Potent Inhibitor Of Platelet Function Research Articles

A Flow Cytometric Analysis of the Inhibition of Platelet Reactivity Due to Nitrite Reduction by Deoxygenated Erythrocytes

Nitric oxide (NO), a small gas molecule, has long been known to be a potent inhibitor of platelet function but the physiological and pathological implications of platelet inhibition by NO have not been well clarified. We recently showed that the addition of nitrite to platelet-rich plasma in the presence of erythrocytes could inhibit platelet aggregation and this inhibitory effect of nitrite + erythrocytes was enhanced by deoxygenation of erythrocytes as measured by P-selectin expression and cGMP production. In order to study the nitrite effect on platelets at different oxygen levels, we used the flow cytometric assays to detect platelet membrane surface markers upon activation. The P-selectin and activated gpIIb/IIIa expression on platelet membranes in response to ADP, collagen and thrombin stimulation was measured at various hematocrit and oxygen levels. Nitrite (0.1 to 1.0 μM) significantly decreased the percentage of these surface markers on the platelet membrane at the hematocrit values above 23% and oxygen levels lower than 49 mmHg. The inhibitory effect of nitrite was augmented by increasing hematocrit values and decreasing oxygen saturation. C-PTIO (an NO scavenger) prevented the platelet inhibition by nitrite + erythrocytes whereas the inhibitors of NO synthase and xanthine oxidoreductase had no effect. These results support the proposal that circulating nitrite decreases platelet reactivity in the presence of partially deoxygenated erythrocytes through its reduction to NO, which may also explain certain differences between arterial and venous thrombosis and support directly the role of deoxyhemoglobin in this process. We believe that our flow cytometric assays offer a possibility to identify the individual molecular process involved in these effects.

A FLOW–CYTOMETRIC ANALYSIS OF THE INHIBITION OF PLATELET REACTIVITY WITH NITRITE REDUCTION BY DEOXYGENATED ERYTHROCYTES

Nitric oxide (NO), a small gas molecule, has long been known to be a potent inhibitor of platelet function but the physiological and pathological implications of platelet inhibition by NO have not been well clarified. We recently showed that the addition of nitrite to platelet-rich plasma in the presence of erythrocytes could inhibit platelet aggregation and this inhibitory effect of nitrite + erythrocytes was enhanced by deoxygenation of erythrocytes as measured by P-selectin expression and cGMP production. In order to study the nitrite effect on platelets at different oxygen levels, we used the flow cytometric assays to detect platelet membrane surface markers upon activation. The P-selectin and activated gpIIb/IIIa expression on platelet membranes in response to ADP, collagen and thrombin stimulation was measured at various hematocrit and oxygen levels. Nitrite (0.1 to 1.0 mM) significantly decreased the percentage of these surface markers on the platelet membrane at the hematocrit values above 23% and oxygen levels lower than 49 mmHg. The inhibitory effect of nitrite was augmented by increasing hematocrit values and decreasing oxygen saturation. C-PTIO (an NO scavenger) prevented the platelet inhibition by nitrite + erythrocytes whereas the inhibitors of NO synthase and xanthine oxidoreductase had no effect. These results support the proposal that circulating nitrite decreases platelet reactivity in the presence of partially deoxygenated erythrocytes through its reduction to NO, which may also explain certain differences between arterial and venous thrombosis and support directly the role of deoxyhemoglobin in this process. We believe that our flow cytometric assays offer a possibility to identify the individual molecular process involved in these effects.

Nitrite Inhibits Platelet Reactivity in Presence of Deoxygenated Red Blood Cells As Measured by Flow Cytometry

