Specific Thrombin Inhibitor Research Articles

Effect of New Direct Oral Anticoagulants on Ex Vivo Platelet and Fibrin Thrombus Formation in Blood Flowing Over Fibrillar Type I Collagen

Abstract 2318 Background.Studies have shown that 40–85% of patients undergoing total knee replacement develop venographically confirmed deep vein thrombosis (DVT) if not given post-operative thromboprophylaxis; approximately 0.1 to 1.7% of these patients will suffer fatal pulmonary embolism (PE). Oral anti-vitamin K anticoagulants are effective for the prevention and treatment of venous thrombosis, but have limitations. In particular, they have multiple food and drug interactions as well as variable pharmacokinetics and pharmacodynamics, such that regular laboratory monitoring and dose adjustments are required to maintain an optimal therapeutic range as defined with the International Normalized Ratio (INR). New oral agents that inhibit coagulation factor Xa or thrombin have been developed and shown to be effective and safe without requiring laboratory monitoring. In view of the relevance of the latter point, we have studied patients treated with an oral anti factor Xa agent (Rivaroxaban) or Coumadin, and evaluated the antithrombotic efficacy of the respective drugs by measuring platelet aggregation and fibrin deposition in patient blood perfused over fibrillar collagen type I. Material and Methods. Blood drawn from an antecubital vein and containing 0.011 M trisodium citrate as anticoagulant was recalcified with 5 mM calcium chloride and immediately perfused through a rectangular chamber mounted on the stage of a confocal microscope and presenting a surface coated with fibrillar collagen type I under laminar flow conditions at the wall shear rate of 300 1/s. Platelets and fibrin were specifically detected in situ through distinct fluorochromes. We tested 8 normal controls, 8 patients treated with Coumadin and a stable INR value between 1.94 and 2.90 (mean 2.34; standard deviation 0.34), and 7 patients treated with Rivaroxaban at between 8 and 16 days (mean 12.14; standard deviation 2.48 days) from the initiation of therapy. The volume of platelet aggregates and fibrin deposited onto the collagen fibrils was measured distinctly from stacks of confocal sections by integrating surface coverage of each thrombus component in consecutive optical planes separated by 2 micrometers in height. Results and Discussion. There was no significant difference in the volume of platelet and fibrin aggregates formed in blood of normal control and patients treated with either Coumadin or Rivaroxaban. This result was surprising because the patients treated with Coumadin had a laboratory demonstration of significantly retarded coagulation. We reasoned, however, that coagulation tests are typically performed in platelet-poor plasma, while in the perfusion assay coagulation occurs in whole blood and on a surface onto which flowing platelets are fully activated, thus increasing the local procoagulant potential. For this reason, we performed a series of experiments in which a variable amount of a highly specific thrombin inhibitor, lepirudin, was titrated into the recalcified blood before perfusion. We thus determined that with 50 nM lepirudin added to blood there was no decrease in the volume of platelet aggregates and fibrin deposited onto collagen in blood of normal individuals, while the volume of fibrin was decreased in patients receiving either Coumadin or Rivaroxaban. The corresponding values for normal controls, Coumadin-treated and Rivaroxaban-treated patients, in the order, were (mean volume ± standard error of the mean in cubic micrometers): Platelet aggregates = 28,592±3,354; 36,959±4,973; 44,448±7,110; Fibrin = 84,190±9,740; 47,298±7,308; 35,780±5,091. The differences in platelet aggregate volumes were not significant, while fibrin volume was significantly smaller in the anticoagulant-treated patients as compared to normal (p<0.01 for Coumadin and p<0.001 for Rivaroxaban); the difference between patients treated with one or the other anticoagulant was not significant. These results show that Rivaroxaban and Coumadin at therapeutically effective dosage have comparable effect in reducing thrombin generation, as evidenced by the reduced volume of fibrin formed in flowing blood exposed to collagen. This, however, is accompanied by an increased volume of platelet aggregates on the highly thrombogenic collagen surface. The relevance of these experimental results with respect to prevention of arterial as opposed to venous thrombosis deserves further investigation. Disclosures:No relevant conflicts of interest to declare.

