Hirudin,a new therapeutic tool?

Summary Hirudin is the most potent natural inhibitor of thrombin known to date. It is gaining popularity as an anticoagulant now that recombinant and synthesized forms are available. It is a monospecific and co-factor-independent thrombin inhibitor with otherwise inert pharmacological properties. Being a surprisingly weak immunogen, its administration has exhibited no side effects, particularly on platelets. Bleeding complications are not to be expected at therapeutic doses. Effective anticoagulatory doses can be easily predicted and laboratory control is no problem. Application of hirudin or derivatives thereof may be indicated for: prophylaxis and treatment of postoperative venous thrombosis and diffuse microthrombosis; prevention of arterial thrombosis, especially in cardiac surgery; enhancement of fibrinolytic therapy and/or angioplasty to prevent reocclusion; extracorporeal circulation; and plastic surgery. Hirudin may be a particularly useful alternative anticoagulatory agent in patients sensitized to heparin or in patients with hereditary or acquired antithrombin III deficiency. However, whether hirudin is really an effective therapeutic tool and whether it can replace heparin in certain clinical indications can be judged only after extended clinical experience has been accumulated.     

Direct and indirect antithrombins. Heparins, low molecular weight heparins, heparinoids, and hirudin

With the eclipse of UH by newer anticoagulants, the field has opened up to search for new and better drugs. Hirulog or bivalirudin is another direct antithrombin that has been used in initial trials. It is smaller than hirudin, at 20 amino acids. Currently under investigation, it seems to have a short half-life, a narrow therapeutic window, and a reverse dose effect, with lower levels achieving better cardiac post-thrombolysis patency than higher doses. Other antithrombins being examined are the hirudisins, where four amino acids of hirudin have been replaced by the RGDS integrin-binding sequence and thrombin receptor antagonist peptides. In addition, many other inhibitors of activated clotting factors are being studied for future therapeutic value. Tick anticoagulant protein studies are underway, as are studies on a group of benzamidine isoxazoline derivates, which are direct Xa inhibitors. We are truly at an age of discovery with the newer anticoagulants and it may take many years until we can distinguish the advantages and disadvantages of all the newer therapies. It looks like an ever more real possibility that medicine may find an antithrombotic regimen that is highly effective, highly reversible, and nontoxic.     

Comparison of Argatroban and Hirudin for the Reperfusion of Thrombotic Arterial Occlusion by Tissue Plasminogen Activator

Despite theoretical advantages of direct thrombin inhibitors, recent clinical studies failed to show the superiority of hirudin over heparin in patients with acute coronary syndromes. However, these inhibitors have important in vitro differences for the inhibition of clot-bound thrombin that may translate into different in vivo relative efficacy. The effects of two direct thrombin inhibitors, argatroban and hirudin, on the reperfusion of thrombotic arterial occlusion by t-PA were compared. In anesthetized rabbits thrombotic occlusion was induced in the femoral artery. t-PA, aspirin, and various doses of argatroban (1.25, 2.5, and 5.0 mg/kg/h) or hirudin (2.5 and 5.0 mg/kg/h) were administered (six animals in each group). Blood flow was measured for 4 hours. Animals treated with 2.5 mg argatroban more rapidly achieved full reperfusion than those treated with high-dose argatroban or hirudin (P < 0.05). At the doses that induced comparable prolongation of bleeding time, argatroban showed a significantly faster and higher level of reperfusion than hirudin. In animals treated with hirudin, there was a positive correlation between the aPTT and the mean reperfusion blood flow (r = 0.70, P < 0.05). In animals treated with argatroban, this correlation did not exist and the high-dose argatroban was paradoxically less effective in promoting thrombolysis despite greater anticoagulation effects. In this animal model of arterial thrombosis, argatroban was more effective than hirudin in inducing rapid, full reperfusion with t-PA. Although they are both direct thrombin inhibitors, these two agents showed important dose-related differences in efficacy and anticoagulant effects.     

Clinical monitoring of hirudin and direct thrombin inhibitors.

