Modified protein z-dependent protease inhibitor and method of use

Abstract

A modif ied protein Z-dependent protease inhibitor (ZPI) comprising an amino acid substitution at position 293 and / or position 240, a method of using the modif ied ZPI for inhibiting or reducing the risk of venous or arterial thrombosis without compromi sing hemostasis, and composition comprising the modif ied ZPI. A method of enhancing in vivo activity of a ZPI by administering the ZPI in combination with protein Z.

Description

UIC0116WO PATENT MODIFIED PROTEIN Z -DEPENDENT PROTEASE INHIBITOR AND METHOD OF USEREFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benef it of priority f rom U. S. Provisional Application Serial Number 63 / 761, 282, f i led February 21, 2025, the content of which is incorporated herein by reference in its entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence list ing (name: UIC0116WO_ST26. xml; si ze: 16, 216 bytes; and date of creation: January 10, 2026 ) is herein incorporated by reference in its entirety.FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0003] This invent ion was made with government support under grant numbers R35HL150797 and R56HL149881 awarded by the National Inst itutes of Health. The government has certain rights in this invention.BACKGROUND OF THE INVENTION

[0004] The protein Z-dependent protease inhibitor (ZPI ) is a natural plasma anticoagulant serpin, which inhibits both activated f actor X ( FXa) and activated factor XI ( FXIa). It has been shown that ZPI may also inhibit act ivated factor IX ( FIXa) (Heeb et al. (2005 ) J. Biol. Chem. 280: 33819 -33825), although other studies fai led to conf irm that f inding (Huang et al. (2008 ) J. Biol. Chem. 283: 29770 - 29783 ). ZPI circulates in the plasma mostly in a tight complex (XD-1. 0 - 10 nM) with its cofactor protein, Protein Z ( PZ) ( Han et al. (2000) Blood 96: 3049 - 3055; Huang et al. (2012 ) Blood 120: 1726 - 1733 ), with ZPI in modest excess in human and PZ in excess in mice (Tabatabai et al. ( 2001 ) Thromb Haemost. 85: 655 - 660; Girard etUIC0116WO PATENT al. (2013 ) J. Thromb. Haemost. 11: 375 - 378 ). PZ is a vitamin K-dependent protein, structurally homologous to factors II, VII, IX, and X, but lacking protease activity due to the absence of serine and hi stidine residues of the catalytic triad (Broze, Jr. & Mi letich ( 1984 ) J. Cl in. Invest. 73: 933 - 938 ). ZPI -PZ complex formation is required for the rapid inact ivat ion of membrane -associated FXa by ZPI in the presence of Ca2+and phospholipids, whi le its inhibit ion of FXIa is PZ - independent (Han et al. (2000) Blood 96: 3049 -3055 ). ZPI inhibits both FXa and FXIa by the same suicide - substrate mechanism, with a fast ka(104-107M-1s-1) and slow kdiss(~0.0002 s-1), typical of serpin-type protein inhibitor interactions with their coagulat ion proteases (Huang et al. ( 2008 ) J. Biol. Chem. 283: 29770 -29783 ). Previous studies have established that the ZPI / PZ complex inhibits membrane -bound FXa by f irst binding to the membrane through the PZ gamma- carboxy glutamic (Gia) domain to promote the irreversible format ion of a stable ZPI - FXa covalent complex that inact ivates FXa ' s catalytic function. After the format ion of the stable ZPI - FXa complex, PZ dissociates from the FXa-bound ZPI to complex with free ZPI, thus maintaining ZPI / PZ levels (Huang et al. (2008 ) J. Biol. Chem. 283: 29770 -29783 ). X- ray crystal structures of the ZPI / PZ complex have suggested how ZPI and PZ interact (Wei et al. (2009 ) Blood 114: 3662 -3667; Huang et al. (2010) J. Biol. Chem. 285: 20399 -20409), with the key interacting sites being further verif ied by mutagenesi s studies (Huang et al. (2012 ) Blood 120: 1726 - 1733; Huang et al. (2015 ) J. Biol. Chem. 290: 9906 - 9918 ). Whereas the interface of ZPI and PZ compri ses a broad area, including electrostatic and hydrophobic interactions, an interaction has been suggested to be between D293 of ZPI and H210 and R298 of PZ (Huang et al. (2012 ) Blood 120: 1726 - 1733; Wei et al. (2009 ) Blood 114: 3662 -3667; Huang et al. (2010 ) J. Biol. Chem. 285: 20399 -20409 ). A single mutation of Asp 293 to Ala increased the KDof the ZPI / PZ complex from 1 -10 nM to 1-4 pM in physiological buf f er andUIC0116WO PATENT appeared to diminish the PZ -dependent inhibit ion of FXa at relatively low levels of ZPI and PZ (~30-50 nM) (Huang et al. (2012 ) Blood 120: 1726 - 1733; Huang et al. (2010 ) J. Biol. Chem.285: 20399 -20409 ).

[0005] The ZPI / PZ complex is a phys iological ant icoagulant (Huang et al. (2019 ) J. Biol. Chem. 294: 7644 - 7657 ). Its importance in vivo was demonstrated by the f inding that combined def iciencies of ZPI or PZ with factor V Leiden ( FVL, an R506Q FV mutant that is resistant to cleavage by activated protein C) result in a severe to mortal thrombosis phenotype in mice as well as in humans (Yin et al. (2000 ) Proc. Na tl. Acad. Sci. USA 97: 6734 - 6738; Zhang et al. (2008 ) Blood 111: 4973 -4978; Kemkes -Matthes et al. (2002 ) Thromb. Res.106: 183 - 185; Kemkes -Matthes et al. (2005 ) Br. J. Haema tol.128: 248 -252; Martinell i et al. (2005 ) J. Thromb. Haemost.3: 2817 - 2819 ). Interestingly, ZPI def iciency produces a more severe prothrombot ic phenotype than PZ def iciency in mice ( Zhang et al. (2008 ) Blood 111: 4973 -4978 ) suggesting the PZ -independent anticoagulant function of ZPI is also physiologically relevant. Indeed, ZPI is a potent natural FXIa inhibitor with the fastest kaamong known FXIa inhibitors in human plasma (Rezaie et al. ( 2006 ) Biochemistry 45: 9427 - 9433 ).

[0006] Anticoagulants have been proven ef fective in the prevention and treatment of thrombotic di seases. However, conventional anticoagulants, including direct oral anticoagulants ( FXa and direct thrombin inhibitors ), are associated with serious side ef fects of bleeding. The key intrinsic pathway component, factor XI (FXI ) or FXIa, has emerged as a promising antithrombotic target with reduced bleeding risks. However, bleeding is still reported in patients given FXI or FXIa inhibitors. Additionally, there are reports of FXIIa - induced activation of FIX and intrinsic pathway via kallikrein, bypassing FXI. Accordingly, needed in the art are ef fective ant icoagulants that can inhibit thrombosi s withUIC0116WO PATENT minimal bleeding risks. The present invention addresses this need in the art.SUMMARY OF THE INVENTION

[0007] This invention provides a method for inhibiting or reducing the risk of venous or arterial thrombosis without compromising hemostasis, comprising administering to a subject in need thereof an effective anticoagulant amount of a modif ied protein Z-dependent protease inhibitor (ZPI) comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1 and an amino acid substitution at position 293 and / or position 240 with reference to SEQ ID NO: 1, thereby inhibiting or reducing the risk of venous or arterial thrombosis without compromising hemostasis.

[0008] The invention also provides a modified protein Z-dependent protease inhibitor (ZPI) comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1; an amino acid substitution at position 293 and / or position 240 with reference to SEQ ID NO: 1; and at least one polyethylene glycol (PEG) group conjugated to the modified ZPI.

[0009] In invention further provide a method of enhancing the activity of a protein Z-dependent protease inhibitor (ZPI) comprising administering to a subject in need thereof a ZPI comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1 in combination with Protein Z (PZ) thereby enhancing the activity of the ZPI.BRIEF DESCRIPTION OF DRAWINGS

[0010] FIG. 1 is a schematic of the modif ied ZPI-based anticoagulation therapy of the invention. X, factor X; Xa, activated factor X; XI, factor XI; Xia, activated factor XI; IX, factor IX; IXa, activated factor IX; XII, factor XII; Xlla, activated factor XII; PK, Prekallikrein, Ka, plasma kallikrein; II, factor II; Ila, activated factor II; PZ, protein Z; WTZPI,UIC0116WO PATENT wild-type protein Z-dependent protease inhibitor; D293A, D293A ZPI; Y240A, Y240A ZPI; D293A / Y240A ZPI; FVIIa, activated factor VII; FVa, activated factor V; PL, phospholipids.

[0011] FIGS. 2A-2F show the effects of wild-type ZPI (WT) and D293A ZPI (D293A) on thrombin generation in pooled human plasma initiated by 33 nM (FIGS. 2A, 2C, and 2E) and 6.5 nM (FIGS. 2B, 2D, and 2F) concentrations of FXIIa. Thrombin generation in human pooled normal plasma (PNP) and factor XI -deficient patient plasma (FXI -DP) activated by various doses of FXIIa was measured by Calibrated Automated Thrombogram (CAT) at pH 7.4, 37°C, as described in Example 1. To 60 pL mixture containing 40 pL Plasma, fluorogenic substrate, and lipid was added 20 pL of wild-type ZPI or D293A ZPI as indicated. Thrombin generation was initiated by adding 20 pL of pre-warmed CaCl2and FXIIa. The final concentration was 420 pM fluorogenic substrate, 25 pM lipids, 20 mM CaCl2, and indicated concentrations of wildtype ZPI or D293A ZPI and FXIIa. Thrombinoscope™ software was used to analyze fluorescence data to obtain the lag time (LT), thrombin peak (TP), time to peak (TTP), and endogenous thrombin potential (ETP). The data represented from three independent measurements is the average of triplicate measurements. Student ' s t-test was used to compare the differences between selected two groups.

[0012] FIGS. 3A-3I show the effects of wild-type ZPI and D293A ZPI on thrombin generation in pooled human plasma initiated by 6 pM (FIGS. 3A, 3D, and 3G), 2 pM (FIGS. 3B, 3E, and 3H), and 0. 5 pM (FIGS. 3C, 3F, and 31) concentrations of tissue factor (TF). Thrombin generation in FXI-DP and PNP activated by various doses of TF was measured by CAT at pH 7.4, 37° C, as described in Example 1. To 60 pL mixture containing 40 pL plasma, fluorogenic substrate, CTI, and lipid was added 20 pL of TF, wild-type ZPI, or D293A ZPI as indicated. Thrombin generation was initiated by adding 20 pL of pre-warmed CaCl2. The final concentration was 420 pM fluorogenic substrate, 30UIC0116WO PATENT pg / mL CTI, 25 pM lipids, 20 mM CaCl2, and indicated concentrations of wild-type ZPI or D293A ZPI and TF. The dotted lines in FIG. 3D- 3 I reproduce the corresponding data of FXI -DP in FIG. 3A-3C. Thrombinoscope™ software was used to analyze f luorescence data to obtain the lag t ime (LT), thrombin peak (TP), time to peak (TTP), and endogenous thrombin potent ial (ETP). The data represented from three independent measurements is the average of triplicate measurements. Student ' s t -test was used to compare the dif ferences between selected two groups.

[0013] FIGS. 4A-4D show the anticoagulant ef f ects of wild-type ZPI or D293A ZPI as determined by Activated part ial thromboplastin time (APTT) or Prothrombin time (PT). APTT and PT analyses were performed as described in Example 1. Data represents the average of 3 - 5 independent measurements, presented as mean±SD. Student ' s t -test was used to compare the di fferences between selected two groups. FIG. 4A, Dose dependent effects of wi ld- type ZPI, D293A ZPI, D293A / Y387D ZPI, BMS -262084, and asundexian ( 0-4 pM) on APTT and PT of PNP. * * *p< 0. 001, wi ld-type or D293A or BMS -262084 vs. asundexian of the same concentrations. FIG. 4B, Dose -dependent effects of BMS -262084 and asundexian ( 0-60 pM) on APTT and PT of PNP. * -* *p values were shown as indicated, * * *p< 0. 001, the APTT of BMS -262084 vs. Asundexian of the same concentrations;AAAp< 0. 001, the PT of BMS -262084 or asundexian vs. no BMS -262084 or asundexian added. FIG. 4C, Effects of wi ld-type ZPI or D293A ZPI (2 pM) in combination with varying concentrations of PZ ( 0 -0. 9 pM) on APTT of PNP in comparison with the ef fect of PZ alone ( 0. 2 - 0. 9 pM). * * *p< 0. 001, WT+PZ vs. D293A+PZ. FIG. 4D, Comparative ef fects of wi ld-type ZPI (2 pM) and D293A ZPI (2 pM) in the absence and presence of PZ ( 1. 4 pM) on PT of PNP. PZ ( 1. 4 pM) alone was also tested as a control. * * *p<0. 001, WT+PZ vs. D293A+PZ.