Neue Antithrombotika

Vitamin K antagonists and heparins have been standard anticoagulation drugs over the past decades. They are effective and safe but they have several drawbacks which has led to the development of new oral anticoagulants. Dabigatran etexilate is a specific oral thrombin inhibitor and rivaroxaban and apixaban are oral inhibitors of factor Xa. These agents produce a predictable anticoagulant response after fixed-dose administration so that routine coagulation monitoring is unnecessary. Currently, dabigatran etexilate, rivaroxaban and apixaban are licensed for thromboprophylaxis after elective total hip or knee replacement surgery. Since august 2011, dabigatran etexilate is licensed for patients with atrial fibrillation, rivaroxaban will follow. However, indications will be expanded e.g. for therapy of venous thromboembolism. It is important to be aware of the pharmacokinetic and pharmacodynamic profiles of these new agents. The drugs considerably influence the global test of coagulation thus making an interpretation of test results difficult. Currently, there is a lack of suitable coagulation tests to monitor anticoagulation in emergency cases, such as bleeding. Specific antidotes are not yet available.

Anticoagulant Effects of Argatroban on the Pre-DIC State in Patients with an Aortic Aneurysm: A Comparative Study of Heparin

We compared the efficacy of argatroban, a new synthetic thrombin-specific inhibitor, with that of heparin in pre-DIC state patients with abdominal aortic aneurysm (AAA). A pre-DIC state was diagnosed by a detection of soluble fibrin monomer complex (FM) and increased levels of thrombin-antithrombin III complex (TAT) of more than 20 ng/ml. Twelve patients showing a pre-DIC condition were treated with argatroban (40 mg/day, n = 6) or heparin (10,000 U/day, n = 6) for 5 days. Coagulation and fibrinolytic profiles were analyzed before and after drug administration. FM became negative in two (33%) patients after the argatroban treatment and in all (100%) of the heparin-treated patients. Plasma levels of TAT were significantly decreased after the heparin treatment, however, there was no significant alteration in this parameter after the argatroban treatment. In conclusion, the anticoagulant effects of heparin were superior to those of argatroban in controlling the pre-DIC state associated with AAA.

Neue orale Antikoagulanzien beim Vorhofflimmern

Atrial fibrillation (AF) causes at least 20% of all ischemic strokes. In large randomized trials of primary and secondary stroke prevention, anticoagulation with vitamin K antagonists (VKA) protected much more efficiently than antiplatelet agents against stroke. Because of the problematic pharmacological properties of VKA only part of the AF patients are currently being treated with oral anticoagulants (OAK). The targeted development of specific oral inhibitors of the central coagulation factors thrombin and factor Xa allows reliable anticoagulation without regular coagulation monitoring. In the present review, pharmacological properties of the different agents are compared. Of the four large randomized phase 3 studies in AF (RELY, ROCKET-AF, ARISTOTLE, ENGAGE-AF) with the primary efficacy endpoint stroke and systemic embolism, the published data from the RELY trial indicate a superior efficacy of dabigatran etexilate (2 × 150 mg/day) and a lower risk of intracranial hemorrhage compared to warfarin. Favorable preliminary results have been demonstrated for the factor Xa inhibitor rivaroxaban. Apixaban was more efficacious than ASA and had a similar risk of hemorrhage in the AVERROES study. Thus, the available data suggest a favorable benefit-risk ratio for the new substances in addition to improved patient comfort. Currently unresolved issues relate to the verification of patient adherence by suitable coagulation tests and to the emergency coagulation diagnostics and therapy in acute ischemic or hemorrhagic strokes under the new OAC.

Determination of biological activity of extract from hirudo by N-benzoyl-L-arginine ethyl ester

As a potent anticoagulant, leech a traditional Chinese medicine, has become increasing topics. Hirudin, which is the primary effective component in leech, is a specific and efficient inhibitor of thrombin, mainly used in prevention and treatment of thrombus on the clinic practice. However, there is still no accurate and convenient method reported about the determination of it's biological activity. This paper reported a method for the determination of the biological activity the of extract from hirudo. The extra thrombin, which was not inhibited by hirudin in the extract from hirudo, reacted with N-benzoyl-L-arginine ethyl ester and was determined. The biological activity of the hirudo extract was determined, indirectly. The linear of calibration curve and accuracy were both perfect, the method was accurate and reliable.

