Array Point Discharge as Enhanced Tandem Excitation Source for Miniaturized Optical Emission Spectrometer.

A new compact tandem excitation source was designed and constructed by using an array point discharge (ArrPD) microplasma for a miniaturized optical emission spectrometer through coupling a hydride generation (HG) unit as a sample introduction device. Three pairs of point discharges were arranged in sequence in a narrow discharge chamber to construct the ArrPD microplasma, for improved excitation capability owing to the serial excitation. Besides, the discharge plasma region was greatly enlarged, therefore, more gaseous analytes could be intercepted to enter into the microplasma for sufficient excitation, for improved excitation efficiency and OES signal. To better understand the effectiveness of the proposed ArrPD source, a new instrument for simultaneous detection of atomic emission and absorption spectral responses was also proposed, designed, and constructed to reveal the excitation and enhancement process in the discharge chamber. Under the optimized conditions, the limits of detection (LODs) of As, Ge, Hg, Pb, Sb, Se, and Sn were 0.7, 0.4, 0.05, 0.7, 0.3, 2, and 0.08 µg L-1, respectively, and the relative standard deviations (RSDs) were all less than 4%. Compared with a commonly used single point discharge microplasma source, the analytical sensitivities of these seven elements were improved by 3-6-fold. Certified Reference Materials (CRMs) were successfully analyzed with this miniaturized spectrometer, which features low power, compactness, portability, and high detectability, and is thereby a great prospect in the field of elemental analytical chemistry.

Microdischarge in Flame as a Source-in-Source for Boosted Excitation of Optical Emission of Chromium.

A compact tandem excitation source-in-source was designed by arranging a point discharge (PD) ignited in argon/hydrogen (Ar/H2) flame and utilized for boosted excitation for the optical emission of chromium. Through a tungsten coil (W-coil) electrothermal vaporizer (ETV) located right under the tandem source without any interface for sample introduction, a miniaturized optical emission spectrometer was realized. Because the discharge gaseous atmosphere of PD was activated in the flame, the energy consumption of PD for breaking down discharge gas and maintenance of plasma was greatly saved. In addition, the flame could partially atomize or keep the atomized state of analyte atoms through its reducing environment. Therefore, the excitation capability of the tandem source was greatly improved, owing to the synergistic effect of PD microplasma and Ar/H2 flame. In addition, part of the analyte was atomized/excited on the W-coil, and thereby, dry, pure, and activated analyte species were released from the W-coil and swept into the tandem source for atomization/excitation. Through the collective effect of W-coil ETV, Ar/H2 flame, and PD microplasma, analytical sensitivity for Cr was greatly enhanced. Under the optimized conditions, with 10 µL sample solution, a limit of detection of 1.5 µg L-1 and a relative standard deviation of 3.6% (20 µg L-1, n = 5) were achieved. Its accuracy was demonstrated by successful analysis of several certified reference materials. Owing to the advantages including high sensitivity, compactness, and cost effectiveness, it is promising to facilitate the miniaturized spectrometer for more elements and potential field analytical chemistry.

A novel, simple and reliable method for the determination of hydronidone and its metabolites M3 and M4 in human plasma and urine by HPLC-MS/MS and its application to a pharmacokinetic study in health Chinese subjects.

In this study, a novel, simple and reliable high-performance liquid chromatography with tandem mass spetrometry method (HPLC-MS/MS) was developed for the determination of hydronidone and its metabolites M3 and M4 in human plasma and urine so as to study the clinical pharmacokinetics of hydronidone. By effectively inhibiting the proliferation of hepatic stellate cells (HSC), hydronidone can reduce collagen synthesis and curbs the process of liver fibrosis, and is currently in the stage of clinical research for anti-liver fibrosis. Hydronidone and its metabolites M3, M4 were extracted from human plasma by protein precipitation, and the urine samples were directly diluted with acetonitrile and analyzed by HPLC-MS/MS. The quantification ranges in plasma were 1.00-1000 ng/mL, 2.00-2000 ng/mL and 4.00-4000 ng/mL, respectively and in urine were 10.0-2000 ng/L, 100-25000 ng/L and 300-75000 ng/L, respectively. Coefficients of variation of less than 15% between intraday and interday accuracy and precision values were observed for hydronidone, M3 and M4. The S/N (signal-to-noise ratio) of the analyte in each Low limit of quantification sample in the analytical batch was greater than 5, indicating good sensitivity. The recovery rates were above 50% for all analytes. The parameters such as linearity, selectivity, lower precision, accuracy, recovery, stability and matrix effects were validated by the methodology and met the requirements specified by the FDA and the European Medicines Agency. The method has been successfully applied to the pharmacokinetics of hydronidone and its metabolites M3 and M4 in healthy Chinese volunteers.