[0014] FIG. 5 shows that wi ld- type ZPI and D293A ZPI inhibit FeCl3- induced mouse carotid artery thrombosis. Carot id arteryUIC0116WO PATENT thrombosis was induced using 7. 5% FeCl3in mice retro-orbitally inj ected with the same volume of control buf fer ( 5 mM sodium phosphate pH 6. 5 containing 0. 16M NaCl, 1 mg / ml mouse albumin, n=11, male 6, female 5), wi ld- type ZPI (n=9, male 5, female 4 ), D293A ZPI (n=8, male 4, female 4 ), and D293A / Y387D ZPI (n=7, male 4, female 3 ) ( 7. 5 mg / Kg). Time to stable occlusion was then monitored as described in Example 1. p values were shown as indicated. D ' Agostino-Pearson normality test was performed before one -way ANOVA testing with Bonferroni correction for multiple testing. The median of each is shown.

[0015] FIG. 6 shows that ZPIs inhibit inf erior vena cava ( IVC) thrombosis induced by 5% FeCl3. Comparison thrombus weights of mice treated with buf f er ( 5mM sodium phosphate pH 6. 5 containing 0. 16 M NaCl, 1 mg / ml mouse albumin, n=9, male 5, female 4 ), 7. 5 mg / kg wi ld- type ZPI (n=8, male 5, female 3 ) and D293A ZPI (n=8, male 5, female 3 ) and 15 mg / kg wild-type ZPI (n=8, male 4, female 4 ) and D293A ZPI (n=8, male 4 f emale 4 ). Buf fer or ZPIs were inj ected retro-orbital ly right before FeCl3application. Thrombus was dissected from IVC and weighted as described in Example 1. p values were shown as indicated. D ' Agostino-Pearson normality test was performed before one -way ANOVA test ing with Bonferroni correct ion for mult iple testing. The median of each is shown.

[0016] FIGS. 7A- 7B show the ef fects of ZPIs on tai l bleeding time and on hemostasis in a saphenous vein model. FIG. 7A, Tai l bleeding times of mice treated with control buff er ( 5mM sodium phosphate pH 6. 5, containing 0. 16M NaCl, 1 mg / mL mouse albumin, n=9, male 6, female 3 ), 7. 5 mg / kg wi ld-type ZPI (n=12, male 7, female 5 ), 7. 5 mg / kg (n=10, male 5, female 5 ) and 15 mg / kg (n=8, male 5, female 3 ) D293A ZPI, and 7. 5 mg / kg D293A / Y387D ZPI (n=9, male 4, female 5 ). FIG. 7B, Ef fects on hemostasis ( indicated by the number of times of formation / ref ormat ion of hemostatic clot is achieved in 20 minutes ) in saphenous vein model treated with buff er (n=10, male 6, female 4 ), 7. 5 mg / kgUIC0116WO PATENT wild-type ZPI (n=11, male 5, female 6), 7. 5 mg / kg (n=10, male 5, female 5) and 15 mg / kg D293A ZPI (n=10, male 4, female 6), and 7. 5 mg / kg D293A / Y387D ZPI (n=8, male 5, female 3 ). See Example 1 for details, p values were shown as indicated. D' Agostino-Pearson normality test was performed before one-way ANOVA testing with Bonferroni correction for multiple testing. The median of each group is shown.

[0017] FIGS. 8A-8C shows that PEGylated ZPI significantly improves pharmacokinetics and effectively decreased thrombosis in vivo. FIG. 8A, D293A ZPI, and PEGylated D293A ZPI similarly prolong APTT but not PT. FIG. 8B, PEGylated D293A ZPI has a significantly prolonged half -life in C57BL / 6 mice circulation than D293A ZPI after the bolus i.v. injections of the same doses of ZPI molecules (7.5 mg / kg ) (n=3). FIG. 8C, PEGylated D293A ZPI inhibited FeCl3(5%) induced IVC thrombosis in a manner similar to that of D293A ZPI, and both were significantly better than the buffer control (5 mM NaH2PO4pH 7.0, 0.2 M NaCl) (n=4, *p<0. 05, **p<0. 01; ns, not signif icant; one-way ANOVA test).DETAILED DESCRIPTION OF THE INVENTION

[0018] This invention is based, in part, on the finding that a modified ZPI including a D293A and / or Y240A mutation exhibits reduced affinity for PZ, diminished FXa inhibitory activity, and selective inhibition of FXIa and the intrinsic coagulation pathway (FIG. 1). In addition, the anticoagulant function of the modif ied ZPI was analyzed in vi tro and in vivo and it was shown that the modified ZPI selectively inhibits the intrinsic coagulation pathway and the ability of the intrinsic pathway to augment thrombin generation induced by low dose tissue factor (TF), one of the mechanisms by which the intrinsic pathway participates in thrombosis. Furthermore, the modified ZPI was shown to be effective in inhibiting FeCl3-induced arterial and venous thrombosis in vivo. Importantly, theUIC0116WO PATENT modified ZPI had a minimal effect on tail bleeding time. In the saphenous vein hemostasis analysis, which is known to be impacted by FXI deficiency, the modified ZPI, at the concentrations that effectively inhibited thrombosis, showed minimal effect on hemostasis in contrast to the same concentration of wild-type ZPI, and only showed an impact at a much higher concentration, demonstrating safety at a therapeutic concentration. Thus, PZ- independent inhibition of FXIa using the modif ied ZPI described herein provides a novel approach for inhibiting thrombosis without exacerbating bleeding. Accordingly, provided herein is a modified ZPI and methods of using the same to inhibit or reduce the risk of thrombosis without compromising hemostasis.

[0019] As used herein, " ZPI" or "protein Z-dependent protease inhibitor" refers to a 423 amino acid protein that is a natural plasma anticoagulant serpin. Wild-type or natural ZPI inhibits both activated factor X (FXa) and activated factor XI (FXIa). The amino acid sequence of a wild-type or natural human ZPI may be found under GENBANK Accession No. NP_001094077. 1 or SEQ ID NO: 1.LAPSPQSPET PAPQNQTSRV VQAPKEEEED EQEASEEKAS EEEKAWLMAS RQQLAKETSN FGFSLLRKIS MRHDGNMVFS PFGMSLAMTG LMLGATGPTE TQIKRGLHLQ ALKPTKPGLL PSLFKGLRET LSRNLELGLT QGSFAFIHKD FDVKETFFNL SKRYFDTECV PMNFRNASQA KRLMNHYINK ETRGKIPKLF DEINPETKLI LVDYILFKGK WLTPFDPVFT EVDTFHLDKY KTIKVPMMYG AGKFASTFDK NFRCHVLKLP YQGNATMLW LMEKMGDHLA LEDYLTTDLV ETWLRNMKTR NMEVFFPKFK LDQKYEMHEL LRQMGIRRIF SPFADLSELS ATGRNLQVSR VLQRTVIEVD ERGTEAVAGI LSEITAYSMP PVIKVDRPFH FMIYEETSGM LLFLGRVVNP TLL (SEQ ID NO: 1)

[0020] In some aspects, a ZPI of the disclosure comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1, e. g., at least about 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1.

[0021] As used herein, "sequence identity" refers to the extent to which two optimally aligned polynucleotide or polypeptide sequences are invariant throughout a window of alignment of components, e.g., nucleotides or amino acids. " Identity" canUIC0116WO PATENT be readily calculated by known methods including, but not limited to, those described in: Computational Molecular Biology (Lesk, A. M., ed. ) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed. ) Academic Press, New York (1993 ); Computer Analysis of Sequence Data, Part I (Griff in, A. M., and Griffin, H. G., eds. ) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinj e, G., ed. ) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds. ) Stockton Press, New York (1991).

[0022] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence (s) relative to the reference sequence, based on the designated program parameters.

[0023] Optimal alignment of sequences for aligning a comparison window are well known to those skilled in the art and may be conducted by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman, and optionally by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA available as part of the GCG® Wisconsin Package® (Accelrys Inc., San Diego, CA). An "identity fraction" for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in the reference sequence segment, e. g., the entire reference sequence or a smaller defined part of the reference sequence. Percent sequence identity is represented as the identityUIC0116WO PATENT fraction multiplied by 100. The comparison of one or more polynucleotide sequences may be to a full - length polynucleotide sequence or a port ion thereof, or to a longer polynucleotide sequence. For purposes of thi s invent ion "percent identity" may also be determined using BLASTX vers ion 2. 0 for translated nucleotide sequences and BLASTN vers ion 2. 0 for polynucleot ide sequences.

[0024] ZPI sequences having at least 70% sequence identity to SEQ ID NO: 1 include, but are not limited to bat ZPI ( e. g., GENBANK Accession No. XP_019601411. 2; 74. 61% sequence identity), beaver ZPI ( e. g., GENBANK Access ion No. XP_020040964. 1; 75. 45% sequence identity), mouse ZPI ( e. g., GENBANK Accession No. NP_659083. 2; 72. 25% sequence ident ity), rat ZPI ( e. g., GENBANK Accession No. NP_598301.2; 74. 14 % sequence identity), baboon ZPI ( e. g., GENBANK Accession No. XP_003902259. 2; 92. 40% sequence identity), guinea pig ZPI ( e. g., GENBANK Access ion No. XP_012999838. 1; 71. 79% sequence identity), chimpanzee ZPI ( e. g., GENBANK Access ion No. XP_054522243. 2; 98. 58 % sequence identity).

[0025] A "modi f ied ZPI" refers to a ZPI of that has been selectively mutated to include one more amino acid substitutions thereby di f ferentiating the modif ied ZPI from a wi ld-type or natural ZPI. In some aspects, a modif ied ZPI includes an amino acid substitution at position 293 and / or position 240 with reference to SEQ ID NO: 1. In some aspects, a modif ied ZPI includes an amino acid subst itut ion at Asp293 and / or Tyr240 with reference to SEQ ID NO: 1, or an amino acid substitution at an Asp at a posit ion analogous to Asp293 and / or at a Tyr at a position analogous to Tyr240 in a ZPI having at least 70% sequence identity to SEQ ID NO: 1 when the proteins are optimally aligned. For example, optimal alignment of human ZPI ( SEQ ID NO: 1 ) with ZPI from mouse (GENBANK Accession No. NP_659083. 2 ), rat (GENBANK Accession No. NP_598301. 2 ), chimpanzee (GENBANK Accession No. XP_054522243. 2 ), and baboonUIC0116WO PATENT (GENBANK Accession No. XP_003902259. 2 ) shows that Asp293 and Tyr240 are conserved in these sequences (Table 1).TABLE 1Region flanking Tyr240 or Asp293 with SEQ ID Organism reference to SEQ ID NO: 1 NO Human DPVFTEVDTFHLDKY240KTIKVPMMYGAGKF 6 Baboon DPVFTEADTFHLDKY240KTIKVPMMYGAGKF 7 Chimpanzee DPVFTEVDTFHLDKY301KTIKVPMMYGAGKF 8 Mouse DPSFTEADTFHLDKY265RAIKVPMMYREGNF 9 Rat DPIFTEADTFHLDKY253KAVKVPMMYREGNF 10 ** ::** ***:**** : :****** *:*Human VVLMEKMGDHLALED293YLTTDLVETWLRNM 11 Baboon VVLMEKMGDHLTLED313YLTTDLVETWLRNM 12 Chimpanzee VVLMEKMGDHLALED354YLTTDLVETWLRNM 13 Mouse VVLMEKTGDYLALED318YLTVDLVETWLQNM 14 Rat VVLMEKSGDHLALED306YLTTDL VEMWLQDM 15 ****:*:**:*:*** ***:***: **::*

[0026] In some aspects, a modified ZPI is a ZPI of SEQ ID NO: 1 that includes an Asp to Ala subsubstituion at position 293 (Asp293Ala) and / or Tyr to Ala subsubstituion at position 240 (Tyr240Ala). In some aspects, a modified ZPI includes an Asp293Ala and / or Tyr240Ala amino acid substituion at an Asp at a position analogous to Asp293 and / or at a Tyr at a position analogous to Tyr240 in a ZPI having at least 70% sequence identity to SEQ ID NO: 1 when the proteins are optimally aligned.

