Proteolytic Activity Of Thrombin Research Articles

Single-Particle Plasmon Sensor to Monitor Proteolytic Activity in Real Time.

We have established a label-free plasmonic platform that monitors proteolytic activity in real time. The sensor consists of a random array of gold nanorods that are functionalized with a design peptide that is specifically cleaved by thrombin, resulting in a blueshift of the longitudinal plasmon. By monitoring the plasmon of many individual nanorods, we determined thrombin's proteolytic activity in real time and inferred relevant kinetic parameters. Furthermore, a comparison to a kinetic model revealed that the plasmon shift is dictated by a competition between peptide cleavage and thrombin binding, which have opposing effects on the measured plasmon shift. The dynamic range of the sensor is greater than two orders of magnitude, and it is capable of detecting physiologically relevant levels of active thrombin down to 3 nM in buffered conditions. We expect these plasmon-mediated label-free sensors to open the window to a range of applications stretching from the diagnostic and characterization of bleeding disorders to fundamental proteolytic and pharmacological studies.

Supra-Physiological Concentrations of C1 Esterase Inhibitor Enhance Thrombin Generation in the Presence of Thrombomodulin

Introduction. Human C1-esterase inhibitor (C1INH) is a multifunctional plasma glycoprotein with a broad spectrum biological activities, best known for its role in regulation of the classical complement pathway, contact activation system and its essential roles in the coagulation and fibrinolytic cascades [1,2]. The main targets of C1INH are C1 esterase, Factors XIa and XIIa, kallikrein, thrombin and plasmin. Low levels of functional C1INH in plasma are considered to be the primary cause for hereditary angioedema (HAE) with symptoms of swelling in the face, throat, hands, feet and abdomen. HAE patients receive replacement therapy with plasma-derived C1INH products recently licensed in the US and EU for prophylactic and acute treatment of HAE [1]. Studies suggest that supra-physiological doses of C1INH can promote thrombotic events.. To date the use of C1INH at plasma concentrations exceeding normal have been proposed in a range of disease states including renal transplant, myocardial infarction and acute lung injury. However, the mechanism and concentration required to augment the risk of thromboembolic events remains unclear and therefore raises questions about the safety of high dose C1INH therapy. We evaluated all possible sites of the C1INH concentration-dependent interaction with the coagulation system and here we focus on a hypothesis that supra-physiological concentrations of C1INH alter the anticoagulant protein C system. To study the interactive role of C1INH and the protein C pathway we performed thrombin generation assays (TGA) on plasmas from both male and female healthy donors in the presence of supra-physiologic concentrations of C1INH and increasing concentrations of thrombomodulin (TM).Methods. Platelet poor plasma (PPP) was isolated by centrifugation from blood of 20 healthy donors and mixed with 16 IU/ml of C1INH or with the same volume of buffer. The used C1INH concentration was 16 times bigger than normal values. Concentrations of TM added to the plasma samples were equal to 3, 6, 9, 12 and 15 nM. 4.5 pm tissue factor-induced thrombin generation was performed by continuous measurement of thrombin proteolytic activity of thrombin-specific substrate Z-Gly-Gly-Arg-AMC added to PPP before induction of coagulation. Thrombin peak was calculated using Origin software. Wilcoxon and sign paired tests with P=0.05 were used to obtain statistics.Results and conclusions. C1INH significantly reduced thrombin peak up to 2 times in the absence and at the lowest concentrations of TM (3 and 6 nM). Addition of 9 nM TM caused disappearance of the difference between the samples with and without C1INH. Finally, in the presence of 12 and 15 nM of TM, C1INH significantly enhanced thrombin peak (Figure 1A, B). Figure 1B demonstrates that reducing C1INH concentration can bring the thrombin peak back to the values obtained without C1INH presence. These results indicate that presence of TM crucially change the action of C1INH on coagulation and explains how supra-physiological concentrations of coagulation inhibitor may cause thrombosis in certain clinical situations.Figure 1. A: Effect of elevation of C1INH concentration on thrombin peak. Statistics was obtained using Wilcoxon (*) and Sign (**) paired tests with P value equal to 0.05 from 20 healthy donors. B: Representative dependence of thrombin peak on TM concentration with and without the presence of 16 IU/ml C1INH. C: representative dependence of Thrombin peak on C1INH concentration in the presence of TM.

