Plasminogen activators (PAs), ()such as t-PA and u-PA, are highly specific serine proteases, which catalyze the hydrolysis of the Arg-Val peptide bond of Glu-plasminogen. The activation product, plasmin, is a potent protease, which digests fibrin and several extracellular matrix proteins. Plasmin also processes the single-chain precursors of t-PA and u-PA to the more active two-chain forms. In contrast to u-PA, the rate of plasminogen activation by t-PA increases by 2-3 orders of magnitude in the presence of fibrin or fibrin(ogen) degradation products (Camiolo et al., 1971; Hoylaerts et al., 1982; Ranby, 1982; Bergum and Gardell, 1992). Both t-PA and its substrate, Glu-plasminogen, bind to fibrin, forming a ternary complex that facilitates the conversion of Glu-plasminogen (Hoylaerts et al., 1982; Ranby, 1982; Fears, 1989). t-PA consists of several structural motifs known by structural homology from other proteins: an N-terminal fibronectin-like finger (F), an epidermal growth factor (E), two kringles (K1 and K2), and a serine protease domain (P) (Pennica et al., 1983; Patthy, 1990). Several authors suggested the F domain and the lysine binding site (LBS) of the K2 domain to be the major contributors to t-PA fibrin affinity and to the observed fibrin-mediated enhancement of plasminogen activation (van Zonneveld et al., 1986; Verheijen et al., 1986; de Munk et al., 1989). Recent results, however, indicate that t-PA interacts with fibrin via a binding region that comprises surface areas of other structural modules as well, including the protease domain (Bennett et al., 1991).

In recent years, thrombolytic treatment with t-PA has emerged as state of the art therapy of acute myocardial infarction (Topol, 1991; Collen and Lijnen, 1991). However, when administered in therapeutic doses, t-PA, due to its limited fibrin selectivity, causes plasminemia that may contribute to bleeding complications (Rao et al., 1988; Arnold et al., 1989). Therefore, considerable efforts have been devoted to the design of new variants of t-PA exhibiting improved fibrin selectivity (Higgins and Bennett, 1990; Lijnen and Collen, 1991). Recently, a novel mutein of t-PA called TNK, which is more fibrin selective than t-PA, has been characterized (Keyt et al., 1994; Collen et al., 1994).

We and others (Gardell et al., 1989; Krätzschmar et al., 1991) have previously reported the cloning, expression, and characterization of plasminogen activators derived from the saliva of vampire bats. A total of four different Desmodus rotundus salivary plasminogen activators (DSPAs), which we named DSPA1, 2, , and have been cloned, expressed, and characterized. DSPA1 and 2 encompass an F, E, K, and P domain, while DSPA lacks the finger module and DSPA contains only a K and a P domain. Apart from these differences, DSPAs are very similar (88.7-99.5% amino acid sequence identity; Krätzschmar et al.(1991)). The amino acid sequence of human t-PA is similarly related (72.3% (DSPA1) and 74.2% (DSPA2) identity; Krätzschmar et al.(1991)). Like u-PA, all DSPAs only contain a single K domain rather than two, as is the case for t-PA. The K module of DSPAs is more similar to the K1 domain of t-PA and does not exhibit an LBS. Furthermore, a plasmin-sensitive activation site, present in the N-terminal region of the t-PA protease domain is absent in DSPAs. Therefore, DSPAs activate plasminogen as single chain molecules (Gardell et al., 1989; Krätzschmar et al., 1991).

Functionally, DSPAs differ from t-PA by their strict requirement for a fibrin cofactor. This was studied in great detail for Bat-PA (equivalent to DSPA2) by Bergum and Gardell(1992) and has been reported for DSPA1 as well (Schleuning et al., 1992). When compared to t-PA, Bat-PA and DSPA1 demonstrated an equal or even higher thrombolytic potency in several animal models of arterial thrombosis (Gardell et al., 1991; Mellot et al., 1992; Witt et al., 1992, 1994; Muschick et al., 1993). Importantly, while being equally effective as t-PA, fibrinogen degradation or 2-antiplasmin consumption were considerably lower with Bat-PA and DSPA1 (Gardell et al., 1991; Mellot et al., 1992; Witt et al., 1992; Muschick et al., 1993).

The present study evaluates the fibrin selectivities of the recombinant forms of all naturally occurring DSPAs and compares these data with those obtained for t-PA. Furthermore, we present data that suggest a molecular mechanism for the unique fibrin selectivity of DSPAs.

MATERIALS AND METHODS

Mutagenesis

Purified Proteins and Substrates

Radioiodination of PAs

Fibrin Binding

The dissociation constant for the binding of DSPAs to fibrin and the numbers of binding sites were calculated by nonlinear regression analysis of the data according to the Scatchard equation employing the programs EBDA (equilibrium binding data analysis) and LIGAND, originally written by Munson and Rodbard(1980) and modified by G. A. McPherson (V 2.0), which were obtained from Elsevier-Biosoft (Cambridge, United Kingdom).