[0027] In some aspects, a modified ZPI comprises, consists essentially of or consists of an amino acid sequence of any one of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.LAPSPQSPET PAPQNQTSRV VQAPKEEEED EQEASEEKAS EEEKAWLMAS RQQLAKETSN FGFSLLRKIS MRHDGNMVFS PFGMSLAMTG LMLGATGPTE TQIKRGLHLQ ALKPTKPGLL PSLFKGLRET LSRNLELGLT QGSFAFIHKD FDVKETFFNL SKRYFDTECV PMNFRNASQA KRLMNHYINK ETRGKIPKLF DEINPETKLI LVDYILFKGK WLTPFDPVFT EVDTFHLDKY KTIKVPMMYG AGKFASTFDK NFRCHVLKLP YQGNATMLW LMEKMGDHLA LEAYLTTDLV ETWLRNMKTR NMEVFFPKFK LDQKYEMHEL LRQMGIRRIF SPFADLSELS ATGRNLQVSR VLQRTVIEVD ERGTEAVAGI LSEITAYSMP PVIKVDRPFH FMIYEETSGM LLFLGRVVNP TLL (SEQ ID NO: 2 )LAPSPQSPET PAPQNQTSRV VQAPKEEEED EQEASEEKAS EEEKAWLMAS RQQLAKETSN FGFSLLRKIS MRHDGNMVFS PFGMSLAMTG LMLGATGPTE TQIKRGLHLQ ALKPTKPGLL PSLFKGLRET LSRNLELGLT QGSFAFIHKD FDVKETFFNL SKRYFDTECV PMNFRNASQA KRLMNHYINK ETRGKIPKLF DEINPETKLI LVDYILFKGK WLTPFDPVFT EVDTFHLDKA KTIKVPMMYG AGKFASTFDK NFRCHVLKLP YQGNATMLW LMEKMGDHLA LEAYLTTDLV ETWLRNMKTR NMEVFFPKFK LDQKYEMHEL LRQMGIRRIF SPFADLSELS ATGRNLQVSRUIC0116WO PATENT VLQRTVIEVD ERGTEAVAGI LSEITAYSMP PVIKVDRPFH FMIYEETSGM LLFLGRVVNP TLL ( SEQ ID NO: 3 )LAPSPQSPET PAPQNQTSRV VQAPKEEEED EQEASEEKAS EEEKAWLMAS RQQLAKETSN FGFSLLRKIS MRHDGNMVFS PFGMSLAMTG LMLGATGPTE TQIKRGLHLQ ALKPTKPGLL PSLFKGLRET LSRNLELGLT QGSFAFIHKD FDVKETFFNL SKRYFDTECV PMNFRNASQA KRLMNHYINK ETRGKI PKLF DEINPETKLI LVDYILFKGK WLTPFDPVFT EVDTFHLDKA KTIKVPMMYG AGKFASTFDK NFRCHVLKLP YQGNATMLW LMEKMGDHLA LEAYLTTDLV ETWLRNMKTR NMEVFFPKFK LDQKYEMHEL LRQMGIRRIF SPFADLSELS ATGRNLQVSR VLQRTVIEVD ERGTEAVAGI LSEITAYSMP PVIKVDRPFH FMIYEETSGM LLFLGRVVNP TLL ( SEQ ID NO: 4 )

[0028] A modi f ied ZPI of the disclosure may be composed of the amino acid residues described herein or may be truncated to remove one or more N- terminal and / or C-terminal amino acid residues. In some aspects, a modi f ied ZPI may have about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or 50 amino acid residues removed f rom the N-terminus of a sequence described herein and / or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or 50 amino acid residues removed from the C- terminus of a sequence described herein.

[0029] In some aspects, a modi f ied ZPI of the disclosure may be composed solely of the amino acid residues described herein. In other aspects, one or more amino acid residues of a modif ied ZPI of the disclosure may be chemically modi f ied. Any amino acid modif ication known in the art may be made to the amino acids of a modif ied ZPI using any method known in the art. For example, a modif ied ZPI of the disclosure may be glycosylated, phosphorylated, sulfateld, amidated, carboxylated, or acetylated. For example, the C-terminus may be modif ied with amidation, addition of peptide alcohols and aldehydes, addit ion of esters, addition of p-nitroaniline and thioesters. The N-terminal and / or s ide chains may be modif ied by PEGylation, acetylation, formylation, addition of a fatty acid, addition of benzoyl, addition of bromoacetyl, addition of pyroglutamyl, succinylation, addition of tetrabutyoxycarbonyl and addit ion of 3 -mercaptopropyl, acylations ( e. g., lipopeptides ), biotinylation, phosphorylation, sulfation, glycosylat ion, introduction of a maleimido group, chelating moiety,UIC0116WO PATENT chromophore or fluorophore. In some aspects, a modified ZPI of the disclosure is glycosylated.

[0030] In some aspects, a modified ZPI comprises at least one polyethylene glycol (PEG) group conjugated thereto. In some aspects, the invention provides a modified protein ZPI comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1; an amino acid substitution at position 293 and / or position 240 with reference to SEQ ID NO: 1; and at least PEG group conjugated to the modified ZPI. In some aspects, a modified ZPI comprises at least one PEG group conjugated to a cysteine residue of the modif ied ZPI. In some aspects, a modified ZPI comprises at least one PEG group conjugated to Cysl69 and / or Cys264 with reference to SEQ ID NO: 1.

[0031] A modified ZPI may be conjugated to a fatty acid, e. g., the modified ZPI may be myristoylated. For example, a fatty acid may be conjugated to the N-terminus of a modified ZPI, such fatty acids include caprylic acid (C8), capric acid (CIO), lauric acid (C12), myristic acid (C14 ), palmitic acid (C16), or stearic acid (C18 ) etc. Furthermore, a cysteine residue of a modified ZPI may be palmitoylated.

[0032] A modified ZPI may be conjugated or linked to another peptide or polypeptide, such as a carrier peptide. The carrier peptide may facilitate cell-penetration, such as antennapedia peptide, penetratin peptide, TAT, tranportan or polyarginine. A modified ZPI may be labeled with heavy isotope, e. g.,15N or13C, labeled with a dye such as FITC, conjugated to an imaging agent, FRET substrate with a fluorophore / quencher pair, DNA, or RNA.

[0033] A modified ZPI may be a component of a fusion protein such as fused to a polypeptide or peptide that promotes oligomerization, such as a leucine zipper domain; a polypeptide or peptide which increases stability or to increase half -life, such as an immunoglobulin constant region; or a polypeptide which has a therapeutic activity different from the modif iedUIC0116WO PATENT ZPI of the invention, a chemotherapeutic agent, an antibody or protein for tissue-specific targeting. Fusions may be made either at the N-terminus or at the C-terminus of modif ied ZPI. The fusion proteins may be direct with no linker or adapter molecule or indirect using a linker or adapter molecule. A linker or adapter molecule may be one or more amino acid residues, typically up to about 20 to about 50 amino acid residues. A linker or adapter molecule may also be designed with a cleavage site for a protease to allow for the separation of the fused moieties. For example, a modified ZPI may be fused to one or more domains of an Fc region of human IgG to increase the half -life of the modified ZPI or the addition of a Fab variable domain to shorten the half -life of the modified ZPI.

[0034] In some aspects, a modified ZPI of the disclosure may be synthesized recombinantly using recombinant DNA techniques. Thus, in another aspect, the invention provides polynucleotides that encode the modified ZPI of the invention. In a related aspect, the invention provides vectors, particularly expression vectors that comprise a polynucleotide encoding the modified ZPI of the invention. In some aspects, the vector provides replication, transcription, and / or translation regulatory sequences that facilitate recombinant synthesis of the modified ZPI in a eukaryotic cell or prokaryotic cell. Accordingly, the invention also provides host cells for recombinant expression of the modified ZPI and methods of harvesting and purifying the modified ZPI produced by the host cells. In some aspects, a host cell for producing a recombinant modified ZPI is a bacterial cell (e. g., E. coli). In some aspects, a host cell for producing a recombinant modified ZPI is an insect cell. In some aspects, a host cell for producing a recombinant modified ZPI is a mammalian cell, e. g., a human cell line capable of glycosylating the modified ZPI. Production and purif ication of a recombinant polypeptide is routinely practiced in the art. A modified ZPI may be purified by any suitable method known inUIC0116WO PATENT the art including without l imitation gel f i ltrat ion and af f inity purif ication. When a modif ied ZPI of the invention i s produced in the form of a fus ion protein, the fus ion moiety ( e. g., an epitope tag) may optionally be cleaved of f us ing a protease before further analysis.

[0035] Alternatively, a modif ied ZPI of the invent ion may be advantageously synthesized by any of the chemical synthesi s techniques known in the art, particularly solid-phase synthesis techniques, for example, using commercially avai lable automated peptide synthesizers. See, for example, Stewart & Young ( 1984 ) Solid Phase Peptide Synthesis, 2nded.. Pierce Chemical Co.; Tam et al. ( 1983 ) J. Am. Chem. Soc. 105: 6442; Merrif ield ( 1986 ) Science 232: 341 -347; Barany et al. ( 1987) Int. J. Peptide Protein Res. 30: 705 - 739; and U. S. Patent No. 5, 424, 398.

[0036] A modif ied ZPI of the disclosure may be administered to any subj ect in which inhibit ion of venous or arterial thrombosis or ri sk of developing a venous or arterial thrombosis would be benef icial. For example, it is contemplated that a modif ied ZPI of the disclosure is particularly useful as an antithrombotic agent that does not compromise hemostas is, and as such is a superior alternative to traditional antithrombotic agents. For example, in the coronary arteries, occlusive thrombus formation often fol lows the rupture of atherosclerotic plaque. This occlusion is the major cause of acute myocardial infarction and unstable angina. Coronary occlusions can also occur following inf ect ions, inflammation, thrombolytic therapy, angioplasty, and graft placements. Similar principles apply to other parts of the arterial vasculature and include, among others, thrombus formation in the carotid arteries, which is the major cause of transient or permanent cerebral ischemia and stroke.

[0037] Venous thrombosis often follows stasis, inf ections, inf lammatory reactions, and major surgery of the lower extremities or the abdominal area. Deep vein thrombosis resultsUIC0116WO PATENT in reduced blood flow from the area distal to the thrombus and predisposes to pulmonary embolism. Pulmonary embolism is a major cause of post-surgical mortality. Disseminated intravascular coagulation (DIC) and acute respiratory distress syndrome (ARDS) where the nonpolymer izable fibrinogen is useful commonly occur within all vascular systems during bacterial sepsis, entry of foreign material into the blood stream following, e. g., trauma and child birth, immune reactions, inflammation, certain viral infections, certain platelet disorders, and cancer. Disseminated intravascular coagulation is a severe complication of many disease conditions and some drug treatments, including, for example, heparin. Thrombotic consumption of coagulation factors and platelets, and systemic coagulation results in the formation of life-threatening thrombi occurring throughout the microvasculature leading to local or widespread hypoxia and organ failure.

