Mechanism of action of thrombin on fibrinogen. On the role of the A chain of bovine thrombin in specificity and in differentiating between thrombin and trypsin.
Explore the source record for details and available documents.
Amino Acids↗
SEARCH · Search PubMed
Results for “THROMBIN”
Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
Inhibition by Argatroban, a specific thrombin inhibitor, of platelet activation by fibrin clot-associated thrombin.
Clot-associated thrombin retains amidolytic activity, and is resistant to inhibition by heparin, but not to low molecular weight thrombin inhibitors. We show that clot-associated thrombin induces platelet aggregation, is resistant to heparin:antithrombin III, less so to recombinant hirudin (rHV2Lys47) but not to argatroban, an active-site directed thrombin inhibitor. Fibrin clots prepared with human fibrinogen and thrombin were used to aggregate rabbit washed platelets assessed by single platelet counting, thromboxane B2 (TXB2) immunoassay and scanning electron microscopy. Fibrin clots decreased platelet counts, and released TXB2. Electron microscopy showed platelet aggregates on the clot surface. Argatroban concentration-dependently inhibited such aggregation with IC50s of 21 nM and 13 nM versus aggregation and TXB2 release respectively. The IC50s of Argatroban against fluid-phase thrombin producing similar aggregation were 12 nM (aggregation) and 33 nM (TXB2). rHV2Lys47 was less active against clot-induced aggregation (IC50 = 1.8 nM) than against fluid-phase thrombin (IC50 = 0.06 nM). Heparin had an IC50 of 0.02 mU/ml against aggregation induced by fluid-phase thrombin, but much greater concentrations are required to inhibit clot-induced aggregation (IC50 = 48 mU/ml). These data provide a basis for the superiority of direct-acting thrombin inhibitors over heparin in platelet rich thrombi.
Animals↗
Thrombin induces IL-6 but not TNFalpha secretion by mouse mast cells: threshold-level thrombin receptor and very low level FcepsilonRI signaling synergistically enhance IL-6 secretion.
Mast cells become activated in multiple diseases wherein thrombin generation is often clinically apparent, but the effect of thrombin on cytokine release by mast cells remains unexplored. Thus, we examined IL-6 and TNFalpha release by thrombin-challenged mast cells. Thrombin and the protease-activated receptor (PAR)-1 peptide TRAP(14) induced these cells to secrete IL-6 in a dose-dependent fashion. Mast cells secreted > or =2800 pg IL-6/10(6) cells over 24 h, but only low levels of serotonin and no significant TNFalpha. Furthermore, at near-background levels of allergen, threshold doses of alpha-thrombin synergistically enhanced the IL-6 response (by up to 100-fold), but high-dose costimulation led to a simple additive response. Both the PI(3)- and sphingosine-kinase signaling pathways contributed importantly to the thrombin response. Our data thus clearly demonstrate that low-level thrombin and FcepsilonRI signaling can synergize to augment mast cell IL-6 responses, and that thrombin also differentially induces cytokine secretion by mast cells.
Animals↗
A thrombin assay based upon the release of fibrinopeptide A from fibrinogen: definition of a new thrombin unit.
An assay for thrombin is presented wherein thrombin-catalyzed hydrolysis at Arg-A alpha-16 to release fibrinopeptide A (FPA) from fibrinogen is measured using high-performance liquid chromatography (HPLC). In this assay one thrombin unit (TU) is defined as that amount of thrombin that will release half of the FPA in one min from one ml of a solution of greater than 90% clottable normal human fibrinogen (less than or equal to 0.35 microM) at 37 degrees C, pH 7.4, /2 0.15. One TU is equivalent to approximately 0.1 NIH unit of thrombin and approximately 1 pmol of pure human thrombin. At 37 degrees C, pH 7.4, and plasma levels of fibrinogen of 3 mg/ml, one TU will catalyze the release of 3.6 nmol FPA min-1. Variability in fibrinogen samples which produce dramatic differences in clotting time assays with the same sample of thrombin, produce little or no variation in the catalytic assay for TU. The assay for TU obviates the need for maintenance of a thrombin reference standard.
