CA2194692A1 - Fibrin(ogen) degradation and clot lysis by fibrinolytic matrix metalloproteinase - Google Patents

Abstract

The invention provides a method of causing the degradation of fibrin(ogen) (i.e., fibrin, fibrinogen, and related substances) by means of a fibrinolytic metalloproteinase, preferably an endogenous metalloproteinase such as MMP-3. The method of the invention can be performed in vitro to provide diagnostic information characterizing fibrin(ogen) and fibrinolytic physiology. The method can also be performed in vivo as a method of thrombolytic therapy in which a fibrinolytic metalloproteinase is administered to a subject to degrade thrombus in situ. The endogenous fibrinolytic metalloproteinase can be administered in conjunction with other active agents, preferably with agents having thrombolytic activity to improve thrombolytic and fibrinolytic therapy. The invention further provides compositions containing a fibrinolytic metalloproteinase for the performance of fibrinolytic or thrombolytic procedures. Also provided are kits which include a fibrinolytic metalloproteinase for performing fibrinolytic or thrombolytic procedures.

Description

2 1 ~ 2 WO 96136227 PCI/US96107~88 FIPT'lN~OGF,N) DEGR~ TlON Al~D Cl.OT LY
BY FlRR~OI,YTlC I~ TRlX ~F~T~ opRoTEll~A~F~

This is a i.. part of application Serial No. 08/446,887, filed on May 17, 1995, the entire disclosure of which is , . ' herein by reference.

BACKGRnlJND OF T~l?, IN V ~ )N
This invention relates to a method of c.~ ' ' breakdown of Sbrinogen and fibrin. More I Ih~ulady~ the invention relates to a method for degrading fibrinogen and causing lysis of fibrin clots tbrough mediation by a fibrinolytic matrix " , ~: The invention further relates to the use of fibrinolytic " r I ' as an ' UlllbUL~ to10 reconstruct s~enotic vessels and remove fibrin deposits.
The clotting of blood is part of the body's natural response to injury or trauma. Blood clot formation derives from a series of events called the r ~ ' cascade, in which the final steps involve the formation of the enzyme thrombin. Thrombin converts circulating fibrinogen into fibrin, a mesh-like structure which forms the insoluble framework of the blood clot. As a 15 part of l-.omnctl~cic" clot formation is often a life-saving process in response to trauma and serves to arrest the 'dow of blood from severed vasculature.
The life-saving process of clot produceion in response to an injury can become life-threatening when it occurs at hlalJ~nu~ iaLI~ places in the body. For example, a clot can obstruct a blood vessel and stop the supply of blbod to an organ or other body part. In 20 addition, the deposition of fibrin contributes to partial or complete stenosis of blood vessels, resulting in chronic diminution of blood 'dow. Equally life-threatening are clots that become detached firom their original sites and fiow through the circulatory system causing blockages at remote sites. Such clots are known as embolisms. Indeed, IJ ~I nlr~ of blood c~ ;. "
such as heart attacks, strokes, and the like, have been estimated to account for alJlJ~wd~aL
~ 25 fifty percent of all hospital deaths.
The formation of fibrin during ~ , tissue repair, or hPmnct~cic, plays only a temporaty role and must be removed when normal tissue structure and function is restored.

WO 96/36227 2 I q 4 6 9 2 PCT/US96/07188 Thus, a fibrin clot that forms quickly to stop I ' _ in an injured blood vessel is remodeled and then removed to restore normal blood fiow as heaGng occurs. The system responslble for fibrin breakdown and clot removal is the fibrinolytic system. Action of the fibrinolytic system is tightly c~u, d ' through the interaction of activators, ,ymogens, 5 en,ymes, as well as through inhibitors of each of these ~ , to provide focused local activation at sites of fibrin deposition ~irancis et al. 1994; Collen 1980; Collen et al. 1991).
The principal mediator of fibrinolysis is plasmin, a trypsin-Gke ~ n ~I~JP. I.~ which cleaves fibrin to dissolve clots and to permit injured tissues to regenerate. Plasmin has also been d ' ' to play a role in degrading proteins involved in cell-cell and cell-matrix as well as in activating other tissue remodeling enzymes such as matrjx ~ " " ' (Murphy et al. 1992). In tur4 control of plasmin activity, as well as these other w.l- a .~ l~ events, is principally mediated by j ' g activators, which convert the inactive zymogen pl : -.,,. to the active enzyme plasmin.
In cGnical settings it is commonly desirable to activate or potentiate the fibrinolytic 15 system. This is particularly necessary in cases of myocardial infarction in which coronary arteries become occluded and require n " ~ has proven somewhat effective in such .~ ' , but p~ ;;,. agents are desired to supplement or replace such invasive procedures to inhibit reocclusion. The study of the intricate system of Ih UmbOI,~ and fibrinolysis has been a rapidly growing field, which has resulted in the 2û de~ ' r ' of a new generation of lh. u...bulyli-, agents.
Previous therapeutic treatments for dissolving life-threatening clots have included injecting into the blood system various enzymes which are known to break down fibrin.
(Collen 1996) The problems with these treatments has been that the enzymes were not site-speciSc, and, therefore, would do more than just cause dissolution of the clot. In 25 additio4 these enzymes interfere with and destroy many vital protein; ~ that serve to keep the body firom bleeding excessively due to the many minor injuries it receives on a daily basis. Destruction of these safeguards by such enzymes can lead to serious 1 1,, and other potentially fatal . ' Currently, the best known therapeutic agents for inducing or enhancing lL.~
3û are ~ which cause the activation of ~ , the so-called "p' ~, activators" (Brakman et al. 1992). These compounds cause the hydrolysis of the _ 21 94~

arg560-val5G1 peptide bond in, ' ,, This hydrolysis yields the wtive two-chain serine protease, plasmin. A number of such ~ ' O activators are known, including serineproteases such as urokinase, ' a,, wtivator (u-PA), tissue-type 1 ' ~, activator(t-PA), ~ t ' (a l,.~J - protein) and ~ ' ylùL~ .. Of these, ~L- ~, ' is ~ 5 the most widely used therapeutic ~ yL;., agent. However, while ~LI, ' ' and the other, ' ,, activators have proven helpful in 1~ " of coronary arteries, their ability to improve mortality is not devoid of side effects and their use still requires stringent control conditions to whieve success in a high percentage of cases (Martin et al. 1994) . In addition, the use of such , ' can cause bleeding, , ' in susceptible 10 individuals. On the other hand, one of the drawbacks of the use of t-PA in clinical trials has been the early ,~ " of the clot after it has been dissolved, resulting in thrombotic .~ ' in some patients.
Numerous studies have t's ' ' the ability of t-PA to initiate or potentiate ~L.u..~bu4ii., ~ ' (Sobel et al. 1987). As a result, t-PA, specifically its 1~. ' 15 form, rt-PA, is becoming more popular as a ~ k. ~ ' ' , rt-PA
does suffer from serious limitations, including extremely high dosage cost, and variable efflcwy. In addition, specific ~pi~ . " inhibitors of t-PA have been identified in human plasma and other fluids (Collen et al. 1987). A further approach to t-PA involves the potential use of gene transfer of and expression of I ~ ' t-PA in endothelial cells (Lee et al.
20 1993). This procedure is ~ " ~'J complex and is not likely to be practical as a LIIIUIIIIJOI~ treatment in the near future.
Enzymes other than plasmin are also known which can degrade fibrin(ogen) to different extents. For example, f ~g~ leukocyte proteases (Bilezikian et al. 1977; Plow et al. 1975), later identified ~ elastase and cathepsin-G (Gramse et al. 1978; Plow 1980; Plow 25 et al. 1982), can partially degrade fibrin(ogen). Exogenous enzymes are also known which degrade fibrin. Such enzymes include hemolytic enzymes collected from the venom of certain snakes, e.g., the families crotalidae and viperidae (Purves et al. 1987; Retzios et al. 1992;
Sanchez et al. 1991). Fibrinolytic enzymes isolated from snakes can be grouped into two differentclasses(Guanetal. 1991). Thoseenzymesthat".Lf~,.. ",y degradetheAa-chain 30 of fibrinogen and also the a- and ~-chains of fibrin are zinc " " (Guan et al.
1991) and all can be inhibited by EDTA. Enzymes in the second class are serine proteinases, Wo 96r~6z27 2 1 9 4 6 ~ 2 PCI/US96107188 andexhibitspecificityforthei3-chainoffibrin(Guanetai. 1991). An. ~ A~ frompuff adder venom (Biiis arie,~ms) can cleave at the y-chain cross-linking site and thereby cleave Fragment D-dimer into a D-iike monomer (Purves et ai. I 9~v7). Fibrinolytic enzymes have aiso been obtained from leeches (Zavalova et ai. 1993; Budzynski 1991), as well as from the S growth medium of a bacterium (Aeromo,7as ~ , v~ ;lu) which was recovered from leech nntestinai tract (Loewy et ai. 1993).
~ i~.io" . ~ matrix " . ~ (M~s)or"matrixins"includethreeclassesof enzymes~ , gelatinases, and vl~u~ ;.,v. MMPs are known to have the capacity to degrade a number of proteins and ~n uleGoi~ which are associated with the ., n ,.. ,.lli , I û matrix (ECM) of connective tissue. They have been shown to break down a number of proteins including coliagen (Types I-IV, Vli and X), laniinin, fibronectin, elastin and MMPs have aiso been identified in leukocytes (Welgus et al. 1990). It has been shown that ~P-2 and MMP-9 possess elastase activity (Senior et al. 1991), to which some of the complex proteolytic activity, initiaiiy observed in O ' - ~ t~,v, could be attributed (Sterrenberg et ai. 1983). MMPs participate in the remodeling oftissues in ~Jh~ OlO~al processes such as ~l~u~iJhvO~ vtv and embryonic d~ (, t, as well as in the patllu~JhJ~;olvOy of wound heaiing, tumor invasio4 and arthritis (Matrisian 1992; Nag_se et ai.
I 991; Woessner 1991; Werb et ai. 1992).
The expression of MMPs and their inhibitors is under extensive and varied molecular 2û and ceiiular control (Kieiner et ai. 1993; Matrisian 1992; Woessner I 991). Known regulating factors include hormones, ~tokines, proto-oncogenes, steroids, and growth factors. MMPs . are blocked by specific inhibitors called "tissue inhibitors of l ~c r ~ ~ " (TlMPs) that can block the activity of each member of the family. An enzyme inhibitor complex is formed and no turnover of coMective tissue takes place if the MMPs are present in excess. The main focus of research on ECM has been to limit ECM fl~L",- l nf~l' by MM?s to interrupt or interferewiththe~luolcvv;v--ofdiseasestates. Severaigroupsof;... ~vfiOaLOlv aremaking small molecules that could inhibit proteinases to alter their destructive activity in arthritis, and as ~ ~ ~ factors to inhibit tumor spread.
Matrix " . . 3 (M~-3) belongs to the stromelysin class of matrix " )~ ~ MhLP-3 is expressed in mature ~ upL.. Oes (Campbell et al. 1931), but aiso in endotheiiai cells, smooth muscle cells and fibroblasts. More recently, MMi'-3 has been 2 1 ~4~L
W O 96/36227 PC~IAUS96/07188 shown to be expressed in llla~ derived foam cells from .,.p.. ' atheroma (Gaiis et al. 1995). The inactive zymogen, proM~-3, is activated by neutrophii elastase, plasma icaiiiicrein, plasmin, ch~.lluL~l trypsin, cathepsin G, and mast cell tryptase, as well as by mercuriai; . ', such as ~ iU acetate (APMA) (Nagase et al. 1992;
S Kieiner et ai. 1993; Nagase et ai. 1990; Nagase 1991). Elevated levels of M~-3 have been found in the joints of patients suffering from, lluilis and ' ' ' artbritis. In IILL~ uacl~ ulh~ plaques there is a large amount of fibrin(ogen)-related antigen (I;~A) consistingofdifferentmolecularforms(Binietai. 1987;Binietai. 1989;Smithetal. 1990;
Vaienzuela et ai. 1992). Two very recent studies have shown the presence of matrix ", ~ 3 in dlh.. u~ ,.ulic plaques (Henney et ai. 1991; Gaiis et al. 1994), but its fiunction in this context has remained ' ' ' Indeed, MMP-3 has been viewed in these studies as a potential negative factor.
The known substrates of ~P-3 inciude ~,. utu~"54, , collagen type IV, fibronectin, and la~ninin. Such substrates are typicai of matrix I " " in general (Dooiittle 15 1987). There has been no suggestion, however, that any ~ ~log ~ " r ut~,;s~; might be involved in the de~ ' ~ of fibrinogen or fibrin. Nor has there been any indication that could be used for fibrinolysis or llu UlllbO4 0;0.
From the foregoing discussion, it becomes clear that significant gaps exist in the ~ of processes involved in thrombus formation and d~ ~ While certain approaches have been identified which permit a measure of control over these processes, these approaches suffer serious defi~ipnripc related to cost, efficacy, or safety. The diagnosis and treatment of disease states associated with pil.~ ~ ' ~ ~ processes involving fibrinogen and fibrin have aiso been found laclcing.
As a result, there exists a need for effective f O ~l u~ and methods for use in iirniting thrombus d~.~.!., and inducing lluullllJu4o;a~
There is a need for methods of disrupting blood clots and alh~uD~ uL;~ plaques, both in vifro, such as for diagnostic purposes, and in vivo, such as for therapeutic treatment of embolism, .llh., uacl~,~ uo;a and other clinicaily imporLant disorders.
In addition, there exists a need for diagnostic and ~ materiais and methods 30 for reveaiing more ~ '' IllaliUconcerning the physical and chemical processes involved in thrombus formation and de~,.aJ~.iol Moreover, there is a need for effective treatment to restore at least some integrity to a damaged vessel wall, to promote regression of alS~ u~ " ul;u plaques, and to aid in ,, , ' ~ and bypass surgery to prevent . .

