soluble fusion proteins that can penetrate cells and inhibit calpain are described herein for the first time . these proteins include a cell - penetrating sequence ( e . g ., the signal sequence of kaposi &# 39 ; s fibroblast growth factor ( kfgf )) and a calpain - inhibiting sequence ( e . g ., calpastatin or a biologically active derivative thereof ). the sequence of one such fusion protein , calpastat ( also known as s - tippeyin ), is provided below ( seq id no : 1 ). the first 16 amino acids of calpastat correspond to the kfgf signal sequence , and the next 24 amino acids correspond to ( i . e ., are identical to ) a biologically active derivative of calpastatin . this fusion protein loses activity when seven particular amino acid residues are replaced with alanine ( as in the peptide s - tippey - ala ( seq id no : 2 )). most of the seven amino acids replaced ( those underlined in the sequence of s - tippy - ala , below ) are highly conserved in calpastatin . nh 2 - aavallpavllallapekl a er a d aaa pe aa ellekktgv - cooh ( seq id no : 2 ) other calpastat - like fusion proteins can contain a biologically active peptide fragment of calpastatin or a biologically active variant of that fragment . moreover , the fusion peptides of the invention ( regardless of whether they contain full length calpastatin peptides or fragments or biologically active variants thereof ) can contain the consensus amino acid residues found in repeat 1 and repeat 4 of mammalian ( e . g ., human , bovine , porcine , rabbit , or rat ) calpastatins ( i . e ., they can be hybrid peptides ). functional variants of calpastatin peptides ( i . e ., biologically active variants ), including hybrid peptides , are within the scope of the invention even when they do not contain all of the amino acid residues present in the consensus sequence . the ability of calpastatin peptides ( including hybrid peptides ) to inhibit calpain can be assessed in cell extracts or intact cells as described in the examples below or in other assays known in the art ( e . g ., in the assay disclosed by bronk et al ., am . j . physiol . 264 : g744 - 751 , 1993 or modified versions thereof ). for instance , calpain activity can be monitored in intact cells by measuring ca 2 + ionophore - specific peptidyl hydrolysis of the peptidyl - 7 - amino bond of a calpain substrate ( e . g ., succinyl - leucyl - leucyl - valyn - tyrosyl - 7 - amino - 4 - methylcoumarin ( suc - llvy - amc ; seq id no : 7 )). to assay calpain activity in this way , cells are washed and re - suspended in hepes - buffered ( 10 mm hepes - naoh , ph 7 . 4 ) hank &# 39 ; s balanced salts solution ( without ca 2 + ) at about 2 . 5 × 10 5 cells / ml and placed on ice . to assay calpain activity , the cell suspension is pre - warmed to 37 ° c . for 10 minutes with stirring in an slm alminco 8000 fluorimeter . at t =− 1 minute , ionomycin in dmso ( at a final concentration of 2 . 5 μm ) or dmso alone ( negative control ) is added to the cells . at t = 0 minute , suc - llvy - amc ( seq id no : 7 ) is added to a final concentration of 50 μm . the initial rate of substrate cleavage , which is linear , is measured by spectroscopy at 2 to 3 minutes . the excitation wavelength is 360 ± 2 nm and the emission detection wavelength is 460 ± 10 nm . the ionomycin - dependent rate of substrate cleavage is subtracted from the ionomycin - independent rate of substrate cleavage to obtain the ca 2 + - dependent rate . amc standard solutions are used to determine moles of amc generated from emission data . cell viability can be monitored during the assay by trypan blue exclusion . calpastat - like fusion proteins can be produced by cultured cells ( e . g ., e . coli , yeast , insect cells , or mammalian cells ) transfected with nucleic acid molecules that encode the fusion protein and have appropriate expression control sequences ( see , e . g ., u . s . pat . no . 5 , 648 , 244 ). the nucleic acid molecules can be introduced into the cultured cells by standard transfection techniques , and the recombinantly produced peptides can then be extracted and purified by techniques well known in the art ( e . g ., immunoaffinity purification ). for an exemplary cloning procedure , see example 15 , below . it is well within the ability of one of ordinary skill in the art to carry out the cloning procedure described in example 15 or any comparable cloning procedure known in the art . calpastat - like fusion peptides can also be produced in significant amounts ( i . e ., in amounts sufficient for commercial or experimental use ) by chemical synthesis . for example , calpastat - like peptides can be synthesized using solid phase n -( 9 - fluorenyl ) methoxycarbonyl / n - methylpyrrolidone ( fmoc ) chemistry ( jacobs et al ., j . biol . chem . 269 : 25494 - 25501 , 1994 ). calpastat , calpastat - ala , and the 24 amino acid calpastatin peptide produced by chemical synthesis were found to be greater than 95 % pure by hplc and to have the correct molecular mass and protein sequence when assayed by mass spectrometry and edman degradation . peptide concentrations can be determined by quantitative amino acid analysis . calpastat - like fusion proteins have numerous uses . they can be used , for example , to reduce coronary thrombosis in coronary bypass surgery , to reduce vascular thrombosis and restenosis in angioplasty , to reduce the progression of an infarct in the event of myocardial infarction or stroke ( including treatment in the acute setting ), and to treat subarachnoid hemorrhage or vasospasm , muscular dystrophy , cataracts , sickle cell crisis , hiv infection , alzheimer &# 39 ; s disease , brain aging , traumatic brain injury , joint inflammation , and arthritis . calpastat - like fusion proteins can be administered to a patient who exhibits symptoms of one or more of the illnesses or conditions listed above , as well as to patients who have not yet exhibited symptoms ( i . e ., the fusion proteins can be administered prophylactically ). for instance , a hybrid fusion protein can be administered to a patient who has undergone angioplasty and shows signs of restenosis . alternatively , the protein can be administered before angioplasty to prevent the occurrence of restenosis . the fusion proteins can be used alone or in combination and can be administered with a pharmaceutically acceptable carrier ( e . g ., physiological saline ). the carrier can be selected on the basis of the mode and route of administration , according to standard pharmaceutical practice . suitable pharmaceutical carriers , as well as useful pharmaceutical necessities , are described in remington &# 39 ; s pharmaceutical sciences ( e . w . martin ), a standard reference text in this field , and in the usp / nf . the therapeutic compositions described above can also include agents that augment or potentiate the therapeutic activity of the fusion proteins . for example , the compositions can include agents that increase the biological stability of the fusion proteins or that increase the ability of the fusion protein to selectively penetrate a target cell . for example , to enhance targeting , fusion peptides can be encapsulated in liposomes coated with ligands that bind cell - surface receptors expressed primarily or exclusively on the target cells . other agents can be selected depending on various considerations , such as the disease or disorder to be treated and the potential for adverse interactions with other drugs prescribed for the patient . therapeutic compositions containing fusion proteins can be administered in dosages determined to be appropriate by one skilled in the art . it is expected that the dosages will vary , depending upon the pharmacokinetic and pharmacodynamic characteristics of the particular agent and its mode and route of administration ; the age , weight , and general health of the recipient ( renal and hepatic function are important considerations ); the nature and extent of the disease or disorder ; the frequency and duration of the treatment ; the type of concurrent therapy ( if any ); and the desired effect . it is expected that a useful dosage will contain between about 0 . 