Lung fibroblast growth-stimulating activity of serine protease

We compared lung fibroblast growth-stimulating activity (FGA) of several serine proteases including thrombin in vitro, and examined the mechanism of FGA. FGA was measured by incorporation of 3H-thymidine into lung fibroblasts (IMR-90). The activities of the enzymes were measured by spectrofluorometric method with synthetic peptides specific for each enzyme, and these enzymes were added to the assay system for FGA at concentrations of 7 x 10(0)-7 x 10(5) unit/ml. Human thrombin, bovine trypsin and bovine alpha-chymotrypsin showed clear FGA, but that of alpha-chymotrypsin was lower than those of thrombin and trypsin. On the other hand, porcine pancreatic elastase and human neutrophil elastase did not show any FGA, and had a cytotoxic effect on fibroblasts. A specific low molecular-weight thrombin inhibitor, argatroban (MW. 562), inhibited not only the enzyme (peptidolytic) activity of thrombin, but also its FGA at the same concentration. These results suggest that serine proteases can be classified into at least two groups, showing FGA and cytotoxic activity, respectively, and that the FGA of the former group is mediated by their catalytic (enzymatic) action.

Thrombin Inhibitors from Different Animals

Venous and arterial thromboembolic diseases are still the most frequent causes of death and disability in high-income countries. Clinical anticoagulants are inhibitors of enzymes involved in the coagulation pathway, such as thrombin and factor Xa. Thrombin is a key enzyme of blood coagulation system, activating the platelets, converting the fibrinogen to the fibrin net, and amplifying its self-generation by the activation of factors V, VIII, and XI. Thrombin has long been a target for the development of oral anticoagulants. Furthermore, selective inhibitors of thrombin represent a new class of antithrombotic agents. For these reasons, a number of specific thrombin inhibitors are under evaluation for possible use as antithrombotic drugs. This paper summarizes old and new interests of specific thrombin inhibitors described in different animals.

Platelets and fibrinogen facilitate each other in protecting tumor cells from natural killer cytotoxicity

The functions of platelets and fibrinogen in protecting tumor cells from natural killer cytotoxicity have been discussed for more than 20 years. However, their exact roles and relationships in the process are still not clear. In this study, we show that tumor cells prefer to adhere to fibrinogen than to platelets, and fibrinogen can enhance the adhesion of tumor cells to platelets. Beta3 integrin plays an important role in the adhesion of B16F10 to platelets enhanced by fibrinogen. In the presence of thrombin, fibrinogen forms dense fibrin(ogen) layers around tumor cells. Tumor cells can induce platelets to aggregate and form thrombin. Platelets, as well as thrombin, can help fibrinogen protect tumor cells from lethal contact with natural killer cells and natural killer cytotoxicity. Hirudin, a specific inhibitor of thrombin, can reverse the effect of platelets on fibrinogen in blocking natural killer cytotoxicity. Our results suggest that fibrinogen helps platelets to adhere to tumor cells, and platelets in turn promote more fibrinogen to aggregate around tumor cells by forming thrombin. They facilitate each other in protecting tumor cells from natural killer cytotoxicity.

Thrombin generation by intimal tissue factor contributes to thrombus formation on macrophage-rich neointima but not normal intima of hyperlipidemic rabbits

Arterial thrombosis occurs in atherosclerotic, but rarely in non-atherosclerotic arteries. The present study investigates how hyperlipidemic condition affects thrombus formation on macrophage-rich neointima or normal intima in rabbits. Rabbits were fed with a 0.5% cholesterol diet, and then the femoral artery on one side of each rabbit was injured with a balloon catheter. Three weeks later, bilateral femoral arteries were similarly injured with a balloon catheter to produce thrombi on neointima and normal intima. We compared the expression and activity of intimal tissue factor (TF) as well as thrombus size and composition between these femoral arteries. 0.5% cholesterol diet combined with a balloon injury induced macrophage-rich neointima in injured arteries. The whole blood coagulation activity or plasma thrombin generation activity did not differ after consuming the 0.5% cholesterol diet for 4 weeks, and an anti-TF antibody did not affect the measured parameters. TF activities were increased in the neointima/media compared with normal intima/media. Balloon injury induced large platelet–fibrin thrombi on macrophage-rich neointima, whereas small platelet thrombi were produced in normal arteries even under hyperlipidemic conditions. Recombinant human tissue factor pathway inhibitor (25 μg/(kg min)) and argatroban (100 μg/(kg min)), a specific thrombin inhibitor, significantly reduced thrombus formation on induced neointima, but not on normal intima. Thrombin generation mediated by TF in intima contributes to thrombus formation on macrophage-rich neointima, but not on normal intima. The TF content in disrupted atherosclerotic plaques might play a more important role than hyperlipidemia in the development of atherothrombosis.

