Human hair derived uPA loaded capsules with dual near-infrared (I and II biowindows) laser responsive capabilities for multi-effective thrombolysis therapy.

Problems such as massive hemorrhage caused by uncontrolled drug dosage are the main significant obstacles in clinical thrombolytic therapy, which are prominently due to the lack of targeting and controlled release ability of efficient thrombolytic drug systems. In recent years, our team demonstrated that the photothermal effect can facilitate the thrombolytic effect of urokinase plasminogen activator (uPA). However, conventional photothermal agents are relatively expensive or contain heavy metals. If drug delivery systems with low toxicity, minimized heavy metal elements and easy accessibility (preferably provided by human self) can be developed, they will be of value in the future related applications. Herein, uPA-loaded human black hair derived nanoparticles with gelatin capsules (uPA@HBHNP@GNCs) were applied for the first time as a thrombolytic system. Upon irradiation by near-infrared I window (NIR-I) laser or II window (NIR-II) laser, the photothermal effect of HBHNP was triggered to promote the melting of the gelatin encapsulated around the outer layer, thereby realizing the targeted release of uPA. The in vitro and in vivo experiments demonstrated that the deep response to NIR (especially II window) of this system exhibited a satisfactory thrombolytic effect with ideal biosafety. Briefly, the proposed hair derived drug delivery system has the characteristics of human source, low cost, minimum heavy metal components, deep response to NIR (II window) laser, and good biocompatibility, which is expected to be expanded to the treatment for some diseases, even in deep tissue areas.

[Thrombolysis with plasminogen activators: use and research of serine proteinases, promise and actuality].

Physiological plasminogen activators of tissue and urokinase type (serine proteinases) are effective thrombolytic agents. Research and development of their novel forms contributed to progress of thrombolytic therapy, advanced treatment of acute coronary syndrome and a marked decrease in the lethal index.

Insight into the structural requirements of Urokinase-Type plasminogen activator inhibitors based on 3D QSAR CoMFA/CoMSIA Models

Urokinase-type plasminogen activator (uPA), a trypsin-like serine protease, has been implicated in large number of malignancies, tumor cell invasion, angiogenesis and metastasis; hence, the potent and selective inhibitors of uPA may therefore be therapeutically useful drugs for treatment of various forms of cancer. A three-dimensional quantitative structure-activity relationship (3D QSAR) study was performed on five different chemical series reported as selective uPA inhibitors employing comparative molecular field analysis (CoMFA)/comparative molecular similarity indices analysis (CoMSIA) techniques to investigate the structural requirements for substrates and derive a predictive model that may be used for the design of novel uPA inhibitors. Inclusion of ClogP did not improve the models significantly and exhibited comparable correlation coefficients with CoMFA steric and electrostatic models. 3D QSAR models were derived for 2-pyridinylguanidines (training set N = 25, test set N = 8), 4-aminoarylguanidines and 4-aminoarylbenzamidines (training set N = 29, test set N = 8), thiophene-2-carboxamindines (training set N = 64, test set N = 19), 2-naphthamidines (training set N = 32, test set N = 8), and 1-isoquinolinylguanidines (training set N = 29, test set N = 7). The CoMFA models with steric and electrostatic fields exhibited r2cv 0.452-0.722, r2ncv 0.812-0.986, r2pred 0.597-0.870, whereas CoMFA ClogP models showed r2cv 0.420-0.707, r2ncv 0.849-0.957, r2pred 0.600-0.870. The CoMSIA models displayed r2cv 0.663-0.729, r2ncv 0.909-0.998, r2pred 0.554-0.855. 3D contour maps generated from these models were analyzed individually, which provides the regions in space where interactive fields may influence the activity. Further, the predictive ability of 3D QSAR models was affirmed by predicting the activity of novel 2-naphthamidines. 3D QSAR models developed may be used in designing and predicting the uPA inhibitory activity of novel molecules.

Cyclo19,31[D-Cys19]-uPA19-31 is a potent competitive antagonist of the interaction of urokinase-type plasminogen activator with its receptor (CD87).

