FIBROLASE

Toxin Reviews, 25:351–378, 2006Copyright C© 2006 Taylor & Francis Group, LLCISSN: 0731-3837 print / 1525-6057 onlineDOI: 10.1080/15569540600567388

FIBROLASE

S. SWENSON AND F. S. MARKLAND JR.

Department of Biochemistry and Molecular Biology and Norris ComprehensiveCancer Center, Keck School of Medicine University of Southern California,

Los Angeles, CA 90033

Snake venoms contain a number of serine and metalloproteinases, includedamong these are the fibrinolytic metalloproteinases. When the fibrinolytic enzymeswere first isolated from viper venoms, it was postulated that there may be a clinicalapplication for these enzymes. In the ensuing years, a substantial body of literaturehas been generated on the identification and characterization of the fibrinolyticenzymes from a broad spectrum of snake species. In this report, we describe thebiological properties and features of the enzyme known as fibrolase.

Fibrolase, a fibrinolytic metalloproteinase, has been isolated from Agkistrodoncontortrix contortrix (southern copperhead) venom. The biochemical, struc-tural, and physiochemical properties of the enzyme are described. In addition, invitro bioactivity studies with a size-altered version of the enzyme and a derivativewith altered binding capabilities are described. The size-modified form of fibrolasewas formed through the adduction of polyethylene glycol to the native protein inan effort to reduce the rate of clearance from the circulation by α2-macroglobulin.Alteration in the binding affinity of the enzyme to platelets was achieved throughcoupling fibrolase to an Arg-Gly-Asp (RGD)-like peptide; this modification im-parts an inhibitory activity on platelet aggregation and thrombus formation,while maintaining full fibrinolytic activity.

Keywords: Fibrolase, Fibrinolytic Metalloproteinase, Agkistrodon contor-trix contortrix.

Introduction

A number of venoms possessing fibrinolytic activity were identi-fied by Didisheim and Lewis (1956). They were among the firstto suggest a clinical use for snake venom fibrinolytic activity inthe dissolution of blood clots. Fibrinolytic activity in the venom of

Address correspondence to Francis S. Markland Jr., Ph.D., Department of Biochem-istry and Molecular Biology, University of Southern California, Keck School of Medicine,Cancer Research Laboratory #106, 1303 N. Mission Rd., Los Angeles, CA 90033. E-mail:[email protected]

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352 S. Swenson and F. S. Markland Jr.

Agkistrodon contortrix contortrix was first described by Kornalik andStyblova (1967). In this work, it was shown that proteinases from A.c. contortrix venom acted directly on fibrin and did not function asa plasminogen activator. Further studies by Bajwa et al. (1982) de-scribed a fibrinolytic protease from A. c. contortrix venom with a molec-ular mass of approximately 25,000; this enzyme is now known as fi-brolase. Fibrolase belongs to a class of metalloproteinases identifiedas α-fibrinogenases. This class of enzyme has been isolated fromthe venom of snakes in the Crotalid, Viperid, and Elapid families.Following this description by Bajwa et al. a more complete biophys-ical analysis determined both the amino acid sequence of fibrolaseand confirmation of the molecular mass as 22,891 Da (Randolphet al., 1992) (E.C.#3.4.24.72, CAS Registry # 116036-70-5). In sub-sequent studies, Markland et al. (1993) reported that two differentisoforms of fibrolase existed, and that they could be separated byweak cation-exchange high-performance liquid chromatography(HPLC). The two identified isoforms have been shown to possessidentical activities and differ in a single amino acid at the aminoterminus (Trika et al., 1994). While the characteristics of fibrolaseare mimicked by enzymes isolated from the venom of a numberof other snakes, fibrolase is the most thoroughly studied of theα-fibrinogenases.

Proteolytic Activity

The Aα chain of fibrinogen is the primary target of the direct-acting fibrinolytic enzyme fibrolase. Initially, fibrolase cleaves atposition Lys413-Leu414 in the Aα chain; it also cleaves the Bβ chain,but at a slower rate. There is no cleavage of the γ chain. Retziosand Markland (1988) reported that fibrolase had no activity witha number of artificial chromogenic substrates commonly cleavedby serine proteinases. The lack of activity against these targetssupports the hypothesis that fibrolase is not a serine protease.Fibrolase activity is completely and rapidly inhibited by the ad-dition of EDTA, tetraethylenepentamine, or 1,10- phenanthroline(Markland et al., 1988). Inhibition by agents that chelate zinc sup-ports the conclusion from atomic absorption spectroscopy (Guanet al., 1991) that fibrolase is a zinc metalloproteinase and not aserine or cysteine proteinase.

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Fibrolase 353

Currently, thrombolytic agents used in the clinic, such as tis-sue plasminogen activator (t-PA) and streptokinase, rely on theactivation of plasminogen to plasmin. By comparison, fibrolase isa direct-acting enzyme. Incubation of fibrolase with plasminogenshows no evidence of plasmin formation (Retzios and Markland,1988). Subsequent incubation of the fibrolase–plasminogen mix-ture with streptokinase yields plasmin, indicating that fibrolasedoes not activate, degrade, or inactivate plasminogen. While fi-brolase degrades fibrin and fibrinogen in vitro, pretreatment offibrolase with plasma inhibits fibrinolytic activity (Markland et al.,1988). Normal human plasma contains a number of protease in-hibitors such as α2-macroglobulin (α2M). More detailed studies onthe interaction of fibrolase with components found in plasma sug-gested that the component responsible for fibrolase inhibition isα2M. Confirmatory experiments utilizing SDS-PAGE showed thatthe 180 kDa subunit of α2M was cleaved into two 90 kDa frag-ments, indicating binding of fibrolase to this proteinase inhibitor(Swenson and Markland, unpublished results). More recent stud-ies have shown that fibrolase interacts with α2M rapidly and withhigh affinity (Swenson and Markland, unpublished data). Fibro-lase has been shown under in vivo conditions to lyse both arte-rial and venous experimental thrombi (Markland, 1996). In vitrofibrolase activity can be measured by several different methods.Two of the most commonly used procedures are the degradationof azocasein and the dissolution of fibrin clots using a fibrin plate(Retzios and Markland, 1992). The azocasein assay is not as spe-cific as the fibrin plate assay, but the fibrin plate assay requiresan incubation of from several hours to overnight and is not verysensitive. Recently, a more sensitive and rapid method of determi-nation of fibrinolytic activity has been developed relying on fluores-cence resonance energy transfer (FRET). Through this method,the evolution of a fluorescent signal from the cleavage of a fluo-rescently quenched synthetic octapeptide (4-amino-benzene-His-Thr-Glu-Lys-Leu-Val-Thr-Ser-dinitrophenol), which contains theamino acid sequence around the fibrolase scissile Lys413-Leu414

bond in the Aα-chain of fibrinogen, is monitored over time. Thedistance along the linear peptide between the amino terminal flu-orophore and the carboxy-terminal quenching moiety as well asthe overlapping absorption and emission spectra limit the fluores-cent emission from the intact peptide (Swenson and Markland,