In addition to heparin, the standard medication for prophylaxis and therapy of thromboembolism, several other substances have been developed and tested for clinical use with the aim of decreasing or eliminating side effects. Most of all, hirudin, a direct antithrombin (AT), has proved to be effective. To define the therapeutic range and to avoid underdosage or overdosage, clinical monitoring is necessary. For monitoring of hirudin, thrombin time (TT) is not suited because of the missing linearity of the standard curve. Activated partial thromboplastin time (aPTT) can be used only in the lower hirudin level range, where the standard curve is quite linear. However, high and toxic hirudin levels cannot be determined using aPTT. Another drawback is a high variation in single measurements and in the normal value of patients. Methods using chromogenic substrates are suited for determination of hirudin in plasma but cannot be used at bedside. Especially for monitoring of hirudin, the ecarin clotting time (ECT) was developed. The standard curve is linear over the entire concentration range. There are no influences by other coagulation parameters or anticoagulants. For both acute clinical situations and long-term monitoring, this method capable of point-of-care therapy (POCT) will be the method of choice.     

Enhancement of fibrinolytic potential in vitro by anticoagulant drugs targeting activated factor X, but not by those inhibiting thrombin or tissue factor.

Tissue factor-induced coagulation leads to the generation of a small amount of thrombin, resulting in the formation of a fibrin clot. After clot formation, thrombin generation continues resulting in the activation of thrombin activatable fibrinolysis inhibitor, leading to downregulation of fibrinolysis. In this study, the effect of anticoagulant drugs targeting different steps in the coagulation cascade on clot formation and subsequent breakdown was investigated using a plasma-based clot lysis assay. All drugs tested significantly delayed clot formation; only those drugs targeting activated factor X (FXa) (tissue factor pathway inhibitor, fondaparinux, and low molecular weight heparin) accelerated fibrinolysis. Anticoagulant drugs targeting tissue factor (active site-inactivated recombinant activated factor VII) or thrombin (hirudin and d-phenylalanyl-l-prolyl-l-arginyl chloromethyl ketone) did not affect clot lysis time. In accordance with these findings, it was shown that total thrombin generation, as quantified by the endogenous thrombin potential, was only affected by anticoagulant drugs targeting FXa when all drugs were used in a concentration resulting in doubling of clotting time. Induction of hyperfibrinolysis by anticoagulant drugs directed against FXa might be beneficial as increased clot breakdown might facilitate thrombolysis or prevent re-occlusion. On the other hand, the induction of hyperfibrinolysis by these compounds might increase the risk of bleeding complications.     

Methods for the monitoring of direct thrombin inhibitors

Direct thrombin inhibitors are available for prophylactic as well as therapeutic purposes. Application of hirudin in therapeutic doses has been shown to require drug monitoring. Currently, most experience is available for recombinant hirudin, but the principle aspects of drug monitoring are the same for all direct thrombin inhibitors. Most frequently, activated partial thromboplastin time (aPTT) and modifications of the activated clotting time (ACT) have been used for the monitoring of hirudin therapy. However, these methods are insensitive at plasma levels higher than 0.6 mg/L of hirudin, so that overdoses may be missed despite monitoring. Correlations between ecarin clotting time (ECT), enzyme immunoassays, and chromogenic substrate assays on one side and global tests on the other side are poor. Fully automated chromogenic substrate-based assays, also available as point-of-care tests (POCT), are more precise and sensitive and are not disturbed by interferents such as heparin and antithrombin. Good correlations can be observed between chromogenic assays and the ECT performed in plasma or whole blood samples. ECT can also be determined with POCT systems. Test characteristics such as imprecision and measuring range are comparable to those of the chromogenic assays. In conclusion, therapy with direct thrombin inhibitors should be monitored with chromogenic assays or ECT.     

Hirudin as alternative anticoagulant--a historical review

Advances in separation techniques and biotechnology have contributed to the development of anticoagulant agents from hematophagous animals. The most potent known natural thrombin inhibitor from blood-sucking leeches ( Hirudo medicinalis), hirudin has served as a standard for designing the natural coagulation inhibitors as an anticoagulant drug. The search for the development of hirudin from leech extract to genetically engineered products as an alternative anticoagulant has been resumed. The pharmacological profiling of the isolated thrombin inhibitor has shown that native hirudin is an antithrombotic agent of high quality. However, its clinical use has remained limited, because the substance has not been available in adequate amounts. The progress in molecular biology has stimulated the interest in the structure and function of hirudin. This development resulted in the production of recombinant hirudins (r-hirudins) through gene technology. The biological properties of hirudin combined with the ready availability of recombinant forms make the specific thrombin inhibitor well-suited for use as an antithrombotic drug. Its use should lead to a decisive progress in the management of thromboembolic diseases of both arterial and venous origin. Clinical trials, especially in diseases in which thrombin plays a crucial role, are in progress.     

