The toxicity assessment of neorudin in cynomolgus monkeys.

In this study, the toxicity effects on circulatory system and respiratory system, and the acute toxicity test of recombinant neorudin (EPR-hirudin, EH) in cynomolgus monkeys were evaluated to provide reference information for clinical studies. Eighteen cynomolgus monkeys were randomly divided into three groups for single intravenous administration of 3, 30 mg/kg EH and normal saline, respectively. The changes of respiratory frequency, respiratory intensity, blood pressure and electrocardiogram before and after administration were recorded. In acute toxicity test, six cynomolgus monkeys were intravenously received EH at a single dose of 171, 257, 385, 578, 867 and 1300 mg/kg respectively. The vital signs, hematology, serum biochemistry, coagulation indexes and electrocardiogram indexes of the animals were determined before administration and on the 7th and 14th day after administration. As the results showed that there were no significant abnormal changes in respiratory frequency, respiratory intensity, blood pressure or electrocardiogram in cynomolgus monkeys after receiving EH at 3 mg/kg and 30 mg/kg, and there was no statistical difference between the treated groups and normal saline group. In the acute toxicity test, no significant abnormalities were observed in vital signs, hematology, serum biochemistry, coagulation indexes and electrocardiogram indexes of six cynomolgus monkeys at day 7 and 14 after EH administration. Furthermore, autopsies of all cynomolgus monkeys showed no abnormalities. The results of toxicokinetics showed that AUClast of the drug increased in proportion to the EH dose in the range of 171-578 mg/kg, and increased in over proportion to the EH dose in the range of 578-1300 mg/kg. The variation of Cmax was basically consistent with AUClast. In a sum, A single intravenous injection of 3 and 30 mg/kg of EH did not affect the circulatory system and respiratory system in cynomolgus monkeys and the maximum tolerated dose of EH in cynomolgus monkey is over 1300 mg/kg (equivalent to 619-1300 times of the proposed clinical equivalent dose).

A long-lasting, plasmin-activatable thrombin inhibitor aids clot lysis in vitro and does not promote bleeding in vivo.

The leech protein hirudin is a potent inhibitor of thrombin, but clinical use of recombinant hirudin is restricted by haemorrhagic risks, and complicated by hirudin's rapid clearance from the circulation. We previously employed albumin fusion to slow hirudin variant 3 (HV3) clearance. In this study, we hypothesized that reconfiguration of the chimera, appending human serum albumin (HSA) to the N-terminus of HV3, with an intervening plasmin cleavage site, would create a slowly cleared, plasmin-activatable HV3. Potential plasmin cleavage sites were screened by expression in Escherichia coli, interposed between glutathione sulfotransferase and HV3 domains. The most reactive sequence (GSGIYR-ITY) was recreated in C-terminally His-tagged albumin fusion protein HSACHV3, expressed in Pichia pastoris yeast and purified by nickel-chelate affinity chromatography. HSACHV3 showed no thrombin inhibitory activity in the absence of plasmin, but liberated active HV3 in a time- and concentration-dependent manner in its presence. In a discontinuous clot assay involving clot-bound thrombin, HSACHV3 assisted clot lysis by limiting clot extension in a tPA- and concentration-dependent manner. Similar results were obtained in plasma at higher concentrations of HSACHV3. The chimeric protein exhibited much slower clearance in mice than unfused HV3, and indistinguishable pharmacokinetics from unfused recombinant HSA. In a mouse tail transection bleeding model, doses of HSACHV3 identical to those of HV3 that elicited a four-fold increase in the volume of shed blood were without effect. Our results suggest that HSACHV3 is a fully latent, plasmin activatable, long-lasting hirudin, of potential benefit in thrombotic disorders resistant to natural or pharmacological clot lysis.

A novel hirudin derivative inhibiting thrombin without bleeding for subcutaneous injection.

