Thrombin Receptor Agonist Peptide Research Articles

Activated Akt Is Associated with Hyperglycemia and Platelet Hyperactivity in a Mouse Model of Diabetes

Platelet hyperactivation has been reported in patients with both Type I and Type II diabetes mellitus and likely contributes to the increased prevalence of thrombotic complications in diabetic patients. However, the mechanisms leading to platelet hyperactivity in diabetes are not fully understood. Our previous studies showed that the Akt signaling pathway positively regulates platelet activity. Here, we evaluate the effect of hyperglycemia on Akt phosphorylation and platelet activation using a Type I diabetic mouse model. Hyperglycemia was induced by intraperitoneal injection of streptozotocin (STZ 50mg/kg for 5 days) in C57BL/6 mice. Blood glucose levels were elevated in 12 mice 4 weeks after STZ injection (mean ± SD: 410 ± 70 mg/ml) compared with 11 mice without STZ injection (212 ± 50 mg/ml). Platelets from hyperglycemic mice showed enhanced agonist-dependent aggregation (max % aggregation: 58% ± 10% in hyperglycemia versus 0% in normoglycemia at 0.6mM AYPGKF, n=3 each), fibrinogen binding (62.2% fibrinogen-bound cells ± 5.4% in hyperglycemic conditions versus 46.1% ± 16.3% in normoglycemia at 2mM AYPGKF) and P-selectin binding (42.1% cells with surface P-selectin ± 3.7% in hyperglycemic platelets, 25.3% ± 5.9% in normoglycemia at 2mM AYPGKF) compared with platelets from mice with normal glucose levels. The blood glucose levels were directly correlated with Alexafluor-fibrinogen binding (r=0.65, p=0.042) when platelets were stimulated with thrombin receptor agonist peptide AYPGKF (1.5mM), and also directly correlated with P-selectin surface exposure (r=0.95, p=0.003) after platelets were stimulated with 1.5mM AYPGKF. To determine whether the Akt pathway is involved in enhanced platelet activation in diabetes, we tested phosphorylation of Akt ser473 by immunoblotting. Akt phosphorylation of this residue was increased (35% ± 4%) in platelets from hyperglycemic mice compared with platelets from nondiabetic mice. In conclusion, platelets from hyperglycemic mice are more sensitive to PAR4 agonist-induced fibrinogen binding and P-selectin exposure compared with nondiabetic platelets. Enhanced activation of Akt in platelets under hyperglycemic conditions may play a role in platelet hyperactivation in diabetes.

Abstract 1710: Pharmacologic Profile of SCH 530348, a Novel Oral Antiplatelet Agent Selective for the Protease-Activated Receptor-1 (PAR-1)

Antiplatelet agents are a cornerstone of therapy for atherothrombotic disease. However, despite the proven efficacy of agents targeting the thromboxane A2 (aspirin) and P2Y12 ADP (clopidogrel, prasugrel) platelet activation pathways, the residual risk for ischemic events remains considerable. Binding of thrombin to the platelet PAR-1 is an important platelet activation pathway not targeted by existing agents. Inhibition of PAR-1 may thus provide incremental clinical benefits over aspirin and ADP receptor antagonists. PAR-1 receptors expressed on endothelial cells, smooth muscle cells, monocytes and neutrophils have been reported to mediate the pro-inflammatory and chemotactic responses to thrombin. In the present study, we report pharmacologic characterization of SCH 530348, a novel thrombin receptor antagonist (TRA) selective for PAR-1, using in vitro assays with human platelet membranes and cultured human coronary artery smooth muscle cells (HCASMC), and ex vivo platelet aggregation assays in cynomolgus monkeys. The affinity of SCH 530348 for the PAR-1 receptor was determined in human platelet membranes. Functional studies involving calcium transients, thymidine incorporation, and receptor kinetics were performed in HCASMC. The oral antiplatelet effects of SCH 530348 in cynomolgus monkeys were evaluated in ex vivo platelet aggregation assays. SCH 530348 exhibited high affinity for PAR-1 receptor. SCH 530348 potently inhibited thrombin-stimulated calcium transients and thymidine incorporation in HCASMC, and displayed slow dissociation from PAR-1 receptor. In cynomolgus monkeys, SCH 530348 administered orally at doses ranging from 0.1 mg/kg to 3 mg/kg, provided rapid, complete, and sustained inhibition of thrombin receptor agonist peptide (TRAP)-induced ex vivo platelet aggregation for 24 hours. Significant inhibition of TRAP-induced platelet aggregation was maintained at 48 hours after dosing of SCH 530348. SCH 530348 is a highly selective, potent, and orally active PAR-1 antagonist. Inhibition of PAR-1 by SCH 530348 may translate into beneficial clinical effects in patients with atherothrombotic disease, and this hypothesis is currently being evaluated in 2 large trials.