Design, synthesis and biological evaluation of hybrid of ubenimex-fluorouracil for hepatocellular carcinoma therapy.

In our previous study, we discovered a ubenimex-fluorouracil (5FU) conjugates BC-02, which displays significant in vivo anti-tumor activity, however, the instability of BC-02 in plasma limits its further development as a drug candidate. Herein, we designed and synthesized four novel ubenimex-5FU conjugates by optimizing the linkers between ubenimex and 5FU based on BC-02. Representative compound 20 is more stable than BC-02 in human plasma and displays about 100 times higher CD13 inhibitory activity than the positive control ubenimex. Meanwhile, the antiproliferative activity of 20 was comparable with 5FU in vitro. The preliminary mechanism study indicated that compound 20 exhibited significant anti-invasion and anti-angiogenesis activities in vitro. Furthermore, compound 20 obviously inhibits tumor growth and metastasis in vivo and prolong the survival time of tumor-bearing mice. Our study may have an important implication reference for the design of more druglike mutual prodrug, and compound 20 can be used as a lead compound for further design and development.

Intracellular tacrolimus concentration correlates with impaired renal function through regulation of the IS-AHR-ABC transporter in peripheral blood mononuclear cells.

BACKGROUNDS: Tacrolimus (TAC) concentration in peripheral blood mononuclear cells (PBMCs) is regarded as a better predictor of its immunosuppressive effect than the TAC concentration in whole blood. However, whether the exposure of TAC in PBMCs or WB was altered in post-transplant recipients with renal impairment remains unclear. METHODS: We investigated the relationship of trough TAC concentration in WB and PBMCs with renal functions in post-transplant recipients. The pharmacokinetic profiles of TAC in PBMCs and WB in the two chronic kidney disease (CKD) rat models were examined using UPLC-MS/MS. Western blotting and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to analyze the expression of proteins and mRNAs related to TAC metabolism and transport, respectively. In addition, the effects of uremic toxins on human PBMCs were investigated using whole-transcriptome sequencing (RNA sequencing [RNA-seq]). RESULTS: We observed a decrease in the trough TAC concentration in PBMCs in the recipients with estimated glomerular filtration rate (eGFR) < 90 mL/min, compared with those of recipients with eGFR > 90 mL/min, but there was no difference in blood based on TAC concentrations (C0Blood). In a 150-patient post-transplant cohort, no significant relationship was observed between PBMCs and WB concentrations of TAC, and the eGFR value was correlated with TAC C0PBMCs but not with TAC C0Blood. In two CKD rat models, the TAC pharmacokinetic profile in the PBMCs was significantly lower than that in the control group; however, the blood TAC pharmacokinetic profiles in the two groups were similar. Transcriptome results showed that co-incubation of human PBMCs with uremic toxins upregulated the expression of AHR, ABCB1, and ABCC2. Compared to control rats, plasma IS increased by 1.93- and 2.26-fold and the expression of AHR, P-gp, and MRP2 in PBMCs was higher in AD and 5/6 nephrectomy (NX) rats, without modifying the expression of other proteins related to TAC exposure. CONCLUSION: The pharmacokinetics of TAC in PBMCs changed with a decline in renal function. Uremic toxins accumulate during renal insufficiency, which activates AHR, upregulates the expression of P-gp and MRP2, and affects their intracellular concentrations. Our findings suggest that monitoring TAC concentrations in PBMCs is more important than monitoring WB concentrations in post-transplant recipients with renal impairment.

Vancomycin relieves tacrolimus-induced hyperglycemia by eliminating gut bacterial beta-glucuronidase enzyme activity.