Enhanced Production of Fibrinolytic Protease from Microalgae Chlorella Vulgaris using Glycerol and Corn Steep Liquor as Nutrient

According to the World Health Organization (2012), in 2030, almost 3.6 million people will die from Cardio Vascular Diseases (CVDs), mainly from heart disease and stroke. One of the pathophysiological Conditions which causes myocardial infarction, stroke and other CVDs, is the formation of a fibrin clot by the proteolytic action of thrombin on fibrinogen, and consequent accumulation of fibrin in the blood vessels usually leads to thrombosis.

Endothelial barrier protective properties of low molecular weight heparin: A novel potential tool in the prevention of cancer metastasis?

Unlabelled Box BackgroundOne of the key events in the progression of cancer metastasis is the trans‐endothelial migration of circulating tumor cells. Moreover, inhibition of tumor‐induced vascular permeability has been shown to inhibit metastasis in vivo. Low molecular weight heparin (LMWH) appears to confer a survival benefit in cancer but the underlying mechanisms are poorly understood. ObjectiveTo characterise LMWH‐mediated endothelial barrier protection and to explore strategies to limit the LMWH‐associated haemorrhagic risk in this setting. MethodsEndothelial barrier function was assessed using in vitro assays of endothelial permeability and tumor cell trans‐endothelial migration. Thrombin‐mediated activation of PAR‐1 signalling was assessed by flow cytometry and western blotting. LMWH anticoagulant activity was assessed by calibrated automated thrombography and plasma anti‐factor Xa activity assay. ResultsLMWH tinzaparin enhanced endothelial barrier function and reduced tumor cell trans‐endothelial migration (73.9±5.7% of baseline; P<.05). Tinzaparin‐mediated attenuation of thrombin‐induced permeability was not mediated through an inhibition of thrombin proteolytic activity. In addition, fractions of LMWH with diminished anticoagulant activity retained endothelial barrier protective properties and a marked synergistic effect on barrier function was observed using combinations of sub‐anticoagulant concentrations of tinzaparin with simvastatin (which exhibits endothelial barrier protective properties in vitro), with almost complete protection against agonist‐induced endothelial barrier permeability achieved (7.9±0.2% of baseline; P<.05). ConclusionCollectively, these results suggest that LMWH supports endothelial barrier function in a manner which does not appear to be dependent on its anticoagulant activity. If replicated in vivo, these findings could represent a novel therapeutic approach to the suppression of metastasis.

Abstract 13427: Treatment With a Direct Thrombin Inhibitor Interrupts the Progression of Obesity-Associated Pathologies in Mice Fed a High Fat Diet (Best of Basic Science Abstract)

Obesity is accompanied by activation of the coagulation cascade in humans and in experimental settings of diet-induced obesity. Genetic and pharmacologic interference with thrombin proteolytic activity has previously been shown to attenuate obesity-associated pathologies, including non-alcoholic fatty liver disease (NAFLD). In this study we sought to determine if intervention with the thrombin inhibitor, dabigatran etexilate (DE), can slow the progression of NAFLD and obesity when administered after onset of disease. Wild-type C57BL/6J mice were fed a control diet (AIN-93M, 10% calories from soybean oil) or a high fat diet (HFD, 60% calories from lard and soybean oil) for 3 months and then switched to identical diets containing 7.5 mg/g DE (AIN-93M+DE and HFD+DE) for an additional 2 months (intervention groups). Additional groups of mice were fed DE-containing diets for all 5 months (prophylactic groups). Terminal plasma DE levels were comparable (~330 ng/ml) in mice fed AIN-93M+DE and HFD+DE. Therapeutic intervention with DE prevented excess body weight gain, marked by no further increase in body fat mass, in HFD-fed mice. DE treatment elicited a selective reduction in micro-, but not macro-vesicular, hepatocellular steatosis in mice with established HFD-induced obesity. In addition, DE treatment prevented progression of liver injury (i.e., serum alanine aminotransferase levels) in HFD-fed mice. HFD-induced hyperglycemia was significantly attenuated in DE-treated mice, suggesting improved insulin sensitivity. DE treatment did not significantly affect these parameters in AIN-93M-fed mice. Analysis of inflammatory cytokines linked to metabolic inflammation revealed a robust increase in plasma KC/Gro, a neutrophil chemokine related to adiposity and insulin resistance, in HFD-fed mice. Notably, both prophylaxis and intervention with DE dramatically reduced plasma KC/Gro levels. Overall, the data suggest that DE can halt the progression of diet-induced obesity and NAFLD in mice fed a HFD. Moreover, the results suggest that thrombin-mediated chemokine induction contributes to HFD induction of obesity and liver disease in mice.

