Recombinant Hirudin Research Articles

Successful renal transplantation in a patient with heterozygous prothrombin gene, factor V Leiden mutation and heparin‐induced thrombocytopenia using r‐hirudin as anticoagulant

Vascular complications remain the most common cause of early renal allograft loss in patients with end-stage renal failure. Underlying thrombophilic disorders increase the risk of early graft thrombosis. A male adolescent with high-risk thrombophilia because of combined heterozygous factor V Leiden (G1691A) and prothrombin gene (G20210A) mutation developed HIT II. Hemodialysis and subsequent renal transplantation were undertaken using recombinant hirudin, a direct and selective thrombin inhibitor, as an anticoagulant. Primary function in the transplanted kidney was excellent. No thrombotic or hemorrhagic events have occurred and follow-up showed excellent long-term graft survival. Patients on HD have an increased risk for the development of HIT, and therefore, they need repetitive screening for the development of acquired thrombotic risk factors (e.g. HIT II or lupus anticoagulant). R-hirudin is efficacious and safe on both HD and following renal transplantation.

Thrombin generation and inhibition in the mediation of ischemic neuronal death

Thrombin is a serine protease with dual effects on cell death and survival: It mediates apoptotic cell death at high concentrations in cultured astrocytes and hippocampal neurons and induces neuroprotection at low concentrations in the same cells. Recent evidence indicates that thrombin plays a role in ischemic cell death, as rats subjected to middle cerebral artery occlusion were protected by the intracerebral injection of hirudin, a selective thrombin inhibitor. We are using an in vitro approach to further characterise the mechanisms involved. Organotypic hippocampal slice cultures were subjected to a 30 minute oxygen (10%) and glucose (1 mmol/L) deprivation (OGD). 24 hours after OGD, there was a marked increase in thrombin immunoreactivity on Western blots, showing for the first time that OGD leads to the activation of prothrombin into thrombin. We could also demonstrate that this ischemia induced activation of thrombin plays an important role in ischemic neuronal death, as both recombinant hirudin and protease nexin-1, an endogenous cerebral thrombin inhibitor, significantly prevented neuronal death, when administered after OGD. Using this in vitro approach we are currently characterising the role of thrombin as a mediator of ischemic neuronal death.

Hirudin-based anticoagulant strategy during isolated limb perfusion in a patient with heparin-induced thrombocytopenia

Heparin-induced thrombocytopenia (HIT) is a rare but potentially fatal complication of heparin therapy. The administration of heparin in patients with HIT causes platelet aggregation, thromboembolism and thrombocytopenia. Therefore, an alternative anticoagulant is recommended in these patients. We describe the use of recombinant hirudin (r-hirudin; Refludan, Pharmion Germany GmbH, Hamburg, Germany) as an anticoagulant in a patient with HIT requiring isolated limb perfusion (ILP) for in-transit metastases of malignant melanoma of the leg; r-hirudin was used in both the extracorporeal and systemic circuits. The coagulation monitoring included the activated partial thromboplastin time (aPTT) and ecarin clotting time (ECT). There were no thrombotic or bleeding complications. The dosage regimen and the strategy of monitoring of the anticoagulant activity are described. It can be concluded that ILP in patients with suspected or confirmed HIT can be safely performed with the use of r-hirudin in both the extracorporeal and systemic circuits. Monitoring of the anticoagulation effect is necessary and should preferably be performed using ECT.

Successful treatment of mesenterial venous thrombosis with recombinant hirudin—a report of five cases

Successful treatment of mesenterial venous thrombosis with recombinant hirudin—a report of five cases

Exosite-interactive Regions in the A1 and A2 Domains of Factor VIII Facilitate Thrombin-catalyzed Cleavage of Heavy Chain