[0038] Thus, in some aspects, a method is provided for inhibiting or reducing the risk of developing thrombosis in a subj ect in need thereof (e. g., a subj ect at increased risk of developing thrombosis) comprising administering to the subject an effective anticoagulant amount of a modif ied ZPI comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1 and an amino acid substitution at position 293 and / or position 240 with reference to SEQ ID NO: 1. In some aspects, the thrombosis is associated with, e. g., follows or is the cause of: ( 1) acute coronary syndromes such as myocardial infarction, unstable angina, refractory angina, occlusive coronary thrombus occurring post -thrombolytic therapy or post-coronary angioplasty; (2) ischemic cerebrovascular syndromes including embolic stroke, thrombotic stroke, or transient ischemic attacks; (3 ) thrombosis occurring in the venous system occurring either spontaneously or in the setting of malignancy, trauma, or surgery, including pulmonary thromboembolism; (4) any coagulopathy including ARDS and DIC, e. g., in the settingUIC0116WO PATENT of sepsis or other infection, surgery, pregnancy, trauma, or malignancy and whether associated with multi -organ failure or not, thrombot ic thrombocytopenic purpura, thromboangi itis obliterans, or thrombot ic disease associated with heparin-induced thrombocytopenia; ( 5 ) thrombot ic compl ications associated with extracorporeal circulation ( e. g., renal dialysis, cardiopulmonary bypass or other oxygenation procedure such as ECMO treatment, and plasmaphoresis ); ( 6 ) thrombotic complications associated with instrumentation ( e. g., cardiac or other intravascular catheteri zation, intraaortic balloon pump, and coronary stent or cardiac valve); and / or ( 7 ) complications associated with f itting of prosthet ic devices. In some aspects, the venous or arterial thrombos is is associated with myocardial infarction, unstable angina, atrial f ibri llation, stroke, renal damage, pulmonary embolism, deep vein thrombosis, percutaneous translumenal coronary angioplasty, disseminated intravascular coagulation, seps is, artif icial organs, shunts, and / or prostheses. In other aspects, the subj ect i s receiving ECMO treatment and / or surgery. In sti ll other aspects, the subj ect has a condition associated with increased ri sk of thrombosis. In some aspects, the thrombosis is a large vascular thrombos is, a smal l vascular thrombosis, or a microvascular thrombosis.

[0039] In accordance with the methods herein, a modif ied ZPI, e. g., isolated and optionally purif ied, may be admini stered to a subj ect in need of treatment. As used herein, "administer, " "administration, " and "administering" ref ers to the introduction or delivery of an agent to a subj ect. Administration may be carried out by any suitable route, including oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra- j oint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intrales ional, intranasal, rectal, vaginal, by inhalation, via an implanted reservoir,UIC0116WO PATENT parenteral ( e. g., subcutaneous, intravenous, intramuscular, intra-articular, intra- synovial, intrasternal, intrathecal, intraperitoneal, intrahepat ic, intralesional, and intracranial injections or infus ion techniques ), and the like. " Concurrent administration, " "administrat ion in combination, " "simultaneous administration, " or "administered simultaneously" as used herein, means that a combinat ion of agents ( e. g., a modif ied ZPI and a second therapeutic) may be administered at the same point in time, overlapping in t ime, or one following the other. In the latter case, the two or more agents may be administered at times suf f iciently close that the results observed are indistinguishable from those achieved when the agents are administered at the same point in time. " Systemic administration" refers to the introducing or delivering to a subj ect an agent via a route which introduces or delivers the agent to extensive areas of the subj ect ' s body ( e. g., greater than 50% of the body), for example through entrance into the circulatory or lymph systems. By contrast, " local administration" refers to the introducing or delivery to a subj ect an agent via a route which introduces or delivers the agent to the area or area immediately adj acent to the point of administration and does not introduce the agent systemically in a therapeutically signif icant amount. For example, locally administered agents are easily detectable in the local vicinity of the point of administration but are undetectable or detectable at negligible amounts in distal parts of the subj ect ' s body. Administrat ion includes self -admini strat ion and the administration by another.

[0040] A "subj ect" herein includes mammals, avians, repti les, amphibians, and f ish. Mammalian subj ects include but are not limited to humans, non-human mammals, non-human primates (e. g., monkeys, chimpanzees, baboons, etc. ), dogs, cats, mice, hamsters, rats, horses, cows, pigs, rabbits, sheep, and goats. In some embodiments, a subj ect is a laboratory animal. HumanUIC0116WO PATENT subj ects include neonates, infants, juveniles, adults, and geriatric subj ects. In some aspects, a subj ect in need of treatment may include those at risk of thrombosis or developing thrombi. A subj ect having an increased risk of thrombosis may include a patient receiving transplanted cells, tissues or organs including hematopoietic transplants, bone marrow transplants, kidney, heart, liver, lung and the like as well as patients receiving certain therapies such as chemotherapy or radiation thereby, surgery or extracorporeal membrane oxygenation (ECMO) treatment.

[0041] " Thrombosis" refers to the formation of a thrombus, meaning a blood clot comprising platelets, fibrin, leukocytes, and red blood cells located within a vascular lumen (. Rubin 's Pathology, Raphael Rubin and David S. Strayer, ed., 5th Ed., Lippincott Williams & Wilkins: 2008, page 233 ). The term encompasses arterial and venous thrombosis, including deep vein thrombosis, portal vein thrombosis, jugular vein thrombosis, renal vein thrombosis, stroke, myocardial infarction, Budd-Chiari syndrome, Paget -Schroetter disease, and cerebral venous sinus thrombosis. A thrombus is distinct from a typical blood clot. While a blood clot results from activation of the coagulation cascade, a thrombus also involves adherence and aggregation of platelets, participation of cellular elements of the immune system, and active participation of endothelial cells of the blood vessel.

[0042] Before injury to a blood vessel, circulating platelets are in a nonadherent state. Injury activates platelet adhesiveness, after which platelets bind to one another to form an aggregate of activated platelets (platelet thrombus). These platelet aggregates occlude injured small vessels and prevent leakage of blood. Once platelets are stimulated to adhere to the vessel wall, their granular contents are released, in part by contraction of the platelet cytoskeleton. In turn, these granules promote aggregation of other platelets. PlateletUIC0116WO PATENT adhesion is enhanced by release of subendothelial von Willebrand factor, which is adhesive for Gplb platelet membrane protein and for fibrinogen. Activated platelets also release ADP and thromboxane A2, which recruit additional platelets to the process. The platelet membrane protein complex GpIIb- IIIc binds to fibrinogen, thereby forming fibrinogen bridges between platelets, enhancing aggregation, and stabilizing the nascent thrombus. Activated platelets in turn release factors that initiate coagulation, thus forming a complex thrombus on the vessel wall. Thrombin itself stimulates further release of platelet granules and subsequent recruitment of new platelets.

[0043] As used herein, "venous thrombosis" refers to a thrombus within a vein. The pathogenesis of venous thrombosis is inherently tied to fibrin matrix formation. Studies have suggested that the configuration of the fibrin matrix itself is a major determinant of clot formation, stability, and ultimately venous thrombosis (Machlus et al. (2011 ) Blood 117 ( 18 ): 4953 -4963; Aleman et al. (2014 ) J. Clin. Invest.124 ( 8 ): 3590 -3600; Byrnes et al. (2015 ) Blood 126 ( 16 ): 1940 - 1948; Aleman et al. (2013 ) Arterioscler. Thromb. Vase. Biol.33 ( 8 ): 1829 - 1836 ). High thrombin and fibrinogen concentrations result in the formation of thin fibrin fibers in a highly dense matrix that promotes RBC retention and suppresses platelet / fibrin-mediated clot contraction, leading to delayed clearance (Aleman et al. (2014 ) J. Clin. Invest. 124 ( 8 ): 3590 -3600; Aleman et al. (2013 ) Arterioscler. Thromb. Vase. Biol.33 ( 8 ): 1829 - 1836; Wolberg et al. (2010 ) Thromb. Res. 125 ( Suppl 1 ): S35 - 37 ). FXIIIa crosslinks fibrin α-chains (Byrnes et al. (2015 ) Blood 126 ( 16 ): 1940 - 1948 ) to establish this dense matrix composed of more elastic fibrin fibers that promote RBC retention and subsequently larger thrombi.

[0044] Risk f actors for venous thrombosis include, without limitation, surgery; trauma; immobili zation; previous thrombosis; cancer; pregnancy; antiphospholipid antibodiesUIC0116WO PATENT syndrome; medical conditions such as atherosclerosis, heart failure, hypertension, dyslipidemia, chronic kidney disease, renal transplant, nephrotic syndrome, microalbuminuria, polycythemia vera, paroxysmal nocturnal hemoglobinuria, hyperhomocysteinemia, Behcet disease, rheumatoid arthritis, systemic lupus erythematosus, antineutrophil cytoplasmic antibodies -associated vasculitis, inf lammatory bowel disease, sepsis, coronavirus disease 2019, tuberculosis, asthma, obstructive sleep apnea, polycystic ovary syndrome, diabetes mellitus; and an inherited hypercoagulable disorder (Factor V Leiden mutation, prothrombin gene mutation, protein C deficiency, protein S def iciency, antithrombin def iciency, dysfibrinogenemia, factor XII deficiency, or hyperhomocysteinemia).

[0045] As used herein, "arterial thrombosis" refers to a thrombus within an artery. Arterial thrombosis is mediated by platelets, which serve as the primary driver of thrombus growth (i. e., arterial thrombi are platelet-rich). The coronary, cerebral, mesenteric, and renal arteries, and arteries of the lower extremities, are the vessels most commonly involved in an arterial thrombosis due to atherosclerosis. Arterial thrombosis may also occur, however, as a result of other disorders, including inflammation of arteries (arteritis), trauma, and blood diseases. Thrombi are also common in aneurysms ( localized-dilations of the lumen) of the aorta and its major branches, in which the distortion of blood flow, combined with intrinsic vascular disease, promotes thrombosis.

[0046] Risk factors for thrombosis in the arterial system include, without limitation, immobilization after surgery or leg casting, obesity, advanced age, previous thrombosis, and cancer. The three factors that are commonly associated with development of thrombosis are: (1) damage to the endothelium, usually by atherosclerosis, which disturbs the anticoagulant properties of the vessel wall and serves as a site of originUIC0116WO PATENT for platelet aggregation and fibrin formation; (2 ) alteration in blood flow, whether from turbulence at the site of an aneurysm, sites of arterial bifurcation, or slowing of blood flow in narrowed arteries; and (3 ) increased coagulability of the blood. Since most arterial thrombi occlude the vessel in which they occur, they often lead to ischemic necrosis of tissue supplied by that artery, i. e., an infarct. Infarction is the process by which coagulative necrosis develops in an area distal to the occlusion of an end-artery. Thrombosis of a coronary or cerebral artery results in myocardial infarct (heart attack) or cerebral infarct (stroke), respectively.

[0047] " Deep vein thrombosis" refers to a thrombus that develops in a deep vein, usually in the lower leg. Deep venous thrombosis often results from one or more of the same causative factors that favor arterial and cardiac thrombosis. Those factors are endothelial injury (e. g., trauma, surgery, childbirth), stasis (e. g., heart failure, chronic venous insufficiency, post-operative immobilization, prolonged bed rest) and a hypercoagulable state (e. g., oral contraceptives, late pregnancy, cancer, inherited thrombophilic disorders, advanced age, venous varicosities, phlebosclerosis).

[0048] Diseases and conditions associated with thrombosis and the risk of developing thrombosis or hypercoagulation include, without limitation, acute venous thrombosis, pulmonary embolism, thrombosis during pregnancy, hemorrhagic skin necrosis, acute or chronic disseminated intravascular coagulation (DIC), clot formation from surgery, long bed rest, long periods of immobilization, conditions that preclude or restrict movement such as partial or complete paralysis, morbid obesity, disorders that impede oxygen uptake and absorption such as lung disorders including lung cancer, chronic obstructive pulmonary disease (COPD), emphysema, drug related fibrosis, cystic fibrosis, venous thrombosis, fulminant meningococcemia, acute thrombotic stroke, acute coronaryUIC0116WO PATENT occlusion, acute peripheral arterial occlusion, massive pulmonary embolism, axillary vein thrombosis, massive iliofemoral vein thrombosis, occluded arterial cannulae, occluded venous cannulae, cardiomyopathy, venoocclusive disease of the liver, hypotension, decreased cardiac output, decreased vascular resistance, pulmonary hypertension, diminished lung compliance, leukopenia, inflammatory diseases, metabolic syndrome, and thrombocytopenia.

