Recombinant Hirudin Research Articles

Hirudin and hirudin analogues as new anticoagulant agents

Recombinant hirudin and hirudin analogues constitute interesting new antithrombotic agents that have distinct advantages over heparin. These agents specifically inhibit thrombin and all of its actions and also suppress further thrombin generation. As opposed to unfractionated heparin, hirudin and hirulog effectively suppress clot-bound thrombin, making these agents of particular interest in the treatment of arterial thrombosis, for example, following thrombolysis or percutaneous transthoracic angioplasty. The recent data derived from clinical trials supporting the use of hirudin and hirulog in the prevention and treatment of thrombotic diseases are reviewed here.

Evaluation of an automated chromogenic substrate assay for the rapid determination of hirudin in plasma

A fully mechanized chromogenic substrate assay method for the rapid and specific determination of recombinant hirudin (r-hirudin) in citrated plasma on clinical chemistry analyzers (Hitachi 911 and Cobas Mira) is described. In a first step, 12 μl sample volume is mixed with the chromogenic substrate. Due to the almost immediate action of hirudin the inhibitory reaction and the cleavage of the substrate is started simultaneously when bovine thrombin is added in excess. This excludes interferences by antithrombin III or heparin cofactor II. The change in absorbance/min is recorded at 405 nm. The measuring range is about 0.2 – 4.0 mg/l r-hirudin on both analyzers. Precision is characterized by intraassay coefficients of variation between 0.63 % and 2.78 % on the Hitachi 911 and 1.51 % and 7.84 % on the Cobas Mira, respectively and interassay coefficients of variation of 3.57 % to 9.15 % (Hitachi 911) and 3.72 % to 12.99 % (Cobas Mira) for the same r-hirudin plasma concentrations. The described determination of r-hirudin correlates well with an enzyme linked immunosorbent assay method for r-hirudin (Hitachi 911: r = 0.964, y = 0.978x + 0.038, n = 323; Cobas Mira: r = 0.964, y = 0.959x − 0.003, n = 323).

Heparin-associated thrombocytopenia: an update.

The use of heparin may be complicated by two types of thrombocytopenia (HAT): type I occurs early, is transient, and has no clinical relevance, while type II may lead to very severe manifestations (arterial or venous thromboses and more rarely bleedings), that are still underestimated by some clinicians. HAT-type II most frequently develops after use of therapeutic doses of unfractionated heparin (UH) but has also been described less frequently after use of very low doses of UH, of low molecular weight heparins (LMWH), and even of polysulfated glycosaminoglycosans devoid of anticoagulant action. The estimation of the incidence of HAT-type II and of related thromboses is a very difficult matter. Recent observations suggest that thromboses (notably venous) may be more frequent than previously estimated. HAT-type II pathophysiology includes the formation of immune complexes at the surface of platelets; the antigen has been shown to be most often platelet factor 4 bound to heparin while the antibody is recognized by platelet Fc gamma RII receptors. Thromboses result most probably from activation of both platelets (leading to the formation of microparticles) and endothelial cells. Several biological tests are presently available for diagnosing HAT-type II but none of them has been shown to be ideal. The prevention of HAT-type II requires history taking preference of LMWH to UH, early start of oral anticoagulation, and platelet monitoring from the fifth day of heparin therapy. The therapy of HAT-type II implies immediate discontinuation of heparin and avoidance of platelet transfusions, unless severe bleeding occurs. If further antithrombotic treatment is deemed necessary (probably in all cases), several options are possible but presently, the most recommended ones are Org 10172 or Ancrod; embolectomy or thrombolysis may also be required if a new thrombotic event has developed. A very difficult dilemma concerns patients previously sensitized to heparin and who present a clinical situation that theoretically mandates re-exposure to UH, such as by-pass surgery; prostacyclin analogues may be successfully used in such cases. Recent developments in the therapy of HAT-type II include recombinant hirudin or synthetic analogs, or use of some intravenous immunoglobulin preparations. Possible candidates are the heparin synthetic pentasaccharide and recombinant tissue factor pathway inhibitor.

Process Development for the Production of Recombinant Hirudin in Saccharomyces cerevisiae: from Upstream to Downstream

Process Development for the Production of Recombinant Hirudin in Saccharomyces cerevisiae: from Upstream to Downstream

Inhibition of clot-bound and free (fluid-phase thrombin) by a novel synthetic thrombin inhibitor (Ro 46-6240), recombinant hirudin and heparin in human plasma.