Thrombin catalyzes the proteolytic activation of factor VIII, cleaving two sites in the heavy chain and one site in the light chain of the procofactor. Evaluation of thrombin binding the reaction products from heavy chain cleavage by steady state fluorescence energy transfer using a fluorophore-labeled, active site-modified thrombin as well as by solid phase binding assays using a thrombin Ser(205) --> Ala mutant indicated a high affinity site in the A1 subunit (K(d) approximately 5 nm) that was dependent upon the Na(+)-bound form of thrombin, whereas a moderate affinity site in the A2 subunit (K(d) approximately 100 nm) was observed for both Na(+)-bound and -free forms. The solid phase assay also indicated that hirudin blocked thrombin interaction with the A1 subunit and had little, if any, effect on its interaction with the A2 subunit. Conversely, heparin blocked thrombin interaction with the A2 subunit and showed a marginal effect on A1 binding. Evaluation of the A2 sequence revealed two regions rich in acidic residues that are localized close to the N and C termini of this domain. Peptides encompassing these clustered acidic regions, residues 373-395 and 719-740, blocked thrombin cleavage of the isolated heavy chain at Arg(372) and Arg(740) and inhibited A2 binding to thrombin Ser(205) --> Ala, suggesting that both A2 domain regions potentially support interaction with thrombin. A B-domainless, factor VIII double mutant Asp(392) --> Ala/Asp(394) --> Ala was constructed, expressed, and purified and possessed specific activity equivalent to a severe hemophilia phenotype. This mutant was resistant to cleavage at Arg(740), whereas cleavage at Arg(372) was not affected. These data suggest the acidic region comprising residues 389-394 in factor VIII A2 domain interacts with thrombin via its heparin-binding exosite and facilitates cleavage at Arg(740) during procofactor activation.

PAR1 Cleavage and Signaling in Response to Activated Protein C and Thrombin

Activated protein C (APC), a natural anticoagulant protease, can trigger cellular responses via protease-activated receptor-1 (PAR1), a G protein-coupled receptor for thrombin. Whether this phenomenon contributes to the physiological effects of APC is unknown. Toward answering this question, we compared the kinetics of PAR1 cleavage on endothelial cells by APC versus thrombin. APC did cleave PAR1 on the endothelial surface, and antibodies to the endothelial protein C receptor inhibited such cleavage. Importantly, however, APC was approximately 10(4)-fold less potent than thrombin in this setting. APC and thrombin both triggered PAR1-mediated responses in endothelial cells including expression of antiapoptotic (tumor necrosis factor-alpha-induced a20 and iap-1) and chemokine (interleukin-8 (il-8) and cxcl3) genes, but again, APC was approximately 10(4)-fold less potent than thrombin. The addition of zymogen protein C to endothelial cultures did not alter the rate of PAR1 cleavage at low or high concentrations of thrombin, and PAR1 cleavage was substantial at thrombin concentrations too low to trigger detectable conversion of protein C to APC. Thus, locally generated APC did not contribute to PAR1 cleavage beyond that effected by thrombin in this system. Although consistent with reports that sufficiently high concentrations of APC can cleave and activate PAR1 in culture, our data suggest that a significant physiological role for PAR1 activation by APC is unlikely.

Role of Lipopolysaccharide and Cecal Ligation and Puncture on Blood Coagulation and Inflammation in Sensitive and Resistant Mice Models

The hemostatic system is severely disturbed during endotoxemia, leading to a hypercoagulable state. However, it remains uncertain to what extent hypercoagulability is the critical factor in determining the clinical course rather than just the consequence of a severe systemic inflammatory response. To answer this question, we evaluated the evolution of hemostatic and inflammatory markers, as well as histological features, in mice sensitive and resistant to two models of endotoxemia: lipopolysaccharide-injection and cecal ligation puncture. Genetic (knockout mice) and pharmacological (PJ34) blockade of the nuclear enzyme PARP-1 was used to achieve resistance to the endotoxemia. In both models, endotoxemia resulted in antithrombin deficiency, decreased platelets, and fibrin deposition in organs, which were similar in all groups of mice. By contrast, proinflammatory mediators, inflammatory cell infiltration (especially that mediated by mononuclear cells), and organ degeneration were more intense in sensitive animals. Further studies supported a negative role for the triggering of the coagulation cascade in the mortality associated with the endotoxic shock. Hirudin had a minor effect on cell infiltration and organ damage, despite causing a potent inhibition of fibrin deposition. On the other hand, a sublethal dose of lipopolysaccharide yielded significant fibrin deposition but weak activation of the inflammatory response. Our results suggest that activation of coagulation by endotoxemia is severe and independent of the inflammatory response. However, such activation may act with fibrin deposition to have a minor influence on survival in sepsis.

