Note: Descriptions are shown in the official language in which they were submitted.
~'. .WO 92/06712 2 ~ 7 1 ~ 7 9 PCI`/US91~7367
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MEGAKARYOCYTE MATURATION FACTORS
The present invention relates to methods and
pharmaceutical compositions for the production of blood
platelet~. More specifically, the invention relates to
treatment of platelet disorders using factors which
increase the levels of circulating blood platelets. Also
encompassed by the invention are pharmaceutical
compositions of factors that promote platelet production.
Backaround of the Invention
Pluripotent hematopoietic stem cells give rise
to different types of terminally differentiated blood
cells. The blood consists of red blood cells
(erythrocytes), white blood cells (leucocytes) and
platelets (thrombocytes). Platelets are derived from
detached fragments of larger cells called megakaryocytes
which reside predominantly in the bone marrow.
Platelets have a central role in blood clotting and
wound healing.
Megakaryocytes undergo various stages of
differentiation to produce mature platelets. A
pluripotent stem cell becomes committed to megakaryocyte
development, then undergoes cellular and nuclear
proliferation to generate a pool of megakaryocyte
progenitor cells. These progenitor cells undergo
endoduplication to form immature megakaryocytes, or
megakaryoblasts, which are characterized by
multilobulated, polyploid nuclei. The development of
mature megakaryocytes from megakaryoblasts involves the
formation of cytoplasmic granules containing platelet
~i; specific proteins. Mature megakaryocytes project
cytoplasmic extensions, termed proplatelets, which
fragment to produce mature platelets.
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W O 92/06712 . PC~r/US91/07367 ~ !
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Several purified factors promote megakaryocyte
differentiation by stimulating the formation of mature
megakaryocytes from megakaryocyte progenitor cells in
vitro. These factors include granulocyte/macrophage
colony stimulating factGr (GM-CSF), granulocyte colony
stimulating factor (G-CSF), erythropoietin (EPO),
interleukin-3 (IL-3), interleukin-6 (IL-6) and
megakaryocyte colony stimulating factor (Meg-CSF)
(Hoffman et al. Blood Cells 1~, 75-86 (1987); Mazur et
al. Exp. Hematol. 15, 1128-1133 (1987); McNiece et al.
Exp. Hematol. 1~, 807-810 (1987); Lu et al. Brit. J.
Hematol. 70, 149-156 (1988); Ishibashi et al. Proc.
Natl. Acad. Sci. U.S.A. 86, 5953-5957 (1989)). A factor
referred to as megakaryocyte stimulating factor (MSF)
has been described in U.S. Patent No. 4,894,440.
Purified MSF is involved in the cytoplasmic maturation
;of megakaryocytes as shown by its ability to stimulate
in megakaryocytes the synthesis of platelet
proteoglycans and platelet specific granule proteins
such as platelet factor IV. Purified IL-6 has been
reported to increase platelet levels i~ vivo (Ishibashi
et al. Blood 74, 1241 (1989); Hill et al. J. Clin.
Invest. 85, 1242-1247 (1990)).
Thrombopoietic stimulating activity has been
found in the plasma, serum and urine of thrombocytopenic
patients and in the culture medium of human embryonic
kidney (HEK) cells. This activity has been attributed
~- to thrombopoietin or thrombopoietic stimulating factor
-~(TSF), a factor which is thought to be an important
controlling element in megakaryocyte maturation
(McDonald Ann. N.Y~ Acad. Sci. 509, 1-24 (1987)). TSF
from HEK cells has been purified (McDonald et al. J. ~ab
Clin. Med. 106, 162-174 (1985)) but the corresponding
activity from thrombocytopenic plasma has not been
purified. The role of TSF in megakaryocyte development
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has not yet been established. However, purified TSF
alone does not stimulate the formation of megakaryocytes
from progenitor cells (Lu et al. Brit. J. Hematol. 70,
149-156 (1988)), suggesting that it plays a role in the
later stages of megakaryocyte d ferentiation.
Inhibition and reversal of megakaryocyte
differentiation and maturation have also been observed.
Platelet factor IV (PF-IV) and transforming growth factor
(TGF)-~ block the development of megakaryocyte progenitor
cells (Ishibashi et al. Blood 69, 1737-1741 (1987);
Gewirtz et al. J. Clin. Invest. ~, 1477-1486 (1989); Han
et al. Blood 75, 1234-1239 (1990)). Various compounds
that affect microtubule formation inhibit proplatelet
formation (Leven et al. Blood 69, 1046-1052 (1987)). In
addition, thrombin, a serum-derived serine esterase,
reverses megakaryocyte maturation by stimulating the
retraction of proplatelet extensions ~Radley et al.,
Thrombosis and Haemotosis ~, 732-736 (1987)).
Under the appropriate culture conditions,
guinea piq megakaryocytes will differentiate in vitro
and form long cytoplasmic extensions which are
precursors to platelets (Leven et al., .supra; Handa~ama
et al., Am. J. Vet. Res. 48, 1192-1146). These
extensions, termed proplatelets, are observed to
differentiate further into small anuclear cells the size
of guinea pig platelets. Proplatelet formation
represents an important event in the development of
megakaryocytes to platelets. Factors influencing t~ 3
process will be important in the production of blood
platelets.
~- As described above, a number of factors have
been identified which stimulate various stages of
megakaryocyte differentiation and maturation and promote
increases in megakaryocyte number and size. However, no
purified factors have been reported to stimulate further
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W092/06712 2 ~ 7 ~ ~ 7 9 PCT/US91/07~7 ~
differentiation of mature megakaryocytes to proplatelet
bearing cells. The identification and isolation of
Eactors which stimulate the formation of proplatelets
will be use~ul in the treatment of excessive bleeding
resulting from platelet disorders.
The cytoplasm of mature megakaryocytes and
platelets contains granules comprising proteoglycans and
platelet specific proteins. Proteoglycans are highly
acidic macromolecules having at least one
glycosaminoglycan chain covalently attached to a protein
core. A proteoglycan was purified from human platelets
by monitoring uronic acid content of glycosaminoglycans
and was found to contain four chondroitin sulfate chains
attached to the protein core (Okayama et al. Biochem. J.
~, 73-81 (1986)). The purified human platelet
proteoglycan protein core was sequenced (Perin et al.
Biochem. J. 255, 1007-1013 (1988); Alliel et al. FEBS
Letters 2~, 123-126 (1988)). The protein was 131 amino
acids long and contained within it an 18 amino acid
region having eight ser-gly repeats. Repeated ser-gly
sequences had been observed in protein core regions of
other proteoglycans and were predicted to be sites for
glycosaminoglycan attachment. Serine residues at
positions 67 and 69 of human platelet proteoglycan were
thought to be modified with,chondroitin sulfate chains
(Alliel et al., ~,U,E~). No biological activity of human
, platelet proteoglycan was measured during or after
purification.
, Genomic and cDNA sequences encoding the
protein core of a secretory granule proteoglycan from
the human promyelocytic leukemia cell line ~L-60 were
disclosed in Stevens et al., PCT Publication No. WO
90/00606, and were also reported by Stellrecht et al.
(Nuc. Acids Res. 17, 7523 (1989)). Based upon these DNA
' 35 sequences, a protein having a molecular weight of 17,600
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WO92/06712 2 0 7 1 4 7 9 PCT/US9l/07367
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was predicted which contained a 131 amino acid mature
polypeptide and a 27 amino acid signal peptide. The
mature human secretory granule proteoglycan had an a- o
acid sequence identical to that reported for the human
S platelet proteoglycan ~Alliel et al., suDra). The
biological activity of human secretory granule
proteoglycan was not disclosed in Stevens et al., suDra.