Clot-bound thrombin remains active and is less accessible to heparin-antithrombin III than fluid-phase thrombin. To determine whether clot-bound human thrombin is more susceptible to inactivation by direct thrombin inhibitors, the activity of a novel synthetic competitive thrombin inhibitor Ro 46-6240, recombinant hirudin and unfractionated heparin were compared with fluid-phase thrombin and clot-bound thrombin. Fibrinopeptide A generated in human plasma was used as an index of thrombin activity. Hirudin was the most potent inhibitor of fluid-phase and clot-bound thrombin. However, Ro 46-6240 inhibited clot-bound thrombin three times more potently than fluid-phase thrombin (IC50 19 vs 56 ng/ml) while hirudin was two times (IC50 8 vs 3 ng/ml) and heparin six times (IC50 1,205 vs 200 ng/ml) less active against clot-bound thrombin compared with fluid-phase thrombin. The relative selectivity for clot-bound thrombin is not a unique property of Ro 46-6240 since two other synthetic thrombin inhibitors tested inhibited clot-bound thrombin more effectively than fluid-phase thrombin and a third was equally active against both forms of thrombin. In contrast, the affinities of two chromogenic substrates were similar for both forms of thrombin. This study shows that direct thrombin inhibitors inhibit clot-bound thrombin more potently than heparin and suggests that an apparent selectivity for clot-bound thrombin can be achieved with some synthetic thrombin inhibitors. Further studies have to show whether the high potency of these direct thrombin inhibitor translates into antithrombotic efficacy in clinical situations with pre-existing clots.

Laboratory Monitoring of New Antithrombotic Drugs

This article examines laboratory methods that are used or may be used to monitor newly developed heparins, low-dose vitamin K antagonists, new thrombin inhibitors such as recombinant hirudin, and oligopeptide thrombin inhibitors (some of which it is hoped will be orally active), thromboxane receptor antagonists, glycoprotein IIb-IIIa inhibitors, and ticlopidine-like structures.

Deamidation of asparagine and glutamine residues in proteins and peptides: structural determinants and analytical methodology.

Non-enzymatic deamidation of asparagine and glutamine residues in proteins and peptides are reviewed by first outlining the well-described reaction mechanism involving cyclic imide intermediates, followed by a discussion of structural features which influence the reaction rate. The second and major part describes analytical techniques that allow studying deamidation in proteins using recombinant human growth hormone and recombinant hirudin as examples. Finally, the significance of non-enzymatic deamidation with respect to the production of pharmaceutical proteins is discussed.

Prevention of Rethrombosis After Coronary Thrombolysis in a Chronic Canine Model. II. Adjunctive Therapy with r-Hirudin

We examined the effectiveness of the direct-acting thrombin inhibitor, recombinant hirudin (r-hirudin), for prevention of coronary rethrombosis after thrombolysis with recombinant tissue plasminogen activator (rt-PA) in a canine model of coronary artery thrombosis. The reocclusion rate of 15-30% associated with thrombolytic therapy emphasizes the need for adjunctive therapy to prevent rethrombosis. We studied r-hirudin for its potential to prevent reocclusion in a model of coronary artery thrombosis/thrombolysis. The circumflex coronary arteries of anesthetized dogs were instrumented with a flow probe, an intraluminal electrode, and a ligature stenosis. The dogs were reanesthetized on the ninth postoperative day, and intimal injury was induced with an anodal current. After occlusive thrombus formation, tissue plasminogen activator (rt-PA) was administered. The animals were allocated to receive either placebo, r-hirudin [5 mg/kg intravenously (i.v.) bolus, 2 mg/kg/h i.v., for 3.5 h] or r-hirudin (5 mg/kg i.v., bolus, 1 mg/kg/h i.v., for 12 h). Neither aspirin nor heparin was used. Ex vivo platelet function and coronary artery blood flow velocity were recorded on each of 5 consecutive days. Infarct size and residual thrombus weight were determined at the end of the protocol. r-Hirudin infusion (3.5 and 12 h) provided little benefit over rt-PA alone. Ex vivo platelet aggregation was not affected by r-hirudin. Little improvement in the incidence of reocclusion and mortality in a model of coronary artery thrombosis/thrombolysis resulted from adjunctive treatment with r-hirudin.

Inhibition of growth of thrombus on fresh mural thrombus. Targeting optimal therapy.