Coagulation Factor Xa inhibits cancer cell migration via Protease-activated receptor-1 activation

Cell migration is critically important in (patho)physiological processes. The metastatic potential of cancer cells partly depends on activation of the coagulation cascade. The aim of the present study was to determine whether coagulation factor X (FXa) can regulate the migration and invasion of cancer cells. Quite unexpectedly, we found that FXa markedly diminished the migration of different cancer cell lines of various origins (breast, lung and colon cancer cells). We showed that FXa mediated inhibition of cancer cell migration was specific, as it was inhibited by TAP (a specific FXa inhibitor) but not by Hirudin (a specific thrombin inhibitor). Moreover, the FXa effect was dose dependent, with a maximal inhibitory effect reached at 0.75 U/ml FXa (corresponding to 130.5 nM). Next, we determined that FXa acted via protease-activated receptor (PAR)-1-dependent signaling, and PAR-1 desensitization, as well as knocking-down PAR-1 expression, abolished the FXa effects. Finally, we showed that Giα was not involved in FXa mediated inhibition of cell migration as its effects were not reverted by pertussis toxin. These results suggest that, beyond its role in blood coagulation, FXa plays a key role in cancer cell migration. They also shed light on an unexpected role of PAR-1, which appears to be a Janus-like receptor in cancer cell biology.

Modulation of human uterine smooth muscle cell collagen contractility by thrombin, Y-27632, TNF alpha and indomethacin

BackgroundPreterm labour occurs in approximately 10% of pregnancies and is a major cause of infant morbidity and mortality. However, the pathways involved in regulating contractility in normal and preterm labour are not fully elucidated. Our aim was to utilise a human myometrial contractility model to investigate the effect of a number of uterine specific contractility agents in this system. Therefore, we investigated the contractile response of human primary uterine smooth muscle cells or immortalised myometrial smooth muscle cells cultured within collagen lattices, to known mediators of uterine contractility, which included thrombin, the ROCK-1 inhibitor Y-27632, tumour necrosis factor alpha (TNF alpha) and the non-steroidal anti-inflammatory indomethacin.MethodsCell contractility was calculated over time, with the collagen gel contraction assay, utilising human primary uterine smooth muscle cells (hUtSMCs) and immortalised myometrial smooth muscle cells (hTERT-HM): a decrease in collagen gel area equated to an increase in contractility. RNA was isolated from collagen embedded cells and gene expression changes were analysed by real time fluorescence reverse transcription polymerase chain reaction. Scanning electron and fluorescence microscopy were employed to observe cell morphology and cell collagen gel interactions. Statistical analysis was performed using ANOVA followed by Tukey's post hoc tests.ResultsTNF alpha increased collagen contractility in comparison to the un-stimulated collagen embedded hUtSMC cells, which was inhibited by indomethacin, while indomethacin alone significantly inhibited contraction. Thrombin augmented the contractility of uterine smooth muscle cell and hTERT-HM collagen gels, this effect was inhibited by the thrombin specific inhibitor, hirudin. Y-27632 decreased both basal and thrombin-induced collagen contractility in the hTERT-HM embedded gels. mRNA expression of the thrombin receptor, F2R was up-regulated in hUtSMCs isolated from collagen gel lattices, following thrombin-stimulated contractility.ConclusionTNF alpha and thrombin increased uterine smooth muscle cell collagen contractility while indomethacin had the opposite effect. Thrombin-induced collagen contractility resulted in F2R activation which may in part be mediated by the ROCK-1 pathway. This study established the in vitro human myometrial model as a viable method to assess the effects of a range of uterotonic or uterorelaxant agents on contractility, and also permits investigation of the complex regulatory pathways involved in mediating myometrial contractility at labour.