[0049] The term "hemostasis" refers to a coordinated mechanism that maintains the integrity of blood circulation following injury to the vascular system. In normal circulation without vascular injury, platelets are not activated and freely circulate. Vascular injury exposes sub-endothelial tissue to which platelets can adhere. Adherent platelets will attract other circulating platelets to form a preliminary plug that is particularly useful in closing a leak in a capillary or other small vessel. These events are termed primary hemostasis. This is, typically, rapidly followed by secondary hemostasis that involves a cascade of linked enzymatic reactions that result in plasma coagulation to reinforce the primary platelet plug. Later, as wound healing occurs, the platelet aggregate and fibrin clots are degraded as wound healing ensues. In accordance with the present invention, hemostasis is not compromised when the function of one or more of platelet activation, protease-activated receptors, FXIII, protein C or thrombin activatable fibrinolysis inhibitor is retained. The term "not compromised, " as used herein with respect to hemostasis, refers to less than a 20% decrease ( e. g., less than about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% decrease) in platelet activation, protease-activated receptors, FXIII, protein C or thrombin activatable fibrinolysis inhibitor function or activity.UIC0116WO PATENT

[0050] The term "coagulation" ref ers to the process of polymerization of f ibrin monomers, resulting in the transformation of blood or plasma from a liquid to a gel phase. Coagulation involves a series of zymogen activation reactions. At each stage, a precursor protein or zymogen, is converted to an active protease by cleavage of one or more peptide bonds in the precursor molecule. The components that can be involved at each stage include a protease from the preceding stage, a zymogen, a non-enzymatic protein cofactor, calcium ions, and an organizing surface that is provided by the damaged blood vessel and platelets in vivo. The f inal protease to be generated is thrombin ( factor Ila).

[0051] In some aspects, administration of a modified ZPI decreases, reduces, or inhibits the incidence or severity of a thrombosis by at least 10%, e. g., by at least 10%, by at least 20%, at least 30%, at least 50%, at least 75 %, at least 100%, at least 200% or more as compared to the incidence or severity of thrombosis in a subject not administered the modif ied ZPI. In some aspects, administration of a modified ZPI provides at least about a 1% ( e. g., about 2 %, 3 %, 4 %, 5 %, 6 %, 7%, 8 %, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45 %, 50%, 55%, 60%, 65% or 70% ) reduction in number of thrombi and / or at least about a 1 mg ( e. g., about 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, or 20 mg) reduction in the mass of individual thrombi as compared to a subj ect with the same or similar condition not receiving treatment with the modif ied ZPI.

[0052] Therapeutic efficacy of a modif ied ZPI in inhibiting thrombosis or reducing the number and / or size of thrombi may be assessed by conventional functional and / or physiological measurements. Exemplary physiological parameters include blood pressure or flow (velocity) inside the vasculature. The pressure and flow measurements may be compared to a reference baseline. The reference baseline may be measurements associatedUIC0116WO PATENT with a normal, healthy population or a reference baseline specific to the individual subject to therapy (e.g., measured pressure / flow levels of the patient prior to the thrombosis). Other assessment indicators include, for example, endovascular echo changes or vascular wall thickness comparisons. For example, vascular wall thickness and intraluminal echo can be assessed by gray-scale sonography, while iliac, femoral vein blood flow and femoral vein valve insufficiency may be evaluated with Doppler ultrasound.

[0053] Assessment of hemostasis may be carried out using any standard template skin bleeding time test (e. g., Surgicutt®, International Technidye Corp). Experimentally, this and similar tests (e. g., Simplate bleeding times) have been shown to be sensitive to the effects of therapeutic anticoagulants, anti-platelet agents, and coagulation abnormalities in humans and non-human primates (Gruber et al., (2007) Blood 109: 3733 -3740; Smith et al. ( 1985) Am. J. Clin. Pathol. 83: 211-215; Payne et al. (2002) J. Vasc. Surg. 35: 1204-1209). For indirect assessment of hemostasis, prothrombin time (PT) and activated partial thromboplastin time (aPTT) measurements may be used.

[0054] As used herein, the term "amount effective, " "effective amount, " "therapeutically effective amount" or "an effective anticoagulant amount" refers to an amount of a modified ZPI of the disclosure or a pharmaceutical composition comprising the modified ZPI sufficient to achieve the stated desired result, for example, treating, inhibiting, or reducing the risk of developing a thrombosis, in particular a venous or arterial thrombosis, without compromising hemostasis. The amount of the modified ZPI which constitutes an "effective anticoagulant amount" or "therapeutically effective amount" may vary depending on the severity of the disease or condition, the condition, weight, or age of the subject to be treated, the frequency of dosing, and / or the route of administration, but may be determined routinely by one of ordinary skill in theUIC0116WO PATENT art. A clinician may titer the dosage or route of administration to obtain the optimal therapeutic effect. Typical dosages may range from about 0.1 μg / kg to up to about 100 mg / kg or more, depending on the factors mentioned above. In some aspects, the dosage may range from 0.1 μg / kg up to about 100 mg / kg, or 1 μg / kg up to about 100 mg / kg, or 5 μg / kg up to about 100 mg / kg. In some aspects, the dosage may range from 5 mg / kg up to about 50 mg / kg. It is recognized that the methods of this disclosure may comprise a single administration of an effective dose or multiple administrations of an effective dose of the modif ied ZPI.

[0055] In some aspects, a modified ZPI of the disclosure is provided in a pharmaceutical composition. In some aspects, the pharmaceutical composition may be specially formulated for intravenous injection. Optimal pharmaceutical compositions may be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format, and desired dosage. See, for example, Remington's Pharmaceutical Sciences, latest edition.

[0056] A modified ZPI of the disclosure may be incorporated in a conventional systemic dosage form, such as an injectable formulation, tablet, capsule, soft gelatin capsule, or elixir. The dosage forms may also include the necessary physiologically acceptable carrier, diluent, or excipient, including, e.g., lubricant, buffer, surfactant, antibacterial, bulking agent (such as mannitol ), antioxidant (ascorbic acid or sodium bisulfite) and the like.

[0057] Acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed. The pharmaceutical composition may contain formulation materials for modifying, maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of theUIC0116WO PATENT composition. Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogensulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA) ); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions ( such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20 and polysorbate 80, Triton, trimethamine, lecithin, cholesterol, or tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents ( such as alkali metal halides, preferably sodium or potassium chloride, mannitol, or sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants. See, for example, Remington's Pharmaceutical Sciences, Id.

[0058] The primary excipient, vehicle, or carrier in a pharmaceutical composition may be either aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier may be water for injection, physiological saline solution, orUIC0116WO PATENT artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. Pharmaceutical compositions may comprise Tris buffer of about pH 7. 0 -8. 5, or acetate buffer of about pH 4. 0-5. 5, which may further include sorbitol or a suitable substitute thereof. Pharmaceutical compositions may be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (Remington's Pharmaceutical Sciences, Id.) in the form of a lyophilized cake or an aqueous solution. Further, a modified ZPI of the disclosure may be formulated as a lyophilizate using appropriate excipients such as sucrose.

[0059] Administration routes for the pharmaceutical compositions herein include orally, through injection by intravenous, intraperitoneal, intracerebral ( intra-parenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional routes; by sustained release systems or by implantation devices. A pharmaceutical composition may be administered by bolus injection or continuously by infusion, or by implantation device. A pharmaceutical composition also may be administered locally via implantation of a membrane, sponge, or another appropriate material onto which the desired molecule has been absorbed or encapsulated. Where an implantation device is used, the device may be implanted into any suitable tissue or organ, and delivery of the desired molecule may be via diffusion, timed-release bolus, or continuous administration. Other routes of administration include epidural or intrathecal administration. In some aspects, topical administration may be performed by applying a modified ZPI-containing dressing and / or catheter to the thrombus area. In some aspects, a modified ZPI may be administered before, during, and / or after other therapies. For example, when used in combination with surgery,UIC0116WO PATENT a modified ZPI may be administered before surgery (e. g., 1, 2, 3, 4, 5, or 6 hours) and / or after surgery (e. g., 1, 2, 3, 4, 5, 6, or 7 days, weeks or months).

[0060] A pharmaceutical composition herein may be delivered parenterally. When parenteral administration is contemplated, a composition for use herein may be in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising the desired modified ZPI in a pharmaceutically acceptable excipient. A particularly suitable vehicle for parenteral injection is sterile distilled water in which a polypeptide is formulated as a sterile, isotonic solution, appropriately preserved. Preparation may involve the formulation of the desired modified ZPI with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads or liposomes, that may provide controlled or sustained release of the modified ZPI which may then be delivered via a depot injection. Formulation with hyaluronic acid has the effect of promoting sustained duration in the circulation. Implantable drug delivery devices may be used to introduce the desired modified ZPI. In some aspects, the invention provides a liposome encapsulating a modified ZPI of the disclosure.

[0061] In some aspects, a modified ZPI of the methods of the disclosure may be administered in combination with a second therapeutic agent. In some aspects, a modified ZPI may be administered in combination with an antithrombotic agent and / or anticoagulant agent. Any such conventional agent may be used including, but not limited to, a direct or indirect thrombin inhibitor, a Factor X inhibitor, a Factor IX inhibitor, a Factor XII inhibitor, a Factor V inhibitor, a Factor VIII inhibitor, a Factor XIII inhibitor, a Factor VII inhibitor, a tissue factor inhibitor, a profibrinolytic agent, a fibrinolytic or fibrinogenolytic agent, a carboxypeptidase B inhibitor, a platelet inhibitor, a selective platelet count reducing agent,UIC0116WO PATENT or a Factor XI inhibitor. Direct thrombin inhibitors include, e. g., argatroban and derivatives or analogs thereof, hirudin and recombinant or synthetic derivatives or analogs thereof, derivatives of the tripeptide Phe - Pro-Arg, chloromethylketone derivatives, lepirudin, bivalirudin, dabigatran, ximelagatran and derivatives, metabolites, or analogs thereof, anion binding exosite inhibitors, and RNA / DNA aptamers. Indirect thrombin inhibitors include, e. g., heparin, enoxaparin, warfarin and other coumarin derivatives, dermatan, and thrombomodulin.

[0062] In aspects pertaining to a combination therapy comprising an ef fective amount of a ZPI { e. g., modif ied ZPI ) and a second therapeutic agent { e. g., an antithrombotic / anticoagulant agent ), the combination therapy may be carried out by administration of the dif ferent active agents in a single composition, by concurrent administration of the dif ferent active agents in dif f erent composit ions, or by sequential administration of the dif f erent act ive agents. Alternatively, or in addition to the use of an antithrombotic / anticoagulant agent, modif ied ZPI may be administered in combination with other drugs including, for example, cardiovascular disease therapeutic drugs, arrhythmia therapeutic drugs, diabetes therapeutic drugs, etc.

[0063] In some aspects, the invention also provides a method of enhancing or increas ing in vivo activity of a ZPI compris ing administering to a subj ect in need thereof { e. g., a subj ect as described herein) a ZPI compris ing an amino acid sequence having at least 70% sequence ident ity to SEQ ID N0: l in combination with Protein Z (PZ ). In some aspects, the ZPI is a wi ld-type ZPI, e. g., a wi ld-type or natural human ZPI as provided under GENBANK Accession No. NP_001094077. 1 or SEQ ID NO: 1. In some aspects, the ZPI is a modif ied ZPI as described herein, e. g., a modif ied ZPI comprising an amino acid substitution at pos ition 293 and / or position 240 with reference to SEQ ID NO: 1. In some aspects, the the ZPI { e. g., wild-typeUIC0116WO PATENT ZPI or modif ied ZPI ) comprises at least one PEG group conj ugated to the ZPI ( e. g., the ZPI is PEGylated).