1 to 300 mg of active ingredient per kilogram of body weight . ordinarily , 1 to 100 mg , and preferably 10 to 50 mg of active ingredient ( nucleic acid or protein ) per kilogram of body weight per day , given in divided doses or in sustained release form , is appropriate . the therapeutic compositions of the invention may be administered to a patient by any appropriate mode . for example , they may be administered orally or parenterally ( e . g ., intraperitoneally or intravenously ). alternatively , it may be desirable to administer the compositions during a surgical procedure . for example , a composition can be applied to the target tissue during angioplasty . the treatments of the invention may be repeated as needed , as determined by one skilled in the art . the therapeutic or prophylactic efficacy of fusion proteins can be assessed using animal models of the diseases or conditions described above . animal models are well known to those of ordinary skill in the art ( see also the examples below ). administration of dna sequences encoding calpastat - like fusion proteins is also within the scope of the invention and is described below ( see , e . g ., example 17 ). calpastat - like fusion proteins can also be used in vitro . for instance , fusion proteins can prevent platelet aggregation and degranulation and can , therefore , be used to preserve platelets stored in blood banks ( e . g ., platelets preserved at temperatures that inhibit the growth of microorganisms , generally 4 ° c .- 15 ° c .). to improve platelet storage , an appropriate amount of a fusion protein ( e . g ., calpastat at about 1 - 100 μm ) can be added to platelet preparations . calpastat - like fusion proteins can also be used to facilitate autologous blood transfusions in sickle cell patients and patients with sickle trait by preventing irreversible sickling during blood storage . thus , fusion proteins can be used to promote storage and use of blood from sickle cell patients or patients with sickle trait ( asakura et al ., blood cells , molecules & amp ; diseases 22 : 297 - 306 , 1996 ; vichinsky et al ., new engl . j . med . 322 : 1617 - 1621 , 1990 ). described below are examples which demonstrate that calpastat , which is pharmacologically active at concentrations ranging from about 5 nm to about 100 μm : ( 1 ) prevents platelet aggregation and degranulation ; ( 2 ) inhibits erythrocyte sickling ; and ( 3 ) inhibits ca 2 + - mediated activation of hiv - 1 provirus . while the studies described in these specific examples use calpastat , other fusion proteins of the invention could be used in a similar manner . the examples also include protocols that can be used to optimize the therapeutic effectiveness of the new fusion proteins in animal models . unless otherwise noted , the materials used in the studies described below were obtained from commercial suppliers . sepharose 2b was obtained from pharmacia biotech ( upsala , sweden ); pure p - calpain ( porcine erythrocyte ) was obtained from chemicon ; succinyl - llvy - amc ( seq id no : 7 ) was obtained from bachem ; a23187 was obtained from lc labs ; human alpha - thrombin was obtained from hematologic technologies ( essex junction , vt . ); anti - p - selectin antibodies conjugated to phycoerythrin were obtained from becton dickinson ( san jose , calif . ); oregon green - phalloidin was obtained from molecular probes ( eugene , oreg .). all other chemicals were acs grade or better and were obtained from sigma chemical company ( st . louis , mo .). the following examples are meant to illustrate the methods and materials of the present invention . suitable modifications and adaptations of the conditions and parameters described below will be obvious to those of ordinary skill in the art and are within the spirit and scope of the present invention . the experiments described below demonstrate that calpastat , but not the calpastat mutant , s - tippey - ala , effectively inhibits platelet aggregation . in one series of experiments , platelets were purified from serum on a sepharose 2b column in pipes buffered saline containing glucose , pre - incubated with 45 μm calpastat or s - tippey - ala for 30 minutes at 37 ° c ., and placed in a cuvette in a biodata aggregometer . the aggregometer measures turbidity of the solution by nephlometry ( light scatter ). aggregation was induced with thrombin ( 0 . 1 to 1 unit / ml ) or the thrombin receptor agonist peptide sfllr ( seq id no : 5 ). calpastat inhibited platelet aggregation , with maximal inhibition achieved at 45 μm calpastat . in contrast , s - tippey - ala had no activity . a version of s - tippeyin lacking the signal sequence similarly had no activity . this result demonstrates that calpastat can effectively inhibit platelet aggregation and that the seven conserved amino acids mutated in s - tippey - ala are essential for calpastatin function . this result also demonstrates that the platelet inhibitory activity of calpastat lies in the calpastatin portion of calpastat , not the amino - terminal kfgf signal sequence which makes it cell - penetrating . in another series of experiments , gel filtered platelets were obtained and tested as follows . blood from healthy , aspirin - free volunteers was collected by venipuncture into 4 % sodium citrate ( 9 : 1 v / v , final concentration 0 . 4 %) and centrifuged at 200 × g for 20 minutes to obtain platelet rich plasma ( prp ). following incubation with apyrase ( 4 units / ml for 5 minutes at room temperature ), platelets were purified from prp by gel - filtration at room temperature using a sepharose 2b column equilibrated in pipes buffer ( 25 mm pipes ph 6 . 8 , 140 mm nacl , 4 mm kcl , and 0 . 1 % glucose ) ( hsu - lin , et al ., j . biol . chem . 259 : 9121 - 9126 , 1984 ). final gel filtered platelet concentrations were about 1 . 5 × 10 8 cells / ml . gel filtered platelets ( 250 μl aliquots at 1 . 6 × 10 8 1 / ml ) were incubated with the indicated concentration of calpastat or mutant calpastat - ala for either 20 or 30 minutes at 37 ° c . in the absence of calcium and stirring . to test peptidyl calpain inhibitors , platelets were preincubated with the indicated concentration of calpeptin , mdl , e64d , dmso ( vehicle control ) or nh 4 cl ( cathepsin control ) for 10 minutes at 37 ° c . following incubation , platelets were recalcified ( cacl 2 , 2 mm ) and stimulated with 0 . 05 - 1 . 