An object of the invention is a method for the
treatment of excessive bleeding comprising the
administration of factors that promote platelet
production. A further object of this invention is the
purification of factors that elevate proplatelet levels,
thereby stimulating platelet formation. Another object
of the invention is the production of pharmaceutical
compositions comprising factors that promote platelet
production.
Summary of the Invention
The subject invention comprises methods for
~ increasing blood platelet levels and treating platelet
; disorders using factors invo -ed in megakaryocyte
maturation and proplatelet formation. Megakaryocytes
mature to form proplatelets which in turn undergo
fragmentation and release platelets. Changes in
proplatelet levels have a direct effect on the levels of
blood platelets produced.
Factors of the invention which stimulate the
` production of proplatelets from megakaryocytes are
referred to as megakaryocyte maturation factor~s) ~MMF).
These factors elevate blood platelet levels and are
useful in the treatment of excessive bleeding. MMF used
;~ in treating platelet disorders may have some or all of
the amino acid sequence of naturally-occurring MMF, may
be the product of procaryotic or eucaryotic expression
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207~79
WO92/~712 PCT/US91/07367
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of an exogenous DNA sequence encoding MMF, and may be
covalently modified with water-soluble polymers such as
polyethylene glycol to increase stability, solubility
and circulating half-life.
Megakaryocyte maturation factors may be used
alone or in combination with other therapeutics for
increasing blood platelet levels. Other factors that
are useful in conjunction with MMF are stem cell factor
(SCF), GM-CSF, G-CSF, IL-3, IL-6, Meg-CSF, MSF, and EPO.
The subject invention provides for a method of
purifying factors which affect megakaryocyte maturation.
A method of purifying megakaryocyte maturation factors
from MMF containing material comprises one or more steps
of ion exchange chromatography.
A method for assaying a megakaryocyte
maturation factor is also provided. The method
comprises incubating MMF (either crude or purified) with
megakaryocytes and monitoring the formation of
proplatelets from megakaryocytes. MMF stlmulates
production of proplatelets in this assay.
The subject invention further relates to
pharmaceutically acceptable compositions of a purified ~ -
and isolated megakaryocyte maturation factor. Also
encompassed by the invention are pharmaceutically
acceptable compositions of a megakaryocyte maturation
factor further comprising pharmaceutically acceptable
compositions of SCF, GM-CSF, G-CSF, IL-3, IL-6, Meg-CSF,
MSF, and EPO.
B~ Description of the Drawinas
Figure lA shows guinea pig megakaryocytes
before proplatelet formation under Megacolor staining.
Figure lB shows guinea pig megakaryocytes after
proplatelet formation under Wright Giemsa staining.
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Figure 2 shows the inhibition of p oplatelet
formation in the in vitro assay by addition of human
serum.
i
Figure 3 shows inhibition of proplatelet
formation by thrombin but not by trypsin, chymotryp~in,
or thrombocytin.
Figure 4 shows the inhibition of proplatelet
formation in the m vitro assay by prothrombin and
thrombin.
Figure 5 shows the retraction of proplatelet
formations in vitro induced by prothrombin and thrombin.
Figures 6A and 6B show the effect of
: inactivating thrombin on inhibition of proplatelet
formation and proplatelet retraction, respectively.
Figure 7 shows DEAE chromatography of human
serum inhibitor of proplatelet formation and
prothrombin.
Figure 8 shows Superose 6 chromatography of
human serum inhibitor of proplatelet formation and
prothrombin.
Figure 9 shows the conversion of prothrombin
to thrombin by megakaryocytes, proplatelets and
platelets.
Figures lOA and lOB show stimulation of
;~ proplatelet formation in the in vitro assay by
guanidinium chloride and CHAPS lysates of human
platelets.
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WO92/06712 2 0 7 1 ~ 7 3 PCT/US91/07367 ~
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Figure 11 shows DEAE chromatography of human
platelets lysed in the presence of guanidinium chloride.
Figure 12 shows Mono Q chromatography of MMF-
III from a guanidinium chloride lysate of humanplatelets.
Figure 13 shows Superose 6 chromatography MMF-
III from a guanidinium chloride lysate of human
platelets.
Figure 14 shows C4 reverse phase HPLC of
MMF-III from a guanidinium chloride lysate of human
platelets.
Figure 15 shows DEAE chromatography of human
platelets lysed in the presence of CHAPS buffer.
Figure 16 shows Mono Q chromatography of MMF-
III from a CHAPS lysate of human platelets.
Figure 17 shows an analysis of MMF-IIIs from
guanidinium chloride and CHAPS lysates by SDS-PAGE.
Figures 18A and 18B shows stimulation of
proplatelet formation and inhibition of proplatelet
retraction, respectively, by MMF1-131 in the in vitro
assay in the presence of increasing thrombin
concentration. -
Figure 19 shows a comparison of MMF1-131 and ' ! `
MMF58-131 activity on proplatelet formation.
Figure 20 shows the activity of MMF1-131 after
removal of chondroitin sulfate.
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Eigure 21 shows platelet levels in mice
receiving 4 ~g day or 20 ~g/day of MMF1-131.
Figure 22 shows platelet levels in mice
receiving 2 ~g/day or 10 ~g/day of human recombinant
IL-6.
Figure 23 shows platelet levels in mice
receiving 20 ~g/day of MMF1-131, 2 ~g/day of IL-6 or a
combination of 20 ~g/day of MMF1-131 and 2 ~g/day of
IL-6.
petailed Description of the Invention
The present invention relates to a class of
megakaryocyte maturation factors (MMF) which stimulate
megakaryocyte maturation and proplatelet formation,
thereby elevating circulating platelet levels. Factors
of the invention have a property of promoting the
production of proplatelets from megaxaryocytes in vitro
when an inhibitory factor is present. As described in
Example 2, one such inhibitory factor is found in human
serum.
MMF is obtained from a variety of sources
including, but not limited to, human serum, urine,
megakaryocytes and platelets. The presence of MMF
- activity in megakaryocytes and platelets is described in
Example 7 and 8. However, any biological material that
; promotes proplatelet formation in vitro may be used as a
source of MMF and the term "MMF containing material"
encompasses said bio~ogical material.
Factors that stimulate proplatelet formation
have not been previously disclosed. Hematopoietic
factors that promote megakaryocyte development such as
G-CSF, GM-CSF, IL-3 and IL-6 were tested in the in vitro
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207~479 1~
WO92/~712 PCT/US91/~7367 ~
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assay and did not stimulate proplatelet formation in the
presence of a serum inhibitor. The activity of these
factors is therefore distinct from the activity of
factors that are the subject of the present application.
A method for assaying a megakaryocyte
maturation factor is also provided. The method is
described in Example l and comprises incubating either
crude or purified MMF with megakaryocytes and monitoring
the formation of proplatelets from megakaryocytes. Said
method is preferably carried out in the presence of an
inhibitor of proplatelet formation. An inhibitor of
proplatelet formation is present in human serum (see
Example 2).
The present invention also relates to factors
which inhibit megakaryocyte maturation and proplatelet
formation and are herein referred to as megakaryocyte
maturation inhibitors. Megakaryocyte maturation
inhibitors have properties of blocking the spontaneous
maturation of megakaryocytes to proplatelets and
stimulating the retraction of proplatelet extensions
vitro. Human serum inhibits megakaryocyte maturation
(Example 2). The inhibitory activity present in human
serum is shown to copurify with prothrombin, an
enzymatically inactive precursor to thrombin
~Example 5). Although both purified prothrombin and
thrombin inhibit proplatelet formation, thrombin has
inhibitory activity at lower concentrations than
prothrombin. It is shown that thrombin is the
megakaryocyte maturation inhibitor present in human
serum and that prothrombin in human serum is converted
to thrombin in order to inhibit megakaryocyte
maturation Isolated megakaryocytes also carry out the
conversion of prothrombin to thrombin (Example 6).
