Combination of a direct thrombin inhibitor and a platelet glycoprotein IIb/IIIa blocking peptide facilitates and maintains reperfusion of platelet-rich thrombus with alteplase.

We sought to determine the efficacy of the combination of argatroban, a direct thrombin inhibitor, and G4120, a platelet glycoprotein (GP) IIb/IIIa blocker, to enhance thrombolysis with alteplase. Platelet-rich thrombus in the rabbit arterial thrombosis model is relatively resistant to alteplase despite the addition of aspirin and heparin. The adjunctive use of either direct thrombin inhibitors or GP IIb/IIIa inhibitors in thrombolysis has been investigated with encouraging, but limited, success. The usefulness of combining both agents as adjunctive therapy to thrombolysis has not been fully explored. Following platelet-rich thrombus formation in the rabbit, argatroban (3 mg/kg), G4120 (0.5 mg/kg), G4120 plus heparin (200 U/kg), or G4120 plus argatroban were intravenously infused over 60 minutes. Alteplase was given as intravenous boluses (0.45 mg/kg) at 15-minute intervals up to 4 doses or until reperfusion. Blood flow and bleeding time were monitored for 2 hours. The combination of G4120 plus argatroban resulted in a persistent patency in 5 of 7 animals compared with 0 of 6 for argatroban alone (p=0.02), 1 of 6 for G4120 alone (p=0.08), and 2 of 6 for G4120 plus heparin (p=0.2). Although during the infusion the bleeding times were longer in the groups that received G4120 (26+/-7.7 minutes vs. 14+/-10 minutes, p<0.05), by the end of the experiment there were no statistically significant differences. Similarly, during the infusion the activated partial thromboplastin times (aPTT) was higher in groups that received heparin or argatroban (99+/-51 seconds vs. 32+/-7.6 seconds, p<0.001), but by the end of the experiment the aPTTs had returned to close to baseline in all groups except the G4120 plus heparin group. These results suggest that lysis of platelet-rich thrombus with alteplase requires the addition of both potent platelet and thrombin inhibitors. Specifically designed agents, G4120 and argatroban, are effective without additional increased risk for bleeding.

A collaborative study to establish a standard for high molecular weight urinary-type plasminogen activator (HMW/u-PA).

An international collaborative study involving eleven laboratories located in eight countries was undertaken to establish an International Standard for high molecular weight urinary-type plasminogen activator (HMW/u-PA). The current International Reference Preparation (IRP code numbered 66/46) for urinary-type plasminogen activator (u-PA) or urokinase (see Nomenclature footnote) is a 66/34 molar ratio mixture of low molecular weight (LMW)--and high molecular weight (HMW)--u-PA's and is considered unsuitable as a standard for homogeneous preparations of HMW/u-PA. The putative standard for HMW/u-PA (code number, 87/594) was compared for potency in a clot lysis assay with the current IRP for u-PA (code number, 66/46) and a lyophilised preparation of single chain urinary-type plasminogen activator (SCuPA), the latter being used in the assay without prior activation by plasmin to its active two chain form (TCuPA). Both the proposed standard for HMW/u-PA (87/594) and the SCuPA compared in a statistically satisfactory manner in parallel line bioassays with the current IRP for u-PA (66/46), thus allowing potency estimates to be obtained for these two materials in relation to defined international units. Data from the eleven laboratories indicated that each ampoule of the proposed standard for HMW/u-PA contained 4,300 i.u. of activity and was stable for over 1 year at 4 degrees C. Most participants indicated that SCuPA expressed only a small amount of its activity without a prior activator step and this suggests that SCuPA assays need to be preceded by a plasmin activation step.(ABSTRACT TRUNCATED AT 250 WORDS)

Phenotypic properties of herpesvirus-transformed cells with high tumorigenic and metastatic ability.

Herpes simplex virus type 2-transformed hamster embryo fibroblasts (333-8-9 cells) produce increased plasminogen activator (PA) compared with normal hamster cells. These cells produce undifferentiated fibrosarcomas at the inoculation site in newborn hamsters, and metastasize to the lungs. Using a direct PA assay, in which 125I-labeled plasminogen is cleaved, the optimum pH and osmolarity for detection of the 333-8-9 extracellular PA were pH 8.9 and approximately 150 mOsmol. Secretion of enzyme did not vary significantly on a per cell basis over cell densities from 0.1 to 8.0 X 10(7) cells/T-75 cm2 flask. This assay demonstrates that the 333-8-9 cells produce at least 20-fold greater levels of PA than normal cell counterparts. Based on the molecular weight (50-58 kDa) of secreted 333-8-9 cells PA and lack of fibrin stimulation, we conclude that it is a urokinase type PA. Subclonal lines of the 333-8-9 cells, selected for an increased PA phenotype were stable in culture, more tumorigenic and probably more metastatic. Correlation of these two events was examined by passaging 333-8-9 cells in vivo to select for greater tumorigenic potential and then determining the production of PA by the in vivo-derived sublines. The metastatic potential of the resulting cells was heterogeneous. Increased PA production upon increased passage in vivo did not always occur, whether the cells were passaged as subcutaneous tumors or as ascites tumors. Thus, while enzyme production correlated with tumorigenicity when selecting cells for an increased protease phenotype, this correlation was not observed when selecting for in vivo tumorigenicity. The results suggest that increased ability to make PA represents only one of multiple selective advantages for tumor growth.