Blood Coagulation Tests↗
Association of thrombin, plasmin, thrombin-antithrombin III complex and plasmin-antithrombin III complex with isolated hepatocytes.
The interaction of thrombin, plasmin or their antithrombin III complexes with isolated mouse hepatocytes was studied. Plasmin bound to hepatocytes in a concentration-dependent manner with an apparent Kd of 6.4.10(-8) M, attaining equilibrium within 10 min, and the interaction was inhibited by 6-amino-n-hexanoic acid. Plasmin treated with diisopropylfluorophosphate (DFP) bound to the cells in similar way as the untreated form of the enzyme. Thrombin bound also to hepatocytes, in a concentration-dependent manner, with a Kd of 5.4.10(-8) M reaching a steady state after 180 min. Thrombin inactivated with DFP, however, was inhibited in its binding to these cells. These data suggest that, whereas the kringle domains of plasmin are responsible for the enzyme-cell interaction, the active center of thrombin may be involved in the binding of this enzyme to hepatocytes. Plasmin-antithrombin III and thrombin-antithrombin III complexes were also associated with hepatocytes in a time-dependent manner, reaching a plateau after 180 min, and the two complexes competed in the interaction. While the interaction of active proteinases plasmin or thrombin with hepatocytes did not result in their internalization, the antithrombin III complexes were taken up by the cells, and thrombin-antithrombin III complex was degraded. These results indicate that hepatocytes may participate in the elimination of proteinase-antithrombin III complexes from the plasma, while the association of plasmin and thrombin with hepatocytes could imply distinct biological importance.
Animals↗
Standard measurement of clot-bound thrombin by using a chromogenic substrate for thrombin.
We have developed an in vitro protocol for the measurement of clot-bound thrombin. This protocol uses a chromogenic substrate for thrombin and a microtiter plate reader and is suitable for screening inhibitors for thrombin that are directed to clot-bound thrombin. Clots were obtained after recalcification of human plasma. For the measurement of clot-bound thrombin, we read the optical density (OD) at 405 nm on a spectrophotometer and compared the results to that obtained with a standard curve of human alpha thrombin. We stopped the amidolytic reaction at 10 min because the optical density was linear until 20 min under our experimental conditions. We suggest that clot-bound thrombin can be measured using a chromogenic substrate specific for thrombin under our experimental condition.
Chromogenic Compounds↗
Platelet activation and its role in thrombin generation in platelet-induced thrombin generation time.
Platelet-induced thrombin generation time (PITT) is a newly developed global coagulation assay in which a small amount of partially anticoagulated platelet-rich plasma (PRP) is rotated in a disc-shaped cuvette within the light beam of a photometer. The time intervals from onset of rotation until aggregation and coagulation of the sample are registered. The aim of our study was to compare platelet activation with generation of thrombin during rotation of PRP in PITT system. Aliquots of PRP were taken before, 1, 3, and 8 min after the onset of rotation as well as at the beginning of aggregation and shortly before coagulation. Thrombin activity was measured with chromogenic substrate S-2238. We have also measured the level of generated prothrombin activation fragment 1+2 (F1+2), which reflects the concentration of liberated thrombin. Platelet activation was assayed by means of platelet factor 4 (PF4) and beta-thromboglobulin (beta-TG) concentration and registration of the aggregation. The concentrations of the F1+2, PF4, beta-TG increased very slowly from the beginning of the test until aggregation occurred. From the start of aggregation, the levels of F1+2 rose rapidly. In contrast to the F1+2 measurements, thrombin activity has not been detected from onset of rotation until the end of the test. Only trace thrombin activity was detectable just after the plasma sample had been clotted in the cuvette. Our results demonstrate that there exists a close relationship between platelet activation and thrombin generation. Viable platelets, which adhered to the cuvette walls, form an active template on which thrombin can be generated from prothrombin.
