Spotlight on Volasertib: Preclinical and Clinical Evaluation of a Promising Plk1 Inhibitor.

Considering the important side effects of conventional microtubule targeting agents, more and more research focuses on regulatory proteins for the development of mitosis-specific agents. Polo-like kinase 1 (Plk1), a master regulator of several cell cycle events, has arisen as an intriguing target in this research field. The observed overexpression of Plk1 in a broad range of human malignancies has given rise to the development of several potent and specific small molecule inhibitors targeting the kinase. In this review, we focus on volasertib (BI6727), the lead agent in category of Plk1 inhibitors at the moment. Numerous preclinical experiments have demonstrated that BI6727 is highly active across a variety of carcinoma cell lines, and the inhibitor has been reported to induce tumor regression in several xenograft models. Moreover, volasertib has shown clinical efficacy in multiple tumor types. As a result, Food and Drug Administration (FDA) has recently awarded volasertib the Breakthrough Therapy status after significant benefit was observed in acute myeloid leukemia (AML) patients treated with the Plk1 inhibitor. Here, we discuss both preclinical and clinical data available for volasertib administered as monotherapy or in combination with other anticancer therapies in a broad range of tumor types.

Looking into the "Garden of the Hesperides": new drugs for hepatocellular carcinoma.

Advanced hepatocellular carcinoma remains an entity that is hard to approach therapeutically and has shown disappointing results in terms of survival. For many years, the only accepted option for this setting was the use of a multikinase inhibitor, sorafenib. Nevertheless, through the years, a deeper knowledge has arisen about how pathogenic pathways correlate with hepatocellular carcinoma. In this review, we provide an update of the most recent data regarding new agents under investigation and new possible targets for future treatments.

Detection and prognostic significance of circulating tumour cells in patients with metastatic breast cancer according to immunohistochemical subtypes.

BACKGROUND: The enumeration of circulating tumour cells (CTCs) with the EpCAM-based CellSearch system has prognostic significance in patients with metastatic breast cancer (MBC). The aim of this study was to explore potential differences in the detection and prognostic significance of CTCs in MBC according to immunohistochemical subtypes of breast cancer. METHODS: CellSearch CTC counts were obtained from 154 MBC patients before first-line systemic treatment between November 2007 and August 2012. Patients were categorised in five subgroups according to immunohistochemical surrogate definitions of intrinsic subtypes in breast cancer based on hormone receptor status, HER2/neu status and histological grade. Differences in progression-free (PFS) and overall survival (OS) were assessed relative to the cut-off value of ≥5 CTCs per 7.5 ml blood. RESULTS: No significant differences were observed in the absolute CTC counts (P=0.120) or in CTC positivity rates according to ≥1 and ≥5 CTCs per 7.5 ml blood detection thresholds (P=0.165 and P=0.651, respectively) between immunohistochemical subtypes. However, very high CTC counts, defined as ≥80 CTCs per 7.5 ml, were observed more frequently in patients with Luminal A and triple negative (TN) breast cancer (P=0.024). In the total study population, the presence of ≥5 CTCs was the single most significant prognostic factor for both PFS and OS in multivariate analysis (P<0.001). A more limited prognostic impact, not reaching statistical significance, was observed in patients with HER2-positive disease as opposed to patients with Luminal A, Luminal B-HER2-negative and TN disease. CONCLUSION: The detection of EpCAM+CTCs was not clearly associated with any of the immunohistochemical subtypes of breast cancer in patients with MBC before first-line treatment. Potentially clinically relevant differences were however observed at very high CTC counts. Furthermore, our data suggest a lower prognostic significance of CTC evaluation in HER2-positive patients with MBC.

Semiautomated isolation and molecular characterisation of single or highly purified tumour cells from CellSearch enriched blood samples using dielectrophoretic cell sorting.

BACKGROUND: Molecular characterisation of single circulating tumour cells (CTCs) holds considerable promise for predictive biomarker assessment and to explore CTC heterogeneity. We evaluate a new method, the DEPArray system, that allows the dielectrophoretic manipulation and isolation of single and 100% purified groups of CTCs from pre-enriched blood samples and explore the feasibility of their molecular characterisation. METHODS: Samples containing known numbers of two cell populations were used to assess cell loss during sample loading. Cultured breast cancer cells were isolated from spiked blood samples using CellSearch CTC and Profile kits. Single tumour cells and groups of up to 10 tumour cells were recovered with the DEPArray system and subjected to transcriptional and mutation analysis. RESULTS: On average, 40% cell loss was observed when loading samples to the DEPArray system. Expected mutations in clinically relevant markers could be obtained for 60% of single recovered tumour cells and all groups of tumour cells. Reliable gene expression profiles were obtained from single cells and groups of up to 10 cells for 2 out of 3 spiked breast cancer cell lines. CONCLUSION: We describe a semiautomated workflow for the isolation of small groups of 1 to 10 tumour cells from whole blood samples and provide proof of principle for the feasibility of their comprehensive molecular characterisation.