Delayed Inhibition of Agonist-Induced Granulocyte-Platelet Aggregation After Low-Dose Sevoflurane Inhalation in Humans

Sevoflurane can be used as sedative-analgesic drug with endothelial protective properties. We tested whether low-dose sevoflurane inhalation provides sustained inhibition of detrimental granulocyte-platelet aggregation in humans. Ten healthy male volunteers were enrolled in this crossover study. Each subject inhaled sevoflurane for 1 h at 0.5-1 vol % end-tidal concentration in oxygen (50 vol %). Inhaling oxygen (50 vol %) alone served as control. Venous blood samples were collected at baseline before inhalation, immediately after inhalation, and 24 h thereafter, and were used for flow cytometry to determine platelet surface marker (CD41, CD42b, CD62P/P-selectin, and PAC-1) on platelets and granulocytes and for kaolin-induced clot formation, as assessed by thromboelastography. In flow cytometry experiments, platelets were stimulated with arachidonic acid (AA, 30 microM), adenosine diphosphate (ADP, 1 microM), and thrombin receptor agonist peptide-6 (TRAP-6, 6 microM). AA, ADP, and TRAP-6 markedly increased the expression of CD62P on platelets, whereas CD42b (shedding) and PAC-1 (heterotypic conjugates) expression decreased. The amount of granulocyte-platelet aggregates increased upon agonist stimulation. Low-dose sevoflurane inhalation reduced ADP-induced CD62P expression on platelets 24 h after inhalation, and inhibited the formation of granulocyte-platelet aggregates under stimulation with AA and ADP after 1 and 24 h, and with TRAP-6 after 24 h compared with control. Inhibition of granulocyte-platelet aggregates was accompanied by reduced clot firmness 24 h after sevoflurane inhalation compared with control. We demonstrated for the first time that inhaling low-dose sevoflurane (<1 vol % end-tidal) inhibits agonist-induced granulocyte-platelet interactions 24 h after administration and thus counteracts thromboinflammatory processes.

Protease activated receptor 1 (PAR-1) activation by thrombin is protective in human pulmonary artery endothelial cells if endothelial protein C receptor is occupied by its natural ligand

We recently demonstrated that the occupancy of endothelial protein C receptor (EPCR) by its natural ligand activated protein C (APC)/protein C switches the protease activated receptor 1 (PAR-1)-dependent signaling specificity of thrombin from a disruptive to a protective effect in cultured human umbilical vein endothelial cells. Given the phenotypic differences between endothelial cells in venular and arterial beds, in this study we evaluated the signaling function of thrombin in human pulmonary artery endothelial cells (HPAECs) before and after treating them with PC-S195A which lacks catalytic activity but exhibits a normal affinity for EPCR. As expected, both thrombin and thrombin receptor agonist peptide (TRAP) enhanced the permeability barrier of HPAECs; however, both PAR-1 agonists exhibited a potent barrier protective effect when the cells were treated with PC-S195A prior to stimulation by the agonists. Interestingly, similar to APC, thrombin exhibited a potent cytoprotective activity in the LPS-induced permeability and TNF-alpha-induced apoptosis and adhesion assays in the PC-S195A treated HPAECs. Treatment of HPAECs with the cholesterol depleting molecule methyl-beta-cyclodextrin eliminated the protective effect of both APC and thrombin. These results suggest that the occupancy of EPCR by its natural ligand recruits PAR-1 to a protective signaling pathway within lipid rafts of HPAECs. Based on these results we conclude that the activation of PAR-1 by thrombin would initiate a protective response in intact arterial vascular cells expressing EPCR. These findings may have important ramifications for understanding the mechanism of the participation of the vascular PAR-1 in pathophysiology of the inflammatory disorders.