[0064] In some aspects, a PZ of the method is a wi ld-type human PZ, e. g., as described under GENBANK Accession No. AAA36501. 1 or SEQ ID NO: 5. In some aspects, a PZ may be a PZ having at least about 60%, e. g., at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% sequence identity to SEQ ID NO: 5.MAGCVPLLQG LVLVLALHRV EPSATSLKER HGLHSDSACT GVQESLFLPA SKANDVLVRW KRAGSYLLEE LFEGNLEKEC YEEICVYEEA REVFENEVVT DEFWRRYKGG SPCISQPCLH NGSCQDS IWG YTCTCSPGYE GSNCELAKNE CHPERTDGCQ HFCLPGQESY TCSCAQGYRL GEDHKQCVPH DQCACGVLTS EKRAPDLQDL PWQVKLTNSE GKDFCGGVI I RENFVLTTAK CSLLHRNITV KTYFNRTSQD PLMIKITHVH VHMRYDADAG ENDLSLLELE WPIQCPGAGL PVCTPEKDFA EHLLIPRTRG LLSGWARNGT DLGNSLTTRP VTLVEGEECG QVLNVTVTTR TYCERSSVAA MHWMDGSWT REHRGSWFLT GVLGSQPVGG QAHMVLVTKV SRYSLWFKQI MN ( SEQ ID NO: 5 )

[0065] PZ sequences having at least 60% sequence ident ity to SEQ ID NO: 5 include, but are not limited to chimpanzee PZ ( e. g., GENBANK Accession No. XP_063648438. 1; 98. 25% sequence identity), rhesus monkey (GENBANK Accession No. XP_077830221. 1; 93. 75% sequence identity), rat (GENBANK Accession No. NP_001296362. 1; 64. 12% sequence identity), and mouse (GENBANK Accession No. NP_080110. 1; 66. 84 % sequence identity).

[0066] In some aspects, the ZPI and PZ are from the same genus and / or species. In some aspects, the ZPI and PZ are f rom dif ferent genus and / or species. In some aspects, the ZPI and PZ are both obtained or i solated from a mammal. In some aspects, the ZPI and PZ are both obtained or isolated from a primate. In some aspects, the ZPI and PZ are both obtained or isolated from a human. In some aspects, the ZPI and PZ are capable of forming a complex in vi tro and / or in vivo. In some aspects, a ZPI / PZ complex is administered to the subj ect.

[0067] In some aspects, an enhancement or increase in in vivo ZPI activity may be assessed by an APTT assay. In some aspects, the activity of a ZPI co-administered with a PZ i s enhanced or increased by at least 1. 2 -, 1. 3 -, 1. 4 -, 1. 5 -, 1. 6 -, 1. 7 -, 1. 8 -UIC0116WO PATENT, 1. 9 -, 2. 0 -, 2. 1 -, 2. 2 -, 2. 3 -, 2. 4 -, or 2. 5 -fold compared to the activity of the ZPI administered in the absence of the PZ. In some aspects, the activity of a ZPI co-admini stered with a PZ is enhanced or increased by at least 1. 5 - fold compared to the activity of the ZPI administered in the absence of the PZ. In some aspects, the activity of a ZPI co-admini stered with a PZ is enhanced or increased by at least 2 -fold compared to the activity of the ZPI administered in the absence of the PZ. In some aspects, the activity of a ZPI co -administered with a PZ is enhanced or increased by at least 2. 5 - fold compared to the activity of the ZPI administered in the absence of the PZ.

[0068] The following non- l imit ing examples are provided to further i llustrate the present invention.Example 1: Materials and Methods

[0069] Animals. The Institutional Animal Care Committee of the University of Illinois at Chicago approved all animal studies. C57BL / 6J mice were obtained from Jackson Laboratory (Bar Harbor, ME) and were maintained in the university-designated facility.

[0070] Proteins. Recombinant human wi ld-type (WT) protein Z -dependent protease inhibitor ( ZPI; SEQ ID NO: 1) and D293A ZPI (SEQ ID NO: 2 ) were expressed in non-mammalian cell systems and purif ied to homogeneity as previously reported (Huang et al. (2008 ) J. Biol. Chem. 283: 29770 -29783; Wei et al. (2009 ) Blood 114: 3662 -3667; Huang (2019 ) J. Thromb. Haemost. 17: 1655 - 1660; Huang et al. (2022 ) J. Biol. Chem. 298: 102022 ). The recombinant ZPIs produced in E. col i and insect cells were used for in vi tro and in vivo assays, respectively. Human plasma protein Z (PZ; SEQ ID NO: 5 ), prothrombin, activated factor X ( FXa), activated factor XI ( FXIa), and corn tryps in inhibitor (CTI ) were purchased from Enzyme Research Laboratories (South Bend, IN). Activated f actor V ( FVa) was from Prolytix (Essex Junction, VT). All proteins were j udged >95% pure by SDS - PAGEUIC0116WO PATENT analysis. Molar concentrations of recombinant ZPI and plasma PZ were determined from the absorbance at 280 nm using absorption coef f icients calculated f rom the amino acid sequence (Huang et al. (2012 ) Blood 120: 1726 - 1733 ).

[0071] Phospholipids. Small, uni lamellar phosphol ipid vesicles ( SUV) were prepared by sonication on ice under nitrogen for 60 minutes from a 7: 3 mixture (by weight) of dioleyl phosphatidylcholine and dioleoyl phosphatidylserine (Avanti Polar Lipids ) as previously described (Huang et al. (2019 ) J. Biol. Chem. 294: 7644 - 7657 ).

[0072] Kinetics of wild- type or D293A inhibi tion of FXa and FXIa. Assays test ing the inhibitory ef fects of ZPI on FXIa, membrane -associated free FXa and prothrombinase -bound FXa were performed as previously described (Huang et al. (2008 ) J. Biol. Chem. 283: 29770 -29783; Huang et al. (2012 ) Blood 120: 1726 - 1733; Huang et al. (2022 ) J. Biol. Chem. 298: 102022 ). In these experiments, purif ied FXa or FXIa were respect ively incubated with various concentrations of ZPI with or without equal molar PZ for increasing lengths of t ime. For every ZPI concentrat ion, at various time points, the FXa- ZPl / PZ or FXIa-ZPI mixtures were quickly di luted into the solution containing the respective f luorescence substrates for FXa or FXIa to determine the residual FXa or FXIa activity by measuring the initial rate of substrate hydrolysi s f luorometrically. The time -dependent inhibition progress curve was generated with the control as zero time point (no ZPI thus full FXa or FXIa activities ) and followed by FXa or FXIa residual activit ies at a series of time points of FXa / FXIa- ZPI incubat ion as described above. The FXa (or FXIa) act ivity vs. time progress curve was f itted using Prism software by a single exponential decay funct ion with a nonzero end point to obtain kobsfor each progress curve at each ZPI concentration (kobs= the f itted rate exponential decay constant ) (Huang et al. ( 2008 ) J. Biol. Chem. 283: 29770 -29783 ). The progress curve has a nonzero end point due to the presenceUIC0116WO PATENT of small amounts (<5%) of partially degraded proteases that were resistant to inhibition by ZPI. After testing several different ZPI concentrations, a series of kobs(each for one ZPI concentration) was obtained. The Apparent second-order associate rate constant (ka) was obtained from the slopes of linear plots of kobsversus the ZPI concentration according to the equation, kobs=kdiss+kax [ZPI]. In this equation, kdissrepresents the intrinsic dissociation rate constant for deacylation of the ZPI-factor Xa / FXIa acyl - intermediate complex, whereas [ZPI]ois the total ZPI concertation. The kdisswas fixed at the experimentally determined value of 2 x 10-4s-1for wild-type and D293A as previously described (Huang et al. (2008) J. Biol. Chem. 283: 29770 -29783; Huang et al. (2012) Blood 120: 1726 -1733; Huang et al. (2022) J. Biol. Chem.298: 102022). The kathus generated for wild-type (WT) or D293A were compared using " Student ' s t -tests to obtain p value. The ratio of mutant ka / WT kawas calculated to indicate the fold change.

[0073] Thrombin generation assay. Certified pooled normal human plasma (PNP, from 30 or more screened normal human donors) and pooled C57BL / 6 plasma were from George King (Kansas) and Innovative Research (MI), respectively. Five different lots of certified FXI-def icient plasma (with FXI activity <1%, George King) with equal volume were pooled to be used as a control. The experiment was conducted as previously described (Huang et al. (2019) J. Biol. Chem. 294: 7644-7657).

[0074] Clotting time. Activated partial thromboplastin time (APTT reagent of Dade Actin-FSL) and Prothrombin time (PT reagent of Dade Innovin) were performed on PNP, and pooled C57BL / 6 mouse (Innovative Research) plasma supplemented with inhibitors or control vehicles (<15% in volume) with an Amelung KC1 analyzer according to the manufacturer' s instructions.

[0075] Determining ZPI and PZ antigen levels in plasma. ZPI and PZ antigen levels in plasma were determined using ELISAUIC0116WO PATENT kits (R& D Systems kit for ZPI; Novus Biologicals kit for PZ ) according to the manuf cturer' s instructions.

[0076] FeCl3-induced mouse carotid artery thrombosis model. The carotid artery ferric chloride model performed on C57BL / 6 mice anestheti zed with 2 -3 % isof lurane inhalation was conducted using 7. 5% FeCl3for 3 minutes to induce thrombosi s in 15 minutes, as previously described (Pang et al. ( 2020 ) Sci. Transl. Med. 12 ). The C57BL / 6 mice were pre - inj ected with control vehicle (n=11, male 6, female 5), wi ld-type ZPI ( SEQ ID NO: 1; n= 9, male 5, female 4 ), D293A ZPI ( SEQ ID NO: 2; n=8, male 4, female 4 ), and D293A / Y387A ZPI (SEQ ID NO: 4; n=7, male 4, female 3 ) at 7. 5 mg / kg via retro-orbital veins.

[0077] FeCl3- induced venous thrombosis. The experiment was conducted on C57BL / 6 mice anesthetized with ketamine ( 100 mg / kg) / xylazine ( 5 mg / kg) using 5 % FeCl3for 3 minutes to induce thrombosis in inferior vena cava ( IVC) in 20 minutes, following previously described procedures (Wang et al. (2006 ) J. Thromb. Haemost. 4: 1982 - 1988 ). The C57BL / 6 mice were pre - inj ected with control vehicle (n=9, male 5, f emale 4 ), wild- type ZPI (n=8, male 5, female 3, for 7. 5 mg / kg; n=8, male 4, female 4, for 15 mg / kg) and D293A ZPI (n=8, male 5, female 3, for 7. 5 mg / kg; n= 8, male 4, female 4 for 15 mg / kg) via retro-orbital veins.

[0078] Tail bleeding assay. The tail bleeding assay on C57BL / 6 mice was conducted as previously described (Pang et al. ( 2020 ) Sci. Transl. Med. 12 ). In brief, mice were anestheti zed with ketamine ( 100 mg / kg) / xylazine ( 5 mg / kg). After cutt ing a 0. 5 cm- long segment of f the di stal tip of the tail, the tai l bleeding time was observed for up to 15 minutes. The C57BL / 6 mice were pre - inj ected with control vehicle (n=9, male 6, female 3 ), wi ld-type ZPI (n= 12, 7. 5 mg / kg, male 7, female 5), D293A ZPI (n=10, male 5, female 5, for 7. 5 mg / kg; n=8, male 5, female 3, for 15 mg / kg), and D293A / Y387A ZPI ( 7. 5 mg / kg, n=9, male 4, female 5 ) via retro-orbital veins.UIC0116WO PATENT

[0079] Saphenous vein hemostasis model. The saphenous vein hemostasis assay on C57BL / 6 mice was conducted as described previously (Ay et al. (2017 ) J. Thromb. Haemost. 15: 1829 - 1833; Pastoft et al. (2012 ) Haemophilia 18: 782 - 788 ). Brief ly, a small cut was made on the wal l of the surgically exposed saphenous vein, which was observed for hemostatic clot formation. Immediately after bleeding stopped at the cut (which was counted as 1 t ime of hemostasis ), the clot was di srupted by stroking a blunted 30 -G needle end in the direction of blood flow to re- initiate a new bleeding episode. The process was then repeatedly performed for the duration of 20 minutes. The total number of times of hemostasis (reformation of hemostatic clot ) was recorded, which ref lects how fast hemostasis occurred. The C57BL / 6 mice were pre - inj ected with control vehicle (n=10, male 6, female 4 ), wi ld-type ZPI (n=ll, male 5, female 6, 7. 5 mg / kg), D293A ZPI (n= 10, male 5, female 5, for 7. 5 mg / kg; n= 10, male 4, female 6, for 15 mg / kg), and D293A / Y387A ZPI ( 7. 5 mg / kg, n=8, male 5, female 3 ) via retro-orbital veins.