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unpublished results). In the uncleaved peptide, the quenchingmoiety blocks the emission of a fluorescent signal, but when thepeptide is cleaved, the two groups become separated, allowing theemission of a fluorescent signal. Cleavage of the peptide can beused both to confirm proteolytic activity and to monitor kineticsof fibrolase activity. This method is rapid and highly sensitive. ThepH optimum for fibrolase activity in both the azocasein and fibrinplate assays is 7.4.

Structural Chemistry

The zinc metalloproteinase identified as fibrolase is a 23 kDasingle-chain protein containing 1 mol of zinc per mol of pro-tein (Guan et al., 1991). The pI of the enzyme, as determined byisoelectric focusing in an immobilized pH gradient, is 6.8 (Guanand Markland, 1988). Structurally, the protein is composed of fiveα-helical regions and four parallel and one antiparallel β sheets(Pretzer et al., 1992). The structure is stabilized by three disulfidebonds (Randolph et al., 1992; Manning, 1995). At least two distinctisoforms of the enzyme exist, as evidenced by separation of fibro-lase by weak cation-exchange HPLC (CM300). The difference be-tween the two isolated isoforms lies at their amino-termini. One ofthe isoforms possesses two Gln residues, while the other has a singleGln residue; in both cases, the amino terminus is blocked (pyroglu-tamate) (Randolph et al., 1992; Loayza et al., 1994). This alterationin the primary sequence of the protein has no apparent effect onenzymatic activity, as both isoforms possess identical activity againstseveral different substrates, and both isoforms are equally inhib-ited when incubated with chelating agents. Fibrolase is a memberof the adamalysin/reprolysin subfamily and shares a high degree(∼60%) of amino acid sequence similarity with other enzymes ofthis class (Bode et al., 1993). Crystals of fibrolase have been grownusing polyethylene glycol as precipitant (Markland et al., 1993),but the structure of fibrolase is yet to be solved. However, a three-dimensional model of the fibrolase structure has been createdbased on the structures of other members of the adamalysin sub-family of proteinases (Bolger et al., 2001). The energy-minimizedmodel reveals an identical structure for fibrolase in the active-sitegroove when compared to enzymes of known structure, and allowsfor the three disulfide bonds in fibrolase compared to two disul-fide bonds in adamalysin (Gomis Ruth et al., 1994) and atrolysin

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Fibrolase 355

C (Zhang et al., 1994) (members of the adamalysin subfamily withknown three-dimensional structures). In addition, the modeledstructure of fibrolase agrees with the solved crystal structure of athree-disulfide-containing snake venom metalloproteinase BaP1,isolated from the venom of Bothrops asper (Watanabe et al., 2002,2003). There is a high degree of structural similarity between fibro-lase and BaP1; the six Cys residues in both enzymes form disulfidebonds with identical partners (Jones et al., 2001).

Sequence similarity in the active-site region of the nonhemor-rhagic fibrinolytic enzymes of known primary structure is shown inFigure 1 and is close to 100%. The conserved amino acids betweenresidues 140 and 166 include the three histidines involved in zinc

FIGURE 1 Comparison of amino acid sequences in the active site region of fivedifferent nonhemorrhagic fibrinolytic venom metalloproteinases. The sequencesof fibrolase, neuwiedase (isolated from Bothrops neuwiedi), Atroxase (isolated fromCrotalus atrox), Alfimeprase (a recombinant form of fibrolase), and brevilysin (iso-lated from Gloydius halys brevicaudus) are shown and the homologies and similar-ities are highlighted (dark shading indicates homology, light shading representsconservative substitutions and no shade shows no similarities; in the consensussequences capital letters represent homology across all groups, lowercase lettersmajority consensus, and no letter no similarity across all sequences). The active-site histidine residues (142, 146, 152) and Met166 of the Met-turn are invariant;and even though the snakes from which these enzymes are purified come fromgeographically isolated areas, they show >90% homology throughout their se-quences.

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ligation and the Met-turn motif, the hallmarks of the metzincins (aprotease superfamily containing an elongated zinc-binding metal-loprotease motif [HEXXHXXGXXH] and presenting a methion-ine residue close to the zinc-binding motif) (Bode et al., 1993).

Fibrolase differs in activity from other protease members ofthe adamalysin subfamily despite a high degree of sequence sim-ilarity. The primary action of fibrolase is the degradation of fib-rin(ogen); it displays no hemorrhagic activity. While similar inprimary and tertiary structure, other members of the subfamilypossess different activities, including hemorrhagic activity.

Preparation

Initially, methods of purification employing open-column chro-matography were utilized to isolate fibrolase from crude venom.(Markland et al., 1988). More recently, HPLC methods have beenutilized, resulting in an enzyme identical to that purified by theopen-column procedure. A three-step HPLC method provides ahigh yield and is both rapid and simple (Loayza et al., 1994). In thispurification strategy, the venom is initially separated by hydropho-bic interaction HPLC followed by hydroxyapatite HPLC. Fractionsthat possess fibrinolytic activity from hydroxyapatite HPLC containone major band when analyzed by SDS-PAGE. The final purifica-tion step by cation-exchange HPLC (CM300) eliminates a minorcontaminant and allows for separation of the two isoforms of fi-brolase. A yield of 15–30 mg per gram of lyophilized venom isachieved when starting with crude Agkistrodon contortrix contortrixvenom.