High-dose erythropoietin alters platelet reactivity and bleeding time in rodents in contrast to the neuroprotective variant carbamyl-erythropoietin (CEPO)

The haematopoietic hormone erythropoietin (EPO) has neuroprotective properties and is currently being explored for treatment of stroke and other neurological disorders. Short-term, high-dose treatment with EPO seems to improve neurological function of stroke patients but may be associated with increased thrombotic risk, whereas alternative non-erythropoietic neuroprotective derivatives of EPO, such as carbamylated EPO (CEPO), may be devoid of such side-effects. We investigated the effects of short-term, high-dose treatment with EPO and CEPO on platelet function and haemostasis in healthy mice and rats. Animals received three daily doses of EPO or CEPO (50 microg/kg), and blood was compared with respect to alterations in haematology and platelet reactivity. In rats, treatment with EPO increased the haematocrit to >50% and the mean platelet volume by 37%, while CEPO had no effect on these parameters. Platelets from EPO-treated rats showed an increased sensitivity to thrombin receptor agonist peptides and elevated plasma levels of soluble P-selectin (sP-selectin) were found in treated mice. Further indicators of platelet hyperreactivity in EPO, but not CEPO-treated animals, were significantly increased aggregatory responses to collagen in whole blood and platelet-rich plasma (PRP). The increased platelet reactivity was paralleled by a decreased bleeding time after tail transection in rats. Samples from EPO-treated rats showed an attenuated response to ADP in whole blood aggregometry and thrombelastography (TEG) platelet mapping but not in apyrase-treated PRP, suggesting involvement of ADP receptor desensitization. These findings suggest that while EPO affects various aspects of platelet function, CEPO is devoid of such effects.

Abstract 3010: Thrombin Receptor Antagonist (TRA;SCH530348) is a Selective, Potent Inhibitor of PAR1 Activity with Predictable Pharmacokinetics

Background: TRA-PCI was a multicenter, randomized, double-blind, placebo-controlled study demonstrating the safety of SCH 530348, a potent thrombin receptor antagonist (TRA), in patients undergoing non-urgent percutaneous coronary intervention (PCI). A secondary endpoint in a sub-study within the primary evaluable patient cohort was inhibition of platelet aggregation (IPA) induced by appropriate agonists relative to baseline. We hypothesized that TRA therapy would selectively inhibit platelet aggregation induced by the thrombin receptor agonist peptide (TRAP) and that sustained inhibition would be observed. Pharmacokinetic (PK) studies were also carried out. Methods: Platelet aggregation responses to TRAP, adenosine diphosphate (ADP), collagen and arachidonic acid (AA) were measured using light transmission aggregometry (LTA) at baseline, 30, 60, 90, 120 min following a loading dose (10, 20 or 40 mg vs placebo) and after a maintenance dose (0.5, 1.0 or 2.5 mg/day) at 30 and 60 days. PK was assessed at 30 and 60 min and 2 hr after loading dose administration. Results: TRA was active in inhibiting 15 μM TRAP-induced platelet aggregation with onset of inhibition directly related to dose. Loading doses of 10, 20 or 40 mg achieved &gt;90% inhibition of platelet aggregation (IPA) 60 –120 min post dose with the 40 mg dose achieving &gt;90% inhibition between 60 and 90 min. Patients receiving maintenance doses of 0.5, 1.0 or 2.5 mg exhibited &gt;90% IPA at 30 and 60 days. Placebo treated patients had on average ≥10% IPA to TRAP. TRA had no significant effects on ADP, collagen or AA -induced platelet aggregation compared with placebo controls. TRA PK was characterized by fast distribution and slow elimination (t1/2 = 165–311 hr) with clear evidence of hysteresis. Conclusion: Among PCI patients treated with TRA, there was a specific, dose-related, sustained inhibition of TRAP-induced platelet aggregation, without an impact on other aggregation related pathways. These data suggest that TRA is a potent, selective inhibitor of PAR-1 activity induced by thrombin activation of platelets, and, in view of this, is a promising therapeutic utility for treatment of thrombosis.