Adult↗
PAR 1-type thrombin receptors are involved in thrombin-induced calcium signaling in human meningioma cells.
Thrombin is known to play a role as regulator in tumor spreading and tumor growth. Proteinase-activated receptor 1 (PAR 1)-type thrombin receptors were identified in different cancer cells including human glioblastoma cells. Thus a function of PAR 1 in brain tumors may be suggested. In this study, the presence of PAR 1-type thrombin receptors was investigated in primary cell cultures established from operated human meningiomas from two 59- and 79-year-old women. Characterization of PAR 1 on binding level was performed using immunofluorescence studies with the monoclonal anti-PAR 1 antibody Mab 61-1 directed against a domain in the NH2-terminus of PAR 1. These binding sites constitute functional thrombin receptors that are involved in thrombin-induced signaling in human meningioma cells as demonstrated by investigation of alpha-thrombin- and PAR 1-activating hexapeptide (TRAP-6)-induced [Ca2+]i mobilization. To our knowledge, this is the first report demonstrating thrombin-induced intracellular signaling in human meningioma cells mediated by the PAR 1-type thrombin receptor.
Aged↗
Effects of baicalein isolated from Scutellaria baicalensis Radix on adhesion molecule expression induced by thrombin and thrombin receptor agonist peptide in cultured human umbilical vein endothelial cells.
We examined the effects of various flavonoids isolated from the roots of Scutellaria baicalensis Georgi on adhesion molecule expression induced by thrombin and thrombin receptor agonist peptide (SFLLRNPNDKYEPF, TRAP) in cultured human umbilical vein endothelial cells. Thrombin and thrombin receptor agonist peptide induced endothelial leukocyte adhesion molecule-1 (ELAM-1) expression. Intercellular adhesion molecule-1 (ICAM-1) expression was also induced by thrombin, but not by TRAP. Baicalein isolated from Scutellariae Radix inhibited ELAM-1 expression induced by thrombin and thrombin receptor agonist peptide dose-dependently, with 50% inhibitory concentrations (IC50) of 5.53 +/- 1.68 microM and 2.44 +/- 1.08 microM, respectively. Furthermore, baicalein inhibited thrombin-induced ICAM-1 expression with an IC50 of 9.67 +/- 1.28 microM. In addition, baicalein inhibited the expressions of ELAM-1 and ICAM-1 stimulated by protein kinase C (PKC) activator phorbol myristate acetate (PMA).
Amino Acid Sequence↗
Thrombin generation and presence of thrombin receptor in ovarian follicles.
Prothrombin, once converted to its enzymatically active form (i.e., thrombin), induces a broad spectrum of cellular responses in both vascular and avascular tissues. Bovine ovarian granulosa cells isolated from healthy follicles of various sizes contain both prothrombin mRNA and immunologically reactive prothrombin that appears to be identical to prothrombin in follicular fluid and plasma. When tissue factor, the primary physiological activator of thrombin generation in plasma, is used to initiate thrombin formation, the profile of prothrombin-to-thrombin conversion is similar in follicular fluid and plasma. The conclusion that biologically functional prothrombin is synthesized by granulosa cells is further supported by evidence that mRNA for gamma-glutamyl carboxylase, an enzyme essential for the vitamin K-dependent posttranslational modification of prothrombin, is expressed in granulosa cells in a manner similar to prothrombin mRNA. Thrombin's biological effects are mediated through selective proteolytic cleavage and activation of specific receptors. Bovine granulosa cells possess thrombin receptor (PAR-1) mRNA, and as seen with prothrombin mRNA and gamma-glutamyl carboxylase mRNA, cells isolated from small follicles possess more PAR-1 mRNA than cells from large follicles. Thrombin receptor expression by cells in close proximity to an active thrombin-generating system suggests that these factors may be important mediators of cellular function in the ovarian follicle.
