close

Вход

Забыли?

вход по аккаунту

?

Патент USA US2408535

код для вставки
2,408,535
_ Patented Oct. 1, 1946
- UNITED ? '? srArEs ?PATENT ' A OFF ICE,
.THROMBOPLASTIN PnonUo'r?ANnraooEss ?
FOR PREPARATION or? SAME
'-
1
Harry ?B. Smith,? lowa. (li?y'g?iowafassignor. to
.
.
Parke, Davis 8: C0mpany,;De.troit, Micl'n, acor'
;I'1)Ql'ation
of
lliichigan
"
Y
I
-
I
N'oDraWingQ
~ Serial-No; 332,397.
Original Divide'd'eand?this
application Amaze-1940,
applicaf-
' tion June 22, 1942, Serial N0.>4,033j _
.
~
f ? ? ? {_ v Y -
"'
~ .
'
?
?5 Claims. (01, liege-24,3
The invention relates to the preparation of
?mixture is centrifugaliz'ed and the. fluid obtained
is diluted with an ?equal volume? of saline. Any
antithrombin-free thromboplastin which is an
prothrcmbin present is removed by adding one
sixth ?volume of magnesium hydroxide suspen
intermediate product useful in the preparation
?of highly active thrombin: preparations capable
of e?ective use in clotting blood.
sion followed by centrifugalization. .
,_ _
2,398,027, issued, April o, 1946, in which the
thrombin preparations and, processes 'ior obtain?
.ing the same are claimed. a
It is known thatextracts of tissues, such as
~made" by adding 25 cc. of concentrated ammoni
um- hydroxide to 100 cc. of 20 %.Vmagnesium chlo
ride, decanting and washing the precipitate sev
10 eral times with water, centrifugalizing .and-gsus
pending the packed precipitate in 30 cc. of saline. _
saline; extracts oiibrain tissueand other body
?After centrifuging off the magnesium hydrox
tissues, e. g. those from the lung and kidneys,
idelwithits adsorbed prothrombin and otherim
purities?, 100 cc;_o�-ammonium sulfate solution
contain a substance or activityrwhich has been
designated "as thromboplastin. The thrombo
plastin preparations previously obtained have
?saturated at v5� C. area'dded to eachlOQ cc. of the
clear solution remaining. A precipitate of throm
boplastin forms, is centrifuged off, and the pre
sipitate dissolved in 100 cc. oi'saline. If desired,
the precipitation can be repeated and the ?nal
I not been of su?icient purity for all purposes, be
cause they contain a certain amount of sub
stances, termed ?antithrombins,? which have
1 the property of destroying thrombin.
previous
thromboplastin
Thus, the
preparations, have
'
? ~ The magnesium hydroxide suspension can? be
This application is_a division of my Patent No.
20 precipitate dissolved in? 15 cc. of saline and dia
proved to be unsatisfactory for use in obtaining
lyzed against saline until free of ammonium sul
stable thrombin preparations. 7
fate.
The saline solution of thromboplastin thus '
obtained is free from antithrombins and can be
Another disadvantage of previously known
used in preparing thrombin as described in my
thromboplastin preparations is that they will not
completely dissolve in saline, for example, physi ~25 Patent No._2,398,077.
The saline thromboplastin solution can be
ological saline (0.85% NaCl). This undesirable
evaporated to dryness,v if desired, in a vacuum.
characteristic of incomplete solubility in saline
The thromboplastin is completely soluble in
is the more likely to be present, the more one
saline. It will be noted that the magnesium hy
tries to purify the'throboplastin preparation and _
droxide suspension used in preparing the throm
eliminate antithrombins from the same. The
boplastin
of this example leaves the solution of
numerous treatments. especially? where these "
thrombo-plastin' at an alkaline pH (about pH 9 to
could not be properly controlled, always tended I
pH 9.5) during the step of precipitation with am
to denature the product and render considerable
monium sulfate. The thromboplastin obtained
amounts of it insoluble in saline.? For this rea
.' by this example is free from antithrombins and
son, it has not been possible heretofore to pre
this can be demonstrated by mixing the product
pare a thromboplastin which is notonly com
with measured amounts of thrombin and testing
pletely free from antithrombins, but which is also
for destruction of thrombin?oy any antithrom
completely soluble in saline.
.
bins, aftera period of 10 hours.
I have found that salt precipitation of a tissue
extract containing throboplastin, when carried 40 Instead of evaporating the saline solution of
thromboplastin of this example to dryness, it can
out at an alkaline pH, preferably pH- about 9,
?rst? be dialyzed against pure water and then