Biodegradable microparticles with immobilized peptide for wound healing

Thrombin receptor agonist peptide (TRAP-6) may effectively replace thrombin for stimulation of damaged tissue regeneration. (Thrombin employment is limited by its high cost, instability and proinflammatory effect at high concentrations.) Immobilization of TRAP-6 into a poly(D,L)-lactide-co-glycolide (PLGA)-based matrix can protect peptides from a destruction by peptidases located in a wound area, and can also provide controlled release of the peptide. PLGA microparticles with immobilized peptide were produced by double emulsion/evaporation technique. An observation of microparticle morphology by scanning electron microscopy highlighted that peptide immobilization resulted in the increase of the microparticle porosity. TRAP-6 release kinetics was characterized by burst increase of TRAP-6 concentration in HEPES buffer solution (pH 7.5) for first 2 hours from the beginning of the experiment, and TRAP-6 complete release occurred for 20 hours. An investigation of TRAP-6 destruction by scanning electron microscopy revealed that the increase of microparticle size and surface porosity were observed already after 1 day of incubation in the buffer solution, and an aggregation of destructing microparticles was obvious by the 7th day of the incubation. Thus, peptide immobilization into PLGA microparticles can allow to develop a novel controlled release drug delivery system.

A familial platelet function disorder associated with abnormal signalling through the glycoprotein VI pathway

The platelet collagen receptor, glycoprotein (GP)VI, of the immunoreceptor family forms a complex with the von Willebrand factor (VWF) receptor, GPIb-IX-V, critical for initiating thrombus formation. GPVI is co-associated with Fc receptor gamma-chain (FcRgamma), which contains a cytoplasmic immunoreceptor tyrosine-based activation motif domain, involved in activation of Syk, and a signalling cascade leading to (i) activation of alpha(IIb)beta(3), which binds VWF and fibrinogen and mediates platelet aggregation, and (ii) metalloproteinase-mediated shedding of the GPVI ectodomain (blocked by Syk inhibitors), a key mechanism for regulating GPVI surface expression. In this study, we report a familial case of abnormal platelet aggregation with dysfunctional signalling through GPVI that uniquely demonstrates divergent alpha(IIb)beta(3)-activating and GPVI-shedding pathways. The patient is a 60-year-old female with a history of immune disorders, excessive bleeding from childhood and a life-threatening haemorrhage post-trauma. Platelet aggregation to ADP, thrombin receptor-agonist peptide or ristocetin/VWF was normal (indicating normal expression and function of alpha(IIb)beta(3)), but platelet aggregation to GPVI agonists, collagen, collagen-related peptide, or convulxin, was defective. Both GPVI/FcRgamma expression and ligand-induced GPVI ectodomain shedding were normal, confirming expression of functional GPVI/FcRgamma, but suggesting a signalling defect downstream of Syk. A genetic defect in GPVI/Fcgamma signalling compromising platelet function is hypothesised in this family.

Platelet reactivity among Asian Indians and Caucasians

Asian Indians are reported to have higher mortality and morbidity from coronary artery disease (CAD) than other ethnic groups. This variation in events cannot be explained only by differences in conventional risk factors. Platelet activation is an important factor in the pathogenesis of CAD, however, there are limited data concerning platelet reactivity in Asian Indians. Therefore, we aimed to examine platelet reactivity in healthy Asian Indians vs. Caucasians. Thirty-five healthy, nonsmoking Asian Indians (mean age 30.1 ± 3.6 years, 31.4% women) were matched for age and sex with 35 healthy, nonsmoking Caucasians (mean age 30.8 ± 5 years, 31.4% women). Platelet reactivity was evaluated by measuring platelet aggregation, platelet leukocyte aggregates (PLA) formation in response to a 6-mer thrombin receptor agonist peptide (TRAP) at a final concentration of 40 µM and flow cytometry determined P-selectin expression induced by ADP, TRAP and arachidonic acid (AA). In addition, P-Selectin glycoprotein ligand-1 (PSGL-1) density on leukocytes was measured. There were no differences in platelet aggregation, basal PLA or PSGL-1 density on leukocytes between the two groups. AA-stimulated P-selectin expression was significantly higher in Asian Indians than in Caucasians (6.1 ± 0.51 vs. 4.2 ± 0.41 MFI, P < 0.02). After stimulation with TRAP, platelets from Asian Indians had increased PLA formation compared with Caucasians (41.6 ± 2.9% vs. 31.4 ± 2.7%, P < 0.02). AA induced P-selectin expression and TRAP stimulated PLA formation is increased in Asian Indians compared with Caucasians. These differences indicate an increase in measures of platelet reactivity among Asian Indians and may help elucidate the reported disparity in cardiovascular disease rates between the two ethnic groups.

