Proteinase-activated receptor agonists stimulate the increase in intracellular Ca2+ in cardiomyocytes and proliferation of cardiac fibroblasts from chick embryos.

We studied activation of cultured cardiomyocytes and cardiac fibroblasts from chick embryos induced by agonists of PAR1 (thrombin and PAR1 peptide agonist) and PAR2 (trypsin, factor Xa, and peptide SLIGRL) by analyzing changes in intracellular Ca2+ concentration ([Ca2+]i) and cardiac fibroblast proliferation. Exposure of cardiomyocytes with thrombin induced immediate permanent dose-dependent increase in [Ca2+]i. Ca2+ response decreased in a calcium-free medium. Peptide agonists of PAR1 and PAR2 also stimulated the increase in [Ca2+]i in cardiomyocytes. Thrombin induced a short-term increase in [Ca2+]i in cardiac fibroblasts and potentiated cell proliferation. PAR2 agonists trypsin and peptide SLIGRL stimulated proliferation of cardiac fibroblasts. Our results indicate that cardiomyocytes and cardiac fibroblasts from chick embryos have at least two types of PAR (types 1 and 2).

[Effect of thrombin on survival of hippocampal neurons].

The effect of thrombin on the rat hippocampal neurons death in model of neurotoxicity induced by hemoglobin or glutamate, was studied. Thrombin (10 nM) was shown to inhibit 100-mkM glutamate--or 10-mkM hemoglobin-induced apoptosis of the rat hippocampal neurons. With the aid of PAR1 (protease-activated receptor1) agonist peptide and PAR1 antagonist, the PAR1 was found to be necessary for protective action of thrombin in hippocampal neurons in models of neurotoxicity induced by hemoglobin or glutamate. Because the prolonged elevation [Ca2+] ib neurons is a critical part of neurodestructive processes in CNS, the effect of thrombin on Ca2+-homeostatis of neurons after its injury by the inducer of neuronal apoptosis: a synthetic agonist of the NMDA receptors N-methyl-D-aspartate (NMDA), was studied. We hypothesized that thrombin via receptors PAR may prove to be neuroprotective for the hippocampus. Thrombin was shown to stimulate via PAR1 a transient increase in [Ca2+] in neurons in a concentration-dependent manner. Thrombin (1 nM) decreased the [Ca2+] signal induced by activation of the NMDA-subtype of glutamate receptors. This thrombin effect may be one of the reasons of the protective action of thrombin in hippocampal neurons.

Evidence for 5-HT2B and 5-HT7 receptor-mediated relaxation in pulmonary arteries of weaned pigs.

This study characterizes the relaxant response to 5-hydroxytryptamine (5-HT) in prostaglandin F(2alpha) (PGF(2alpha))-precontracted pulmonary arteries of weaned pigs. In arterial rings with intact endothelium, the relaxation to 5-HT was biphasic. The high affinity component of relaxation to 5-HT (0.1-10 nM) was abolished by mechanical removal of the endothelium or after the addition of L: -NAME (200 microM), and was inhibited by the 5-HT(2B/2C) receptor antagonist SB 206553 (1 microM), but not the 5-HT(2C) receptor antagonist SB 242084 (0.1 microM). Endothelium-intact arteries were also relaxed by the selective 5-HT(2B) receptor agonist BW 723C86 (pD(2) 7.7). The relaxant response to BW 723C86 was inhibited by 1 microM SB 206553 (pK(B) 6.8). The low affinity component of relaxation to 5-HT (>/=30 nM) remained unaffected after mechanical removal of the endothelium or the addition of L: -NAME. In endothelium-denuded arterial rings, 5-HT, 5-carboxamidotryptamine (5-CT), 5-methoxytryptamine (5-MeOT), and frovatriptan produced monophasic relaxations with pD(2) values of 6.5, 7.5, 5.9, and 4.7 respectively. Relaxant responses to the agonists were antagonized by the selective 5-HT(7) receptor antagonist SB 269970 (pK(B) 8.2-8.9). The relaxant response to the potent 5-HT(7) receptor agonist 5-CT was also antagonized by methiothepin (pK(B) 9.6), pimozide (pK(B) 8.2), mesulergine (pK(B) 7.7), methysergide (pK(B) 7.4), clozapine (pK(B) 7.6), and spiperone (pK(B) 7.4). The estimated pK(B) values argue in favor of an involvement of 5-HT(7) receptors in the direct vasorelaxant action of 5-HT in the pulmonary arteries of weaned pigs. The relaxant response to 5-CT was associated with an increase in cAMP that was surmountably antagonized by SB 269970 (pK(B) 8.6). The present in vitro bioassay can be used to characterize new drugs with potential agonist or antagonist properties at functional 5-HT(7) receptors.