SUMMA RY OF 1~, INVF l~TIOI'I
The present invention provides a method for degrading Sbrin, fibrinogen, and related substances ~ne., "fibrin(ogen)") by means of a fibrinolytic " r ~ ' (F~) Preferably, the fibrinolytic " r ~ ' ' iS ' ~ c '''I ~t more preferably, a stromelysin.
Most preferably, the fibrinolytic ", ~ includes matrix ", ut~,;..,w.,-3 (MMP-3). The fibrinolytic " r ~ ' ' preferably cleaves or degrades fibrin(ogen) at peptide bond designated yGly404-Ala405.
The inventive method can be performed in vitro. In vilro, the method involves contacting a tissue sample, such as blood or plasma, with at least one fibrinolytic matrix ", ~ In this method, fibrin may be degraded as a constituent of clots and/or ,.u~ulu. uli.. plaques for purposes of . . i~ ,, the structure of such materials, as well as 15 forfurther .~ ~iO~ofthe ' offibrinolysisandLlu Ul~lbul,~ . Fibrinogenmay be degraded for ~p. ' or diagnostic purposes related to the formation of fibrin or to prevent potential growth of a .' ' " fibrin mesh.
In a preferred diagnostic method, the method includes contacting a sample containing fibrin(ogen) with at least one ~ fibrinolytic " r ~ ' ' to provide ~1~ L~20 products. Preferably, the method includes analyzing the ~I c,. ~ products to ~IICIICU1t;1 i~., the fibrin(ogen). Such analysis typically includes di~.,..,..t; '!~ separating the various products. The d~ ;.... products can be identified or measured by various means. For example, the firagments can be detected through antibodies which specifically bind to or associate with particular region(s) of fibrin(ogen), or fail to associate with them due to 25 loss of epitope induced by enzymatic d .6. ~ Preferably, such antibodies are , more preferably ' ' Synthetic and/or chimeric antibodies may be used, as may antigen binding regions such as Fab and F(ab')2. Meclaul~ of specific association bet veen such antibodies and dc5. aJdtiu.. firagments can provide qualitative, or, ~, ' about the fibrin(ogen) sample being analyzed. Such antibodies can be detectably 30 labeled to aid in the l..W..II ~ of the types and amounts of the du5. adaliùl, products.

WO 96136217 2 î ~ 4 ~ ~ 2 ; PCT/US96/07188 Altematively, antibodies fixed to a substrate can be employed to aid in the separation or " ~ of d~ fragments produced by a fibrinolytic " ~
The invention also provides a method of performing 11.1 UlllbCII~ lic, embolytic, or atherolytic therapy in a vertebrate subject, prefierably a primate, more preferably a human In 5 this . bc ' t, the invention involves ~ to a subject a Lh.,. r '- ~ effective amount of at least one ~ r~ fibrinolytic matrix r '~ Typically, the fibrinolytic " r ~ ~ is: ' ~ ~ cd hlatherapeutic r--' - comprisingthe ", ~ and a ~ acceptable carrier or diluent. Optionally, the r I ~ ~ ' Cd . ~'- can further include one or more other active ingredients as an 10 adjuncttothefibrinolyticactivityofthe~ )r ~' ~ Suitableadjunct~ . ' include ~ u~ having ll.-u--~bulyt;-, or fibrinolytic activity. For example, such adjunct ' can be a i ' ~ activator, hirudin, an enzyme inhibitor, an _ ' t, an antibody or synthetic peptide specrfic for plateiet gpllb/lIla receptor, or a ' thereo~
The method can be used prevent or ameliorate 1, ' - associated with 15 ~IL. u~,lu. u~i~ (i.e., to degrade fibrinogen and fibrin in plaques before occlusion occurs), as well as to prevent 1~r ' following ll~ Ull~bUIy~ therapy, e.g., with t-PA, following myocardial infarction. In fact, t-PA is fast-acting, while a preferred " " t~ ~ ~IP-3 has a slower and IJlU~.t~ . action. Thus, the compounds can be beneficially used in ' ~ including sequential or concurrent usage. The method can also be used as a 20 p. ur.hJ' agent to inhibit restenosis followillg surgical hll~l ,,. ~ , such as bypass surgery or lg r~ ty. Alternatively, the method can be used in atherolytic therapy, to inhibit the initial formatio4 or promote the regression, of ~lh~.~ u ~ uL;~, plaques.
In additio4 the invention provides diagnostic and therapeutic kits which include a fibrinolytic matrix r ~ The fibrinolytic ", is preferably 25 . ---1-.c. --- ,..~ Preferably, the " ~, ~ includes a stromelysin, more preferably ~5P-3. It is also preferred that the ", UtU;~lr ~r, cleave or hydrolyze fibrin(ogen) at the yGly404-Ala405 peptide bond. Such kits can include one or more containers, as weU as additional reagent(s) and/or active and/or inert ingredient(s) for performing any variations on - the inventive method. Exemplary reagents include, without limitation, synthetic substrates to 30 test enzymatic activity, and antibodies (preferably , ~~, e.g., ~' ') to measure increase or decrease of antigen level. Preferred kits include at least one Ih~ JL;~lly W O 96136227 PC~rnUS96/07188 effective unit dose of a f brinolytic " r I ' ' Also preferred are kits which include means for ' _, preferably 1~ ClltCI '!~, more preferably a~ JU:~Iy~ a f,~
containing a fibrinolytic " ~, ~ The kits can include one or more other active ingredients as adjuncts, such as L ' ,, activators, hirudin, or anti-coagulants such as 5 heparin or aspirin. These other ingredients can be included in separate ~,~....l...~:l;....~ in separate reagent containers, or can be included with each other and/or the fibrinolytic ", ~,tc;.ld~e in a single reagent container. The kits can also include U~ l, formrxing or combining ingredients and or use of the kit according to the invention.
The invention fiurther provides a method of controlling formation of thrombus caused 10 by medical-related apparatus. In this ~ ~ - " t, the method includes contacting a medical-related apparatus with a f....l..- ~;.. which includes a fibrinolytic matrix " , ~ , preferably MMP-3 The " . .: desirably adheres or binds to a surface of the apparatus~ The method can be used to modify blood-contacting surfaces of . ' ' ' - prosthetic devices such as cannulae, catheters, grafts, stents, filters, coils, valves, 15 and the like, to provide surfaces which inhibit the formation of clots or plaques. Alternatively, the method enables the fibrinolytic ' ~ of apparatus such as needles, blood collection tubes, culture 'dasks, test plates, pipets, reagent containers, tubing, membranes, and the like, to promote fibrinolysis and to inhibit the pol~ ~ of fibrinogen and the formation of thrombus which might otherwise interfere with the (.A~ .ltdl protocols. Likewise, the 20 invention provides medical-related apparatus such as; 'I.'l - - a i~ , labware, and other devices for in vivo and in vi~ro uses, which apparatus has been modified to include adhered fibrinolytic r ~
;'n stiD another 'h ' t, the invention provides a method of enhancing the regulation of fibrinolysis in a subject in need of such therapy. In this C113 ~' t, the method 25 of the invention includes inducing enhanced regulation of an 1~ fibrinolytic matrix " l ~ ~ in a subject. Preferably, the method involves increasing the activity orexpression of an ~ l~.c. . ~ fibrinolytic matrix " r ~ ' ' by treating the subject with somatic ceD gene transfer therapy. Alternatively, the method involves decreasing the activity or expression of an ~ fibrinolytic matrix " r ut~,;..~., by treating the subject 30 with somatic ceD gene transfer therapy. Other therapeutic approaches can be employed which 2t9~

involve I ' ' ~ y -.~ for either promoting or inhibiting the activity or the expression of an e ~o5. --- ~ fibrinolytic matrix ", I
These and other advantages of the present invention will be r~ ' ' J from the detailed description and examples which are set forth herein. The detailed description and examples enhance the I ' " ., of the invention, but are not intended to limit the scope of the invention.

p, Dlli F DE~(~R~PTIO~ OF T~, DR ~WINGS
Preferred: ' " of the invention have been chosen for purposes of illustration and ~cr ipti~ln, but are not intended in any way to restrict the scope of the present invention.
The preferred . ' - " of certain aspects of the invention are shown in the a~u.
drawings, wherein:
Figure 1 shows an cl.,.,l~ 1. ' t:L;~, analysis of fibrinogen treated with MMP-2 or MMP-3, shovl~ing differential d~ ;. - of fibrinogen by each of the enzymes.
Figure 2A is a graph illustrating COIul~al~liv~ fibrin clot Iysis by MMPs and plasmin as measured by the percentage of l.~diOa.,livily in the sample ~,.~
Figure 2B is a graph illustrating . , ~, fibrin clot Iysis by MMPs and plasmin as measured by the percentage of ladiO~livily in the residual clots.
Figure 3 shows an clc~.l, I, ' ~li~, analysis of fibrin treated with MMP-2, MMP-3, and plasmin, showing differential dc~j. alaLull of fibrinogen by each of the enzymes.
Figure 4A shows an "( of the dr~ . products of fibrin digestion by MMP-3 and plasmin, as measured with MoAb/4-5 (y392-406).
Figure 4B shows an ' ' of the dc~. aJal;OII products of fibrin digestion by MMP-3 and plasmin, as measured with MoAb/45 (y397-411).
Figure 5 shows an ' ' of the d~ ~laliull products of fibrin digestion by MMP-3, as measured with MoAb/T2G1 (B~15-42) and MoAb/lD4 (Aa349-406).
Figure 6A shows an Cl~ upl~uli li-, analysis (non-reducing conditions) of D-dimer treated with MMP-3, showing time- and: dependent d~,5. hdaliU~ of D-dimer by MMP-3.
Figure 6B shows an .,h,~,l. UIJIIVI t~ , analysis (reducing conditions) of D-dimer treated with MMP-3, showing time- and al;u.. -dependent d ~,. r 1~ of D-dimer by MMP-3.

