Neutralization of Recombinant Hirudin: Some Practical Considerations

OVERVIEW The development of recombinant hirudin (r-hirudin) has added a new dimension to the area of therapeutic and surgical anticoagulation.1-3 This potent anticoagulant protein is a specific inhibitor of thrombin and thrombinmediated protease pathways that contribute to the overall thrombotic process. On a weight basis, r-hirudin is a three to five times stronger anticoagulant than heparin; however, the relative anticoagulant actions of this inhibitor are assay dependent. Unlike heparin, r-hirudin does not require any endogenous cofactors such as heparin cofactor II (HC II) and antithrombin III (AT III) for mediating its anticoagulant effects. Furthermore, none of the endogenous inhibitors of heparin, such as the platelet factor 4, histidine-rich glycoprotein, and vitronectin, inhibits the action of this agent. Because of its defined molecular structure, r-hirudin is now obtained through biotechnologic methods. Studies in both the experimental and clinical settings are currently being carried out on different r-hirudin preparations. Several r-hirudins are currently being commercially developed for medical and surgical use. A list of various companies interested in the development of recombinant and functional hirudin preparations is given in Table 1. At the present time, most of these companies are developing r-hirudin for the following indications.

From the Departments of Pathology and Thoracic and Cardiovascular Surgery, Loyola University Medical Center, Maywood, Illinois. Reprint requests: Dr. Fareed, Department of Pathology, Loyola University Medical Center, 2160 South First Avenue, Maywood, IL 60150.

1. 2. 3. 4.

Surgical anticoagulation Medical anticoagulation Adjunct agents with thrombolytic agents Anticoagulation during pulmonary transluminal coronary angioplasty (PTCA) 5. Hemodialysis

The dosage of r-hirudin used for these indications will vary and should be determined in valid clinical trials. If the products from different manufacturers differ significantly, the circulating anticoagulant activity will also vary. However, if the products are similar, the level of anticoagulation with different products is expected to be comparable and the clinical effects of these agents may also be comparable. Regardless of the product variations, because of the potent anticoagulant nature, it may be necessary to have a pharmacologic antagonist for r-hirudin and related drugs. One of the major questions that require clear answers is related to the possible hemorrhagic effects of r-hirudin. All anticoagulants are known to have some hemorrhagic effects. Because of the lack of clinical trails at this time, the true hemorrhagic potential of r-hirudin is unknown. As depicted on Table 2, there are several functional and immunologic methods available for the assay of r-hirudin. None of these methods is satisfactory for the broad range of circulating levels achieved with r-hirudin and can be used in the prediction of bleeding effects of this agent. The relatively short half-life, single site of action, defined composition, and clearance by the kidneys are useful in predicting the endogenous behavior of this agent. However, the nonavailability of an antagonist for the anticoagulant/hemorrhagic effects of r-hirudin is a timely issue that needs to be addressed at this early stage if r-hirudin is to be considered clinically.

Copyright © 1991 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, NY 10016. All rights reserved.


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SEMINARS IN THROMBOSIS AND HEMOSTASIS—VOLUME 17, NO. 2, 1991 TABLE 1. Manufacturers of Natural and Recombinant Hirudins and Related Agents Company 1. Biophram, Ltd., Swansea, UK

Natural hirudin

2. Ciba-Geigy Pharmaceuticals, Basel Switzerland

r-Hirudin (Escherichia coli and yeast)

3. Farmitalia Carlo Erba, Milan, Italy

Newer r-hirudins

4. Genbiotec, Heidelberg, Germany

r-Hirudin (E coli)

5. Hoechst AG, Frankfurt am Main, Germany

r-Hirudin (yeast), other expressions

6. Knoll Pharmaceuticals, Ludwigshafen, Germany

r-Hirudin (E coli), r-hirudin conjugates

7. Merrell Dow, Cincinnati, OH

r-Hirudin, synthetic fragments

8. Mitsui Co., Tokyo, Japan

r-Hirudin (Bacillus subtilis)

9. Pentapharm, Ltd., Basle, Switzerland

Natural hirudin Natural and r-hirudin (E coli)

11. SRI International, Menlo Park, CA

r-Hirudin, hirudin fragments

12. Transgene (Sanofi), Strasbourg, France

r-Hirudin (yeast)

The manufacturers have been active in the isolation, characterization, and pharmaceutical development of hirudin and related agents. Several other companies are also involved in the development of hirudin-like anticoagulants.