In Vivo exposure of lipopolysaccharide (LPS) but not estrogen decreases platelet reactivity in early postmenopausal women

Infection and oral estrogen treatments are risk factors for thrombosis. Experiments were designed to evaluate in vitro effects of priming doses of LPS and physiological doses of estrogen on platelets from healthy, newly menopausal women. Blood was collected from early postmenopausal women (42–56 years old) with a menopausal age between 6 months to 3 years into anticoagulants (citrate, hirudin and TAP). Platelet rich plasma was prepared and preincubated with LPS (ultrapure LPS from E. Coli: 10ng-1000ng/mL) and/or 17β-estradiol(10−10M/mL) Four groups were studied: control, LPS, 17β-estradiol, 17β-estradiol+LPS). Platelet aggregation was measured by lumiaggregometry. Thrombin receptor agonist peptide (TRAP 10μM) was used as an agonist. Platelet dense granules ATP secretion was measured by luciferin bioluminescence. Only the highest concentration of LPS (100ng/ml) decreased the maximum and rate of ATP secretion from dense granules and aggregation. At physiological concentrations, 17β-estradiol did not affect platelet aggregation alone or in combination with LPS. These data suggest that LPS and estrogen may not increase thrombotic risk by direct activation of platelets; effects most likely require the interaction of the platelets with other cells in the circulation. (Supported by NIH HL78638, the Mayo Foundation, Kronos Longevity Institute and Japanese Menopause Society)

Thrombin receptor agonist peptide entrapped in poly(D,L)-lactide-co-glycolide microparticles: Preparation and characterization

Thrombin receptor agonist peptide (TRAP-6) could advantageously replace thrombin in terms of accelerating wound healing being less expensive and more stable. To promote TRAP-6 pharmacological action as a tissue reconstruction stimulator this study investigated its entrapment within poly(D,L)-lactide-co-glycolide (PLGA) microparticles. Due to its low molecular weight and water solubility, TRAP-6 microencapsulated form is expected to be more useful. This paper reports TRAP-6 microencapsulation by a double (w/o/w) emulsion-evaporation technique. TRAP-6 release kinetics were evaluated by both chemical (HPLC) and biological assays in vitro. The results revealed a high level of TRAP-6 sensitivity to physico-chemical events during the microencapsulation. The surface morphology difference between control microparticles (without TRAP-6) and microparticles with entrapped TRAP-6 during in vitro degradation highlighted a particular role of TRAP-6. The results can allow one to optimize the microencapsulation procedure and to encounter a new promising approach to development of biodegradable polymer drug delivery systems for wound healing.

Evaluation of platelet activation and cytokine release during storage of platelet concentrates processed from buffy coats either manually or by the automated OrbiSac system

This study aimed to compare platelet (PLT) quality during storage of buffy coat (BC) PLT concentrates (PCs), prepared either manually or by the automated OrbiSac system (Gambro BCT). Following overnight storage at 20 to 22 degrees C, five BCs were pooled with 300 mL of PLT additive solution. Twenty-one PCs were produced manually (M-PCs) and 21 by the automated OrbiSac system (A-PCs). Swirling, PLT count, mean PLT volume, blood gas analyses, potassium, glucose, and lactate were assessed. Expression of the activation markers CD42a, CD62P, and PAC-1 was analyzed by flow cytometry on resting PLTs and PLTs stimulated with thrombin receptor agonist peptide (TRAP). Levels of CCL5 and transforming growth factor-beta1 (TGF-beta1) were measured by enzyme-linked immunosorbent assay. The A-PCs had significantly larger volume and higher PLT yield, PLT recovery, and white blood cell concentration than the M-PCs, whereas the red blood cell content was significantly highest in the M-PCs. pH levels were between 6.9 and 7.2 in all PCs. Neither glucose consumption nor lactate production differed significantly over time. A-PCs had, compared to M-PCs, significantly higher expression of CD62P on resting PLTs, lower capacity for up regulating CD62P on TRAP-stimulated PLTs, and higher levels of CCL5 during storage. TRAP-stimulated A-PCs had a significantly higher potential for down regulation of CD42a than M-PCs. No difference was found in TGF-beta1 levels or TRAP-induced up regulation of PAC-1. The levels of CCL5 and the expression of CD62P in resting and stimulated PLTs indicate that PLTs in A-PCs are slightly more activated than in M-PCs, but the clinical importance of this finding is yet unknown.