, Factors other than thrombin and prothrombin that inhibit
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proplatelet formation and induce retraction of
proplatelets are readily detected in the Ln vitro assay.
A method for purifying MMF is also provided.
This method comprises the steps of lysing human
platelets and subjecting the human platelet lysate to
two steps of ion exchange chromatography (e.g., DEAE and
Mono Q). Throughout the procedure, the presence of MMF
is detected by Ln vitro maturation of megakaryocytes to
proplatelets in the presence of an inhibitor, either
human serum or purifi~d thrombin. Platelets are lysed
in the presence of either guanidinium chloride or CHAPS
buffer (Example 8) although other methods suitable for
platelet lysis may also be used. As shown in Example 9,
platelet lysates obtained by either method are subjected
to DEAE chromatography and three distinct peaks of
proplatelet formation activity are observed ~Figs. 11
and 15). The three peaks are designated MMF-I, MMF-II
and MMF-III depending upon the salt concentration
required for elution from the column. In Fig. 15,
fractions containing MMF-I are not assayed for
proplatelet formation. The biological activities of
MMF-I, MMF-II and MMF-III are summarized in Example 12.
MMF-III is further purified by Mono Q chromatography
(Figs. 12 and 16).
Two different biologically active forms of
MMF-III are purified from human platelets. As shown in
Example 10, lysis of platelets in the presence of
guanidinium chloride to inactivate platelet proteases
results in purified MMF-III having an amino terminal
sequence starting with Y-P-T-Q. Lysis of platelets in
the presence of CHAPS buffer results in purified MMF-III
having an amino terminal sequence starting with R-I-F-P.
The sequence of 16 amino acids originating from the
amino terminus of MMF-III from the-guanidinium chloride
- 35 lysate is identical to the sequence of 16 amino acids
2~71~79
W092/06712 PCT/US91/07~7
- 12 -
originating from the amino terminus of a purified human
platelet proteoglycan (Alliel et al., ~La; Perin et
al., ~La). The complete 131 amino acid long sequence
of human platelet proteoglycan (Alliel et al., ~L~)
also contains within it a nine amino acid internal
sequence extending from residues 58 to 67 which is
identical to the first nine amino acids of MMF-III from
a CHAPS lysate. MMF-III from the guanidinium chloride
lysate is hereafter referred to as MMF1-131 and is
identical to the human platelet proteoglycan. MMF-III
from the CHAPS lysate is a truncated form of human
platelet proteoglycan representing the carboxy terminal
half of the full-length protein and is hereafter
; referred to as MMF58-131. Purified MMF1-131 stimulates
proplatelet formation in ~i~LQ in the presence of
thrombin and blocks thrombin-induced retraction of
proplatelets (Figs. 18A and 18B).
The ability of a factor having part or all of
the amino acid sequence of human platelet proteoglycan to
promote megakaryocyte maturation to proplatelets has not
been disclosed previously. Tt has been suggested that
human platelet proteoglycan may act as a carrier for
delivery of platelet factor IV to sites of blood vessel
injury or as an inhibitor of complement sub-component Clq
(Okayama et al., S~L~; Perin et al., suDra). However,
no Ln vitro or in ~i~Q biological activity of human
platelet proteoglycan has been disclosed, nor has any
therapeutic benefit resulting from the administration of
human platelet proteoglycan been described.
The present invention also encompasses
megakaryocyte maturation factors having part or all of
the amino acid sequence of MMF1-131 and having the
property of promoting proplatelet formation from mature
megakaryocytes. The factors described herein include
; 35 biologically active peptide fragments and amino acid
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variants of naturally-occurring MME1-131. Said
biologically active peptides are generated by
proteolysis of MMF1-131 either by the action of cellular
proteases in sitU or by protease treatment of full-
S length purified MMF1-131 to generate protein core
fragments having the ability to stimulate proplatelet
formation. For example, purified MMF58-131 has
equivalent proplatelet formation activity, based upon
amount of uronic acid, compared to full-length MMF1-131
(Example 11).
In a preferred embodiment, MMF is the product
of procaryotic or eucaryotic expression of exogenous
DNA, that is, MMF is preferably recombinant MMF.
Recombinant mouse MMF and human MMF1-131 are described
in Example 13. Exogenous DNA is obtained from genomic
or cDNA cloning or from gene synthesis. Expression of
MMF is carried out in procaryotic (bacteria) or
eucaryotic (yeast, plant, insect or mammalian cells)
host cells.
Analogs of MMF are also provided. Such
analogs are produced by the manipulation of DNA seq~ences
encoding the protein core of MMF1-131 to produce
deletions, additions, or subs_itutions of nucleotides
within the coding sequence so as to generate altered
amino acid sequences. Such analogs are prepared using
published procedures known to those skilled in the art.
Purified MMF having a carbohydrate structure
- different from that of naturally-occurring MMF is also
encompassed by the invention. The presence of
glycosaminoglycan side chains on MMF1-131 is essential
for megakaryocyte maturation activity as indicated by
the loss of this activity upon treatment of purified
MMF1-131 with chondroitinase ABC to remove attached
carbohydrate chains (Example 11). Variation in
carbohydrate structure can give rise to MMF molecules
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2~71~79
WO92/~712 PCT/US91/07367
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di.ffering in overall charge which are termed isoforms.
Isoforms of MMF are separated from each other and
purified by techniques such as isoelectric focusing or
chromatofocusing which have been described in the art.
The invention also provides for chemically
modified forms of MMF which may exhibit increased
solubility, stability and/or circulating half-life
compared to unmodified MMF. The covalent attachment of !
a water soluble polymer to MMF is an example of one such
chemically modified form. The water soluble polymer may
be polyethylene glycol or a copolymer of polyethylene
glycol and polypropylene glycol and said polymer is
unsubstituted or substituted at one end with an alkyl
group. These and related modifications are described in
U.S. Patent No. g,179,337 hereby incorporated by
reference.
Antibodies specifically binding to purified
MMF are also comprehended by the invention. Such
antibodies are directed to multiple antigenic
determinants (polyclonal) or are directed to a single
determinant (monoclonal) and are prepared using
procedures known to those skilled in the art. Polyclonal
and monoclonal antibodies are raised to purified
glycosylated or deglycosylated MMF1-131 and MMF58-131.
Antibodies to MMF may be used in affinity chromatography
to selectively remove MMF from media, serum, or urine.
In addition, antibodies specifically binding to MMF so as
to inhibit proplatelet formation In yitro may be used to
treat conditions resulting from excessive platelet
production by stabilizing or decreasing circulating
platelet levels.
The invention provides for the use of MMF
alone or in combination with other therapy in the
treatment of platelet disorders. The methods and
compositions of the subject invention are useful in
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'~WO92/~712 2 0 7 1 `I 7 9 PCT~US91/~7~7
treating thrombocytopenia, a condition marked by
subnormal platelet levels in the circulating blood and
the most common cause of abnormal bleeding.
Thrombocytopenia results from three processes~
deficient platelet production; (2) accelerated platelet
destruction; and (3) abnormal distribution of platelets
within the body. A compilation of specific disorders
related to thrombocytopenia is shown in Table 1 (see ! - :
Wintrobe et al.(1981) In Clinical Hematology, Eighth
edition, pp. 1090-1127).