A collaborative study of a proposed international standard for tissue plasminogen activator (t-PA).

An international collaborative study involving seven laboratories was undertaken to assess which of three lyophilised preparations might serve as an International Standard (I.S.) for tissue plasminogen activator (t-PA). Two of the preparations were isolates from human melanoma cell cultures while one was of pig heart origin. A clot lysis assay was used by all participants in the study. The data suggested that both preparations of human cell origin were comparable, in that their log dose-response lines were parallel, while that of the porcine preparation was not. Accelerated degradation studies indicated that one melanoma extract (denoted 83/517) was more stable than the other and it was decided to recommend preparation 83/517 as the standard for t-PA. The International Committee for Thrombosis and Haemostasis (Stockholm 1983) has recommended the use of this material as a standard and it has been established by the Expert Committee on Biological Standardization of the World Health Organization as the International Standard for tissue plasminogen activator, with an assigned potency of 1000 International Units per ampoule.

Heterogeneity of plasminogen activator expression in various Moloney virus-induced tumor cell lines. Lack of correlation with tumor growth and cell phenotype.

The aberrant expression of a plasminogen activator (PA) by Moloney virus (MuLV)-transformed mouse lymphocytes and its relation to cell phenotype and tumor growth have been studied. Nine cultured cell lines were established from neoplastic splenic and thymic tissues obtained from B10 congeneic mice inoculated with MuLV and killed when overtly leukemic. Cell surface markers were assayed by microcytotoxicity tests, the concentration of MuLV p30 and group-specific MuLV gp 70 was determined by radioimmunoassays and the expression of PA activity was assessed in a fibrin-agar plate method. PA activity of 24 h serum-free culture supernatant, intact cells or cell lysates (2 X 10(5) cells/ml) was expressed in International Units by reference to a urokinase standard curve. Tumor extension and cell morphology were investigated by histologic and cytomorphologic analysis. In all cases the cell lines were derived from T cells. PA activity is not expressed by normal lymphocytes, but variations in PA expression were observed in the transformed cells. Five out of nine transformed cell lines showed PA activity with a range of 1.3 to 9.9 IU/ml. No PA activity could be detected in the other cell lines. No correlation was found between PA expression and the cell-surface-expressed phenotype, neither was there any correlation between the PA content, the cytopathological features and the degree and type of organ infiltration. This lack of correlation indicates that there is no relation between PA activity and the expression of the transformed phenotype, and that the presence of PA activity seems to be irrelevant to the tumorigenic capacities of the transformed cell lines.

Possible use of monoclonal antibodies to standardize the production and use of human urokinase.

Urokinase, a plasminogen activator found in urine is used in the treatment of pulmonary embolism, and is supplied commercially by several companies in Europe, U.S.A. and Japan. It exists in two forms, of molecular weights 50-55 000 and 33 000 daltons, which may differ in their therapeutic efficiency. A variety of assay systems exist to measure plasminogen activator activity--clot lysis, casein hydrolysis, amidolytic cleavage of synthetic peptide substrates. These assays are time-consuming, relatively insensitive, and cannot accurately assess the amount of the two molecular species in any preparation of urokinase. Standardization therefore presents a problem to manufacturers, control agencies, and clinicians. We have prepared a variety of monoclonal antibodies to human urokinase and have assessed their value as standardizing different commercial preparations of urokinase and measurement of urokinase for therapeutic monitoring.

The stability of tissue plasminogen activator (t-PA).

Since tissue-type plasminogen activator (t-PA) derived from tissue culture and recombinant DNA procedures has been proposed for use in thrombolytic therapy in man, it is essential that the t-PA molecule should display reasonable stability in a lyophilised state to facilitate its usefulness. In this study, four laboratories compared the potencies of three preparations of t-PA following storage at 4 degrees, 20 degrees, 37 degrees and 45 degrees C, using each t-PA stored at -20 degrees C as a reference (100% activity) in each case. A pig heart extract of t-PA was the most stable, losing no activity when stored for 30 days at 37 degrees C, while two melanoma cell tissue culture extracts varied in their storage behaviour. One was quite stable at 37 degrees C (losing about 3% of its activity) while the other lost about 16%. Thus both the pig heart t-PA and one t-PA from melanoma cell culture proved suitable for the further development of reference standards for t-PA activity.