Immobilized thrombin receptor agonist peptide accelerates wound healing in mice.

To accelerate the healing processes in wound repair, attempts have been repeatedly made to use growth factors including thrombin and its peptide fragments. Unfortunately, the employment of thrombin is limited because of its high liability and pro-inflammatory actions at high concentrations. Some cellular effects of thrombin in wound healing are mediated by the activation of protease activated receptor-1 (PAR-1). The thrombin receptor agonist peptide (TRAP:SFLLRN) activates this receptor and mimics the effects of thrombin, but TRAP is a relatively weak agonist. We speculated that the encapsulated peptide may be more effective for PAR-1 activation than nonimmobilized peptide and developed a novel method for TRAP encapsulation in hydrogel films based on natural and synthetic polymers. The effects of an encapsulated TRAP in composite poly(N-vinyl caprolactam)-calcium alginate (PVCL) hydrogel films were investigated in a mouse model of wound healing. On day 7 the wound sizes decreased by about 60% under TRAP-chitosan-containing PVCL films, as compared with control films without TRAP. In the case of TRAP-polylysine-containing films no significant decrease in wound sizes was found. The fibroblast/macrophage ratio increased under TRAP-containing films on day 3 and on day 7. The number of proliferating fibroblasts increased to 150% under TRAP-chitosan films on day 7 as compared with control films. The number of [3H]-thymidine labeled endothelial and epithelial cells in granulation tissues was also enhanced. Thus, the immobilized TRAP to PVCL-chitosan hydrogel films were found to promote wound healing following the stimulation of fibroblast and epithelial cell proliferation and neovascularization. Furthermore, TRAP was shown to inhibit the secretion of the inflammatory mediator PAF from stimulated rat peritoneal mast cells due to augmentation of NO release from the mast cells. The encapsulated TRAP is suggested to accelerate wound healing due to the anti-inflammatory effects and earlier development of the proliferative phase of wound healing.

Endothelium-dependent relaxation induced by cathepsin G in porcine pulmonary arteries.

Serine proteinases elicit profound cellular effects in various tissues mediated by activation of proteinase-activated receptors (PAR). In the present study, we investigated the vascular effects of cathepsin G, a serine proteinase that is present in the azurophil granules of leukocytes and is known to activate several cells that express PARs. In prostaglandin F2alpha (3 microM)-precontracted rings from porcine pulmonary arteries with intact endothelium, cathepsin G caused concentration-dependent relaxant responses (pEC(50)=9.64+/-0.12). The endothelium-dependent relaxant effect of cathepsin G could also be demonstrated in porcine coronary arteries (pEC(50)=9.23+/-0.07). In pulmonary arteries the cathepsin G-induced relaxation was inhibited after blockade of nitric oxide synthesis by L-NAME (200 microM) and was absent in endothelium-denuded vessels. Bradykinin- and cathepsin G-induced relaxant effects were associated with a 5.7 fold and 2.4 fold increase in the concentration of cyclic GMP, respectively. Compared with thrombin and trypsin, which also produced an endothelium-dependent relaxation in pulmonary arteries, cathepsin G was 2.5 and four times more potent, respectively. Cathepsin G caused only small homologous desensitization. In cathepsin G-challenged vessels, thrombin was still able to elicit a relaxant effect. The effects of cathepsin G were blocked by soybean trypsin inhibitor (IC(50)=0.043 microg ml(-1)), suggesting that proteolytic activity is essential for induction of relaxation. Recombinant acetyl-eglin C proved to be a potent inhibitor (IC(50)=0.14 microg ml(-1)) of the cathepsin G effect, whereas neither indomethacin (3 microM) nor the thrombin inhibitor hirudin (5 ATU ml(-1)) elicited any inhibitory activity. Due to their polyanionic structure defibrotide (IC(50)=0.11 microg ml(-1)), heparin (IC(50)=0.48 microg ml(-1)) and suramin (IC(50)=1.85 microg ml(-1)) diminished significantly the relaxation in response to the basic protein cathepsin G. In conclusion, like thrombin and trypsin, cathepsin G is able to induce endothelium-dependent vascular relaxation. It can be released from activated leukocytes at sites of vascular injury and inflammation and, therefore, sufficiently high concentrations might be reached locally in the vascular space to induce vasodilatation.

