Ralph Pannell

Effective and fibrin-specific clot lysis by a zymogen precursor form of urokinase (pro-urokinase). A study in vitro and in two animal species.

A single-chain 55,000-mol wt form of urokinase (UK), similar to that previously isolated from urine, was purified from a transformed kidney cell culture medium and characterized; and its fibrinolytic properties were evaluated. The preparation immunoprecipitated with UK antiserum, had a low intrinsic amidolytic activity that was 0.1% of its active derivative, and resisted diisopropyl fluorophosphate treatment and inactivation by plasma inhibitors. The single-chain UK was therefore designated pro-UK. In the presence of plasmin and during clot lysis, activation by conversion to two-chain, 55,000-mol wt UK (TC-UK) was demonstrated. This did not occur during blood clotting nor on incubation with purified thrombin. Clot lysis in plasma consistently occurred in 2-5 h with 50-100 IU per ml of pro-UK, whereas comparable lysis was inconsistently achieved by 500-1,000 IU of UK. Pro-UK, in sharp contrast to UK, caused no fibrinogen degradation at fibrinolytic concentrations. In the absence of a clot, pro-UK in plasma was stable for more than 2 d. When a clot was added after incubation (37 degrees C) for 50 h, activation to full lytic activity took place. The findings in vivo were comparable but the rapid clearance of pro-UK required that it be given by a constant infusion despite its plasma stability. In rabbits, a UK-resistant species, pro-UK was significantly (P less than 0.001) more efficacious than TC-UK but neither induced significant fibrinogen degradation. In dogs, a more sensitive species, the high specificity of thrombolysis by pro-UK contrasted with the defibrinogenation and uncontrollable bleeding that accompanied thrombolysis by UK. It was concluded that clot lysis by pro-UK is more effective and specific than UK. The advantage of pro-UK is in the limitation of its activation to the site of a clot. This can be explained by an activation mechanism that is dependent, under physiological conditions, on fibrin-stabilized plasmin.

Complementary modes of action of tissue-type plasminogen activator and pro-urokinase by which their synergistic effect on clot lysis may be explained.

Tissue plasminogen activator (t-PA) and/or pro-urokinase (pro-UK) induced lysis of standard 125I-fibrin clots suspended in plasma was studied. Doses were kept below the concentration at which a nonspecific effect was seen, i.e., where fibrinogenolysis and major plasminogen consumption were observed. Small amounts of t-PA potentiated clot lysis by pro-UK by attenuating the lag phase characteristic of pro-UK, and causing a much earlier transition to the rapid phase of lysis. Similar promotion of the fibrinolytic effect of pro-UK was obtained when clots were pretreated with UK or with a little plasmin (less than 1% clot lysis). Promotion by plasmin was nullified by a subsequent treatment of the clot with carboxypeptidase B, indicating that the plasmin effect was related to the exposure of carboxy terminal lysine residues on fibrin. These lysine termini, absent in undegraded fibrin, are known to be essential for the high affinity binding of plasminogen to fibrin. In contrast, clot lysis by t-PA was unaffected by plasmin pretreatment and little affected by carboxypeptidase B treatment of the fibrin substrate. Therefore, plasminogen bound to lysine termini on fibrin, although found to be essential for pro-UK, did not appear to serve as a substrate for t-PA. Selective activation of fibrin bound plasminogen has been attributed to the conformational change in Glu-plasminogen that occurs as a result of binding. The present findings suggest that this conformational change occurs when plasminogen is bound to a terminal lysine but not to an internal lysine. Plasminogen bound to the latter site on fibrin was activated by t-PA and therefore is involved in the ternary complex. This initiates lysis of the undegraded clot and exposes the plasminogen binding sites required by pro-UK. By their complementary activation of fibrin bound plasminogen, t-PA followed by pro-UK induces efficient and synergistic fibrinolysis, whereas each is relatively inefficient when used alone.