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WO92/06712 PCT/US91/07367
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TABLE 1
Platelet Disorders
I. Deficient Platelet Production
A. ~yDoDlasia or suppression of meaakaryQ~y~es
Chemical and physicaL;agents (ionizing
radiation, antineoplastic drugs), aplastic
anemia, congenital megakaryocytic hypoplasia -
myelophthisic processes, some viral infections
B. Ineffective thrombo~oiesis ::
Disorders due to deficiency of vitamin B12 or
folic acid
C. Disordered control mechanisms
Deficiency of thrombopoietin, cyclic
thrombocytopenia
D. Miscellaneous
Many hereditary forms
II. Accelerated Platelet Destruction
A. ~Due to immunoloaic ~rocesses
Idiopathic Thrombocytopenia Purpura, drug-
. induced antibodies, various hemolytic anemia,
fetomaternal incompatibility, post-transfusion
B. pue to nonimmunoloaic processes
Kasabach-Merritt syndrome, thrombotic
thrombocytopenic purpura, infections ~viral,
- bacterial, protozoan), massive transfusions
: 30
. III. Abnormal Platelet Distribution
.,~ ' .
A. Disorders of the spleen
B. Hypothermia anesthesia
:~ 35
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Advantageous applications of the subject
invention are to thrombocytopenia resulting from
deficient platelet production and, in some cases, from
accelerated platelet destruction. In instances where
levels of mature megakaryocytes are normal but platelet
levels are low, MMF is used alone to stimulate
proplatelet formation leading to increased platelet
production. In cases where depressed platelet levels
result from low levels of megakaryocytes, MMF is used in
combination with one or more additional hematopoietic
factors such as stem cell factor (SCF), G-CSF, GM-CSF,
IL-3, IL-6, Meg-CSF, MSF, and EPO to elevate both
megakaryocyte and platelet levels.
Deficient platelet production results from a
number of processes. The most common are those that
depopulate the stem cell or megakaryocyte compartments,
such as marrow injury by myelosuppressive drugs or
irradiation, aplastic anemia, congenital megakaryocytic
hypoplasia or myelodysplastic syndrome. A purified
factor termed stem cell factor (SCF) has the ability to
stimulate the formation of early hematopoietic
progenitor cells, including megakaryocyte progenitor
; cells. SCF is described in U.S. Patent Application Ser.
No. 573,616 hereby incorporated by reference. Patients
suffering from thrombocytopenia as a result of depleted
stem cell levels are treated by administration of a
pharmaceutically effective amount of SCF in combination
with a pharmaceutically effective amount of MMF.
Thrombocytopenia resulting from depleted megakaryocyte
levels is treated by administration of a therapeutically
effective amount G-CSF, GM-CSF, IL-3, IL-6, Meg-CSF,
MSF, or EPO in combination with a therapeutically
effective amount of MMF.
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W092/~7~ 2 0 7 1 ~ 7 9 PCT/~S91/07367 ~
- 18 -
Deficient platelet production may also result
from ineffective thrombopoiesis where levels of mature
megakaryocytes are normal or even elevated but platelet
production is insufficient, as in, for example,
megaloblastic hematopoiesis. Under these conditions, a
therapeutically effective amount of MMF alone is
sufflcient to raise platelet levels. In addition,
abnormalities related to thrombopoietic control, such as
cyclic thrombocytopenia, are treated with a
therapeutically effective amount of MMF.
Accelerated platelet destruction results in
thrombocytopenia due to a more rapid rate of platelet
turnover than platelet production by meqakaryocyte
maturation. Disorders such as idiopathic
thrombocytopenic purpura (ITP), which are characterized
by accelerated platelet destruction via an autoimmune
response, may show reduced rates of platelet production.
In these instances, ITP is treated with a
therapeutically effective amount of MMF.
Also comprehended by the invention are
pharmaceutical compositions comprising therapeutically
effective amounts of MMF together with suitable
diluents, adjuvants, solubilizers, preservatives and/or
carriers. A therapeutically effective amount of MMF is
that amount sufficient to elevate circulating platelet
levels in a mammal. A therapeutically effective amount
of MMF in a pharmaceutical composition can be determined
by the ordinary artisan taking into account such
variables as the half-life of MMF preparations, route of
administration and the clinical condition being treated.
Pharmaceutical compositions of MMF include diluents of
various buffers (e.g. Tris-HC1, acetate, phosphate)
having a range of pH and ionic strength that is
compatible with MMF, solubilizers (e.g., Tween,
Polysorbate), preservatives, (e.g., Thimerosol, benzyl
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- 19 - .
alcohol) and carriers (e.g., human serum albumin).
Compositions comprising MMF may be administered by any
route appropriate to the condition being treated, for
example, by continuous infusion, sustained release
formulation, or injection. The preferred route will be
apparent to one skilled in the art.
The invention also comprises compositions of
MMF and one or more additional hematopoietic factors
such as SCF, G-CSF, GM-CSF, IL-3, IL-6, Meg-CSF, MSF, `
0 and EPO.
Megakaryocyte maturation inhibitors are used
to stabilize or decrease blood platelet levels.
Excessive platelet concentrations can lead to extensive
blood ~tting, a situation observed in deep venous
thrombc~.s and in thrombosis associated with post-
surgery recovery. Maturation inhibitors are used alone
or in combination with other therapeutics as anti-
coagulants. Heparin and aspirin are currently used in
anti-coagulation therapy.
Th~ following examples are offered to more
fully illustrate the invention, but are not to be
construed as limiting the scope thereof.
EXAMPLE 1
Assay for ProDlatelet Formation
An in vitro assay for the formation of
platelet precursors from megakaryocytes was developed
based upon observations of Radley et al., ~eL~ and
Leven et al., supra. Guinea pig megakaryocytes were
purified from bone marrow as previously described (Leven
et al., suDra). Approximately 5,000 megakaryocytes
(counted using a hemocytometer) were placed into wells
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W092/~7t2 2 0 7 1 ~ 7 9 PCT/US91/07~7 f'
- 20 -
of flat-bottomed 96-well microtiter plates (Falcon) in
100 ~1 of Iscoves media (Gibco) supplemented with S0 ~M
2-mercaptoethanol and 100 ~g/ml heat inactivated bovine
serum albumin ~Sigma). After 18 hours incubation at
37C in 7% CO2 the cells were fixed in 10 mM EDTA, 0.37%
formaldehyde and examined under bri`ght field microscopy
for the number of cells in each;~,well that had developed
proplatelet formations. The data are expressed as the
number of proplatelet formations per well (PPF/well).
Under these conditions, guinea pig megakaryocytes
elaborate cytoplasmic extensions (proplatelets) without
any other additions to the media. Photomicrographs of
developing megakaryocytes before and after incubation
are shown in Figures lA and lB.
EXAMPLE 2
Serum Inhibition of Proplatelet Formation
The addition of increasing volumes of human
serum ~Gibco) to guinea pig megakaryocytes prepared and
incubated in medium at 37C for 18 hours as described in
Example 1 inhibited proplatelet formation ~n vitro
~Fig. 2). Human serum present at 0.03% or greater
resulted in complete inhibition of proplatelet
; formation.
The inhibition of proplatelet formation by
human serum in the i~ vitrQ assay was transient. As
` shown in Table 2, megakaryocytes incubated in 0.1% human
serum do not develop proplatelet formations when
incubated for 18 hours at 37C, but do so after 42 hours
incubation at 37C.
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- 21 -
TABLE 2
Transient Inhibition of
Proplatelet Formation by Human Serum
PPF/well
Cells cultured in: 18 hours 42 hours
Human serum 0 189
media 371 322
~' 10
EXAMPLE 3
Inhibition of Proplatelet Formation
by Prothrombin and Thrombin
Thrombin, a serum-derived serine protease, was
tested for inhibition of proplatelet formation ~n vitro.