A novel hirudin derivative characterized with anti-platelet aggregations and thrombin inhibition

Background Hirudin is an anti-coagulative product of the salivary glands of the medicinal leech Hirudo medicinalis. It is a powerful and specific thrombin inhibitor. Peptides containing the RGD motif competitively inhibit the binding of fibrinogen to GP IIb/IIIa on the platelets, thus inhibiting platelet aggregation. Results We have constructed a recombinant RGD-hirudin (r-RGD-hirudin) by fusing the tripeptide RGD sequence to the native hirudin (wt-hirudin). The r-RGD-hirudin was expressed at high levels in Pichia pastoris, and was purified to ~97% homogeneity. The specific anti-thrombin activity of purified r-RGD-hirudin is 12,000 ATU/mg, which is equivalent to wt-hirudin, but only r-RGD-hirudin can inhibit platelet aggregation. The biological effects of r-RGD-hirudin on Thrombin Time (TT), Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT), Bleeding Time (BT), maximum platelet aggregation (PAGm) induced by ADP were studied in rabbit model and compared with that of wt-hirudin. The rabbits were infused r-RGD-hirudin had prolonged TT, PT, and aPTT which were similar to that of wt-hirudin; but only r-RGD-hirudin was capable of inhibiting PAGm. Histopathological analyses showed that r-RGD-hirudin was two to three times more effective than wt-hirudin in preventing thrombosis. Conclusions r-RGD-hirudin can potentially be used as a novel anti-coagulant for the prevention of thrombosis after carotid artery anastomosis or in other thrombotic events.

Prevention of stroke in patients with atrial fibrillation: current strategies and future directions

The morbidity and mortality associated with atrial fibrillation (AF) are related mainly to ischaemic stroke, and the prevention of thrombo-embolism is an important component of the patient management. The choice of optimum antithrombotic therapy for a given patient depends on the risk of thrombo-embolism, and the assessment of thrombo-embolic risk using validated stratification schemes, such as the CHADS2 score, is a critical step. Improved stratification schemes are needed that take into account the risk of intracerebral haemorrhage, which is the most worrisome complication of anticoagulant therapy. The pattern of AF (paroxysmal, persistent, or permanent) should not influence the selection of antithrombotic treatment. Similarly, successful rhythm control is not a sound basis for withdrawing antithrombotic treatment, and whether this situation differs after successful catheter ablation of AF has not been established. At present, oral vitamin K antagonists alone are recommended for patients with AF at moderate-to-high risk of stroke. A combination of anticoagulant and antiplatelet drugs is necessary in patients with AF undergoing percutaneous coronary intervention and stent implantation, but the optimal therapeutic management of these patients has not been defined. The development of new antithrombotic agents that are easier to use and have a superior benefit-to-risk ratio will extend treatment to a greater proportion of the AF population at risk. The large number of phase III trials currently investigating specific inhibitors of thrombin or factor Xa that do not require laboratory monitoring suggests that this goal is within reach.

Thrombin Up-regulates Cathepsin D which Enhances Angiogenesis, Growth, and Metastasis