Kinetics of Plasminogen Activation

Kinetics of S-2765 Hydrolysis

RESULTS

DSPAs 1, 2, , and were expressed in BHK cells as described (Krätzschmar et al., 1992). Recombinant proteins were purified to homogeneity from cell culture supernatants by affinity chromatography on immobilized Erythrina trypsin inhibitor (Heussen et al., 1984). As judged from SDS-PAGE analysis, preparations of recombinant DSPAs 1, 2, , and were homogeneous, and the proteins displayed an apparent molecular mass of 52, 52, 46, and 44 kDa, respectively (Fig. 1, lanes 1-4).

Figure 1: SDS-PAGE of purified recombinant DSPAs and t-PA. Prior to electrophoresis on a SDS-gel containing 12.5% polyacrylamide (Laemmli 1970), all samples were reduced by the addition of dithiothreitol (12.5 mM). Approximately 3 µg of each protein was loaded. The gel was stained with Coomassie Brilliant Blue (G250). Proteins were produced as outlined under ``Materials and Methods.'' Lane 1, rDSPA1; lane 2, rDSPA2; lane 3, rDSPA; lane 4, rDSPA; lane 5, marker proteins (M) whose molecular mass is indicated on the right; lane 6, rt-PA (Actilyse®); lane 7, [R275H,I276S,K277T] t-PA lacking the plasmin-sensitive site; lane 8, [H189R,S190I,T191K] DSPA1 containing a plasmin-sensitive site.

DSPA Affinity for Fibrin

Figure 2: Scatchard analysis of the interaction between DSPA1 or DSPA2 and forming fibrin clots. A, Scatchard analysis of fibrin binding data obtained for I-DSPA1. The plot is based on a model involving one type of binding site. Inset, equilibrium binding of I-DSPA1 to fibrin. Binding was determined at variable concentrations of unlabeled DSPA1 as described under ``Materials and Methods.'' Each point is represented by the results of three independent determinations. The average standard error for the equilibrium binding curve was approximately 7%. Specific binding was calculated by subtracting nonspecific binding (e.g. in the presence of excess unlabeled DSPA1) from total binding. Nonspecific binding was 4% of total cpm. B, Scatchard plot of fibrin binding data of I-DSPA2. The plot is based on a model involving a sole type of binding site. Inset, equilibrium binding of I-DSPA2 to fibrin. Binding was determined as described under ``Materials and Methods.'' The results of three independent experiments are displayed. The average standard error for the equilibrium binding curve was about 6%. Specific binding was calculated by subtracting nonspecific binding (e.g. in the presence of excess unlabeled DSPA2) from total binding. Nonspecific binding was 6% of total cpm.

Glu-Plasminogen Activation by DSPAs and by t-PA in the Absence and Presence of a Fibrin(ogen) Cofactor

In the presence of fibrin, however, the catalytic efficiency of DSPAs was augmented by several orders of magnitude. The steepest increase by a factor of 10, resulting in a k/K value of 684,000 M s, was observed for DSPA1. The bimolecular rate constant of DSPA2 was raised to 517,000 M s, corresponding to a 53,000-fold enhancement (Table 1). DSPAs and exhibited considerably smaller catalytic efficiencies of 9900 M s and 3510 M s, reflecting a 1650- and 800-fold increase in catalytic activity, respectively. This demonstrates that binding of the DSPAs to fibrin significantly contributes to enhanced plasminogen activation. For DSPA1 and 2, the fibrin-mediated enhancement of catalytic efficiency resulted from both a moderate decrease (25- and 34-fold) in K and a concomitant profound increase (4300- and 1550-fold) in k, respectively. Interestingly, despite their high degree of homology (89% identity in amino acid sequence) (Krätzschmar et al., 1991), the unstimulated activity of DSPA2 was slightly higher than that of DSPA1, which, however, was more active in the presence of fibrin (Table 1). The 2-fold higher fibrin-mediated enhancement of the catalytic efficiency of DSPA1 was due to a steeper increment in the k value of DSPA1 rather than a more pronounced decrease in its K (Table 1). The reduction in K and in particular the increase in k was significantly smaller for DSPAs and (Table 1).

In comparison to the absence of a cofactor, fibrinogen promoted the catalytic efficiency of DSPAs by 7-9-fold, resulting in k/K values ranging from 39 to 79 M s, which were several orders of magnitude smaller than those observed in the presence of fibrin (Table 1). The ratio of catalytic efficiencies in the presence of fibrin to the corresponding values in the presence of fibrinogen, which serves as a measure of ``fibrin selectivity'', amounted to 12,900 for DSPA1. The bimolecular rate constant of DSPA2 in the presence of fibrin was 6550-fold higher than that in the presence of fibrinogen. The respective values for DSPAs and were 235 and 90 (Table 1). DSPA1 therefore exhibited the highest fibrin selectivity, and this was mainly attributable to its superior stimulation by fibrin.