[0080] Statistical analysis. Student ' s t -tests were used to compare the di f ferences between two treatments or two treatment time points for in vi tro assays. D ' Agost ino-Pearson normality test was performed before the signif icance tests. In animal models, dif ferences between buf fer, wild-type, and mutant ZPIs were tested by one-way analysis of variance (ANOVA) (with Bonferroni correction for multiple testing). GraphPad Prism version 6. 0 for Windows was used to perform data analys is. A value of p < 0. 05 was considered statistically signif icant.Example 2: Mutation of ZPI selectively abolishes PZ -dependent inhibition of FXa without af fecting PZ- independent inhibition of FXIa

[0081] To determine whether D293A mutation could diminish PZ -dependent ZPI inhibition of FXa and prothrombinase at highUIC0116WO PATENT D293A ZPI and PZ concentrations relevant to pharmacological application, the FXa inhibitory effects of wild-type and D293A ZPI together with equimolar PZ up to 300 nM were analyzed. As shown Table 2, D293A mutation caused a 20-fold decrease in kaof PZ-dependent inhibition of FXa (not complexed with FVa) as compared with wild-type ZPI (85.0±5.7 x103M-1s-1(D293A) vs.1787.0±197.3x103M-1s-1(WT), n=3, p<0.001), although there were residual inhibitory effects, as compared with the D293A / Y387D double mutation, which shows no detectable inhibition. Under the physiological condition when FXa is bound to membrane-associated FVa to form prothrombinase, the kaof prothrombinase inhibition by D293A mutant ZPI was further reduced to ~50-fold slower than that of WT ZPI (6.8±0.5x103M-1s-1vs. 296.4±24.9 x103M-1s-1, Table 2, n=3, pcO. OOl), which already had a -6. 0-fold decrease in kacompared with that in the absence of FVa (296.4±24. 9 x M^S’1vs. 1787. 0±197. 3xlO3M-1s-l, Table 2, n=3, p< 0.001). Consistently, the D293A mutation diminished the PZ-dependent inhibitory effect on the prothrombinase function of the FVa / FXa complex, a key component of the common pathway of blood coagulation. In the absence of PZ and lipids, however, both WT and D293A ZPI similarly showed only residual FXa inhibitory effects (ka-1 / 190 of PZ-dependent inhibition by WT) (9. 2 + 0.4 xlO3M- 1s-1and 9. 5±0. 4xlO3M- 1s- 1for WT and D293A FXa inhibition, respectively, vs 1787. 0±197. 3xlO3M_ 1s’l of WT FXa inhibition in the absence of FVa, Table 2, n=3, pcO. OOl), confirming that the FXa inhibitory effect of ZPI is predominantly PZ-dependent. Importantly, D293A ZPI and wildtype ZPI similarly inhibited FXIa in the absence of PZ and lipids ( 112.1±5x103M-1s-1and 86.2±3.6x103M-1s-1, respectively, for FXIa inhibition, Table 2), indicating that the function of ZPI to inhibit FXIa is not negatively affected by the D293A mutation. Thus, D293A ZPI is a selective inhibitor of FXIa with minimal effect on FXa.UIC0116WO PATENT TABLE 2ka(x103M-1s-1)ZPI+PZ - FVa: FXa +PZ+FVa: FXa - PZ: FXa - PZ: FXIa 1787. 0 ±197. 3 296.4±24.9 9. 2 ± 0. 4 86. 2 ±3. 6 WT( 1. 0 ) ( 1. 0 ) ( 1. 0 ) ( 1. 0 ) D 85. 0 ±5. 7 6. 8 ± 0. 5 9. 5± 0. 4293A 112. 1 + 5. 0( 0. 05 ) ( 0. 02 ) ( 1. 0 ) ( 1. 3 )2. 6± 0. 2 D293A / Y387D - - -( 0. 03 ) Second-order association rate constants (ka) were measured in 50 mM Tri s Buf fer pH 7. 4, 0. 1 M NaCl, 1 mg / mL BSA, 2.5 mM CaCl2at 25°C, and in the presence or absence of equimolar PZ, 25 pM lipids, and 16 nM FVa, as described in Example 1. The results represent three independent measurements, presented with mean+SD (n=3 ). The student ' s t - test was used to compare the dif ferences between selected two groups. The p values were shown in the results. The normal i zed values vs. wi ld- type ( as 1. 0 ) were shown in brackets.Example 3: Inhibitory ef fect of wild-type and D293A ZPI on thrombin generation induced by activated factor XII (FXIIa) in the Calibrated Automated Thrombogram (CAT) assay

[0082] Coagulation can be act ivated via the FXIIa- and FXIa- dependent intrinsic pathway and the tissue f ctor- initiated extrinsic pathway, both of which activate FX and the common prothrombinase pathway of thrombin generation. The CAT assay was used to quanti fy the ef fects of wild- type and D293A ZPI on thrombin generation. Human FXIIa was used to initiate the intrinsic pathway of thrombin generation in both human and C57BL / 6 mouse plasma, either with a high dose ( 33 nM for humans, 6. 5 nM for mice) or a low dose ( 6. 5 nM for humans, 1. 5 nM for mice) FXIIa. In PNP, both wi ld-type ZPI and D293A ZPI caused a potent dose -dependent inhibition of thrombin generat ion induced by FXIIa. In contrast, inact ive ZPI mutant D293A / Y387A had no detectable ef f ect on thrombin generation. Wi ld-type ZPI caused a more potent inhibitory ef fect than D293A on lag t ime (LT), thrombin peak (TP), and endogenous prothrombin potential (ETP) at lower concentrations of ZPI ( 0. 2 - 0. 7 pM, FIGS. 2A- 2D) when both wild- type and D293A only part ially inhibited FXIIa - induced thrombin generation. However, when the concentrat ion wasUIC0116WO PATENT increased incrementally from 0. 7 pM to 1 pM, wild-type and D293A ZPI similarly achieved complete inhibition (>40 minutes for LT or 0 for TP and ETP) when the assay was induced by either low or high concentrations of FXIIa. Considering that D293A differs from wild-type only in its def iciency in PZ-dependent FXa inhibition and is similar to wild-type in inhibiting FXIa, these data indicate that the deficient FXa inhibitory function of D293A manifests in the CAT assay only when the intrinsic pathway is not completely abolished and that similar complete inhibition of thrombin generation at the same high dose likely reflects their similar function in inhibiting the intrinsic pathway. Interestingly, in contrast to the complete inhibition of intrinsic pathway in PNP by high concentrations of wild-type or D293A ZPIs, FXI deficiency in FXI-def icient plasma (FXI-DP) showed only partial reduction in FXIIa- induced thrombin generation (FIGS. 2E-2F). This partial inhibition of FXIIa-induced thrombin generation in FXI-DP may be due to the presence of a low level of FXI -dependent activity in FXI-DP or / and due to the presence of FXI - independent activation of the intrinsic pathway by FXIIa. In this respect, there were reports that FXIIa- induced activation of FIX and thus intrinsic pathway via kallikrein (Visser et al. (2020) Arterioscler. Thromb. Vase. Biol. 40: 103 -111; Kearney et al. (2021) Proc. Natl. Acad. Sci. USA 118 ), bypassing FXI. The data demonstrate that both wildtype and D293A ZPI can completely inhibit FXIIa-induced activation of intrinsic pathway of thrombin generation. In mouse plasma, dose -dependent inhibitory effects of wild-type and D293A ZPIs (ZPI concentrations ranged from 0 -6 pM) were also seen on high and low dose FXIIa-induced thrombin generation, although they were much less potent than in normal human plasma, possibly due to species differences. Also, the inhibitory effect of wild-type ZPI in mouse plasma was more potent relative to D293A ZPI, possibly due to a higher PZUIC0116WO PATENT concentration in mouse plasma and the effect of endogenous PZ concentration in limiting the function of excess ZPI.Example 4: Inhibitory effect of Wild-Type and D293A ZPI on thrombin generation induced by tissue factor (TF) in the CAT assay

[0083] It has been shown that FXI and, consequently, the intrinsic pathway can be activated by thrombin. This thrombin-mediated feedback activation loop is crucial in amplifying thrombin generation via the extrinsic -common pathway induced by low initial concentrations of TF, FXa, and thrombin. The effects of exogenously added wild-type and D293A ZPI on human and C57BL / 6 mouse plasma thrombin generation activated by various doses of TF (in the presence of 30 pg / mL CTI to prevent FXII activation) was analyzed. At a high dose of TF (6 pM), PNP and FXI-DP had a similar profile of thrombin generation (FIGS.3A). However, at lower doses of TF (2 pM and 0.5 pM), FXI -DP exhibited significantly decreased thrombin generation as indicated by prolonged LT, and decreased TP and ETP, consistent with the role of FXI in promoting low-dose TF-initiated coagulation (FIGS. 3B-3C). Exogenous wild-type and D293A ZPIs both had minimal to modest impacts on LT and ETP in both human and mouse plasma at the high TF concentration, although wildtype ZPI still potently reduced TP (FIGS. 3D and 3G). At low TF concentrations (0.5-2 pM), wild-type ZPI more potently inhibited thrombin generation in both human plasma (FIG. 3E vs. FIG. 3H and FIG. 3F vs. FIG. 31) and mouse plasma than D293A ZPI and FXI-DP (in human). However, D293A ZPI still dose-dependently inhibited thrombin generation initiated by lower doses of TF, reaching or exceeding FXI -DP at the high concentrations of D293A (FIGS, 3H and 31). Together, these data indicate that D293A ZPI, while having a reduced inhibitory effect compared with wild-type ZPI due to the loss of the PZ-dependent inhibitory effect on FXa, still effectively inhibitsUIC0116WO PATENT the amplification oflow dose TF-induced thrombin generation similar to FXI-DP.Example 5: Effect of wild-type ZPI and D293A ZPI on activated partial thromboplastin time (APTT) and prothrombin time (PT) in human plasma

[0084] To compare the PZ-dependent and independent inhibitory function of D293A ZPI and wild-type ZPI on coagulation triggered through intrinsic and extrinsic pathways, standard APTT and PT assays were performed. The PNP used in the experiments had an APTT and PT of -30 seconds and -10 seconds, respectively. Adding wild- type ZPI or D293A ZPI to plasma increased APTT dose -dependently and to a similar extent for every concentration employed (0 - 4 pM) (FIG. 4A). At 4 μM wild-type ZPI or 4 μM D293A ZPI added, both APTTs were increased by 2.5-fold as compared with control plasma. In contrast, neither wild-type ZPI nor D293A ZPI affected PT (FIG. 4A). Thus, both wild-type ZPI and D293A ZPI potently inhibit the intrinsic / common coagulation pathway as indicated by APTT but not the extrinsic / common coagulation pathway induced by very high concentrations of TF as indicated by PT.

[0085] Next, the effects of wild-type ZPI and D293A ZPI on APTT and PT were compared with that of asundexian (MedChemExpress, Monmouth Junction, NJ), a small molecule inhibitor of human FXIa currently in clinical trials (Heitmeier et al. (2022) J. Thromb. Haemost. 20: 1400-1411), and BMS-262084 (MedChemExpress), a known potent FXIa inhibitor (Mader et al. (2024) J. Thromb. Haemost. 22: 199-212). Interestingly, the dose response curve of asundexian' s effect on APTT was not saturated even at 60 pM and was weaker than BMS-262084 (p < 0. 01- 0.05, FIG.4B). The dose response curve for asundexian and BMS-262084 on PT, however, had an initial lack of effect (up to 4 pM) followed by a moderate but significant increase in inhibitory effect at higher concentrations (FIG. 4B), consistent withUIC0116WO PATENT previous reports showing that although asundexian and BMS-262084 potently inhibit FXIa, they may also have additional targets (Mader et al. (2024) J. Thromb. Haemost. 22: 199-212; Wong et al. (2011) J. Thromb. Thrombolysis 32: 129 -137). The inhibitory effects of wild-type ZPI and D293A ZPI were compared with asundexian and BMS-262084 at concentrations that only prolonged APTT but not PT. Both D293A ZPI and wild- type ZPI showed similar inhibitory effects to that of BMS-262084 but were significantly more potent than that achieved by the same concentrations of asundexian (FIG. 4A, p<0. 001). These data demonstrate that ZPI (wild-type or D293A) is superior in selective inhibition of intrinsic pathway-mediated coagulation as compared with asundexian, but similar to BMS-262084.Example 6: Effects of WT and D293A on APTT and PT in mouse plasma and the role of PZ in regulating the pharmacological effect of ZPI.