~ighly purified thrombin (Sigma) was an effective
inhibitor of proplatelet formation at concentrations
less than 10 pM and complete inhibition was observed at
25 pM (Fig. 3). The inhibitory effect of thrombin was
specific and was not observed with equivalent
concentrations of the serine proteases trypsin (human
pancreatic from Calbiochem) or chymotrypsin ~human
;~ pancreatic from Calbiochem). Nanomolar levels of
trypsin and chymotrypsin were lethal to megakaryocytes
while similar levels of thrombin did not affect
megakaryocyte viability even though differentiation was ~
30 blocked. In addition, thrombocytocin, a thrombin-like -
serine protease from ~othro~s atrox venom (Kirby et al.
Biochemistry 1~, 3564-3570 (1979), obtained from Sigma)
was tested for inhibition of proplatelet formation. No
inhibition was observed and cell viability was
maintained up to 2.8 nM.
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2071~79
WO92/~712 -PCT/US91/07~7
- 22 -
Thrombin activity was detected in a
c:hromogenic assay using chromogenic substrates S-2238
~Sigma) or Chromozyme-Pca (Boehringer Mannheim) as
clescribed (Lottenberg et al.~-~ethods Enzymol. 80, 341-
361 ~1981)). When thrombin:-was present in complex
mixtures, the specificity of the reaction was confirmed
by the addition of hirudin, a specific anti-thrombin
reagent. Using this assay, thrombin was detected in
lots of human serum that inhibited proplatelet
formation, but the amount present was too low to account
for the extent of inhibition that was observed ~see Fig.
2). However, prothrombin, the unprocessed precursor of
thrombin, is reported to be present in human serum at 1-
2 ~M ~Mann et al. Methods Enzymol. 80, 286-303 ~1981)).
Purified prothrombin inhibited proplatelet formation L~
vitro when added to 2-5 nM ~Fig. 4). Complete
inhibition was observed at 5 nM.
As with human serum, proplatelet inhibition by
either thrombin or prothrombin is transient. No
proplatelet formations were seen after 18 hours
incubation at 37C in the presence of either 0.35% human
serum, 25 pM thrombin, or 5 nM prothrombin. However, by
42 hours the inhibition had been overcome (Table 3).
TABLE 3
Transient Inhibition of Proplatelet
Formation by Thrombin and Prothrombin
PPF/well
Inhibitor 18 hours 42 hours
Human Serum 3 252
Thrombin 0 227
Prothrombin 0 163
None 229 312
. .
.
2071~79 `
WO92/~712 PCT/US91/0~7
- 23 -
In addition to blocking proplatelet formation,
thrombin and prothrombin induced the dedifferentiation
of proplatelets. Approximately 5000 guinea pig
megakaryocytes were incubated a~ described in Example 1
to form proplatelets, thrombin or prothrombin was then
added to 66 pM or 5.7 nM respectively, and the cultures
were returned to 37C. The number of proplatelets
remaining were counted at the times indicated in Fig. 5.
ExAMæLE 4
Effect of Inactivatin~ Thrombin on
proplatelet Inhibition Functions
Purified thrombin (12.5 ~g/ml) in 40 mM Tris,
pH 8.0, 100 mM NaCl, 2 mM ^aCl2 and 150 ~g/ml bovine
serum albumin was reactec 1th 4.2 mM final
concentratlon of diisopropyl fluorophosphate (DFP,
obtained from Sigma) for two hours at room temperature
to inactivate serine esterase activity. After
incubation, the DFP-reacted thrombin was dialyzed
extensively against 40 mM Tris, pH 8.0, 100 mM NaCl ar;
2 mM CaCl2 before use. Inactivation of serine esterase
activity was confirmed by the inability of DFP-reacted
thrombin to use chromozyme-Pca as a substrate.
DFP-reacted thrombin was compared to unreacted
thrombin for its ability to block proplatelet formation
(Fig. 6A) and induce retraction of proplatelet
extensions (Fig. 6B). Inactivate thrombin had 2% and
1.5~ of the activity of thrombin in preventing
proplatelet formation and inducing proplatelet
retraction, respectively.
In addition to chemical inactivation of
thrombin, thrombin inhibition of proplatelet formation
is prevented by the addition to the proplatelet assay of
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2071~7~ ` I
WO92/~712 PCT/US91/07~7
- 24 -
agents which neutralize thrombin or prevent conversion
of prothrombin to thrombin. As shown in Table 4,
addition of 2.5 mM EDTA, 0.04 unit,s/ml heparin, 0.04
units/ml hirudin, or 0.10 units/ml antithrombin III
: 5 allows proplatelet formation in ~lhrQ~
TAPLE 4
10Agents Which Neutralize Thrombin
Prevent Inhibition of Proplatelet Formation
Neutralizing PPF
: Ex~. Inhibitor Aaent well
1 None None 120
Human Serum None 2
Human Serum 2.5 mM EDTA 106
':
20 2 None None 230
Human Serum, None 2
Human Serum 0.04 units/ml Heparin 214 ..
. . ,
3 None None 162
' 25 Human Serum None 0
Human Serum 0.04 units/ml Hirudin 143
., .
., 4 None None 255
Human Serum None 24
Human Serum 0.10 units/ml , 278
`~ Antithrombin III
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2071~79
;W092/~712 PCT/US91/07~7
- 2S -
EXAMPLE 5
Purification of Human Serum Inhibitor
and Com~ariqon With Prothrombin
The inhibitory activity present in human serum
was purified by DEAE-Sepharose chromatography ~Fig. 7)
and Superose 6 chromatography (Fig. 8). Prot~-ombin
levels were measured by conversion of prothro~in to
thrombin with snake venom prothrombinase (Owen et al.
Thrombosis Res. ~, 705-714 (1973), obtained from Sigma)
and assayed as described in Example 3 for thrombin. 20
ml of human serum were dialyzed against 40 mM Tris-HCl,
pH 8.0 and loaded at 2 ml/min onto a 300 ml bed volume
DEAE-Sepharose column (5 cm x 15 cm) equilibrated with
the same buffer. Proteins bound to the column were
e:.~ted with a linear NaCl gradient from 0 to 1 M in the
same buffer. As shown in Figure 7, the peak of
proplatelet inhibitory activity coincided with the peak
obtained by the prothrombin assay. Fractions
corresponding to this peak were pooled, concentrated and ~;
loaded at 0.75 ml/min onto a Superose-6 gel filtration
column equilibrated in 20 m~ Tris-HCl, 0.1 M NaCl, 0.01%
polyethylene glycol 600, pH 7Ø As shown in Figure 8,
; 25 the proplatelet inhibitory activity eluted as a single
broad peak having a molecular weight slightly higher
than bovine serum albumin and coinciding with the peak
obtained by prothrombin assay.
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WO9~/067l2 207 1 ~7 9 PCT/U591/07~7
- 26 -
:
~ EXAMPLE 6
Conversion of prothrom~in
to Thrombin by Meaakaryocytes
s
Prothrombin is biologically inert until it is
enzymatically converted to thrombin. The ability of
megakaryocytes to convert prothrombin to thrombin is
shown in Fig. 9.
Megakaryocytes were prepared as described in
Example 1. Megakaryocytes with proplatelet formations
- (PPF-megs) were prepared by incubating megakaryocytes as
described in Example 1. Platelets were isolated from
guinea pig marrow by differential centrifugation; they
remain in the supernatant after centrifugation at
500 x g for ten minutes and are pelleted at 1,500 x g. .
Prothrombin was added to the indicated number of guinea
pig platelets, megakaryocytes ~megs) or megakaryocytes
with proplatelet formations (PPF-megs) to 143 ~gJml
final concentration and the cultures were incubated for
one hour at 37C. The culture supernatants were
recovered and assayed for thrombin using the chromophore
S-2238 as described in Example 3. Thrombin was
generated under these conditions only when cells and
;~ 25 prothrombin were both present. Megakaryocytes and
megakaryocytes with proplatelet formations were equally
effective at the conversion of prothrombin to thrombin
while several hundred times more platelets than
megakaryocytes were needed for the conversion.