Currently, anticoagulants would be used to prevent thrombosis. Thrombin is an effector enzyme for haemostasis and thrombosis. We designed a direct thrombin inhibitor peptide (DTIP) using molecular simulation and homology modelling and demonstrated that the C-terminus of DTIP interacts with exosite I, and N-terminus with the activity site of thrombin, respectively. DTIP interfered with thrombin-mediated coagulation in human, rat and mouse plasma (n=10 per group) and blocked clotting in human whole blood in vitro. When administered subcutaneously, DTIP showed potent and dose-dependent extension of aPTT, PT, TT and CT in rats (n=10 per group). The antithrombotic dose of DTIP induced significantly less bleeding than bivalirudin determined by transecting distal tail assay in rats. Furthermore, DTIP reached peak blood concentration in 0.5-1 hour and did not cause increased bleeding after five days of dosing compared to dabigatran etexilate. The antithrombotic effect of DTIP was evaluated in mice using lethal pulmonary thromboembolism model and FeCl3-induced mesenteric arteriole thrombus model. DTIP (1.0 mg/kg, sc) prevented deep venous thrombosis and increased the survival rate associated with pulmonary thromboembolism from 30 % to 80 %. Intravital microscopy showed that DTIP (1.0 mg/kg, sc) decelerated mesenteric arteriole thrombosis caused by FeCl3 injury. These data establish that DTIP is a novel antithrombotic agent that could be used to prevent thrombosis without conferring an increased bleeding risk.

Toxicology Assessment of a Dual-Function Peptide 5rolGLP-HV in Mice.

5rolGLP-HV had an ideal therapeutic potential in the prevention of hyperglycemia in type 2 diabetes and delay of the thrombosis. The objective of the study was to investigate the toxicology effects of 5rolGLP-HV and guarantee its safety. In acute toxicity test, the mice were orally receiving 5rolGLP-HV at a single dose of 300 mg/kg or 2000 mg/kg. For sub-chronic toxicity study, the mice received 5rolGLP-HV at doses of 800 mg/kg or 1600 mg/kg for 9 weeks. No significant adverse effects were evident in acute and sub-chronic toxicity tests, indicating that the LD50 value is greater than 2000 mg/kg. Although the liver and kidney exhibited a little abnormal in sub-chronic toxicity study, they could recovery to normal after withdrawal 5rolGLP-HV for 2 weeks. In micronucleus assay, the mice received 5rolGLP-HV at doses of 250, 500, or 1000 mg/kg for two consecutive days. The micronucleus numbers and the polychromatic erythrocytes to normochromatic erythrocytes (PCE/NCE) ratios among 5rolGLP-HV groups were within the normal range. Similarly, sperm aberration test demonstrated that 5rolGLP-HV had no teratogenic effect on the mice sperm. In conclusion, the combined results clearly demonstrated the safety of 5rolGLP-HV and support its use as a drug to treat diabetes and thrombosis.

[Studies on the nasal epithelium toxicity of adjuvants and recombination hirudin (rHV2) nasal spary].

OBJECTIVE: To investigate the nasal epithelium toxicity of adjuvants and rHV2 nasal spary(HVS). METHOD: Ciliary movement were evaluated with in situ toad palate model; The histology assessment of nasal epithelium were carried out after long-lasting and repeated use of HVS. RESULT AND CONCLUSION: Adjuvants included SDS, Brij 35, azone, lecithin, EDTA, menthol, nipagin and thiomersal were able to significantly inhibited the ciliary movement, while tween80, glycyrrhizic acid monoammonium salt, benzalkonium bromide, sodium benzoate and adhensive materials investigated had less influence on it. HVS was able to damaged the nasal epithelium, but this effect recovered soon after stopping administration. It was demonstrated that SDS, Brij 35, azone,lecithin, EDTA, menthol, nipagin and thiomersal. It had significant cilitoxity, while tween80, glycyrrhizic acid monoammonium salt, benzalkonium bromide, sodium benzoate and adhensive materials investigated had no significance; Chitosan co-administration with some adjuvants may make the cillitoxity severer; It is available that rHV2 be administered by nasal spary.

Pharmacological studies on the antithrombotic action of hirudin in experimental animals.

The pharmacodynamics and pharmacokinetics of hirudin were studied in dogs, rabbits and rats. Hirudin proved to be a well tolerated substance with low toxicity. After intravenous injection it was eliminated with a half time of 50 to 60 min. It was nearly completely excreted through the kidneys in biologically active form. The efficacy of hirudin in preventing venous thrombosis, vascular shunt occlusion and disseminated intravascular coagulation in rats was demonstrated.

Pharmacological effects of a novel recombinant hirudin, CX-397, in vivo and in vitro: comparison with recombinant hirudin variant-1, heparin, and argatroban.