with a plurality of such salt precipitations not to
dried to give a puri?ed, stable, antithrombin-free
exceed about 5 times, gives a product which is
pro-duct which is completely soluble in saline.
not only free from antithrombins but which is
As previously stated, the new antithrombin
completely soluble in dilute sodium chloride so 45
free thromboplastin is of particular utility in the
lution. Such a product is extremely valuable,
since it can be used to prepare stable thrombin ' manufacture of highly active thrombin prepara
tions as set forth in my application, Serial No.
preparations which, prior to the present inven
tion, could not be obtained by the use of the
50
known thromboplastin extracts.
Example.--Preparation of thromboplastin
332,397.
_
What I claim as my invention is:
1._Process for the preparation of antithrom
loin-free thromboplastin completely soluble in
saline which comprises ?rst removing part of im
100 grams of fresh ground beef lung are mixed
purities with magnesium hydroxide and then
with 100 cc. of saline and allowed to stand with
occasional stirring, for 48 hours at 5� C. The 55 adding a protein-precipitating salt to precipitate
2,408,535
3
a tissue extract containing thromboplastin at an
alkaline pH for a plurality of times not to exceed
about ?ve times.
2. Process for the preparation of antithrom
bin-free thromboplastin completely soluble in
physiological saline solution which comprises ?rst
removing part of the impurities from an animal
tissue extract containing thromboplastin by ad-'
sorption of said impurities on magnesium hy
droxide, separating the magnesium hydroxide
and adsorbed impurities from the remaining solu
tion of unadsorbed thromboplastin andv eliminat
ing additional impurities by adding a protein
precipitating salt to the partially puri?ed throm
boplastin solution in thromboplastin precipitat
ing proportions at an alkaline pH,?separating the
resulting precipitate of thromboplastin from the >
..
c, i
_'
4
nium sulphate to the partially puri?ed thrombo
plastin solution in thromboplastin precipitating
proportions at an alkaline pH, separating the re
sulting precipitate of thromboplastin from the
solution containing unprecipitated impurities, re
dissolving the thromboplastin precipitate in sa
line solution and repeating the precipitation at
alkaline pH and separation from unprecipitated
impurities a number of times not to exceed about
four, and thereafter dialyzing a solution of the
puri?ed thromboplastin against physiological
saline solution.
4. Process for the preparation of antithrom
loin-free thromboplastin completely soluble in
saline which comprises ?rst removing part of
the impurities from an extract containing throm
boplastin with magnesium hydroxide, adding a
solution containing unprecipitated impurities, re?
protein-precipitating salt, separating and re
dissolving the thromboplastin precipitate in saline
dissolving the precipitate in physiological saline
solution and repeating the precipitation at alka 20 solution and removing said protein-precipitating
line pH and separation from unprecipitated im
salt by dialysis of said solution against a physio
purities a number of times not? to exceed about
logical saline solution.
four,'and thereafter dialyzing a solution of the
5. Thromboplastin in stable puri?ed form, de
puri?ed thromboplastin against physiological
rived from animal tissue, completely soluble in
saline solution.
'
3. Process for the preparation of antithrom
bin-free thromboplastin completely soluble in
physiological saline solution which comprises ?rst
removing part of the impurities from an animal
?tissue extract containing thromboplastin by ad
sorption of said impurities on magnesium hy
droxide, separating the magnesium hydroxide
and adsorbed impurities from the remaining so
lution of unadsorbed thromboplastin and elimi
nating additional impurities by adding ammo
physiological saline solution, insoluble in con
centrated solution of ammonium sulphate, free
from antithrombin and antithrombin substances
associated with thromboplastin in said animal tis
sue from which said thromboplastin is derived,
and which is an intermediate product useful in
the preparation of a stable and highly active an
tithrombin-lfree thrombin capable of effective use
in clotting blood.
HARRY P. SMITH.
Документ
Категория
Без категории
Просмотров
0
Размер файла
262 Кб
Теги
1/--страниц
Пожаловаться на содержимое документа