Cathepsin D (CD) up-regulation has been associated with human malignancy and poor prognosis. Thrombin up-regulated CD mRNA and protein in eight tumor cell lines as well as in human umbilical vascular endothelial cells (HUVEC). Thrombin increased the secretion of CD by 3- to 8-fold and enhanced chemotaxis ( approximately 2-fold) in 4T1 murine mammary CA cells, which was completely inhibited with the knockdown of CD. Secreted 4T1 CD induced neoangiogenesis by 2.4-fold on a chick chorioallantoic membrane, which was blocked in CD-KD cells. The addition of pure CD (2 ng) to the chick chorioallantoic membrane increased angiogenesis by 2.1-fold, which was completely inhibited by Pepstatin A (Pep A). CD enhanced human HUVEC chemotaxis and Matrigel tube formation by 2-fold, which was then blocked by Pep A. CD enhanced HUVEC matrix metalloproteinase 9 (MMP-9) activity by approximately 2-fold, which was completely inhibited by Pep A as well as a generic MMP inhibitor, GM6001. The injection of CD-KD 4T1 cells into syngeneic mice inhibited tumor growth by 3- to 4-fold compared with empty vector (EV) cells. Hirudin, a specific thrombin inhibitor, inhibited the growth of wild-type and EV cells by 2- to 3-fold, compatible with thrombin up-regulation of CD. CD and thrombin also contributed to spontaneous pulmonary metastasis; 4-fold nodule inhibition with CD versus EV and 4.6-fold inhibition with hirudin versus EV (P < 0.02). Thus, thrombin-induced CD contributes to the malignant phenotype by inducing tumor cell migration, nodule growth, metastasis, and angiogenesis. CD-induced angiogenesis requires the proteolytic activation of MMP-9.

Hirudin versus heparin for use in whole blood in vitro biocompatibility models

Heparin has traditionally been a widely used anticoagulant in blood research, but has been shown to be inappropriate for work with the complement system because of its complement-interacting properties. In this work, we have compared the effects of heparin with those of the specific thrombin inhibitor hirudin on complement and blood cells in vitro. Whole blood collected in the presence of hirudin (50 microg/mL) or heparin (1 IU/mL) was incubated in the slide chamber model. The plasma was analyzed for complement activation markers C3a and sC5b-9, and the polyvinylchloride test slides were stained for adhering cells. The integrity of the complement system was tested by incubating serum and hirudin-treated plasma in the presence of various activating agents. In contrast to heparin, the addition of hirudin generally preserved the complement reactivity, and complement activation in hirudin plasma closely resembled that in normal serum. Importantly, immunochemical staining of surface-bound cells demonstrated the inducible expression of tissue factor on bound monocytes from hirudin-treated blood, an effect that was completely abolished in heparin-treated blood. Our results indicate that hirudin as an anticoagulant produces more physiological conditions than heparin, making hirudin well-suited for in vitro studies, especially those addressing the regulation of cellular processes.

Combination of a SAW-biosensor with MALDI mass spectrometric analysis

A S-sens® K5 surface acoustic wave biosensor was coupled with mass spectrometry (SAW-MS) for the analysis of a protein complex consisting of human blood clotting cascade factor α-thrombin and human antithrombin III, a specific blood plasma inhibitor of thrombin. Specific binding of antithrombin III to thrombin was recorded as a function of time with a S-sens® K5 biosensor. Two out of five elements of the sensor chip were used as references. To the remaining three elements coated with RNA anti-thrombin aptamers, thrombin and antithrombin III were bound consecutively. The biosensor measures mass changes on the chip surface showing that 20% of about 400fmol/cm2 thrombin formed a complex with the 1.7-times larger antithrombin III. Mass spectrometry (MS) was applied to identify the bound proteins. Sensor chips with aptamer-captured (1) thrombin and (2) thrombin–antithrombin III complex (TAT-complex) were digested with proteases on the sensor element and subsequently identified by peptide mass fingerprint (PMF) with matrix assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry. A significant identification of thrombin was achieved by measuring the entire digest with MALDI-ToF MS directly from the sensor chip surface. For the significant identification of both proteins in the TAT-complex, the proteolytic peptides had to be separated by nano-capillary-HPLC prior to MALDI-ToF MS. SAW-MS is applicable to protein interaction analysis as in functional proteomics and to miniaturized diagnostics.

A Recombinant Peptide, Hirudin, Potentiates the Inhibitory Effects of Stealthy Liposomal Vinblastine on the Growth and Metastasis of Melanoma