Thrombin Inhibits IFN-γProduction in Human Peripheral Blood Mononuclear Cells by Promoting a Th2 Profile

Thrombin, the key enzyme of the coagulation cascade, is involved in inflammation. It was proposed recently that thrombin activity may play an important role in allergic inflammation. Interferon-gamma (IFN-gamma) is a potent Th1-related cytokine secreted by activated T cells and is usually downregulated in allergic inflammation. We recently demonstrated that thrombin enhances interleukin-10 (IL-10) in peripheral blood mononuclear cells (PBMC). Thus, we hypothesized that thrombin may promote a Th2 profile. We here report that human alpha- thrombin downregulates IFN-gamma expression at both protein and mRNA levels in activated PBMCs. The use of proteolytically inactive thrombin and of the specific thrombin receptor agonist peptide, SFLLRN, shows that this downregulation is thrombin specific and requires thrombin proteolytic activity. The addition of an anti- IL-10 monoclonal antibody (mAb) to thrombin-treated PBMCs abolishes IFN-gamma downregulation, suggesting that thrombin exerts its effect through IL-10, a Th2-related cytokine. Furthermore, IFN-gamma reduction was accompanied by increased IL-4 release, as well as by an increase in the proinflammatory cytokine IL-1. In conclusion, the observation that thrombin affects the production of IFN-gamma (Th1 profile) and IL-4 (Th2 profile) provides further evidence for the role played by thrombin in modulating Th1/Th2 cytokine balance, which could be particularly relevant in allergic inflammation.

Arginase Activity Is Increased by Thrombin: A Mechanism for Endothelial Dysfunction in Arterial Thrombosis

Earlier observations implicate arterial thrombosis causing endothelial dysfunction by decreasing nitric oxide (NO) levels. NO levels are restored by regional L-arginine supplementation in animal models. The purpose of this study was to investigate the roles of thrombus components in NO generation. Human umbilical vein endothelial cells were harvested and cultured. The thrombus components thrombin, thrombin receptor agonist peptide (TRAP), and fibrin were added to a media of confluent human umbilical vein endothelial cells. Endothelial nitric oxide synthase (eNOS) activity was assayed by measuring conversion of L-arginine to L-citrulline. Endothelial NOS mRNA levels were quantitated using real-time polymerase chain reaction. Cellular membrane transport of L-arginine through the y+ channel was assayed with (14)C-labeled L-arginine. Arginase activity was determined as the conversion of (14)C L-arginine to (14)C urea and trapped as Na(2)(14)CO(3) for scintillation counting. Arginase protein amounts were assessed using Western blotting. Endothelial cells exposed to thrombin for 4 hours led to increased arginase activity. Thrombin (10 U/mL) caused a 1.6-fold increase compared with that in controls (320+/-29 microM urea/min versus 194+/-10 microM urea/min, p=0.03), and thrombin (30 U/mL) increased arginase activity 2.1-fold (398+/-27 microM urea/min, p < 0.001, versus controls); thrombin at 1 U/mL and fibrin had no effect. TRAP (50 microM) had an effect similar to that of thrombin 10 U/mL (316+/-21 microM urea/min, p < 0.01, versus controls). Protein amounts of arginase corresponded with activity levels. Neither eNOS nor inducible nitric oxide synthase (iNOS) activities were affected by exposure to thrombin and TRAP for 4 hours. Similarly, quantification of eNOS, iNOS, and endothelin-1 mRNA did not change, although CL-100, a known thrombin-inducible gene, was upregulated. Finally, transport of L-arginine into endothelial cells was unaffected by thrombin, TRAP, and fibrin exposure. Endothelial cells exposed to thrombin have increased arginase enzymatic activity, and the remainder of NO generation capability is unaffected. L-arginine supplementation or arginase blockade may counteract endothelial dysfunction in the setting of acute arterial thrombosis.