The data summarized in Table 1also depict how the kinetic parameters of DSPAs compare to those obtained for t-PA. In the absence of a fibrin(ogen) cofactor, t-PA was 260-fold more efficient in activating Glu-plasminogen than DSPA1. In the presence of fibrin, however, both plasminogen activators were similarly effective (Table 1). The enhancement of the bimolecular rate constant of t-PA in the presence of fibrin was only 550-fold as compared to 10-fold for DSPA1. In the presence of fibrinogen, the catalytic efficiency of t-PA was increased to 13,600 M s, which was 260- and 170-fold higher than the respective values measured for DSPA1 and 2 (Table 1). Fibrin increased the catalytic efficiency of t-PA by only 72-fold over that in the presence of fibrinogen, meaning that DSPA1 was about 180-fold more fibrin selective than t-PA. DSPA2 exhibited a 90-fold higher fibrin selectivity than t-PA, and even the finger-deficient variant DSPA was still 3-fold more fibrin selective. The latter strongly indicates that fibrin selectivity is not merely a consequence of the plasminogen activator's affinity for fibrin.

Fibrin Stimulation and Fibrin Selectivity Depend on the Presence of a Plasmin-sensitive Site

Figure 3: Plasmin-mediated conversion of [R275H,I276S,K277T] t-PA and [H189R,S190I,T191K] DSPA1. Recombinant proteins were prepared as described under ``Materials and Methods.'' Approximately 5 µg each of t-PA, [R275H,I276S,K277T] t-PA, and [H189R,S190I,T191K] DSPA1 were incubated for 30 min at 37 °C in the absence (lanes 2, 4, and 6) or presence of Sepharose-immobilized plasmin (lanes 3, 5, and 7). The samples were analyzed by SDS-PAGE as outlined in the legend to Fig. 2. Lane 1, marker proteins (M) whose molecular mass is indicated on the left; Lanes 2, 4, and 6, t-PA, [R275H,I276S,K277T] t-PA, and [H189R,S190I,T191K] DSPA1, respectively, incubated in the absence of plasmin, or as shown in lanes 3, 5, and 7, treated with plasmin.

In the absence of a stimulator, the DSPA1 mutein exhibited a bimolecular rate constant of 135 M s, which reflected a 20-fold increase over that of wild-type DSPA1. The catalytic efficiency of sct-PA was reduced, in comparison to t-PA, by 50-fold to 34 M s (Table 1), which is in good agreement to the activity decrease observed previously (Andreasen et al., 1991; Petersen et al., 1988; Tate et al., 1987). Fibrinogen raised the k/K value of plasmin-sensitive DSPA1 mutein to 516 M s, which in comparison to the wild type, corresponded to a 10-fold increase. The respective value (638 M s) of sct-PA was 20-fold decreased. In the presence of fibrin, however, both muteins displayed catalytic efficiencies (DSPA1 mutein, 565,000 M s, and t-PA mutein, 525,000 M s) that were similar to the respective wild-type proteins (Table 1). In comparison to fibrinogen, fibrin promoted the catalytic efficiency of plasmin-sensitive DSPA1 by 1100-fold, which was 12-fold less than that of the uncleavable wild-type enzyme. In case of t-PA, the absence of the cleavage site resulted in an 11-fold increase of its fibrin selectivity. Importantly, DSPA1 and DSPA2 were still about 16- and 8-fold more fibrin selective than uncleavable sct-PA, and plasmin-sensitive DSPA1 was 15-fold more fibrin selective than t-PA (Table 1), implying that other features apart from the lack of the plasmin-sensitive cleavage site must contribute to the superior fibrin selectivity of DSPAs.

Hydrolysis of S-2765 by DSPAs and t-PA in the Presence and Absence of Fibrin or Fibrinogen

In the absence of a fibrin(ogen) cofactor, DSPA1 and -2 exhibited 17 and 37.5%, respectively, of t-PA catalytic efficiency. While fibrinogen had only a very small effect on the k/K of t-PA, fibrin promoted its bimolecular rate constant by 4-fold. In the presence of the latter the activity of DSPA2 was equivalent to that of t-PA, whereas S-2765 hydrolysis by DSPA1 was 2-fold less efficient (Table 2).