Thrombin Drives Tumorigenesis in Colitis-Associated Colon Cancer

The established association between inflammatory bowel disease and colorectal cancer underscores the importance of inflammation in colon cancer development. On the basis of evidence that hemostatic proteases are powerful modifiers of both inflammatory pathologies and tumor biology, gene-targeted mice carrying low levels of prothrombin were used to directly test the hypothesis that prothrombin contributes to tumor development in colitis-associated colon cancer (CAC). Remarkably, imposing a modest 50% reduction in circulating prothrombin in fII+/- mice, a level that carries no significant bleeding risk, dramatically decreased adenoma formation following an azoxymethane/dextran sodium sulfate challenge. Similar results were obtained with pharmacologic inhibition of prothrombin expression or inhibition of thrombin proteolytic activity. Detailed longitudinal analyses showed that the role of thrombin in tumor development in CAC was temporally associated with the antecedent inflammatory colitis. However, direct studies of the antecedent colitis showed that mice carrying half-normal prothrombin levels were comparable to control mice in mucosal damage, inflammatory cell infiltration, and associated local cytokine levels. These results suggest that thrombin supports early events coupled to inflammation-mediated tumorigenesis in CAC that are distinct from overall inflammation-induced tissue damage and inflammatory cell trafficking. That prothrombin is linked to early events in CAC was strongly inferred by the observation that prothrombin deficiency dramatically reduced the formation of very early, precancerous aberrant crypt foci. Given the importance of inflammation in the development of colon cancer, these studies suggest that therapeutic interventions at the level of hemostatic factors may be an effective means to prevent and/or impede colitis-associated colon cancer progression.

Thrombin inhibitory activity of some polyphenolic compounds

Thrombin, also known as an active plasma coagulation factor II, belongs to the family of serine proteases and plays a crucial role in blood coagulation process. The process of thrombin generation is the central event of the hemostatic process and regulates blood coagulant activity. For this reason, thrombin inhibition is key to successful novel antithrombotic pharmacotherapy. The aim of our present study was to examine the effects of the well-known polyphenolic compounds on the activity of thrombin, by characterization of its interaction with selected polyphenols using different biochemical methods and biosensor BIAcore analyses. Only six compounds, cyanidin, quercetin, silybin, cyanin, (+)-catechin and (−)-epicatechin, of all examined in this study polyphenols caused the inhibition of thrombin amidolytic activity. But only three of the six compounds (cyanidin, quercetin and silybin) changed thrombin proteolytic activity. BIAcore analyses demonstrated that cyanidin and quercetin caused a strong response in the interaction with immobilized thrombin, while cyanin and (−)-epicatechin induced a low response. Lineweaver–Burk curves show that used polyphenol aglycones act as competitive thrombin inhibitors. Our results suggest that polyphenolic compounds might be potential structural bases and source to find and project nature-based, safe, orally bioavailable direct thrombin inhibitors.

Caveolar Uptake and Endothelial-Protective Effects of Nanostructured Lipid Carriers in Acid Aspiration Murine Acute Lung Injury

