Methods of inhibiting clot formation

Details Methods of inhibiting clot formation Methods of inhibiting clot formation

1999-05-28

2003-03-18

Carlson, Karen Cochrane (Department: 1652)

Drug, bio-affecting and body treating compositions

Radionuclide or intended radionuclide containing; adjuvant...

In an organic compound

C530S380000, C530S350000

Reexamination Certificate

active

06534035

ABSTRACT:

BACKGROUND OF THE INVENTION
This invention relates to inhibition and detection of blood clot formation.
The resistance of thrombi to fibrinolysis induced by plasminogen activators is an impediment to the successful treatment of thrombotic diseases. Fibrinolytic resistance is evident in patients with acute thrombotic coronary occlusion, where treatment with plasminogen activators results in full coronary reperfusion in only 33-55% of patients at 90 minutes (Lincoff et al., 1995, Am. J. Cardiol. 75:871-876; Karagounis et al., 1992, J. Am. Coll. Cardiol. 19:1-10). The resistance of thrombi to lysis by plasminogen activators may be even more marked in patients with venous thromboembolism. In deep venous thrombosis treated with tissue plasminogen activator (TPA), nearly two thirds of patients have minimal or no significant lysis evident on repeat venography at 24 hours (Salzman et al., 1994, The epidemiology, pathogenesis and natural history of venous thrombosis, in Hemostasis and Thrombosis: Basic Principles and Clinical Practice, 3rd ed., Philadelphia, Pa.: Lippincott, pp 1275-1296; Goldhaber et al., 1990, Am. J. Med. 88:235-240). In patients with pulmonary embolism, TPA restores blood flow within 24 hours to only about a third of occluded lung segments, as judged by serial perfusion scanning (The Urokinase Pulmonary Embolism Trial. A national cooperative study, 1973, Circulation 47:1-108; Goldhaber et al., 1986, Lancet 2:886-889). Improved thrombolysis may reduce mortality and morbidity associated with thrombotic disease.
SUMMARY OF THE INVENTION
The invention provides methods of improving therapeutic thrombolysis, detecting blood clots in vivo, and inhibiting clot formation using alpha-2 antiplasmin (&agr;2AP) polypeptides.
To detect blood clot formation in a mammal, a diagnostically effective amount of a detectably labeled alpha-2 antiplasmin (&agr;2AP) polypeptide is administered to the mammal, and association of the polypeptide with a blood clot determined. Association of the &agr;2AP polypeptide with a vascular obstruction, e.g, via &agr;2AP-fibrin crosslinking, is an indication of the presence blood clot formation at the site of the obstruction. Since &agr;2AP crosslinks with fibrin in actively forming blood clots but not at the site of old (i.e., not actively forming) blood clots, the method is useful to characterize blood vessel obstructions as pre-existing or actively forming. Newly forming thrombi are detected by virtue of the formation of &agr;2AP polypeptide-fibrin crosslinks mediated by activated factor XIIIa. Thus, the present method provides an advantage over conventional visual detection techniques such as scintiphotography and angiography with which such characterization is difficult or unachievable.
The polypeptides of the invention are substantially pure. Polypeptides or other compounds encompassed by the invention are said to be “substantially pure” when they are within preparations that are at least 60% by weight (dry weight) the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. Purity can be measured by any appropriate standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
The &agr;2AP polypeptide preferably contains the amino acid sequence of X
1
QX
2
X
3
X
4
X
5
PLX
6
LLK (SEQ ID NO:1), wherein X
1
=N or A, X
2
=E or Q, X
3
=Q or K, X
4
=V or L, X
5
=P or S, and X
6
=T, S or A (the Q residue indicated in bold type is involved if &agr;2AP-fibrin crosslinking). For example, the polypeptide comprises the amino acid sequence of &agr;2AP
13-24
NQEQVSPLTLLK (SEQ ID NO:2) or &agr;2AP
1-24