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EXAMPLE 7
~Y~aL ~o-f Serum Inhibition by a Meaakaryocyte Factor
5 Either human serum at O . 035 %, thrombin at
25 pM,or prothrombin at 5 nM were incubated for 42 hours
at 4C or 37C under conditions described for the ln
vitro assay. Approximately 5,000 megakaryocytes were
added and the number of proplatelets formed after 18
hours was determined. No proplatelet formation was
observed (Table 9). However, when human serum, thrombin
or prothrombin were first incubated with megakaryocytes
for 42 hours at 37C and 50 ~l of the reaction
supernatant was then transferred to fresh megakaryocyte
cultures, extensive proplatelet formation occurred after
18 hours (Table 5). The inhibitory activity of human
serum, thrombin and prothrombin had been neutralized by
prior incubation with megakaryocytes.
TABLE 5
Effect of Inhibitor Pretreatment
on Proplatelet Formation
Pretreatment of Inhibitor
PPF/well
42 hrs, 37C,
Tnh;hitor42 hrs. 4C 42 hrs. 37Cwith ~egs
- Human Serum 0 62 327
. .
Thrombin 0 0 218
None 279 298 343
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WO92/06712 2071479 PCT/US91/07~7 ~
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Megakaryocytes were incubated in medium as
described in Example l in the absence of inhibitor for
42 hours at 37C. The conditioned medium was harvested,
concentrated six-fold by centrifugation through a
Centricon-lO membrane filter, and 50 ~l of the
concentrated medium was incubated with an equal volume
of fresh megakaryocytes and either human serum or
thrombin for 18 hours at 37C. Proplatelet formation
was observed when megakaryocyte conditioned medium was
used, whereas inhibition occurred in the presence of
unconditioned medium (Table 6~.
TABLE 6
Stimulation of Proplatelet Formation
by Megakaryocyie Conditioned Medium
Megakaryocyte
Inhibitor Conditioned Medium PPF/Well
None No 20S
Human Serum No l5
Human Serum Yes 258
Thrombin No l8
25 Thrombin Yes 223
These experiments indicated that
megakaryocytes produce and secrete soluble factors that
: 30 neutralize or functionally override the inhibition of
-.~ proplatelet formation by human serum or purified
` thrombin. These factors are referred to as
megakaryocyte maturation factors (MMF).
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EXAMPLE 8
Stimulation of ProDlatelet Formation
by Human Platelet Lysate~
The presence of megakaryocyte maturation
factors in platelets was determined by preparing human
platelet lysates and assaying for in vitro proplatelet
formation (Fig. 10).
Human platelets from normal donors were
obtained in plateletpheresis packs containing
approximately 3-4 x 1011 platelets in approximately 200
ml of platelet rich plasma ~PRP, obtained from
HemaCare). Platelets were used within 24 hours of the
- 15 draw. Apyrase (Sigma) was added directly to the blood
bag to a final concentration of 2 units/ml and incubated
at 37C for 20 minutes. PRP was transferred to 50 ml
polypropylene tubes and centrifuged at 120 x g for 8
minutes at room temperature to remove contaminating
blood cells. The supernatant was transferred to
polycarbonate tubes and centrifuged at 1,500 x g for 20
minutes to pellet platelets.
For platelet lysis in CHAPS, the pellet was
washed three times by centrifugation at 1,500 x g for 20
- 25 minutes at room temperature and resuspension in the
following buffers: Wash 1, 280 ml of Tyrodes buffer (137
mM NaCl, 2.7 mM KCl, 12 mM NaHC03, 0.4 mM NaH2P04, lmM
MgCl2, 2 mM CaC].2, 5.5 mM dextrose, pH 7.35)
. . .
supplemented with ~.4% human serum albumin and
2 units/ml Apyrase; Wash 2, 140 ml Tyrodes buffer and
` 2 units/ml Apyrase; Wash 3, 140 ml Tyrodes buffer -
followed by the final centrifugation. Platelets were
lysed in 5 mM 3-(3-cholamidopropyl)-dimethyl-ammonio-1-
propanesulfonate (CHAPS, obtained from Calbiochem) at
1.6 x 101 platelets/ml for one hour on ice. The lysate
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2071~79
WO92/~12 PCT/US91/07367
- 30 -
was centrifuged at 2,200 x g and dialyzed against four
changes of 90 mM Tris, pH~8 (4 liters each change). The
platelet lysate was~c~arified by centrifugation at
150,000 x g for 60 minutes.
For platelet lysis in guanidinium chloride,
the pellet was washed se~uentially in the following
buffers: Wash 1, 280 ml Tyrodes buffer supplemented with
22 mM trisodium citrate, 0.4% human serum albumin and 2
units/ml apyrase, pH 6.5; Wash 2, 140 ml Tyrodes buffer
supplemented with 22 mM trisodium citxate and 2 units/ml
apyrase, pH 6.5; Wash 3, 140 ml Tyrodes buffer and 22 mM
trisodium citrate, pH 6.5 followed by the final
centrifugation. Platelet pellets were solubilized in
6 M guanidinium chloride in 50 mM sodium acetate, 10 mM
EDTA, 1 mM phenylmethylsulfonyl fluoride and 10 mM
6-amino hexanoic acid, pH 6.0 at 8 x 109 platelets/ml
for 3 hours at 4C with gentle stirring. The solution
was then dialyzed against four changes of 4 liters each
of 40 mM Tris, pH 8, 1 mM PMSF, and clarified by
20 centrifugation at 150,000 x g for 60 minutes.
EXAMPLE 9
purification of Meaakaryocyte Maturation Factors
From Human Platelets
.`: -
MMF from human platelets was pur.ified by thefollowing procedures. The presence of MMF during
purification was detected by proplatelet formation
j 30 Ln vitro in the presence of a maturation inhibitor.
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~;~WO9~/~712 2 0 7 1 ~ 7 9 PcT/Usgl/o7~
. Purification of MMF from guanidinium chloride
extracted platelets. i
A guanidinium chloride lysate of human
platelets (160 ml containing 240 mg of protein) was
prepared according to Example 8. The lysate was
equilibrated with 90 mM Tris-HCl, pH 8.0 and loaded onto
a 220 ml (2.6 cm x 40 cm) DEAE-Sepharose column at a
flow rate of 1 ml/min. The column was washed with the
Tris buffer and developed with a linear NaCl gradient
from 0 to 1 M in the same buffer (total gradient volume
was 800 ml). As shown in Figure 11, assay of column
fractions for proplatelet formation activity revealed
three distinct peaks, designated M~F-I, MMF-II and MMF-
III, eluting at different NaCl concentrations.
The fractions corresponding to MMF-III were
pooled and dialyzed against 5 mM sodium citrate, 0.01%
PEG 600, pH 5Ø The dialyzed pool (237 ml at 0.012
A2go/ml) was loaded onto a Mono-Q FPLC column (0.5 x 5
cm) equilibrated with 5 mM sodium citrate, 0.01% PEG
600, pH 5Ø The flow rate was adjusted to 0.5 ml/min.
After washing with the same buffer, the column was
developed with a linear NaCl gradient from 0 to 1 M
~total gradient volume was 60 ml) followed by a 1 M NaCl
wash. As shown in Figure 12, a broad peak corresponding
to proplatelet formation corresponds with a peak of
- absorbance at 280 nm. The fractions corresponding to
this peak were combined and the resulting pool (18 ml at
- 0.072 A2go/ml) were analyzed for purity, molecular
weight, and amino acid sequence.