The metastasis of tumor cells is one of the major obstacles to successful clinical therapy. A treatment strategy by incorporating a specific inhibitor of thrombin, recombinant hirudin with stealthy liposomal vinblastine, was used in this study for inhibiting the metastasis of tumor cells and enhancing the efficacy of anti-tumor agents. In vitro cytotoxicity, cell adhesion to extracellular matrix (ECM) proteins, and cell invasion and migration assays were performed on human A375 melanoma cell line. In vivo measurement of coagulation parameters, inhibition of tumor growth, and inhibition of metastasis were assessed in female BALB/c mice. In vitro, vinblastine or stealthy liposomal vinblastine alone was effective to inhibit the growth of A375 cells. On the contrary, hirudin had no influence on either cytotoxicity when treating with hirudin alone or hirudin plus vinblastine. In addition, in vitro results showed that hirudin had no impact on the adhesion of tumor cells to extracellular matrix proteins, and metastasis and invasion of tumor cells. In mice, hirudin significantly inhibited the activity of thrombin. Furthermore, administered at the initial implantation of murine B16 melanoma cells, hirudin evidently delayed the growth of tumor, and depressed the occurrence of experimental lung metastasis. A subsequent administration of stealthy liposomal vinblastine resulted in further inhibiting growth and metastasis of tumor, indicating that hirudin plus stealthy liposomal vinblastine exhibited a significant anti-metastasis effect and slightly potent effect against tumor growth as compared with stealthy liposomal vinblastine alone. In conclusion, administration of recombinant hirudin followed by giving stealthy liposomal vinblastine may be beneficial for inhibiting the growth and metastasis of melanoma in vivo. The likely mechanism could be associated with inhibition of thrombin after administration of hirudin.

The thrombin inhibitor Argatroban reduces breast cancer malignancy and metastasis via osteopontin-dependent and osteopontin-independent mechanisms

Osteopontin (OPN) has been clinically and experimentally associated with breast cancer metastasis. Proteolytic cleavage of OPN by thrombin has been reported to increase its biologic activity. The purpose of this study was to determine if inhibition of thrombin could reduce the malignancy-promoting effects of OPN on breast cancer cell behavior in vitro and in vivo. MDA-MB-468 human breast cancer cells were stably transfected to overexpress OPN (468-OPN) or a control vector (468-CON) and compared for functional differences in malignant/metastatic behavior in response to treatment with the thrombin-specific inhibitor Argatroban. Western blot analysis revealed that both 468-CON and 468-OPN cells produce thrombin and the thrombin-related protein tissue factor, and express very low levels of thrombin receptor (PAR-1). In vitro assays demonstrated that Argatroban treatment (25 microg/ml) of 468-OPN cells resulted in decreased cell growth, colony-forming ability, adhesion, and migration relative to untreated controls (P < 0.05), but did not have a significant effect on 468-CON cells. Following mammary fat pad injection, treatment with Argatroban (9 mg/kg/day) increased the in vivo tumor latency of both 468-CON and 468-OPN cells, and reduced primary tumor growth of 468-OPN cells (relative to untreated controls; P < 0.05). Furthermore, Argatroban treatment significantly decreased lymphatic metastasis of both 468-CON (P < 0.04) and 468-OPN (P < 0.01) cells relative to untreated controls. These novel findings indicate that inhibition of thrombin can reduce malignant and metastatic behavior of MDA-MB-468 breast cancer cells using both OPN-dependent and OPN-independent mechanisms, and suggest that thrombin inhibitors such as Argatroban may hold potential as therapeutic agents to combat breast cancer progression.

Thrombin Contributes to the Tumor Promalignant Phenotype by Upregulation of Cathepsin D Which Stimulates Angiogenesis.