0 unit / ml thrombin , while being stirred ( 1400 rpm ) at 37 ° c . the sensitivity of platelet preparations to thrombin decreased over the course of a given experiment . the thrombin concentration used was to elicit robust aggregation and varied between 0 . 05 and 1 . 0 u / ml . aggregation was measured using a bio - data lumi - aggregometer . percent inhibition of aggregation was measured at 4 minutes following the onset of aggregation . the initial rate of aggregation during the first 15 seconds for treated vs . untreated samples was used to calculate the ic 50 for each inhibitor . calpastat effectively inhibited the extent of platelet aggregation by 60 to 95 % at four minutes following the onset of aggregation ( fig1 ). in contrast , calpastat - ala ( fig1 ) or the calpastatin 24 - mer had no measurable effect on platelet aggregation ( table 2 ; ni = no inhibition ). by comparing the initial rate of thrombin - induced aggregation at various concentrations of inhibitor , the ic 50 of calpastat was determined to be 50 ( table 2 ). three immediately acting peptidyl calpain inhibitors ( calpeptin , mdl and e64d ) also inhibited thrombin - induced platelet aggregation . following a 10 minute pre - incubation period , calpeptin , mdl , and e64d inhibited the initial rate of platelet aggregation with ic 50 &# 39 ; s of 150 , 240 , and 340 μm , respectively ( table 2 ). at high micromolar concentrations , these peptidyl compounds also inhibit members of the cathepsin family of lysosomal proteases . to test whether the peptidyl calpain inhibitors prevent aggregation by inhibiting cathepsins as well as calpain , platelets were pre - incubated with 10 mm nh 4 cl , which maximally inhibits cathepsin function ( hopgood et al ., biochem j . 164 : 399 - 407 , 1977 ). pretreatment of platelets with 10 mm nh 4 cl does not affect platelet aggregation ( table 2 ). thus , inhibition of aggregation by peptidyl calpain does not occur through inhibition of cathepsins . inhibition of aggregation by calpastat and by peptidyl calpain inhibitors together demonstrate that calpain plays a regulatory role in the intracellular events leading to platelet aggregation . to determine whether s - tippeyin could inhibit α - granule secretion in platelets , surface expression of p - selectin , which indicates α - granule secretion , was induced with the thrombin peptide sfllr ( seq id no : 5 ) and analyzed by flow cytometry using an antibody against p - selectin . blood from healthy volunteers was collected into sodium citrate ( 4 % w / v ) and centrifuged at 200 × g for 10 minutes to prepare platelet - rich plasma ( prp ), from which platelets were subsequently purified by gel filtration using a sepharose 2b column in pipes buffer . twenty μl of platelets ( approximately 2 × 10 8 platelets / ml ) were aliquoted into eppendorf tubes and one μl of s - tippeyin , tippeyin ( lacking the signal peptide sequence ), or s - tippey - ala was added to achieve final concentrations of 6 . 25 , 12 . 5 , 25 , 50 , or 100 μm . the platelets were then incubated for 2 hours at 37 ° c . and subsequently exposed to either 1 μl pbs ( unstimulated ) or 1 μl of sfllr ( seq id no : 5 ; stimulated ) at the indicated concentration . after 30 seconds , a 7 . 5 μl aliquot was transferred to an eppendorf tube containing non - immune serum having antibodies that were conjugated to phycoerythrin . following a 20 minute incubation at room temperature , the mixture was diluted with 1 ml of 2 % paraformaldehyde and incubated at 4 ° c . for two hours . antibody binding to platelet surface p - selectin was quantified using a facscan flow cytometer ( becton dickerson , san jose , calif .). results were reported as the geometric mean of the relative fluorescence . as shown in fig2 stimulation of gel - filtered platelets with sfllr ( 50 μm ) results in an approximately 10 - fold increase in surface p - selectin expression ( compare the bars marked respectively “ unstimulated ” and “ sfllr ”). pre - incubation with s - tippeyin resulted in only a 5 - fold increase in p - selectin surface expression ( compare the bars marked respectively “ unstimulated ” and “ s - tippeyin / sfllr ” ( seq id no : 5 )). in contrast , pre - incubation with tippey , a corresponding peptide lacking the kfgf signal sequence , had little effect on sfllr ( seq id no : 5 )- induced p - selectin surface expression ( see the bar marked “ tippey / sfllr ” ( seq id no : 5 )). similarly , a peptide in which the seven conserved residues of calpastat were mutated to alanine had no effect on sfllr ( seq id no : 5 )- induced p - selectin surface expression ( see the bar marked “ s - tippey - ala / sfllr ” ( seq id no : 5 )). the inhibitory effect of s - tippeyin did not result from inhibition of the p - selectin antibody by surface p - selectin , since incubation of platelets with s - tippeyin following exposure to sfllr ( seq id no : 5 ) had no effect on the binding of antibody to surface expressed p - selectin . as shown in fig3 s - tippeyin inhibits the surface expression of p - selectin in a dose - dependent manner . half - maximal inhibition occurred at about 15 - 20 μm , and maximal - inhibition occurred at about 40 - 60 μm . s - tippeyin also inhibited p - selectin surface expression induced by the ionophore a23187 . to further confirm that calpain regulates the surface expression of p - selectin , the inhibitory effects of three rapidly acting , but less specific , peptidyl calpain inhibitors ( calpeptin , mdl , and e64d ) were also tested . platelets were pre - incubated with increasing concentrations of calpeptin , mdl , e64d , or dmso ( vehicle control ) for ten minutes prior to stimulation with sfllr . calpeptin , mdl , and e64d each inhibited p - selectin surface expression with ic 50 &# 39 ; s between 200 and 300 μm . in contrast , calpastat inhibits p - selectin expression in a dose - dependent manner with half - maximal inhibition occurring at a concentration of 30 μm . these results , together with those derived using the specific inhibitor calpastat , demonstrate a novel role for calpain in the regulation of α - granule exocytosis . to investigate whether calpastat inhibits calpain activity in platelets , the ionophore - dependent proteolysis of actin binding protein ( abp ; about 280 kda ) and talin ( about 240 kda ), both of which are substrates of both μ - and m - calpains , was examined as follows . platelets were pre - incubated with zllychn 2 , dmso ( solvent for zlly - chn 2 ), calpastat , or hepes buffer ( buffer for calpastat ), and then treated with or without ionophore a23187 for 5 , 8 , or 10 minutes . the platelets were then gel - purified as described above . whole cell lysates were prepared and fractionated by sds polyacrylamide gel electrophoresis . the data showed that both zlly - chn 2 and calpastat inhibited a23187 - dependent calpain degradation of abp and talin in platelets at all three time points examined . nonetheless , only calpastat inhibited platelet aggregation and secretion . calpastat was examined for its ability to inhibit sickling of red blood cells isolated from sickle cell patients . s - tippey - ala , which demonstrates no inhibition of purified μ - calpain , was used as a control peptide . to examine sickling , a wet preparation of red blood cells ( as opposed to a dried smear ) was made by placing blood under a coverslip on a glass slide . the slide was placed under a light microscope , and the number of cells displaying