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W092/~712 2 0 714 7 9 PCT/US91/07~7 ~
- 32 -
B. Analysis of purified MMF from guanidinium chloride
lysates.
2 ml of the Mono-Q pool was concentrated to
200 ~l by ultrafiltration using a Centricon-10
filtration device and loaded onto a Superose-6 gel
filtration column (1 cm x 30 cm) at a flow rate of 0.5
ml/min in 40 mM Tris-HCl, O.lmM NaCl, 2 mM CaCl2, pH
8Ø As shown in Figure 13, a peak of activity
corresponding to proplatelet formation activity
coincides with a peak and shoulder measured by
absorbance at 230 nm appearing in the void volume. This
indicates that the MMF-III preparation in heterogeneous
in size, but the different forms of MMF-III have similar
levels of activity.
2 ml of the Mono-Q pool were dialyzed against
0.1~ trifluoroacetic acid (TFA) and concentrated to 200
~l by ultrafiltration using a Centricon-10 filtration
device. The concentrated sample was loaded onto a C4-
reverse phase high pre~sure liquid chromatography column(0.46 x 25 cm Vydac C4 column 214TP54) in 0.1% TFA at
~- 0.75 ml/min and the column was developed with an
acetonitrile gradient in 0.1% TFA. As shown in Figure
14, a broad peak of proplatelet formation activity
coincided with a peak of absorbance at 214 nm, again
indicating different forms of active MMF-III.
C. Purification of MMF from CHAPS lysates of human
platelets.
A platelet lysate extracted with CHAPS buffer
as described in Example 8 and equilibrated with 40 mM
; Tris-HCl, pH 8.0, was purified by DEAE-Sepharose and
Mono-Q column chromatography as described above for the
; 35 guanidinium chloride extracted platelets. As shown in
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2071479
!' ,~ ,~0 92/~712 PCT/US9l/07~7
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- 33 -
E`igure 15, two distinct peaks of proplatelet formation
activity designated MMF-II and MMF-III were obtained by
~EAE-Sepharose chromatography. A third peak of
activity, designated MMF-I, is present in the flow-
through fractions but was not assayed in thispreparation. The peak of activity around fraction 38
(corresponding to MMF-III from the guanidinium lysates)
was pooled and applied to a Mono-Q column. As shown in
Figure 16, proplatelet formation activity was eluted in
a broad peak from fractions 28 to 36.
D. Analysis of purified MMF-IIIS by SDS-PAGE.
Aliquots of the Mono-Q pools from guanidinium
chloride and CHAPS lysates were treated with 0.1 unit of
chondroitinase ABC at room temperature for 24 hours in
40 mM Tris-HCl, pH 8Ø The samples were dried in a
speed-vac and analyzed along with untreated samples on a
12.5% SDS-polyacrylamide gel (Fig. 17). Samples that
had not undergone chondroitinase ABC treatment were not
detected in the gel, suggesting that purified MMF-III
from CHAPS or guanidinium chloride lysates had a
substantial amount of covalently attached carbohydrate
that prevented entry into the gel. Chondroitinase-
treated samples migrated as several bands on SDS-PAGE,
; suggesting that not all carbohydrate could be removed
from the protein core even after exhaustive digestion.
.
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2071~73
W O 92/06712 PC~r/US91~07367
- 34 -
EXAMPLE 10
N-terminal Amino Acid Se~uences of
MMF-IIL_~om Guanidinium Chloride and CHAPS Lysates
of Human Platel~
MMF-III purified from guanidinium chloride and
CHAPS lysates of human platelets were subjected to
N-terminal sequencing using Applied Biosystems Models
470A and 973A sequencers with on-line PTH analysis using
the manufacturer's high pressure liquid chromatography
systems. Sequence assignments were made by comparison
of the cycle to cycle chromatograms. The following
sequences were assigned:
MMF-III from guanidinium chloride lysate:
Y-P-T-Q-R-A-R-Y-Q-W-V-R-X-N-P-D
MMF-III from CHAPS lysate:
R-I-F-P-L-S-E-D-Y
The N-terminal amino acid sequence determined
for MMF-III from the guanidinium chloride lysate was
identical to the N-terminal sequence of human platelet
proteoglycan (Alliel et al., supra; Perin et al.,
supra). MMF-III from the guanidinium chloride lysate,
; which is identical to human platelet proteoglycan, is
~`~ referred to as MMF1-131.
The N-terminal sequence of MMF-III from the
CHAPS lysate was identical to the sequence of amino
acids 58 to 66 of human platelet proteoglycan (Alliel et
al., ~L~; Perin et al., supra). MMF-III from the CHAPS
- lysate is identical to the carboxy terminal 64 amino
- acid fragment of human platelet proteoglycan (and
MMF1-131) and is referred to as MMF53-131.
` 35
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2071479 `
WO92/~712 PCT~US9~/07~7
- 35 -
EXAMPLE 11
A~Yi~Y of Purif;ed MMF1-131 and 58-131
MMF1-131 and MMF58-l3l were assayed for uronic
acid content as described (Bitter and Muir, Anal.
Biochem. 4, 330-339 (1962)). Protein concentrations
were determined by theoretical extinction coefficients
based upon the amino acid sequence data of each form and
amino acid yields obtained during sequencing. MMF58-13
had approximately 140 ~g uronic acid/~g protein and
MMF1-131 had approximately 80-lO0 ~g uronic acid/~g
protein.
MMF1-131 was assayed for its ability to
prevent thrombin-induced inhibition of proplatelet
formation ~Fig.18A). Purified thrombin was serially
diluted in Iscoves media or in an MMF1-131 preparation,
added to culture wells and incubated at 37C for 3
hours. MMF1-131 was present at 0.1 ~g/ml protein and 10
~g/ml uronic acid and thrombin was present from 0.35-100
pM. Approximately 5000 megakaryocytes per well were
added and the number of proplatelets in each well was
counted after 18 hours.
MMF1-131 was assayed for its ability to
prevent thrombin-induced proplatelet retraction ~Fig.
18B). Purified thrombin and an MMF1-131 preparation
were distributed into culture wells as described above
and incubated at 4C for 18 hours. The contents of the '
- wells were transferred to wells containing proplatelets
and the number of proplatelets remaining were counted
; after 10 minutes.
MMF1-131 and MMF58-131 were added to the
ln vitro proplatelet formation assay described in
Example 1 at equivalent uronic acid concentrations and
proplatelet formations were determined ~Fig. 19).
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MMF1-131 and MMF5~8 131 were equally active (per ~g of
uronic acid) in this assay.
The role of attached carbohydrate ~chondroitin
sulfate) in the biological activity of MMF-III was
determined. MMF58-131 from DEAE chromatography was
incubated in 40 mM Tris, 90 mM Na acetate, pH 8.0 in the
presence or absence of 0.1 unit/ml chondroitinase ABC
(Boehringer Mannheim) for 18 hours at 37C. Treated
MMF58-131 was exchanged into Iscoves media and added at
up to 50$ of the volume the proplatelet assay. The
results in Fig. 20 show that MMF53-131 treated with
chondroitinase ABC lacks detectable proplatelet
formation activity.
EXAMPLE 12
Pro~erties of Meaakaryocvte Maturation Factors
.Separated bv DEAE ChromatoaraDhy
Table 7 shows a comparison of the biological
activities of MMF-I, MMF-II and MMF-III which were
obtained by lysis of human platelets in CHAPS buffer as
described in Example 8 and DEAE chromatography as
described in Example 9.
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WO92/06712 PCT/US91/07;~7
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EXAMPLE 13
., .