Animals↗
Interaction of thrombin des-ETW with antithrombin III, the Kunitz inhibitors, thrombomodulin and protein C. Structural link between the autolysis loop and the Tyr-Pro-Pro-Trp insertion of thrombin.
X-ray diffraction studies of human thrombin revealed that compared with trypsin, two insertions (B and C) potentially limit access to the active site groove. When amino acids Glu146, Thr147, and Trp148, adjacent to the C-insertion (autolysis loop), are deleted the resulting thrombin (des-ETW) has dramatically altered interaction with serine protease inhibitors. Whereas des-ETW resists antithrombin III inactivation with a rate constant (Kon) approximately 350-fold slower than for thrombin, des-ETW is remarkably sensitive to the Kunitz inhibitors, with inhibition constants (Ki) decreased from 2.6 microM to 34 nM for the soybean trypsin inhibitor and from 52 microM to 1.8 microM for the bovine pancreatic trypsin inhibitor. The affinity for hirudin (Ki = 5.6 pM) is weakened at least 30-fold compared with recombinant thrombin. The mutation affects the charge stabilizing system and the primary binding pocket of thrombin as depicted by a decrease in Kon for diisopropylfluorophosphate (9.5-fold) and for N alpha-p-tosyl-L-lysine-chloromethyl ketone (51-fold) and a 39-fold increase in the Ki for benzamidine. With peptidyl p-nitroanilide substrates, the des-ETW deletion results in changes in the Michaelis (Km) and/or catalytic (kcat) constants, worsened as much as 85-fold (Km) or 100-fold (kcat). The specific clotting activity of des-ETW is less than 5% that of thrombin and the kcat/Km for protein C activation in the absence of cofactor less than 2%. Thrombomodulin binds to des-ETW with a dissociation constant of approximately 2.5 nM and partially restores its ability to activate protein C since, in the presence of the cofactor, kcat/Km rises to 6.5% that of thrombin. This study suggests that the ETW motif of thrombin prevents (directly or indirectly) its interaction with the two Kunitz inhibitors and is not essential for the thrombomodulin-mediated enhancement of protein C activation.
Amino Acid Sequence↗
Specific inhibition of thrombin-induced cell activation by the neutrophil proteinases elastase, cathepsin G, and proteinase 3: evidence for distinct cleavage sites within the aminoterminal domain of the thrombin receptor.
The aim of this study was to investigate the inhibitory effects of human leukocyte elastase (HLE), cathepsin G (Cat G), and proteinase 3 (PR3) on the activation of endothelial cells (ECs) and platelets by thrombin and to elucidate the underlying mechanisms. Although preincubation of ECs with HLE or Cat G prevented cytosolic calcium mobilization and prostacyclin synthesis induced by thrombin, these cell responses were not affected when triggered by TRAP42-55, a synthetic peptide corresponding to the sequence of the tethered ligand (Ser42-Phe55) unmasked by thrombin on cleavage of its receptor. Using IIaR-A, a monoclonal antibody directed against the sequence encompassing this cleavage site, flow cytometry analysis showed that the surface expression of this epitope was abolished after incubation of ECs with HLE or Cat G. Further experiments conducted with platelets indicated that not only HLE and Cat G but also PR3 inhibited cell activation induced by thrombin, although they were again ineffective when TRAP42-55 was the agonist. Similar to that for ECs, the epitope for IIaR-A disappeared on treatment of platelets with either proteinase. These results suggested that the neutrophil enzymes proteolyzed the thrombin receptor downstream of the thrombin cleavage site (Arg41-Ser42) but left intact the TRAP42-55 binding site (Gln83-Ser93) within the extracellular aminoterminal domain. The capacity of these proteinases to cleave five overlapping synthetic peptides mapping the portion of the receptor from Asn35 to Pro85 was then investigated. Mass spectrometry studies showed several distinct cleavage sites, i.e., two for HLE (Val(72)-Ser73 and Ile74-Asn75), three for Cat G (Arg41-Ser42, Phe55-Trp56 and Tyr69-Arg70), and one for PR3 (Val(72)-Ser73). We conclude that this singular susceptibility of the thrombin receptor to proteolysis accounts for the ability of neutrophil proteinases to inhibit cell responses to thrombin.