Selective antimetastatic effect of heparins in preclinical human melanoma models is based on inhibition of migration and microvascular arrest

Use of heparin derivatives in several cancer types revealed that anticoagulant therapies have a beneficiary side effect: delay of tumor progression. Since there are no data on human melanoma, we have analyzed the effect of heparins in preclinical models. Neither unfractionated heparin (UFH), nor its low molecular weight derivative (LMWH) influenced in vitro or in vivo growth of HT168-M1 human melanoma cells. However, heparins significantly inhibited lung colony formation and liver metastasis development in the concentration range of 20-200 IU/kg, whereas recombinant hirudin was ineffective. The antimetastatic effect was due to an early (5-60 min) inhibition of tumor cell arrest in the lung microvasculature. Analysis of the molecular mechanism of the antimetastatic effect of heparins indicated a specific inhibition of tumor cell migration and matrix invasion. The presented experimental data suggest that heparins have specific antimetastatic effect in the case of human melanoma, which is independent from the coagulation cascade.

Successful Use of Recombinant Hirudin and Its Monitoring by Ecarin Clotting Time in Patients with Heparin-Induced Thrombocytopenia Undergoing Off-Pump Coronary Artery Revascularization

Refludan (lepirudin-rDNA for injection) is the first direct thrombin inhibitor approved by the United States FDA for anticoagulation to patients with heparin-induced thrombocytopenia (HIT). It was monitored by ecarin clotting time (ECT) assay in patients with HIT. Case histories and clotting parameters for three patients undergoing off-pump coronary artery revascularization procedure are discussed. The first patient received r-hirudin at a dose of 0.2 mg/kg intravenous (IV) bolus followed by 0.15 mg/kg/hour infusion. The second patient received 0.4 mg/kg IV bolus followed by infusion of 0.15 mg/kg/hour infusion. The third patient with renal failure received 0.2 mg/kg IV bolus followed by an infusion of 0.02 mg/kg/hour. Blood samples were drawn at baseline, 5 minutes post bolus and every 15 minutes during the coronary artery revascularization procedure. ECT was performed immediately on the citrated whole blood samples using the ECT cards in conjunction with the point-of-care, the thrombolytic assessment system (TAS) Analyzer (Pharmanetics, Raleigh, NC). The plasma samples were then analyzed for APTT and liquid ECT assay performed on a kinetic centrifugal analyzer (ACL 300 Plus). The ECT by cards was ideally maintained above 600 seconds during the surgical procedure. Additional boluses of Refludan were given as and when necessary (ECT < 600 sec) in order to maintain adequate anticoagulation. The calculated circulating concentrations of Refludan, following a bolus administration, based on the ECT cards, liquid ECT and APTT were 3.20 +/- 1.3, 3.51 +/- 1.35 and 2.02 +/- 1.19 microg/mL, respectively.

Pharmacodynamics and pharmacokinetics of recombinant hirudin via four non-parenteral routes

One of recombinant hirudin variants, rHV2, a polypeptide used as an anticoagulant agent in clinic, was administered to anesthetized rats via intratracheal, buccal, nasal and rectal routes. Prolongation in clotting time and thrombin time was measured to calculate pharmacological bioavailability. Plasma concentration of rHV2 was determined using a chromogenic thrombin substrate assay and pharmacokinetic parameters were obtained on the basis of a non-compartmental model. Intravenous administration was also performed as the gold standard by which the other routes were compared. Difference in pharmacological bioavailability (P.A.), bioavailability ( F) and absorption rate of rHV2 was found for the four non-parenteral routes. The rank order for both P.A. and F was intratracheal > nasal > buccal > rectal. Absorption was more rapid after both intratracheal and rectal administration ( t max ∼ 20–40 min), compared with that after nasal and rectal administration. It is evident that the pulmonary route is preferable to other three routes for successful systemic delivery of rHV2.

Hirudin ameliorates intestinal radiation toxicity in the rat: support for thrombin inhibition as strategy to minimize side‐effects after radiation therapy and as countermeasure against radiation exposure

The small bowel is a dose-limiting normal tissue in radiation therapy of malignancies in the abdomen and pelvis, as well as an important determinant of survival after non-therapeutic radiation exposure. Irradiation of normal tissues, including intestine, causes loss of vascular thromboresistance and upregulation of thrombin receptors. Radiation-induced endothelial dysfunction is thought to be involved in both early and delayed radiation responses. Hence, thrombin may be a potential target for ameliorating normal tissue radiation toxicity. To assess direct thrombin inhibition as a protective strategy against small bowel radiation toxicity. Rat small intestine was exposed to localized orthovoltage X-radiation. Recombinant hirudin, a direct thrombin inhibitor, or vehicle was infused from 2 days before irradiation to 14 days after irradiation. Structural, cellular, and molecular aspects of intestinal radiation injury were assessed at 2 weeks (early toxicity) and 26 weeks (chronic toxicity) after irradiation. Compared with unirradiated intestine, irradiated intestine showed increased expression of tissue factor, increased immunoreactivity for enzymatically active thrombin, and increased extravascular fibrin(ogen) deposition. Hirudin treatment significantly attenuated radiation-induced mucosal damage (P = 0.04), reactive intestinal wall thickening (P = 0.02), transforming growth factor-beta immunoreactivity levels (P = 0.0002), and collagen III deposition (P = 0.003). The differences between hirudin-treated and control rats were more pronounced at 2 weeks than at 26 weeks after irradiation. Hirudin treatment did not affect postradiation granulocyte infiltration. Short-term thrombin inhibition attenuates important aspects of intestinal radiation toxicity. Thrombin is a promising target for minimizing normal tissue injury after radiation therapy of cancer, as well as for protecting normal tissues from the adverse effects of non-therapeutic radiation exposure.