Clonina and Ex~ression of Mouse and Human ~MFl-131 Genes
Except where noted, recombinant DNA procedures
described in Maniatis et al. (Molecular ~lQn1n~, Cold
Spring Harbor Laboratory, pp. 212-246 (1982)) were used.
A. Amplification and Cloning of the Mouse MMF cDNA.
RNA was purified from the murine cell line
MC/9.5, a subclone of MC/9 (ATCC No. CRL 8306) using the
cesium trifluoroacetate pelleting protocol (Okayama et
al. Meth. Enzym. 154, 3-28 (1987)). Oligonucleotide
primers M1-M4 were designed from the published cDNA
sequence of a mouse mast cell secretory granule
proteoglycan (Avraham et al. Proc. Natl. Acad. Sci. USA
86, 3763-3767 (1989)) and synthesized on an Applied
Biosystems DNA synthesizer.
First strand cDNA synthesis was derived from
MC/9.5 RNA as template and the antisense primer
5'-CTGAATACATTGTTCCACATGG-3' (Ml) -
whose sequence is complementary to a portion of the cDNA
sequence of mouse mast cell secretory granule
proteoglycan at the 3' side of the protein coding
region. cDNA synthesis was carried out with M-MLV
` reverse transcriptase using procedures supplied by the
manufacturer (Bethesda Research Laboratories,
Gaithersburg, MD).
First strand cDNA from about 60 ng of RNA was
; used as template for polymerase chain reaction (PCR)
; amplification (Saiki et al. Science 239, 487-491 (1988))
using the oligonucleotide primer
5'-CTAATCCAGAGGCTGAGTGGA-3' (M2)
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a sense strand pr~mer positioned at the 5' side of the
coding region. The product of this PCR amplification
was further amplified using the nested primers
5'-GACGGATCCAAGCTTCCACCATGCAGGTTCCCGTCGGCA-3'
(M3) and
5'-GTGAGTCGACAGAGACCGTCACATTCA-3' ~M9).
Primer M3 contains the sequence 5'-CCACC-3' immediately
preceding the coding sequence for murine MMF-III, such a
sequence having been shown previously to be optimal for
translational efficiency (Kozak, Nuc. Acid Res. 1~,
8125-8148 (1987)).
The products of PCR amplification using
primers M3 and M4 were digested with BamHI and SalI and
ligated into pDSR~2, a derivative of vector pCD (Okavama
et al., Mol. Cell. Biol. 3, 280-298 (1983)), yieldir.
the recombinant plasmid pDSR~2 (muMMF). The DNA
sequence of murine MMF insert was determined by the
dideoxy method (Sanger et al. Proc. Natl. Acad. Sci.,
USA ~, 1934-1938 ~1977)). The sequence of murine MMF
was identical to that reported for the mouse mast cell
; secretory granule proteoglycan (Avraham et al., ~upra).
.
B. Amplification and Cloning of the Human1~131 cDNA.
RNA was purified from a human leukemic cell ~;
line (HEL, ATCC No. TIB 810) using procedures described
j~ above. Oligonucleotide primers Hl-H4 were designed from
the sequence of the human secretory granule proteoglycan
(Stevens et al., supra).
- First strand cDNA synthesis was derived from
HEL RNA as template and the human MMF antisense primer
5'-TGCTAACTAATTGCCTGGTGT-3' (Hl).
PCR amplification was performed with primers H1 and
5'-GAGAGCTAGACTAAGTTGGTCA-3' (H2).
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The product of PCR was further amplified using the
nested primers
5'-GAGGATCCAAGCTTCCATGATGCAGAAGCTAC-3' tH3)
and
5'-GCCGTAGTCGACAACCTGGGAAAACCTCTT-3' (H4)
which contain the restriction sites HindIII and SalI,
respectively.
The product of PCR amplification using primers
H3 and H4 were digested with HindIII and SalI and
ligated into pDSRa2 as described above yielding the
recombinant plasmid pDSRa2 (hUMMFl-l3l). The DNA
sequence of human MMF was determined by the dideoxy
method (Sanger et al. supra) following irreversible
denaturation of supercoiled DNA. The sequence of human
MMF was identical to that reported for the human
secretory granule proteoglycan (Stevens et al., sy~La)-
C. Expression of murine and human MMF-III.
For expression of mouse and human MMF, plasmid
pDSRa2 (huMMF-III) or pDSRa2 (muMMF) was transfected
into COS cells by electroporation (Potter et al. Proc.
Natl. Acad. Sci. ~SA ~1, 7161-7165 (1984)) or into
Chinese Hamster Ovary (CHO) cells by calcium phosphate
25 coprecipitation (Wigler et al. Cell 11, 223-232 (1977)).
Transfected COS cells were grown for 2-5 days at 37 in
Dulbecco's modified essential medium (DMEM) supplemented
with 1% fetal calf serum (FCS). Conditioned media is
harvested and assayed for proplatelet formation in Yi~LQ
- 30 as described in Example 1. Transfected CHO cells were
seeded at a low density (-105 cells/100 mm dish) and
grown for 10-14 days at 37C in DMEM supplemented with
nonessential amino acids and 10% dialyzed FCS. Colonies
` were picked or cells were treated with trypsin and
transferred to fresh media for an additional 10-14 days.
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Conditioned media is harvested and assayed for
proplatelet formation ~n vitro as described in
E,xample 1. Transfected CHO cell cultures that stimulate
proplatelet formation are then grown in the presence of
methotrexate to amplify MMF expression.
EXAMPLE 14
Effect of MMF1-l3l on Blood Platelet Levels
Experiments designed to determine the effects
of administering MMF1-13l on circulating platelet levels
were performed on female Balb/c mice (Charles River) 6-8
weeks old. All animals within an experiment were from
age-matched litters.
MMF1-131 was purified from human platelets as
described in Example 9. Human recombinant IL-6 was
purified from CHO cell conditioned media. Mice were
injected subcutaneously with 200 ~l of either MMF1-13
or IL-6 in 150 mM NaCl, 0.1% bovine serum albumin ~BSA)
- two times per day at eight hour intervals for a total of
ten injections. Three hours after the final injection,
a 20 ~l blood sample was taken from each animal through
a small incision in the lateral tail vein using
calibrated microcapillary tubes. The samples were
~ diluted directly into a diluent required for analysis in
`~ a Sysmex microcell counter F-800 (TOA Medical
Electronics Co.). The resulting data were analyzed by
Scheffe's F-test using that Statview 512+ software
program. Data having significance at greater than 95%
are indicated by an asterisk.
MMFl-13l increased platelet levels when
administered at 4 ~g/day or 20 ~g/day ~Fig. 21). A
statistically significant increase of 21% in platelet
levels was observed when MMF1-131 was administered at a
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207~9
W092/06712 ` PCT/US91/07~7
- 42 -
dose of 20 ~g/day. IL-6 also increased platelet levels
when administered at 2 ~g/day or l0 ~g/day with a
statistically significant increase of 34% observed at a
dosage of l0 ~g/day (Fig. 22). A combination of
MMF1-l3l at 20 ~g/day and IL-6 at ~2-~g/day resulted in a
40% increase in platelet levels. -This increase is
statistically significant compared to the levels
obtained upon administration of only MMF1-131 at 20
~g/day or only IL-6 at 2 ~g/day (Fig. 23). Under the
conditions of the experiment, the doses of MMF1-131 and
IL-6 used did not, by themselves, raise platelet levels
to significantly higher levels. Other hematological
parameters such as white and red blood cell counts and
hematocrit were unaffected by MMF1-131 or IL-6
treatments.
* * *
While the present invention has been described
in terms of the preferred embodiments, it is understood
that variations and modifications will occur to those
skilled in the art. Therefore, it is intended that the
appended claims cover all such equivalent variations
` ~ which come within the scope of the invention as claimed.
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