AbstractAbstract 5146Nitric oxide (NO), a small gas molecule with multiple functions in the body, has been shown to have several precursors in body, the most abundant of which is nitrite. In the last decade several studies show that nitrite is reduced to NO by various enzymatic and non-enzymatic reductive processes, especially at low oxygenation conditions. These pathways are alternative mammalian sources of NO as compared to its enzymatic production by a family of nitric oxide synthase enzymes (NOS). NO has long been known to be a potent inhibitor of platelet function but the physiological and pathological implications of this has not been well clarified. We recently showed that addition of nitrite into plasma in the presence of red blood cells inhibits platelet aggregation and that this effect, as measured by cGMP production is enhanced by deoxygenation of erythrocytes. In order to study these effects more thoroughly on the molecular level, we have been developing flow-cytometric assays to study molecular processes on the platelet cell membrane known to correlate with platelet aggregation. We focused on P-selectin (CD62P) and activated GpIIb/IIIa (PAC-1) levels in platelets exposed to three activators, ADP, collagen and thrombin, in the presence of various concentrations of red blood cells and at variable levels of oxygen. We found that nitrite at physiological concentration (0. 1 to 1. 0 uM) significantly inhibited exposure of both of these surface markers on the outer membrane of platelets at levels of hematocrit above 23% and mean oxygen levels lower than 49 mmHg. The effect increased with increasing hematocrit and decreasing oxygen saturation into the hypoxic range. Addition of an NO scavenger prevented nitrite-related platelet inhibition, but addition of an inhibitor of NOS or of xanthine oxidoreductase had no effect. These results indicate that physiological levels of nitrite ions in the blood can modulate platelet reactivity in the presence of partially deoxygenated erythrocytes by the reduction to NO and that this process can modulate platelet activation and aggregation levels and confirm and extend our previous report (Srihirun et al, PLoS One 7, e30380, 2012), done primarily using aggregation assays. Our flow cytometry assays not only allows a more detailed and systematic study of the kinetics and equilibrium aspects of the aggregation process, but also offers a possibility to identify the individual molecular processes involved in these effects. The results may also explain certain differences between arterial and venous thrombosis. Disclosures:Schechter:NIH: Employment, co-inventor of NIH patent on use of nitrite in disease, co-inventor of NIH patent on use of nitrite in disease Patents & Royalties.

N-octanoyl-dopamine is a potent inhibitor of platelet function

Dopamine (DA) is a co-agonist for platelet activation; yet, donor DA treatment is associated with improved transplantation outcome in renal and heart recipients. Recently, N-octanoyl-dopamine (NOD) was developed which displays superior effects compared to DA in terms of graft protecting properties. Whereas DA is a known platelet co-agonist, the effect of NOD on platelet function is unknown. This is a hypothesis generating study with the aim to assess the effects and molecular mechanisms of NOD and NOD-like compounds on platelet function. The influence of DA, NOD, and NOD-like compounds on platelet responses to classical agonists (adenosine 5′-diphosphate (ADP), U46619) was investigated in six healthy donors by applying whole blood aggregometry (Multiplate®) and flow cytometry for Pac-1, CD62P, and CD63 expression. Changes in platelet cAMP concentrations were assessed by ELISA. While DA showed synergy in platelet activation by ADP and U46619, NOD caused significant inhibition of platelet function both in whole blood aggregometry and flow cytometry. The inhibitory effect of NOD was not mediated via cAMP levels. The nonredox-active NOD-analog N-octanoyl-tyramine had no effects on platelet function. Acetylated NOD conferred to NOD by intracellular esterases showed similar inhibitory effects as NOD. In contrast to DA, NOD is a potent inhibitor of platelet function most likely through intracellular redox-active processes. This adds to the overall protective effect of NOD on pre-transplantation injury and makes NOD an attractive candidate compound for donor or organ conditioning prior to transplantation.

Neue Ansätze und Indikationen zur Plättchenfunktionsdiagnostik in der Kardiologie

Inhibiting platelet function is a key therapeutic principle in cardiology because platelets play a pivotal role in triggering cardiovascular events. In addition to acetylsalicylic acid, a cyclooxygenase inhibitor, ADP-receptor blockers are frequently used for anti-platelet therapy. This therapy does not abolish platelet activation and aggregation. Platelets may still be activated by alternative routes such as the thrombin receptor-mediated pathway. New, more potent inhibitors of platelet function continue to lower the risk of ischaemic events but several trials and clinical registries have also shown that this advantage was frequently offset by an increased risk of bleeding complications. As a consequence, the individual risk of ischaemia and bleeding of a patient must be taken into consideration to select the platelet inhibitor offering the best benefit-risk ratio. Modern laboratory diagnostics may help to achieve this goal by complementing functional platelet tests with pharmacogenomic analyses consistent with the idea of "personalized medicine".

Effects of Dabigatran, a Direct Thrombin Inhibitor, as Compared to the Direct Factor Xa Inhibitors, Rivaroxaban and Apixaban, on Tissue Factor-Induced Human Platelet Aggregation in Platelet Rich Plasma.