Residual mural thrombus on severely damaged arterial wall is very thrombogenic. We tested the hypothesis that direct thrombin inhibition will block thrombus growth on fresh thrombus better than indirect thrombin inhibition, cyclooxygenase inhibition, or both. A fresh mural thrombus was formed by directly perfusing fresh porcine blood for 5 minutes over severely damaged arterial wall at a high shear rate in a well-characterized ex vivo perfusion system. The average platelet (P) and fibrinogen (F) deposition (D) achieved in 5 minutes were 382 +/- 32 x 10(6) platelets/cm2 and 296 +/- 36 x 10(12) fibrinogen molecules/cm2, respectively. Thrombus growth on the fresh mural thrombus was quantitated by directly perfusing blood from pigs with 111In-labeled platelets and 125I-labeled fibrinogen for an additional 5 minutes over the preformed mural thrombus. Treatment included recombinant hirudin (1 mg/kg per hour IV) as a probe for thrombin, aspirin (5 mg/kg IV) as a platelet inhibitor of cyclooxygenase, heparin (moderate, 100 IU/kg per hour IV; high-dose, 250 IU/kg per hour IV) as an indirect thrombin inhibitor, and heparin (100 IU/kg per hour) plus aspirin (5 mg/kg IV). Thrombus growth as measured by labeled PD (x 10(6)/cm2) and FD (x 10(12) molecules/cm2) was mildly but not significantly reduced by aspirin (1034 +/- 92 and 436 +/- 78, respectively) compared with baseline (1113 +/- 67 and 545 +/- 52, respectively). Inhibition of thrombus growth with heparin was dose dependent. A regression analysis showed an inverse correlation of PD and FD with mean plasma heparin concentrations (r = -.81, P = .0001 and r = -.49, P = .0007, respectively). Recombinant hirudin led to a profound inhibition of thrombus growth (PD, 30 +/- 12; FD, 109 +/- 21), which was significant compared with all groups, even the highest dosage of heparin (250 IU/kg per hour). Specific thrombin inhibition markedly inhibits platelet and fibrinogen deposition onto fresh mural thrombus at a high shear rate. Aspirin alone or in combination with heparin has little effect on evolving thrombosis. Heparin dose dependently reduces thrombus growth, but even the highest dosage is less effective than hirudin. Thrombin appears to be the primary activator of platelets by fresh thrombus.

Randomized trial of intravenous heparin versus recombinant hirudin for acute coronary syndromes. The Global Use of Strategies to Open Occluded Coronary Arteries (GUSTO) IIa Investigators.

Although intravenous heparin is routinely used in the treatment of patients with acute coronary syndromes, this anticoagulant requires antithrombin III as a cofactor, has no affinity to clot-bound thrombin, and is bound or inactivated by several plasma proteins and platelet factor 4. Recombinant hirudin, the prototypic direct thrombin inhibitor, has been demonstrated in pilot studies to yield improved angiographic and clinical outcomes compared with heparin. We compared these two antithrombins in a large-scale randomized trial. At 275 participating hospitals in 12 countries, patients within 12 hours from the onset of ischemic chest discomfort with an abnormal ECG were randomly assigned to receive a 72- to 120-hour infusion of heparin (5000-U bolus and 1000- to 1300-U/h infusion, adjusted to activated partial thromboplastin time (APTT) of 60 to 90 seconds or hirudin (0.6-mg/kg bolus and 0.2-mg/kg per hour infusion without aPTT adjustment) on a double-blind basis. Although recruitment of 12,000 patients was planned, the trial was stopped earlier because of an excess of intracerebral hemorrhagic events after 2564 patients were enrolled. The overall incidence of hemorrhagic stroke tended to be higher for patients receiving hirudin (1.3%) compared with heparin (0.7%), P = .11, but the incidence was significantly higher in patients receiving thrombolytic therapy (1264 patients, 1.8%) compared with those who did not (1168 patients, 0.3%), P < .001. The hemorrhagic stroke rate varied by the thrombolytic and antithrombin combination: tissue-type plasminogen activator and heparin, 0.9%; with hirudin, 1.7%; streptokinase with heparin, 2.7%; with hirudin, 3.2%. All these rates are higher than the overall incidence of hemorrhagic stroke in the patients receiving thrombolytic therapy and intravenous heparin in the GUSTO I trial (30,892 patients with rate of 0.7%, 95% CI of 0.6 to 0.8%). Among the 26 patients who had intracerebral hemorrhages, the aPTT was significantly elevated compared with the event-free patients (110 +/- 46 versus 87 +/- 36 seconds at 12 hours of therapy, respectively), P = .03. At the dose of hirudin tested, there was a trend of an excess of hemorrhagic stroke compared with heparin. Heparin, at a slightly higher dose than previously used in a large-scale trial (approximately 20% increase) was accompanied by a twofold risk of hemorrhagic stroke in patients receiving thrombolytic therapy. With both thrombin inhibitors, the aPTT appears to be a useful index for predicting risk of hemorrhagic stroke in patients receiving thrombolytic therapy.