Urokinase-type plasminogen activator (uPA) represents a central molecule in pericellular proteolysis and is implicated in a variety of physiological and pathophysiological processes such as tissue remodelling, wound healing, tumor invasion, and metastasis. uPA binds with high affinity to a specific cell surface receptor, uPAR (CD87), via a well defined sequence within the N-terminal region of uPA (uPA19-31). This interaction directs the proteolytic activity of uPA to the cell surface which represents an important step in tumor cell proliferation, invasion, and metastasis. Due to its fundamental role in these processes, the uPA/uPAR-system has emerged as a novel target for tumor therapy. Previously, we have identified a synthetic, cyclic, uPA-derived peptide, cyclo19,31uPA19-31, as a lead structure for the development of low molecular weight uPA-analogues, capable of blocking uPA/uPAR-interaction [Burgle et al., Biol. Chem. 378 (1997), 231-237]. We now searched for peptide variants of cyclo19,31uPA19-31 with elevated affinities for uPAR binding. Among other tasks, we performed a systematic D-amino acid scan of uPA19-31, in which each of the 13 L-amino acids was individually substituted by the corresponding D-amino acid. This led to the identification of cyclo19,31[D-Cys19]-uPA19-31 as a potent inhibitor of uPA/uPAR-interaction, displaying only a 20 to 40-fold lower binding capacity as compared to the naturally occurring uPAR-ligands uPA and its amino-terminal fragment. Cyclo19,31[D-Cys19]-uPA19-31 not only blocks binding of uPA to uPAR but is also capable of efficiently displacing uPAR-bound uPA from the cell surface and to inhibit uPA-mediated, tumor cell-associated plasminogen activation and fibrin degradation. Thus, cyclo19,31[D-Cys19]-uPA19-31 represents a promising therapeutic agent to significantly affect the tumor-associated uPA/uPAR-system.

A peptide derived from the non-receptor-binding region of urokinase plasminogen activator inhibits glioblastoma growth and angiogenesis in vivo in combination with cisplatin.

The urokinase plasminogen activator system is involved in angiogenesis and tumor growth of malignant gliomas, which are highly neovascularized and so may be amenable to antiangiogenic therapy. In this paper, we describe the activity of A6, an octamer capped peptide derived from the non-receptor-binding region of urokinase plasminogen activator. A6 inhibited human microvascular endothelial cell migration but had no effect on the proliferation of human microvascular endothelial cells or U87MG glioma cells in vitro. In contrast, A6 or cisplatin (CDDP) alone suppressed subcutaneous tumor growth in vivo by 48% and 53%, respectively, and, more strikingly, the combination of A6 plus CDDP inhibited tumor growth by 92%. Such combination treatment also greatly reduced the volume of intracranial tumor xenografts and increased survival of tumor-bearing animals when compared with CDDP or A6 alone. Tumors from the combination treatment group had significantly reduced neovascularization, suggesting a mechanism involving A6-mediated inhibition of endothelial cell motility, thereby eliciting vascular sensitivity to CDDP-mediated toxicity. These data suggest that the combination of an angiogenesis inhibitor that targets endothelial cells with a cytotoxic agent may be a useful therapeutic approach.

Single-chain urokinase-type plasminogen activator bound to its receptor is relatively resistant to plasminogen activator inhibitor type 1.

Urokinase-type plasminogen activator (uPA) is synthesized as single-chain protein (scuPA) with little intrinsic activity. scuPA is activated when it is converted to two-chain urokinase (tcuPA) by plasmin or when it binds as a single-chain molecule to its cellular receptor (uPAR). Previous data indicate that complexes between scuPA and its receptor have somewhat higher affinity for plasminogen than does tcuPA. The current study indicates that plasminogen activator activity of scuPA bound to recombinant, soluble uPAR (suPAR) is also fivefold less sensitive to inhibition by plasminogen activator type 1 (PAI-1) than is soluble or receptor-bound tcuPA. Binding of PaI-1 to suPAR/scuPA complexes is totally reversible and can be overcome by increasing the concentration of plasminogen, suggesting a competitive mechanism of inhibition (Ki = 18 nmol/L). Binding of scuPA to suPAR also retards its cleavage by plasmin. These results indicates that binding of single-chain urokinase to its receptor promotes its activity, retards its inhibition, and protects it from conversion to a two-chain form of the enzyme, a step that may precede its inactivation and clearance from cell surfaces. These results are consistent with a physiologic role for receptor-bound single-chain urokinase as a cellular plasminogen activator.

Thrombin inhibitory and clot-specific fibrinolytic activities of the urokinase variant, M23 (rscu-PA-40 kDa/Hir).

The recombinant bifunctional urokinase variant, M23 (rscu-PA-40 kDA/Hir), comprising the kringle and protease domain of single-chain urokinase-type plasminogen activator and a C-terminal fragment of hirudin in one single-chain molecule, was evaluated for its thrombin-inhibitory and fibrinolytic properties in vitro and in vivo. M23 inhibited thrombin-activated coagulation of human blood and thrombin-induced aggregation of human platelet rich plasma in a concentration-dependent manner. The ADP-induced aggregation of human platelet rich plasma was not influenced by M23. In contrast, recombinant single-chain urokinase-type plasminogen activator (saruplase) inhibited neither blood coagulation nor platelet rich plasma aggregation. M23 and saruplase both lysed radiolabelled human thrombi immersed in human plasma (Chandler Loop system) with equal potency. However, there was a significantly lower systemic generation of plasmin (measured as consumption of alpha 2-antiplasmin) by M23 compared to saruplase. In anaesthetized non-heparinized rabbits, experimental femoral artery thrombosis was treated with intravenous bolus injections of M23 or saruplase (6 mg/kg, each). Thrombolytic restoration of arterial blood perfusion was significantly higher in M23- than in saruplase-treated rabbits. Plasma fibrinogen concentrations were decreased markedly in saruplase-treated animals, but remained at significantly higher levels in M23-treated rabbits. In conclusion, the bifunctional molecule, M23, showed thrombin inhibitory and fibrinolytic properties in human in vitro systems and exerted superior thrombolytic effects to saruplase in rabbit femoral artery thrombosis. In vitro and in vivo data indicate that the fibrinolytic activity of M23 is highly clot-specific.