Direct thrombin inhibitors (DTIs) have been shown to be very potent inhibitors of platelet function when platelets are activated with thrombin. This action does not occur by direct binding of the DTI to the platelet PAR-1/-4 receptor, but indirectly, by reducing thrombin concentrations and thereby reducing the interactions of thrombin with its receptor on the platelet. It was hypothesized that both thrombin and factor Xa inhibitors could inhibit platelet aggregation, if the stimulus to initiate aggregation was higher in the cascade than factor Xa, such as tissue factor. Thus, dabigatran, a DTI, and the direct factor Xa inhibitors, rivaroxaban and apixaban were tested. Free flowing whole blood (60 ml) was obtained from an antecubital vein using an 18 gauge needle from healthy human volunteers. Blood was collected in tubes containing 3.13% sodium citrate (1 in 10 dilution with whole blood). Blood was centrifuged at 200x g to obtain platelet rich plasma (PRP). Samples (300 μL PRP) were placed in a 6-channel aggregometer, equilibrated for 5 min at 37°C and calibrated with PPP from same individual (0–1 Volts). Photometric tracings were continuously digitally recorded over 5 min following the addition of tissue factor and curves were evaluated as AUC over this time interval. Each PRP sample was incubated with 2 mg/ml Pefabloc®FG (Gly-Pro-Arg-Pro) to prevent fibrin polymerisation, 5 mM CaCl2 and increasing concentrations of dabigatran or factor Xa inhibitor. Tissue factor stimulus (range, 5–27 μl of 10 ml Innovin solution) was tailored for each individual, so that the minimum concentration that resulted in maximum aggregation was used. As positive controls, aggregation was also performed after stimulating with ADP (10 μM), collagen (2 μg/ml), TRAP (20 μM) or ecarin (0.1 U/ml). All substances inhibited tissue factor-induced platelet aggregation in a concentration-dependent manner. Dabigatran was the most potent inhibitor of platelet aggregation among the substances tested, with an IC50 of 35 nM, rivaroxaban and then apixaban followed, with IC50s of 312 and 817 nM, respectively. All substances had no effect on platelet aggregation induced by ADP, collagen and TRAP. Dabigatran was a potent inhibitor of ecarin-induced platelet aggregation, while the factor Xa inhibitors had no effect, as expected from their mechanism of action. Thus, these studies demonstrate that both direct thrombin inhibitors (by inhibiting thrombin) and direct factor Xa inhibitors (by preventing thrombin generation) indirectly inhibit platelet aggregation, though dabigatran was more potent than rivaroxaban and apixaban under these experimental conditions. Thus, these substances may not only be effective in venous/stasis thrombotic episodes where fibrin formation plays an important role, but may also be effective in more platelet dominant, arterial thrombosis settings.

Extracellular fibrinogen binding protein, Efb, from Staphylococcus aureus as an antiplatelet agent in vivo

Staphylococcus aureus produces and secretes a protein, extracellular fibrinogen binding protein (Efb), which contributes to virulence in wound infection. We have previously shown that Efb is a potent inhibitor of platelet function in vitro. We confirm here that this is also the case in vivo. Pre-treatment with Efb resulted in a significant prolongation of bleeding time in a mouse model. Furthermore, Efb was capable of rescuing animals from death caused by the administration of potent platelet agonists. This antiplatelet effect may explain the retardation of wound healing associated with Efb in S. aureus wound infections. These results are important not only in terms of understanding S. aureus pathogenesis, and consequently identifying new treatment strategies, but also with regard to the development of potential, novel antiplatelet agents for the prevention of thrombosis.

Regulation of Endogenous Reactive Oxygen Species in Platelets Can Reverse Aggregation

While much is known about the normal activation of platelets, there have been few observations demonstrating reversibility of the aggregation process. Inhibition of phosphoinositide 3-kinase (PI3-kinase) has been shown to cause platelet disaggregation. In addition, NO is a known potent inhibitor of platelet function. In this study, the role of PI3-kinase in the regulation of endogenous platelet NO and the relevance to platelet function was determined. Incubation of platelets with PI3-kinase inhibitors led to a dose-dependent increase in platelet NO and cGMP levels that were temporally related to the period of platelet disaggregation. Addition of ferroheme myoglobin eliminated both the augmented NO release and disaggregation. PI3-kinase inhibition decreased the functional activation of NADPH oxidase and this corresponded to decreased superoxide release. To confirm these findings, platelets from NOS III-deficient mice were studied. These platelets did not release NO, and PI3-kinase inhibition led to decreased superoxide but not platelet disaggregation. Overall, these results indicate that platelet-derived NO contributes to the process of platelet disaggregation. PI3-kinase plays a role in regulating NADPH oxidase-generated superoxide in platelets and, by altering the bioactivity of platelet NO, may be a potential method for reversing platelet aggregation and thrombus formation.

