmin in the control of platelet function.

Stimulus-response coupling in a cell-free platelet membrane system. GTP-dependent release of Ca2+ by thrombin, and inhibition by pertussis toxin and a monoclonal antibody that blocks calcium release by IP3.

The Ca2+-mobilizing action of thrombin was demonstrated in a cell-free platelet membrane system consisting of open sheets of plasma membrane plus sealed membrane vesicles that accumulate Ca2+ and release Ca2+ in response to IP3. Thrombin plus GTP, acting on plasma membrane (not vesicles), produced a soluble factor (destroyed by alkaline phosphatase) that released Ca2+ from the vesicles. This effect of thrombin/GTP was blocked by a monoclonal antibody that binds to vesicles and prevents Ca2+ release by IP3.

Regulation of intracellular pH in human platelets. Effects of thrombin, A23187, and ionomycin and evidence for activation of Na+/H+ exchange and its inhibition by amiloride analogs.

Intracellular pH (pHi) of human platelets was measured with the fluorescent dye 2',7'-bis(carboxyethyl)5,6-carboxyfluorescein under various conditions. Stimulation by thrombin at 23 degrees C caused a biphasic change in pHi (initial pHi 7.09); a rapid fall of 0.

Effects of thrombin, phorbol myristate acetate and prostaglandin D2 on 40-41 kDa protein that is ADP ribosylated by pertussis toxin in platelets.

Intact platelets were stimulated with thrombin and the amount of GTP-binding protein (G-protein) oligomers was assessed by measuring ADP ribosylation of 40-41 kDa protein by pertussis toxin in isolated membranes. The toxin substrate fell by 57-62% in 10-60 s, but then returned towards normal over 5 min. Recovery was greatly enhanced by removal of thrombin from receptors with hirudin.

Phorbol esters and oleoyl acetoyl glycerol enhance release of arachidonic acid in platelets stimulated by Ca2+ ionophore A23187.

Washed human platelets prelabeled with [14C]arachidonic acid and then exposed to the Ca2+ ionophore A23187 mobilized [14C]arachidonic acid from phospholipids and formed 14C-labeled thromboxane B2, 12-hydroxy-5-8,10-heptadecatrienoic acid, and 12-hydroxy-5,8,10,14-eicosatetraenoic acid. Addition of phorbol myristate acetate (PMA) by itself at concentrations from 10 to 1000 ng/ml did not release arachidonic acid or cause the formation of any of its metabolites, nor did it affect the metabolism of exogenously added arachidonic acid. When 1 microM A23187 was added to platelets pretreated with 100 ng of PMA/ml for 10 min, the release of arachidonic acid, and the amount of all arachidonic acid metabolites formed, were greatly increased (average 4.

Phorbol myristate acetate inhibits thrombin-stimulated Ca2+ mobilization and phosphatidylinositol 4,5-bisphosphate hydrolysis in human platelets.

The tumor-promoting phorbol diester 4 beta-phorbol 12-myristate 13-acetate (PMA) inhibited mobilization of intracellular Ca2+ in platelets by thrombin (also trypsin and 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphocholine). PMA was effective over the same concentration range that activates protein kinase C in intact platelets; IC50 vs. thrombin = 2 ng/ml, 3.