Unfractionated Heparin Reduces the Anti-Platelet Effects of Abciximab but not Eptifibatide During PCI

In 29 patients undergoing percutaneous coronary intervention (PCI), we obtained blood samples at baseline, 10 minutes after standard weight-based abciximab (n=15) or double-bolus eptifibatide (n=14) and 5 minutes after unfractionated heparin (UFH; 70 U/kg bolus). The median percent inhibition was significantly higher in the eptifibatide group compared with the abciximab group both before (96.5% [94-100] vs. 85% [77-89.5] [adenosine diphosphate; ADP]; 89.5% [84-95] vs. 59% [37.5-76.5] [thrombin receptor agonist peptide; TRAP], p<0.001 for both) and after UFH (95% [93-100] vs. 79% [68.8-87.5] [ADP]; 82% [77-93] vs. 51% [34.5-71.3] [TRAP], p<0.001 for both). Addition of UFH significantly reduced platelet inhibition in the abciximab group (85% [77-89.5] vs. 79% [68.8-87.5] [ADP]; 59% [37.5-76.5] vs. 51% [34.5-71.3] [TRAP], p<0.05 for both) but not in the eptifibatide group (96.5% [94-100] vs. 95% [93-100] [ADP]; 89.5% [84-95] vs. 82% [77-93] [TRAP], p=ns for both). Eptifibatide achieved superior platelet inhibition before but especially after UFH compared with abciximab.

Mesotrypsin, a brain trypsin, activates selectively proteinase‐activated receptor‐1, but not proteinase‐activated receptor‐2, in rat astrocytes

Proteinase-activated receptors (PARs), a subfamily of G protein-coupled receptors, which are activated by serine proteases, such as trypsin, play pivotal roles in the CNS. Mesotrypsin (trypsin IV) has been identified as a brain-specific trypsin isoform. However, its potential physiological role concerning PAR activation in the brain is largely unknown. Here, we show for the first time that mesotrypsin, encoded by the PRSS3 (proteinase, serine) gene, evokes a transient and pronounced Ca(2+) mobilization in both primary rat astrocytes and retinal ganglion RGC-5 cells, suggesting a physiological role of mesotrypsin in brain cells. Mesotrypsin mediates Ca(2+) responses in rat astrocytes in a concentration-dependent manner, with a 50% effective concentration (EC(50)) value of 25 nm. The maximal effect of mesotrypsin on Ca(2+) mobilization in rat astrocytes is much higher than that observed in 1321N1 human astrocytoma cells, indicating that the activity of mesotrypsin is species-specific. The pre-treatment of cells with thrombin or the PAR-1-specific peptide TRag (Ala-pFluoro-Phe-Arg-Cha-HomoArg-Tyr-NH(2), synthetic thrombin receptor agonist peptide), but not the PAR-2-specific peptide, reduces significantly the mesotrypsin-induced Ca(2+) response. Treatment with the PAR-1 antagonist SCH79797 confirms that mesotrypsin selectively activates PAR-1 in rat astrocytes. Unlike mesotrypsin, the two other trypsin isoforms, cationic and anionic trypsin, activate multiple PARs in rat astrocytes. Therefore, our data suggest that brain-specific mesotrypsin, via the regulation of PAR-1, is likely to be involved in multiple physiological/pathological processes in the brain.

Thrombin receptor agonist peptide immobilized in microspheres stimulates reparative processes in rats with gastric ulcer

The effect of synthetic thrombin receptor (PAR1) agonist peptide encapsulated in microspheres made of lactic and glycolic acid copolymer on tissue reparation was studied in rats with acetate-induced ulcer. PAR1 agonist peptide was immobilized in biodegraded lactic and glycolic acid microspheres by double emulgation, the kinetics of peptide release was analyzed, and the dynamics of ulcer healing was studied in experimental (administration of microspheres with the peptide into the stomach) and two control groups (administration of saline or spheres without peptide). Thrombin receptor agonist peptide gradually released from lactic and glycolic acid microspheres into the stomach shortened the inflammation phase and shifted the proliferation phase to the earlier period, thus accelerating healing of experimental ulcers in rats.

Protease‐activated receptor‐1 protects rat astrocytes from apoptotic cell death via JNK‐mediated release of the chemokine GRO/CINC‐1