Current role of platelet glycoprotein IIb/IIIa inhibitors in acute coronary syndromes.

The central role of platelet-rich thrombus in the pathogenesis of acute coronary syndromes (ACSs) is well-known. Glycoprotein IIb/IIIa (Gp IIb/IIIa) receptor antagonists are potent inhibitors of platelet function that may be expected to affect favorably the natural history of ACSs. To define the optimal role of Gp IIb/IIIa inhibitors in treatment strategies for ACSs. A MEDLINE search was performed to identify all English-language articles regarding use of Gp IIb/IIIa inhibitors in ACSs published between 1966 and June 2000. In addition, relevant abstracts from the annual meetings of the American Heart Association, American College of Cardiology, and the European Society of Cardiology were reviewed. Only studies of 500 or more patients were included. Of 15 studies identified, 10 randomized, placebo-controlled, double-blind trials of Gp IIb/IIIa inhibitors in ACSs were selected for review. Data quality was determined by publication in the peer-reviewed literature or presentation at an official cardiology society-sponsored meeting, as well as by verification with the primary author. Three members of this class of drugs are available for intravenous use. Abciximab, eptifibatide, and tirofiban hydrochloride, each have data demonstrating their value in improving the outcomes of patients presenting with ACSs. Current evidence supports use of these drugs in both conservative and invasive treatment strategies. Glycoprotein IIb/IIIa-blocking therapy is safe, and with proper precautions, bleeding risks can be minimized. Biological differences exist among these agents, but as of yet, no head-to-head comparisons have been made of their clinical efficacy. Unlike intravenous Gp IIb/IIIa inhibitors, available data regarding any role of oral Gp IIb/IIIa inhibitors are not favorable. Current data indicate that intravenous Gp IIb/IIIa inhibitor therapy merits a prominent role in the initial management of patients with ACSs. JAMA. 2000;284:1549-1558.

Calcium antagonists and ACE inhibitors. Effect on endothelium and vascular smooth muscle.

The effects of cardiovascular drugs on endothelium and vascular smooth muscle function are important for the prevention of cardiovascular disease. Changes in endothelial function are an early event in most forms of cardiovascular disease and, later in the disease process, vascular smooth muscle cells are functionally altered and begin to migrate to and proliferate in the intima. Calcium antagonists and angiotensin converting enzyme (ACE) inhibitors are widely used in patients with cardiovascular disease and are thought to have vascular protective effects. ACE, an enzyme located in the endothelial cell membrane, activates angiotensin I and angiotensin II, and deactivates bradykinin. Bradykinin activates endothelial bradykinin (B2) receptors, which results in the formation of nitric oxide and prostacyclin. Hence, ACE inhibitors not only prevent the formation of angiotensin II, but also increase the local levels of bradykinin and in turn nitric oxide and prostacyclin. These compounds are vasodilators and potent inhibitors of platelet function, and therefore may mediate important protective effects of ACE inhibitors. Furthermore, nitric oxide may have antiproliferative effects in vascular smooth muscle cells. Calcium antagonists do not appear to affect the release of endothelium-derived relaxing factors or any other endothelial product. However, they facilitate endothelium-dependent relaxation and reduce the contracting effects of endothelin-1 at the level of smooth muscle. Indeed, in some blood vessels, e.g. the large coronary arteries and the human forearm circulation, verapamil and nifedipine antagonise endothelin-induced contractions. In addition, calcium antagonists inhibit the effects of platelet-derived growth factor and may have antiproliferative effects in vascular smooth muscle cells. In conditions involving progressive dysfunction of the endothelium, vascular deposition of platelets increases the local levels of platelet-derived growth factor, and the antiproliferative effects of calcium antagonists may thus be particularly important.