Fibrin-targeted recombinant hirudin inhibits fibrin deposition on experimental clots more efficiently than recombinant hirudin.

Although the indirect thrombin inhibitor heparin and the more potent direct inhibitor hirudin are useful in preventing thrombosis, a substantial opportunity remains for improving the thrombus selectivity of thrombin inhibitors. To explore the effect of targeting an antithrombin to the surface of a clot, we covalently linked recombinant hirudin to the Fab' (or IgG) of a monoclonal antibody (59D8) that selectively binds to an epitope on fibrin that becomes exposed only after thrombin cleaves fibrinopeptide B. Antibody-coupled hirudin bound to an immobilized peptide of the fibrin beta-chain amino-terminal sequence and inhibited the peptidolytic activity of thrombin more efficiently than free hirudin. Thrombin inhibition dependent on binding to immobilized fibrin monomer was enhanced 1100-fold (P < .0001). Hirudin-59D8 Fab' was 10 times more effective than hirudin in inhibiting fibrin deposition on experimental clot surfaces in fibrinogen solution (P < .0001) and human plasma (P < .0001). The more effective inhibition of thrombin by the conjugate was supported by significantly diminished concentrations of fibrinopeptide A in the plasma supernatant of the clot (P = .0001). Inhibition of clotting by an uncoupled mixture of hirudin and 59D8 Fab' was indistinguishable from that by hirudin alone, indicating that the conjugate's greater inhibitory activity was due to the covalent linkage between antibody and hirudin. Fibrin-targeted hirudin (in comparison with unmodified hirudin) significantly reduces fibrin deposition on the surface of experimental clots.

Thrombolytic therapy in acute myocardial infarction—Selected recent developments

Thrombolytic therapy is the established treatment of choice for most eligible patients with acute myocardial infarction. Early initiation of treatment and early, complete and maintained patency of the infarct-related coronary artery are desirable, because these variables correlate with a reduction in mortality. As a consequence, considerable efforts have been undertaken to develop new pharmacological agents that serve these purposes. Among these, new plasminogen activators such as reteplase (r-PA), saruplase (scuPA), and staphylokinase are in clinical development, and DSPA (bat t-PA) and antibody-targeted plasminogen activators (ScuPA-59D8) have undergone extensive animal testing. Anticoagulants such as recombinant hirudin, hirulog, argatrobane, and Factor Xa inhibitors, as well as antiplatelet agents on the basis of monoclonal antibody 7E3 offer promise as adjunctive therapy to thrombolysis or to invasive intracoronary procedures.

Recombinant hirudin as haemodialysis anticoagulant

Recombinant hirudin as haemodialysis anticoagulant

Enzymic O‐sulfation of tyrosine residues in hirudins by sulfotransferase from Eubacterium A‐44

The enzymic O-sulfation of Tyr residues in a recombinant hirudin variant-1 (rHV-1) and its analog in which Glu61 and Glu62 were replaced by Tyr, [E61Y, E62Y]rHV-1, was carried out by use of sulfotransferase isolated from an anaerobic bacterium from the human intestine, Eubacterium A-44. Although rHV-1 was not sulfated by this enzyme, the sulfation of [E61Y, E62Y]rHV-1 was observed, and three kinds of sulfated analog, whose C-terminal six amino acid residues were -PYY(SO3H)YLQ, -PYYY(SO3H)LQ, and -PYY(SO3H)Y(SO3H)LQ, were obtained. Among the sulfated hirudin analogs tested here, the Tyr62 and Tyr63 bisulfated [E61Y, E62Y]rHV-1 showed the strongest thrombin inhibition with the inhibition constant (Ki) of 0.0430 pM, followed by the Tyr63 monosulfated analog (Ki = 0.0593 pM) and the Tyr62 monosulfated one (Ki = 0.158 pM). The Tyr63 monosulfated analog and Tyr62 and Tyr63 bisulfated one were more potent inhibitors of thrombin than unsulfated rHV-1. The increase in affinity caused by sulfation was predominantly due to an increase in the association-rate constant.