Inborn oxidative phosphorylation defect as risk factor for propofol infusion syndrome.

Propofol is an anesthetic agent widely used for induction and maintenance of anesthesia, and sedation in children. Although generally considered as reliable and safe, administration of propofol can occasionally induce a potentially fatal complication known as propofol infusion syndrome (PRIS). Mitochondrial dysfunction has been implicated in the pathogenesis of PRIS. We report on an adult patient with Leber hereditary optic neuropathy (LHON) who developed PRIS. He was a carrier of the m.3460G>A mutation, one of the major three pathogenic point mutations associated with LHON. The propositus was blind and underwent propofol sedation after severe head injury. Five days after start of propofol infusion, the patient died. The activity of complex I of the oxidative phosphorylation (OXPHOS) system was severely deficient in skeletal muscle. Our observation indicates that fulminate PRIS can occur in an adult patient with an inborn OXPHOS defect and corroborates the hypothesis that PRIS is caused by inhibition of the OXPHOS system.

Atypical adenolymphoma and glomus caroticum tumour: a rare coincidence.

We describe the rare simultaneous appearance of an atypical adenolymphoma with a glomus caroticum tumour on the same side of the neck in a middle-aged man. This case report is the first to describe this coexistence. Due to the atypical, cyst-like presentation of the Warthin's tumour, a final diagnosis was made only after surgical resection and histopathological examination. Both the adenolymphoma and glomus caroticum tumour were successfully removed surgically.

PRECISION: the Belgian molecular profiling program of metastatic cancer for clinical decision and treatment assignment.

PRECISION is an initiative from the Belgian Society of Medical Oncology (BSMO) in collaboration with several stakeholders, encompassing four programs that aim to boost genomic and clinical knowledge with the ultimate goal to offer patients with metastatic solid tumors molecularly guided treatments. The PRECISION 1 study has led to the creation of a clinico-genomic database. The Belgian Approach for Local Laboratory Extensive Tumor Testing (BALLETT) and GeNeo studies will increase the number of patients with advanced cancer that have comprehensive genotyping of their cancer. The PRECISION 2 project consists of investigator-initiated phase II studies aiming to provide access to a targeted drug for patients whose tumors harbor actionable mutations in case the matched drug is not available through reimbursement or clinical trials in Belgium.

Circulating tumour cells in the central and the peripheral venous compartment in patients with metastatic breast cancer.

BACKGROUND: The enumeration of circulating tumour cells (CTC) has prognostic significance in patients with metastatic breast cancer (MBC) and monitoring of CTC levels over time has considerable potential to guide treatment decisions. However, little is known on CTC kinetics in the human bloodstream. METHODS: In this study, we compared the number of CTC in both 7.5 ml central venous blood (CVB) and 7.5 ml peripheral venous blood (PVB) from 30 patients with MBC starting with a new line of chemotherapy. RESULTS: The number of CTC was found to be significantly higher in CVB (median: 43.5; range: 0-4036) than in PVB (median: 33; range: 0-4013) (P=0.001). When analysing samples pairwise, CTC counts were found to be significantly higher in CVB than in PVB in 12 out of 26 patients with detectable CTC. In contrast, only 2 out of 26 patients had higher CTC counts in PVB as compared with CVB, whereas in 12 remaining patients no significant difference was seen. The pattern of CTC distribution was independent of the sites of metastatic involvement. CONCLUSION: A substantial difference in the number of CTC was observed between CVB and PVB of patients with MBC. Registration of the site of blood collection is warranted in studies evaluating the role of CTC assessment in these patients.

Cross-border reproductive care in Belgium.