Thrombin at low doses is an endogenous mediator of protection in ischaemic and haemorrhagic models of stroke. However, the mechanism of thrombin-induced protection remains unclear. Recently accumulating evidence has shown that astrocytes play an important role in the brain after injury. We report that thrombin and thrombin receptor agonist peptide (TRag) up-regulated secretion of the chemokine growth-regulated oncogene/cytokine-induced neutrophil chemoattractant-1 (GRO/CINC-1) in primary rat astrocytes in a concentration-dependent manner. However, we found no increase of interleukin (IL)-6, IL-1beta and tumour necrosis factor-alpha secretion. Protease-activated receptor 1 (PAR-1)-induced GRO/CINC-1 release was mainly mediated by c-Jun N-terminal kinase (JNK) activation. Extracellular signal-regulated kinase 1/2 might be partially involved, but not p38 mitogen-activated protein kinase. Further studies demonstrated that PAR-1 activation, as well as application of recombinant GRO/CINC-1, protected astrocytes from C(2)-ceramide-induced cell death. Protection occurred with suppression of cytochrome c release from mitochondria. The inhibition of cytochrome c release was largely reduced by the antagonist of chemokine receptor CXCR2, SB-332235. Importantly, a specific JNK inhibitor significantly abolished the protective action of PAR-1. These results demonstrate for the first time that PAR-1 plays an important role in anti-apoptosis in the brain by regulating the release of chemokine GRO/CINC-1, which gives a feedback through its receptor CXCR2 to preserve astrocytes from toxic insults.

Biological properties of a specific Gαq/11 inhibitor, YM‐254890, on platelet functions and thrombus formation under high‐shear stress

1 The effects of YM-254890, a specific Galpha(q/11) inhibitor, on platelet functions, thrombus formation under high-shear rate condition and femoral artery thrombosis in cynomolgus monkeys were investigated. 2 YM-254890 concentration dependently inhibited ADP-induced intracellular Ca(2+) elevation, with an IC(50) value of 0.92+/-0.28 microM. 3 P-selectin expression induced by ADP or thrombin receptor agonist peptide (TRAP) was strongly inhibited by YM-254890, with IC(50) values of 0.51+/-0.02 and 0.16+/-0.08 microM, respectively. 4 YM-254890 had no effect on the binding of fibrinogen to purified GPIIb/IIIa, but strongly inhibited binding to TRAP-stimulated washed platelets. 5 YM-254890 completely inhibited platelet shape change induced by ADP, but not that induced by collagen, TRAP, arachidonic acid, U46619 or A23187. 6 YM-254890 attenuated ADP-, collagen-, TRAP-, arachidonic acid- and U46619-induced platelet aggregation with IC(50) values of <1 microM, whereas it had no effect on phorbol 12-myristate 13-acetate-, ristocetin-, thapsigargin- or A23187-induced platelet aggregation. 7 High-shear stress-induced platelet aggregation and platelet-rich thrombus formation on a collagen surface under high-shear flow conditions were concentration dependently inhibited by YM-254890. 8 The antithrombotic effect of YM-254890 was evaluated in a model of cyclic flow reductions in the femoral artery of cynomolgus monkeys. The intravenous bolus injection of YM-254890 dose dependently inhibited recurrent thrombosis without affecting systemic blood pressure or prolonging template bleeding time. 9 YM-254890 is a useful tool for investigating Galpha(q/11)-coupled receptor signaling and the physiological roles of Galpha(q/11).

A novel Syk-dependent mechanism of platelet activation by the C-type lectin receptor CLEC-2

AbstractThe snake venom rhodocytin has been reported to bind to integrin α2β1 and glycoprotein (GP) Ibα on platelets, but it is also able to induce activation independent of the 2 receptors and of GPVI. Using rhodocytin affinity chromatography, we have identified a novel C-type lectin receptor, CLEC-2, in platelets that confers signaling responses to rhodocytin when expressed in a cell line. CLEC-2 has a single tyrosine residue in a YXXL motif in its cytosolic tail, which undergoes tyrosine phosphorylation upon platelet activation by rhodocytin or an antibody to CLEC-2, but not to collagen, thrombin receptor agonist peptide (TRAP), or convulxin. Tyrosine phosphorylation of CLEC-2 and other signaling proteins by rhodocytin is inhibited by the Src family kinase inhibitor PP2. Further, activation of murine platelets by rhodocytin is abolished in the absence of Syk and PLCγ2, and partially reduced in the absence of LAT, SLP-76, and Vav1/Vav3. These findings define a novel signaling pathway in platelets whereby activation of CLEC-2 by rhodocytin leads to tyrosine phosphorylation of its cytosolic tail, binding of Syk and initiation of downstream tyrosine phosphorylation events, and activation of PLCγ2. CLEC-2 is the first C-type lectin receptor to be found on platelets which signals through this novel pathway.