Comparative study of the efficacy and specificity of tissue plasminogen activator and pro-urokinase: demonstration of synergism and of different thresholds of non-selectivity

Clot lysis and non-specific plasminogen activation in human plasma by tissue tissue plasminogen activator (t-PA) and/or pro-urokinase (pro-UK) were studied. The fibrinolytic activity of pro-UK was expressed as latent units, i.e. measured after activation with plasmin on a fibrin plate against the reference standard. The t-PA unitage was assigned on a weight basis of a similar equivalence of 100,000 IU/mg. To simplify comparison, both activators were expressed in IU (1 IU = approximately 10 ng). At low concentration (1-50 IU/ml), t-PA induced more effective and more linear clot lysis, whereas pro-UK induced lysis was preceded by a lag phase. The two activators were equivalently effective at higher concentrations and saturated at the same lysis rate. Clots made from platelet rich plasma or whole blood were more responsive to lysis by pro-UK but not t-PA than corresponding platelet poor clots. At very low concentrations (2.5-5 IU/ml) of t-PA combined with moderate concentrations (25-50 IU/ml) of pro-UK, a synergistic effect on clot lysis, which was fibrin-specific, was observed. Plasminogen and fibrinogen and the appearance of plasmin-inhibitor complexes in plasma were measured after incubation with either activator with and without a clot present. Non-specific plasminogen activation occurred above a certain concentration of either activator but was found at lower concentrations of t-PA than pro-UK. In the absence of a clot, plasmin generation occurred with t-PA at about 30% of the concentration at which pro-UK induced a corresponding effect. It is concluded that there are important differences in the fibrinolytic and clot selective properties of t-PA and pro-UK, and that some of these properties may be complementary resulting in a fibrin specific, synergistic fibrinolytic effect.

Characterization of the intrinsic fibrinolytic properties of pro-urokinase through a study of plasmin-resistant mutant forms produced by site-specific mutagenesis of lysine(158).

Two plasmin-resistant mutant forms of pro-urokinase (pro-UK) constructed by site-directed mutagenesis of Lys158 to Val158 and Met158 were used to evaluate the intrinsic enzymatic and fibrinolytic properties of pro-UK as distinct from those of its two-chain UK (TC-UK) derivative. Both mutants, while resistant to plasmin activation, were as sensitive as pro-UK to degradation by thrombin. Since thrombin cleaves a peptide bond only two residues from the activation site, the integrity of this loop was maintained in the two mutants. The amidolytic and plasminogen-activating activities of the mutants averaged 0.14 and 0.12% that of TC-UK, respectively. The fibrin plate activities were 2,400 IU/ml and 700 IU/mg for the Met158 and Val158 mutants or about 1.5% that of TC-UK. These findings attest to a discrete but low intrinsic activity for pro-UK and suggest that the higher values reported in the literature may be related to UK contaminants or plasmin-induced TC-UK generation during the assay. Clot lysis by the mutants required doses greater than 100-fold higher than those of pro-UK to induce a comparable effect. From this it appears that pro-UK activation is a major determinant of the rate of clot lysis occurring with pro-UK. Clot lysis by the mutants was potentiated by plasmin pretreatment of the fibrin and by the addition of small amounts of TC-UK or tissue plasminogen activator (t-PA). Combinations of t-PA and the mutants were synergistic in their fibrinolytic effects. These findings mirror those previously obtained with pro-UK. We concluded that the previously described potentiation of pro-UK-induced clot lysis by UK or t-PA is mediated primarily by pro-UK itself rather than by a promotion of its activation.

Pro-urokinase and prekallikrein are both associated with platelets Implications for the intrinsic pathway of fibrinolysis and for therapeutic thrombolysis

The contact‐dependent intrinsic pathway of fibrinolysis involving factor XII, prekallikrein (PK) and pro‐urokinase (pro‐UK) remains poorly understood. Casein autography of washed, intact platelets revealed both PK and pro‐UK. Accordingly, platelets may mediate physiological thrombolysis by this pathway since factor XIIa activates PK and kallikrein activates pro‐UK. Acid washing dissociated PK but not pro‐UK from platelets. Exogenous pro‐UK was specifically incorporated by platelets from the ambient fluid and similarly could not be dissociated from intact platelets. Therefore, platelets may also mediate an effect from therapeutically administered pro‐UK by prolonging its half‐life.