2 1 94~92 wo 96136227 Pcr/ltss6l07l88 Figure 7 is a schematic diagram illustrating the cross-linking region in cross-linked fibri4 showing cleavage sites of MMP-3 and CNBr.
Figure 8 shows an eii~l~ u~Lo~ , analysis of the products of dr ~, . t ~ of fibrinogen and cross-iinked fibrin by MMP-3.

S DF.TAn.~n DF~'R~iON OF T~17 INVF.NTION
As noted above, two very recent studies have identified the presence of MMP-3 in~Lh~..... ...ui~ ,.uli., plaques (Henney et al. 1991; Galis et al. 1994). These studics have regarded the presence of ~fPs in the plaques as a negative factor which might favor fissure of the plaques. The present inventio4 however, is consistent with an entirely different ~
lû of the function of MMP-3 . The invention relates to the unexpected role which MMP-3 has been discovered to play in the 1~ ;" of fibrinogen and fibrin.
For purposes of more clearly and accurately describing the invention herei4 certain u..~ fio~ have been adopted in the following discussion. These ~u~ Liuils are intended to provide a practical means for enhancing description of the inventio4 but are 15 not intended to be limiting, and the skilled artisan will appreciate that other and additional, albeit not i t, ;..t."~ L~ILiu.... can be implied.
For example, the invention relates to the use of a matrix " l ~ to degrade fibrin and fibrinogen. The matrix " r ~ ' ' useful according to the invention must exhibit some enzymatic activity against fibri4 fibrinogen, and or related proteins, p~ ,Li i~,s, 20 or ~ g,,~ Matrix ", ~ which exhibit this activity are termed L~
matrix " r ~ or ~r,b. ;llolyLi~, MMPs".
The fibrinolytic matrix ", ut~,;..~c can be exogenous or ~ e ,..~ It is preferred that the fibrinolytic ", ~ be P~ f~ ~ As used herein, the term "- ~o,.,.. - ~" means that the fibrinolytic " r Ut~.;.l~. have an origin in the species in 25 which the method of the invention is to be performed. The species can be any vertebrate, preferably a manunal, and more preferably a primate. Most preferably, the rnethod is performed in a human subject Accordingly, when the method of the invention is empioyed in a human subject, it is preferred that the active " r ~ ' be ~ ~rlog~ to humans.
i.e., of human origi4 whether employed as a l ' ' . or resulting from30 therapy designed to improve the subject's internal control of his/her fibrinolytic system. For in WO 96136227 2 1 9 4 6 9 2 PCT/I~S96/07188 vitro procedures, the origin ofthe fibrinolytic " " ~ is less critica~, but it is preferred that the " . ~ ~ have an ofigin as close as possible to the species origin of the biological sample being examined. In such methods, the ~ " I ut~ e is ~ ,g. -to the sample if it is derived from the species to which the tested sample belongs.
Preferred . ---1~ .f.,. --- .-- - fibrinolytic " r ~ ~ include any of the stromelysin class of ", ~ Preferred ~L-u..l.,l~. include MMP-3 (~.u....,4 ~ I), MMP-I0(~1., '~. ~ 2), and MMP~ L~u...~ ~ 3). A highly preferred ~ f.t~ fibrinolytic MMP is MMP-3. We have found that ~DMP-3 hydrolyzes fibrin(ogen) at the yGly404-Ala405 peptide bond in the cross-linking region of the gamma chain.
It is known that, due to the fluidity and complexity of the physiology of fibrinformation and J L~ ~ . many forms of fibrin and fibrinogen are present in the circulating blood as well as in thrombotic and alh~,.u~.h,. uLiC lesions. The many forms of these molecules result from continual assault by proteolytic enzymes which vafiously cleave the molecules.
The method of the invention is performed by means of a fibrinolytic MMP which has activity 15 against at least one fibrinogen- or fibrin-related compound. If a fil,. ;,0 i~rived molecule has previously been cleaved or modified to delete all ~P cleavage sites, then that molecule is no longer capable of acting as a substrate for a fibrinolytic MMP. Substrates for ~IP-3 have now I , "~, been found to include, inter alia, native fibrinogen and fibrin, and would also be expected to include modified, synthetic, and , .,th~..i., forms of these c ~ . as well as a large number of cleavage products of these ~ The class of substances which are derived from or related to fibrinogen and/or fibrin can be termed "fibrin(ogenr'. The method of the invention can, therefore, be performed using any MMP
which acts to degrade a fibrin(ogen) moiety. ~lhile such enzymes are generally termed ~' ~ l~t;.,", they can also be more precisely eermed "fibrin(ogen)olytic" ~Ps.
Fibrinogen (also abbre~,iated herein as "Fg") is known to be a I ' protein, in which each monomer includes three ' '1~ og~ poly~ iJc chains, identified as the a (alpha), p (beta), and y (gamma) chains. For a review see Doolittle (1987). Thus, fibrinogen has the structure (a~y)~. f;~ll three fibrinogen subunits have coiled domains which permit the subunits to engage one another to form a "coiled coil" region in the fibrinogen monomer. In addition, the beta and gamma chains each have a globular domain, while the alpha chain is present in two forms; a 1~ c ' ~ form having no .,u.. ~ globular domain (a), and a less prevalent form in which a globular domain is present (aE) (Fu et al.
1994). Accordingly, because fibrinogen is I - " and because two forms of the alpha subunit have been identified, two principal forms of fibrinogen are known: (a~y)2 and (aE~y)2.
Both forms of fibrinogen are considered to be substrates of fibrinolytic MMPs according to 5 the invention. Artificial }..,t.,. ~ " a of fibrinogen, as well as . ' forms are also within the class of fibrinolytic MMP substrates.
As noted, fibrin (also ahb~ ' herein as "Fb") is generated through an induced and controlled pvl~ ..i~fiu.. of fibrinogen (Fu et al. 1994). Given that various forms of fibrinogen are known in circulating blood, it is known that various pGI ~ " structures 10 for fibrin occur. Fibrin structure can affect the processes of fibrinolysis (Gabriel et al. 1992).
A fibrinolytic " r ~ ' ' has now been found to effectively Iyse fibrin. It appears, therefore, that fibrinolytic " r ut~,;... ses are active against fibrin without being lirnited by ~ ~ " of fibrin cross-linking. Accordingly, fibrin is considered to be an ~P substrate according to the invention. Thus, fibrin which occurs naturally in a 15 subject is suitable for d ~, ~ l - n .... according to the invention, as is fibrin induced in vi~o.
Thus, clots which are induced in blood ex viw, e.g., in a blûod sample, can be degraded according to the invention. In such in vi~ro,, ' ' , a fibrinolytic ~ ,Lallo~Jlut~ l~G can be employed as a coating on a container such as blood collection tube. Also, artificial fibrin, formed from natural, synthetic, ~i.. i~.~..il.~,li~", c ' and/or other types of fibrinogen can also be degraded by the method described herein.
Under I b, ~;olog;c conditions, plasmin is the central enzyme which acts to degrade fibrin. Plasmin action is restricted to the site of fbrin deposition by plasma control that prevent proteolysis of circulating proteins. However, under pathologic conditions, plasmin is known to degrade plasma proteins, especially fibrinogen.
Degraded fibrinogen can be separated by ion ~ into five fractions (A, B, C, D, and E), of which fragments D and E are the major end products of the original molecule. The i~l ~t;~ - - and ~ I .-- Irl ;~ -- of the transient i..i~,. "
fragments X and Y engendered the insight for the d.,v .,h~,n.. l of an asymmetric scheme of fibrinogen d~ ad~ u~ (Francis et al. 1994).
Classically, fibrinogen structure is bilaterally ~ al~ including a central globular domain E which is a "knot" made up of the N-terminal regions of all six chains in the fibrinogen molecule. From E extend two coiled coils each of which contains portions of one set of a, ~, and ~ chains. At the other ends of the coiled coils are globular domains D.
Extending from the D domains, are the Aa chain extensions, which, in the a, subunit only, terminate in snother globular domain.
Under proteolytic attack by plasmin, initial cleavages liberate the carboxy-terrninal, polar appendage of the Aa chain, and a peptide from the N-terminal portion of the B~ chain (B~ 1-42). The remaining major fragment is Fragment X. Cleavages of all three pol~ t,.,"Ld~.
chains along one coiled coil connecting the central N-terminal knot (E) and a terminal (D) domain of fragment X split it ~ . The result is one fragment D molecule, which consists of carboxy-terminal portions of the three chains, and a fragment Y moiety, consisting of central and terminal domains still connected by a coiled coil. 8~hseq~ cleavage of the coiled coil of fragment Y produces a second fragment D and a firagment E moiety. Fragment X is slowly coagulable by thrombin, but fragments Y and D have potent 'i, ~ '.~
effects, due mostly to disruption of the proper alignment and ~ of build-up of tbe protofibrils of fibrin.
Knowledge of the .,u..~. ' r. .~ ;. ." of fibrinogen assists in providing a conceptual framework against which to compare the activity of other potential fibrinolytic enzymes. Moreover, antibodies have been developed which are specifically reactive with or specifically bind to only some of the fragments, thereby pern~itting molecular i~i -; i~. . ~ ;, ..~ of 20 fragments with great accuracy and precision (E~udryk et al. 1989a) Using this knowledge.
fibrinolytic activity of an f' lrlg, .... ~ " . Ut~,;.l~C has now been .. ~ identified, thereby enabling the d v .1~ of the method of the invention.
The inveDtion provides, inter alia, a method of degrading fibrin(ogen) Generally, the method requires the use of an _. lr~" - - ~ matrix " r ~ ' ~ which possesses 25 enzymatic activity against, i.e., csn hydrolyze, fibrin and/or fibrinogen. The method includes contacting fibrin or fibrinogen with an effective amount of a fibrinolytic matrix . . , preferably MMP-3.
The method typically involves contacting fibrinogen or fibrin with a matrix I 8~ - - including a fibrinolytic ~ " " can also include 30 other active and/or inert ~ ~ ~ ' Other active ingredients can include such ingredients as inbibitors of MMPs, I ' ~ ,, activators, other fibrinolytic enzymes, anti-c~