The novel recombinant hirudin analog CX-397 was investigated with respect to its pharmacological activity and antithrombin profiles in vivo and in vitro. In three different types of thrombosis models in rats, including stasis and thrombin-induced venous, glass surface-activated arterio-venous shunt, and ferric chloride-induced arterial thrombosis models, CX-397 and rHV-1 elicited potent antithrombotic effects, where the minimum effective doses of rHV-1 tended to be higher than those of CX-397 in the arterio-venous shunt and arterial thrombosis models. The hemorrhagic risk of CX-397 in template bleeding in rats was not higher than that of rHV-1, indicating that CX-397 is superior to rHV-1 for treating the platelet-dominant type of thrombosis. However, no differences were detected between CX-397 and rHV-1 in their effects on in vitro coagulation times and thrombin-induced platelet aggregation, suggesting the possibility that some unknown mechanisms other than simple thrombin inhibition are also involved in their antithrombotic actions.

Comparison of enoxaparin, hirulog, and heparin as adjunctive antithrombotic therapy during thrombolysis with rtPA in the stenosed canine coronary artery.

A canine model of electrolytic injury-induced coronary artery thrombosis and rtPA-induced thrombolysis was used to evaluate the relative antithrombotic efficacy of enoxaparin (a low molecular weight heparin), conventional therapy (heparin or heparin plus aspirin), and hirulog (a direct thrombin inhibitor), when used as adjunctive therapy during thrombolysis. After 60 min of clot aging, adjunctive therapy was begun at doses which elevated APTT approximately 2-fold over baseline. Fifteen minutes after the start of adjunctive therapy, recombinant tissue plasminogen activator (rtPA) was administered (100 microg/kg i.v. bolus + 20 microg/kg/min for 60 min). Adjunctive therapy continued for 1 h after termination of rtPA and blood flow was monitored for two additional hours. Enoxaparin (1 mg/kg i.v. bolus + 30 microg/kg/min, n = 10 for each treatment group) was the only adjunctive treatment that significantly increased the total minutes of flow (143 +/- 25 min out of a possible 240 min, vs 54 +/- 25 min for vehicle, p <0.05) and decreased thrombus mass (6.0 +/- 1.3 mg vs 11.8 +/- 3.2 mg for vehicle). Although hirulog (2 mg/kg i.v. bolus + 40 microg/kg/min) did not significantly increase the minutes of flow (120 +/- 27 min, p <0.06) or decrease thrombus mass (8.7 +/- 1.7 mg) compared to vehicle, these values were not significantly different than those measured in the enoxaparin group. However, the results with hirulog were achieved at the expense of a significantly greater increase in template bleeding time than that measured during enoxaparin treatment. Minutes of flow for heparin (50 U/kg i.v. bolus + 0.6 U/kg/min) and heparin plus aspirin (5 mg/kg i.v. bolus) were 69 +/- 20 and 60 +/- 23 min, respectively; thrombus masses were 8.2 +/- 1.3 and 7.3 +/- 1.0 mg, respectively. In summary, enoxaparin was more effective than conventional therapy in this model in terms of vessel patency and thrombus mass, and was as effective as hirulog, at least at a dose of hirulog that only modestly impaired hemostasis. Therefore, enoxaparin may prove to be a safe and effective alternative agent for adjunctive therapy during thrombolysis with rtPA.

Haemostyptic effects of batroxobin with regard to hirudin treatment.

In contrast to thrombin the fibrinogen coagulant effect of the thrombin-like enzyme batroxobin in vitro and in vivo is not inhibited by the specific thrombin inhibitor hirudin. The haemostyptic effect of batroxobin has been studied in rats after bleeding had been induced by corresponding hirudin dosages. Dependent on batroxobin concentration bleeding time was shortened by local application of batroxobin containing solutions. Strong bleeding induced by i.v. injection of 5 mg r-hirudin/kg was stopped almost immediately when a batroxobin concentration of 40 BU/ml was used. Thrombin was less active to stop bleeding after r-hirudin administration than batroxobin.

An animal model of fibrinolytic bleeding based on the rebleed phenomenon: application to a study of vulnerability of hemostatic plugs of different age.