BACKGROUND: Cross-border reproductive care indicates the cross-border movements made by patients to obtain infertility treatment they cannot obtain at home. The problem at present is that empirical data on the extent of the phenomenon are lacking. This article presents the data on infertility patients going to Belgium for treatment. METHODS: A survey was conducted among the centres for reproductive medicine that are allowed to handle oocytes and create embryos (B-centres). Data were collected on the nationality of patients and the type of treatment for which they attended during the period 2000-2007. RESULTS: Sixteen of 18 centres responded to the questionnaire. The flow of foreign patients has stabilized since 2006 at approximately 2100 patients per year. The majority of foreign nationals seeking treatment in Belgium were French women for sperm donation. The next highest group was patients entering the country to obtain ICSI with ejaculated sperm. CONCLUSIONS: There are clear indications that numerous movements are motivated by the wish to evade legal restrictions in one's home country, either because the technology is prohibited or because the patients have characteristics, which exclude them from treatment in their own countries.

Hotspot DAXX, PTCH2 and CYFIP2 mutations in pancreatic neuroendocrine neoplasms.

Mutations in DAXX/ATRX, MEN1 and genes involved in the phosphoinositide-3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway have been implicated in pancreatic neuroendocrine neoplasms (pNENs). However, mainly mutations present in the majority of tumor cells have been identified, while proliferation-driving mutations could be present only in small fractions of the tumor. This study aims to identify high- and low-abundance mutations in pNENs using ultra-deep targeted resequencing. Formalin-fixed paraffin-embedded matched tumor-normal tissue of 38 well-differentiated pNENs was sequenced using a HaloPlex targeted resequencing panel. Novel amplicon-based algorithms were used to identify both single nucleotide variants (SNVs) and insertion-deletions (indels) present in >10% of reads (high abundance) and in <10% of reads (low abundance). Found variants were validated by Sanger sequencing. Sequencing resulted in 416,711,794 reads with an average target base coverage of 2663 ± 1476. Across all samples, 32 high-abundance somatic, 3 germline and 30 low-abundance mutations were withheld after filtering and validation. Overall, 92% of high-abundance and 84% of low-abundance mutations were predicted to be protein damaging. Frequently, mutated genes were MEN1, DAXX, ATRX, TSC2, PI3K/Akt/mTOR and MAPK-ERK pathway-related genes. Additionally, recurrent alterations on the same genomic position, so-called hotspot mutations, were found in DAXX, PTCH2 and CYFIP2. This first ultra-deep sequencing study highlighted genetic intra-tumor heterogeneity in pNEN, by the presence of low-abundance mutations. The importance of the ATRX/DAXX pathway was confirmed by the first-ever pNEN-specific protein-damaging hotspot mutation in DAXX. In this study, both novel genes, including the pro-apoptotic CYFIP2 gene and hedgehog signaling PTCH2, and novel pathways, such as the MAPK-ERK pathway, were implicated in pNEN.

ALK immunohistochemistry positive, FISH negative NSCLC is infrequent, but associated with impaired survival following treatment with crizotinib.

OBJECTIVE: Metastasized non-small cell lung cancer (NSCLC) with an anaplastic lymphoma kinase (ALK) rearrangement is usually sensitive to a range of ALK-tyrosine kinase inhibitors. ALK-positive NSCLC have been identified in pivotal phase III trials with fluorescence in situ hybridization (ALK FISH+). These tumors are also expressing the fusion product (ALK immunohistochemistry (IHC)+). However, discrepant cases occur, including ALK IHC + FISH-. The aim of this study was to collect ALK IHC + cases and compare within this group response to crizotinib treatment of ALK FISH + cases with ALK FISH- cases. MATERIALS AND METHODS: In this European prospective multicenter research study patients with Stage IV ALK IHC + NSCLC treated with crizotinib were enrolled. Tumor slides were validated centrally for ALK IHC and ALK FISH. RESULTS: Registration of 3523 ALK IHC tests revealed a prevalence of 2.7% (n = 94) ALK IHC + cases. Local ALK FISH analysis resulted in 48 concordant (ALK IHC+/FISH+) and 16 discordant (ALK IHC+/FISH-) cases. Central validation revealed 37 concordant and 7 discordant cases, 5 of which had follow-up. Validation was hampered by limited amount of tissue in biopsy samples. The PFS at 1 year for ALK concordant and discordant was 58% and 20%, respectively (HR = 2.4; 95% CI: 0.78-7.3; p = 0.11). Overall survival was significantly better for concordant cases than discordant cases after central validation (HR=4.5; 95% CI= 1.2-15.9; p=0.010. CONCLUSION: ALK IHC + FISH- NSCLC is infrequent and associated with a worse outcome on personalized treatment. A suitable predictive testing strategy may be to screen first with IHC and then confirm with FISH instead of considering ALK IHC equivalent to ALK FISH according to the current guidelines.