sickled morphology was counted . na 2 s 2 o 5 was used to increase the rate of sickling . when edta - anticoagulated peripheral blood from a sickle cell patient was preincubated with calpastat for 10 minutes prior to exposure to sodium metabisulfite ( na 2 s 2 o 5 ), there was a significant delay ( about 30 minutes ) in sickling . the time course of sickling is shown in fig4 for this patient . there was no detectable difference in the final percentage of sickled cells . in a second experiment , the effect of calpastat on recovery from sickling was measured . blood from the same patient was incubated with na 2 s 2 o 5 for two hours in a microfuge tube . the supernatant plasma was then removed from the settled erythrocytes and the erythrocytes were diluted in four volumes of saline . the washed erythrocytes were then incubated in saline , and wright - giemsa stained smears were made 60 minutes following the washout of na 2 s 2 o 5 . the percentage of sickled cells in the microfuge tube preparation was found to be at least 2 - fold greater than in the microscope slide preparation . sixty minutes following the na 2 s 2 o 5 washout , the percentage of discocytes was two - fold greater and the percentage of sickled cells two - fold lower in calpastat - treated cells as compared to s - tippey - ala - treated cells . the percentages of discocytes , sickled cells , and echinocytes are shown in fig5 . these observations suggest that calpastat delays the onset of sickling in erythrocytes and facilitates the recovery of discocytes following sickling in vitro . thapsigargin ( tpg ), an inhibitor of the ca 2 +- dependent atpase , has been demonstrated to induce intracellular ca 2 + leakage and activation of hiv - 1 proviral transcription and virion release . calpastat was examined for its ability to inhibit thapsigargin - induced hiv - 1 activation in u1 promonocytes . u1 is a human promonocytic cell line that is chronically infected with hiv - 1 and that constitutively expresses low levels of hiv - 1 . u1 cells are derived from u937 cells , which are acutely infected with hiv - 1 , and possess two integrated copies of hiv - 1 pro - viral dna . in the present experiment , 1 × 10 6 cells / ml were treated with various dosages of calpastat and incubated for one hour at 37 ° c . after incubation , various concentrations of tpg were added to the calpastat - treated cells and aliquoted into a 24 - well plate . to observe the stimulation of hiv - 1 activity by tpg , equivalent numbers of cells were treated with tpg alone . the cells were then incubated at 37 ° c . for up to four days , and supernatants from the cell culture were collected each day to measure hiv - 1 activity . the viability of cells treated with both tpg and calpastat were determined each day by trypan - blue exclusion test . the hiv - 1 activity in the collected supernatants was determined by measuring the hiv - 1 p24 core antigen protein by elisa ( dupont medical products , boston , mass .) according to the manufacturer &# 39 ; s instructions . the data showed that tpg - induced activation of hiv - 1 occurred over a narrow concentration range , with minimal induction at 1 nm ( 1 . 37 - fold induction of virus at 24 h ) and maximal induction at 5 nm ( 20 - fold induction at 24 h ). increasing doses of tpg correlated with decreasing cell viability after 24 hours of incubation ( fig6 ). for instance , u1 cell viabilities at 96 hr were about 50 % and 5 % in the presence of 4 . 0 and 50 nm tpg , respectively . five μm calpastat decreased mean fold viral production by 47 , 44 , 55 and 61 % at 24 , 46 , 72 and 96 hours of tpg stimulation , respectively ( fig7 a - 7 d ). these results were highly statistically significant and indicated that calpastat inhibits hiv - 1 release from infected cells by about 50 %. in another series of experiments , u1 cells , which are latently infected with hiv - 1 provirus , were plated at 10 6 cells / well in 1 ml of medium and treated with varying concentrations of either calpastat ( fig7 e ) or calpeptin ( benzyloxycarbonyl - leucyl - norleucinal ) ( fig7 f ) for 45 minutes before addition of thapsigargin ( 4 nm final concentration ). the viability of u1 monocytes was 90 % at 48 hours of thapsigargin ( 4 nm ) treatment , in the presence or absence of calpain inhibitors . after 48 hours of stimulation , hiv - 1 p24 antigen levels were determined in 200 μl of tissue culture supernatant , using a p24 elisa ( dupont ). all assays were performed on triplicate cultures . mean values and standard deviations are shown for each concentration of inhibitor ( fig7 e - 7 f ). u1 viability studies confirmed that inhibition of hiv - 1 activation correlates with increase in cell viability . calpastat , at a concentration of 5 μm , was shown to increase the viability of u1 cells in 4 nm tpg from 40 % to 90 % at 96 hr ( fig8 ). this result demonstrates that calpastat prevents cell death caused by hiv - 1 activation . to assess the ability of calpastat to inhibit calpain activity , a fluorometric calpain assay was performed . the assay determined specific peptidyl hydrolysis of the peptidyl 7 - amino bond of suc - llvy - amc ( seq id no : 7 ) by calpain . the assay was performed in a 0 . 5 ml reaction volume at 37 ° c . the reaction buffer contained 50 mm kcl , 10 mm tris - hcl ( ph 7 . 5 ), 1 mm edta , and 1 mm dtt . two hundred μm suc - llvy - amc ( seq id no : 7 ) was added along with 5 mm cacl 2 . calpastat or s - tippey - ala was then added at the given concentrations ( fig9 ). at time zero , 4 . 0 μg of purified μ - calpain was added , resulting in a final concentration of 70 nm enzyme . the initial rate of amc production was measured by fluorimetry for the first few minutes . fluorescent excitation was at 360 ± 2 nm and emission detection was at 460 ± 10 nm . amc standard solutions were used to determine moles of amc generated from emission data . the initial rate of substrate cleavage was given as picomoles of amc released / microgram calpain / minute . this assay showed that calpastat inhibits μ - calpain cleavage of suc - llvy - amc ( seq id no : 7 ) with an ic 50 of 50 nm ( fig9 ). in contrast , s - tippey - ala had no inhibitory activity in this assay even at a concentration of 10 μm . another calpain inhibition assay entails measurement of the inhibition of calpain cleavage of a protein substrate methylcasein in a cell - free system . methylcasein is a substrate of both μ - and m - calpain ( see , e . g ., demartino et al ., j . biol . chem . 261 : 12047 - 12052 , 1986 ; croall et al ., physiol . rev . 71 : 813 - 847 , 1991 ). the assay was performed in a 0 . 1 ml reaction volume at 30 ° c . the reaction buffer contained 50 mm mops ( ph 7 . 5 ) and 1 . 4 mm dtt . the substrate was added , along with 10 mm ca 2 + , to a final concentration of 0 . 5 mg / ml . calpastat or s - tippey - ala was added at the given concentrations ( fig1 ). purified μ - calpain was then added to the reaction mix to give a concentration of 2 . 7 μm . the reaction was allowed to proceed for 10 minutes , and was stopped with 0 . 