Plasmin is present in blood to prevent unwanted clotting by catalysing the breakdown of the fibrin polymer that provides the framework of a blood clot. Plasmin is formed from plasminogen, a process that occurs after plasminogen has been activated by forming a complex with fibrin. After certain tissue fragments had been shown to possess plasminogenactivating ability, a soluble fraction possessing this activity was extracted and purified. Cloning and expression of complementary DNA (cDNA) for human tissue-type plasminogen activator permitted commercial production of alteplase.
Alteplase was shown to be a potent thrombolytic agent by virtue of its ability to activate plasminogen, thus breaking down fibrin in the thrombus. An early clinical study in patients with coronary occlusion following myocardial infarction showed that it was an effective drug for dissolving clots in coronary arteries. It was marketed for this purpose in 1988.
A 5 gallon drum of frozen plasma oxalated with a known anticoagulant
quantity and proportion of oxalic acid and sodium oxalate as described in US
Patent 2,394,566 is permitted to stand at room temperature (24° to 26°C) for
24 hours after which the remaining unmelted portion is broken up with an ice
pick and a stainless steel warming coil containing running warm water at
about 40°C is inserted into the mixture and the mixture stirred. The
remaining frozen material is rapidly melted. The warming is then continued
with vigorous agitation.
When the temperature of the plasma reaches about 5° to 8°C, the calculated
quantity of calcium chloride solution is added in amount which is from 0.2 to
0.3% in excess of that needed to react with and precipitate the anticoagulant.
The temperature of the plasma is allowed to rise to about 24°C. At 18° to
24°C strands of fibrin begin to appear and the vigor of stirring is increased to
prevent a gel of fibrin from forming. Stirring is continued or 30 minutes after
the fibrin is whipped out to allow for complete conversion of all prothrombin to
thrombin and for the antithrombin to completely destroy all thrombin. At the
end of this time the stirring is stopped, the fibrin allowed to rise to the surface
and the clear serum siphoned off.
If, through failure to stir with enough vigor, a gel forms instead of strands of
fibrin, when the temperature reaches about 18°C, the serum can also be
obtained from the fibrin by working and kneading the gel in a cheesecloth bag
while draining off the clear serum. However, this method is time-consuming
and it is preferred to prevent gel formation by very vigorous stirring of the
mixture.
The clear serum of this example is an amber liquid free from prothrombin,
thrombin, fibrinogen and fibrin. It contains profibrinolysin and is excellently
suited to further purification by salt precipitation fractionation, as given below.
The special serum is brought to a temperature of about 4° to 6°C (preferably
5°C) and saturated ammonium sulfate solution added drop by drop with
constant stirring to about 24 to 26% of saturation (preferably 25%). The
precipitated protein impurities are then centrifuged off and the supernatant
brought to about -1° to +1°C (preferably 0°C). The degree of its saturation is
then brought to about 28 to 31% of saturation (preferably 29%) by further
addition of ammonium sulfate solution with stirring. This further degree of
saturation precipitates the profibrinolysin which is collected by centrifugation
and separated from soluble impurities. By washing the profibrinolysin several
times with ammonium sulfate solution of a strength which is 29% of
saturation a practically white solid is obtained which can be freeze-dried
(frozen and dried under reduced pressure) to give a dry, white, product
containing purified profibrinolysin free from thromboplastin, prothrombin,
thrombin, fibrinogen and fibrin, (from US Patent 2,624,691), which is then
activated to fibrinolysin.