Systemic vascular effects of thrombin and thrombin receptor activating peptide in rats.

The proteolytic enzyme thrombin activates its receptor by cleavage of a peptide from the extracellular N-terminus. The newly generated N-terminus acts as a tethered ligand to activate the receptor. Receptor-mediated cellular effects of thrombin can be mimicked by synthetic peptides, which correspond to the amino acid sequence of the newly formed N-terminus. The aim of the present study was to investigate vascular effects of thrombin and the thrombin receptor activating peptide (TRAP: SFLLRN) in vitro and in vivo in rats. In precontracted rat aortic rings, both thrombin (0.3, 1, 3 U/ml) and TRAP (1, 3, 10, 20, 40 microM) induced endothelium-dependent relaxant responses. In anaesthetized rats, the mean arterial blood pressure (MAP) was measured continuously in the carotid artery by a pressure transducer. Thrombin and TRAP were administered as intravenous bolus injection via the femoral vein. Thrombin at doses of 3-100 U/kg, as well as TRAP at doses of 0.1-0.6 mg/kg i.v., caused a reversible decrease in MAP. Administration of TRAP at doses of 0.3 and 0.6 mg/kg led to a triphasic response in most of the animals treated (50% and 75%, respectively), i.e. a short drop of MAP was followed by an increase and finally a longer lasting decrease in MAP. Pretreatment with the nitric oxide (NO)-synthase inhibitor N(G)-nitro-L-arginine-methylester (L-NAME) suppressed the dose-dependent vasodilator effects of thrombin. Heparin and hirudin also inhibited the hypotensive response to thrombin. The TRAP-induced triphasic reaction on MAP was not affected by the serotonin antagonists ketanserin and tropisetron, as well as the aminopeptidase inhibitor amastatin. Pretreatment with L-NAME led to an inhibition of hypotension induced by TRAP at 0.1 mg/kg, as well as of the initial transient fall in blood pressure at doses of 0.3 and 0.6 mg/kg. The studies suggest that the thrombin- and TRAP-induced vasodilation in vitro and in vivo is in part due to the release of endothelial NO. In the blood pressure response to TRAP, additional effects seem to be involved.

In vitro dose response to different GPIIb/IIIa-antagonists: inter-laboratory comparison of various platelet function tests.

AIMS: The aim of this study was to assess the inter- and intra-laboratory variation of the concentration-response to the GPIIb/IIIa-antagonists abciximab and eptifibatide on platelet aggregometry and to compare results with flow cytometric tests as well as the rapid platelet function analyser (RPFA). METHODS: In five different laboratory sites, blood from three to five healthy donors was spiked with abciximab or eptifibatide, followed by the assessment of: (1) aggregometry (anticoagulant: sodium citrate 3.18% or hirudin 5 microg/ml); (2) flow cytometry (fibrinogen binding or PAC1-expression), or (3) RPFA. Dose-response curves were established on the basis of a sigmoidal Imax)-model [I=(Imax)*Cg)/(IC50g + Cg)]. RESULTS: For citrated blood, aggregation induced by 20 microM ADP was blocked up to 100% by both GPIIb/IIIa-antagonists, IC50 values varied between 0.11-0.22 microg/ml for eptifibatide and 1.25-2.3 microg/ml for abciximab. I(max) of the response to 5 microg/ml collagen ranged from 46% to 100%, and IC50 values varied between 0.28-0.34 microg/ml for eptifibatide and 2.3-3.8 microg/ml for abciximab. In hirudinized blood, IC50 values for eptifibatide were 1.5- to 3-fold higher than those obtained with citrated plasma. Inhibition of PAC1-expression by abciximab (IC50) 0.84 microg/ml) showed results similar those of the RPFA (approx. 1.0 microg/ml); larger differences between PAC1 and RPFA results were observed for eptifibatide. Based on aggregometry, eptifibatide concentrations for 80% inhibition varied from 0.27 to 0.55 microg/ml, and were considerably less when the RPFA was taken as basis (0.15 or 0.22 microg/ml). A similar pattern was observed for abciximab. CONCLUSIONS: We found quite a low inter- and intra-laboratory variation in the in vitro pharmacodynamic characterization of GPIIb/IIIa-antagonists by aggregometry, making results of these tests obtained from different laboratories during clinical trials at least comparable. The RPFA exhibits a higher sensitivity to inhibitory GPIIb/IIIa-effects, in keeping with the "real" inhibition of the activated receptor (PAC1) as assessed with more elaborate flow cytometry.