Laboratory monitoring of new anticoagulants

Maintaining a balance between bleeding and clotting has always been a challenge in treating coagulation disorders. A perturbation in that balance can be associated with substantial morbidity and mortality. As a result, anticoagulant monitoring is extremely important, and inappropriate testing may lead to complications. There are now a variety of new anticoagulant drugs in clinical use including several direct thrombin inhibitors (DTIs), such as argatroban, bivalirudin, and hirudin, as well as a Factor Xa inhibitor, fondaparinux. There are pitfalls associated with some of the currently used laboratory monitoring tests, and newer alternative laboratory monitoring tests have been investigated (Walenga and Hoppensteadt, Semin Thromb Hemost 2004;30:683–695). In addition, laboratory testing can assist with transitioning patients from DTI to warfarin therapy. Am. J. Hematol. 2010. © 2009 Wiley‐Liss, Inc.     

Determination of dabigatran in human plasma samples

The oral direct thrombin inhibitor dabigatran effectively prevents arterial and venous thromboembolism using fixed doses without the need for adjustment according to laboratory results. Dabigatran is eliminated from the circulation by ～80% through the kidneys. However, the in vitro anticoagulant effect of dabigatran may be necessary to determine in special patient populations such as in the elderly, for renal impairment, before operations, bleeding or thrombotic episodes, and to monitor self-compliance. Several clotting and thrombin-specific chromogenic substrate assays are available to analyze the biological activity of dabigatran. All of them are prolonged in the presence of dabigatran. This article reports the effects of dabigatran on clinical routine assays and the potential usefulness for determination in special risk groups of patients when overdose or lack of compliance are suspected.     

Inhibition of coagulation and thrombin-induced platelet activities by a synthetic dodecapeptide modeled on the carboxy-terminus of hirudin

A synthetic, tyrosine-sulfated, dodecapeptide (BG8865) modeled on residues 53-64 of hirudin was found to elevate the activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) of human plasma in a dose-dependent manner. The most sensitive assay was the TT, which was prolonged 2 and 3 times control values at 2.2 and 4.1 micrograms/mL hirudin peptide, respectively. The sulfated dodecapeptide exhibited no dependency on antithrombin III as monitored by the APTT in the presence of sheep anti-human antithrombin III antibodies, and its activity was not neutralized by platelet releasates or platelet factor 4. In studies of thrombin-induced platelet activation, the hirudin peptide was found to block aggregation, serotonin release and thromboxane A2 generation. At thrombin concentrations of 0.25 U/mL, the IC50 (concentration resulting in 50% inhibition) for inhibition of platelet aggregation was 0.72 micrograms/mL peptide. Inhibition of TXA2 generation and serotonin release correlated closely with inhibition of aggregation. Using platelets from patients with clinically documented heparin-induced thrombocytopenia anticoagulant doses of heparin were found to induce platelet aggregation and thromboxane A2 generation. In sharp contrast, anticoagulant-equivalent doses of hirudin peptide had no effect on patient platelets, as evidenced by a lack of platelet aggregation and thromboxane A2 generation. These data provide compelling in vitro evidence that the hirudin peptide has several potential advantages over heparin, namely effective inhibition of thrombin-induced platelet activities, co-factor independence, insensitivity to endogenous heparin-neutralizing factors, and an apparent lack of direct or immune-mediated platelet stimulating properties.     

Antithrombotic effects of three thrombin inhibitors in a rat model of laser-induced thrombosis

The antithrombotic effects of three thrombin inhibitors (hirudin, NAPAP and argidipine) were investigated in an experimental thrombosis model using laser lesions of rat mesenteric venules. Furthermore, their in vitro anticoagulant activity (partial thromboplastin time, thrombin time, Heptest, inhibition of factor IIa and factor Xa) in human platelet poor plasma (PPP) and their in vitro and ex vivo activities were studied in rat plasma. All three thrombin inhibitors showed significant and dose-dependent antithrombotic effects after intravenous injection, if venules were damaged, which lasted for more than 4 but less than 8 h. The anticoagulant effect observed in vitro did not differ much between human and rat PPP. The two synthetic thrombin inhibitors NAPAP and argidipine were about as effective as hirudin in vitro; however, the ex vivo effect after intravenous injection of hirudin in rats was more pronounced than that observed with the two synthetic thrombin inhibitors. The antithrombotic effect of all three thrombin inhibitors in the laser model lasted much longer than the anticoagulant activity. This fact needs further investigations in the future.     