Amino Acid Sequence↗
The isomorphous structures of prethrombin2, hirugen-, and PPACK-thrombin: changes accompanying activation and exosite binding to thrombin.
The X-ray crystal structure of prethrombin2 (pre2), the immediate inactive precursor of alpha-thrombin, has been determined at 2.0 A resolution complexed with hirugen. The structure has been refined to a final R-value of 0.169 using 14,211 observed reflections in the resolution range 8.0-2.0 A. A total of 202 water molecules have also been located in the structure. Comparison with the hirugen-thrombin complex showed that, apart from the flexible beginning and terminal regions of the molecule, there are 4 polypeptide segments in pre2 differing in conformation from the active enzyme (Pro 186-Asp 194, Gly 216-Gly 223, Gly 142-Pro 152, and the Arg 15-Ile 16 cleavage region). The formation of the Ile 16-Asp 194 ion pair and the specificity pocket are characteristic of serine protease activation with the conformation of the catalytic triad being conserved. With the determination of isomorphous structures of hirugen-thrombin and D-Phe-Pro-Arg chloromethyl ketone (PPACK)-thrombin, the changes that occur in the active site that affect the kinetics of chromogenic substrate hydrolysis on binding to the fibrinogen recognition exosite have been determined. The backbone of the Ala 190-Gly 197 segment in the active site has an average RMS difference of 0.55 A between the 2 structures (about 3.7 sigma compared to the bulk structure). This segment has 2 type II beta-bends, the first bend showing the largest shift due to hirugen binding. Another important feature was the 2 different conformations of the side chain of Glu 192. The side chain extends to solvent in hirugen-thrombin, which is compatible with the binding of substrates having an acidic residue in the P3 position (protein-C, thrombin platelet receptor). In PPACK-thrombin, the side chain of Asp 189 and the segment Arg 221A-Gly 223 move to provide space for the inhibitor, whereas in hirugen-thrombin, the Ala 190-Gly 197 movement expands the active site region. Although 8 water molecules are expelled from the active site with PPACK binding, the inhibitor complex is resolvated with 5 other water molecules.
Amino Acid Chloromethyl Ketones↗
Thrombin inhibitors and anti-coagulants on thrombin-induced embolisation in rabbit cranial vasculature.
111Indium-labelled platelets were continuously monitored in the cranial vasculature of anaesthetised rabbits and thrombin inhibitors and anti-coagulants were tested on the sustained platelet accumulation induced by intracarotid injection of thrombin (90 U/kg). Pretreatment, commencing 30 min prior to thrombin, with a 1-h intracarotid infusion of D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK; 0.25-1.0 micrograms/kg per min), unfractionated heparin (Multiparin; 5-20 U/kg bolus + 0.75-3.0 U/kg per min infusion) or low molecular weight heparin (Fragmin; 2.4-9.6 U/kg per min) produced dose-related reductions in platelet accumulation. Continuous infusion of acetyl-D-phenylalanyl-prolyl-boroarginine (DuP-714 ester; 30 micrograms/kg per min) for 30 min induced marked accumulation of platelets in the pulmonary circulation in the absence of thrombin. Bolus intracarotid injection, 1 min before thrombin, of Hirulog (0.05-0.2 mg/kg), PPACK (10-30 micrograms/kg), Multiparin (25-100 U/kg), Fragmin (150 U/kg) or DuP-714 ester (15-30 micrograms/kg) caused significant reductions in platelet accumulation. When injected 1 min after thrombin, Hirulog (1 mg/kg), PPACK (100 micrograms/kg), Fragmin (150 U/kg) and DuP-714 ester (30 micrograms/kg) had no significant effect and Multiparin (100 U/kg) increased platelet accumulation. The results demonstrate that pretreatment with a range of thrombin inactivators, acting via different mechanisms, can inhibit thrombin-induced cerebral thromboembolism in the rabbit.