7 ml of 10 % tricholoracetic acid containing 2 mg / ml bovine serum albumin . the reaction mix was then incubated on ice for 1 hour and centrifuged at 1 , 500 × g for 10 minutes . two hundred μl of the supernatant was counted for 14 c cpm in 5 ml of scintillation fluid . calpain activity was determined by subtracting ca 2 + - independent acid soluble cpm ( in the presence of edta ) from ca 2 + - dependent acid soluble cpm ( in the presence of excess ca 2 + ) the ic 50 of calpastat in this assay was 500 nm . this example provides a protocol for determining the effectiveness of a calpastat - like fusion protein in reducing cerebral infarction in rats that have undergone ischemia surgery . this model mimics a focal ischemic episode ( or “ stroke ”). experimental rats are anesthetized ( e . g ., with chloral hydrate ) prior to surgery and their body temperatures are maintained at 37 ± 0 . 50 ° c . using a heating blanket connected to a temperature feedback monitor . the right femoral artery is cannulated with a silicon catheter for measurement of blood pressure and blood gases . blood samples can also be obtained through the catheter and used to determine fusion protein levels in sera . the right femoral vein is similarly cannulated for infusions . to induce ischemia , a 4 - 0 nylon suture with a flame - rounded tip is inserted into the right external carotid artery just distal to the right common carotid bifurcation and advanced 18 . 5 - 19 . 5 mm ( depending on the animal &# 39 ; s weight ) through the internal / intracranial carotid arteries until the tip occludes the origin of the mca . the suture is left in place for 2 hours and then withdrawn into the external carotid artery . immediately following restoration of blood flow , animals are continuously infused for 3 hours with either saline ( 0 . 5 ml / hr ), a calpastat - like fusion protein , or a control peptide . arterial blood gases , ph , and hematocrit are monitored before vascular occlusion and at the end of drug infusion . mean arterial blood pressure is measured before and during occlusion and throughout drug infusion . blood samples for determination of the fusion protein serum levels ( e . g ., by western blotting ) are drawn 2 . 5 hours after the start of drug infusion and 30 minutes following drug termination . to measure the extent of infarction , the rats are sacrificed 48 hours after the onset of occlusion and cerebral infarct volume is determined by computer image analysis of coronal brain slices stained with 2 % 2 , 3 , 5 - triphenyltetrazolium chloride ( bioquant , r + m biometrics , nashville tenn ). infarct volume is calculated using the “ indirect ” method , in which the infarcted area of a brain slice is first determined by subtracting the undamaged area of ipsilateral hemisphere from the total area of the contralateral hemisphere ( swanson et al ., j . cereb . blood flow metab . 10 : 290 - 293 , 1990 ). the infarcted area is then multiplied by section thickness ( 2 mm ) to obtain infarct volume for that slice . total brain infarct volume is finally obtained by summing the volumes of the series of seven brain slices prepared from each animal . statistical analysis of treatment groups is performed using one - way anova followed by a two - tailed unpaired t - test . calpastat - like fusion proteins can be used to treat alzheimer &# 39 ; s disease and brain aging . fusion proteins can be tested using a murine model of alzheimer &# 39 ; s disease ( see , e . g ., games et al ., nature 373 : 523 - 27 , 1995 ; sisk et al ., j . neurosci . 16 : 5795 - 5811 , 1996 ; irizarry et al ., j . neurosci . 17 : 7053 , 1997 ; johnson - wood et al ., proc . natl . acad . sci . usa 94 : 1550 - 1555 , 1997 ). calpastat - like fusion proteins can be used to treat cataracts . the fusion proteins can be screened for effectiveness using animal models of cataract disease ( see , e . g ., zigler , exp . eye res . 50 : 651 - 657 , 1990 ; shearer et al ., current eye res . 6 : 289 - 300 , 1987 ). calpastat - like fusion proteins can be used to treat sickle crisis . the fusion proteins can be screened for effectiveness using animal models of sickle cell disease ( see , e . g ., ryan et al ., science 278 : 873 - 876 , 1997 ; pászty et al ., science 278 : 876 - 878 , 1997 ). calpastat - like peptides can inhibit chemokine production in vitro . the effect of calpastat on chemokine production was tested as follows . samples of thapsigargin - stimulated u937 cells were incubated with 5 nm calpastat or control buffer . the chemokines ( mip - 1α , mip - 1p and rantes ) in the supernatants of u937 cells were assayed using quantikine ™ kits and as described by r & amp ; d systems ( minneapolis , minn .). fig1 depicts the results of these experiments and shows that calpastat incubation reduced the production of all three chemokines by about one third . thus , calpastat can be used to reduce production of inflammatory cytokines and treat inflammatory disorders . calpastat - like fusion peptides ( e . g ., those including the peptides shown in table 1 ) can be used to treat inflammatory disorders including , but not limited to , rheumatoid arthritis , skin inflammation ( e . g ., polyarteritis nodosa ), glomerulonephritis , asthma , pneumonitis ( e . g ., pneumonitis and pulmonary eosinophilia ), and uveltis . calpastat can significantly inhibit chemokine production ( and thereby inhibit inflammatory processes ) at concentrations below those required to substantially inhibit platlet activation . one of the animal models that can be used to examine the effect of calpastat - like fusion peptides on inflammation is the rabbit model of hemorrhagic vasculitis described by argenbright and barton ( j . clin . invest . 89 : 259 - 72 , 1992 ). inhibitors of calpain , including calpastat , can be used as immunosuppressants . accordingly , calpastat - like fusion proteins can be used to treat graft - vs - host disease ; to otherwise reduce the risk of transplant rejection ( e . g ., renal transplant rejection , cardiac transplant rejection , liver transplant rejection ); and to treat other conditions associated with an unwanted immune response ( e . g ., autoimmune diseases ). thus , calpastatin - like fusion peptides ( or gene constructs encoding those peptide ) can be administered to treat autoimmune diseases such as non - obese diabetes , systemic lupus erythematosus , sclerodermia , sjögren &# 39 ; s syndrome , dermatomyositis or multiple sclerosis , rheumatoid arthritis , artheriosclerosis , and psoriasis , asthma , rhinitis , fibrosis , chronic bronchitis , hepatitis , post - infectious anergy , acquired immune deficiency diseases such as aids , and post traumatic immunological anergy . currently , platelets cannot readily be stored in the cold . for example , platelets in citrate dextrose plasma ( cdp ) undergo an irreversible disk - to - sphere transition when they are stored in the cold , which results in loss of viability following transfusion . moreover , platelets can be stored for only about five days in cdp due to growth of contaminating bacteria . as a result , many units of platelets are discarded . calpastat - containing fusion peptides can be used to protect platelets from the disk - to - sphere shape change and thereby improve platelet viability . to examine the effects of calpastat on shape change , platelet rich plasma ( prp ) was preincubated with calpastat ( 50 mm ) or edta for about 24 hours at 4 ° c . disk - to - sphere shape change was then assayed using the extent of shape change ( esc ) assay ( holme et al ., transfusion 38 : 31 - 40 , 1998 ). briefly , samples of stored prp