At the present time, r-hirudin is primarily being developed for surgical anticoagulation and adjunct thrombolytic agents. As such, it may take the place of heparin in certain indications or be used when an anticoagulant is needed but heparin is contraindicated. A comparison of r-hirudin and heparin as anticoagulants is given in Table 3. Heparin is generally used in terms of units for anticoagulation (0.5 to 5.0 U/ml); r-hirudin produces comparable anticoagulant effects, as measured by global clotting tests, at 10 to 30 µg/ml. In treatment, heparin is generally administered via the intravenous route, whereas r-hirudin can be administered via an intravenous bolus or by slow infusion methods. Although

TABLE 2. Laboratory Assays for the Determination of Hirudin Functional assays Whole blood clotting time Bleeding time Prothrombin time Partial thromboplastin time Thrombin time Heptest time Anti-IIa (amidolytic) Anti-Xa (amidolytic) Thrombin generation assay Fibrinopeptide A generation Thrombin - AT complex generation F1+2 generation D-dimer level Nonfunctional assays High-performance liquid chromatographic methods Immunoasays (RIA, EIA, IRMA) Physical methods

protamine can effectively neutralize heparin, it has almost no effect on the anticoagulant action of r-hirudin. Heparin treatment can be associated with such adverse effects as thrombocytopenia, arterial thrombosis, and allergic reactions. To date, none of these complications has been reported with the use of r-hirudin in Phase I clinical trials. Heparin is known to produce bleeding; however, at equivalent anticoagulant doses, as studied in animal models, the potential bleeding effect of r-hirudin is less than that observed for heparin. Because hemodilution results in a marked drop in AT III and HC II, the overall anticoagulant profile of blood changes. Although such changes decrease the anticoagulant potency of heparin, they would not alter the anticoagulant action of r-hirudin. Heparin is metabolically transformed into nonanticoagulant forms, whereas r-hirudin is relatively inert and its effect is not altered by endogenous enzymes.

HEMORRHAGIC POTENTIAL OF r-HIRUDIN A comparison of the hemorrhagic potential of heparin and r-hirudin is given in Table 4. Surface interactions are known to play a very important role in the hemostatic balance. Heparin is known to bind to endothelium and some of its therapeutic effects are attributable to this interaction.4 At this time there is no available evidence that r-hirudin interacts with surfaces. Both r-hirudin and heparin are known to impair platelet adhesion in in vitro system. 5-7 However, the exact implication of this interaction on the bleeding effects of these two agents is unknown at this time. Heparin and its

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10. Plantorgan Werk, Bad Zwischenahn, Germany



Monocomponent protein with single target (thrombin)

Polycomponent glycosaminoglycan-based drug with multiple sites of action

Thrombin-mediated amplification of coagulation is affected only under certain conditions. Thrombin-mediated protein C activation is inhibited

Thrombin and Factor VIIa feedback amplification of clotting is affected. Fibrinolytic and platelet function is affected

No known interaction with endothelium other than blocking thrombin-thrombomodulation medited activation of protein C

Significant interactions with endothelium. Both physical and biochemical modulation of endothelial function

Ultra short half-life via intravenous route

Short half-life via intravenous route.

Functional bioavailability is vriable and is dependent on the composition of hirudin

Functional bioavailability is 20 to 30%. Low molecular weight heparins are better absorbed than higher molecular weight

Endogenous factors such as platelet factor 4, Factor VIII, and other proteins do not alter its antithrombotic action

Marked modulation by endogenous factors. Several factors may alter the anticoagulant action

Relatively inert proteins not altered by metabolic processes

Transformed by several enzyme systems that reduce its anticoagulant actions

Information on cellular uptake and depot formation is not known at this time

Significant cellular uptake and depot formation

components are known to inhibit the generation of thrombin on surfaces. However, r-hirudin is a relatively weaker inhibitor of the generation of thrombin as is evident from its weak effect on the global clotting tests, such as the prothrombin time (PT) and activated partial thromboplastin time (APTT).1,2 More recent evidence suggests that the protease generation during the thrombotic activation process is affected by r-hirudin to a much lesser extent than heparin. Thus, during the thrombotic activation process such as in PTCA, the effect of hirudin at identical dosage may be weaker in comparison to heparin.