Anti-Xa and Anti-IIa Drugs Alter International Normalized Ratio Measurements: Potential Problems in the Monitoring of Oral Anticoagulants

Several of the newly developed anti-Xa and anti-IIa agents have been shown to influence the International Normalized Ratio (INR) values. During phase I trials with normal healthy volunteers and phase II study patients who were given warfarin and concomitant anti-IIa or anti-Xa agents, it has been reported that INR values were falsely elevated. It is of critical importance to know of the effects of these agents on INR to avoid dosage errors. To study the influence of these agents on INR, we used several anti-IIa agents (argatroban, recombinant hirudin, efegatran, and PEG-hirudin) and anti-Xa drugs (pentasaccharides such as fondaparinux and idraparinux, DX-9065a and JTV-803). The anti-IIa drugs were supplemented in citrated plasma at a concentration of 0 to 1 microg/mL level and anti-Xa drugs in the range of 0 to 25 microg/mL. The IC(50) values for each of these agents were calculated. Four different commercially available prothrombin time (PT) reagents were used to perform the PT assays and to calculate the relative INR values. Direct synthetic factor IIa and Xa inhibitors exhibited a concentration-dependent increase in the INR values. Hirudin, efegatran, and PEG-hirudin showed a weaker effect, whereas argatroban showed a much higher elevation of the INR values. Synthetic indirect anti-Xa agents such as the pentasaccharide did not show any effect on the INR values. Furthermore, prothrombin time reagents with high ISI values exhibited disproportionally higher INR values for both the direct anti-Xa and anti-IIa agents. Elevation of INR values has therapeutic implications when non-oral anticoagulant drugs are used in combination with drugs such as warfarin. Because of the false elevation of INR values with some of the non-oral anticoagulant drugs, patients who are on concomitant warfarin therapy should be carefully evaluated for their corresponding INR values for proper dosing. To avoid dosing errors it is best not to use the INR values in the therapeutic monitoring of anti-Xa and anti-IIa agents either in the monotherapeutic or polytherapeutic modalities. These data also warrant the development clinically relevant methods for the monitoring of the concomitant use of newly developed anti-Xa and anti-IIa drugs with oral anticoagulants.

Management der akuten heparininduzierten Thrombozytopenie Typ II während der Schwangerschaft und peripartal

In pregnancy and in puerperium heparin is prescribed only for acute thromboembolism and/or for female patients with a clearly increased risk of venous thromboembolism. Since treatment changed from unfractionated heparin to low-molecular-weight heparin, the incidence of heparin-induced thrombocytopenia type II (HIT type II) is therefore a rare event in this collective. HIT type II is a life threatening side effect of heparin which must immediately lead to the stop of heparin administration. Experiences with alternative anticoagulation in pregnancy and puerperium are limited. Oral anticoagulation with coumarin (vitamin K antagonists) is contraindicated for the initial therapy of HIT II. The treatment with these substances is dangerous, especially in the phase oforganogenesis, because it can cause malformation and because of the elevated maternal and the child intra- and peripartal bleeding risk in the 2nd half of the third trimenon. Danaparoid Sodium and recombinant Hirudin represent treatment options, whereby because of the better data and the intravenous and especially the subcutaneous application form the application of Danaparoid in the therapy of HIT typ II in pregnancy would appear to be more favourable. In the puerperium Warfarin or Phenprocoumon represent the therapy of choice. Breast-feeding is possible with this option. We report on a female patient with an acute thrombosis of the cerebral sinus vein during early pregnancy who developed a HIT type II under heparin treatment. The long-term anticoagulation with Danaparoid Sodium and the peripartale management are presented.