Biological Aspects

It is assumed in nature that the role of fibrolase upon envenoma-tion is to remove fibrin and fibrinogen from the blood of the prey tomaintain an anticoagulated state that will enable the rapid spreadof other venom components throughout the circulatory system. Fi-brolase is present in the venom of the southern copperhead snake,Agkistrodon contortrix contortrix, at approximately 5% of the totalvenom protein. Fibrolase shares a high degree of sequence simi-larity with a number of other venom metalloproteinases (Figure 1),

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Fibrolase 357

both nonhemorrhagic and hemorrhagic. Fibrolase, however, dis-plays only a specific fibrin(ogen)olytic activity in the cleavage ofthe α and β chains of fibrin and Aα and Bβ chains of fibrinogenand does not exhibit hemorrhagic activity. α2M inhibits fibrolasein vivo, but because the enzyme is a metalloproteinase, it is notinhibited by serine proteinase inhibitors in the blood. Fibrolasedoes not possess thrombin-like activity nor does it activate or de-grade plasminogen (Retzios and Markland, 1988). Recombinantfibrolase has been successfully produced, and this protein has beenshown to possess activity identical to that of the purified naturalenzyme. Both the natural sequence and a modified sequence (themodified enzyme produced by a yeast expression system is calledalfimeprase) have been prepared recombinantly (Loayza et al.,1994; Jones et al., 2001). From genetic analysis, it is known that fi-brolase is expressed as a preproenzyme in the snake venom gland;no other information is available concerning the gene coding forfibrolase in the southern copperhead venom gland.

Fibrolase recognizes specific peptide bonds in the cleavage offibrin(ogen), but the minimal sequence requirements for efficientcleavage are not known. Target peptides based on the Lys413-Leu414

bond cleaved in the α chain sequence of fibrin were synthesized inan attempt to address this question. Octapeptides representing theHis410-Thr-Glu-Lys-Leu-Val-Thr-Ser417 sequence of the Aα-chain offibrinogen including peptides with various substitutions for Lys413

or Leu414, and peptides containing permutations of the Leu11-Val-Glu-Ala-Leu-Tyr-Leu-Val18 sequence cleaved by fibrolase in theoxidized B chain of insulin, were evaluated as potential substratesfor fibrolase.

Peptide substrates at different concentrations were incubatedwith a constant amount of fibrolase. Following digestion for a fixedperiod of time, the reaction was stopped, and fibrolase was in-activated. The degree of peptide hydrolysis was characterized byreverse-phase HPLC according to a modified method of Guan et al.(1991). Samples of each reaction were injected onto a C18 reverse-phase HPLC column. After washing with the equilibration buffer,the column was eluted with a linear gradient of increasing acetoni-trile concentration in 0.01% TFA. The column eluate was analyzedat 214 nm with an ultraviolet (UV) detector, and the amount ofmaterial in the intact peptide and digestion product peaks werequantitated by peak integration.

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TABLE 1 Enzymatic Turnover Rates for Cleavage of Peptides ContainingSubstitutions for Lys413 in the His410-Ser417 Sequence of the Aα-Chain ofFibrinogen by Fibrolase

Peptide Substrate Turnover Rate

410His-Thr-Glu-Lys-Leu-Val-Thr-Ser417 2.23His-Thr-Glu-Arg-Leu-Val-Thr-Ser 1.97His-Thr-Glu-Ala-Leu-Val-Thr-Ser 5.00His-Thr-Glu-Leu-Leu-Val-Thr-Ser 0.16His-Thr-Glu-Phe-Leu-Val-Thr-Ser 0.45His-Thr-Glu-His-Leu-Val-Thr-Ser 5.19His-Thr-Glu-Pro-Leu-Val-Thr-Ser 1.59His-Thr-Glu-Glu-Leu-Val-Thr-Ser 0.46His-Thr-Glu-Asn-Leu-Val-Thr-Ser 65.7His-Thr-Glu-Ser-Leu-Val-Thr-Ser 3.98

Leu11-Val-Glu-Ala-Leu-Tyr-Leu-Val18 0.09(oxidized insulin β chain)

Kinetic values for fibrolase cleavage were calculated from theintegrated values of the peak areas of the intact peptide and thedigestion products. The rate of digestion was determined by di-viding the area of the individual digestion peaks by the total areaunder the peaks (intact + digestion peak). The percentage of di-gestion was then converted to the actual amount of peptide di-gested per unit time (mol/sec). The reciprocal of the velocity ofreaction was then plotted versus the reciprocal of the substrateconcentration on a Lineweaver–Burke plot from which the Km andVmax values were determined through standard linear regression.

Table 1 compares the activity of fibrolase against His-Thr-Glu-Lys-Leu-Val-Thr-Ser, the octapeptide containing the Lys413-Leu414

cleavage site of the Aα-chain of fibrinogen and octapeptides withsingle amino acid substitutions for the lysine residue. Fibrolase ex-hibits almost 300-fold differences in cleavage kinetics for variouspeptides with Lys413 substitutions, but cleaves them all. In addi-tion, fibrolase cleaves Leu11-Val-Glu-Ala-Leu-Tyr-Leu-Val18, the oc-tapeptide containing the Ala14-Leu15 cleavage site in the oxidizedinsulin B chain. These results suggest that fibrolase-mediated cleav-age is directed to an X-Leu bond. In contrast, for the octapep-tides with single amino acid substitutions for the Leu414 residue(Table 2), only the phenylalanine-substituted peptide serves as afibrolase substrate. This observation is consistent with the com-mon hydrophobic characteristics of phenylalanine and leucine.

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Fibrolase 359

TABLE 2 Enzymatic Turnover Rates for Cleavage of Peptides ContainingSubstitutions for Leu414 in the His410-Ser417 Sequence of the Aα-Chain ofFibrinogen by Fibrolase

Peptide Substrate Turnover Rate

410His-Thr-Glu-Lys-Leu-Val-Thr-Ser417 2.23His-Thr-Glu-Lys-Arg-Val-Thr-Ser No cleavageHis-Thr-Glu-Lys-Ala-Val-Thr-Ser No cleavageHis-Thr-Glu-Lys-Phe-Val-Thr-Ser 0.20His-Thr-Glu-Lys-His-Val-Thr-Ser No cleavageHis-Thr-Glu-Lys-Pro-Val-Thr-Ser No cleavageHis-Thr-Glu-Lys-Glu-Val-Thr-Ser No cleavageHis-Thr-Glu-Lys-Asn-Val-Thr-Ser No cleavageHis-Thr-Glu-Lys-Ser-Val-Thr-Ser No cleavage

These results suggest that the fibrolase cleavage specificity may bedirected toward both X-Leu and X-Phe.