Although the association of thrombosis and occult cancer has been recognized for >40 years, the role of thrombin in enhancing the malignant phenotype has only been recently appreciated. To better understand the mechanisms involved we performed an Affymetrix gene display on 2 tumor cell lines, B16F10 and UMCL to look for genes which could be activated by thrombin and contribute to the promalignant phenotype. Cathepsin D (CD) was upregulated 2–4 fold, confirmed by mRNA and protein analysis. We focused on CD because its upregulation has been associated with human malignancy and poor prognosis, particularly with breast CA as well ovarian, prostate, bladder and melanoma. Thrombin (0.5 u/ml for 24 hrs) upregulated mRNA 1.5 – 2.3 fold and protein 1.5 – 4.2 fold in 8 different cell lines: human breast MCF7 and MDAMB-231, cervical HeLa, prostate PC3, primary HUVEC and brain microvascular endothelial cells as well as murine melanoma B16F10 and mammary 4T1. Thrombin also increased secretion of CD 3.1 – 8.2 fold in MCF7, HeLa and PC3 cell lines. We next examined the effect of CD (shRNA) knock down (KD) and thrombin in 4T1 cells. Thrombin (0.5 u/ml, 24 hrs) enhanced chemotaxis ∼2 fold in 4T1 cells. This was completely inhibited when CD was knocked down with siRNA but not with empty vector (EV) or scrambled vector (SV). We next tested the effect of secreted tumor cell CD (4T1 conditioned media) on angiogenesis (neoangiogenesis branching) following addition to a chick chorioallantoic membrane (CAM). The angiogenesis index from 24 hr conditioned media of wild type (WT) and KD cells was 46 ± 4 and 19 ± 3 respectively, n=4, p=0.03. A 2.1 fold inhibition was also obtained with anti-CD Ab vs irrelevant IgG, n=3, p=0.001. This prompted testing for the effect of pure enzymatically-active CD (2 ng) on the CAM. This resulted in a 2.1 fold increase in angiogenesis which was completely inhibited by the proteolytic inhibitor, Pepstatin A (Pep A), n=10, p<0.005. Pep A alone had no effect. To further define the mechanism of enhanced angiogenesis, we measured the effect of CD on HUVEC chemotaxis in the presence and absence of Pep A. CD enhanced chemotaxis of HUVEC 2.2 fold, which was also completely inhibited by Pep A, n=4, p<0.01. CD KD in a HUVEC-matrigel tube assay revealed similar results, n=3. Since proteolysis was required for angiogenesis we hypothesized that CD could be activating metalloproteinases (MMP) required for endothelial tube formation. We therefore performed zymography. This revealed an ∼2 fold upregulation of MMP-2 and MMP-9 compared to PBS which was completely inhibited by Pep A as well as a generic MMP inhibitor, GM6001, n=3. CD activation of angiogenesis and 4T1 tumor cell chemotaxis suggested that in vivo growth and metastasis could be affected by CD. Accordingly, 1 × 105 4T1 cells were injected into the flank of syngeneic mice. CD KD revealed a 3–4 fold inhibition of tumor growth on days 19 and 25 respectively, n=5. Hirudin, a specific thrombin inhibitor significantly inhibited WT and EV cells 2–3 fold on days 19 and 25 respectively (n=5), which is compatible with thrombin upregulation of CD. Both CD and thrombin also contributed to spontaneous metastasis with CD KD vs EV vs (EV + hirudin) of 6.4 ± 0.9, 1.6 ± 0.5 and 1.4 ± 0.4 nodules respectively on day 27, n=5, p<0.02. We conclude that thrombin-induced CD contributes to the malignant tumor phenotype by inducing tumor cell migration, growth, metastasis and angiogenesis. CD-induced angiogenesis requires its proteolytic activation of MMP-2 and MMP-9.

Atrial fibrillation: insights from clinical trials and novel treatment options

Atrial fibrillation (AF) is the most common encountered sustained arrhythmia in clinical practice. The last decade the result of large 'rate' versus 'rhythm' control trials have been published that have changed the current day practise of AF treatment. It has become clear that rate control is at least equally effective as a rhythm control strategy in ameliorating morbidity as well as mortality. Moreover, in each individual patient the risk of thromboembolic events should be assessed and antithrombotic treatment be initiated. There have also been great advances in understanding the mechanisms of AF. Experimental studies showed that as a result of electrical and structural remodelling of the atria, 'AF begets AF'. Pharmacological prevention of atrial electrical remodelling has been troublesome, but it seems that blockers of the renin angiotensin system, and perhaps statins, may reduce atrial structural remodelling by preventing atrial fibrosis. Clinical studies demonstrated that the pulmonary veins exhibit foci that can act as initiator and perpetuator of the arrhythmia. Isolation of the pulmonary veins using radiofrequency catheter ablation usually abolishes AF. The most promising advances in the pharmacological treatment of AF include atrial specific antiarrhythmic drugs and direct thrombin inhibitors. In the present review we will describe the results of recent experimental studies, discuss the latest clinical trials, and we will focus on novel treatment modalities.