The primary bleeding time is prolonged when tested during the infusion of both plasminogen activators and anticoagulants, and such sites frequently exhibit rebleeding after initial hemostatic control. This study describes an animal (rabbit) model which distinguishes fibrinolytic from anticoagulant hemorrhage and further applies the model to the study of hemostatic plugs of increasing age. In this model, rebleeding occurred from hemostatically-stable ear puncture sites induced prior to infusion of streptokinase (SK) or recombinant tissue-plasminogen activator (rt-PA), but not of heparin or hirudin. This distinction was apparent even for lesions induced only 15 minutes prior to the infusion and fibrinolytic bleeding was observed in such lesions induced up to 24 hours earlier. Post-infusion sites bled more quickly than did pre-infusion sites, and there was a gradual decrease in susceptibility of such prior trauma sites for rebleeding, evidenced not only by a lower proportion of sites that rebled, but also by a longer lag time after starting SK or rt-PA before such rebleeding occurred. At the dosages tested, SK showed a trend (not statistically significant) toward more sites that rebled, while rt-PA showed a trend towards a longer duration of rebleeding. Thus, this animal model of rebleeding appears to be unique for fibrinolytic agents and allows for more detailed study of the physiological mechanisms of such bleeding and for a multifaceted comparison of the bleeding potential of plasminogen activators.

Comparative study on the use of anticoagulants heparin and recombinant hirudin in a rabbit traumatic anastomosis model.

Antithrombotic drugs, such as heparin, have been used in the clinics for a long time. Heparin acts by binding with antithrombin III to form a complex thereby enhancing the activity of antithrombin III to inactivate coagulation factors IIa, IXa, Xa, XIa and XIIa. Hirudin is a new antithrombotic agent and is reported to be much more powerful than heparin on a gravimetric basis. When both are administered systemically, one of the common complications seen is bleeding. Some previous studies have shown that local vascular endothelial concentrations of heparin are 30 to 7500 times greater than those found in the circulating blood. In order to avoid such complications, topical administration of antithrombotic drugs may be an ideal route of administration. The rabbit ear arterial crush-avulsion thrombosis model was used in this study. The animals were divided into five groups: one control group and four treatment groups which received varying concentrations of heparin and hirudin. In the saline control group, the patency rate was 19.23% at 24 hrs and 15.38% at 7 days. A higher patency rate at 7 days was obtained in groups treated with high concentration of heparin and hirudin. ACT, PT and APTT performed on samples drawn one hour after drug administration were within the normal range in both the control and the treatment groups. Scanning electron microscopy revealed the different extent of the clots on the injured intimal surfaces of the vessels in different groups. The results indicate that high concentrations of topically administered heparin or hirudin minimize the systemic complications and maximize the antithrombotic effects.

An objective perspective on recombinant hirudin: a new anticoagulant and antithrombotic agent.

Leeches have been in medical use for many years. Hirudin, the anticoagulant obtained from the medicinal leech has been purified, characterized and can now be produced by recombinant (r) technology. R-hirudin is a potent inhibitor of thrombin and is therefore a potentially valuable anticoagulant and antithrombotic drug. This article reviews the current status of r-hirudin in this role and compares the pharmacokinetics, mechanism of action and clinical efficacy of this agent with heparin. The methods available for laboratory assessment and clinical monitoring of r-hirudin and the possible ways of antagonizing its effects are also discussed. Finally, the potential clinical applications of r-hirudin are outlined, although further laboratory and clinical studies, together with a fall in the cost of this compound are required before r-hirudin can be more widely accepted as an anticoagulant and antithrombotic agent.

Factor VIII and DDAVP reverse the effect of recombinant desulphatohirudin (CGP 39393) on bleeding in the rat.

The infusion of a high dose of recombinant desulphatohirudin HVI (CGP 39393) for 40 min at 30 micrograms/kg/min, resulted in a prolongation of bleeding time in the rat when evaluated using transection of the tail. The prolonged bleeding was evident both immediately, and 30 min after cessation of the infusion of hirudin (CGP 39393). Bleeding time returned to normal after 60 min. The effect of several agents, reported to be successful in reducing bleeding tendencies in man, were evaluated in this rat model. The agents were administered immediately following cessation of the CGP 39393 infusion and their ability to normalize the prolonged bleeding-time, observed at 30 min after cessation of the CGP 39393 infusion, determined. Desmopressin (DDAVP), recombinant factor VIII and Vueffe reduced the bleeding time to the control range but did not exert any significant effects on the bleeding time in rats which did not receive CGP 39393. Epsilon-aminocaproic acid (EACA) and recombinant factor VII were ineffective, at the doses used. In conclusion, DDAVP, factor VIII and Vueffe are effective in reversing the effect of direct thrombin inhibition on bleeding in the rat.