Nanostructured lipid carriers (NLC), nanosized phospholipids/triglyceride particles developed for drug delivery, are considered biologically inactive. We assessed the efficacy of unloaded NLC as experimental treatment for acute lung injury (ALI). To induce ALI, C57Black/6 male mice received intratracheal injections of HCl or saline; A single dose of 16mg/Kg NLC or saline was injected intravenously concomitantly with HCl challenge. NLC uptake mechanisms and effects on endothelial permeability and signaling were studied in cultured endothelial cells and neutrophils. NLC pre-treatment attenuated pulmonary microvascular protein leak, airspace inflammatory cells, thrombin proteolytic activity and histologic lung injury score 24h post insult. Using fluorescence measurements and flow cytometry in mouse lung microvascular endothelial cell culture homogenates, we determined that NLC rendered fluorescent by curcumin labeling are taken up by endothelial cells from mice expressing caveolin-1, the coat protein of caveolar endocytic vesicles, but not from caveolin-1 gene-disrupted mice, which lack caveolae. In contrast, conventional emulsions (CE), consisting of larger particles, were not incorporated. In addition, NLC pre-treatment of cultured human lung microvascular endothelial cells abrogated thrombin-induced activation of p44/42, albumin permeability response, actin cytoskeletal remodeling and interleukin-6 production. Finally, NLC but not CE abrogated lipopolysaccharide-triggered interleukin-8 release. NLC are engulfed by endothelial caveolae and possess endothelial-protective effects. These novel properties may be of potential utility in ALI.

Thrombin inhibits osteoclast differentiation through a non-proteolytic mechanism

Thrombin stimulates expression of interleukin 6 and cyclooxygenase 2 by osteoblasts, both of which enhance osteoblast-mediated osteoclast differentiation by increasing the ratio of receptor activator of nuclear factor κB ligand (RANKL) expression to that of osteoprotegerin (OPG) in osteoblasts. We hypothesised that thrombin would also increase this ratio and thereby stimulate osteoclast differentiation in mixed cultures of osteoblastic cells and osteoclast precursors. In primary mouse osteoblasts, but not in bone marrow stromal cells, thrombin increased the ratio of RANKL to OPG expression. Thrombin inhibited differentiation of osteoclasts, defined as tartrate-resistant acid phosphatase (TRAP)-positive cells with three or more nuclei, in mouse bone marrow cultures treated with osteoclastogenic hormones; this effect was not mediated by the major thrombin receptor, protease-activated receptor 1, nor did it require thrombin's proteolytic activity. Thrombin also caused a decrease in the number of TRAP-positive cells with fewer than three nuclei. Thrombin (active or inactive) also inhibited osteoclast differentiation and bone resorption, respectively, in cultures of mouse spleen cells and human peripheral blood mononuclear cells induced to undergo osteoclastogenesis by treatment with RANKL and macrophage colony-stimulating factor. Osteoclast differentiation in spleen cells was inhibited when they were exposed to thrombin from days 0 to 3 or 3 to 5 of culture but not days 5 to 7 when most fusion occurred. Thrombin inhibited expression of RANK by spleen cells. These observations indicate that, although thrombin stimulates production of osteoclastogenic factors by osteoblastic cells, it inhibits the early stages of RANKL-induced osteoclast differentiation through a direct effect on osteoclast precursors that does not require thrombin's proteolytic activity.

Thrombin Activity Associated with Neuronal Damage during Acute Focal Ischemia

Mechanisms of ischemic neuronal and vascular injury remain obscure. Here we test the hypothesis that thrombin, a blood-borne coagulation factor, contributes to neurovascular injury during acute focal ischemia. Stroke was induced in adult Sprague Dawley rats by occluding the middle cerebral artery. Intra-arterial thrombin infusion during ischemia significantly increased vascular disruption and cellular injury. Intravenous infusion of argatroban, a direct thrombin inhibitor, alleviated neurovascular injury. Immunostaining showed thrombin on neurons in the ischemic core. Using an activatable cell-penetrating peptide engineered to detect thrombin activity, we discovered that thrombin proteolytic activity was specifically associated with neuronal damage during ischemia. Protease activated receptor-1, the presumptive thrombin receptor, appeared to mediate ischemic neurovascular injury. Furthermore, rats receiving thrombin during ischemia showed cognitive deficit, whereas rats receiving argatroban retained intact learning and memory. These results suggest a potential role for thrombin contributing to neurovascular injury and several potential avenues for neuroprotection.