Type I and Type IV allergy to unfractionated heparin and low‐molecular‐weight heparin with no reaction to recombinant hirudin

Type I and Type IV allergy to unfractionated heparin and low‐molecular‐weight heparin with no reaction to recombinant hirudin

Fondaparinux: a suitable alternative in cases of delayed‐type allergy to heparins and semisynthetic heparinoids? A study of 7 cases

Hypersensitivity to unfractionated and low-molecular-weight heparins and semisynthetic heparinoids is increasingly common. 7 female patients between 30 and 74 years with delayed-type allergy to heparins and semisynthetic heparinoids were investigated for (cross)-reactivity to fondaparinux, a new pentasaccharide with selective factor Xa inhibition. All patients showed delayed-type reactions to heparins and some additional cross-reaction to a heparinoid on intracutaneous testing. 6/7 tolerated fondaparinux on intradermal testing as well as on subcutaneous challenge testing. However, the 7th patient developed a characteristic delayed-type reaction to both skin tests with fondaparinux. Fondaparinux is a new synthetic pentasaccharide with a molecular weight of 1.728 Da. In some patients with cross-reactivity between various heparins and semisynthetic heparinoids, lepirudin, a recombinant hirudin, may be a safe and effective alternative. However, combined allergy to hirudin and heparins has been reported. Sometimes, intravenous administration of heparins or heparinoids may be tolerated. However, these patients are at risk of developing a systemic reaction. The pathogenesis of heparin hypersensitivity is not fully understood. Heparins may act as haptens by binding to dermal and/or subcutaneous structural proteins. The chemical structures of heparins and fondaparinux are different concerning their alpha- and beta-configuration and the molecular weight. However, some of their functional groups are nearly identical and therefore similar chemical and pharmacological reactivity is to be expected. Fondaparinux seems to be a valuable alternative in most cases of heparin and hirudin hypersensitivity. The clearly rare cross-reaction between fondaparinux and heparins, now confirmed by us, may be due to differences in the response to haptens.

Recombinant hirudin enhances cardiac output and decreases systemic vascular resistance during reperfusion after cardiopulmonary bypass in a porcine model

Objective Cardiopulmonary bypass and surgical stress are accompanied by a systemic inflammatory response and activation of coagulation. Thrombin forms fibrin and activates platelets and neutrophils. Consequently, disseminated microthrombosis might increase capillary vascular resistance and thus impair reperfusion. We hypothesized that recombinant hirudin, a direct inhibitor of thrombin, could attenuate coagulation and enhance microvascular flow during reperfusion. Methods Twenty pigs undergoing 60 minutes of aortic clamping and 75 minutes of normothermic perfusion were randomized in a blinded setting to receive an intravenous bolus of recombinant hirudin (10 mg, 0.4 mg/kg; n = 10) or placebo (n = 10) 15 minutes before aortic declamping and then continued with an intravenous 135-minute infusion of recombinant hirudin (3.75 mg/h, 0.15 mg/kg) or placebo. Thrombin-antithrombin complexes, activated clotting times, and several hemodynamic parameters were measured before cardiopulmonary bypass, after weaning from cardiopulmonary bypass, and at 30, 60, 90, and 120 minutes after aortic declamping. Intramucosal pH and P co 2 were measured from the luminal surface of ileum simultaneously with arterial gas analysis at 30-minute intervals. Results Recombinant hirudin inhibited thrombin formation after aortic declamping; at 120 minutes, thrombin-antithrombin complexes levels (μg/L, mean ± SD) were 75 ± 21 and 29 ± 44 ( P < .001) for placebo and pigs receiving recombinant hirudin, respectively. When compared with the placebo group, pigs receiving recombinant hirudin showed significantly higher stroke volume, cardiac output, and lower systemic vascular resistance at 60 and 90 minutes after aortic declamping ( P < .05). Based on arteriomucosal P co 2 and pH differences, progressive worsening of intestinal microcirculatory perfusion occurred in the placebo group but not in the recombinant hirudin group. Conclusion Infusion of thrombin inhibitor recombinant hirudin during reperfusion was associated with attenuated postischemia left ventricular dysfunction and decreased vascular resistance. Consequently microvascular flow was improved during ischemia-reperfusion injury. Control of thrombin formation during reperfusion may be a feasible approach to improve oxygen delivery to reperfused vascular beds.