Fluorescent probes as tools to assess the receptor for the urokinase-type plasminogen activator on tumor cells.

Flow cytofluorometric protocols (FACScan) are described for the rapid and quantitative real-time analysis of binding of FITC-pro-u-PA to cell surface receptors (u-PAR) on living, resting, and also on PMA-stimulated human monocytic U937 cells. Binding of pro-u-PA was visualized by CLSM. This fairly new technique is superior over conventional fluorescence microscopy and is an alternative to electron microscopic approaches. Both flow cytofluorometry and confocal laser scanning microscopy allow the analysis quantitatively and with high-sensitivity binding of FITC-pro-u-PA to single suspended or adherent cells. By CLSM u-PA/u-PAR were found to be located in heterogeneously distributed discrete patches at the cell surface on U937 and not inside the cell. This is in agreement with previous studies by Hansen et al, who applied radioiodinated u-PA and electron microscopy to locate u-PAR on microvilli of fixed U937 cells. By flow cytofluorometry, it was possible to quantify the time-dependent and temperature-dependent binding of FITC-pro-u-PA to living single U937. Apparent saturation of u-PAR was achieved at 5 nM FITC-pro-u-PA for both nonstimulated and PMA-stimulated U937 cells. Half saturation of u-PAR was also determined. Nonstimulated U937 was 0.7 nM, and PMA-stimulated U937 was 1.1 nM of FITC-pro-u-PA. This increase in half-saturation concentration in PMA-stimulated cells is paralleled by a steep increase in binding sites (3.6-fold). The use of fluoresceinated reference beads is recommended to verify changes in affinity and binding sites. Using CLSM or flow cytofluorometry, it is also possible to study the structure relationship of u-PA/u-PAR in the presence of competitive binding analogues or inhibitors. Fluorescence techniques will also permit the identification of u-PAR-positive cells in blood, ascitic fluid, or biopsies obtained from cancer patients.

Insertion of fibrin peptides into urokinase enhances fibrin affinity.

Low molecular weight two-chain urokinase is a 33-kD plasminogen activator, which has no innate affinity for fibrin and consequently, its use to facilitate lysis of blood clots may lead to systemic activation of plasminogen. In order to impart clot affinities to this urokinase form (UK) we have generated two novel fibrin-binding derivatives by partially reducing UK and exchanging the native disulfide-linked peptide A with peptide A analogs. The peptide A analogs contained the fibrin-adherent fibrin-derived sequences, GPRP (derived from positions 17-20 of the fibrinogen alpha chain) or QAGDV (407-411 sequence of the fibrinogen gamma chain), each coupled through amino-hexanoic acid to a synthetic peptide, LKFQCGQK, containing the Leu 144-Lys 158 sequence of the urinary plasminogen activator A Chain. The resultant derivatives contained about 0.4 moles peptide analog/mole UK, were 75% active toward synthetic UK substrates, and were recovered in a nearly 80% yield. The two fibrin peptide derivatives had a five-fold greater affinity for the clots.

Reduction of disulfides in urokinase and insertion of a synthetic peptide.

Abbokinase is a commercially available two-chain lower molecular weight urinary plasminogen activator (u-PA) with low affinity for fibrin. Therefore, therapeutic use of this u-PA form (UK) may cause activation of not only clot-bound plasminogen but also systemic plasminogen. Since this form of UK contains a 13-residue peptide remnant of the A chain disulfide-linked to the catalytic B chain, disulfide-exchange could allow replacement of this native peptide with peptides of desired clot-directed properties. We report here a method in which native peptide was partially removed by subjecting UK to 1-100 mM dithiothreitol (DTT), in the presence of arginine. Arginine (250 mM) was found to slow the rate of release of the peptide by 2-fold, to slow the loss in enzymatic activity by about 9-fold and to limit the extent of disulfide reduction. Upon dialysis to remove DTT from the reduced UK mixture, the disulfides reformed and enzymatic activity was regained. Synthetic peptide added at this point became incorporated to the level of about 0.4 mol/mol UK. This method should be very useful for developing UK derivatives with altered affinities toward fibrin clots.