Loss of protein phosphatase 2A regulatory subunit B56δ promotes spontaneous tumorigenesis in vivo.

Protein Phosphatase 2A (PP2A) enzymes counteract diverse kinase-driven oncogenic pathways and their function is frequently impaired in cancer. PP2A inhibition is indispensable for full transformation of human cells, but whether loss of PP2A is sufficient for tumorigenesis in vivo has remained elusive. Here, we describe spontaneous tumor development in knockout mice for Ppp2r5d, encoding the PP2A regulatory B56δ subunit. Several primary tumors were observed, most commonly, hematologic malignancies and hepatocellular carcinomas (HCCs). Targeted immunoblot and immunohistochemistry analysis of the HCCs revealed heterogeneous activation of diverse oncogenic pathways known to be suppressed by PP2A-B56. RNA sequencing analysis unveiled, however, a common role for oncogenic c-Myc activation in the HCCs, independently underscored by c-Myc Ser62 hyperphosphorylation. Upstream of c-Myc, GSK-3ß Ser9 hyperphosphorylation occurred both in the HCCs and non-cancerous B56δ-null livers. Thus, uncontrolled c-Myc activity due to B56δ-driven GSK-3ß inactivation is the likely tumor predisposing factor. Our data provide the first compelling mouse genetics evidence sustaining the tumor suppressive activity of a single PP2A holoenzyme, constituting the final missing incentive for full clinical development of PP2A as cancer biomarker and therapy target.

Results of a national survey on drug resistance among pulmonary tuberculosis patients in Rwanda.

BACKGROUND: One of the principal objectives of tuberculosis (TB) control is to minimise the emergence of drug resistance. The first national survey was conducted in Rwanda to determine the prevalence of M. tuberculosis drug resistance. METHODS: Sputum samples were collected from all new and retreatment cases in the health districts from November 2004 to February 2005. Drug susceptibility testing of isolates against first-line drugs was performed by the proportion method. RESULTS: Of 616 strains from new cases, 6.2% were resistant to isoniazid, 3.9% to rifampicin and 3.9% were multidrug-resistant TB. Among 85 strains from previously treated cases, the prevalence of resistance was respectively 10.6%, 10.6% and 9.4% (MDR-TB strains). Eight MDR cases showed additional resistance to ethambutol and streptomycin. CONCLUSION: The level of MDR-TB among TB patients in Rwanda is high. The main reasons of this emergence of MDR-TB can be attributed to the disorganisation of the health system, migration of the population during the 1994 civil war and poor success rates, with a high number of patients transferred out and lost to follow-up. On the other hand, the use of treatment regimens administered twice weekly during the continuation phase could be another important factor and merit further investigations.

Endometrial safety after 5 years of continuous combined transdermal estrogen and intrauterine levonorgestrel delivery for postmenopausal hormone substitution.

OBJECTIVE: To investigate endometrial histology and thickness of the endometrium after long-term use of continuous transdermal estrogen substitution combined with intrauterine release of levonorgestrel (LNG) in postmenopausal women. DESIGN: A 5-year non-comparative prospective clinical trial. SUBJECTS: Out of 182 symptomatic postmenopausal women using estrogen substitution therapy (EST) combined with a novel T-shaped LNG-releasing intrauterine system (Femilis Slim LNG-IUS), to prevent endometrial proliferation and bleeding, only those women (n=102) who used two consecutive LNG-IUSs, were isolated with the aim to study the long-term effects on the endometrium. The mean age of the women was 57 years (range 47-71). The majority of women received percutaneous 17beta estradiol, 1.5mg daily, or an equivalent dose by patch or orally, on a continuous basis. MAIN OUTCOME MEASURES: Endometrial histology and ultrasonographic evidence of endometrial suppression, after a period of approximately 5 years of use. The mean duration of use of the regimen was 70 months (range 25-98). RESULTS: The dominant endometrial histologic picture was that of inactive endometrium characterized by glandular atrophy and stroma decidualization (Kurman classification 5b). No cases of endometrial hyperplasia were found. On transvaginal ultrasound, this corresponds with a thin endometrium (< or = 5 mm). CONCLUSION: The results of this 5-year study in 102 postmenopausal women using EST demonstrates that the LNG-IUS effectively opposes the estrogenic effect on the endometrium resulting in strong suppression during the entire period of EST. Due to its high efficacy and absence of systemic effects on organ tissues (e.g., breasts), target delivery in the uterine cavity could be a preferred route to administer a progestagen in women using EST.