A comparison of the rates of clot lysis in a plasma milieu induced by tissue plasminogen activator (t-PA) and rec-pro-urokinase: Evidence that t-PA has a more restricted mode of action

Abstract Tissue plasminogen activator (t-PA) and pro-urokinase (pro-UK) were previously found to have distinct and complementary mechanisms of action in a plasma milieu. The t-PA activated primarily plasminogen bound to intact fibrin (internal lysine-bound), whereas pro-UK activated plasminogen bound to partially degraded fibrin (C-terminal lysine-bound). These observations therefore suggested that each of these activators was restricted in its fibrin-dependent lytic effect. At least this is true at activator concentrations where activation of plasminogen is strictly fibrin dependent; at some high concentration all plasminogen is activated including free plasminogen in the ambient plasma. However, since some pro-UK is converted to UK at the plasmin rich fibrin surface during the course of lysis and since UK is a non-selective plasminogen activator, the pro-UK/UK system should not be similarly restricted. The present study was therefore designed to examine whether this is the case by determining the highest rates of lysis achievable at the threshold of specificity for t-PA and for pro-UK/UK respectively. Maximal rates of clot lysis by t-PA (0.1–5 μg/ml) and by rec-pro-UK (0.5–5 μg/ml) were determined and correlated with the degree of preservation of fibrinogen. The highest rate of clot lysis induced by a fibrin-specific ( 80% fibrinogen degradation. The highest fibrin-specific rate of lysis achievable with t-PA was only 42%/h. When a small amount of t-PA was added at intervals during clot lysis induced by rec-pro-UK, the t-PA significantly foreshortened the lag phase, but it did not enhance the maximal rate of clot lysis. By contrast, when rec-pro-UK was added during the course of clot lysis induced by a large, but fibrin-specific amount of t-PA, rec-pro-UK accelerated the rate of lysis thereby demonstrating the presence of some fibrin-bound plasminogen not activated by t-PA. The study indicates that in a plasma milieu t-PA is more restricted in its action and activates only about half the fibrin-bound plasminogen which is activated by rec-pro-UK/UK.

Spontaneous clot lysis in whole human plasma by endogenous tissue type and urokinase type plasminogen activators: Demonstration of a promoting effect by t-PA and by platelets on urokinase

Endogenous pro-urokinase (pro-UK)/urokinase (UK) has been reported not to contribute significantly to spontaneous lysis of clotted plasma. Accordingly, its importance to physiological fibrinolysis has been questioned. However, since pro-UK was recently shown to be rapidly inactivated by thrombin, its role in fibrinolysis may have been underestimated in these experiments. To avoid thrombin inactivation of pro-UK, we studied spontaneous exolysis of clots rather than endolysis. In fresh plasma, lysis of clots prepared from fibrinogen or platelet poor plasma occurred progressively over 6–10 and 8–15 days respectively, and was inhibited by aprotinin (⩾ 200 KIU/ml) or by antibody to tissue plasminogen activator (t-PA) but not by antibody to UK. Supplementation with exogenous pro-UK indicated a threshold concentration in plasma of about 20 ng/ml before pro-UK had a measurable fibrinolytic effect against these clots. The progressive fibrinolytic effect of supplemental pro-UK (20–200 ng/ml) was abolished or inhibited by t-PA antibody, indicating that endogenous t-PA had a strong promoting effect on fibrinolysis by exogenous pro-UK. Some acceleration of clot lysis occurred after addition of dextran sulfate (1 ELM). When platelet rich plasma (PRP) clots were used, t-PA antibody inhibited but did not abolish clot lysis, but the latter was inhibited by UK antibody. Therefore, endogenous pro-UK/UK had an independent fibrinolytic effect against PRP clots. When clot lysis was studied in plasma preincubated (37°C) for up to 24 h, lysis of platelet poor clots was only slightly inhibited after 4 h incubation, and still went to completion even in plasma preincubated for 24 h. This lysis was abolished by t-PA antibody. Therefore, endogenous t-PA was surprisingly stable in plasma, having t12 to inhibition of > 24 h, in contrast to the t12 of exogenous t-PA in plasma which is < 2 h. It was concluded that endogenous pro-UK/UK contributes to fibrinolysis in intact plasma, but that its activity is dependent on platelets and is strongly promoted by endogenous t-PA, or more likely, by t-PA:inhibitor (PAI-1) complex.

The effect of the carboxy-terminal lysine of urokinase on the catalysis of plasminogen activation.