Evidence for functionally active protease-activated receptor-4 (PAR-4) in human vascular smooth muscle cells.

1. This study investigates, whether in addition to the protease-activated receptor-1 (PAR-1), PAR-4 is present in vascular smooth muscle cells (SMC) of the human saphenous vein and whether this receptor is functionally active. PAR-1 and PAR-4 are stimulated by thrombin and by the synthetic peptides SFLLRN and GYPGQV, respectively. 2. mRNAs for both, PAR-1 and PAR-4, were detected in the SMC by using reverse transcriptase polymerase chain reaction (RT - PCR). 3. Treatment of the SMC with GYPGQV (200 microM) resulted in a transient increase in free intracellular calcium. This calcium signal was completely abolished after a preceding challenge with thrombin (10 nM), indicating homologous receptor desensitization. 4. Stimulation of the SMC with 10 nM thrombin or 200 microM SFLLRN caused a time-dependent activation of the extracellular signal-regulated kinases-1/2 (ERK-1/2) with a maximum at 5 min. In contrast, 100 nM thrombin as well as 200 microM of GYPGQV induced a prolonged phosphorylation of ERK-1/2 with a maximum at 60 min. These data suggest that PAR-1 and PAR-4 are activated by thrombin at distinct concentrations and with distinct kinetics. 5. GYPGQV stimulated [(3)H]-thymidine incorporation in SMC. At 500 microM, the peptide increased DNA synthesis 2.5 fold above controls. A comparable mitogenic effect was obtained after stimulation of the SMC by 10 nM thrombin or 100 microM SFLLRN, respectively. 6. These data indicate that a functionally active PAR-4 is present in SMC and, in addition to PAR-1, might contribute to thrombin-induced mitogenesis.

Factor Xa acts as a PDGF-independent mitogen in human vascular smooth muscle cells.

This study investigates the mitogenic effect of the coagulation factor Xa in smooth muscle cells (SMC) from human saphenous vein and the procoagulant activity of these cells. Factor Xa elicited a concentration-dependent increase in [3H]thymidine incorporation. This mitogenic effect of factor Xa was inhibited by DX-9065a and BABCH, indicating the requirement of proteolytic activity of the enzyme. Factor Xa activated the MAP kinases ERK1/2 concentration- and time-dependently. PDGF-neutralizing antibodies neither inhibited the increase in [3H]thymidine incorporation nor ERK-1/2 phosphorylation in factor Xa-stimulated cells, suggesting that factor Xa-induced signaling and mitogenic activity in human venous SMC are independent of PDGF. Exposure of SMC to recalcified plasma resulted in a significant thrombin generation which was inhibited by anti-tissue factor antibody, tissue factor pathway inhibitor, inactivated factor VIIa and DX-9065a. These data indicate that interaction of SMC with the clotting system may contribute to venous graft disease, i.e. thrombus formation and intimal hyperplasia.

Further evidence that 5-HT-induced relaxation of pig pulmonary artery is mediated by endothelial 5-HT(2B) receptors.