WO 96136227 2 i 9 4 6 q 2 PCT/US96/07188 reagents, etc. Should anotha active ingredient be employed, it is preferred to include in the J~ a ~ ,. activator. It has been found that matrix ", . 3. in particular, does not compete ' ".~, with or ~ , inhibit t-PA or plasmin.
Accordingiy, the method of the invention can include p~ activators such as u-PA, S t-PA, Dlaph.~' ' , D~ or ~1 ' t, synthetic, or , ' forms thereof.
The invention aiso now permits the . _~tio~lliu~ of the interaction of MMIP-3 with i ' ~ O activators, i ' ~ ,_ activator inhibitors (e.g., PAI-I), and native For e~cample, while it is icnown that piasmin can activate MMP-3, it is not icnown whether MiviP-3 activates t-PA or u-PA, or if it might be activated by them instead.
10 Nor is it icnown if ~MP-3 might activate native I ' ~ ,.
It may aiso be desirable to include an ~ ' agent such as heparin, to inhibitor prevent re-- .,~;"1-:;. .., Such measures would be less criticai in in vitro Pl ' but could be ~~ helpful in in vivo ,, ' ~ The c ~ ~;, u u" of t-PA with.heparin to define a duai-functional Liuu~nlJùlyLic c.. ~l -.- ~;-~" is iiiustrated in U.S. Patent No. 5,130,143.
In one preferred ' " t, the method of the invention permits f brinolytic therapyof a ' , preferably human, subject. In this c,ul., " t, the method includes the I ' ~ - to a subject of a Lh~ , effective amount of a matrix ", UtC..lllDCwhichdegrades fibrin(ogen). The " r ~ preferablyincludesan ", utu;..~, more preferably a DLlu..._l~ , and most preferably MMP-3. The 20 therapeutic method can be empioyed for ~Iu, ' '~.;., or for prevention of IJluOlca~;u~l and faciiitation of regression of alh~.. uD"I~,. uli" plaques. Thus, the method can be performed for acute or emergency therapy or for prolonged or chronic therapy to reduce the likei ihood of or inhibit the J~ IU~ L of abnormai thrombi, emboli or aLL~ u~,h,.ulic plaques.
Modes of ~ ' aL;ùll of such a ~ are icnown in the art, and are related to those tecbniques employed in the ' ~ of Cu..._~;;m~di LluullliJulyt;c agents. Such methods include, without iimitation, parenteral methods, preferably ~ uul~ methods such as ~ ~ u:~ injection, intraarterial injection, and aJ,.;u iDL- aLiull by catheter.
The d~,t~,., ~ of the effective amount of a , ~ ~ of the invention is within 30 the discretion of the sicilled clinician. Specific ~" u~,h ~ ' ~ or therapeutic dosages and the timing of ~ ' ~ ~ c an be selected depending upon prevailing conditions to achieve wo 96ri6227 2 ~ ? ~ 6 9 ~ PCrllJS96/07188 clinically acceptable treatment The skilled clinician will take into account such factors as the age, sex, weight, and condition of the subject, as well as the route of - ' ~ ~ ' The skilled clinician wiU also recognize that the fibrinolytic activity of MMPs can be enhanced by the collaterai ~ ' ~ ~ ~ (e.g., co-: ' ~ ~ ' ' or sequential a ' ~ ' ' ' ) of other S acfive and/or inert substances. For example, it can be desirable to administer adjunct agents having i' ~ ' 'yti., activity. These agents can have direct fibrinolytic activity or can be regulators or modulators of fibrinolysis in the system in which they are employed. For example, agents having Llllu.ubul~ , activity include I ' ' ,, activators, hirudin, enzymes (e.g., proteases derived firom snake venom), enzyme inhibitors, ~ ' . ' (e.g., hepari4 10 aspirin), antibodies (preferably ~.. .".~r.lr~ antibodies) or synthetic peptides specific for platelet gpIlbmIa receptor, or a cr~ thereof Various such LLI u..lbul~ ,ic agents are described in Collen (1996). These agents can be ~ ' ~ ~ cd together with or ancillary to the ~ ~ of an MMP-containing ~ u. ,.l Thus, such other agent or agents can be included m the " r ~ ~ ~ -containing , ' . or can be ' ' ' cJ as part of 15 another . ' In another; bc " t~ the invention includes targeted fibrinolytic ll~t~_~JIn~ ' i.e., ' " r ~ ~ which are bound to moieties having specificity for a biological target molecule. For example, a .l.~Llk)~ ~ ~ can be bound to an antibody by methods known in the art for attaching proteins to antibodies. In this way a ' " r ut~.;.la:~ can be 20 ~ ,f~ directed to a fibrin(ogen) substrate for improving fibrin(ogen)olytic efficacy.
Thus, a fibrinolytic " r ut~,;.,a~; such as MMP-3 can be linked to antibodies having specificity for fibrin or a ,i. ~,.,..i -~;.,.. product thereof, to platelets, specifically to P-selectin, to oxidized 'i,,, ~ , etc.
The invention also provides a diagnostic method for the ~,La a ~ rd ~ - of fibrinogen.
25 In tbis method, fibrin(ogen) is contacted with an . ---1..~,. --- ..,~ matrix " ~
preferably MMP-3, to produce d~E,. adafiull products. The ~Ir ~ products are then analyzed to determine the types and amounts of cleavage products generated by the activity of the MMP.
TypicaUy, the method involves the differential separation of d~ ~1al;ull products, such 30 as separation of the products by gel .~ , ' t~ >. The products are then measured such as by S".,~,;r.., staining to reveal quantities of products of different sizes. Alternatively, the 2~ 94692 products can be identified by contacting the products with antibodies which are specifically reactive with or specificaily associate with one or more domains of fibrin(ogen) (Kudryk et ai.
1989a). Preferably, such antibodies are specifically reactive with a single de~5~Lioll product, tbereby permiffing ~ of the product in relation to other products.
New antibodies, usefifl according to the diagnostic method of the invention, can be developed and detectably labeled with any detectable marker group. Suitable marker groups include, for example, fluorescent labels, enzyme labels, and radioactive labels. Detector groups useful according to the invention include, for example, duorescein as a duorescent label, horseradish peroxidase as an enyme label, and lodine-125 (l2'I) as a radioactive label.
Additionai fluorescent labels which can be utiiized in the invention include, but are not limited to, rhodamine, I/L~.u~,~yi' ~ and additionai compounds emitting fluorescent energy.
Additional enzyme labels which can be utiiized in tbis invention include, but are not limited to, glucose oxidase and aikaline r ~ .1 Additional radioactive labels which can be utilized in this invention include, but are not limited to, Iodine-13 1 (l3lI) and Indium-l 11 ("'In).
Suitable detectable labels can be seiected from among those known in the art, such as radioactive labels, enymes, specific binding pai m ~ , colloidal dye substances, fl..u.. ' u....,~, reducing substances, latexes, d;gr~Y;g~ nin~ metais, I,alL;~ k.L~, dansyl Iysine, antibodies, protein A, protein G, electron dense materiais, ~ uum~ Ol~a~ and the iike.
Eflfectively, any suitable label, whether directly or indirectly detectable, can be empioyed. One 20 skilled in the art wiil clearly recognize that these labels set forth above are merely illustrative of the different labels that could be utilized in the diagnostic method of the invention.
Fibrinogen subunit-reactive antibodies can aiso be derivatized by l~onjnePfi~n to biotin, and used, upon addition of species of avidins which have been rendered detectable by ; ,, to fluorescent labels, en2yme labels, radioactive labels, electron dense labels, etc., 25 in a multipGcity of ~ ' ~ ' and i -~ ~ g Aiternatively, the method of the invention can be performed using antibodies which bave been attached or bound to substrates materiais according to methods known to those skilled in the art. Such materials are generaily ' "~, solid and relatively insoluble, imparting stability to physical and chemical disruption of the antibodies, and permitting the 30 antibodies to be arranged in specific spatial J; ,L. ;bUL;U.~ Among substrate materiais, materials can be chosen according to the artisan's desired ends, and include materials such as W O 96/36227 2 ~ q 4 6 ~ 2 PC~rlUS96/07188 gels, hydrogels, resins, beads, ~ " ' , nylon filters, microtiter plates, culture flasks, polyn eric materials, and the like, without limitation.
The method of the present invention can involve ~ ~ ~O ' assays to deter nine the presence of fibrin(ogen) breakdown products in tissue samples from human or animal 5 suyects. Biopsy and necropsy samples of subjects, as well as samples firom tissue libraries or blood banks, can be evaluated for the presence of fibrin(ogen) MMP breakdown fragments using an anti-fibrinogen antibodies. Moreover, suitable ,UII, '- can be devised for in vivo use, such as for the ~ ' ~ of fibrinogen or '" ~ _ containing substances and structures in a living subject. In this way the IJl uO.~ ~ of fibrinolysis induced by MMPs can 10 be assessed in situ.
In one such: ' ' t, an ~ fibrinolytic MMP, preferably MMP-3, is bound to a substrate material such as a membrane, blood collection tube, microtiter plate, culture flask or the like. In this manner, the method of the invention can be per~ormed in the absence of soluble MMP, to induce fibrin(ogen)olysis in a fluid sample. Alternatively, this 15 approach is useful in coating membranes and prosthetic devices.
Indeed, in another; ' ' t, the invention provides a method of controlling formation of clots or plaques caused or induced by medical-related apparatus. In this L ' t, the method includes contacting a medical-related apparatus with a u~ ~l u ' ;~
which includes a fibrinolytic matrix ", . ~ , preferably MMP-3. This method can be 20 used to cause a " r ~ ~ to bind or adhere to a sur~ace. It is believed that any apparatus which would contact blood can be so modified by rnethods known in the art which permit the attachment of proteins to substrate nnaterials. For example, the method can be used to modify blood-contacting surfaces of ~ . ' ' k prosthetic devices such as cannulae, catheters, grans' stents, filters, coils, valves, and the like, to provide surfaces wbich inhibit the 25 formation of thrombus. Alternatively, the method enables the fibrinolytic ' ~ of apparatus such as blood collection tubes, culture flasks, test plates, pipets, reagent containers, tubing, ' , and the bke, to promote fibrinolysis and to inhibit the formation ofthrombus which might otherwise interfere with the ~ ,. ' protocols. Likewise, the invention provides medical-related apparatus such as , ' ' ' , labware, and other devices 30 for in vivo and in vitro uses which possess the capacity or inhibiting tbrombus formation by promoting the d~ of fibrin(ogen). The " ~ , can be adhered to an V~O 96/36227 2 1 9 4 6 ~? 2 PCI/US96/07188 apparatus eitber p. 1~ or reversibly, such as for delivery of " r ~ from an apparatus into solution.
Tbe invention also provides a method of enhancing regulation of fibrinolysis in a subject in need of such therapy. In this ' ' ~, the method of the invention includes S inducing enhanced regulation of an c loc, ~ fibrinolytic matrix " r ~ in asubject. Preferably, the method involves increasing or decreasing the activity or expression of an ---l~.~. --- ...~ fibrinolytic matrix " " ~ by treating the subject with somatic cell gene transfer therapy. Any gene therapy approach can be employed according to this .~ I ' Thus, up-regulation of MMP expression can be a , ' ' I by vJu~,;..g a10 gene for a fibrinolytic MMP by ex vivo or in vivo gene transfer techniques. Alternatively, up-regulation of an ~MP can be a , ' ' ~ ' by inhibiting the expression of an MMP
inhibitor via anti-sense technology. Down-regulation of ~P activity can be r , ' ' ' by these techniques, the design and . ' - of which are within the skill of those in the art. A brief overview of several gene therapy methods is provided in Glick et al. (1994), 15 which is ~,, ' herein by reference. Other therapeutic approaches can be employed which involve L ' ' ~ , for either promoting or inhibiting the activity or the expression of an .. 1O~,,.. ~--- fibrinolytic matrix " I ~Jt~,;.l~;;. Given the complexity of the fibrinolytic regulation system, and given the unexpected role of MMPs in that regulatory scheme, it would appear to those skilled in the art that many potential avenues exist for 20 adjustment of the fibrinolytic regulatory status of a subject.
Il~e following examples are intended to assist in a further, ' ' ,, of the invention. The particular materials and conditions employed are intended to be further illustrative of the invention and are not limiting upon the reasonable scope thereof In the following examples, we describe our ~., which have revealed the role 25 that M~s, particularly MMP-3, play in the d~,. ,- l ';~).~ of fibrinogen (Fg) and cross-linked fibrin (~Fb). We have studied the following aspects: I) d~.aJcl~iO~ of Fg; 2) effect of M:~s-digestion of Fg on its subsequent clottability with thrombin; 3) Iysis of XL-Fb and purified Fragment D-dimer (DD); 4) NH2-terminal analyses of chain fragments of selected digests; 5) reactivity of the cleaved fragments with a number of specific 30 antibodies. We show the ability of MMP-3 to degrade Fg and to Iyse XL-Fb clots. Based on this work, we conclude that both matrix " r ~Jt~ ;~lr~ ) and matrix 2 (MMP-2) can partially degrade Fg, and that MMP-2 has a limited capacity to degrade XL-Fb. We have also determined that, in XL-Fb, the yGly404-Ala405 bond is a major MMP-3 cleavage site, leading to the formation of a Fragment D-like monomer. This indicates a specific mechanism of fibrin dcg. adal;Oil, different from plasmin, by an . ~ 2~ ~ -5 enzyme.
The following ~ r ' ' procedures are relevant to Examples 1-9, below:
Proteins~ndOtherReagents. r~ ~ ~ g fireearldfib~ - freeFg(Fg 2 95~/0 clottable) or Iyophilized human Fg were purchased (American Diagnostica Inc., Greenwich, CT). I ~~ and fibronectin were removed by affinity ' . . - ~ h~ on 10 Iysine-Sepharose and gelatin-Sepharose, essentially as described by others (Deutsch et al.
1970; Engvall et al. 1977; Procyk et al. 1985). The amount of Factor XIIl in these ~,. . is 0.1 -0.2 Loewy units/mg of Fg according to the '' ti. . Stock solutions of Fg (12 mg/mL in TNE buffer (0.05 M Tris-HCI (pH 7.4), containing 0. I M NaCI, 0.001 M
EDTA and 100 KIU/mL aprotinin)) were stored at -70~C until used. Fg (.~ tl was 15 measured ~ r~ in alkaline-urea using extinction coefficient (1%, I cm) =
16.5 at 282 nm. Human GL. . ~ ,, (I U/0.5 mg) was from Imco (Stockholm, Sweden).
Slll, ' ' (4500 u/mg solid), bovine serum albumin (BSA, Fraction V, RIA-grade), J' ~,ufi-, acetate (APMA) and EDTA were from Sigma Chemical Company (St.
Louis, MO). Aprotinin was from Mobay Chemical Corp (New York, NY). Human 20 a-thrombin (2300 U/mg) was a generous gift of Dr. J. Fenton. 'ZsI-Fg, labeled by the iodogen method (specific activity I .5 x I o6 cpm/llg protein) was a generous gift of Drs. M. Nag and D.
Banerjee, Laboratory of Membrane r- ~ ~ ~ II, The New York Blood Center.
Pro-MMP-I, pro-MMP-2, and pro-MMP-3 were purified as previously described by others (Okada et al. 1986; 0kada et al. 1990; Suzuki et al. 1990). All other reagents were of 25 analytical grade and were purchased firom Fisher Scientific (S, , ~ ' d, NJ).Gel e~h" ~ ,c~ .O. Samples of Fg and XL-Fb degraded with plasmin or MMPs were subjected to SDS-PAGE using both reducing and non-reducing conditions. Reduced samples were prepared in 62.5 mM Tris buffer, pH 6.8, containing 4~/O
- SDS, 8 M urea, 5~/O DTT, 10% glycerol and 1% b~ . ' ' blue. Non-reduced samples 30 were made in the same buffer without DTT. SDS-PAGE was performed using 5-15%
gradient or 12.5% pOI~a~ ' ' gels in Tris-glycine buffer (Laemmli 1970) or with 5% and Wo96136227 21 94692 Pcrll7S96/07188 7.5~/0 mini gels in phosphate buffer (McDonagh et ai. 1972) following general procedures.
Prestained molecular weight standards used were myosin (200 kDa), phoD~Jl.vlyL~De B (97.4 kDa), BSA (68 kDa), ovaibumin (43 kDa), a-l,h,..l~L~yl g (25.7 icDa), ~
(18.4 kDa) and Iysozyme (14.3 kDa) (Bethesda Research Laboratories, ~ ;, MD).
S Transfer to ~ u.~eiiuluD~i membr~mes for ' ' analyses was perforrned as described by Towbin et ai. (1979) with few " ~ (Kudryk et ai. 1 989b). In some ~~,.,. ;ll..".,D, membranes were stained with colloidai gold prior to ' ' ~ (Colloidal Gold Totai Protein Stain, BioRad, Hercules, CA). r ~ ' were blocked with 5% dry milk (Carnation, Nestle, Glendaie, CA) or with 5% BSA, incubated overnight with a selected primary antibody (Table I) and then probed with a second antibody. Rabbit anti-mouse-horseradish peroxidase (RAM-HRPO) was prepared as described by Goding(1986) using RAM purchased from Dako (Carpinteria, CA) and HRPO (type Vl) from Sigma.
Bound peroxidase complexes were detected using the ' ' substrate Luminol (ECL Western blotting detection system, Amersham Life Science, Arlington Heights, IL).
Light emitted from the hydrolysis of the added Luminol substrate exposed the provided film ~odak x-Omat RP, Eastman Kodak Company, Rochester, NY) in 10 to 30 seconds.
EX~MPLE I
~ ~liu,. of ~ibrinogen. An experiment was performed to evaiuate whether MMPs possess fbrin(ogen)olytic activity. Fibrinogen (Fg) (120 llg, 3.5 ~lM) was incubated with MMP-2 or MMi?-3 (6 uglmL or 1:20 E:S ratio) at 37 ~C for different time intervals.
ProMMP-2 and pro-Mi~?-3 (in 50 mM Tris-HCI, pH 7.5, 0.15 M NaCi, 0.05~/0 Brij 35, O,OSD/o NaN3) were activated with I mM APMA at 37 ~C for 45 min and 24 hr" ~DIJ~.~,fi ~,ly, prior to addition to the Fg solutions. Ail reactions were in the presence of 10 mM CaClz at 37 ~C. Digestions were terminated by addition of EDTA (25 mM final, Reaction products were mixed with reducing or non-reducing buffer and subjected to SDS-PAGE separation (12.5%). The gels were stained for protein with Coomassie blue.
Figure I shows the time-dependent digestion of fibrinogen by MMP-2 and MMP-3 by ,.. to no.A d;,~,~..t~.l fibrinogen. The enzymes were used at E:S = 1:20 (w/w) and incubation was for 1, 2, 4 and 24 hrs. The key to Figure I is as follows:

WO 96/36227 2 I q ~ 6 q ~ PCI~/US96/07188 Lane No. Sample Non-digested fibrinogen 2 ~P-2 digest of Fg (I hr) 3 MMP-2 digest of Fg (2 hrs) 4 MMP-2 digest of Fg (4 hrs) MMP-2 digest of Fg (24 hrs) 6 MMP-3 digest of Fg (I hr) 7 MMP-3 digest of Fg (2 hrs) 8 MMP-3 digest of Fg (4 hrs) 9 MMP-3 digest of Fg (24 hrs) It is clear from Figure I that, using M~-2 and M~-3 in . ~I,h, amounts (6 15 ,ug/120 ,ug Fg), both the Aa- and B~-chains of Fg were extensively degraded in I hr (lanes 2 and 6). A longer (24 hr) incubation resulted in fiurther cleavage of both chains (lanes 5 and 9).
Degradation of Fg y-chains with MMP-3 was extensive at 24 hr (Figure 8, lane 9) and different from that with MMP-2 (lane 5). Significant dcg.~daL~ with M~-2 and MMP-3 was also obtained at lower l )r ~ of enzyme (0.2-0.6 pg/120 ,ug Fg, data not shown).
20 MMP-I, was also tested using this protocol, and, at the highest conc~i ' (6 ,ug/120 ,ug Fg), showed apparently intact B,3- and y-chains and only partial d~a~ .Id~.iio.. of the Aa-chains (data not shown).

C'~.~,-7 7;~;~of~ibrinogenDegrac.7edwifhMMP-3. Digests(I.5 and3 hrs)of fibrinogen with MMP-2 and MMP-3 were prepared as described above. A plasmin digest of Fg (18-20 hr) (Gardlund et al. 1972) was used as control. Intact Fg and Fg digests were clotted with thrombin as described by Bini et al. (1994). Briefly, 1.2 mg Fg/mL, or any of the above MMP-2 and MMP-3 digests of fibrinogen, was clotted with thrombin (0.4 NIH U/mL) in the presence of 20 mM CaC12. Clotting time was deter nined as an increase in turbidity and read at 350 nm (Blomback et al. 1982). Co~g ' ' ' t~ was determined from the plot of turbidity versus time. A tangent was drawn to the steepest part of the curve; its ~
with the time axis is defined as clotting time (Blomback et al. 1994). In all cases, the gels were formed without any observed IJlt~ J;t.liiOll. Clot ~u~ ldtalliS were run on HPLC
(Kudryk et al. 1989b) in order to determine release of ~ p Id;ll~ 5 A (FPA) and B (FPB).
These results are tabulated in Table I, below.

w096/36227 2 1 q 4 6 q 2 Pcr/uss6/07188 TABLE I

Samplc E:5 Incubahon Thrombin Turbidi~y Turbidit,y Time Time w/uJ(hrs) (same day)(ncrL dau) Fg control a - 68" 0.841 0973 b En~yrncs:
MMP-2 1:'00 1; > 10 min 0.433 0.963 ~ ~ 0.377 0.823 3 >10 min 0.681 1.039 0.7s8 1.12~
MMP-3 1:600 15> 10 min C - 0.025 0.03~
3 ~ - 0.004 ~ n _ 0.029 1200 1.5> 10 min d - 0.001 ~ _ 0.000 3 ~ - 0.008 0.012 Piasrnin 1:1200 15 1075- 03g6 103.9~ 0.399 3 139.1" 0502 1:240 15unciottable a Fbrinogen (Fg~ ~, .... u ~ ~ ;" ., in all samples: 120 llg/ml br~
c soft clot the next day at both 15 and 3 hrs d no ciot the next day F8 digested with MMP-2 (1.5 and 3 hr) did not clot within IO min (arbitrarily taicen as maximum time), but was stiil capable of forming a fibrin gel afrer overnight incubation with thrombin as shown by the turbidity data (Table 1). By contrast, both MMP-3- and 5 plasrnin-digests, at compsrable time and - , were unclottable even after overnight incubation. Turbidity data indicated that gelation was obtained only from reaction mixtures of Fg digested with M~-2 and with the iowest plasmin ~ r... Ih~....ul ~, turbidity values of the same ~ ,~ aliu.~ measured the next day showed that reaction mixtures of Fg and M~-2 were similar to control while Fg digests generated by MMP-3 or plasrnin had low W O 96136227 2 1 9 ~ 6 9 2 PC~rrUS96/07188 or no turbidity (Table I). HPLC profiles of ~ from all digests showed normal release of r~ A and B with thrombin (not shown).