Therapeutic Implications of a Specific Murine Monoclonal Antibody (7E3) to the Platelet Receptor GPIIb/IIIa

The murine monoclonal antibody 7E3 blocks the platelet glycoprotein IIb/IIIa receptor, and is a potent inhibitor of platelet function in both animals and man. Animal models of the acute coronary syndromes suggest that 7E3 abolishes the in vivo formation of platelet thrombi, accelerates thrombolysis with tissue plasminogen activator, and prevents subsequent reocclusion. Human studies with 7E3 suggest that complete inhibition of platelet function may be safely undertaken for periods of up to 36 h, and preliminary studies indicate effectiveness in the therapy of clinical unstable angina. Potential problems with its use include immunogenicity and thrombocytopenia. The outcome of the acute coronary thrombotic syndromes, which include unstable angina, acute myocardial infarction and abrupt closure following coronary angioplasty, may be significantly improved with 7E3 therapy.

Endothelium-derived vasoactive factors and their role in the coronary circulation

The endothelium—due to its strategic anatomic position between the circulating blood and vascular smooth muscle—plays an important functional role in the coronary circulation. Endothelial cells release factors interfering with coagulation, platelet function, vascular tone, and growth. Endothelium-derived nitric oxide (NO) is the endogenous nitrovasodilator that is a potent inhibitor of platelet function and vasodilator. Together with prostacyclin, NO plays an important protective role in preventing platelet adhesion, aggregation, and coronary vasospasm. Endothelial cells also are a source of contracting factors such as endothelin-1, thromboxane A 2, and endoperoxides. Cardiovascular risk factors (hyperlipidemia, hypertension, and diabetes) inhibit the formation of endothelium-derived relaxing factors and promote that of contracting factors. In coronary arteries with advanced atherosclerosis, endothelial function is severely impaired. The reduced release of endothelium-derived NO is associated with an increased platelet-vessel-wall interaction and, in turn, platelet activation and vasospasm, both known events in coronary artery disease.

Infusion of a stable prostacyclin analogue, iloprost, to patients with peripheral vascular disease: lack of antiplatelet effect but risk of thromboembolism

purpose: Prostacyclin, a potent inhibitor of platelet function and vasodilator, has been used to treat peripheral vascular disease. The aim of this study was to monitor the thrombotic status of patients treated by infusion of a stable prostacyclin analogue, iloprost. patients and methods: Thirteen patients with peripheral vascular disease underwent iloprost infusion for 3 days (8 hours each day) in a dose ranging from 0.5 to 2 ng/kg/minute. Variable parameters of thrombosis such as platelet reactivity (shear-induced hemostatic plug formation and thrombus formation on a collagen fiber), coagulation, and spontaneous thrombolysis (dislodgment of hemostatic plugs) were measured from non-anticoagulated blood samples by hemostatometry immediately before and 1 hour after the infusion and on the last day, 4 hours after initiation of the infusion. results: Analysis of data from all patients 1 hour after the infusion showed no changes in platelet reactivity and spontaneous thrombolysis, but coagulation was significantly enhanced. In four patients, significant platelet hyperreactivity was observed after the infusion. Four of the five patients tested while undergoing iloprost infusion showed an enhanced thrombotic reaction and markedly enhanced coagulation. Iloprost employed in vitro in a concentration that corresponds to the therapeutic peak blood level caused no inhibition of platelet function but significantly enhanced coagulation. The threshold in vitro iloprost concentration at which antiplatelet effect and increased spontaneous thrombolysis were observed was twice that of the therapeutic blood level. conclusions: These findings challenge the view that antagonism of platelet function is an important factor of iloprost therapy. Furthermore, platelet hyperreactivity in some patients and markedly enhanced coagulation during and after infusion of iloprost in general, represent a risk of thromboembolism, especially as patients are already in a prethrombotic condition.

Extracellular adenine compounds, red blood cells and haemostasis: facts and hypotheses.