The endothelial 5-HT receptor mediating relaxation of pig pulmonary artery has been characterized using the selective 5-HT(2B) receptor agonist BW 723C86 and a variety of structurally diverse 5-HT receptor antagonists. If arterial rings with intact endothelium were precontracted with prostaglandin F(2alpha) (3 microM), BW 723C86 caused concentration-dependent relaxation with a pEC(50)=8.21+/-0.03 and E(max)=89+/-4% relative to 5-HT. The relaxant responses to BW 723C86 were inhibited by the 5-HT(2B) receptor antagonist SB 204741, the 5-HT(2B/2C) receptor antagonist SB 206553 and the antimigraine drug pizotifen, yielding pA(2) values of 6.68, 7.20 and 8.32, respectively. The pA(2) values against BW 723C86 were similar to those determined against 5-HT. The relaxant effect of 5-HT was antagonized by a variety of 22 compounds of diverse chemical structures. Based on the calculated mean pA(2) values the order of the most potent antagonists was ritanserin (9.38) > methysergide (8. 86) > pizotifen (8.47) >/= methiothepin (8.32) > LY 53857 (7.84) >/= amoxapine (7.80) >/= loxapine (7.73) >/= metergoline (7.64) >/= mianserin (7.51) >/= rauwolscine (7.39). Compounds with weak blocking potency were yohimbine (6.37), spiperone (5.88) and ketanserin (5.85). Correlation analysis between the affinities of the antagonists in pig pulmonary artery and those from radioligand binding studies at human and rat 5-HT(2B) receptors showed a highly significant correlation (r=0.95 and 0.84, P<0.002 and <0.005). Correlation with 5-HT(2C) receptors was much lower (r=0.57, P=0.035), and no correlations were obtained with 5-ht(6) and 5-HT(7) receptors. It is concluded that the 5-HT receptor mediating endothelium-dependent relaxation of pig pulmonary artery is of the 5-HT(2B) subtype.

Modulation of mast cell activity by a peptide agonist of the thrombin receptor: role of nitric oxide.

The effect of a thrombin receptor agonist peptide (TRAP-6) on the release of nitric oxide (NO) and platelet activating factor (PAF) from resting and calcium-ionophore (A23187)-activated rat peritoneal mast cells (RPMC) was studied using a platelet aggregation bioassay. RPMC spontaneously released NO, which inhibited TRAP-6-, ADP-, and PAF-stimulated platelet aggregation. This effect of NO was abolished by the addition of an NO binding agent, oxyhemoglobin (oxyHb), to the platelet suspension. The RPMC-induced suppression of platelet aggregation was completely inhibited by the NO-synthase inhibitor L-NAME. TRAP-6 and its high affinity analog haTRAP stimulated the rapid release of NO from RPMC. The effect of TRAP-6 was inhibited by pretreatment of the RPMC with L-NAME or with the inhibitor of the constitutive NO-synthase isoform (cNOS) calmidazolium. TRAP-6 inhibited PAF release from A23187-activated RPMC via an NO-dependent mechanism. Platelet aggregation induced by PAF release from activated RPMC was also confirmed in experiments using the PAF receptor antagonist ginkgolide B. Thus, TRAP-6 is a rapidly acting modulator of mast cell reactivity; it stimulates NO release and inhibits PAF secretion.

Evidence for proteinase-activated receptor-2 (PAR-2)-mediated mitogenesis in coronary artery smooth muscle cells.

1. This study investigates, whether in addition to the thrombin receptor (PAR-1), the proteinase-activated receptor-2 (PAR-2) is present in vascular smooth muscle cells (SMC) and mediates mitogenesis. PAR-2 is activated by low concentrations of trypsin and the synthetic peptide SLIGRL. 2. Stimulation of bovine coronary artery SMC by trypsin (2 nM) caused a 3 fold increase in DNLA-synthesis. A similar effect was observed with 10 nM thrombin. Trypsin-induced mitogenesis was inhibited by soybean trypsin inhibitor, indicating that the proteolytic activity of the enzyme was required for its mitogenic effect. 3. The specific PAR-2-activating peptide SLIGRL or the PAR1-activating peptide SFFLRN did not elicit mitogenesis. 4. When the SMC were exposed to SLIGRL (40 nM), a homologous desensitization of cytosolic Ca2+ mobilization was found after subsequent stimulation with trypsin (40 nM) but not thrombin (15 nM). 5. Trypsin (2 nM) as well as SLIGRL (100 microm) activated the nuclear factor KB (NFkappaB) with a maximum response 2 h after stimulation of the SMC. This suggests that both agonists acted via a common receptor, PAR-2. Maximum activation of NFkappaB by thrombin (10 nM) was detected after 4-5 h. 6. These data suggest that PAR-2 is present in coronary SMC and mediates a mitogenic response. Activation of NFkappaB via either PAR-1 or PAR-2 does not predict mitogenesis.