As observed for t-PA and DSPA1, the extent of fibrin-mediated stimulation of S-2765 hydrolysis was dependent on whether they occurred in their single or two-chain forms. The single chain forms of DSPA1 and t-PA were more highly stimulated than their two-chain counterparts (Table 2). The intrinsic activity of tct-PA was not significantly stimulated by fibrin, while tc DSPA1's catalytic efficiency was still enhanced (2.6-fold), albeit 5-fold less than that of the single chain molecule. This effect was attributed to an increased k, which was not observed for t-PA. By comparison to the wild-type molecule, the tc DSPA1 mutein exhibited a 4-fold higher catalytic efficiency in the absence of a fibrin(ogen) cofactor (Table 2).

Influence of EACA on Plasminogen Activation by DSPAs and t-PA

Since alterations in plasminogen conformation only accounted for a small portion of fibrin's stimulatory effect, it is evident that the major contribution to fibrin stimulation is mediated by the ternary complex formation and/or the interaction of DSPA1 and 2 with fibrin (Hoylaerts et al., 1982; Wu et al., 1990). To discriminate between these two mechanisms, we have measured enzyme kinetics in the presence of both fibrin and EACA. Under these conditions, only the plasminogen activator is able to bind to fibrin, whereas binding of plasminogen to fibrin is inhibited (Lucas et al., 1983; Nesheim et al., 1990). Binding of t-PA is solely mediated by the finger domain because kringle 2-dependent binding of t-PA to fibrin is inhibited (van Zonneveld et al., 1986; Nesheim et al., 1990).

By comparison to their catalytic efficiencies in the presence of fibrin alone, the additional presence of EACA diminished the catalytic activities of DSPAs 1, 2, tc DSPA1, and t-PA by 90-24-fold (Table 4). As expected, this decrease was entirely due to an increase in the apparent K, since the k values were similar to that obtained in the presence of fibrin alone ( Table 1and Table 4). Hence, the dissociation of plasminogen from the fibrin template caused similar decrements, within the same order of magnitude, of plasminogen activator catalytic efficiencies. However, a comparison of the bimolecular rate constants in the presence of fibrin and EACA to those in the presence of EACA alone revealed striking differences between DSPAs and t-PA. The addition of fibrin to the reaction mixture that already contained EACA raised DSPA1's k/K from 26 to 7790 M s (about 300-fold), whereas that of t-PA was promoted by only 6-fold from 7480 to 41,270 M s ( Table 3and Table 4). In accordance with our earlier observation, the activity of DSPA1 was 2-fold more highly promoted by the addition of fibrin than that of DSPA2 (Table 4). In the presence of fibrin and EACA, the catalytic efficiency of the plasmin-sensitive DSPA1 mutein was enhanced to 11,020 M s, which represented an only 12-fold increase over that, 890 M s, in the presence of EACA alone.

DISCUSSION

There are three plausible mechanisms pertinent to fibrin-mediated stimulation of plasminogen activation, all based on protein-protein interactions: 1) a template-mediated rendezvous mechanism furthering the physical encounter of both enzyme and substrate, 2) the exposure of the activation site of plasminogen, following a conformational change induced by fibrin binding, and (3) a stabilizing effect of fibrin on the active site of plasminogen activators, probably mediated by domain-domain interactions.

We have attempted to attribute the observed stimulatory effects to one or the other of these mechanisms. In contrast to t-PA, the major contribution of fibrin to its overall stimulatory effect on plasminogen conversion by DSPAs 1 and 2 is mediated by its interaction with the plasminogen activator itself (Table 4). The template effect appears to be less important, whereas it is paramount to fibrin-mediated enhancement of plasminogen activation by t-PA (Hoylaerts et al., 1982). Corroborating the results from direct measurements of fibrin binding, an interaction of DSPA1 or 2 and fibrin is also demonstrated by the enhancement of S-2765 hydrolysis. Upon binding to fibrin, the catalytic activities of DSPA1 and 2 were raised by about 1 order of magnitude, whereas those of DSPAs and were increased only marginally (Table 2). Therefore, in case of DSPA and , the increase in the plasminogen activation rate is most likely due to a conformational change in plasminogen induced by its interaction with partially degraded fibrin (Suenson et al., 1984), although domain-domain interactions occurring within the DSPA molecules might also be involved.

The striking difference between DSPAs and t-PA, as far as fibrin stimulation is concerned, is not a consequence of disparate fibrin affinities. The K values of DSPA1 and DSPA2 (Fig. 2) are within the range of values published for t-PA (0.13-0.58 µM) (Higgins and Vehar, 1987; Husain et al., 1989; Nesheim et al., 1990; Bergum and Gardell, 1992; Horrevoets et al., 1994). The data are particularly consistent, if only finger-dependent binding of t-PA is analyzed. Under these conditions, Nesheim et al.(1990) measured a K of 0.13 µM and a molar binding ration of 0.6, values that are almost identical to those of DSPA1 and 2 (Fig. 2). Furthermore, the dependence on the fibrin concentration was very similar for DSPAs and t-PA. Half-maximal velocities were achieved at 25 ± 3, 31 ± 5, and 13 ± 2 µg/ml for DSPA1, DSPA2, and t-PA, respectively (data not shown).