Previously, the role of adenine nucleotides was thought to be confined to the intracellular space of the cell. Research of the last decades has revealed that nucleotides also occur in the extracellular milieu. This survey deals with extracellular adenine compounds in the blood, focussing on their role as chemical mediators in the haemostatic effect of red cells. Erythrocytes may act as pro-aggregatory cells by at least two chemical mechanisms. Firstly, they can enhance platelet aggregation by releasing adenosine diphosphate (ADP), a well known platelet stimulatory substance. ADP is set free when red cells are stressed mechanically, for instance by shear forces generated in the blood stream; ample experimental evidence supporting this view is summarized. Secondly, erythrocytes efficiently take up extracellular adenosine via their nucleoside transporters, thereby removing a potent inhibitor of platelet function. Extracellular adenosine occurs in the blood stream, either directly released from various tissues or as the end product of extracellular adenine nucleotide metabolism, e.g. after degradation of red cell-born ADP or ATP. Finally, a novel mechanism of action of the antithrombotic drug dipyridamole, which has very recently been put forward, is demonstrated. Dipyridamole inhibits platelet function indirectly by blocking the uptake of extracellular adenosine via the nucleoside transporter of red cells; increased adenosine levels in turn are responsible for the antiaggregatory effect of dipyridamole.

Ticlopidine inhibits platelet thrombus formation studied in a flowing system

We have studied the effect of ticlopidine on platelet function. This effect was assessed by aggregation studies and by the Baumgartner perfusion system as an ex vivo approach to study modifications in the interaction of platelets with vascular subendothelium. Platelets from volunteers, that were given 250 mg of ticlopidine twice a day showed a significantly decreased aggregation of platelets induced by several agonists. In the perfusion studies a marked reduction in the parameters that measure platelet interaction with subendothelium was also observed. The decrease in thrombus formation,and the diminished size of platelet aggregates, clearly indicated that ticlopidine impaired platelet-platelet interaction in this experimental flowing system. Our results suggest that ticlopidine is a potent inhibitor of platelet function and that its antiplatelet activity might be related to the mechanisms that regulate the interaction between platelets at the membrane level.

Cultured endothelial cells synthesize both platelet-activating factor and prostacyclin in response to histamine, bradykinin, and adenosine triphosphate.

Cultured human endothelial cells synthesize prostacyclin (PGI2), a potent inhibitor of platelet function, when stimulated with histamine, bradykinin, or ATP. Paradoxically, we report that these agonists also induced the rapid and sustained synthesis of platelet-activating factor (PAF) by endothelial cells. In fact, the synthesis of this potent activator of platelets and neutrophils was induced by stimulation of the same receptor subtype that induced PGI2 synthesis: stimulation of a histamine H1 or a bradykinin B2 receptor induced both PAF and PGI2 synthesis. However, two physiologically important differences exist between the production of PAF and PGI2 by endothelial cells. The synthesis of PGI2 proceeded for only 7.5 min before the abrupt termination of synthesis, whereas the synthesis of PAF was clearly detectable even 45 min after stimulation. Although maximal accumulation of PAF occurred after 10-15 min of stimulation, the prolonged synthesis resulted in the presence of PAF for up to 1 h after stimulation. Secondly, whereas PGI2 was released from the cell monolayer, PAF remained cell-associated without significant release to the external medium. Endothelial cell-generated PAF, therefore, does not function as a hormone. The prolonged association of this potent activator of platelets and neutrophils with endothelial cells may mediate some of the inflammatory properties of histamine and bradykinin. It may also be a factor in the formation of a thrombogenic vascular surface, an event suggested to play a primary role in the pathogenesis of thrombosis and atherosclerosis.

Alpha tocopherol quinone (αTQ): A potent inhibitor of platelet function

Alpha tocopherol quinone (αTQ): A potent inhibitor of platelet function

The Effect of Prostacyclin (PGI2) on Platelet Behaviour. Thrombus Formation in Vivo and Bleeding Time

Prostacyclin (PGI2) infused intravenously into anaesthetized rabbits inhibited electrically-induced thrombus formation in the carotid artery, increased bleeding time and inhibited ex vivo platelet aggregation induced by ADP or arachidonic acid. The increase in bleeding time and the inhibition of ex vivo platelet aggregation lasted for as long as the infusion of PGI2 was maintained but rapidly disappeared after infusion was stopped. Prostacyclin is a more potent inhibitor of platelet function, in vivo than prostaglandin E1 (PGE1) or prostaglandin D2 (PGD2). The effects of prostacyclin on all parameters studied except blood pressure were potentiated by the concomitant administration of theophylline, a phosphodiesterase inhibitor.