Drotrecogin alfa activated (recombinant human activated protein C) in combination with heparin or melagatran: effects on prothrombin time and activated partial thromboplastin time.

Recombinant human activated protein C (rhAPC) has recently been demonstrated to be a promising candidate to improve the outcome for patients with severe sepsis. Plasma-derived activated protein C and unfractionated heparin (UH) exert anticoagulant synergy due to mechanisms that simultaneously decrease thrombin generation. Melagatran, a new direct thrombin inhibitor, does not bind to plasma proteins or requires antithrombin as a cofactor. The latter is often consumed in patients with severe sepsis. We investigated the anticoagulant efficiency in combined administration of rhAPC and UH or melagatran in terms of prolongation of the standard clotting assays activated partial thromboplastin time (aPTT) and prothrombin time (PT) in pooled plasma samples in vitro. RhAPC dose-dependently prolonged the aPTT but not the PT. The ability of UH and melagatran to prolong the aPTT was significantly enhanced in combination with rhAPC. The combined administration of rhAPC and melagatran, but not UH, resulted in additive prolongation of the PT. In control measurements the capability of rhAPC to suppress prothrombin fragment 1.2 generation dose-dependently increased in combination with heparin and melagatran. Our study demonstrates the respective effects of rhAPC, UH, melagatran and further different additive effects in combined administration of rhAPC and UH or melagatran on the prolongation of the aPTT and PT clotting assays usually used to monitor anticoagulant treatment.     

Ancylostoma caninum anticoagulant peptide: a hookworm-derived inhibitor of human coagulation factor Xa.

Human hookworm infection is a major cause of gastrointestinal blood loss and iron deficiency anemia, affecting up to one billion people in the developing world. These soil-transmitted helminths cause blood loss during attachment to the intestinal mucosa by lacerating capillaries and ingesting extravasated blood. We have isolated the major anticoagulant used by adult worms to facilitate feeding and exacerbate intestinal blood loss. This 8.7-kDa peptide, named the Ancylostoma caninum anticoagulant peptide (AcAP), was purified by using a combination of ion-exchange chromatography, gel-filtration chromatography, and reverse-phase HPLC. N-terminal sequencing of AcAP reveals no homology to any previously identified anticoagulant or protease inhibitor. Single-stage chromogenic assays reveal that AcAP is a highly potent and specific inhibitor of human coagulation, with an intrinsic K*i for the inhibition of free factor Xa of 323.5 pM. In plasma-based clotting time assays, AcAP was more effective at prolonging the prothrombin time than both recombinant hirudin and tick anticoagulant peptide. These data suggest that AcAP, a specific inhibitor of factor Xa, is one of the most potent naturally occurring anticoagulants described to date.     

The effect of different anticoagulants on thrombin generation.

Decrease in thrombin generation is the key effect in anticoagulation. The aim of the present study was to investigate the effect of anticoagulants on thrombin generation and the relation to platelet count. Plasma samples from 10 healthy volunteers (mean age 43.0 +/- 9 years) were incubated at preset platelet counts with different doses of the anticoagulants lepirudin, fondaparinux and low molecular weight heparins. Thrombin generation was measured in a tissue factor-mediated assay using a fluorometer and a slow-reacting fluorogenic substrate. The endogenous thrombin potential, the lag phase, the maximum reaction velocity (Vmax) and the concentration of a given anticoagulant required for 50% inhibition of thrombin generation (IC50) are presented. All three anticoagulants decreased endogenous thrombin potential and prolonged the lag phase in a dose-dependent manner. Fondaparinux and low molecular weight heparins, but not hirudin, decreased Vmax in a concentration-dependent manner. With increasing platelet count, the IC50 increased but the extent of this increase was not uniform for the three anticoagulants and the three variables investigated. The influence of anticoagulants on thrombin generation is variable, depending on their basic mechanism of action. In defining and comparing their effects, the endogenous thrombin potential, the lag phase and the maximum reaction velocity should be considered together. Platelets have a considerable influence on the magnitude of thrombin generation.     