Previous studies involving intra-arterial administration of thefibrolase derivative alfimeprase in a guinea pig model of thrombo-sis indicated a transient hypotension (Toombs, personal commu-nication), this was prevented by a bradykinin antagonist. In orderto determine the target protein causing the fibrolase-mediated hy-potension, the catalytic activity of fibrolase against proteins in thebradykinin synthetic pathway was assessed.

Bradykinin is produced via two biochemical routes (Figure 2)(Burch et al., 1990). High-molecular-weight kininogen can bedirectly cleaved by plasma kallikrein to generate bradykinin. Al-ternatively, low-molecular-weight kininogen can be cleaved by tis-sue kallikrein to form kallidin. Kallidin, a decapeptide, has itsN-terminal lysine removed by plasma aminopeptidase to formbradykinin. In completion of the bradykinin cycle, the breakdownof bradykinin is mediated by kininase II, yielding the inactiveheptapeptide Arg-Pro-Pro-Gly-Phe-Ser-Pro. Interestingly, fibrolasecan generate kallidin from LMW kininogen, convert kallidin tobradykinin, while at the same time cleave bradykinin in a mannersimilar to kininase II.

Various proteins and peptides of the bradykinin pathway de-scribed above were examined for cleavage by fibrolase (Table 3).Characterization of the resultant digestion products shows thatfibrolase promotes bradykinin formation through cleavage oflow-molecular-weight kininogen with the formation of kallidin.

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FIGURE 2 Effect of fibrolase on bradykinin biosynthesis and metabolism. Shownare the two independent pathways for the production of bradykinin, a peptidecritical to regulation of blood pressure. At the points indicated, fibrolase playsa role in both the production (through action on LMWK and kallidin) of anddegradation of bradykinin (a hypotensive peptide), which results in a transientdrop in blood pressure following systemic administration of fibrolase at concen-trations exceeding that of α2-macroglobulin.

Kallidin is a relatively poor substrate for fibrolase, as comparedto bradykinin, and the inefficiency of cleavage of kallidin by fi-brolase suggests that plasma aminopeptidase is required for theconversion of kallidin to bradykinin. Fibrolase does, subsequently,cleave bradykinin to the inactive heptapeptide Arg-Pro-Pro-Gly-Phe-Ser-Pro. Thus, the production of kallidin and its subsequentconversion to bradykinin accounts for the initial drop in bloodpressure, while the degradation of bradykinin (in combinationwith the rapid elimination of fibrolase by α2M, described later)results in the rapid rise to normal blood pressure. The tran-sient nature of the production and subsequent degradation ofbradykinin and its precursors mimics findings observed on blood

TABLE 3 Turnover Rates of Fibrolase-Mediated Cleavage of Peptides of theBradykinin Synthetic Pathway

Substrate

Turnover rate(mol substrate/mol enzyme sec)

Low-molecular-weight kininogen 8.60 × 10−3

High-molecular-weight kininogen No cleavageKallidin Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg 2.30 × 10−2

Bradykinin Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg 6.03

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Fibrolase 361

pressure fluctuations in animals treated with fibrolase. In sum-mary, fibrolase promotes bradykinin generation through the low-molecular-weight kininogen pathway (Figure 2). Fibrolase cleaveslow-molecular-weight kininogen, while having no effect on high-molecular-weight kininogen. Cleavage of low-molecular-weightkininogen by fibrolase results in the generation of kallidin. Kallidinis a poor substrate for fibrolase. However, fibrolase was effective inthe cleavage of bradykinin. Mass spectrometry confirms that fibro-lase cleaves bradykinin between residues Pro7-Phe8 to form theinactive heptapeptide Arg-Pro-Pro-Gly-Phe-Ser-Pro. These actionspresumably result in the transient hypotensive response followingfibrolase treatment.

Distinguishing Features

Fibrolase has been used as an experimental thrombolytic agent inartificially created thrombi in both rabbit and canine models. Theefficacy of fibrolase in lysing occlusive thrombi in the carotid arter-ies of anesthetized dogs was compared to anisoylated plasminogenstreptokinase activator complex (APSAC) (Markland et al., 1994).Briefly, electrolytic injury in both the right and left carotid arterieswas utilized to create a stable thrombus. Thirty minutes followingocclusion, the vessels were infused with either fibrolase (4 mg/kgover 5 min) or physiological saline (vehicle, over 5 min). Addi-tionally, in two separate groups of dogs, APSAC (0.1 U/kg) wasinfused into the occluded vessel for comparison with fibrolase. Inthe artery infused with fibrolase, all of the dogs exhibited patencywithin 6 ± 1 min of infusion (P < 0.05 versus vehicle-treated artery;Fisher’s exact test). The occlusion was maintained throughout theexperimental protocol in all thrombi treated with vehicle alone.APSAC alone lysed the thrombus in each vessel within 27 ± 3 min.Five minutes after the end of fibrolase administration and in oneof the groups administered APSAC, a glycoprotein (GP) IIb/IIIaantibody, 7E3 (0.8 mg/kg IV), was administered to prevent reoc-clusion of the patent artery. After 7E3 administration, the vesseltreated with fibrolase remained patent in four of five dogs, andsix of six APSAC-treated vessels were patent for the remainder ofthe observation period (2 h) (Figure 3). These studies demon-strated that local administration of fibrolase lyses a carotid arterialthrombus rapidly without excessive hemorrhage or hemodynamic

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FIGURE 3 In vivo analysis of thrombolytic efficiency. Thrombi were inducedin canine carotid arteries (six per group) through electrolytic injury to the in-timal surface of the vessel. Once thrombi were established, they were aged for30 min to ensure stability of the clot. Using a catheter established at the surfaceof the clot, thrombolytic agents, fibrolase (4 mg/kg), or activated streptokinase(0.1 U/kg), were introduced over a 5 min injection. Time to lysis and confirma-tion of occlusion of the artery were determined via Doppler flow probe. Averagelysis times are shown for each treatment group. Because this model results in apersistently reoccurring thrombus, the antithrombotic agent 7E3 was adminis-tered with APSAC and fibrolase following clot lysis; an APSAC control without7E3 was also included. The arteries were followed for an additional 2 h followingthrombolytic administration. Fibrolase rapidly cleared the thrombi in all cases.(∗Note: Reocclusion was observed in one of the six fibrolase dogs immediatelyfollowing following successful lysis.)

compromise. No side effects or adverse reactions were evident fol-lowing both gross and histological examination of the treated ani-mals after lysis of thrombi. Due to its direct mode of action, fibro-lase shows promise as a therapeutic agent. Both t-PA and uroki-nase act indirectly in thrombolysis relying on the conversion ofplasminogen to plasmin which can lead to systemic side effects.Fibrolase acts directly on the thrombus and is then inactivated byα2-M, thereby eliminating potential negative systemic side effectsat clinically useful doses.