A supersulfated low-molecular-weight heparin (IK-SSH) increases plasma levels of free and total tissue factor pathway inhibitor after intravenous and subcutaneous administration in humans.

Unfractionated as well as low-molecular-weight heparins (LMWH) are known to cause an increase in blood levels of tissue factor pathway inhibitor (TFPI). To study the effect of a newly developed supersulfated LMWH (IK-SSH, Iketon Farmaceutici) on TFPI concentrations in human plasma, the compound was injected into volunteers at doses of 0.14, 0.33 and 0.66 mg/kg intravenously or 0.33, 0.66 and 1.0 mg/kg subcutaneously. At certain known times blood was drawn and plasma levels of both total and free TFPI were measured using enzyme-linked immunosorbent assay methodology. Baseline plasma concentrations of TFPI were 72.2+/-3.1 ng/ml for total and 10.8+/-0.8 ng/ml for free TFPI. Intravenous or subcutaneous injection of IK-SSH led to a strong and long-lasting rise in TFPI levels which were increased more than 5-fold for total TFPI and more than 30-fold for free TFPI. Maximum TFPI levels were reached 5-10 min after intravenous and 60 min after subcutaneous administration. IK-SSH caused prolongation of ex-vivo clotting times in the APTT and Heptest assay, whereas thrombin time was not affected. Anticoagulant actions of IK-SSH showed a significant correlation to plasma concentrations of TFPI and they are thought to be based at least partially on the release of TFPI from vascular sites.

Inhibition of thrombin-induced contractile responses by protein kinase inhibitors in porcine pulmonary arteries.

The clotting enzyme thrombin is known to cause receptor-mediated contractile effects in isolated blood vessels. In the present studies the influence of protein kinase inhibitors on the contractile response of porcine pulmonary arteries to thrombin (3 U/ml) was investigated. Endothelium-denuded rings (2-3 mm) from small arteries were placed in organ baths for isometric tension recording. The vessels were preincubated for 30 min with the inhibitors before inducing contractions. In the presence of the protein kinase C (PKC)-inhibitors staurosporine, BIM I (bisindolyl-maleimide I), chelerythrine and Ro 31-8220 (1 microM each), the contractile responses to the PKC activator phorbol 12,13-dibutyrate (PDBu; 50 nM) were diminished by 70-100%. However, for inhibition of thrombin-induced contractions generally higher concentrations of the inhibitors were required. Only staurosporine at 1 microM inhibited the thrombin effect by about 75%. The tyrosine kinase inhibitor erbstatin (30 microM) did not significantly alter the thrombin effect, whereas genistein at 10 microM caused a significant inhibition of contractile responses to both thrombin and PGF2alpha. At 100 microM genistein also inhibited the contractile effects of PdBu and KCl. These studies suggest that activation of both PKC and non-receptor tyrosine kinases seems to be involved in the signal transduction pathways of thrombin-induced contractile effects in isolated vessels.

Thrombin-induced mitogenesis in coronary artery smooth muscle cells is potentiated by thromboxane A2 and involves upregulation of thromboxane receptor mRNA.

BACKGROUND: Previous studies have shown that thrombin is a potent though slow-acting mitogen for vascular smooth muscle cells (SMC). Because thrombin generation in vivo is accompanied by platelet activation, it has been suggested that platelet-derived factors might enhance thrombin-induced SMC proliferation. No information is available so far on the possible role of thromboxane A2. METHODS AND RESULTS: Thrombin (1 U/mL) caused a threefold to fourfold increase of DNA synthesis in cultured bovine coronary artery SMC as assessed from [3H]thymidine incorporation. U 46619, a stable thromboxane A2 mimetic, had only a minor stimulating effect on its own but potentiated the thrombin effect sixfold to sevenfold above control (P<.05). These findings were paralleled by a 52+/-5% (P<.05) increase in cell number at 48 hours after addition of both mitogens as compared with 24+/-5% with thrombin alone and no change with U 46619 alone. Thromboxane A2 receptor mRNA was found to be upregulated sixfold 20 minutes after thrombin stimulation. Pretreatment of SMC with thrombin for 4 hours markedly increased U 46619-induced mitogen-activated protein kinase activity, indicating thrombin-induced upregulation of functional thromboxane receptors in SMC. CONCLUSIONS: Thrombin-induced proliferation of SMC is markedly enhanced by thromboxane A2. This might result in an enhancement of SMC proliferation by platelet-derived thromboxane A2 in vivo.

