Hr. Lijnen et al., FUNCTIONAL-PROPERTIES OF A RECOMBINANT CHIMERIC PROTEIN WITH COMBINEDTHROMBIN INHIBITORY AND PLASMINOGEN-ACTIVATING POTENTIAL, European journal of biochemistry, 234(1), 1995, pp. 350-357
A chimeric protein (rscu-PA-40-kDa/Hir), consisting of the C-terminal
amino acids 53-65 of hirudin (Hir), fused via a 14-amino-acid linker s
equence to the C-terminal of a 40-kDa fragment (Ser47-Leu411) of recom
binant (r) single-chain (sc) urokinase-type plasminogen activator (rsc
u-PA), was produced by expression of the corresponding chimeric cDNA i
n Escherichia coli cells. The thrombin inhibitory potential of purifie
d rscu-PA-40-kDa/Hir was confirmed by complete inhibition of the coagu
lant activity of thrombin at 20-30-fold molar excess of the chimera, a
nd by the resistance of rscu-PA-40-kDa/Hir to proteolytic cleavage by
thrombin. rscu-PA-40-kDa/Hir prolonged the thrombin time of normal hum
an plasma in a dose-dependent way (reduction of the apparent thrombin
concentration to 50% with 95 nM chimeric protein as compared to 4.7 nM
hirudin), and inhibited thrombin-mediated platelet aggregation (reduc
tion of the apparent thrombin concentration to 50% with 40 nM chimeric
protein). The chimera had a specific activity on fibrin films of 5700
0 IU/mg as compared to 95000 IU/mg for rscu-PA. The urokinase-like ami
dolytic activity of the single-chain protein was only 220 IU/mg but in
creased to 169000 IU/mg after treatment with plasmin, which resulted i
n quantitative conversion to a two-chain (tc) derivative (rtcu-PA-40-k
Da/Hir). Corresponding values for rscu-PA were 270 and 226000 IU/mg. T
he catalytic efficiencies for plasmin-mediated conversion to two-chain
molecules were comparable for rscu-PA-40-kDa/Hir and rscu-PA (0.63 an
d 0.65 mu M(-1). s(-1), respectively). The plasminogen-activating pote
ntial of the single-chain chimera was comparable to that of rscu-PA; t
he catalytic efficiencies for plasminogen activation by their two-chai
n counterparts were also similar (0.55 and 0.73 mu M(-1). s(-1), respe
ctively). In 2 h, 50% lysis of I-125-fibrin-labeled clots prepared fro
m platelet-poor human plasma and immersed in normal plasma was obtaine
d with 1.3 mu g/ml rscu-PA-40-kDa/Hir and with 0.67 mu g/ml rscu-PA, w
ith corresponding residual fibrinogen levels of 74% and 87%, respectiv
ely. In the absence of fibrin, 50% fibrinogenolysis in 2 h in normal h
uman plasma required 2.1 mu g/ml rscu-PA, but 7.9 mu g/ml rscu-PA-40-k
Da/Hir. Thus, the chimera rscu-PA-40-kDB/Hir has maintained the specif
ic fibrinolytic and plasminogen acti vating activity of rscu-PA as wel
l as its fibrinolytic potency in plasma: whereas it displayed a simila
r or somewhat better fibrin specificity. In addition, the fibrinolytic
ally active concentration in a plasma medium is severalfold lower than
the concentration required for thrombin inhibition, which may limit s
ystemic anticoagulant activity. Therefore, further evaluation of the t
hrombolytic and antithrombotic potential of such chimeric molecules se
ems to be war-ranted.