Chimeric Derivative of Fibrolase

In an effort to improve the activity of fibrolase through a reductionin rethrombosis in fibrolase-treated vessels, the enzyme has beencoupled with an RGD-like peptide to form a platelet avid chimera(Swenson et al., 2000). This chemical coupling does not affectfibrinolytic activity but imparts a novel activity to the enzyme—the

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Fibrolase 363

ability to inhibit platelet aggregation by binding to the fibrinogenreceptor (GPIIb/IIIa) on the platelet surface.

Because rethrombosis is a serious complication experiencedin a significant percentage of patients treated with thrombolyticagents to remove occlusive vascular thrombi, and the involve-ment of platelets in the initiation of rethrombosis is well known,a chimera has been constructed with both fibrinolytic and an-tiplatelet activity (Sanchez et al., 1997; Swenson et al., 2000). Pep-tides containing the Arg-Gly-Asp (RGD) motif have been shownto inhibit rethrombosis following thrombus dissolution by plas-minogen activator (Baker et al., 1992; Rote et al., 1993; Udvardyet al., 1995). In an effort to create a more effective fibrinolyticenzyme by targeting the enzyme to platelet-rich thrombi, therebydecreasing the potential for rethrombosis, a chimeric derivative offibrolase was produced (Swenson et al., 2000). A possible disad-vantage in the use of fibrolase as a thrombolytic is that the enzymedoes not contain a thrombus-targeting motif (such as a kringledomain), like the tissue plasminogen activators (Randolph et al.,1992). The introduction of a targeting motif should facilitate thelocalization of fibrolase to the site of the thrombus; this repre-sents a potential benefit from forming a platelet-avid derivative offibrolase. GPIIb/IIIa, the fibrinogen receptor on the platelet sur-face is activated, enabling platelets to bind fibrin when circulatingplatelets are recruited into a growing thrombus. In an attempt toproduce a chimeric derivative of fibrolase with platelet avidity, anRGD-like peptide was covalently linked to a surface residue of fi-brolase distant from its active site (Figure 4). By using an RGD-likepeptide to form the chimera with fibrolase, the peptide is availableto bind to the platelet fibrinogen receptor. The platelet-bindingability of the chimeric enzyme would aid in its localization at thesite of the growing thrombus and the enzymatically active portionof the chimera would be available for fibrinolysis. Formation of abifunctional chimeric enzyme with the ability not only to partici-pate in thrombus dissolution but also to bind to activated plateletsand prevent rethrombosis would represent a unique approach tothrombolysis.

A number of different cross-linking agents are available thatcan bind to different functional groups in proteins (Fujiwaraet al., 1988; Carter, 1994). N -(γ -maleimidobutyryloxy) sulfosuccin-imide ester (S-GMBS) was selected to covalently couple fibrolase to

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FIGURE 4 Structure of RGD-like peptide covalently attached to fibrolase.(A) The cyclic nonapeptide P734 contains an RGD-like sequence. Two nonstan-dard amino acids are included in or near the RGD-like sequence—D-Tyrosineand 4-amidinophenylalanine (Amp)—arginine in the RGD loop is substitutedby 4-amidinophenylalanine. Cyclization of the peptide is accomplished throughan internal thioether linkage. A free sulfhydryl is contained in the peptide thatis used for covalent attachment to the cross-linking agent. (B) Predicted three-dimensional structure of fibrolase. The active site of fibrolase spans amino acids139–159 and contains a zinc atom (represented by the sphere), which is com-plexed by three histidine residues (represented by the pentagons). This three-dimensional representation is positioned in order to see the active site. The po-sition of adduction of both the heterobifunctional cross-linker and polyethyleneglycol (Lys183) is identified by the red arrow. This Lys residue is distant from theactive site, and its modification does not effect activity.

the RGD-like peptide. This heterobifunctional cross-linking agentcontains both N -hydroxysuccinimide ester (NHS) and maleimidegroups. To engineer the chimera, advantage was taken of theknowledge that fibrolase contains free primary amines (the aminoterminus and the ε-amino groups of the seven lysine residues) andno free thiols. By contrast, the peptide contains no free aminesand a single free thiol. Covalent adduction of S-GMBS to fibro-lase through reaction of NHS with available primary amines isthe first step in chimera formation. This was then followed by theaddition of peptide to the enzyme-bound cross-linker using thefree thiol in the peptide and the reactive maleimide group inthe cross-linker. This two-step reaction was optimized by altering

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Fibrolase 365

the time of reaction as well as the stoichiometry of the reactants. Co-valent modification of fibrolase with the RGD-like peptide did notinhibit fibrinolytic activity or the platelet aggregation inhibitoryactivity of the chimera. The chimera not only retained the samelevel of enzymatic activity as native fibrolase but also acquired theability to inhibit platelet aggregation by binding to the fibrinogenreceptor (GPIIb/IIIa) on platelets.

The amino acid sequence of fibrolase indicates that there areseven ε-amino groups of lysine available for attachment of the cross-linker. Although fibrolase contains a number of lysine residues,there are no free thiols in the enzyme, because all six cysteineresidues are involved in intramolecular disulfide bonds. Thus, themaleimide of S-GMBS was restricted to reacting with the free-sulfhydryl of the synthetic RGD-like peptide. Our data indicatedthat the cross-linker reacts with fibrolase at close to equimolarstoichiometry, and that S-GMBS-fibrolase is fully reactive with theadded sufhydryl-containing RGD peptide. Tryptic peptide map-ping allowed for the determination of the site of RGD-like peptideattachment to a single lysine residue remote from the enzyme ac-tive site. The effect on the overall structure of fibrolase causedby forming the peptide adduct has been analyzed using a three-dimensional model of fibrolase (Bolger et al., 2001), which wasconstructed based on homology with the known structures of othermembers of the metzincin family of proteinases (Gomis Ruth et al.,1994; Zhang et al., 1994; Bode et al., 1996; Kumasaka et al., 1996).As observed from this model (Figure 4B), covalent attachment ofthe RGD-like peptide to Lys 183 distances the added peptide fromthe active site of the enzyme. While the model of fibrolase indicatesthat all of the lysine residues lie on the surface of the molecule(with extended incubation, virtually all of the lysines are capableof reacting with the S-GMBS), lysine at position 183 is shown tobe the most reactive with S-GMBS. From the structural model offibrolase, the predicted position of this lysine is on an extendedloop held in position by two disulfide bonds, making it an idealcandidate for reaction. In addition, the predicted positioning oflysine 183 as being distant from the active site correlates with ourfindings, which show essentially no loss of intrinsic fibrinolytic ac-tivity in the chimeric molecule.