All DSPAs exhibited only marginal activity in the absence of a fibrin(ogen) cofactor (Table 1). Upon addition of fibrinogen, their second order rate constants increased similarly by roughly 1 order of magnitude, which is in contrast to the markedly diverging stimulatory effect mediated by fibrin. For instance, in case of DSPA1 and DSPA, the extent of fibrin stimulation differs by a factor of 62 (Table 1). These data therefore suggest that the stimulatory effect exerted by fibrinogen is not conferred via the DSPAs but is rather mediated by an interaction between plasminogen and fibrinogen (Lucas et al., 1983).

The ratio of the bimolecular rate constants of plasminogen activation in the presence of fibrin versus fibrinogen is defined as fibrin selectivity. Since DSPA1, 2, and t-PA exhibited very similar bimolecular rate constants in the presence of fibrin, the significant difference in fibrin selectivity of DSPA1/2 and t-PA is mainly caused by their unequal catalytic efficiencies in the presence of fibrinogen (Table 1).

To further understand the underlying structure-function relationship, we have analyzed the properties of a mutein of DSPA1, whose protease domain contained a plasmin-sensitive site (Table 1Table 2Table 3Table 4). In the presence of fibrin, the catalytic efficiency of plasmin-sensitive DSPA1 was strikingly less (24-fold) increased than that of the wild-type protein (Table 1). This decrease in fibrin stimulation was entirely attributable to a diminished stimulation via the plasminogen activator protease domain because upon prevention of the template effect by addition of -amino caproic acid, fibrin stimulated the catalytic efficiency only 12-fold as opposed to 300-fold as observed for DSPA1 (Table 4). Since the bimolecular rate constants of DSPA1 and its plasmin-sensitive mutein were almost identical in the presence of fibrin (Table 1), the decreased stimulatory effect was a consequence of the mutein's higher basal activity. Further, the fibrin selectivity of cleavable DSPA1 was decreased about 12-fold (Table 1) and the fibrin-stimulated intrinsic catalytic activity about 5-fold (Table 2). Hence, the lack of a plasmin-sensitive site within the DSPA1 protease domain contributes significantly to both the impressive stimulation by fibrin and its fibrin selectivity. Abolition of the plasmin-sensitive site of t-PA brings a quantitatively similar effect into bearing, namely a 28-fold increase in fibrin stimulation (Table 1). Concomitantly, the fibrin selectivity is improved 11-fold, again quantitatively equal to the effect observed for the cleavable DSPA1 mutein. The role of the t-PA plasmin cleavage site for fibrin stimulation has been investigated previously, and similar results have been obtained (Tate et al., 1987; Petersen et al., 1988; Higgins et al., 1990). Nienaber et al.(1992) suggest that the interaction of the single-chain form of t-PA with fibrin(ogen) induces a conformational change at the active site stabilizing an ``active locked'' conformation. Since the influence on fibrin stimulation and on fibrin selectivity of a plasmin-sensitive site in the protease domain of DSPA1 or t-PA is almost identical, it is likely that the stimulation of the DSPA1 protease domain by fibrin is due to a similar mechanism.

Notably, plasmin-sensitive DSPA1 was still 15-fold more fibrin selective than t-PA, and DSPA1 discriminated more than 7-fold better between fibrin and fibrinogen than sct-PA (Table 1). This difference is also corroborated by amidolytic data. While fibrin raised the intrinsic catalytic activity of tc DSPA1 by 2-3-fold over that in the presence of fibrinogen, the k/K of tct-PA remained essentially unchanged (Table 2). Therefore, the heterotropic effect conferred by fibrin does not depend solely on the lack of the plasmin-sensitive cleavage site but involves other, yet unknown, determinants within the molecule.

Only DSPA2 (Bat-PA) and t-PA have been evaluated in a way comparable to this study by other authors. Pertaining to t-PA, our data are similar to those previously reported (Rånby, 1982; Urano et al., 1988; de Vries et al., 1991; Bergum and Gardell, 1992). Our values were generally higher than those published for Bat-PA (corresponding to DSPA2) by Bergum and Gardell(1992). This difference can be explained by use of different k values for plasmin-mediated hydrolysis of the chromogenic substrate (FlavigenPli versus SpectrozymePl). The relative stimulation factors, however, are in good agreement (the Bat-PA k/K 43500-fold increase in the presence of fibrin, DSPA2 52,700-fold). Fibrin (fibrin II) stimulated DSPA2 catalytic efficiency 10,900-fold more than fibrinogen as compared to the ratio of 6550 determined in our system. Our data, however, do not confirm that in the absence of a stimulator the Bat-PA K was 0.6 µM and therefore smaller than in the presence of fibrinogen (Bergum and Gardell, 1992). By contrast, DSPA2 affinity for Glu-plasminogen was very low as indicated by a K of 12.3 µM, a value very similar to those determined for DSPA1 (9.5 µM) and sct-PA (17.7 µM). Also, our unstimulated K value for t-PA, 5.2 µM, was similar to the 6.7 µM reported by Bergum and Gardell(1992) and agreed very well with the values of 7.6 and 9 µM published by Rånby(1982) and Urano et al.(1988), respectively.