Up to 40% of transplant recipients treated long-term with tacrolimus (TAC) develop post-transplant diabetes mellitus (PTDM). TAC is an important risk factor for PTDM, but is also essential for immunosuppression after transplantation. Long-term TAC treatment alters the gut microbiome, but the mechanisms of TAC-induced gut microbiota in the pathogenesis of PTDM are poorly characterized. Here, we showed that vancomycin, an inhibitor of bacterial beta-glucuronidase (GUS), prevents TAC-induced glucose disorder and insulin resistance in mice. Metagenomics shows that GUS-producing bacteria are predominant and flourish in the TAC-induced hyperglycemia mouse model, with upregulation of intestinal GUS activity. Targeted metabolomics analysis revealed that in the presence of high GUS activity, the hydrolysis of bile acid (BAs)-glucuronic conjugates is increased and most BAs are overproduced in the serum and liver, which, in turn, activates the ileal farnesoid X receptor (FXR) and suppresses GLP-1 secretion by L-cells. The GUS inhibitor vancomycin significantly eliminated GUS-producing bacteria and inhibited bacterial GUS activity and BAs levels, thereby enhancing L-cell GLP-1 secretion and preventing hyperglycemia. Our results propose a novel clinical strategy for inhibiting the bacterial GUS enzyme to prevent hyperglycemia without requiring withdrawal of TAC treatment. This strategy exerted its effect through the ileal bile acid-FXR-GLP-1 pathway.

Immune Checkpoint-Modulating Photosensitizer That Targets BRD4 for Cancer Photoimmunotherapy.

Photodynamic therapy is an efficient approach to promote cytotoxic T lymphocyte tumor infiltration to convert immunologically cold tumors into hot tumors through the induction of immunogenic cell death . However, tumors usually overexpress immune checkpoints such as PD-L1 to suppress T lymphocyte antitumor activity and evade immune surveillance. Therefore, the design of efficient photosensitizers to overcome checkpoint-mediated immune evasion is highly necessary. In this work, we report the design of BRD-PS, a BRD4-targeting photosensitizer, as a new class of immunomodulatory photosensitizer termed an immune checkpoint-modulating photosensitizer, to solve this issue. On one hand, BRD-PS induces immunogenic pyroptosis and ferroptosis to promote the activation and tumor infiltration of cytotoxic T cells. On the other hand, BRD-PS suppresses the expression of PD-L1 to avoid immune evasion. This work demonstrated the feasibility of utilizing a single photosensitizer to simultaneously induce immunogenic cell death and PD-L1 downregulation for synergistic cancer photoimmunotherapy.

Nuclear PARP1-Targeted Photosensitizer as a Dual-Mode DNA-Damaging Agent and Immune Activator for Tumor Ablation.

Photodynamic therapy is a promising cancer therapeutic method that can damage DNA via photoinduced reactive oxygen species production. However, tumor cells can initiate DNA repair pathways to resist oxidative damage. In this study, a nuclear-targeted photosensitizer PARP-PS with a poly (ADP-ribose) polymerase 1 (PARP1) inhibitory effect is developed based on the reported PARP1 inhibitor, rucaparib. As a dual-mode DNA-damaging agent, PARP-PS damages DNA upon photoirradiation and enhances oxidative DNA damage by blocking the DNA repair pathway via PARP1 inhibition and degradation. Both in vitro and in vivo investigations demonstrate that PARP-PS exhibits high antitumor activity with few side effects in breast cancer. In addition, PARP-PS can act as an immunogenic cell death inducer to activate immune responses characterized by the promotion of cytotoxic T lymphocyte activation and tumor infiltration. Therefore, PARP-PS is a potential multimodal antitumor agent with synergistic phototherapeutic, chemotherapeutic, and immunotherapeutic effects.

General principle and optimization of magneto-acousto-electrical tomography based on image distortion evaluation.