Hirudin in acute myocardial infarction

Summary The central role of thrombosis in the pathogenesis of acute myocardial infarction has led to intense interest in developing more effective thrombolytic-antithrombotic regimens. Hirudin is 65 amino acid polypeptide that binds in a 11 relationship with thrombin, thereby inhibiting the final step in the coagulation cascade. Hirudin has several potential advantages over the current antithrombin agent heparin: It is a direct inhibitor that does not require a cofactor, it has no known inhibitors that would attentuate its anticoagulant effects, and it can inhibit clot-bound thrombin, thereby achieving an antithrombotic effect at the site of potential rethrombosis. Initial clinical trials have shown promising results: Hirudin, as compared with heparin, provided a more consistent level of anticoagulation, as gauged by the activated partial thromboplastin time. As an adjunct to thrombolytic therapy in acute myocardial infarction, hirudin improved indices of coronary reperfusion and patency. Initial results with clinical end points, including death or myocardial infarction, also have shown favorable results for hirudin compared with heparin. In the first phases of the larger phase III trials, the rate of hemorrhagic events, including intracranial hemorrhage, was higher than expected in both the hirudin and heparin arms, indicating that a safety ceiling had been reached. The TIMI 9B and GUSTO IIb trials are using lower doses of intravenous hirudin and heparin, which should allow testing of the thrombin hypothesis: that more potent inhibition of thrombin will translate into improved clinical outcome for patients with acute MI.     

Oral antithrombins and the future of antithrombotic therapy

The coagulation cascade leads, via thrombin activation, to the formation of fibrin (from its precursor fibrinogen) and platelet thrombus. Several drugs are able to interfere with some of the enzymatic factors involved in thrombin activation, but thrombin inhibitors (heparin, hirudin and hirulog [bivalirudin]) are of particular interest because they disrupt the coagulation cascade by acting upon one of its final steps. Among the thrombin inhibitors, oral antithrombins (ximelagatran) appear to hold great promise for the control of thrombogenicity in a number of clinical contexts. Oral antithrombins do not require the participation of cofactors to exert their action, and in contrast to thrombin inhibitors administered parenterally, they bind specifically to the active site of thrombin without interfering with other portions of the molecule. Their specificity makes these drugs safe and obviates the need to monitor coagulability. Moreover, they show a low rate of interaction with other drugs or with foods, and the response to fixed doses is predictable. A number of clinical studies have investigated antithrombins for prophylaxis prior to orthopedic surgery, in the prevention of cerebrovascular accidents associated with atrial fibrillation, and in the control of coronary artery disease. These studies have shown that antithrombins are superior to other habitually used treatment options. Surprisingly, their use in long-term studies has not been associated with an increased rate of bleeding events. Their interesting molecular characteristics and considerable therapeutic efficacy and safety suggest that oral antithrombins will in the near future become a valuable therapeutic option.     

Inhibition of endothelial cell-mediated generation of activated protein C by direct and antithrombin-dependent thrombin inhibitors.

The present study investigated the effect of the thrombin inhibitors antithrombin (AT) (with and without unfractionated heparin or low molecular weight heparin), hirudin, inogatran and melagatran on thrombin-thrombomodulin-mediated generation of activated protein C (APC), in solution and on endothelial cells. Sequential incubation with thrombin, thrombin inhibitors and protein C was followed by measurement of APC by an amidolytic assay. The approximate concentrations resulting in 50% inhibition of endothelial cell-mediated APC generation for AT, AT-unfractionated heparin, AT-low molecular weight heparin, hirudin, melagatran and inogatran were 200, 4, 9, 1, 8 and 60 nmol/l, respectively. The normal plasma level of AT is 2800 nmol/l and relevant therapeutic concentrations from clinical trials are 200 nmol/l for hirudin, 500 nmol/l for melagatran and 1000 nmol/l for inogatran. The present study indicates that clinically relevant concentrations of the tested thrombin inhibitors interfere with endothelial-mediated APC generation, which may offer an explanation for the lack of a dose-response effect in clinical trials with thrombin inhibitors.     