~1,~, r~ ' of Cross-Linked Fibrin. Fibrin clots were made from purified fibrinogen S according to the method of Bini et al. (1994) and the references cited therein. ~ n~ A
clots were made with 0.1 mL purified Fg (1.2 mglmL in TNE buffer) containing l25I-Fg (20,000 cpm) in the presence of 20 mM CaCI2. Thrombm (1.5 N~ UlmL, final ) was added and samples were incubatcd at 37 ~C for 18-20 hrs (Bini et al.
1994). Active MMP-I, MMP-2, or MMP-3 were added in different amounts (2-60 ,ug/mL, cu.. ~ r ' ~to 1:600-1:20E:Sratio)inthepresenceoflOmMCaCI2. Plasminwas generated by addmg l ' ~ g (50 ,uglmL) and ~L-I, ~ ' ~ (1080 U/mL) to the fibrinclots (ayyluAill.dt~ 0.02-0.5 UlmL plasmin, &na B ,lliu.., .,c,.", " ~ to 1:1200-1:48 E:S). Clots were gently dislodged from the wall of the test tube with a wooden stick and the content lightly vortexcd after addition of each enzyme. Incubation times were from 1-48 hrs at 37 ~C. Digests with MMPs were terminated by addition of EDTA (25 mM
fimal ~ . .1 "~l;o ), and those with plasmin were terminated with 5,000 KlU/mL aprotinin.
Clots were separated from ~ by ~,e.ltl'r ~' ,, at 13,000 rpm for 20 min in a Sorvall RCL-B (SS-34 rotor). Fibrinolysis was measured both by relcase of .~dio~ ity into the ~y~,~llr~ L (A) and by residual ladiu~ ivily in each clot (B). Samples were counted in a Packard Auto-y-5000 Series Gamma Counter. Control fibrin clots, with and withoutdigestion with the different enzymes, were made at the same time to be used for SDS-PAGE.
Data represent mean values of 2-4 separate c Ay.
The results of this experiment are presented in Figures 2A and 2B, which show the percent radioactivity in the ~ (Fig. 2A) and in the residual clots (Fig. 2B). Asshown, after 24 hours of incubation, both the MMP-3 (6 ,ugll 20 llg Fg) and plasmin (0.02 IUlmL) samples showed release of over 80% of the radioactivity into the ;.~
(average of three CAIJ. ~ ~ ) (Fig. 2A). Rcsidual 1 ~UI;O~1;V;IY in the clots at 24 hrs was less than 10% (Fig. 2B), and the clots appcared dissolved by both MMP-3 and plasmin. See Figures 2A and 2B. The .u ~ . of plasmin used in the Iysis CAIJ. ' ' was chosen on the basis of obtaining a slow lysis (Liu et al. 1986). In preliminary CAIJ~ ' ', similar release of.aJ;o~ ;.;;ywasmeasuredinthe , usingplasminat 1:240and 1:1200(E:S, WO 96136227 2 1 ~ 4 6 q 2 PCIIUS96/07188 w/w), which was used throughout the study. Degradation with MMP-2 at highest ~ at 24 hr, was similar to that obtained with MMP-3 at l :200. Degradation with MMP-I at highest , after 48 hr, was similar to MMP-3 at l: 1200. Controls, with no addition of enzyme as well as a plasmin digest of XL Fb, are also shown.
Incubation with a n~ixture of MMP-3 and plasmin in the sample produced results similar to those produced by either enzyme alone (data not shown). This indicates that the two enzymes do not interfere with one another. Identical l~ were perfommed using clots made from plasma, and similar results were obtained (data not shown).

Chain C~ , of XL-Fb Degraded by A~fMPs and Plasrnin The digestion of cross-linked fibrin by plasmin, MMP-2 and MMP-3 was analyzed. Samples of fibrin d~ g " ~ . by each of the enzyrnes were reduced and subjected to SDS-PAGE (5- 15%
gradient) ~Laemmli et al. 1973; McDonagh et al. 1972). Following cle~ u~ ol~,O;., resolved proteins were transferred to u~,elluloOe. Protein was stained with colloidal gold on the u~,elluluO_ membrane. The pattems of dose-dependent and time-dependent ~l. g, A- 1 -~ ;l ~-- of fibrinogen and fibrin by MMP-2, MMP-3 and plasmin are shown in Figure 3.
The key to Figure 3 is as follows:

Lane No. Sample Fibrin degraded with plasmin (1:1200) for 24 brs 2 Fibrin degraded with plasmin ( I :24û) for 24 hrs 3 Fibrin degraded with MMP-2 (I :200) for 24 hrs 4 Fibrin degraded with MMP-2 (1:20) for 24 hrs Fibrin degraded with MMP-3 (I :600) for 24 hrs 6 Fibrin degraded with MMP-3 (I :200) for 24 hrs 7 Fibrin degraded with MMP-3 (I :20) for 24 hrs 8 Fibrin degraded with MMP-3 (I :10) for 24 brs Figure 3 shows plasmin d~,gl _J_Liu,. of cross-linked fibrin y-dimer chain (94 kDa) into DD y-dimer chain (76 kDa) at higher E:S ratio (lanes I and 2). No d~ .AJ~l;u~ ofcross-linked fibrin y-dimer was observed with MMP-2, at highest E:S (I :20) (lane 4).
Significant l~ ;.... of the y chain was obtained with MMP-3 (E:S = 1 :20) at 24 hrs (lane 7) and complete d~,~r- ' was obtained by increasing E:S two-fold (lane 8). The pattern of 35 A L ~ of cross-linked fibrin y-dimer chain by MMP-3 is different from that obtained W O 96136227 2 ~ 9 4 6 ~ 2 PC~rrUS96/07188 with plasmin. In fact, plasmin decreases the molecular weight of the y dimer, but does not ~ it (lanes 1, 2) even at bigher E:S ratio (I :240).
The XL-Fb clots were gradually degraded by MMP-3 and resulted in near complete lysis at 24 hr. The amount of d b~ with MMP-3 (1.20) at 24 hrs was ~ . ' ' to that produced by plasmin (1: 1200). Therefore, MMP-3 is a slower fibrinolytic enzyme than plasmin. The rates of clot ' ' ' with MMP-I and MMP-2 were much slower: at the same E:S ratio (I :20), only 34% and 58% ~ ,4, was solubilized after 24 hr, whereas 84% was degraded by MMP-3. In digests with MMP-3 and plasmin, residual clot l~ul;o~ ;ty was ~ 10%. These results indica~ed that digestion of ~-Fb with MMP-3progressed further, possibly with a different and more specific mechanism than that obtained with either MIMP-I or MMP-2. However, the weaker activity of MMP-2 may be due to rapid autolysis of the enzyme after activation by APMA (Okada et al. 1990).

I J3dP-3-1~ced Cleawge of y-Chain Cross-Link Dontain. T ~ ~ performed using standard t~bniques were performed on plasmin and " r ~ ' ~ digests of fibrin with a panel of ' ' antibodies (MoAbs) (see Table 11) to identify how the three chains of fibrin are cleaved by MMP-3 in cl~mparison with plasmin. 1~.' ' ' antibodies were prepared according to techniques known in the art.

Antibody Isotype C. . ~ c With MoAb/4A5 IgG1,k y397-311, native fibrinogen and fragments D/D-dimer (Gift) (Matsueda et al. 1988) MoAb/4-2 IgGl,k y392-406,fibrinogenandfragmentsD/D-dimerbutonlv r,8 ~-n~ . (Kudryk et al. 1991) MoAb/T2GI IgG1,k Bpl 5-42, fibrin Il, hl t r~-t fihrjnr~ nl~
(Kudryk et al. 1984) MoAb/lD4 IgG1,k A~349~06, fibrinogen, fibri4 and plasmin digests of both(Procyketal. 1991) Samples were incubated either with or without enzyme for 24 or 48 hr. Incubated sarnples were then subjected to SDS-PAGE (7~/O gels) under reducing conditions. After elU~llUI~IlUlU~ .., samples were transferred to u~,ellulu~i membranes, and the membranes were blotted with selected antibodies. Specific antibody-bound fibrin(ogen) chains were détected using RAM-HRPO and the ' ' substrate, as described above The results are iiiustrated in Figures 4-5.
Figure 4A shows ' ' of the drO, ~ - products of fibrin digestion by MMP-3 and plasmin with MoAb/4-2. This antibody is specific to an epitope in the y-chain:
y392-406. This antibody reacts with fibrinogen and tbe D and D-dimer firagments oniy after .. . ..
Cross-iinked fibrin and fibrinogen were each incubated without enzyme at 37 ~C, for 24 hr. Aiso, XL-Fb was incubated with plasmin (24 hr) and with MMP-3 (24 hr and 48 hr) The digests were examined under reducing conditions. The key to Figure 4A is as follows:

I,nne No. Snmple I XL Fb 2 Fg 3 Plasmin digest of 7~-Fb (24 hr) 4 ~viMP-3 (1 :20) digest of XL-Fb, residual clot (24 hr) S MMP-3 (I :20) digest of XL-Fb, totai digest (48 hr) As seen in Figure 4A, MoAb/4-2 (anti-y392-406) reacted with both undigésted XL-Fb y-dimer (94 kDa, lane 1) and undigested Fg y-chain (47 icDa, lane 2). Residuai y-chain monomer in the cross-linked fibrin preparation aiso reacted with MoAb/4-2 (lane 1). In the plasmin digest of XL~Fb, DD y-dimer (76 kDa) and Fragment D y-chain monomer (known to result from both Fg and ~-Fb plasmin digests (Siebeniist et al. 1992)) aiso reacted equaily with MoAbl4-2 (lane 3). A 24 hr digest of XL-Fb generated by MMiP-3 showed reduced DD
y-dimer chain in the residuai clot, but significant amounts of Fragment D monomer-iike y-chain (36 kDa, lane 4). A longer incubation (48 hr) showed oniy Fragment D monomer-iike y-chain in the totai digest. Digests with both enzyrnes bound MoAb/4-2.
Cross-iiniced fibrin was aiso examined by ' ' ,, with MoAb/4A5, which is specific for the y-chain at y397-411, an epitope close to that resctive with MoAb/4-2.. As a benchmark, XL Fb clot was incubated without enzyme at 37~C for 48 hr. XL Fb was aiso incubated with MMP-3 or plasmin for 24 hr. The results of this; - ~ -- .i .l. .l are shown in Figure 4B. The key to Figure 4B is as follows:

wo96136227 2 ~ 92 PCT/US96/07188 Lane No. Sample Undigested XL-Fb clot 2 MMi'-3 (l :20) diges~ of XL-Fb, residuai clot (24 hr) 3 MMP-3 (1:20) digest of XL-Fb, clot ~ (24 hr) 4 MMi'-3 (I :20) digest of XL-Fb, totai digest (24 br) Plasmin digest of XL-Fb Thus, as seen in Figure 4B, MoAb/4A5 (anti-y397-411) showed reactivity oniy withthe y-dimer band from both intact and l ' ~ d;~Ct~ XL-Fb (ianes 1, 5) and with residuai DD y-dimer from digests generated by MMP~3 (lanes 2, 4). The Fragment D monomer-Cke y-chain (36 kDa), fuily reactive with MoAb/4-2 ~Figure 4A, lanes 4, 5), faiied to bind MoAb/4A5 (l~igure 4B, lanes 2, 4). r "(: analysis of the same samples under non-reducing conditions showed similar loss of ~ ~,~ivily with MoAb/4A5 (data not shown).
These ~,. ~ show that the pattem of XL,Fb d L,., ~ ) with MMi'-3 is different from that obtained with plasmin. In digests with MMP-3 (1 20), oniy very smail amounts of y-dimer remain. At higher levels of this same enzyme, no dimers can be detected.
MMP-2 does not seem to affect the d g. ,~ ~ of y-dimer ", ~~ 'y. Two antibodies, MoAb/4-2 and MoAb/4A5, reactive with different epitopes in the sequence y392-411, were used to define the regions of cleavage of XL Fb by MMP-3 in c. . .~ ,. . to plasmin. This segment ofthe chain contains residues (yGln398 and yLys406) that participate in covaient cross-linking (fomling ~-(y-Glu)-Lys isopeptide bonds) on n.,;ghb, ,, molecules leading to fibrin mediated by Factor XIlla (Chen et al. 1971). MoAb/4A5 recognizes an epitope in the COOH-temlinai region ofthis peptide (y397-411), while MoAb/4-2 reacts with its NH2-terminai end (y392-406). Both antibodies bind to microtiter plates coated with the plasmin-derived digest products Fragments D and DD. Only MoAb/4A5 competes with such fragments when each is in solution (Kudryk et ai. 1991). In a 24 hr MMP-3-digest of ~Fb, both residual DD y-dimer chain and Fragment D
monomer-iike y-chain were reactive with MoAb/4-2. Longer (48 hr) digests resulted in Fragment D monomer-like y-chain only, which was still reactive with MoAb/4-2. Analysis of these same digests with MoAb/4A5 (anti-y397-411) showed only the DD y-dimer band to be reactive. The Fragment D monomer-like y-chain failed to bind MoAb/4A5. These results WO 96/36227 2 ~ 9 4 6 9 2 PCTNS96/07188 suggested that a major MMP-3 cleavage site was witbin the y-chain cross-link domain resulting in the dc~5. adal;ùn of the y-dimer. Purified Fragment DD, was also cleaved with MMP-3 to a D-monomer like fragment.
T '' ' ofthe ~Irc~ ;.. productsoffibrindigestionbyMMP-3 andplasmin 5 was also perforrned using MoAb/T2GI and MoAb/lD4 under reducing conditions, as shown in Figure 5. The ~Ap. ~ ' ~ protocol was as described above for the other antibodies. The key to Figure 5 is as follows:

Llme No. Smnple MMP-3 (1:200) digest of XL-Fb (24 hr) 2 MMP-3 (I :20) digest of XL-Fb (24 hr) 3 Undigested XL-Fb (24 hr) 4 MMP-3 (1:200) digest of XL-Fb (24 hr) MMP-3 (1:20) digest of XL Fb (24 hr) 6 Undigested XL-Fb (24 hr) 7 Undigested Fg (24 hr) As indicated in Figure 5, lanes 1-3 were blotted with MoAb/T2GI, while lanes 4-7 were 20 blotted with MoAb/lD4.
MoAb/T2GI isspecificforB,B15-42,butonlyoffibrinII, notoflil,.i..u~,_,Jfil,.; I.
MoAb/lD4 is specific for Aa349-406 in fibrinogen and fibrin, as well as in their plasmin digests. As shown in Figure 5, MoAb/T2GI ~ ~.~l;vity was lost in the digests of fibrin by MMP-3 both at lower (I :200) and higher (I :20) . (lanes 4, 5), whjle it is present in intact fibrin (lane 6). MoAb/lD4 ~.,L;v;ly is still partially preserved in the fibrin digest at lower C~Liu~l of MMP-3 (lane 1), but it is completely lost in digests at higher of MMP-3 at both 24 hr (lane 2) and 48 hours (not shown), while it is clearly present in the control sample (lane 3).
Accordingly, MMP-3 d~"5. ad~.Liùn products of ~-Fb were probed with MoAbs, T2GI
(anti-B,B15-42) and ID4 (anti-Aa349-406), -~ ,L;~ . Both epitopes are present in XL-Fb.
In plasmin digests of XL-Fb, many different size bands (220 kDa) react with MoAb/lD4, while aD MoAb/T2GI reactivity is lost. Even relatively low, of MMP-3 (I :200) resulted in digests which failed to react with these antibodies on ' ' _ This result means that Aa349-406 has been cleaved from fibrin by MMP-3. No 1~..,Livity with wo s6rO2622~ 2 ~ 9 ~ 6 9 ~ Pcrluss6/07l88 MoAbllD4 in MMP-3-digests of XL-Fb could be detected by . ' ELISA. This antibody reacts identically with A~349-406 and Hi2-DSK (Aa241-476) before and afte complete digestion with trypsin.
EXAMl~LE 6 Digestion of Fragment D-Dimer (I)D) byM~P-3. E,.l,.,. were carried out to see whether MMP-3 would cleave purified D-dimer (DD) into D monomer. Purified fragment DandD-dirnerwereincubated37 ~Cfor4and24hourswithlower(1:20)andhigher(1:200) of MMP-3. The reactions were terminated with 25 mM EDTA. The digests were resolved on PAGE (7~/0 phosphate). The samples were transferred to ' u~.~,llulose 1 ' and stained with Coomassie blue. The results are shown in Figure 6. Samples were run under non-reducing (Fig. 6A) and reducing ~Fig. 6B) conditions. The key to Figures 6A and 6B is as follows:

Lane No. Sample Plasmin digested Fg (D monomer 93 kDa) 2 Plasmin digested XL Fb (D dimer 186 kDa) 3 MMP-3 digest (I :200) of purified D dimer (4 hr) 4 MMP-3 digest (I :20) of purified D dimer (4 hr) MMP-3 digest (I :20) of purified D dimer (24 hr) As shown in Figures 6A and 6B, ~,. .,,5. .,0O; ~c (time-dependent) d~,b. ad~.;oii of D dimer (186 kDa) into D monomer-like fragment (lanes 4, 5). The size of the resulting monomer was slightly larger than Fragment D (93 kDa), obtained during plasmin deb~_ ' of Fg (lane 1).
Also, as this gel was made 2 months after preparation of these samples, this proves that the reaction has been stopped effectively and has not progressed any further The results indicate that " occur even at 4 hours with the lower amount of enzyme (I :200) (lane 3).
Increasing the amount of enzyme ten-fold increases the at 4 hours (lane 4) and almost completely converts the substrate at 24 hours (lane 5). This is supported in Fig.
6B, which shows the conversion of y dimer (78 kDa) to y monomer-like chain (38 kDa).
EXA~fPLE 7 Analysis of MMP Digestion Products by ELISA. ELISA binding assays were performed using generally accepted methods. Polyvinyl microtiter plates were coated with ~IJIl r ' ' dilutions of different digests and assayed for antibody binding (Kudryk et al.

21 946~

1984). In accord with the results shown in Figure 4, digests with increasing, ofMMP-3 failed to bind MoAb/4A5 (anti-y397-411). This means that the fibrinogen cleavage site of MMP-3 is in the region of this epitope (the fibrin cross-unks occur in the region yGly397-Lys406). This type of cleavage does not occur with plasmin. However, the digests S were found to be reactive with MoAb/4-2 (y349406). No ' ~,~LiviLy with MoAb/lD4 in MMP-3 digests of XL-Fb could be detected by . .~ ELISA, suggesting that the ID4-reactive epitope is destroyed in such digests. MoAb/lD4 binds equally to plasmin digests of Fg and XL-Fb.
Thus, the ELISA (direct binding) performed on digests of Fg and XL-Fb with MMP-3showed that the linear sequence epitope y392-406 (MoAb/4-2-reactive), but not y397-411 (MoAb/4A5-reactive), could be detected by this method. This confirmed the results obtained with ' ' ' ' ~ and further suggested that MMP-3 cleaves within the sequence y397-411. NH2-terminal sequence analysis of the dipeptide (isolated from CNBr de~ aVali of XL-fibrin digested with MMP-3) indicated that yAla405 is the first residue of the second sequence (See Examples 8-9, below). We note that MoAb/4-2 also binds a synthetic peptide whose sequence cu~ r ' to y392-400. MoAb/4A5 reacts with synthetic peptides UUII t~ to y392-411 and y397-411, which is lost when either peptide is cleaved with trypsin at yLys406-Gln407. This feature is consisten~ with the observed lack of reactivity of MoAb/4A5 with the dipeptide.

Size and Sequence Analysis of Chains of MMP-3-Diges~ed Fg and XL-F~. Fg and XL-Fb digested with MMP-3 were separated under reducing conditions on a 12.5%
SDS-PAGE (Laemmli 1970) and c~ ,L-ublvll~id to a pul~ ,,,e difluoride membrane (PVDF) (1' ~ ' _ 1987). The portion of the membrane that contained the y-chain fragment was excised and subjected to automated sequencing on a model 477A Applied Biosystems Inc. pulsed liquid phase sequencer with a model 120A on-line ~h~ Lhiuhyla~Lu;
(Pl~l) arnino acid analyzor.
The digests of both Fg and XL-Fb yielded a y-chain sequence Leu-Lys-Ser-Arg-Lys (SEQ ID NO: 1), indicating that MMP-3 cleaves both Fg and XL-Fb at the yThr83-Leu84 bond. This same band from the XL-Fb digest, however, also gave a second y-chain sequence WO 96/36227 2 1 9 ~ 6 9 2 PCI/US96107188 Ala-X-Gln-Ala-Gly-Asp (SEQ ID NO:2) indicating M~-3-induced hydrolysis at the yGly404-Ala405 bond, in the y-chain cross-link region.

Cl~.,.,f~, of a CNBr-derived y~hainfragmentfromMMP-3-digesfs of 5 ~. To confirm the results obtained from sequencing the y-chain of XL-Fb degraded with MMP-3, the same digest was treated with CNBr (Blomback et al. 1968) and the fragrnent reactive with MoAbl4-2 (anti-y392-406) was isolated, as follows. MMP-3-cleaved fragments were purified by reverse-phase HPLC using a Vydac C-4 column (1.0 x 25 cm, The S-,~,lallal Group, Hesperia, CA). The column was developed at room t~,...~,.,.~Lu.t; using the following gradient ~,u~ u~,l~l with 0.05% L~inuu~u~.,etic acid (TFA, solvent A) and 50%
acetonitrile in A (solvent B): at 5% Bl0.5 min; 5 to 50% Blat 50 min; 50 to lOO0/D B/at 70 min;
100% B/at 75 min. Column fiow rate was 1.0 mL/min and fractions (I mL) were monitored for readivity with MoAb/4-2 (anti-y392-406). The antibody-reactive fraction was further purified by FPLC using the Superdex Peptide 1~ 10/30 column (I .0 x 30-31 cm, Pharmacia 15 Biotech, Piscataway, NJ). The column was developed at room t~,~,v. ~lu. ~ using 20 mM
phosphate buffer (pH 7.2), s~ containing 0.25 M NaCI. The fraction reacting withMoAb/4-2 was pooled, desalted by passage over Sep-Paklg) (Millipore Corp., Milford, MA) and subjected to sequence analysis.
The sequenced fragment cull~ r ~ ~ to a cross-linked dipeptide with two distinctNH2-terminals: yLys385, resulting from CNBr cleavage, and yAla405 (Figure 7). The two sequences were recovered in u.. ~, --,.l,l~ molar quantities. In cycle 3 NH2-terminal analyses showed no PTH residues (due to a machine error), but evidence for Gln and ne in cycle 3 was seen in cycle 4 as lag. These data support our conclusion that MMP-3 cleaves at yGly404-Ala405 within the cross-link region of fibrin y-chain.
Table m shows the partial sequence for the cross-linked dipeptide isolated from XL-Fb digested with MMP-3 after CNBr d~ The y cross-linking domain, with the sites of MMP-3 digestion and CNBr cleavage, is shown ' "~ in Figure 7. The resulting dipeptide contains a single ~-(y-Glu)-Lys bond, with one NH2-terminal as yLys385, resulting from CNBr cleavage at yMet384-Lys385. The presence of the second NH,-terminal, yAla405, supported our conclusion that MMP-3 cleaves at yGly404-Ala405 in the cross-link region of the y-chain.