BACKGROUND: As a novel non-invasive multi-physics imaging methodology, the magneto-acousto-electrical (MAE) technology is capable of detecting electric conductivity changes for biological tissues, exhibiting prosperous perspectives in medical applications. However, the acoustic beam was often simplified to a straight line or a focused one, being perpendicular to layered boundaries of tissues in previous studies. Linear-scanning measurements were carried out to reconstruct B-mode MAE images for layered models without considering the radiation pattern of transducers. Obvious image distortions in both shape and brightness were observed in experiments without any reasonable explanation. PURPOSE: This study aims to establish a general physical model for MAE measurements and solve the problem of B-mode image distortion, and hence provide theoretical and technical supports for the improvement of MAE imaging in practical applications. METHODS: By considering the radiation pattern of actual transducers and the inclined angle of electric conductivity boundaries, a general principal of MAE measurements applicable for objects of arbitrary shapes is proposed based on the theories of acoustic radiation, Hall Effect and electrical detection. The influences of inclined conductivity boundaries and transducer directivities are numerically analyzed with Matlab programming and also demonstrated by experimental measurements. To evaluate the degree of B-mode image distortion, the deformation length (3 dB amplitude decrease) of approximate straight lines for a circular model is defined as L = dtan(ßm /2), with d and ßm being the measurement distance and the half radiation angle of the main-lobe, respectively. The rotary-scanning-based MAE tomography (MAET) is employed to reduce the image distortion, and the rotation angle step is further optimized based on the criterion of the boundary radius fluctuation coefficient <0.01 mm. RESULTS: The experimental results of MAE signals and B-mode images as well as MAETs show good agreements with simulations. It is demonstrated that, as the increase of the inclined angle, the MAE decreases rapidly with an extended time interval and reaches the 20 dB amplitude decrease when the angle exceeds 12°. Meanwhile, the deformation length of B-mode MAE imaging increases with the increase of the radiation angle for the transducer with a weaker radiation pattern, and hence results in a more serious image distortion. A smaller rotation angle step should be adopted to the MAET system with a longer deformation length, and the optimized maximum angle step of 12° is also achieved for the omnidirectional radiation of point sources with a long deformation length. CONCLUSION: The image distortion is originated from the amplitude decrease, the time shift and the time interval expansion of MAE signals introduced by the deformation length and the incident angle. The favorable results demonstrate that the fast high-resolution imaging can be accomplished by the minimum rotations of the rotary-scanning-based MAET using an actual transducer, and also provide an optimized scheme for the rotary-based MAET without scanning using a linear array of point sources.

Lysosome-targeting phenalenones as efficient type I/II photosensitizers for anticancer photodynamic therapy.

Development of safe and effective photosensitizers is important for enhancing the efficacy of photodynamic cancer therapy. Phenalenone is a type II photosensitizer with a high singlet oxygen quantum yield; however, its short UV absorption wavelength hinders its application in cancer imaging and in vivo photodynamic therapy. In this study, we report a new redshift phenalenone derivative, 6-amino-5-iodo-1H-phenalen-1-one (SDU Red [SR]), as a lysosome-targeting photosensitizer for triple-negative breast cancer therapy. SDU Red produced singlet oxygen (Type II reactive oxygen species [ROS]) and superoxide anion radicals (Type I ROS) upon light irradiation. It also exhibited good photostability and a remarkable phototherapeutic index (PI > 76) against triple-negative breast cancer MDA-MB-231 cancer cells. Additionally, we designed two amide derivatives, SRE-I and SRE-II, with decreased fluorescence and photosensitizing capabilities based on SDU Red as activatable photosensitizers for photodynamic cancer therapy. SRE-I and SRE-II could be further converted into the active photosensitizer SDU Red via carboxylesterase-catalyzed amide bond cleavage. Moreover, SDU Red and SRE-II induced DNA damage and cell apoptosis in the presence of light. Therefore, SRE-II can act as a promising theranostic agent for triple-negative breast cancer.

Ligand-Directed Photodegradation of Interacting Proteins: Oxidative HER2/HER3 Heterodimer Degradation with a Lapatinib-Derived Photosensitizer.

In this work, we described a photocatalytic approach, termed ligand-directed photodegradation of interacting proteins (LDPIP), for efficient protein-protein heterodimer degradation. This LDPIP approach utilizes a combination of a photosensitizing protein ligand and appropriate light and molecular oxygen to induce oxidative damage to the ligand-binding protein as well as its interacting protein partner. As a showcase study, a photosensitizing HER2 ligand HER-PS-I was rationally designed based on the FDA-approved HER2 inhibitor lapatinib to efficiently degrade HER2 together with its interacting protein partner HER3, which is thought to induce HER2-targeted therapy resistance and difficult to target by small molecules. HER-PS-I exhibited excellent anticancer activity against drug-resistant MDA-MB-453 cells and its three-dimensional multicellular spheroids. We hope that this LDPIP approach would find more applications in degrading proteins that are thought undruggable or difficult to drug.