Amino Acid Chloromethyl Ketones↗
Refined 2.3 A X-ray crystal structure of bovine thrombin complexes formed with the benzamidine and arginine-based thrombin inhibitors NAPAP, 4-TAPAP and MQPA. A starting point for improving antithrombotics.
Well-diffracting crystals of bovine epsilon-thrombin in complex with several "non-peptidic" benzamidine and arginine-based thrombin inhibitors have been obtained by co-crystallization. The 2.3 A crystal structures of three complexes formed either with NAPAP, 4-TAPAP, or MQPA, were solved by Patterson search methods and refined to crystallographic R-values of 0.167 to 0.178. The active-site environment of thrombin is only slightly affected by binding of the different inhibitors; in particular, the exposed "60-insertion loop" essentially maintains its typical projecting structure. The D-stereoisomer of NAPAP and the L-stereoisomer of MQPA bind to thrombin with very similar conformations, as previously inferred from their binding to bovine trypsin; the arginine side-chain of the latter inserts into the specificity pocket in a "non-canonical" manner. The L-stereoisomer of 4-TAPAP, whose binding geometry towards trypsin was only poorly defined, is bound to the thrombin active-site in a compact conformation. In contrast to NAPAP, the distal p-amidino/guanidino groups of 4-TAPAP and MQPA do not interact with the carboxylate group of Asp189 in the thrombin specificity pocket in a "symmetrical" twin N-twin O manner, but through "lateral" single N-twin O contacts; in contrast to the p-amidino group of 4-TAPAP, however, the guanidyl group of MQPA packs favourably in the pocket due to an elaborate hydrogen bond network, which includes two entrapped water molecules. These thrombin structures confirm previous conclusions of the important role of the intermolecular hydrogen bonds formed with Gly216, and of the good sterical fit of the terminal bulky hydrophobic inhibitor groups with the hydrophobic aryl binding site and the S2-cavity, respectively, for tight thrombin active site binding of these non-peptidic inhibitors. These accurate crystal structures are presumed to be excellent starting points for the design and the elaboration of improved antithrombotics.
Amidines↗
The inhibitory effect of melagatran, the active form of the oral direct thrombin inhibitor ximelagatran, compared with enoxaparin and r-hirudin on ex vivo thrombin generation in human plasma.
INTRODUCTION: The effect of the oral direct thrombin inhibitor (DTI) ximelagatran (Exanta, AstraZeneca) on the endogenous thrombin potential (ETP) of activated plasma was investigated ex vivo using a thrombin generation assay and compared with recombinant (r)-hirudin and enoxaparin. MATERIALS AND METHODS: 120 healthy male volunteers were randomized to one of six treatment groups (n=20 in each): oral ximelagatran (15, 30, or 60 mg), intravenous r-hirudin (0.4 mg/kg bolus, 0.15 mg/kg/h infusion for 2 h, followed by 0.075 mg/kg/h infusion for 2 h), subcutaneous enoxaparin (100 IU/kg), or control (tap water administered orally). Venous blood was collected predose and at 2, 4, and 10 h postdosing. Thrombin generation was triggered by the addition of tissue factor to platelet-poor plasma, and the ETP and time to peak thrombin generation were measured. RESULTS AND CONCLUSIONS: A significant and dose-dependent reduction in ETP was observed 2 and 4 h after the administration of ximelagatran 30 mg (70.3% of predose, 95% confidence intervals 63.0-78.5, P<0.0001 at 2 h) and 60 mg (49.8%, 43.2-57.4, P<0.0001 at 2 h), r-hirudin (19.5%, 10.1-37.6, P<0.0001 at 2 h), and enoxaparin (34.2%, 21.4-54.7, P<0.0001 at 2 h). Ximelagatran (30 mg, 3.79 min, 3.52-4.08 at 2 h), r-hirudin (6.23 min, 4.93-7.86 at 2 h), and enoxaparin (4.68 min, 3.30-6.64 at 2 h) also delayed the lag phase before the thrombin generation burst compared to placebo (2.92 min, 2.71-3.25 at 2 h). The oral DTI ximelagatran, in its active form melagatran, is a potent thrombin inhibitor that efficiently decreases ETP and delays the generation of thrombin in plasma in this ex vivo model.