Heparin‐induced thrombocytopenia during renal replacement therapy

There is increasing awareness that antibodies to heparin/platelet factor 4 complex can develop in both those with acute renal failure treated with continuous forms of renal replacement therapy and, to a lesser extent, regular hemodialysis patients. Clinical manifestations include premature clotting of the extracorporeal circuit and increased central venous thrombosis with indwelling venous dialysis catheters, associated with thrombocytopenia and increased platelet activation. Nevertheless, a minority of patients who develop heparin/platelet factor 4 antibodies do not have thrombocytopenia or thrombosis. When systemic anticoagulation is also required to treat venous thrombosis, then synthetic heparinoids or the direct thrombin inhibitors argatroban or recombinant hirudins are the agents of choice. Neither the synthetic heparinoids nor hirudin, however, are without their own problems, in that a minority of patients may have cross-reacting antibodies against the currently available heparinoids, and antibodies may similarly develop against recombinant hirudin, in this case leading to a potentiation of anticoagulant activity and increased risk of hemorrhage.

Lepirudin for therapeutic use in heparin-induced thrombocytopenia

Heparin-induced thrombocytopenia (HIT) type II is an antibody mediated severe adverse event to heparin with a paradoxical decrease of platelet count and an increased risk for thromboembolic complications. The antibodies are directed against a neoepitop of platelet factor 4 after its binding to heparin. The incidence of HIT type II is lower with low-molecular-weight heparin compared to unfractionated heparin and lower in not operated patients compared to those after major surgery. In patients with HIT type II alternative anticoagulation has to be performed immediately due to the high thrombogenicity of the antibodies. The recombinant hirudin lepirudin (Refludan) is the anticoagulant drug of choice. A long-term anticoagulation has to be performed depending on the concomitant risk factors, intravenous administration followed by subcutaneous lepirudin overlapping with vitamin K antagonists.

Pharmacokinetics of recombinant hirudin in healthy horses.

Recombinant (r)-hirudin is a specific inhibitor of thrombin that is independent of the activity of antithrombin. To evaluate pharmacokinetic properties and coagulatory changes of r-hirudin in healthy horses. Two clinically healthy horses received a single i.v. bolus of 0.4 mg/kg bwt r-hirudin and 6 clinically healthy horses received the same dose subcutaneously (subcut.) q. 12 h for 3 days. Coagulation times and r-hirudin plasma concentration were determined over 720 mins and 3 days after i.v. and subcut. administration, respectively. In all horses, treatment with r-hirudin was not associated with systemic or local side effects. After i.v. injection, the 2 horses showed an elimination half-life of 58 and 80 mins, respectively. After subcut. administration, maximum plasma concentration of r-hirudin occurred at 128 +/- 55 mins and declined with a terminal half-life of 561 +/- 364 mins. Maximum response of activated partial thromboplastin time (aPTT) occurred 1.5 h after administration of r-hirudin. A prolongation of 1.9 +/- 0.2 times the pretreatment value was noted. Pharmacokinetics of r-hirudin in healthy horses were similar to those in man and other animal species. The results of this study indicate that r-hirudin can be used in horses, but further studies should be performed in order to prove its effectiveness in diseased horses.

Antibodies against lepirudin are polyspecific and recognize epitopes on bivalirudin

AbstractBivalirudin is a synthetic antithrombin sharing a sequence of 11 amino acids with the recombinant hirudin lepirudin. We investigated whether antilepirudin antibodies recognize epitopes on bivalirudin. Antilepirudin antibody–positive sera of 43 patients, treated with lepirudin for heparin-induced thrombocytopenia, were analyzed. Lepirudin- and bivalirudin-coated microtiter plates were used for antibody testing in an enzyme-linked immunosorbent assay (ELISA) system. Of the 43 sera-containing antibodies binding to lepirudin, 22 (51.2%) contained antibodies that also recognized bivalirudin. Binding of these antibodies to bivalirudin was inhibited by more than 70% by preincubation with high doses of bivalirudin. However, if lepirudin-coated microtiter plates were used, high concentrations of bivalirudin inhibited only 2 of the 43 positive sera by more than 30%. Therefore antihirudin antibodies must be polyspecific. The clinical consequences of this cross-reactivity are unknown but bivalirudin, targeted by antibodies of patients treated with lepirudin previously, could potentially boost antibody titers in such patients or even trigger an immune response by itself. Clinically significant antibody formation in response to bivalirudin monotherapy has not been observed, however. Yet, as lepirudin and antilepirudin antibodies have recently been implicated in severe anaphylactic reactions, caution is warranted when using bivalirudin in patients previously treated with lepirudin.