[Primary and acquired resistance to antituberculous drugs in strains of Mycobacterium tuberculosis isolated in Rwanda]. / Résistance primaire et acquise aux antituberculeux des souches de Mycobacterium tuberculosis isolées au Rwanda.

This study was undertaken within the framework of a surveillance project on the resistance of Mycobacterium tuberculosis to first-line antituberculosis drugs in four provinces of Rwanda with a high prevalence of tuberculosis (TB). The purpose was to determine the prevalence of primary and acquired resistance of M. tuberculosis to major antituberculosis drugs. A cohort of patients (n=710) with pulmonary TB documented by positive microscopic examinations of exhaustive samples was recruited at 7 treatment centers. Sputum samples were cultured on Löwenstein-Jensen and Coletsos media. Sensitivity to antituberculosis drugs was tested using a BACTEC 460 radiometric system. M. tuberculosis was isolated in 644 of the 710 patients (90.7%). A total of 296 out of 573 tested for HIV infection (51.7%) were positive. Primary resistance to one, two, three or four antituberculosis drugs was observed in 3.5%, 2.9%, 1.4% and 5.7% respectively. The prevalence of acquired resistance to antituberculosis drugs was 11.2%. Primary monoresistance to streptomycin was the most prevalent (2.3%) followed by resistance to ethambutol (1%). The combined rate of multiresistance was 11.6% with 7% involving new cases and 25.5% involving retreatment. This study showed that the rates of primary and acquired resistance to first-line antituberculosis drugs were high and that TB was associated with HIV infection. The National TB Control Program must implement measures to coordinate diagnosis and management of TB and HIV infection.

Exosomal microRNAs in liquid biopsies: future biomarkers for prostate cancer.

Prostate cancer is the second most diagnosed cancer in males in the world. Plasma quantification of prostate-specific antigen substantially improved the early detection of prostate cancer, but still lacks the required specificity. Clinical management of prostate cancer needs advances in the development of new non-invasive biomarkers, ameliorating current diagnosis and prognosis and guiding therapeutic decisions. microRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene expression at the post-transcriptional level. These miRNAs are expressed in the cells and are also present in cell-derived extracellular vesicles such as exosomes. Exosomes have been shown to act as mediators for cell to cell communication because of the regulatory functions of their content. High levels of exosomes are found in several body fluids from cancer patients and could be a potential source of non-invasive biomarkers. In this review, we summarize the diagnostic and prognostic utility of exosomal miRNAs in prostate cancer.

Circulating cell-free nucleic acids and platelets as a liquid biopsy in the provision of personalized therapy for lung cancer patients.

Lung cancer is the predominant cause of cancer-related mortality in the world. The majority of patients present with locally advanced or metastatic non-small-cell lung cancer (NSCLC). Treatment for NSCLC is evolving from the use of cytotoxic chemotherapy to personalized treatment based on molecular alterations. Unfortunately, the quality of the available tumor biopsy and/or cytology material is not always adequate to perform the necessary molecular testing, which has prompted the search for alternatives. This review examines the use of circulating cell-free nucleic acids (cfNA), consisting of both circulating cell-free (tumoral) DNA (cfDNA-ctDNA) and RNA (cfRNA), as a liquid biopsy in lung cancer. The development of sensitive and accurate techniques such as Next-Generation Sequencing (NGS); Beads, Emulsion, Amplification, and Magnetics (BEAMing); and Digital PCR (dPCR), have made it possible to detect the specific genetic alterations (e.g. EGFR mutations, MET amplifications, and ALK and ROS1 translocations) for which targeted therapies are already available. Moreover, the ability to detect and quantify these tumor mutations has enabled the follow-up of tumor dynamics in real time. Liquid biopsy offers opportunities to detect resistance mechanisms, such as the EGFR T790M mutation in the case of EGFR TKI use, at an early stage. Several studies have already established the predictive and prognostic value of measuring ctNA concentration in the blood. To conclude, using ctNA analysis as a liquid biopsy has many advantages and allows for a variety of clinical and investigational applications.

Preclinical and clinical studies on afatinib in monotherapy and in combination regimens: Potential impact in colorectal cancer.