were treated to induce disk - to - sphere shape change by cold storage and the addition of edta ( to prevent aggregation ). adp was then added as a stimulus to measure esc , and shape change was measured by determining light transmission through the sample . if the initial storage conditions caused considerable disk - to - sphere shape change , there would be little additional shape change upon addition of edta and adp . conversely , if the initial storage conditions caused little disk - to - sphere shape change , there would be considerable additional shape change upon addition of edta and adp . the results of three experiments in which 24 hour storage of platelets at 4 ° c . was tested , in the presence and absence of calpastat , are shown in fig1 . the bracket labelled “ fresh cdp ” indicates the esc that can be expected for fresh prp in cpd , and the bracket marked “ cold edta - treated prp ” indicates the esc that can be expected for prp subjected to cold storage in edta . as fig1 shows , calpastat significantly improved cold storage of prp compared to edta ( esc is greater upon addition of edta and adp for calpastat treated cells than non - treated cells ). fig1 shows the results of an esc assay performed on prp stored at 4 ° c . for five days in the presence of calpastat or control vechicle / edta . the day o control is an assay performed on fresh prp with no cold storage in standard buffer . calpastat provided considerable protection against disk - to - sphere shape change even after five days of cold storage . because calpastat is a reversible inhibitor of calpain and platelet activation and because calpastat can be washed out of platelets , allowing them to aggregate in the presence of thrombin , calpastat and related calpain inhibitors are useful for preservation of platelets in the cold . platelet activation on surfaces is a major problem in many different medical procedures ( e . g ., angioplasty , cardiopulmonary bypass , extracorporeal oxygenation , and dialysis ). platelet activation on surfaces can be inhibited by calpastat or calpastat - like fusion proteins . to study the effect of calpastat on platelet activation on surfaces , platelets were preincubated with 100 mm calpastat ( s - tippeyin ) or s - tippey - ala for 90 minutes at 37 ° c . the platelets were then spread on glass surfaces for 20 minutes , and then fixed . platelet spreading was assayed by oregon green - phalloidin staining , followed by fluorescence micrography . fig1 a depicts the distribution of platelet spread size in the presence of s - tippeyin , and fig1 b depicts the distribution platelet spread size in the presence of s - tippeyin - ala . calpastat ( s - tippeyin ) pretreatment resulted in a 70 % reduction in spreading on the glass surface . in a related series of experiments , gel filtered platelets were preincubated with either calpastat or calpastat - ala ( 100 μm concentrations for 30 , 60 , and 90 minutes at 37 ° c .) or with the low molecular weight inhibitors calpeptin , mdl , or e64d ( 50 - 500 μm for 10 minutes at 37 ° c .). an aliquot of the platelet suspension ( 80 μl ) was plated on a glass coverslip and the platelets were allowed to settle and spread for 20 minutes at 37 ° c . in a tissue culture incubator ( revco ultima ™ incubator ; revco scientific , inc ., asheville , n . c .). the platelets were then fixed in 3 . 7 % formaldehyde , permeabilized with triton x - 100 ( 0 . 1 % for four minutes ), and blocked in pipes buffered saline with 3 % bovine serum albumin . the coverslips were stained with oregon - green - phalloidin and photographed on kodak tri - x film by photomicrography using a nikon optiphot 2 fluorescence microscope ( 100 × objective ). the cellular area of spread platelets was determined by computer assisted image analysis using nih image 1 . 61 . treatment of platelets with 3 % dmso led to a complete inhibition of spreading and provided baseline cell area for unspread platelets . percent inhibition of spreading was calculated from the increase in mean cell area for untreated and treated samples vs . unspread dmso controls . pre - incubation of platelets with calpastat ( 100 μm ) for 30 , 60 , or 90 minutes resulted in 0 , 46 , or 80 % inhibition of platelet spreading ( i . e ., increase in mean platelet area ), respectively , relative to the calpastat - ala control ( fig1 ). calpastat - ala results in minimal inhibition of platelet actin remodeling , including filopodial and lamellipodial protrusion formation . thus , the calpastatin motif is responsible for inhibition of spreading . moreover , the extent of inhibition depends on the pre - incubation time and 30 - 60 minutes is required . calpastat does not act as rapidly as the peptidyl calpain inhibitors calpeptin , mdl , and e64d . in contrast , zllychn 2 , which takes up to 14 hours to inhibit spreading in fibroblasts , has no inhibitory activity in this assay . the ic 50 of calpastat for inhibition of platelet spreading is 35 μm . notably absent in the calpastat - treated cells is the peripheral rim of f - actin , which otherwise appears at the leading edge of each lamellae . half of the platelets pre - treated with calpastat ( 100 μm ) for 1 . 5 hours are round and unspread on glass . the disc - like , unspread platelets demonstrate no ( or few ) filopodia and lack lamellipodial protrusions . these findings suggest that calpain is involved in an early process in spreading , rather than in retraction following spreading ( the unspread platelets lack the pattern of retraction fibers together with extended filopodia that are seen in platelets undergoing cytoskeletal retraction ). to confirm the role of calpain in platelet spreading , the peptidyl calpain inhibitors calpeptin , mdl , and e64d were tested for their ability to inhibit spreading . platelets were pre - incubated for 10 minutes with varying concentrations of calpeptin , mdl and e64d ( 50 - 500 μm ) and then spread on glass , as described above . all three compounds effectively inhibited platelet spreading ( table 3 ). the ic 50 s for inhibition of spreading by calpeptin , mdl , and e64d are 200 , 200 , and & gt ; 250 μm , respectively ( table 3 ). these concentrations are at least 6 - fold higher than the ic 50 of calpastat . the spreading morphology of platelets treated with peptidyl calpain inhibitors is similar to that of calpastat - treated platelets . regardless of the inhibitor , there was a notable absence of the peripheral rim of f - actin at the leading edges of the lamellae . under these conditions ( spreading on glass , as described above ), 30 % of platelets pre - treated at the ic 50 for calpeptin or mdl ( 200 μm ) are round and unspread , with a thin cortical layer of f - actin . as occurs with calpastat treatment , the unspread platelets have no ( or few ) filopodia and lack lamellipodia . about 20 % of the platelets pretreated at the ic 50 of e64d ( 250 μm ) are round and unspread . inhibition of platelet spreading by peptidyl calpain inhibitors does not occur through inhibition of lysosomal cathepsins because treatment with 10 mm nh 4 cl has no effect on platelet spreading ( table 3 ). to prevent platelet spreading on a surface , calpastat , a related calpain inhibitor , or a calpastat - like fusion protein would be attached to the surface non - covalently , for example , in a polyethylene glycol ( peg ) material , without loss of function . the proteins could also be attached covalently to surfaces by conservative substitution of the lysine residues with arginine ( except for the last residue ), with subsequent n - hydroxysuccinamide ester chemistry used to attach the inhibitor to surfaces via aliphatic linker arms . the inhibitor ( s ) could be applied to stents ( e . g ., those used for angioplasty ), tubing ( e . g ., cardiopulmonary bypass tubing or dialysis tubing ), oxygenation devices , catheters ( e . g ., those used for angiographic procedures , swan - ganz catheters , hickman catheters , or vascath catheters ), gortex vascular prostheses , and artificial heart valves . a polymerase chain reaction ( pcr ) product , 160 basepairs in length , that encodes the met - calpastat gene therapy construct has been generated and gel - purified . this construct encodes a calpastat molecule , met - calpastat , which has an amino - terminal methionine ( fig1 ). topo ™ cloning ( invitrogen , carlsbad , calif .) will be used to insert the pcr product into a suitable eukaryotic expression vector polylinker . prc / cmv or pcdna3 ( invitrogen , carlsbad , calif .) are suitable vectors . the polylinker insert containing the met - calpastat minigene can then be inserted into any of the naked dna or viral gene therapy systems indicated below . topo ™ ta cloning kits available from invitrogen ( carlsbad , calif .) provide a highly efficient and rapid cloning strategy for the direct insertion of taq polymerase - amplified pcr products into a plasmid vector . no ligase , post - pcr procedures , or pcr primers containing specific sequences are required . the invitrogen product literature provides an excellent description of this procedure , should further explanation be desired . a dna sequence that can be used to express calpastat is shown in fig1 . the sequence encodes a polypeptide that contains an amino - terminal methionine , which is hydrophobic and does not interfere with the membrane penetrating function of signal sequences . however , the polypeptide can be produced without the intial methionine using various techniques known to those of skill in the art . generally , these techniques produce a polypeptide with an initial methionine and then remove it . because the signal peptidase cleavage site of the kfgf signal sequence is deleted , calpastat should be secreted intact , which would facilitate purification from eukaryotic tissue culture supernatants or bacterial growth medium . for bacterial expression , the calpastat or calpastatin sequence could be inserted in an iptg - regulated expression vector . for eukaryotic expression , the calpastat or calpastatin sequence could be inserted in a tetracycline - induced expression vector system . thus , the invention encompasses nucleic acid molecules that encode calpastat - like fusion peptides ( e . g ., a peptide shown in table 1 fused to a suitable cell penetration peptide ). the nucleic acid molecules can be inserted into vectors , such as those described below , which will facilitate expression of the gene . accordingly , expression vectors containing such nucleic acid molecules and cells transfected with these vectors are within the scope of the invention . a transformed cell is any cell into which ( or into an ancestor of which ) a nucleic acid molecule encoding a polypeptide of the invention has been introduced ( e . g ., by recombinant dna techniques ). an isolated nucleic acid molecule is a nucleic acid molecule that is separated from the 5 ′ and 3 ′ coding sequences with which it is immediately contiguous in the naturally occurring genome of an organism . isolated nucleic acid molecules include nucleic acid molecules which are not naturally occurring , e . g ., nucleic acid molecules created by recombinant dna techniques . nucleic acid molecules include both rna and dna , including cdna and synthetic dna ( i . e ., chemically synthesized dna ). the expression systems that can be used to produce calpastatin - like fusion proteins include , but are not limited to , microorganisms such as bacteria ( for example , e . coli and b . subtilis ) transformed with recombinant bacteriophage dna , plasmid dna , or cosmid dna expression vectors containing the nucleic acid molecules of the invention ; yeast ( for example , saccharomyces and pichia ) transformed with recombinant yeast expression vectors containing the nucleic acid molecules of the invention ; insect cell systems infected with recombinant virus expression vectors ( for example , baculovirus ); or mammalian cell systems ( for example , cos , cho , bhk , 293 , vero , hela , mdck , wi38 , and nih 3t3 cells ) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells ( for example , the metallothionein promoter ) or from mammalian viruses ( for example , the adenovirus late promoter and the vaccinia virus 7 . 5k promoter ). in bacterial systems , a number of expression vectors may be advantageously selected depending upon the use intended for the gene product being expressed . such vectors include , but are not limited to , the e . coli expression vector pur278 ( ruther et al ., embo j . 2 : 1791 , 1983 ), in which the coding sequence of the insert may be ligated individually into the vector in frame with the lacz coding region so that a fusion protein is produced ; pin vectors ( inouye and inouye , nucleic acids res . 13 : 3101 - 3109 , 1985 ; van heeke and schuster , j . biol . chem . 264 : 5503 - 5509 , 1989 ); and the like . pgex vectors may also be used to express foreign polypeptides as fusion proteins with glutathione s - transferase ( gst ). in general , such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione - agarose beads followed by elution in the presence of free glutathione . the pgex vectors are designed to include thrombin or factor xa protease cleavage sites so that the cloned target gene product can be released from the gst moiety . in an insect system , autographa californica nuclear polyhidrosis virus ( acnpv ) can be used as a vector to express foreign genes . the virus grows in spodoptera frugiperda cells ( for example , see smith et al ., j . virol . 46 : 584 , 1983 ; smith , u . s . pat . no . 4 , 215 , 051 ). in mammalian host cells , a number of viral - based expression systems can be utilized . in cases where an adenovirus is used as an expression vector , the nucleic acid molecule of the invention can be ligated to an adenovirus transcription / translation control complex , for example , the late promoter and tripartite leader sequence . this chimeric gene can then be inserted in the adenovirus genome by in vitro or in vivo recombination . specific initiation signals may also be required for efficient translation of inserted nucleic acid molecules . these signals include the atg initiation codon and adjacent sequences . the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert . these exogenous translational control signals and initiation codons can be of a variety of origins , both natural and synthetic . the efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements , transcription terminators , etc . ( see bittner et al ., methods in enzymol . 