21 ~46~2 TABLE m ~1~ PTH A A~ ' / Yi ' '_( ') K (215) A (673) 2 I (648) 3 I* Q*
4 P (406) A (438) F C352) G (565) 6 N(219) D(202) 7 R (200) V (115) 8 L (313) 9 T(179) ---I (349) Data obtained from sequence analysis of MMP-3 digests of Fg and XL-Fb showed proximity of cleavage sites with plasmin on these same substrates. Plasmin cleaves at yLys62-Ala63 and more slowly at yLys85-Ser86 (Collen et al. 1975). Thus, fibrinogen digested by plasmin results in two Fragment D species, i.e., yAla63-Val411 and ySer86-Val411. By contrast, MMP-3 cleaves both Fg and ~-Fb at yThr83-Leu84. In addition, since MMP-3 hydrolyzes the yGly404-Ala405 peptide bond, the y-chain has a molecular weight which is ' ~ similar to that of ~ Gd Fragment D
y-chain. However, in the digests of XL-Fb with MMP-3, this product is not a real monomer since the y405-411 domain is cross-linked to the adjacent y-chain having the sequence yLeu84-Gly404. It should be noted that the recovery of similar quantities of the two sequences from the y-chains of the CNBr-generated dipeptide indicated that, albeit slow, the d~ c ~ of XL-Fb by MMP-3, resulting in formation of the D monomer-like fragment, was near complete.

CJiu~ u~, f ~ ul~u. . of Other Cleavage Pro~ucts of Fg and XL-Fb by MMP-3. MMP-3also generates other cleavage products of Fg and XL-Fb, specifically cutting not only the y-chain, but also the a- and ,~-chains. Fg and XL-Fb were digested using MMP-3 (E:S =
1 :20) for 24 hr, separated, and sequenced using the methods described in Example 9. As indicated in Figure 8, both Fg (lane 1) and XL-Fb (lane 2) were specifically cleaved. In Figure 8, band I was found to include two fragments: a y-chain fragment having an NH2-terminal sequence of Leu-Lys-Ser-Arg-Lys (SEQ ID NO:I), again CUllC:>,UU~.d;..,~, to cleavage y84; and a ~-chain fragment (,B,) having an NH2-terrninal sequence Lys-Arg-Gln-Lys-Gln (SEQ ID
NO:3) cull~-r ' ~ to cleavage at ~127. Band 2 was found to contain another ~-chain 2 1 946q2 WO 961~6227 PCI/US96107188 firaglnent (i~32) having the NH2-terminal sequence Tyr-Ser-Ser-Glu-Leu (SEQ ID NO:4), to cleavage at i3142. Band 3 is the enzyme ~-3 remaining in the digest qJ4laliun~ Band 4 was found to contain an a-chain fragment having an NH2-terminal sequence Asn-Arg-Asp-Asn-Thr (SEQ ID NO:5), c~ to cleavage at alû3. (Bamd5 4 was aiso found to contain fragments cleaved at r I and a414 in both fibrinogen and fibrin digests.) Band 5 contained fragments cleaved at ~51 and ,~52 in both fibrin and fibrinogen digests with MMP-3.
Accordingly, the evidence presented in Examples 1-10, above, establishes that fibrinogen becomes thrombin-unclottable when treated with matrix " r ~ ' ' 3 10 (MMP-3, ~L~ ~ 1), but not when treated with matrix ", ut~ .., 2 (MMP-2, gelatinase A). Incubation of XL,-Fb clûts (made with 'Zsl-Fg) with MMP-3 results in complete Iysis after 24 hrs. A D monomer-like fragment is generated by MMP-3 de~ aJ~.IiO.. of fibrinogen, ~Fb and Fragment DD. T l,a~ /ity with ' ' antibody MoAb/4-2 (anti-y392-406), but not with MoAb/4A5 (anti-y397-411), suggested that a major cleavage 15 site was within the sequence ~ l Li.,;~,..lill~ in the cross-linking of two y-chains. NH2-terminal sequence of the y-chain of the D monomer-like fragment and of a dipeptide isolated from the MMP-3 digcst of XL -fibrin, identified the hydrolysis of yGly404-Ala405 peptide bond. These data indicate that the -~ " -l 1;~ , of Fg and ~Fb by MM[P-3 is specific and different from plasmin. This mechanism of fibrinolysis is of relevance in wound healing, 20 ..~,.u .~ ,.u~ , y, renai disease, and other I ' , ' J~.:olo~5i.,~i processes.
Inasmuch as MMP-3 cleaves near the site at which fibrin cross-linking occurs, a strong t~ g;. 1 argument is readily made that MMP-3 plays a singular role in fibrinolysis.
Coordinated regulation of the " r ~ ' ' and l ' ~ activators could, therefore, result m synergistic effects and complete d~v~ of both the ~ matrix and the 25 fibrin meshwork resulting from wound heaiing, ~ '' , thrombosis, cancer, renal disease, or other I ' r~ ;;llO~;;.,~i processes. Accordingly, it is: . ' ' that the co-~ ' ~ ~ of MMP-3 with another il.. ~ ' 'yfic agent such as t-PA would aiso achieve an adjunctive therapeutic effect, in which each component r Ij ' or potentiates the - other.
Thus, while there have been described what are presently believed to be the preferred ' - ' of the present invention, those skilled in the art will realize that other and further 2 I q4~q2 wo 96136227 ~cr~ss6lo7l88 be '- can be made without departing from the spirit of the invention, and it is intended to include all such further I ' ~ ~ and changes as come within the true scope of the claims set forth herein.

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Claims (45)

WHAT IS CLAIMED IS:

1. A method of degrading fibrin(ogen), comprising contacting fibrin(ogen) with an amount of a fibrinolytic metalloproteinase effective for cleaving the fibrin(ogen)..

2. The method of Claim 1, wherein said metalloproteinase is endogenous.

3. The method of Claim 1, wherein said fibrinolytic metalloproteinase is a stromelysin.

4. The method of Claim 1, wherein said fibrinolytic metalloproteinase is MMP-3.

5. The method of Claim 1, wherein said fibrinolytic metalloproteinase cleaves the fibrin(ogen) at the .gamma.Gly404-Ala405 peptide bond.

6. The method of Claim 1, wherein said method further comprises:-contacting said fibrin(ogen) with an effective amount of an adjunct compound having thrombolytic activity.

7. The method of Claim 4, wherein said adjunct compound having thrombolytic activity is selected from the group consisting of a plasminogen activator, hirudin, an enzyme inhibitor, and enzyme, an anticoagulant, an antibody or synthetic peptide specific for platelet gpIIb/IIIa receptor, or a combination thereof.

8. The method of Claim 7, wherein said plasminogen activator is selected from the group consisting of u-PA, t-PA, streptokinase, staphylokinase, and combinations thereof.

9. The method of Claim 1, wherein said method is performed in vivo.

10. A method of fibrinolytic therapy, comprising:
administering to a subject in need of fibrinolytic therapy a therapeutically effective amount of a fibrinolytic metalloproteinase.

11. The method of Claim 10, wherein said fibrinolytic metalloproteinase is an endogenous fibrinolytic metalloproteinase.

12. The method of Claim 10, wherein said fibrinolytic metalloproteinase is a stromelysin.

13. The method of Claim 10, wherein said fibrinolytic metalloproteinase is MMP-3.

14. The method of Claim 10, wherein said fibrinolytic metalloproteinase cleaves fibrin(ogen) at the .gamma.Gly404-Ala405 peptide bond.

15. The method of Claim 10, wherein said method further comprises:
administering to said subject a therapeutically effective amount of an adjunct compound having thrombolytic activity.

16. The method of Claim 15, wherein said adjunct compound having thrombolytic activity is selected from the group consisting of a plasminogen activator, hirudin, an enzyme inhibitor, an enzyme, an anticoagulant, an antibody or synthetic peptide specific for platelet gpIIb/IIIa receptor, or a combination thereof.

17. The method of Claim 16, wherein said plasminogen activator is selected from the group consisting of u-PA, t-PA, streptokinase, staphylokinase, and combinations thereof.

18. The method of Claim 10, wherein said method is performed following thrombolytic therapy to inhibit vascular reocclusion.

19. The method of Claim 10, wherein said method is performed following surgical intervention to inhibit restenosis.

20. The method of Claim 10, wherein said method is performed to inhibit initial formation, or promote regression, of atherosclerotic plaques.

21. A composition for thrombolytic therapy, comprising:
a fibrinolytic metalloproteinase which degrades fibrin(ogen), and a pharmaceutically acceptable carrier.

22. The composition of Claim 21, wherein said fibrinolytic metalloproteinase is an endogenous fibrinolytic metalloproteinase.

23. The composition of Claim 21, wherein said fibrinolytic metalloproteinase is a stromelysin.

24. The composition of Claim 21, wherein said fibrinolytic metalloproteinase is MMP-3.

25. The composition of Claim 21, wherein said fibrinolytic metalloproteinase cleaves fibrin(ogen) at the .gamma.Gly404-Ala405 peptide bond.

26. The composition of Claim 21, wherein said composition further comprises an adjunct compound having thrombolytic activity.

27. The composition of Claim 26, wherein said adjunct compound having thrombolytic activity is selected from the group consisting of a plasminogen activator, hirudin, an enzyme inhibitor, an enzyme, an anticoagulant, an antibody or synthetic peptide specific for platelet gpIIb/IIIa receptor, or a combination thereof.

28. The composition of Claim 27, wherein said plasminogen activator is selected from the group consisting of u-PA, t-PA, streptokinase, staphylokinase, and combinations thereof.

29. A kit for performing thrombolytic therapy, comprising:
a composition comprising a fibrinolytic metalloproteinase, and a container.

30. The kit of Claim 29, wherein said fibrinolytic metalloproteinase is MMP-3.

31. The kit of Claim 30, wherein said kit further comprises an adjunct compound having thrombolytic activity, selected from the group consisting of a plasminogen activator, hirudin, am enzyme inhibitor, an enzyme, an anticoagulant, an antibody or synthetic peptide specific for platelet gpIIb/IIIa receptor, or a combination thereof.

32. The kit of Claim 30, further comprising means for administering a therapeutically effective amount of said composition.

33. The kit of Claim 32, wherein said administering means includes means for administering said composition parenterally.

34. A diagnostic method for characterizing fibrin(ogen), comprising:
contacting fibrin(ogen) with a fibrinolytic metalloproteinase to provide characteristic degradation products of said fibrin(ogen).

35. The method of Claim 34, wherein said fibrinolytic metalloproteinase is MMP-3.

36. The method of Claim 34, wherein said method further comprises:
contacting said degradation products with at least one antibody which specifically associates with a domain of fibrin(ogen), and measuring specific association of said antibody with said separated degradation products.

37. The method of Claim 36, wherein said antibody is detectably labeled with a detectable marker moiety.

38. The method of Claim 36, wherein said antibody is a monospecific antibody.

39. A method of inhibiting thrombus formation by a medical-related apparatus, comprising:
contacting a medical-related apparatus with a composition comprising a fibrinolytic metalloproteinase to provide a thrombus-inhibiting surface on said medical-related apparatus.

40. The method of Claim 39, wherein said medical-related apparatus is selected from the group consisting of blood collection tubes, culture flasks, test plates, pipets, reagent containers, tubing, membranes, needles, cannulae, catheters, grafts, stents, filters, coils, valves, and the like.

41. A medical-related apparatus having thrombus- inhibiting properties, comprising:
a medical-related device having provided thereto a thrombus-inhibiting amount of a composition comprising a fibrinolytic metalloproteinase.

42. The apparatus of Claim 41, wherein said medical-related apparatus is selected from the group consisting of blood collection tubes, culture flasks, test plates, pipets, reagent containers, tubing, membranes, needles, cannulae, catheters, grafts, stents, filters, coils, valves, and the like.

43. A method of enhancing regulation of fibrinolysis in a subject in need of such therapy, comprising:
inducing enhanced regulation of an endogenous fibrinolytic metalloproteinase in a subject.

44. The method of Claim 43, wherein said inducing step comprises increasing the activity or expression of an endogenous fibrinolytic metalloproteinase by treating said subject with somatic cell gene transfer therapy.

45. The method of Claim 43, wherein said inducing step comprises decreasing the activity or expression of an endogenous fibrinolytic metalloproteinase by treating said subject with somatic cell gene transfer therapy.