In summary, we have provided a biochemical rationale for the striking fibrin selectivity of DSPAs: finger-dependent fibrin binding confers a heterotropic effect, which is conceivably mediated by domain-domain interactions on the protease domain to stabilize a preformed active site. Introduction of a plasmin-sensitive cleavage site partially obliterates the requirement for the fibrin cofactor. Further understanding of the molecular details of this interaction will depend on the results of structural analysis, which is currently underway.

FOOTNOTES

*

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence should be addressed. Tel.: 49-30-468-2130; Fax: 49-30-4691-6707.

()

The abbreviations used are: PA, plasminogen activator; EACA, -amino caproic acid; PAGE, polyacrylamide gel electrophoresis; DSPA, D. rotundus salivary plasminogen activator; LBS, lysine binding site; pNA, p-nitroaniline; tcDSPA1, two-chain DSPA1; tct-PA, two-chain t-PA.

ACKNOWLEDGEMENTS

REFERENCES

1. Andreasen, P. A., Petersen, L. C., and Danø, K. (1991) Fibrinolysis 5, 207-215 [CrossRef]
2. Arnold, A. E. R., Bowr, R. W., Collen, D., van Es, G.-A., Lubsen, J., Serruys, P. W., Simoons, M. L., and Verstraete, M. (1989) J. Am. Coll. Cardiol. 14, 581-588 [Abstract]
3. Artelt, P., Morelle, C., Ausmeier, M., Fitzek, M., and Hauser, H. (1988) Gene (Amst.) 68, 213-219 [CrossRef][Medline] [Order article via Infotrieve]
4. Bearden, J. C., Jr. (1978) Biochim. Biophys. Acta 533, 525-529 [Medline] [Order article via Infotrieve]
5. Bennett, W. F., Paoni, N. F., Keyt, B. A., Botstein, D., Jones, A. J. S., Presta, L., Wurm, F., and Zoller, M. J. (1991) J. Biol. Chem. 266, 5191-5201 [Abstract/Free Full Text]
6. Bergum, P. W., and Gardell, S. J. (1992) J. Biol. Chem. 267, 17726-17731 [Abstract/Free Full Text]
7. Blombäck, B. (1958) Ark. Kemi. 12, 99-113
8. Boose, J. A., Kuismanen, E., Gerard, R., Sambrook, J., and Gething, M.-J. (1989) Biochemistry 28, 635-643 [CrossRef][Medline] [Order article via Infotrieve]
9. Camiolo, S. M., Thorsen, S., and Astrup, T. (1971) Proc. Soc. Exp. Biol. Med. 138, 277-280 [Medline] [Order article via Infotrieve]
10. Collen, D., and Lijnen, H. R. (1991) Blood 78, 3114-3124 [Free Full Text]
11. Collen, D., Stassen, J.-M., Yasuda, T., Refino, C., Paoni, N. F., Keyt, B., Roskams, T., Guerrero, J. L., Lijnen, H. R., Gold, H. K., and Bennett, W. F. (1994) Thromb. Haemostasis 72, 98-104 [Medline] [Order article via Infotrieve]
12. de Munk, G. A. W., Caspers, M. P. M., Chang, G. T. G., Pouwels, P. H., Enger-Valk, B. E., and Verheijen, J. H. (1989) Biochemistry 28, 7318-7325 [CrossRef][Medline] [Order article via Infotrieve]
13. Devereux, J., Haeberli, P., and Smithies, O. (1984) Nucleic Acids Res. 12, 387-395
14. de Vries, C., Veerman, H., Nesheim, M. E., and Pannekoek, H. (1991) Thromb. Haemostasis 65, 280-285 [Medline] [Order article via Infotrieve]
15. Drapier, J. C., Tenu, J. P., Lemaire, G., and Petit, J. F. (1979) Biochimie (Paris) 61, 463-471 [Medline] [Order article via Infotrieve]
16. Fears, R. (1989) Biochem. J. 261, 313-324 [Medline] [Order article via Infotrieve]
17. Gardell, S. J., Duong, L. T., Diehl, R. E., York, J. D., Hare, T. R., Register, R. B., Jacobs, J. W., Dixon, R. A. F., and Friedman, P. A. (1989) J. Biol. Chem. 264, 17947-17952 [Abstract/Free Full Text]