A Pharmacokinetic Drug-Drug Interactions Study between Entecavir and Hydronidone, a Potential Novel Antifibrotic Small Molecule, in Healthy Male Volunteers.

INTRODUCTION: Hepatic fibrosis is an inevitable process of hepatic sclerosis, malignancy, and insufficiency, and hydronidone is an innovative antifibrosis drug. This study focus on the pharmacokinetic interaction of hydronidone and entecavir in healthy Chinese male subjects. METHODS: An open-label, three-period, multiple-dosage, self-controlled clinical trial was executed in 12 healthy male subjects. In period 1, the subjects took hydronidone 60 mg, q8h, for 7 days. In period 2, they were given entecavir 0.5 mg once daily for 9 days. Then, hydronidone and entecavir were given in combination for 6 days (days 20-26). Blood samples were taken up to 24 h post-dosing, while pre-dose blood samples were drawn on days 7, 19, and 26. RESULTS: The area under the curve (AUC)0-t_ss of entecavir slightly increased from 15.56 ± 2.67 to 16.17 ± 2.77 ng h/ml with coadministration with hydronidone, while the other pharmacokinetic parameters of hydronidone and entecavir were comparable between monotherapy and combination therapy. The geometric mean ratios (GMRs) [90% confidence intervals (CIs)] of Cmax_ss, AUC0-t_ss, and AUC0-∞_ss of entecavir after coadministration compared with entecavir alone were 107.21% (97.04-118.45%), 103.85% (100.94-106.83%), and 110.81% (97.19-126.33%), respectively. And the GMRs and 90% CIs of Cmax,ss, AUC0-t_ss, and AUC0-∞_ss for combination therapy compared with the hydronidone monotherapy group were 102.72% (84.21-125.29%), 106.52% (97.06-116.90%), and 108.86% (96.42-122.89%), respectively. CONCLUSIONS: There was no drug-drug interaction between hydronidone and entecavir in healthy male volunteers. However, multiple doses of hydronidone have a risk with increasing exposure to entecavir in vivo, which needs to be further clarified. REGISTRATION NUMBER: ChiCTR2200059683 (retrospectively registered).

Pharmacokinetics and bioequivalence evaluation of omeprazole and sodium bicarbonate dry suspensions in healthy Chinese volunteers.

Omeprazole and sodium bicarbonate dry suspension are effective treatments for acid-related disorders. This study compared the bioequivalence and safety of the two formulations of omeprazole and sodium bicarbonate powder and assessed how CYP2C19 gene polymorphisms affect pharmacokinetics (PK). A single-center, randomized, single-dose, 2-sequence and 2-period crossover method was performed in forty healthy Chinese subjects. Blood samples were collected after a single dose for PK (AUC0-∞, AUC0-t, and Cmax) analysis. The concentrations of Omeprazole in human plasma were determined by HPLC-MS/MS. Besides, the gene polymorphisms of CYP2C19 were assessed by Sanger sequencing. The geometric mean ratios (90% confidence interval) [GMR (95% CI)] of Test/Reference preparation for Cmax: 95.2% (88.48%, 102.43%), AUC0-t: 97.47% (94.4%, 101.02%), AUC0-∞: 97.68% (94.27%, 101.21%) were within the range of 80.00-125.00%. The non-parametric test showed no statistical difference in Tmax between the two groups (p > 0.05). All drugs were well tolerated, no severe adverse reactions occurred, and no significant differences in adverse events between the two drugs. For CYP2C19 gene polymorphisms, the results showed that of 40 subjects, 12 subjects were extensive metabolizers, 24 were intermediate metabolizers, and 4 were poor metabolizers, the frequency of metabolic genotypes were 30%, 60%, and 10%. And the allele distributions for CYP2C19 were *1, *2, and *3 at 60%, 38.75%, and 1.25%. Both the CYP2C19 alleles and metabolic genotypes were consistent with other studies in Chinese. The results of PK parameters showed that different genotypes of CYP2C19 lead to significant differences in t1/2, AUC0-t, AUC0-∞ and Cmax, but no significant differences in Tmax in each group. At the same time, we confirmed that the PK parameters of the test and reference had no differences between the males and females. This study has shown that the pharmacokinetic parameters of the two formulations are not significantly different, which showed bioequivalence and exemplary safety. CYP2C19 gene polymorphism significantly differed in the PK parameters of omeprazole sodium bicarbonate powder.