The RGD-like peptide (P734, Figure 4A) inhibits platelet ag-gregation with an IC50 of 76 nM, whereas the peptide-fibrolase

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FIGURE 5 Fibrolase-P734 chimera retains inhibitory activity on ADP-inducedplatelet aggregation. Chimeric fibrolase inhibits platelet aggregation at levelscomparable to P734 alone. Complete inhibition of platelet aggregation is ob-served at concentration >120 nM. Native fibrolase and S-GMBS-fibrolase areplotted on the x axis, because neither displayed any effect on platelet aggrega-tion, even at very high concentrations (>1,600 nM).

conjugate has an IC50 of 105 nM. Concentrations of native fibrolasealone ranging from 100 nM to 1.6 µM showed no inhibition ofADP-induced platelet aggregation. As expected, a dose-responsecurve of inhibition of platelet aggregation is generated in responseto increasing concentrations of the chimeric protein (Figure 5),with complete inhibition at concentrations greater than 120 nM.The shape of the inhibition curve is normal for dose-responsepharmacokinetic binding curves. The biphasic nature of the ag-gregation inhibition curve is due to the requirement to block afinite number of RGD binding sites on platelets before there iselimination of platelet aggregation.

While it is evident that the chimera inhibits platelet aggre-gation, a thorough evaluation of the fibrinolytic activity of thechimera was also carried out. Kinetics of hydrolysis of the syntheticoctapeptide substrate containing the Lys413-Leu414 cleavage sitefrom the Aα-chain of fibrinogen displayed essentially no changein cleavage kinetics as compared to native unmodified fibrolase;Km and Vmax for the native enzyme are indistinguishable fromthose of the chimeric protein. Although it was expected that ad-duction of the RGD-like peptide to fibrolase would alter the ki-netics of inhibition of fibrolase by α2-macroglobulin (α2M), thiswas not observed. Preincubation of fibrolase with physiological

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Fibrolase 367

concentrations of α2M, using either fresh plasma or purified α2M,and assay of fibrinolytic activity by the fibrin plate method indi-cates that formation of the chimera does not significantly alterthe ability of α2M to inhibit fibrinolytic activity. Thus, similar lossof fibrinolytic activity was observed following incubation of eitherchimeric or native fibrolase with α2M.

We hypothesized that the mechanism of inhibition of plateletaggregation by the chimera was due to the RGD-like peptide in thechimera being targeted to platelet GPIIb/IIIa. When 125I-fibrolase-chimera was incubated with washed platelets in the absence of theplatelet agonist ADP, we found no platelet binding of chimera ornative fibrolase. However, after ADP-induced activation, plateletsbound the chimeric protein but not native fibrolase. Proof of theinvolvement of GPIIb/IIIa was shown by the finding that 125I-fibrolase-chimera binding to activated platelets was completely in-hibited following preincubation of platelets with a large excess ofthe GPIIb/IIIa antagonist, GRGDSP. The finding that inhibitionof platelet aggregation by chimeric fibrolase involves binding toGPIIb/IIIa supports our hypothesis that the covalent attachmentof the platelet-avid peptide to fibrolase confers platelet-binding ca-pacity and platelet aggregation inhibitory activity to the chimericprotein.

The chimeric derivative of fibrolase is a potent inhibitor ofplatelet aggregation; formation of the chimera does not signifi-cantly alter the platelet aggregation inhibitory activity of the pep-tide. Platelet aggregation is one of the central events in the for-mation of an arterial thrombus (Mustard et al., 1990; Ruggeri,1997). When platelets become activated, a complex cascade is ini-tiated that terminates with the formation of a thrombus consist-ing of platelets tightly bound to fibrinogen through GPIIb/IIIaunder normal flow conditions. Degradation of the thrombus canbe accomplished either by plasminogen activators or by direct-acting agents such as fibrolase. It is generally felt that target-ing of the thrombolytic agent to the occlusive thrombus shouldavoid systemic fibrin(ogen)olysis. Thus, in the formation of thechimeric fibrolase derivative, a targeting mechanism is introducedthat should lower the potential for systemic fibrin(ogen)olysis fol-lowing intravenous administration. Our approach of creating adual-acting agent that not only degrades fibrin but simultane-ously targets fibrolase to the platelet-rich thrombus, should lead

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to inhibition of platelet aggregation and thrombus reformation.The fibrolase-RGD-like peptide conjugate carries out these dualroles at efficiencies close to those of the individual components.Neither the peptide nor fibrolase significantly blocks activity ofthe other component of the chimera. In a reoccluding arterialthrombosis model using selective administration of the chimera,the combined thrombolytic/antithrombotic approach should notonly degrade the thrombus but should also prevent reocclusion.

Limitation of α2M Inactivation by Formation of Size-EnhancedForms of Fibrolase

Fibrolase in vivo is efficiently and rapidly inactivated by α2M, ageneral proteinase inhibitor found in blood (Toombs, 2001). Inorder to allow for longer circulatory half-life and to enable morecomplete thrombus dissolution in vivo, a modification to the en-zyme is necessary to block its rapid inactivation by α2M, which ispresent in the blood at fairly high concentrations (∼=3 µM) (Laureland Jeppson, 1975). α2M functions by binding to and sequester-ing small proteinases and removing them from the circulation viathe formation of a covalent bond between the proteinase and the720 kDa tetrameric inhibitor. Proteinases larger than ∼40 kDa ex-hibit diminished interactions with α2M due to steric limitations inthe proteinase trap (Werb et al., 1974; Kolodziej et al., 1998). Thesteric limitation is exemplified by the observation that a 68 kDahemorrhagic metalloproteinase from Crotalus atrox venom is notinhibited by α2M, while a 23 kDa metalloproteinase, with a highdegree of sequence homology to the 68 kDa metalloproteinase, israpidly and effectively bound and inhibited by α2M, (Baramovaet al., 1990). Upon proteinase binding, a structural alteration isinduced in α2M, and the transformed complex exposes receptor-binding domains (RBDs) that allow its rapid endocytosis by cell-membrane receptors principally displayed by hepatocytes but alsoby a variety of other cells (Sottrup-Jensen, 1989; Ashcom et al.,1990; Baramova et al., 1990; Strickland et al., 1991). Although thesequestered proteinase is not able to act on large target molecules,covalent attachment between α2M and the proteinase does notinactivate the active site of the entrapped enzyme (Barrett andStarkey, 1973). Studies in which small peptide substrates are incu-bated with metalloproteinase, previously incubated with purified

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Fibrolase 369

α2M, show the proteinase is able to cleave the small peptides butis inactive against large protein substrates. This indicates that theenzyme is still catalytically active but is sterically restricted fromaccess to large substrates (Barrett and Starkey, 1973).