Administration, Oral↗
Effect of oligodeoxynucleotide thrombin aptamer on thrombin inhibition by heparin cofactor II and antithrombin.
'Thrombin aptamers' are based on the 15-nucleotide consensus sequence of d(GGTTGGTGTGGTTGG) that binds specifically to thrombin's anion-binding exosite-I. The effect of aptamer-thrombin interactions during inhibition by the serine protease inhibitor (serpin) heparin cofactor II (HCII) and antithrombin (AT) has not been described. Thrombin inhibition by HCII without glycosaminoglycan was decreased approximately two-fold by the aptamer. In contrast, the aptamer dramatically reduced thrombin inhibition by >200-fold and 30-fold for HCII-heparin and HCII-dermatan sulfate, respectively. The aptamer had essentially no effect on thrombin inhibition by AT with or without heparin. These results add to our understanding of thrombin aptamer activity for potential clinical application, and they further demonstrate the importance of thrombin exosite-I during inhibition by HCII-glycosaminoglycans.
Antithrombins↗
Effects of ximelagatran, an oral direct thrombin inhibitor, r-hirudin and enoxaparin on thrombin generation and platelet activation in healthy male subjects.
OBJECTIVES: The effects of ximelagatran, an oral direct thrombin inhibitor (DTI), recombinant hirudin (r-hirudin) and enoxaparin on thrombin generation and platelet activation were studied in humans. BACKGROUND: Recombinant hirudin (parenteral DTI) and enoxaparin (low molecular weight heparin) have been demonstrated to be clinically effective in acute coronary syndromes. Ximelagatran is currently under investigation for the prevention and treatment of thromboembolism. The shed blood model allows for the study of thrombin generation and platelet activation in humans in vivo. METHODS: This was an open-label, parallel-group study involving 120 healthy male volunteers randomized to receive one of three oral doses of ximelagatran (15, 30 or 60 mg), r-hirudin (intravenous) or enoxaparin (subcutaneous) at doses demonstrated to be clinically effective in acute coronary syndromes, or to serve as a control. Thrombin generation (prothrombin fragment 1+2 [F1+2] and thrombin-antithrombin complex [TAT]) and platelet activation (beta-thromboglobulin [beta-TG]) biomarkers were studied using a shed blood model involving blood collection from skin incisions made using standardized bleeding time devices. RESULTS: Oral ximelagatran, intravenous r-hirudin and subcutaneous enoxaparin rapidly and significantly (p < 0.05) decreased F1+2, TAT and beta-TG levels in shed blood, indicating inhibition of thrombin generation and platelet activation. Statistically significant concentration (melagatran, the active form of ximelagatran)-response relationships for F1+2 (p = 0.005), TAT (p = 0.005) and beta-TG (p < 0.001) levels, with IC(50)s of 0.376 (F1+2), 0.163 (TAT) and 0.115 (beta-TG) micromol/l, were detected. Melagatran showed dose-proportional pharmacokinetics with low variability. All drugs were well tolerated. CONCLUSIONS: Oral administration of the DTI ximelagatran resulted in a rapid inhibition of both thrombin generation and platelet activation in a concentration-dependent manner using a human shed blood model. The inhibition of thrombin generation by 60 mg ximelagatran was comparable to that observed with doses of r-hirudin and enoxaparin demonstrated to be effective for the treatment of acute coronary syndromes.
Administration, Oral↗