TABLE 4. A Comparison of the Hemorrhagic Potential of r-Hirudin and Heparin Parameter



Surface interaction

+ ++ +

+++ +++ +++



Arachidonic acid

++ ± — — ± ±

++ ++ ± ++ ++ +++ +++

Platelet-adhesion effect Thrombin generation on surfaces Platelet aggregation antagonism

Platelet release Thrombocytopenia Platelet activation Plasmatic modulation Endothelial function

The characteristics of platelet-rich plasma (PRP) prepared in r-hirudinized and heparinized systems are reported to be different than those prepared in citrated systems. r-Hirudin blocks the aggregation of platelets induced by epinephrine, whereas heparin produces a relatively weaker inhibitory effect. Both heparin and r-hirudin produce partial antagonism of the arachidonic acid-induced aggregation of platelets. How these inhibitory effects of r-hirudin and heparin translate to physiologic responses are unknown at this time. Both r-hirudin and heparin have agonist-dependent inhibitory effects on the platelet release response. In both animal models and in in vitro screening systems, heparin is known to produce thrombocytopenic and platelet activation responses. However, in both systems, r-hirudin does not produce any effect. Many plasma proteins bind to heparin; however, such information on r-hirudin is not available at this time. Based on the available pharmacologic data, it is evident that the hemorrhagic potential of r-hirudin and heparin differs in terms of the components of the hemostatic system that are affected by either agent. Thus, different pharmacologic approaches than that used for heparin may be needed in the development of an antagonist for r-hirudin. Furthermore, an appropriate method to predict bleeding effects of r-hirudin is not available at this time. Bleeding in surgical patients is a complex phenomenon. Beside the antihemostatic effect of a drug, many other factors inherent to the patient's physiology and the surgical procedure contribute to the overall bleeding complications. For example, as shown in Table 5,

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TABLE 3. r-Hirudin Versus Heparin


TABLE 5. Predisposing Factor in Which r-Hirudin May Augment the Hemorrhagic Potential During Surgery A. Hemostatic deficit Factor deficiency Qualitative or quantitative platelet defects Vascular defects Acquired deficit Fibrinolytic activation B. Vascular deficit C. Liver dysfunction D. Kidney ailments E. Aging F. Hypoprothrombinemia G. Postpartum state

hemostatic defects related to blood coagulation factor deficiency, qualitative and quantitative disorders of platelets, vascular deficit, defects in coagulation and platelet function, and endogenous fibrinolytic activation may be involved. In addition, liver and kidney dysfunction, aging, hypoprothrombinemia, and postpartum state may contribute to bleeding complications. It is therefore important to consider the patient's own hemostatic status to determine the potential bleeding risks associated with anticoagulants such as r-hirudin. These considerations are crucial for the development of neutralization protocol for the bleeding effects of r-hirudin. Another important consideration to identify the sites that may be responsible for the observed bleeding effects is that different approaches may be required to antagonize the anticoagulant effects of r-hirudin. It is not known at this time whether the plasmatic or platelet-dependent processes are involved in the mediation of hemorrhagic effects.

ANTAGONISM OF r-HIRUDIN Although the current data on the pharmacology of r-hirudin are limited at this time, it is clear that the molecular and biochemical characteristics of r-hirudin