18. Gardell, S. J., Ramjit, D. R., Stabilito, I. I., Fujita, T., Lynch, J. J., Cuca, G. C., Jain, D., Wang, S., Tung, J., Mark, G. E., and Shebuski, R. J. (1991) Circulation 84, 244-253 [Abstract/Free Full Text]
19. Heussen, C., Joubert, F., and Dowdle, E. B. (1984) J. Biol. Chem. 259, 11635-11638 [Abstract/Free Full Text]
20. Higgins, D. L., and Vehar, G. A. (1987) Biochemistry 26, 7786-7791 [CrossRef][Medline] [Order article via Infotrieve]
21. Higgins, D. L., and Bennett, W. F. (1990) Annu. Rev. Pharmacol. Toxicol. 30, 91-121 [CrossRef][Medline] [Order article via Infotrieve]
22. Higgins, D. L., Lamb, M. C., Young, S. L., Powers, D. B., and Anderson, S. (1990) Thromb. Res. 57, 527-539 [CrossRef][Medline] [Order article via Infotrieve]
23. Horrevoets, A. J. G., Smilde, A., de Vries, C., and Pannekoek, H. (1994) J. Biol. Chem. 269, 12639-12644 [Abstract/Free Full Text]
24. Hoylaerts, M., Rijken, D. C., Lijnen, H. R., and Collen, D. (1982) J. Biol. Chem. 257, 2912-2919 [Abstract/Free Full Text]
25. Husain, S. S., Hasan, A. A. K., and Budzynski, A. Z. (1989) Blood 74, 999-1006 [Abstract/Free Full Text]
26. Jones, A. J. S., and Meunier, A. M. (1990) Thromb. Haemostasis 64, 455-463 [Medline] [Order article via Infotrieve]
27. Keyt, B. A., Paoni, N. F., Refino, C. J., Berleau, L., Nguyen, H., Chow, A., Lai, J., Peña, L., Pater, C., Ogez, J., Etcheverry, T., Botstein, D., and Bennett, W. F. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 3670-3674 [Abstract/Free Full Text]
28. Krätzschmar, J., Haendler, B., Langer, G., Boidol, W., Bringmann, P., Alagon, A., Donner, P., and Schleuning, W.-D. (1991) Gene (Amst.) 105, 229-237 [CrossRef][Medline] [Order article via Infotrieve]
29. Krätzschmar, J., Haendler, B., Bringmann, P., Dinter, H., Hess, H., Donner, P., and Schleuning, W.-D. (1992) Gene (Amst.) 116, 281-284 [CrossRef][Medline] [Order article via Infotrieve]
30. Laemmli, U. K. (1970) Nature 227, 680-685 [CrossRef][Medline] [Order article via Infotrieve]
31. Lewis, M. K., and Thompson, D. V. (1990) Nucleic Acids Res. 18, 3439-3443 [Abstract/Free Full Text]
32. Lijnen, H. R., and Collen, D. (1991) Thromb. Haemostasis 66, 88-110 [Medline] [Order article via Infotrieve]
33. Lucas, M. A., Fretto, L. J., and McKee, P. A. (1983) J. Biol. Chem. 258, 4249-4256 [Free Full Text]
34. Mangel, W. F., Lin, B., and Ramakrishan, V. (1990) Science 248, 69-73 [Abstract/Free Full Text]
35. Mellot, M. J., Stabilito, I. I., Holahan, M. A., Cuca, G. C., Wang, S., Li, P., Barrett, J. S., Lynch, J. J., and Gardell, S. J. (1992) Arterioscler. Thromb. 12, 212-221 [Abstract/Free Full Text]
36. Munson, P. J., and Rodbard, D. (1980) Anal. Biochem. 107, 220-239 [CrossRef][Medline] [Order article via Infotrieve]
37. Muschick, P., Zeggert, D., Donner, P., and Witt, W. (1993) Fibrinolysis 7, 284-290 [CrossRef]
38. Nesheim, M., Fredenburgh, J. C., and Larsen, G. R. (1990) J. Biol. Chem. 265, 21541-21548 [Abstract/Free Full Text]
39. Nienaber, V. L., Young, S. L., Birktoft, J. J., Higgins, D. L., and Berliner, L. J. (1992) Biochemistry 31, 3852-3861 [CrossRef][Medline] [Order article via Infotrieve]
40. Nieuwenhuizen, W., Voskuilen, M., Traas, D. W., Nobel, B. H., and Verheijen, J. H. (1985) in Fibrinogen: Structural Variants and Interactions (Henschen, A., and Hessel, B., eds) pp. 331-341, Walter de Gruyter & Co., Berlin
41. Paoni, N. F., Chow, A. M., Peña, L. C., Keyt, B. A., Zoller, M. J., and Bennett, W. F. (1993) Protein Eng. 6, 529-534 [Abstract/Free Full Text]