Modulation of expression of innate immunity markers CXCL5/ENA-78 and CCL20/MIP3α by protease-activated receptors (PARs) in human gingival epithelial cells

Protease-activated receptors (PARs) are G-protein-coupled receptors with an active role in host defense. The two most highly expressed members of the PAR family in gingival epithelial cells (GECs) are PAR1 and PAR2. The major virulence factors of periodontal pathogen Porphyromonas gingivalis are its proteases which can activate PAR2. However, little is known about the function of PARs in GECs when they are activated by their endogenous agonist enzymes. The purpose of this study was to characterize how the expression of innate immune markers is modulated when PAR1 and PAR2 are activated by their agonist enzymes, thrombin and trypsin, respectively. Here, we report that activation of PAR1 and PAR2 induces cell proliferation at low concentration. Activation of PAR via proteolytic activity of thrombin and trypsin induces expression of CXCL5/ENA-78 and CCL20/MIP3alpha in a concentration-dependent manner. Induction of CXCL5 via PAR1 was inhibited in the presence of PAR1 cleavage blocking antibodies and by PAR1 siRNA. The induction of CXCL5 and CCL20 via PAR2 was inhibited by PAR2 siRNA. These findings indicate an active role in innate immune responses by PAR1 and PAR2 in GECs. Modulation of innate immunity by PARs may contribute to co-ordinated and balanced immunosurveillance in GECs.

Receptor Dimerization with EPCR Redirects PAR‐1 Signaling in Endothelial Cells

Activated protein C (APC) is the only therapeutic shown to reduce mortality in patients with severe sepsis, however its mechanism of action remains unknown. An important possibility is that APC influences the activation of the G‐protein coupled thrombin receptor (PAR‐1) in the endothelium to promote barrier function. We propose that PAR‐1 signaling is regulated by direct interactions with the single transmembrane endothelial protein C receptor (EPCR), and that this co‐factor, not the proteolytic activity of thrombin or APC per se, determine whether PAR‐1 signaling is endothelial barrier‐disruptive or barrier‐protective. We postulate that the interaction of PAR‐1 with EPCR alters the G‐protein specificity of PAR‐1 by stabilizing a discrete active conformation. Toward this end we show here that PAR‐1 constitutively interacts with EPCR. However, the PAR‐1/EPCR complex is maintained only when PAR‐1 is activated by APC and not by thrombin. Hence EPCR may function as a critical molecular switch redirecting PAR‐1 signaling from endothelial barrier‐disruptive to barrier‐protective.

Thrombin-induced cell proliferation and platelet-derived growth factor-AB release from A172 human glioblastoma cells.

In a previous study, we found that thrombin induced proliferation of TM-1 and T98G human glioma cells and that the mitogenic effect was abolished by hirudin. We investigated thrombin's effects on the proliferation of A172 human glioblastoma cells and the induction of growth factors. Furthermore, we examined whether or not the expression of heparin cofactor II (HCII) in A172 cells using adenovirus vector could suppress thrombin's effects. The effect of thrombin on cell proliferation was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. The amount of growth factors in the conditioned medium was measured by enzyme-linked immunosorbent assay. The level of platelet-derived growth factor (PDGF)-B mRNA was assessed by reverse transcriptase-polymerase chain reaction analysis. Thrombin-induced proliferation of A172 cells primarily depended on the enhanced secretion of PDGF-AB by thrombin. The action of thrombin depended on its proteolytic activity. However, thrombin-induced PDGF-AB secretion was not abolished by anti-protease-activated receptor (PAR) antibody. The PAR-1 agonist peptide had no effect on cell growth and PDGF-AB levels. Thrombin did not increase PDGF-B gene expression. Expression of HCII effectively suppressed thrombin-induced PDGF-AB release. These results indicate that thrombin may play an important role in the proliferation of A172 cells by inducing PDGF-AB secretion and that thrombin's action is mediated by its proteolytic activity. Inhibition of thrombin's proteolytic activity may be a new therapeutic method for gliomas.

6A.1 Fibrin formation disorders and pregnancy loss

Fibrinogen, the soluble clottable protein in the blood, is converted into fibrin by the proteolytic action of thrombin. The insoluble polymerized fibrin then becomes cross-linked by activated factor XIII resulting in mechanically stabilized fibrin protected from fibrinolysis. Severe deficiency of fibrinogen and factor XIII are associated with severe bleeding diathesis and with pregnancy loss. Both factor XIII and fibrinogen play an essential role in placental implantation and maintenance of pregnancy. Investigation of pregnancy loss in fibrinogen and factor XIII knock-out mice demonstrated that miscarriages are due to the improper anchoring of cytotrophoblasts after their invasion of the endometrium and to the uncontrolled hemorrhage along the planes of placental-decidual attachment. It is also clear that maternal fibrinogen and factor XIII are required to maintain pregnancy, and factor XIII present in the plasma and not in the cells is essential. Supplementation therapy with factor concentrates is advocated throughout pregnancy.