Tailoring haemostatic treatment to patient requirements – an update on monitoring haemostatic response using thrombelastography

Summary.  Currently, there is no single haemostasis laboratory test that has the capacity to accurately illustrate the clinical effects of procoagulant or anticoagulant interventions. Although the time course of thrombin generation in plasma and the endogenous thrombin potential (ETP) may be useful coagulation parameters, clotting involves components other than thrombin (e.g. platelets, fibrinogen). The continuous coagulation profiles of thrombelastography may provide a more accurate reflection of in vivo biology, covering initiation, development and final clot strength during whole blood clot formation. This method has helped to clarify the mechanism of action of whole blood clot formation, demonstrating the differences from clotting in plasma, and the importance of platelets and tissue factor titrations. It has also been used to investigate hypocoagulation (in haemophilia A, rare coagulation disorders, anticoagulant therapy and dilutional coagulopathy), hypercoagulation and the ex vivo testing of haemostatic interventions. Thrombelastography has been shown to reflect the clinical efficacy of activated prothrombin complex concentrate (aPCC) and recombinant activated factor VII (rFVIIa) in patients with haemophilia A with inhibitors and in patients with acquired haemophilia. Overall, tailoring laboratory assays to illustrate and correlate with clinical phenotypes is essential for effective coagulation monitoring. Applying an algorithm of preoperative, perioperative and postoperative tests, including thrombelastography, may enable physicians to achieve this.     

Determination of prothombinase activation after adding human purified prothrombin to human clot: comparison of hirudin, an activated factor II inhibitor, with DX9065a, an activated factor X inhibitor, on clot-associated thrombin and on prothrombin activation.

Clot-associated prothrombinase and thrombin activities may contribute to thrombus extension after thrombolytic and anticoagulant treatment. We studied prothrombin activation after adding human purified prothrombin to human clot. By using two different drugs with an exclusive direct anti-activated factor X activity (DX9065a) or anti-activated factor II activity (r-hirudin), we tried to determine whether clot-bound thrombin and prothrombinase could be inhibited in our experimental system when human purified prothrombin was added. Standard clots were prepared from platelet-poor human plasma after addition of calcium. We measured clot-bound thrombin or free thrombin using a direct simple chromogenic assay. In parallel, prothrombin fragment 1+2 measurement was used to monitor prothrombin activation. For this, two protocols were used. We introduced the direct inhibitors before starting the activation process (protocol A) or at the time of the activation process (protocol B). We found a direct correlation between thrombin generation and prothrombin fragment 1+2 with an increase of thrombin activity on clots and in the incubation mixtures when clots were incubated in human purified pothrombin alone. Two protocols were used: in the first, clots were pre-incubated in presence of drugs before adding prothrombin; and in the second, clots were incubated in the presence of prothrombin and drugs. Prothrombin activation was not inhibited when clots were incubated with r-hirudin and consequently thrombin generation still occurred. However, added r-hirudin blocks thrombin activity on the clots and in the incubation mixture, but does not prevent prothrombin activation, as shown by the increase of prothrombin fragment 1+2. In contrast, DX9065a did not suppress clot-bound thrombin. However, DX9065a blocks prothrombin activation whichever protocol was used. The results show that hirudin is a poor inhibitor of thrombin generation in contrast to DX9065a. On the other hand, DX9065a cannot inhibit thrombin bound to clot in contrast to hirudin.     

Effect of hirudin on tissue plasminogen activator induced clot lysis

The effect of recombinant hirudin in the in vitro tPA fibrinolytic and thrombolytic activity was investigated. The activity was evaluated by following lysis of radiolabelled fibrin or plasma clot formed in the presence of tPA alone or with hirudin. The results obtained indicate that increasing concentrations of hirudin had a potentiating effect, with faster clot lysis rates and reduced time to complete lysis. However, when radiolabelled plasma or whole-blood clots were immersed in autologous plasma in the presence of tPA and hirudin, no significant difference in the lysis rates and time to complete lysis was observed. The findings suggest that hirudin or hirudin-thrombin complex interferes with the forming fibrin, thereby making clots more susceptible to lysis, while the presence of hirudin in the surrounding medium during lysis of formed clots helps to rapidly neutralize active thrombin released during clot lysis, thereby preventing further activation of coagulation. Thus, use of hirudin as an anticoagulant during thrombolytic therapy may prove to be helpful in reducing the incidence of reocclusion.