[0086] The effect of exogenously added wild-type ZPI and D293A ZPI on APTT in pooled C57BL / 6 mouse plasma was also tested. Wild-type ZPI or D293A ZPI increased the APTT of mouse plasma dose-dependently. Different from human plasma APTT, however, wild-type ZPI exhibited a stronger inhibitory effect than D293A ZPI at all ZPI concentrations, suggesting the relative importance of PZ-dependent inhibitory effects of ZPI on mouse APTT. This is consistent with the results of thrombin generation assays above (FXIIa triggered CAT). C57BL / 6 mouse plasma has a higher concentration of endogenous PZ (-240 nM) than the PNP used (-37 nM, similar to previous reports; Han et al. (2000) Blood 96: 3049-3055; Miletich et al. (1987) Blood 69: 1580-1586). As the PZ-dependent inhibitory function of ZPI on FXa is abolished in D293A ZPI, it was hypothesized that the more potent effect of wild-type ZPI vs. D293A ZPI on APTT in mice may be due to the presence of higher PZ concentrations in mouse plasma. Indeed, adding exogenous PZ (0.2-0.9 μM) togetherUIC0116WO PATENT with exogenous ZPI to human plasma, but not adding PZ alone, significantly enhanced the effect of exogenous wild-type ZPI in prolonging APTT without affecting the inhibitory effect of D293A ZPI (FIG. 4C). These data indicate that the enhanced effects of exogenously added wild-type PZ in prolonging APTT relative to the effect of D293A ZPI is PZ-dependent and is limited by the relatively low PZ concentrations in plasma. This supports the notion that PZ-dependent ZPI function is diminished in D293A ZPI and that the anticoagulant effect of D293A is mostly PZ- independent. These results also indicate that due to the limiting effect of endogenous PZ on the wildtype ZPI -mediated inhibition of the FXa-dependent common pathway, the wild-type ZPI-mediated inhibitory effect on clotting time (especially in human plasma) mostly resulted from the PZ- independent inhibitory effect of ZPI on the intrinsic pathway, as displayed by the effect of D293A. Interestingly, exogenous wild-type ZPI or D293A ZPI ( 0-4 pM), in the absence of exogenous PZ, minimally affected PT in C57BL / 6 mouse plasma, which is similar to human plasma (FIG. 4A). Adding PZ along with ZPI selectively induced prolongation of the PT only in wild-type ZPI-treated human and mouse plasma, but not D293A-treated mouse and human plasma (FIG. 4D). Thus, the inability of even wild-type ZPI to prolong PT is not only due to the large amount of TF in the PT assay (that likely overwhelms the inhibitory effects of ZPI by activating FXa via the extrinsic pathway) but also due to the limiting effect oflow endogenous PZ concentrations on the PZ-dependent function of exogenous wild-type ZPI to inhibit Xa. These results are consistent with the results obtained from the CAT assays, indicating the minimally changed LTs of wild-type and D293A in high dose TF triggered CAT assays (FIGS. 3D and 3G), although thrombin generation is still more significantly inhibited by wild-type ZPI than D293A ZPI (TPs in the same FIGS. 3D and 3G).UIC0116WO PATENT Example 7: Comparison of antithrombotic ef fects of wild- type and D293A ZPIs using the mouse carotid artery thrombosis model

[0087] Clinical anticoagulation therapies usually have an APTT target of 1.5- to 2.5-fold of normal (Bates et al. (2001) Arch. Intern. Med. 161:385-391), equivalent to that achieved by 1~2 μM of ZPIs. The studies showed that retro-orbital injection of 7.5 mg / kg of ZPIs gave the plasma concentrations of ~2.0 μM in C57BL / 6 mice at 3-4 minutes, and the concentration went below 1.0 μM after ~20 minutes. Thus, 7.5 mg / ml i.v. injection was selected as the concentration to be used in the in vivo studies.

[0088] To compare the abi lity of wild-type ZPI and D293A ZPI to inhibit arterial thrombosis in vivo, the FeCl3-induced carotid artery thrombosis model was used. After induction of injury to the carotid artery with 7.5% FeCl3for 3 minutes, C57BL / 6 mice formed stable occlusive thrombi with a median time of 330 s. The occlus ion time was signif icantly delayed ( - 525 s ) in mice inj ected with a one -time bolus of wi ld-type ZPI ( 7. 5 mg / kg retro-orbital ly, p< 0. 001, wi ld-type vs. buf fer, FIG. 5). Interestingly, despite the absence of FXa inhibitory function, the same dose of D293A ZPI had a potent inhibitory ef fect ( -480 s, p=0. 0043, D293A vs. buf f er, FIG. 5 ), only slightly lower than that of wild- type ZPI, though no signi f icant di fference was detected. In contrast, the loss of function mutant ZPI, D293A / Y387A, at the same dose, did not af fect the occlusion time.Example 8: Effect of D293A on venous thrombosis

[0089] The FeCl3- induced inferior vena cava ( IVC) inj ury model was used to evaluate the eff ect of D293A on venous thrombosis in vivo. In this experiment, D293A and wi ld-type administered at the same dose that inhibited carotid artery thrombosis ( 7. 5 mg / kg) decreased venous thrombus s i ze signif icantly p < 0. 001, FIG. 6, 7. 5 mg / kg D293A or wi ld-type vs. buf fer). A double dose of D293A and wild- type ( 15 mg / kg) further decreased thrombusUIC0116WO PATENT si ze (D293A 7. 5 mg / kg vs. 15 mg / kg, p < 0. 001; wild- type 7. 5 mg / kg vs. 15 mg / kg, p = 0. 0017, FIG. 6 ). At the same concentration, the inhibitory eff ect of wi ld-type ZPI showed no signif icant dif ferences compared with D293A, although showing a trend of enhanced inhibition (p=0. 082, FIG. 6 ). Thus, D293A ZPI and wi ld-type ZPI inhibit both arterial and venous thrombosis in vivo.Example 9: Effect of wild-type ZPI and D293A ZPI on mouse tail bleeding time and vena saphenous hemostasis model

[0090] Mouse tai l bleeding time analysis and a saphenous vein hemostasis model were used to determine the ef fect of wild- type ZPI and D293A ZPI on hemostasis. Wi ld-type ZPI, when inj ected at the same dose ( 7. 5 mg / kg) that potent ly inhibited thrombosis, increased tai l bleeding time (p=0. 028, wi ld-type vs. buf f er, FIG. 7A) and decreased hemostasis incidence of inj ured saphenous vein (p < 0. 001, WT vs. buf fer or D293A / Y387D, FIG. 7B). In contrast, D293A at the same dose ( 7. 5 mg / kg) that potently inhibited thrombosis did not increase the tai l bleeding time and did not decrease hemostasis incidence in saphenous vein bleeding model ( FIG. 7B). As a control, the functionally dead D293A / Y387D double mutant of ZPI also had no ef fect on tail bleeding time and saphenous vein bleeding. To determine whether even higher concentrations of D293 could af fect hemostasis, the D293A dose was doubled to 15 mg / kg. Again, D293A did not increase the tai l bleeding time ( FIG. 7A) but moderately decreased saphenous vein hemostasis incidence (p=0. 0081 or 0. 0054, D293A 15 mg / kg vs. buf fer or D293A / Y387D, FIG. 7B).UIC0116WO PATENTExample 10: PEGylation significantly improves the pharmacokinetics of recombinant ZPI

[0091] The recombinant ZPI made from non-mammalian cell systems used for these studies had a half -life of only ~20 minutes in mouse circulation, contrary to a previous study that showed ZPI made from mammalian cell systems had a much longer half -life in mouse circulation (Girard et al. (2013 ) J. Thromb. Haemost. 11: 375-378). The recombinant ZPIs displayed a much smaller size than human plasma ZPI on SDS-PAGE (~50 kDa vs. ~72kDa), indicating glycosylation deficiency in these ZPIs, as ZPI has 5 N-linked glycosylation sites (Han et al. (1999) Biochemistry 38: 11073 -11078). The results herein also indicate that glycosylation has a significant effect on pharmacokinetics. It was found that the cysteine residues (C169 and C264 of SEQ ID NO:2) in the recombinant D293A ZPI can react with thiol-reactive methoxy-(PEG)n-maleimide (~20 kDa) (PEG, polyethylene glycol). This click chemistry reaction results in a ZPI-PEG molecule of ~90kDa on SDS-PAGE, indicating that each of the two cysteine residues was labeled with a 20 kDa thiol reactive PEG moiety. Importantly, this PEG-modified ZPI derivative maintained anticoagulation function (selectively inhibiting APTT; FIG. 8A) and significantly improved its pharmacokinetics in mouse circulation (increasing the half-life from ~20 minutes to ~70 minutes; FIG. 8B). In addition, it effectively inhibited the thrombosis formation in FeCl3-induced IVC DVT model in mice, similarly to unmodified D293A ZPI (FIG.8C).

Claims

UIC0116WO PATENT WHAT IS CLAIMED IS:

1. A method for inhibiting or reducing the risk of venous or arterial thrombosis without compromising hemostasis, comprising administering to a subj ect in need thereof an effective anticoagulant amount of a modified protein Z-dependent protease inhibitor (ZPI) comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1 and an amino acid substitution at position 293 and / or position 240 with reference to SEQ ID NO: 1, thereby inhibiting or reducing the risk of venous or arterial thrombosis without compromising hemostasis.

2. The method of claim 1, whereing the modified ZPI comprises the amino acid sequence of any one of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

3. The method of claim 1, wherein the modified ZPI comprises at least one polyethylene glycol (PEG) group conjugated thereto.

4. The method of claim 3, wherein the at least one PEG group is conjugated to a cysteine residue of the modif ied ZPI.

5. The method of claim 1, wherein the venous or arterial thrombosis is associated with myocardial infarction, unstable angina, atrial fibrillation, stroke, renal damage, pulmonary embolism, deep vein thrombosis, percutaneous translumenal coronary angioplasty, disseminated intravascular coagulation, sepsis, artificial organs, shunts, or prostheses.

6. The method of claim 1, further comprising administering to the subj ect an effective amount of protein Z.UIC0116WO PATENT 7. A modified protein Z-dependent protease inhibitor (ZPI) comprising:an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1;an amino acid substitution at position 293 and / or position 240 with reference to SEQ ID NO: 1; andat least one polyethylene glycol (PEG) group conjugated to the modified ZPI.

8. The modified ZPI of claim 7, whereing the modified ZPI comprises the amino acid sequence of any one of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

9. The modified ZPI of claim 7, wherein the at least one PEG group is conjugated to a cysteine residue of the modif ied ZPI.

10. A composition comprising an effective anticoagulant amount of the modified ZPI protein of claim 7 and a pharmaceutically acceptable excipient.

12. The composition of claim 10, further comprising protein Z.

13. A method of enhancing in vivo activity of a protein Z-dependent protease inhibitor (ZPI) comprising administering to a subj ect in need thereof a ZPI comprising an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 1 in combination with Protein Z (PZ) thereby enhancing the in vivo activity of the ZPI.

14. The method of claim 13, wherein the ZPI is a wild-type ZPI comprising the amino acid sequence of SEQ ID NO: 1.UIC0116WO PATENT 15. The method of claim 13, wherein the ZPI is a modi f ied ZPI comprising an amino acid substitut ion at position 293 and / or position 240 with reference to SEQ ID NO: 1.

16. The method of claim 13, wherein the ZPI comprises at least one polyethylene glycol ( PEG) group conj ugated to the ZPI.

17. The method of claim 13, wherein the PZ comprises an amino acid sequence of SEQ ID NO: 5.

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