In order to limit the interaction between fibrolase and α2M,we have introduced a covalent modification of the enzyme witha water-soluble polymer of polyethylene glycol (PEG). This yieldsan enzyme that should be sterically limited in its interaction withα2M. We have introduced this modification without altering thefibrinolytic activity of the enzyme in vitro. The size-modified fi-brolase, due to its increased bulk and rotational size, is shownto interact with α2M with slower kinetics and thus, should havea longer circulatory half-life. Slowing the interaction of fibrolasewith α2M may allow for more rapid and effective thrombolysis bythe fibrinolytic enzyme and at lower administered dosages. This iscounterbalanced by potential side effects related to a circulatingactive proteolytic enzyme.

The method for the construction and biochemical character-ization of PEG-modified fibrinolytic enzyme is briefly describedbelow. The degree of polymer adduction and site(s) of attach-ment have been determined. Finally, the retention of fibrinolyticenzyme activity following polymer adduction, both in the presenceand absence of α2M, has been determined.

Activated polyethylene glycols (PEG) that contain an N-hydroxy succinimide (NHS) ester are commercially available, andthese polymers can be readily adducted to primary amines (Clarket al., 1996). ε-Amino groups of lysine are generally used for NHSmodification, as the α-amino group in some proteins is not avail-able for reaction. This is the case with fibrolase, in which there isa cyclized Gln at the NH2-terminus (Randolph et al., 1992). NHSester adduction has been successfully used for the modificationof fibrolase, resulting in the attachment of polyethylene glycols ofmolecular weights 5,000 and 20,000 to a surface lysine residue(s).PEGylated fibrolase is created through the reaction of PEG of thedesired molecular mass with fibrolase in different stoichiometriesdepending on the extent of PEGylation desired. A single predom-inant form of the modified protein was produced following cross-linking for a short time with a 10:1 molar ratio of amine reactivePEG to fibrolase. PEGylated-fibrolase was purified by size-exclusionchromatography, using molecular sieve HPLC. On the first pass

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over the sizing column, a nearly homogenous preparation of thepredominant form of PEGylated fibrolase was obtained. Final pu-rification was accomplished by a second run through the sizingcolumn using the same conditions. From the second pass throughmolecular sieve HPLC, we obtained a fraction containing PEGy-lated fibrolase that was determined to have a mass of of 43,331 Da,as determined by matrix-assisted laser desorption ionization massspectrometry (MALDI-MS). This mass corresponds to the adduc-tion of one NHS-PEG (20 kDa) molecule per molecule of fibrolase.Alteration of both time of incubation and PEG:fibrolase stochiom-etry resulted in a number of different adducted species. Using theconditions described above, we found that the purified product ofthe PEGylation reaction migrated on SDS-PAGE with an apparentmolecular weight of 60,000 Da, different from the mass obtainedby mass spectrometry and from that expected for a globular pro-tein. Nonetheless, the PEGylated species existed predominatelyas a single band by SDS-PAGE. This anomaly can be explainedby the SDS-PAGE method, where SDS is incorporated along thepeptide backbone in proportion to the mass of the protein. ThePEG adducted species would be larger but without the sameadded negative charge imparted from the SDS, so in an electricfield, the protein would run with an apparently larger molecularmass.

As already noted, there are seven lysine residues in the pri-mary structure of fibrolase. From our model of fibrolase, each ofthese lysine residues lies sufficiently outside the active site so thatmodification of any of these lysine residues should have no ef-fect on activity of the enzyme (Bolger et al., 2001). Purified PEG-ylated fibrolase has been analyzed by SDS-PAGE to determine ho-mogeneity, but as indicated above, the true mass of the modifiedfibrolase has been determined through mass spectrometry. Exactmass determination allows for a calculation of the number of PEGmolecules attached to the fibrinolytic enzyme. The site of attach-ment of PEG in fibrolase has been determined by site-specific pro-teolytic cleavage of PEGylated fibrolase followed by amino acidanalysis of the PEGylated peptide fragments.

Using a 10 min coupling reaction at room temperature anda PEG:fibrolase ratio of 10:1, the PEG adduction is primarily lo-cated at Lys183 (Figure 6). This lysine residue is part of a Lys-Lyssequence located on a flexible loop distant from the active site with

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TABLE 4 PEGylated Fibrolase Retains Proteolytic Activity

Specific Activity

Enzyme SpeciesAzocassein(units/µg)

%Control

Human Fibrin(units/µg)

%Control

Fibrolase 1.26 ± 0.03 100 11.3 ± 0.1 100PEGylated-Fibrolase 1.20 ± 0.03 95 10.9 ± 0.1 97

full exposure to the surrounding solvent, based on the proposedfibrolase model (Bolger et al., 2001). This reactive lysine has alsobeen identified as that adducted by an amine reactive heterobi-functional cross-linking reagent (Swenson et al., 2000).

Following determination of the number of PEG adducts andthe position of the PEG attachment, purified PEGylated-fibrolasewas analyzed for fibrinolytic activity. The in vitro assays comparedthe relative fibrinolytic activity of either PEGylated fibrolase or na-tive enzyme, alone or in the presence of α2M. PEGylated-fibrolasehas been assayed for general proteolytic and fibrinolytic activ-ity utilizing a colorimetric azocasein assay and a fibrin plate as-say (Table 4). Quantitation of specific activity by the fibrin platemethod is made through a previously described method that relieson a determination of the change in optical density (OD 405 nm)of the fibrin plug per unit weight of enzyme applied to the fib-rin clot (Patton et al., 1993; Swenson et al., 2000). By comparingPEG-modified fibrolase and unmodified enzyme (Table 4), it isobvious that there is no loss of proteolytic activity upon PEGylationof fibrolase (using the NHS cross-linking method). In the fibrinplate assay, 97% of native protein activity is retained showing thatPEGylation does not alter the structure or function of the activesite of fibrolase.