153 : 516 - 544 , 1987 ). in addition , a host cell strain may be chosen that modulates the expression of the inserted sequences , or modifies and processes the gene product in the specific fashion desired . different host cells have characteristic and specific mechanisms for the post - translational processing and modification of proteins and gene products . appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed . to this end , eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript , glycosylation , and phosphorylation of the gene product may be used . expression contructs capable of expressing calpastat , calpastat related calpain inhibitors , or calpastat - like fusion proteins can be prepared using methods known to those of ordinary skill in the art . such constructs can be used for gene therapy , for example , for paracrine production of calpastat at a site of inflammation , within a tumor or within the vicinity of potential cancer cell movement , or at the site of an angioplasty . a dna sequence encoding a calpastat related calpain inhibitor containing the human calpastatin repeat 1 sequence is shown in fig1 . calpastatin , related calpain inhibitors , and calpastat - like fusion peptides can be used to inhibit the motility of cancer cells , which would in turn inhibit their ability to spread to other tissues or parts of the body , as well as the survival of cancer cells . without any intention to limit the invention to proteins that function by any particular mechanism , it is believed that calpastatin , related inhibitors , and calpastat - like proteins inhibit actin remodeling . calpastatin , related inhibitors , and calpastat - like fusion proteins can be administered in a variety of ways to a patient who has cancer . for example , calpastat - like fusion proteins can be delivered via a liposomal delivery system that selectively targets tumor cells . such delivery systems are known in the art , and are not limited to use for the treatment of cancer ; liposomal delivery systems can be used to administer calpastatin , related inhibitors , and calpastat - like fusion proteins to treat the other conditions described herein ( e . g ., sickle cell crisis , hiv infection , cerebral ischemia , alzheimer &# 39 ; s disease , cataracts , inflammatory disorders , and disorders or conditions in which immunosuppression is required ). for example , the pegylated stealth liposome system , which has been used to deliver doxorubicin ( vail et al ., clinical cancer res . 4 : 1567 - 71 , 1998 ) to cancer cells could be used . the compositions of the invention could be delivered alone , in combination , or together with chemotherapeutic agents . because calpastat can be secreted intact with its signal sequence , and can act in a paracrine fashion , it is an ideal candidate for gene therapy applications . again , the application of calpastatin , related inhibitors , and calpastat - like fusion proteins via gene therapy is not restricted to any particular type of disease or condition . gene therapy can be employed in any situation in which calpastatin , related inhibitors , or calpastat - like fusion proteins are effectively administered . without limiting the invention to calpastat - like fusion proteins that function by a particular mechanism , it is believed that calpastatin , like other protease inhibitors , may block the degradation of wild type p53 and thereby promote apoptosis of cancerous cells ( see , e . g ., pariat et al ., mol . and cell . biol . 17 : 2806 - 2815 , 1997 ; zhang et al ., oncogene 14 : 255 - 263 , 1997 ; braun et al ., int . j . cancer ( pred . oncol ): 84 : 6 - 9 , 1999 ; kimura et al ., nature medicine 4 : 915 - 922 , 1998 ; and zhu et al ., biochem . & amp ; biophys . res . com . 214 : 1130 - 1137 , 1995 ). because the calpastatin - like fusion peptides of the invention can inhibit calpains that are ubiquitously expressed , these peptides can be used to inhibit the motility or reduce the survival of numerous types of cancer cells . for example , calpastatin - like fusion peptides can be used to treat cancers of mesenchymal origin such as sarcoma , e . g ., fibrosarcoma , myxosarcoma , liposarcoma , chondrosarcoma , osteogenic sarcoma or chordosarcoma , angiosarcoma , endotheliosarcoma , lymphangiosarcoma , synoviosarcoma or mesothelioma ; leukemias and lymphomas such as acute myelogenous leukemia ( aml ), acute lymphocytic leukemia ( all ), chronic myelogenous leukemia ( cml ), myelodysplastic syndrome ( mds ), chronic lymphocytic leukemia , prolymphocytic leukemia , non - hodgkin &# 39 ; s lymphoma , aids - related lymphoma , kaposi &# 39 ; s sarcoma , multiple myeloma , hodgkin &# 39 ; s disease ; sarcomas like leiomyosarcoma or rhabdomyosarcoma , tumours of epithelial origin ( carcinomas ) such as squamous cell carcinoma , basal cell carcinoma , sweat gland carcinoma , sebaceous gland carcinoma , esophagel carcinoma , gastric carcinoma , breast carcinoma , colon adenocarcinoma , pancreatic adenocarcinoma , adenocarcinoma of the thyroid , papillary carcinoma , papillary adenocarcinoma , cystadenocarcinoma , medullary carcinoma of the thyroid , undifferentiated carcinoma , non - small cell lung carcinoma , small cell lung carcinoma , bronchogenic carcinoma , melanoma , renal cell carcinoma , hepatoma - liver cell carcinoma , bile duct carcinoma , cholangiocarcinoma , papillary carcinoma , transitional cell carcinoma choriocarcinoma , semonoma or embryonal carcinoma , prostate , uterine , or ovarian carcinomas , or other carinomas of the reproductive tract , bladder carcinoma , squamous cell carcinoma of the neck and head region , and tumours of the central nervous system like glioma , meningoma , medulloblastoma , schwannoma or ependymoma . introduction of calpastat , a related calpain inhibitor , or a calpastat - like fusion peptide at the time of angioplasty can potentially have a lasting effect on restenosis . the peptide could be introduced at the time of angioplasty by catheter , balloon , or stent . a gene therapy vector could also be included , for later expression , beginning about one day following the procedure . it is to be understood that while the invention has been described in conjunction with the detailed description thereof , that the foregoing description is intended to illustrate and not limit the scope of the invention , which is defined by the scope of the appended claims . for example , the fusion proteins may be used to inhibit activation of nf - κb regulated viruses , for example , cytomegaloviruses , hepatitis b virus , herpes viruses , adenoviruses , htlv - i , sendai virus , human herpes virus 6 , and hsv type 1 ( see , e . g ., baeuerle , biochem . biophys . acta 1072 : 63 - 80 , 1991 ). other aspects , advantages , and modifications are within the scope of the following claims . asp lys leu gly glu arg asp asp thr ile pro pro glu tyr arg his asp lys leu gly glu arg asp asp thr ile pro pro lys tyr gln his asp lys leu gly glu arg asp asp thr ile pro pro glu tyr arg his asp lys leu gly glu arg asp asp thr ile pro pro glu tyr arg his glu lys leu gly glu arg asp asp thr ile pro pro glu tyr arg his gccgcc atg gcc gcg gta gcg ctg ctc ccg gcg gtc ctg ctg gcc ttg 48 ctg gcg ccc gaa aag ctg ggt gag aga gac gac aca att cct cca gag 96 tac agg gaa ctt ctg gag aaa aaa aca ggg gtt tga 132 gccgcc atg gcc gcg gta gcg ctg ctc ccg gcg gtc ctg ctg gcc ttg 48 ctg gcg ccc gag gaa ttg ggt aaa aga gaa gtc aca att cct cca aaa 96 tat agg gaa cta ttg gct aaa aag gaa ggg atc tga 132