Targeting the epidermal growth factor receptor (EGFR) with monoclonal antibodies (mAbs) or tyrosine kinase inhibitors (TKI) has been an interesting therapeutic strategy because aberrant activation of this receptor plays an important role in the tumorgenesis of many cancer types, including colorectal cancer (CRC). After the initial promising results of EGFR-targeted therapies, therapeutic resistance is a major clinical problem. In order to overcome resistance to these EGFR-targeted therapies, new treatment options are necessary. In contrast to first generation EGFR inhibitors, afatinib (BIBW2992) is a second-generation irreversible ErbB family blocker that inhibits EGFR as well as HER2 and HER4. Consequently, treatment with afatinib may result in a distinct and more pronounced therapeutic benefit. Preclinical studies have reported promising results for afatinib in monotherapy as well as in combination with other drugs in CRC model systems. Furthermore, clinical studies examining afatinib as single agent and in combination therapy demonstrated manageable safety profile. Nevertheless, only limited antitumor activity has been observed in CRC patients. Although several combination treatments with afatinib have already been investigated, no optimal combination has been identified for CRC patients yet. As molecular tumor characteristics have gained increased importance in the choice of treatment, additional studies with biomarker-driven patient recruitment are required to further explore afatinib efficacy in CRC.

Effectiveness of crizotinib in a patient with ALK IHC-positive/FISH-negative metastatic lung adenocarcinoma.

We report a case of crizotinib effectiveness in a heavily pretreated patient with a metastatic NSCLC initially considered IHC-positive and FISH-negative for ALK rearrangement. After repeated analyses of tumor samples, borderline ALK FISH-positivity (18.5% positive cells) was demonstrated.

Comparative thrombolytic properties of tissue-type plasminogen activator (t-PA), single-chain urokinase-type plasminogen activator (u-PA) and K1K2Pu (a t-PA/u-PA chimera) in a combined arterial and venous thrombosis model in the dog.

The chimeric molecule K1K2Pu, comprising the two kringle domains (K1 and K2) of tissue-type plasminogen activator (t-PA) and the COOH-terminal region with the serine protease domain (Pu) of urokinase-type plasminogen activator (u-PA), was previously shown to have a 5- to 10-fold reduced clearance rate with maintained specific thrombolytic activity, resulting in an increased thrombolytic potency in animal models of venous and arterial thrombosis. To document the thrombolytic potential of K1K2Pu, the thrombolytic potency and fibrin specificity were studied in a combined platelet-rich arterial eversion graft thrombosis and venous whole blood clot model in heparinized dogs (100 U/kg bolus and 50 U/kg per h infusion). Dose-response effects of bolus injections of K1K2Pu (0.032 to 0.25 mg/kg) were compared with those of recombinant t-PA (rt-PA) and of recombinant single chain u-PA (rscu-PA) (0.25 to 1.0 mg/kg each) in groups of five or six dogs, each given heparin with or without the thromboxane synthase inhibitor/prostaglandin endoperoxide receptor antagonist ridogrel. Heparin and ridogrel in the absence of a thrombolytic agent did not produce arterial reflow or venous clot lysis in five dogs. Addition of K1K2Pu, rt-PA or rscu-PA resulted in a dose-dependent induction of arterial reflow and of venous clot lysis in the absence of systemic fibrinolytic activation and fibrinogen breakdown. Consistent arterial reflow required 0.063 mg/kg of K1K2Pu and 0.5 mg/kg of rt-PA or of rscu-PA. The thrombolytic potency for venous clot lysis, expressed as percent lysis per mg compound administered per kg body weight, was (mean +/- SEM) 750 +/- 160 for K1K2Pu, 68 +/- 17 for rscu-PA (p less than 0.001 vs. K1K2Pu) and 110 +/- 29 for rt-PA (p less than 0.001 vs. K1K2Pu). The plasma clearance rates were significantly lower for K1K2Pu than for rscu-PA and rt-PA. In the absence of ridogrel, arterial reflow was significantly slower and was followed by cyclic reocclusion and reflow; however, venous clot lysis was unaffected. Template bleeding times were not significantly altered in the absence but were markedly prolonged in the presence of ridogrel. These results confirm and establish that, when given as a bolus injection, K1K2Pu has an approximately 10-fold higher thrombolytic potency for arterial and venous thrombolysis than does rt-PA or rscu-PA. Thrombolysis with K1K2Pu is obtained in the absence of systemic fibrinolytic activation and fibrinogen breakdown. These properties suggest that K1K2Pu offers potential for thrombolytic therapy by bolus administration in patients with thromboembolic disease.