differ from that of heparin. 1-6,8,9 In particular, although both agents are effective anticoagulants, the safety margin, in terms of bleeding, is markedly different for r-hirudin than for heparin. Regardless of these pharmacologic characteristics of r-hirudin, it is desirable to have appropriate means available to neutralize any unexpected bleeding complications. Like heparin, several factors influence the anticoagulant action of r-hirudin. These include dosage, route of administration, type of r-hirudin used, physiologic target of the drug, including plasmatic and cellular sites, and the clinical condition of the patient. Similarly, the antagonism or neutralization of the anticoagulant/ bleeding effect would also be dependent on the same parameters. At the present time, there is no known antagonist of the anticoagulant action of r-hirudin; however, monoclonal antibodies, selective increase in the generation of thrombin through the use of proteases, and physical methods have been considered.8,9 None of these methods has yet been applied in clinical settings. Table 6 depicts the comparative antagonism profile of r-hirudin and heparin. Although both heparin and r-hirudin are acidic in nature, polybasic proteins readily neutralize heparin, but they do not have an effect on the anticoagulant action of r-hirudin. At equivalent anticoagulant levels, heparin produces a marked inhibitory action on platelet function, whereas r-hirudin only produces relatively weak actions. Heparin exhibited a relatively longer half-life due primarily to endogenous receptor interactions, whereas r-hirudin has a shorter half-life. The question of r-hirudin binding to endogenous thrombin remains unresolved. Heparin exerts its anticoagulant actions at multiple sites, whereas r-hirudin is directed toward a single target. The specific interaction of r-hirudin with thrombin is a biochemically defined process, and it is not affected by endogenous inhibitors. In addition to the inhibition of thrombin, heparin produces several indirect effects involving the prostaglandins, the fibrinolytic system, and other physiologic systems. At this time, the effects of r-hirudin on these pathways are not known. Table 7 shows the results of the antagonism profile

TABLE 6. Heparin Versus Recombinant Hirudin Antagonism Profile Polycomponent highly charged mucopolysaccharide. Neutralizable by basic proteins

Highly acidic protein with specific composition. Not neutralizable by basic proteins

Marked inhibitory effects on platelets as supraanticoagulant levels

Minimal inhibitory effects on platelets at supraanticoagulant levels

Relatively longer half-life; binding to endogenous receptors

Relatively shorter half-life; no endogenous receptors except thrombin

Multiple sites of action, requiring neutralization

Single site of action, may not require neutralization

Profibrinolytic effects

No known profibrinolytic effects

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TABLE 7. Comparative Antagonism of Recombinant Hirudin and Heparin Agent



Protamine sulfate

— — — ± ± ±

++++ ++++ ++++

++ +++ +++

± ++ +

Platelet factor 4 Polylysine Thrombin Thrombin- α-macroglobulin complex Activated prothrombin complex concentrate (Feiba) Immunosorption Exchange transfusion Replacement therapy

— — ±

of r-hirudin by various substances and physical tech­ niques. As can be seen, protamine sulfate, platelet factor 4, and polylysine (substances routinely used to neutralize the anticoagulant/bleeding effect of heparin) do not have any effect on the anticoagulant action of r-hirudin. Noncoagulant forms of thrombin, namely 7-thrombin and thrombin-α2-macroglobulin complexes produce some degree of neutralization. Both of these agents can readily complex with r-hirudin, thus blocking the way for an interaction with the more potent α-thrombin. Acti­ vated prothrombin complex concentrates such as Feiba (Immuno, Vienna, Austria) produced partial antagonism of the anticoagulant effects of r-hirudin by generating higher than normal levels of thrombin. Affinity columns utilizing antihirudin antibodies, exchange transfusion, and replacement therapy neutralized the effects of rhirudin; however, these techniques require stepwise validation in preclinical and clinical settings. In studies from our laboratory, a commercially available activated prothrombin complex concentrate was studied for its effect on the neutralization of the antiplatelet and bleeding effects of r-hirudin. Table 8


shows the effect of an activated prothrombin complex concentrate (Feiba) on the restoration of the epinephrineinduced aggregation PRP (collected in r-hirudin). As can be seen in the r-hirudinized PRP (10 µg/ml), epinephrine-induced aggregation was completely blocked. How­ ever, preincubation of the r-hirudinized PRP with 1.25 to 5.0 U/ml Feiba produced a reversal of the inhibitory effect in a concentration-dependent manner. These stud­ ies suggest that activated prothrombin complex concen­ trates may be useful in the reversal of the antiplatelet action of r-hirudin. The exact physiologic mechanism of this process is unknown at this time. Similar results have been obtained with other activated prothrombin complex concentrates. Table 9 shows the effect of Feiba on the r-hirudininduced bleeding in a rabbit ear model of blood loss. r-Hirudin produced an increase in blood loss at a 2.0 mg/kg dosage (circulating level

Neutralization of recombinant hirudin: some practical considerations.

SEMINARS IN THROMBOSIS AND HEMOSTASIS—VOLUME 17, NO. 2, 1991 Neutralization of Recombinant Hirudin: Some Practical Considerations OVERVIEW The devel...
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