Effects of a supersulfated low molecular weight heparin (IK-SSH) on different hemostatic parameters.

In a phase I trial effects of a new supersulfated low molecular weight heparin (IK-SSH) on different hemostatic parameters were investigated in healthy volunteers. Parameters studied were activated partial thromboplastin time (aPTT), thrombin time, Heptest, anti-activated factor II (anti-FIIa) and anti-activated factor X (anti-FXa) activity, platelet adhesion, platelet count, platelet-induced thrombin generation time (PITT), bleeding time, antithrombin III, fibrinogen and several safety parameters. After single intravenous (i.v.) injections of IK-SSH (0.14, 0.33 and 0.66 mg/kg) aPTT, Heptest and PITT were strongly and dose-dependently prolonged. After ascending subcutaneous (s.c.) doses of IK-SSH (0.33, 0.66 and 1 mg/kg) aPTT, Heptest and PITT were prolonged in a dose-dependent manner. Repeat s.c. injections of 1 mg/kg IK-SSH for 5 days markedly prolonged aPTT, Heptest and PITT. No cumulative effects were observed. Anti-FIIa and anti-FXa activity were not or only slightly increased. Bleeding time, thrombin time and platelet adhesion were not significantly changed after i.v. and s.c. injections of IK-SSH. However, tissue factor pathway inhibitor (TFPI) concentration was markedly increased after each injection of IK-SSH and returned to the preinjection value 24 h later. IK-SSH prolongs aPTT, Heptest and PITT in a similar manner as other low molecular weight heparins but without significantly affecting thrombin time, FIIa and FXa activity. The release of TFPI may well be responsible for the prolongation of aPTT, Heptest and PITT. IK-SSH may be further developed as an antithrombotic agent.

Inhibition of thrombin-mediated cellular effects by triabin, a highly potent anion-binding exosite thrombin inhibitor.

Triabin, a 17 kDa protein from the saliva of the assassin bug Triatoma pallidipennis is a potent thrombin inhibitor interfering with the anion-binding exosite of the enzyme. The recombinant protein, produced by the baculovirus/insect cell system, was used to study the inhibitory effect on thrombin-mediated cellular responses. The thrombin (1 nM)-stimulated aggregation of washed human platelets and the rise in cytoplasmic calcium in platelets were inhibited by triabin at nanomolar concentrations. In contrast, the rise in calcium induced by the thrombin receptor-activating peptide (10 microM) was not suppressed by triabin. In isolated porcine pulmonary arteries, preconstricted with PGF 2 alpha thrombin (2 nM) elicited an endothelium-dependent relaxation which was inhibited by triabin in the same concentration range as found for the inhibition of platelet aggregation. Higher concentrations of triabin were required to diminish the contractile response of endotheliumdenuded pulmonary vessels to thrombin (10 nM). In cultured bovine coronary smooth muscle cells, the mitogenic activity of thrombin (3 nM), measured by [3H]thymidine incorporation, was also suppressed by triabin. In all these assays, the inhibitory effect of triabin was dependent on the thrombin concentration used. These studies suggest that the new anion-binding exosite thrombin inhibitor triabin is one of the most potent inhibitors of thrombin-mediated cellular effects.

Activation of NFkappaB is essential but not sufficient to stimulate mitogenesis of vascular smooth muscle cells.

This study investigates the role of the transcription factor NFkappaB in thrombin- and thrombin receptor activating peptide (TRAP, SFLLRNPNDKYEPYF)-induced mitogenesis of cultured bovine coronary artery smooth muscle cells (SMC). Stimulation of resting cells by thrombin (10 nM) or TRAP (10-100 microM) resulted in a comparable time-dependent activation of NFkappaB as detected by Western blotting and electrophoretic mobility shift assay (EMSA) of nuclear extracts. The NFkappaB activation was antagonized by N-acetyl-L-cysteine (20 mM) and pentoxifylline (0.5 mM). Thrombin caused a 3-4-fold increase in [3H]thymidine incorporation within 24 h which was prevented by inhibitors of NFkappaB activation. In contrast, TRAP did not cause any mitogenic response. These results demonstrate that activation of NFkappaB is an essential but not a sufficient signal for SMC mitogenesis.