Recombinant hirudin: A specific thrombin inhibiting anticoagulant for hemodialysis

The first experience with hirudin as an alternative anticoagulant for heparin in hemodialysis is reported. Recombinant hirudin (HBW 023) was administered in 20 patients as a bolus before dialysis with low flux polysulfone dialyzers (PS400), the dosage being adapted stepwise from patient to patient by 0.02 mg/kg to the occurrence of clotting or bleeding. Four different administration schedules were studied. The first three schedules (0.02 mg/kg, N = 1; 0.04 mg/kg, N = 1; 0.06 mg/kg, N = 4) were discontinued because of clotting. The 0.08 mg/kg schedule was maintained without clotting event in 14 patients. Bleeding was not observed. Plasma hirudin averaged 503.9 +/- 214.0 and 527.7 +/- 217.1 ng/ml after two and four hours of dialysis, and decreased during an interdialytic interval of 44 hours to 223.2 +/- 86.2 ng/ml. Modified antithrombin III (P < 0.05) and activated partial thromboplastin times were lower (P < 0.01) under hirudin compared to heparin; these coagulation parameters were closer to normal during hirudin treatment. The patients developing clotting could be distinguished from those without clotting by the registration of the activated clotting times (9.2 +/- 3.0 vs. 18.7 +/- 3.2 min after 2 hr, P < 0.01; 8.1 +/- 3.0 vs. 16.2 +/- 3.8 min after 4 hr of dialysis, P < 0.05); cut-off value below which clotting is to be expected was 12 min). It is concluded that administration of hirudin as a bolus before the start of dialysis, at a dosage of 0.08 mg/kg, is not complicated by clotting or by bleeding. Coagulation tendency can optimally be monitored by the registration of the activated clotting time.(ABSTRACT TRUNCATED AT 250 WORDS)

Pilot trial of recombinant hirudin in deep vein thrombosis

Pilot trial of recombinant hirudin in deep vein thrombosis

Fibrinolytic compromise by simultaneous administration of site-directed inhibitors of thrombin

Newly developed synthetic and recombinant thrombin inhibitors possess strong anticoagulant effects. Despite these effects, interactions of these agents with enzymes in the fibrinolytic network result in the modulation of such proteases as t-PA, u-PA and streptokinase. The inhibitory spectrum of several thrombin inhibitors [D-Phe-Pro-Arg-H (GYKI 14166), D-MePhe-Pro-Arg-H (GYKI 14766), Boc-D-Phe-Pro-Arg-H (GYKI 14451), Ac-D-Phe-Pro-boroArg-OH (DuP 714), recombinant hirudin (r-Hir) and unfractionated porcine mucosal heparin complexed with antithrombin III (Heparin/AT-III)] was studied towards various serine proteases such as tissue plasminogen activator (t-PA), plasmin, plasminogen/streptokinase complex, urokinase and kallikrein. Aprotinin was also studied in the same systems as the thrombin inhibitors. All four tripeptide derivatives were found to inhibit t-PA, plasmin and plasminogen/streptokinase complex at micromolar concentrations (IC 50: 0.57 mM - 3.3 μM). Boc-D-Phe-Pro-Arg-H and Ac-D-Phe-Pro-boroArg-OH also inhibited urokinase, while Ac-D-Phe-Pro-boroArg-OH inhibited kallikrein as well (IC 50: 0.15 mM - 16 μM). In contrast, r-Hir and Heparin/AT-III did not inhibit any of these enzymes at millimolar concentrations (IC 50≥ 1 mM). Aprotinin inhibited plasmin, plasminogen/streptokinase complex and kallikrein at micromolar concentrations (IC 50: 3.1-0.85 μM). In a rabbit thrombolysis model, where pre-formed clots are lysed by streptokinase, simultaneous administration of D-MePhe-Pro-Arg-H or Ac-D-Phe-ProboroArg-OH, at concentrations ∼ 1 μmol/kg, I.V. resulted in complete inhibition of the fibrinolytic process. Aprotinin at 0.1 μmol/kg, I.V. produced similar inhibition. These results demonstrate that thrombin inhibitors may exert significant antiprotease actions against various fibrinolytic enzymes.