42. Patthy, L. (1990) Blood Coagul. & Fibrinolysis 1, 153-166
43. Pennica, D., Holmes, W. E., Kohr, W. J., Harkins, R. N., Vehar, G. A., Ward, C. A., Bennett, W. F., Yelverton, E., Seeburg, P. H., Heyneker, H. L., and Goeddell, D. V. (1983) Nature 301, 214-221 [CrossRef][Medline] [Order article via Infotrieve]
44. Petersen, L. C., Johannessen, M., Foster, D., Kumar, A., and Mulvihill, E. (1988) Biochim. Biophys. Acta 952, 245-254 [CrossRef][Medline] [Order article via Infotrieve]
45. Ponting, C. P., Marshall, J. M., and Cederholm-Williams, S. A. (1992) Blood Coagul. & Fibrinolysis 3, 605-614
46. Rao, A. K., Pratt, C., Berke, A., Jaffe, A., Ockene, I., Schreiber, T. L., Bell, W. R., Knatterud, G., Robertson, T. L., and Terrin, M. L. (1988) J. Am. Coll. Cardiol. 11, 1-11 [Abstract]
47. Ranby, M. (1982) Biochim. Biophys. Acta 704, 461-469 [CrossRef][Medline] [Order article via Infotrieve]
48. Rijken, D. C., Hoylaerts, M., and Collen, D. (1982) J. Biol. Chem. 257, 2920-2925 [Free Full Text]
49. Robbins, K. C., and Summaria, L. (1970) Methods Enzymol. 19, 184-199
50. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467 [Abstract/Free Full Text]
51. Schleuning, W.-D., Alagon, A., Boidol, W., Bringmann, P., Petri, T., Krätzschmar, J., Haendler, B., Langer, G., Baldus, B., Witt, W., and Donner, P. (1992) Ann. N. Y. Acad. Sci. 667, 395-403 [Abstract]
52. Suenson, E., Lutzen, O., and Thorsen, S. (1984) Eur. J. Biochem. 140, 513-522 [Medline] [Order article via Infotrieve]
53. Tate, K. M., Higgins, D. L., Holmes, W. E., Winkler, M. E., Heyneker, H., and Vehar, G. A. (1987) Thromb. Res. 27, 175-183
54. Topol, E. (1991) Prog. Cardiovasc. Dis. 34, 165-178 [CrossRef][Medline] [Order article via Infotrieve]
55. Urano, T., de Serrano, V. S., Gaffney, P. J., and Castellino, F. J. (1988) Biochemistry 27, 6522-6528 [CrossRef][Medline] [Order article via Infotrieve]
56. van Zonneveld, A. J., Veerman, H., and Pannekoek, H. (1986) J. Biol. Chem. 261, 14214-14218 [Abstract/Free Full Text]
57. Verheijen, J. H., Caspers, M. P. M., Chang, G. T. G., de Monk, G. A. W., Pouwels, P. H., and Enger-Valk, B. E. (1986) EMBO J. 5, 3525-3530 [Medline] [Order article via Infotrieve]
58. Waller, E. K., and Schleuning, W.-D. (1985) J. Biol. Chem. 260, 6354-6360 [Abstract/Free Full Text]
59. Wirth, M., Schuhmacher, L., and Hauser, H. (1991) Protein Glycosylation: Cellular, Biotechnical, and Analytical Aspects (Conradt, H. S., ed) Vol. 15, pp. 49-52, VCH Publishers, Weinheim, Germany
60. Witt, W., Baldus, B., Bringmann, P., Cashion, L., Donner, P., and Schleuning, W.-D. (1992) Blood 79, 1213-1217 [Abstract/Free Full Text]
61. Witt, W., Maaß, B., Baldus, B., Hildebrand, M., Donner, P., and Schleuning, W.-D. (1994) Circulation 90, 421-426 [Abstract/Free Full Text]
62. Wu, H.-L., Chang, B.-I., Wu, D.-H., Chang, L.-C., Gong, C.-C., Lou, K.-L., and Shi, G.-Y. (1990) J. Biol. Chem. 265, 19658-19664 [Abstract/Free Full Text]

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				J. P. Lopez-Atalaya, B. D. Roussel, C. Ali, E. Maubert, K.-U. Petersen, V. Berezowski, R. Cecchelli, C. Orset, and D. Vivien Recombinant Desmodus rotundus Salivary Plasminogen Activator Crosses the Blood-Brain Barrier Through a Low-Density Lipoprotein Receptor-Related Protein-Dependent Mechanism Without Exerting Neurotoxic Effects Stroke, March 1, 2007; 38(3): 1036 - 1043. [Abstract] [Full Text] [PDF]