(MEPLGWQLTSGPNQEQVSPLTLLK; SEQ ID NO:16). Polypeptides derived from human &agr;2AP-include those which contain a sequence that is 80-100% identical to the amino acid sequence of MEPLGXQLTS GPNQEQVSPL TLLKLGNQEP GGQTALKSPP GVCSRDPTPE QTHRLARAMM AFTADLFSLV AQT (SEQ ID NO:3), where “X” represents a residue that can differ among human &agr;2AP variants. Human N-terminal &agr;2AP polypeptides include those which contain the amino acid sequence MEPLGWQLTS GPNQEQVSPL TLLKLGNQEP GGQTALKSPP GVCSRDPTPE QTHRLARAMM AFTADLFSLV AQT (SEQ ID NO:4) or MEPLGRQLTS GPNQEQVSPL TLLKLGNQEP GGQTALKSPP GVCSRDPTPE QTHRLARAMM AFTADLFSLV AQT (SEQ ID NO:5). Alternatively, the polypeptide may contain a sequence that is 80-100% identical to the amino acid MEPLDLQLMD GQAQQKLPPL SLLKLDNQEP GGQIAPKKAP EDCKLSPTPE QTRRLARAMM TFTTDLFSLV AQS (SEQ ID NO:6), corresponding to an N-terminal fragment of naturally-occurring bovine &agr;2AP, or VDLPGQQPVS EQAQQKLPLP ALFKLDNQDF GDHATLKRSP GHCKSVPTAE ETRRLAQAMM AFTTDLFSLV AQT (SEQ ID NO:7), corresponding to an N-terminal fragment of naturally-occurring mouse &agr;2AP . An &agr;2AP polypeptide is a peptide with at least 80-100% sequence identity to a portion of a naturally-occurring &agr;2AP protein but having a length that is shorter than the length of the naturally-occurring mature full-length &agr;2AP protein. Human &agr;2AP variants within the invention include those with the amino acid sequence of SEQ ID NO:11, 13, 15, or 17.
The invention also features methods of preventing the development of clots in patients at risk for thrombosis and methods of treating patients with thrombotic conditions such as stroke, myocardial infarction, pulmonary embolism, and deep venous thrombosis. For example, a method of inhibiting blood clot formation in a mammal is carried out by administering to the mammal a therapeutically effective amount of an &agr;2AP polypeptide. A method of preventing and lysing blood clots is carried out by co-administering to a mammal a therapeutically effective amount of an &agr;2AP polypeptide and a thrombolytic agent such as a plasminogen activator, e.g., tissue plasminogen activator (t-PA). Thrombolytic agents such as prourokinase, urokinase, streptokinase, staphylokinase, and vampire bat-derived plasminogen activator may be co-administered with an &agr;2AP polypeptide to increase the effectiveness of the thrombolytic agent.
&agr;2AP polypeptides and peptide mimetics thereof are useful in the diagnostic and therapeutic methods described above. Preferably the &agr;2AP polypeptide is derived from the N-terminus of a mature, naturally-occurring mammalian &agr;2AP protein. For example, the polypeptides may be derived from human, bovine, or mouse &agr;2AP proteins, the amino acid sequences of which are shown in Tables 1-3, respectively (the first amino acid of the mature protein is indicated with an arrow).
TABLE 1
HUMAN &agr;2AP
                          →
  1
LWGLLVLSWS CLQGPCSVFS PVSAMEPLGR QLTSGPNQEQ VSPLTLLKLG NQEPGGQTAL
 61
KSPPGVCSRD PTPEQTHRLA RAMMAFTADL FSLVAQTSTC PNLILSPLSV ALALSHLALG
121
AQNHTLQRLQ QVLHAGSGPC LPHLLSRLCQ DLGPGAFRLA ARMYLQKGFP IKEDFLEQSE
181
QLFGAKPVSL TGKQEDDLAN INQWVKEATE GKIQEFLSGL PEDTVLLLLN AIHFQGFWRN
241
KFDPSLTQRD SFHLDEQFTV PVEMMQARTY PLRWFLLEQP EIQVADFPFK NNMSFVVLVP
301
THFEWNVSQV LANLSWDTLH PPLVWERPTK VRLPKLYLKH QMDLVATLSQ LGLQELFQAP
361
DLRGISEQSL VVSGVQHQST LELSEVGVEA AAATSIAMSR MSLSSFSVNR PFLFFIFEDT
421
TGLPLFVGSV RNPNPSAPRE LKEQQDSPGN KDFLQSLKGF PRGDKLFGPD LKLVPPMEED
481
YPQFGSPK (SEQ ID NO:8)
TABLE 2
BOVINE &agr;2AP
                             →
  1
MALLWGLLVL SWSCLQGPCS VFSPVSAMEP LGRQLTSGPN QEQVSPLTLL KLGNQEPGGQ
 61
TALKSPPGVC SRDPTPEQTH RLARAMMAFT ADLFSLVAQT STCPNLILSP LSVALALSHL
121
ALGAQNHTLQ RLQQVLHAGS GPCLPHLLSR LCQDLGPGAF RLAARMYLQK GFPIKEDFLE
181
QSEQLFGAKP VSLTGKQEDD LANINQWVKE ATEGKIQEFL SGLPEDTVLL LLNAIHFQGF
241
WRNKFDPSLT QRDSFHLDEQ FTVPVEMMQA RTYPLRWFLL EQPEIQVAHF PFKNNMSFVV
301
LVPTHFEWNV SQVLANLSWD TLHPPLVWER PTKVRLPKLY LKHQMDLVAT LSQLGLQELF
361
QAPDLRGISE QSLVVSGVQH QSTLELSEVG VEAAA

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