Pharmacological survey of recombinant hirudin.

The pharmacological properties of a genetically engineered recombinant hirudin (r-hirudin) were studied in animal experiments. r-Hirudin proved to be a well tolerated substance. I.v. injection of up to 200 mg/kg did not lead to perceptible functional or morphological changes. There were no treatment-related effects on the cardiovascular system of dogs and rats after administration of up to 10 mg/kg. After long-term treatment (4 weeks, 1.0 mg/kg daily), no r-hirudin-related histopathological, haematological or biochemical changes could be found. Formation of antibodies was not detectable. Absorption, distribution, and elimination of r-hirudin were studied in dogs and rats. Pharmacokinetics could be best described by an open two-compartment model with first-order kinetics. After i.v. injection in dogs, r-hirudin is distributed into the extracellular space and eliminated through the kidneys in active form by glomerular filtration. After i.v. administration, a half-life of about 1 h was estimated; s.c. administration prolonged the apparent half-life. Pulmonary absorption was shown. Enteral absorption, placental transfer as well as transfer through the fetal integument were very low. r-Hirudin did not pass the blood-brain barrier. The efficacy of r-hirudin in preventing both venous and arterial thrombosis, vascular shunt occlusion or disseminated intravascular coagulation was demonstrated in rats. Depending on the dose, r-hirudin was able to prevent or reduce stasis-induced venous thrombosis, prolong the patency of an extracorporeal arteriovenous shunt, reduce the incidence of arterial thrombosis caused by vascular wall lesions as well as of microthrombosis induced by thrombin infusion.(ABSTRACT TRUNCATED AT 250 WORDS)

New derivative of staphylokinase SAK-RGD-K2-Hirul exerts thrombolytic effects in the arterial thrombosis model in rats.

SAK-RGD-K2-Hir and SAK-RGD-K2-Hirul are recombinant proteins that are derivatives of r-SAK (recombinant staphylokinase). They are characterized by their fibrin-specific plasminogen activation properties and their antithrombin and antiplatelet activities. The difference between these proteins is the presence of the antithrombotic fragment (hirudin or hirulog) in the C-terminal portion of the r-SAK. The aim of the present study was to examine the thrombolytic potentials of SAK-RGD-K2-Hir and SAK-RGD-K2-Hirul in an electrically induced carotid artery thrombosis model in rats and to compare the potentials to that of r-SAK. We determined that a bolus injection of SAK-RGD-K2-Hirul was more effective than one of r-SAK in the improvement and maintenance of carotid patency and in arterial thrombus weight reduction; however, it had the same potency as SAK-RGD-K2-Hir. The bleeding time, prothrombin time and activated partial thromboplastin time were significantly prolonged in the animals that were treated with either dose (1.5 or 3.0 mg/kg) of SAK-RGD-K2-Hir or SAK-RGD-K2-Hirul, whereas no changes were observed in the plasma fibrinogen concentration or the α2 plasmin inhibitor level. r-SAK alone did not change the bleeding time or coagulation parameters. In conclusion, our findings demonstrate the thrombolytic activity of intravenous bolus injection of the novel thrombolytic agent SAK-RGD-K2-Hirul in rats. Although this protein compares favorably with r-SAK, we were unable to show the presence of any beneficial effects of SAK-RGD-K2-Hirul over those of SAK-RGD-K2-Hir. Furthermore, our results suggest that high doses of SAK-RGD-K2-Hirul bear the risk of bleeding.

Bivalirudin: pharmacology and clinical applications.