When single-chain pro-UK is activated by plasmin or kallikrein, the Lys158-Ile159 bond is cleaved, leaving a C-terminal lysine on the A-chain (Lys-UK). Two-chain, high molecular weight urokinase (UK) purified from urine, however, has been shown to contain a phenylalanine residue as the C-terminal of the A-chain (Phe-UK). Since C-terminal lysine residues have a strong binding affinity for plasminogen that may promote its activation, we undertook kinetic studies comparing plasminogen activation by Lys- and Phe-UK. A two-stage method was employed in order to minimize factors known to interfere with plasminogen activation and plasmin determination. The Lys-UK was prepared by plasmin activation of pro-UK purified from human fetal kidney cell culture medium. The Phe-UK was prepared by carboxypeptidase B (CpB) treatment of Lys-UK. Removal of the C-terminal lysine of Lys-UK by CpB produced small but significant increases in the Michaelis constants for the activation of both Glu- and Lys-plasminogen. The apparent Michaelis constants for Glu-plasminogen activation by Lys- and Phe-UK were 3.7 microM +/- .36 microM and 5.9 microM +/- .70 microM, respectively and the Michaelis constants for Lys-plasminogen activation by Lys- and Phe-UK were 5.4 microM +/- .72 microM and 15.2 microM +/- 1.4 microM, respectively. The catalytic efficiency (kcat/Km) of Lys-UK was approximately 2-fold greater than that of Phe-UK for the activation of either Glu- or Lys-plasminogen. When the fibrinolytic activities of Lys- and Phe-UK were compared in a plasma milieu no significant differences were detected. In conclusion, the findings indicate that the C terminal lysine on the A-chain of UK significantly promotes the catalysis of plasminogen in a purified system. However, the higher catalytic efficiency of Lys-UK was not found to induce significant acceleration of clot lysis at pharmacological concentrations in plasma.

The Selective Uptake of High Molecular Weight Urokinase-type Plasminogen Activator by Human Platelets

Summary In a previous study it was shown that about 20% of the endogenous pro-urokinase (pro-UK) in whole blood was associated with platelets and that there was uptake of pro-UK by platelets from the ambient plasma or buffer. In the present study, the association of urokinase-type plasminogen activator (u-PA) with platelets was further characterized. Isolated platelet suspensions were exposed to high and low molecular weight u-PA or tissue plasminogen activator (t-PA), then washed and analyzed by SDS gel electrophoresis and zymography. Only the high molecular weight u-PA was found associated with platelets and this association resisted acid washing, aggregation by thrombin, or osmotic swelling of platelets to obliterate the open canalicular system. Ultrasonic disruption of platelets and isolation of the membrane portion indicated that u-PA was associated with the membrane. The catalytic site of u-PA remained available on the platelet surface as evidenced by its inhibition by the specific inhibitor glutamyl-glycyl-arginyl chloromethylketone (GGAck). Uptake by platelets, but not red cells, of pro-UK from whole blood was demonstrated, and this was detectable at physiological concentrations of pro-UK suggesting that endogenous u-PA in platelets may originate from the ambient plasma. A Western blot of platelet membrane proteins with antibody to u-PA receptor (u-PAR) showed no evidence of u-PAR. It was postulated that u-PA binds to a platelet membrane protein distinct from u-PAR, via the A-chain of u-PA, and that this interaction becomes stabilized resisting dissociation.

Evidence for the expression of urokinase-type plasminogen activator by human venous endothelial cells in vivo.

Endothelial cells (ECs) in culture synthesize and secrete urokinase-type plasminogen activator (u-PA), but the normal vascular endothelium is believed to synthesize only tissue plasminogen activator (t-PA), which is thought to be responsible for intravascular fibrinolysis. More recently, animal studies have shown that the biological role of u-PA in fibrinolysis has been underestimated, prompting a re-examination of its synthesis by the endothelium. In this study, we investigated whether u-PA was synthesized by non-atherosclerotic endothelial cells in vivo by testing ECs dislodged by venipuncture from 12 normal volunteers and 17 patients admitted for plasmapheresis. The ECs were isolated with an anti-endothelial monoclonal antibody coupled to immunomagnetic beads and characterized by morphology and by labelling for vWF, CD31, and UEA-1 binding. U-PA antigen was found in 50% of the ECs from the normal subjects and in 60% of those from patients. U-PA enzymatic activity on zymograms was detected in 50% of the normal samples and 60% of the patient samples, with the latter being more frequently and more strongly positive. U-PA mRNA was found in all the normal and patient samples tested. The results indicate that u-PA is synthesized by the venous endothelium in vivo but that its expression is highly variable.