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.

Activation of Microglial Cells by Thrombin: Past, Present, and Future

In addition to its role in the coagulation cascade, the serine proteinase thrombin (factor IIa) activates cell surface proteinase-activated receptors (PARs) both within and outside the vascular system. PARs are expressed in the central nervous system and mediate thrombin-induced cellular responses in a variety of neural cell types, including microglial cells. Microglial activation by thrombin was reported to induce proliferation, cytokine release, and intracellular calcium signaling. Recently, additional experiments questioned whether these effects are mediated either by thrombin's proteolytic activity or by thrombin itself. Analysis of commercially available plasma-derived thrombin frequently used in the earlier studies showed that cyto/chemokine release-activating properties were not residing with thrombin but were with high molecular weight contaminant(s). In the absence of such contamination, no microglial activation was seen. We compared commercial-grade plasma-derived thrombin to pharmaceutical-grade recombinant thrombin devoid of any measurable contamination. The pharmaceutical-grade thrombin displayed a much more limited profile of microglia-activating properties, triggering only intracellular calcium signals and small changes in surface antigen expression. The signals induced by the pharmaceutical-grade thrombin were completely abolished by proteolytic inhibition, indicating that they are proteolysis-dependent, are most likely PAR mediated, and reflect thrombin's true microglia-activating potential. Prior reports using nonpharmaceutical-grade thrombin need to be reinterpreted critically given these new findings.

2-(2-Br-phenyl)-8-methoxy-benzoxazinone (HPW-RX2), a direct thrombin inhibitor with a suppressive effect on thromboxane formation in platelets

2-(2-Br-phenyl)-8-methoxy-benzoxazinone (HPW-RX2), a newly synthetic benzoxazinone derivative, has previously been shown to inhibit rabbit platelet aggregation caused by thrombin and arachidonic acid. In the present study, the mechanism for the antiplatelet effect of HPW-RX2 was further investigated. In human platelets, HPW-RX2 concentration-dependently inhibited platelet aggregation, ATP release, P-selectin expression, and intracellular calcium mobilization caused by thrombin. In contrast, HPW-RX2 had no significant effect on either SFLLRN- or GYPGKF-induced platelet aggregation, indicating that HPW-RX2 did not interfere with platelet thrombin receptors. Moreover, HPW-RX2 inhibited the amidolytic activity of thrombin and prolonged the fibrinogen clotting time. These results suggest that the inhibitory effect of HPW-RX2 on thrombin-induced platelet aggregation is via direct inhibition of thrombin proteolytic activity. Besides the inhibition on thrombin, HPW-RX2 also prevented platelet aggregation, ATP release, and increase in [Ca 2+] i caused by arachidonic acid and low concentration collagen. In a parallel manner, both arachidonic acid-induced thromboxane B 2 and prostaglandin D 2 formations were decreased in platelets treated with HPW-RX2. This indicates that HPW-RX2 is able to inhibit the arachidonic acid cascade at the cyclooxygenase level. This is the first report of a benzoxazinone derivative possessing both thrombin and cyclooxygenase inhibitory properties. The dual effect of HPW-RX2 might provide extra therapeutic benefits for treatment of arterial thrombosis.

Thrombin mediated migration of osteogenic cells

Given that thrombin is ubiquitously expressed at sites of vascular injury, and that osteogenic cells express receptors for thrombin, we questioned whether thrombin could attract osteogenic cells to a bony wound. Using a scratch wound assay, thrombin stimulated a significant increase in migration of osteogenic cultures of primary marrow cells. This effect was dependent on thrombin proteolytic activity; however, thrombin was unable to stimulate the migration of a more differentiated marrow-derived osteogenic cell line. To better understand the role of thrombin in osteoprogenitor migration, we developed an osteoprogenitor migration assay that combines a modified Boyden chamber with a bone nodule assay. Primary cells that migrated through the transwell filter in the presence of thrombin formed significantly more bone nodules compared to the condition without thrombin. This was not due to proliferation or differentiation effects of thrombin. In contrast, thrombin was unable to stimulate an increase in the number of nodules for the more differentiated osteogenic cell line. Thus, our results suggest that thrombin exhibits differential motogenic effects on osteogenic cells depending on their differentiation state. The cell migration/bone nodule assay described here is the first assay that can be specifically used to examine the effects of factors on the migration of osteoprogenitor cells, particularly those derived from primary populations.