Bivalirudin (Hirulog, Angiomax) is a specific, reversible and direct thrombin inhibitor with a predictable anticoagulant effect. It is cleared by both proteolytic cleavage and renal mechanisms, predominantly glomerular filtration. Bivalirudin inhibits both circulating thrombin and fibrin bound thrombin directly by binding to thrombin catalytic site and anion-binding exosite I in a concentration-dependent manner. Bivalirudin prolongs activated partial thromboplastin time, prothrombin time, thrombin time and activated clotting time (ACT). ACT levels with bivalirudin do not correlate with its clinical efficacy. Bivalirudin with a provisional GpIIb/IIIa inhibitor is indicated in elective contemporary percutaneous coronary intervention (PCI). In respect to combined ischemic and hemorrhagic endpoints of death, myocardial infarction, unplanned urgent revascularization and major bleeding during PCI (including subgroups of patients with renal impairment and diabetes) bivalirudin is not inferior to unfractioned heparin and planned GpIIb/IIIa inhibitors. In addition, bivalirudin has been consistently shown to have significantly less in-hospital major bleeding than heparin alone or heparin in combination with a GpIIb/IIIa inhibitor. Bivalirudin appears to be also safe and effective during PCI in patients with heparin-induced thrombocytopenia. Finally, data from PCI studies support the safety and efficacy of bivalirudin, although its direct randomized comparison with unfractionated heparin is lacking.

The haemorrhagic effect of low molecular weight heparins, dermatan sulphate and hirudin.

In a randomized, blind study the primary effect on haemostasis after intravenous administration of dermatan sulphate (DS), recombinant hirudin (r-hirudin) and four commercial low molecular weight heparins (LMWHs) (nadroparine, enoxaparin, dalteparin and tinzaparin) was investigated in rats and compared with saline (control). The tail bleeding time, the bleeding from the gastric mucosa [the mucosal bleeding time (min) and the mucosal bleeding (microliter)] as well as changes in activated partial thromboplastin time, antifactor IIa and Xa activities were investigated. DS and r-hirudin were investigated in a dose potentially suitable in thomboprophylaxis and the LMWHs in doses recommended by the manufacturers for thromboprophylaxis, adjusted to body weight. All substances significantly prolonged the mucosal bleeding time. Dalteparin, tinzaparin, DS and r-hirudin increased the mucosal bleeding when compared with controls whereas nadroparine and enoxaparin did not. The effect of r-hirudin was also significantly more pronounced compared with other treatments. Moreover, r-hirudin prolonged the tail bleeding time significantly whereas the other substances did not. The antifactor Xa activity in plasma correlated well with the given dose of the LMWHs (rs = 0.7). However, the monitored bleeding parameters in the LMWH groups did not correlate with the plasma activities of antifactor IIa or Xa. The results indicate that the tested LMWHs are not equipotent in their effect on haemostasis in this model and that antifactor IIa or Xa activities do not directly correlate with their effect on haemostasis although increased haemorrhage was observed in the LMWHs with lower antifactor Xa/antifactor IIa ratios.(ABSTRACT TRUNCATED AT 250 WORDS)

Toxicological studies with recombinant hirudin.

After daily administration of 1 mg/kg over a period of 4 weeks, hirudin did not influence growth and general behaviour of the animals. There was no indication of treatment-related effects on the liver and the kidneys, on number and function of thrombocytes, on number and differentiation of white cells or on the erythrocytic system. Haemorrhagic complications did not occur. Formation of antibodies was not detectable. Histopathological changes due to application of r-hirudin were not found. LD50 is greater than 50 mg/kg body weight for intravenous application.

Recombinant hirudin (HBW 023): biological data of ten patients with severe venous thrombo-embolism.

This study reports on the biological data of ten patients with acute venous thrombo-embolism. They were treated for 5 days with continuous intravenous infusion of a fixed dose (0.05 mg/kg/hr) of a recombinant hirudin (r-H HBW 023 Behringwerke, Germany). The plasma level of r-H (HBW 023), assessed by an anti-factor IIa amidolytic activity, was stable after Day 2 and showed considerable individual variations. It correlated with APTT ratio, suggesting that this test is a reliable tool to monitor therapy. In contrast, thrombin time was constantly over 120 sec (control 15 sec) and consequently was not a useful parameter. Prothrombin time showed a slight, but significant, prolongation, which was correlated with the increase of APTT ratio. There was no bleeding time prolongation, platelet count, or ATIII level decrease. Levels of thrombin-antithrombin III complexes, and D-dimers, which were high in all patients on admission, decreased during the course of the treatment but remained abnormal on Day 5, showing an ongoing hemostasis and fibrinolysis activation: this is consistent with the delayed, but only slightly decreased thrombin generation evidenced by thrombin generation test performed on Day 3. These results suggest that thrombin inhibition by rH-hirudin at this dosage is only partial, which allows the generation of traces of thrombin needed for the feed-back thrombin production generated by factor V and VIII activation.