Two-site immunoassay of recombinant hirudin based on two monoclonal antibodies

Two monoclonal antibodies (mAbs), 10-2 and 10-5, both directed against recombinant hirudin variant 2-Lys47 (rHV2), were selected for their high affinity and epitopic specificities to develop a two-site immunoassay of rHV2. The mAb concentrations, incubation time, and temperature were optimized. The immunoassay has a detection limit for rHV2 of 45 ng/L in plasma and 30 ng/L in urine. The reactivity of the mAbs was tested against rHV2 and several forms of this protein truncated in the carboxyl terminus. The capture mAb 10-2 was found to be mainly directed against rHV2, whereas tracer mAb 10-5 was independent of the carboxyl-terminal region of the protein. This explains the high specificity of the immunoassay for the 65-amino acid form of hirudin.

Mapping of the Thrombin des-ETW Conformation by Using Site-Directed Mutants of Hirudin. Evidence for the Induction of Nonlocal Modifications by Mutagenesis

Deletion of Glu146, Thr147, and Trp148 from thrombin dramatically alters its interactions with substrates, ligands, and inhibitors, and the changes appear to result from nonlocal modification of thrombin's structure [Le Bonniec, B. F., Guinto, E. R., & Esmon, C. T. (1992) J. Biol. Chem. 267, 19341-19348]. In an attempt to localize conformational change in this thrombin mutant (des-ETW), its affinity for 43 site-directed mutants of hirudin have been compared with that of native thrombin. The inhibition constants (KI) of recombinant and natural hirudin with des-ETW were found to be approximately 2300-fold higher than those with thrombin. The reduced affinity was predominantly the result of an increase in the dissociation rate constant rather than a decrease in the association rate constant. For most of the hirudin mutants tested, the difference in binding energy between thrombin and des-ETW [delta delta Gbo(IIa-ETW)] was about 19.9 kJ mol-1 and comparable to that of hirudin; in particular, all mutations in the C-terminal region of the inhibitor did not affect delta delta Gbo(IIa-ETW). Thus, the conformation of thrombin's anion-binding exosite seems unaltered by the des-ETW mutation. In contrast, hirudin substitutions involving Val1', Val2', Thr4', Asp5', and Ser19', as well as the addition of an N-terminal glycine, resulted in values of delta delta Gbo(IIa-ETW) which were 2 to 10 kJ mol-1 lower than that for hirudin. Furthermore, a Leu15'-->Ala hirudin mutant behaved with des-ETW, as a partial competitive inhibitor rather than a tight-binding inhibitor, in contrast to all other hirudin variants tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Combination of platelet fibrinogen receptor antagonist and direct thrombin inhibitor at low doses markedly improves thrombolysis.

We evaluated the effects of a novel platelet fibrinogen receptor antagonist, Integrelin, and a direct thrombin inhibitor, recombinant hirudin, given together with recombinant tissue plasminogen activator (rTPA) in a canine experimental model of intracoronary thrombosis. We tested the hypothesis that combination of both agents at low doses would have an additive antithrombotic effect, resulting in a significant improvement in the efficacy of rTPA. Thirty-two dogs with an electrically induced coronary thrombus were treated with rTPA (1 mg/kg over 20 minutes) together with one of the following adjunctive treatments in a random fashion. Eight dogs received saline for 90 minutes; Integrelin (5 micrograms.kg-1.min-1 for 90 minutes) was given to 8 dogs; 8 dogs received recombinant hirudin (20 micrograms.kg-1.min-1 for 90 minutes); and 8 dogs were treated with a low-dose combination of Integrelin (2.5 micrograms.kg-1.min-1) plus recombinant hirudin (10 micrograms.kg-1.min-1) for 90 minutes. Integrelin or recombinant hirudin, when given as single adjunct to rTPA, enhanced the lysis of the occlusive thrombus, causing full restoration of coronary blood flow (100% of its baseline value) for 29 +/- 16 and 26 +/- 5 minutes, respectively, whereas coronary blood flow was fully restored for only 5 +/- 1 minutes in dogs receiving rTPA plus saline (both P < .05). However, either Integrelin or recombinant hirudin failed to modify the reocclusion rate (57% and 63%, respectively) compared with saline (83%; all P = NS). Conversely, the low-dose combination therapy led to complete restoration of coronary blood flow for 92 +/- 19 minutes (P < .01 versus all treatments) and significantly reduced the reocclusion rate (25%; P < .05 versus saline). These data show that inhibition of specific pathways of platelet and thrombin activity improves the extent and duration of rTPA-induced thrombolysis in the electrolytic canine model. Furthermore, our findings suggest that low doses of platelet IIb/IIIa and direct thrombin antagonists in combination may be used successfully during thrombolysis.