Thrombin-mediated IL-10 up-regulation involves protease-activated receptor (PAR)-1 expression in human mononuclear leukocytes

Thrombin, the key enzyme of the coagulation cascade, exerts cellular effects through activation of the protease-activated receptors (PARs). Interleukin (IL)-10, besides its anti-inflammatory properties, is considered a major denominator of the immunosuppressive effect during human endotoxemia. We have recently shown that thrombin inhibits IL-12 production in human mononuclear cells and that such inhibition is accompanied by IL-10 up-regulation. To our knowledge, there are no data available to show that thrombin mediates IL-10 production by its interactions with PAR-1. We here report that human alpha-thrombin enhances IL-10 expression in human peripheral blood mononuclear cells and in established monocytic cell lines and that this up-regulation requires PAR-1 expression. The use of proteolytically inactive thrombin reveals that such enhancement requires thrombin proteolytic activity. Addition of PAR-1 agonist peptides, such as SFLLRN, results in a significant increase of IL-10 production. PAR-1 expression is required for thrombin-induced IL-10 production, as shown by experiments performed with antisense or sense PAR-1 oligonucleotides. Treatment with thrombin or SFLLRN of monocytic cell lines, such as U937 and Mono Mac-6, results in an increased IL-10 production. This suggests that the observed IL-10 up-regulation may be the result of a direct interaction with monocytes. The observation that thrombin-mediated up-regulation of IL-10 may require the expression of the PAR-1 receptor identifies a new, functional link between inflammation and coagulation. Our results may also contribute to better design therapeutic strategies to treat several disorders, characterized by the presence of inflammatory as well as coagulant responses.

RWJ-58259: a selective antagonist of protease activated receptor-1.

Protease activated receptor-1 (PAR-1) is a key mediator of the cellular actions of alpha-thrombin. Thus, antagonism of this unique G-protein coupled receptor with a small molecule represents a means of selectively inhibiting thrombin's cellular actions without inhibiting its proteolytic activity. RWJ-58259 (alphaS)-N-[(1S)-3-amino-1-[[(phenylmethyl)- amino]carbonyl]propyl]-alpha-[[[[[1-(2,6-dichlorophenyl)methyl]-3-(1-pyrrolidinylmethyl)-1H-indazol-6-yl]amino]carbonyl]amino]-3,4-difluorobenzenepropanamide) is a potent and selective inhibitor of PAR-1 identified as part of a synthetic chemistry program based upon a de novo design approach. RWJ-58259 inhibited thrombin-induced platelet aggregation in human platelets with an IC50 of 0.37 microM without inhibiting thrombin's proteolytic activity or aggregation induced by other agonists. RWJ-58259 was not effective in guinea pig models of thrombosis. This reflected the presence of a second thrombin-sensitive receptor system in guinea pigs (PAR-3/4) and the selectivity of RWJ-58259 for PAR-1. However, RWJ-58259 was effective in a non-human primate model of thrombosis. Because human platelets have a PAR expression profile similar to the non-human primate, PAR-1 antagonism has the potential to be antithrombotic in humans. RWJ-58259 also inhibited thrombin-induced intracellular calcium signaling and proliferation in rat vascular smooth muscle cells. Perivascular application of RWJ-58259 in vivo significantly inhibited arterial injury-induced stenosis in a rat model of balloon angioplasty. These preclinical results suggest a potential clinical utility of RWJ-58259 for treatment of thrombotic disorders and vascular injury associated with acute coronary interventions and atherosclerosis. Given the potential role of PAR-1 in thrombin's actions in other cell types and disease states, RWJ-58259 provides a means for assessing additional clinical utilities of PAR-1 antagonism in disease conditions such as inflammation, cancer and neurodegeneration.