Effects of intestinal microbiota on pharmacokinetics of cyclosporine a in rats.

Background: Intestinal microbiota has been confirmed to influencing the pharmacokinetic processes of a variety of oral drugs. However, the pharmacokinetic effects of the gut microbiota on cyclosporine A, a drug with a narrow therapeutic window, remain to be studied. Method: Twenty-one rats were randomly divided into three groups: (a) control group (CON), (b) antibiotic treatment group (ABT) and (c) fecal microbe transplantation group (FMT). The ABT group was administrated with water containing multiple antibiotics to deplete microorganisms. FMT was with the same treatment, followed by oral administration of conventional rat fecal microorganisms for normalization. Result: The bioavailability of CSA increased by 155.6% after intestinal microbes were consumed by antibiotics. After intestinal microbiota reconstruction by fecal transplantation, the increased bioavailability was significantly reduced and basically returned to the control group level. Changes in gut microbiota alter the protein expression of CYP3A1, UGT1A1 and P-gp in liver. The expressions of these three proteins in ABT group were significantly lower than those in CON and FMT groups. The relative abundance of Alloprevolleta and Oscillospiraceae UCG 005 was negatively correlated with CSA bioavailability while the relative abundance of Parasutterella and Eubacterium xylanophilum group was negatively correlated with CSA bioavailability. Conclusion: Intestinal microbiota affects the pharmacokinetics of CSA by regulating the expression of CYP3A1, UGT1A1 and P-GP.

Construction and Expression of Mouse-human Chimeric Antibody SZ-51 Specific for Human Activated Platelets.

SZ-51, a murine monoclonal antibody (McAb) specific for alpha-granule membrane protein (GMP-140) on the surface of the activated human platelets, has shown promise for thrombus imaging and thrombolysis. In order to reduce the immunogenicity of the murine McAb SZ-51 in man and to obtain a high level of antibody production, we constructed two chimeras (alpha-Lys17-51BVK/Hu, alpha-Lys30-51VH/Hu) by joining the variable regions gene of mouse antibody to the constant regions gene of human immunoglobulin (Ig)(gamma1,k). Both chimeric genes were cloned into two selectable expression vectors separately, which were co-transfected into a non-Ig secreting murine myeloma cell line SP2/0 with the Lipofectin reagent. One transfectoma, which showed stable antigen (GMP-140) binding ability and a high level expression of 5 mg/L, was obtained. Immunoblotting analysis demonstrated that the chimeric antibody in the supernatant, like the native mouse SZ-51, had the characteristic of binding to GMP-140. In addition, the chimeric antibody can bind competitively to activated platelets with (125)I-labeled mouse SZ-51. Therefore, the SZ-51 chimeric antibody may be a potential agent for the diagnosis and therapy of thrombotic diseases in future.

Construction and Expression of a Recombinant Antibody-targeted Plasminogen Activator Composed of a Humanized Monoclonal Antibody against Activated Human Platelets and a Single-chain Urokinase.

To obtain a thromblytic agent with high efficacy and specifity, we have engineered a recombinant chimeric plasminogen activator SZ51Hu-scuPA, which was composed of a humanized monoclonal antibody (SZ51Hu) directed against activated human platelets and a single-chain urokinase (scuPA). The cDNA sequence encoding scuPA was fused through 5' end to 3' end of the CH(3) of SZ51Hu heavy-chain sequence in the expression vector alphalys30(alphalys30-SZ51VH/Hu-scuPA) by PCR. This construct was transfected into a murine myeloma line secreting SZ51Hu light chain with Lipofectin reagent and three lines which showed stable integration and high level expression with concentration of 5 mg/L in supernatant, were selected in the end. Purified SZ51Hu-scuPA migrated as a 160 kD band on non-reduced SDS/PAGE and had the same characteristic of binding to the activated platelets compared with its parent murine antibody molecule SZ51 by Western-blot analysis. The fibrinolytic activity of purified products was 39 000 IU/mg. Thus, by recombinant techniques, an expressed fusion protein was capable of specific binding to activated platelets and plasminogen activation. These observations laid a solid foundation for further study of targeting of thrombolysis in vitro and in vivo.