While it is apparent that fibrinolytic activity is retained by PEG-ylated fibrolase, the effects of PEGylation on kinetics of substratehydrolysis are of interest. Cleavage kinetics were determined forboth PEGylated and native fibrolase on the synthetic target peptidewith the sequence HTEKLVTS containing the scissile bond cleavedby fibrolase in the α-chain of fibrin. Cleavage was assessed by moni-toring the formation of peptide fragments and the loss of the intactpeptide. Comparison between the rate of peptide substrate cleav-age by PEGylated and native fibrolase indicates that there is no dif-ference in the rate of substrate cleavage following PEG adduction.

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Fibrolase 373

From the Lineweaver–Burke plot, it was determined that Vmax andKm were identical for native and PEGylated fibrolase. The best-fitlines on the Lineweaver–Burke plots for PEGylated fibrolase andnative fibrolase superimpose, indicating that there is no changein either the velocity of the reaction or the efficiency of substrateutilization. This kinetic data show that PEGylated fibrolase cleavesthe synthetic substrate efficiently and rapidly, indicating that thebulky PEG group has no impact on enzymatic activity with eithera large (fibrin plate assay) or a small peptide substrate.

Having established that PEGylation does not alter fibrinolyticactivity, we determined the rate of interaction between α2M and na-tive and PEGylated fibrolase. As already indicated, the main mech-anism of fibrolase clearance from the circulation is by binding toα2M. The interaction of α2M and fibrolase is mediated by the cleav-age of a peptide bond in the bait region of α2M by fibrolase. Thiscleavage leads to a conformational change in α2M and irreversiblecovalent bond formation with the enzyme after cleavage of the baitregion. SDS-PAGE can be used to observe the kinetics of bindingof native and modified fibrolase to α2M, but in the case of nativefibrolase, the in vitro interaction is so rapid that the kinetics of thisinteraction could not be effectively evaluated via this method.

Although SDS-PAGE is an important method for observingthe binding of either modified or native fibrolase to α2M, theinteraction of the fluorescent compound 2-(p-toluidinyl) naphtha-lene-6-sulfonic acid (TNS) with conformationally changed α2Mcan be used to monitor kinetics of the rapid interaction. TNSbinds to native α2M with very low affinity, but the affinity increasesdramatically when the conformation of α2M is changed by cleav-age of its bait region (Strickland et al., 1991). The kinetics of thiscleavage were monitored by recording the increase in the fluores-cence signal of TNS as fibrolase or PEGylated fibrolase is bound byα2M (Bjork et al., 1989). PEGylated-fibrolase shows a significantdecrease in the rate of interaction with α2M as compared to theextremely rapid entrapment of native fibrolase, but there is notcomplete elimination, only a slowing of proteolytic entrapment(Swenson and Markland, unpublished data).

The ability of α2M, either the purified or plasma form of theprotein, to inhibit the activity of PEGylated and unmodified fibro-lase was determined. Using the TNS method, we found that na-tive fibrolase is quickly and efficiently inactivated by α2M. We then

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TABLE 5 PEGylation of Fibrolase Significantly Reduces Proteolytic Inhibitionby α2-Macroglobulin

Fibrin plate clearance (in mm2) By Fibrolase or PEGylatedFibrolase Incubated for 45 Sec in the Presence of

Enzyme Species Saline Human Plasma α2-Macroglobulin

Native fibrolase 11.3 ± 0.02 0.0 ± 0.2 0.0 ± 0.2PEGylated fibrolase 10.9 ± 0.2 10.4 ± 0.2 10.1 ± 0.2

determined the difference in the extent of inhibition of PEGylatedand native fibrolase by α2M. Both forms of fibrolase at known con-centration were incubated with known amounts of α2M, either pu-rified or in plasma (concentration of α2M in plasma was assumedto be the literature value of 2.9 µM). The mixtures were incubatedfor 45 sec at 37◦C and then placed directly on the fibrin plate inthe presence of methylamine, which prevents α2M from bindingto proteinases (Sottrup-Jensen, 1989). As is evidenced by Table 5,native fibrolase is fully inactivated by both purified and plasmaforms of α2M during the 45 sec incubation. However, PEGylatedfibrolase shows little or no loss of activity when incubated withα2M under these conditions. Thus, it is apparent that PEGylationprovides partial protection of fibrolase from rapid inactivation byα2M.

Conclusion

In recent years, a number of clinically useful agents have beendeveloped from natural products, and research in this field hasreceived increasing attention. Didisheim and Lewis described theclinical potential of components found in snake venom, includ-ing fibrinolytic activity, nearly 100 years ago. These snake venomfibrinolytics garner particular attention in that they bypass one ofthe problems associated with current thrombolytics, systemic plas-minogen activation. Fibrolase from southern copperhead venomeffectively and directly degrades fibrin(ogen) based on cleavage ofa Lys-Leu bond in the Aα-chain, and several bonds in the Bβ chain.While fibrolase, in a slightly altered recombinant form, continuesto make its way to the clinic, further study has shown that modifica-tion of the enzyme can impart interesting and useful functions to

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the molecule. The covalent modifications of fibrolase have beenaimed at imparting additional biologically relevant activities to theenzyme. Importantly, the introduction into clinical trials of a fib-rinolytic enzyme that has biochemical and biophysical propertiesnearly identical to the venom enzyme, is an illustration of the pow-erful capabilities of modern biotechnology. The technical studiesnecessary to bring a modified form of fibrolase into clinical useillustrate the complicated path that needs to be followed in or-der to introduce new clinical agents (see paper by Dietcher andToombs, this volume). Snake venoms have evolved over millions ofyears, and through the study of the components of venom, naturalevolution can shape the development of novel pharmaceuticals.

Acknowledgments

The authors wish to thank the National Institutes of Health for fi-nancial support given during the development of fibrolase and thechimeric derivatives (NIH STTR Grant 2R42 HL052995 to FSM).In addition, the authors wish to thank Hasmik Agadjanian for tech-nical assistance in the preparation of purified venom components.

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FIBROLASE