Molecular mechanism of action of newer thrombolytic agents

Recombinant tissue-type plasminogen activator (rt-PA) and single chain urokinase-type plasminogen activator (scu-PA) are thrombolytic agents, characterized by a high but not absolute degree of fibrin specificity that is mediated through different molecular mechanisms. Both activators are still under clinical investigation but it has become apparent that their therapeutic dose in humans is high and associated with a variable degree of systemic activation of the fibrinolytic system and fibrinogen breakdown. Therefore, the quest for further improvement of agents and therapeutic schemes continues. Research is being pursued in this area along the following lines: 1) tissue-type plasminogen activator (t-PA) and single chain urokinase-type plasminogen activator in molar ratios of 4:1 to 1:4 do not act synergistically on thrombolysis in a plasma environment in vitro, but display significant synergism in animal models of thrombosis. In pilot studies in patients with coronary artery occlusion, rt-PA and scu-PA are markedly synergistic and efficient thrombolysis can be obtained with a fivefold lower combined dose than that of the separate agents. The combined dose does not seem to induce systemic fibrinogen breakdown. 2) Deletion mutants of rt-PA can be constructed with a significantly prolonged half-life in vivo, and a better thrombolytic potential after bolus intravenous injection. 3) Cleavage site-specific mutants of scu-PA that abolish the conversion to urokinase may have a higher fibrin specificity. The mutants constructed thus far, however, seem to have a lower specific thrombolytic activity. 4) Chimeric molecules obtained by fusion of cDNA encoding the NH2-terminal region of t-PA, responsible for its fibrin affinity and cDNA encoding the COOH-terminal region of scu-PA, responsible for its enzymatic properties, combine both mechanisms of fibrin specificity, at least to some extent.It is anticipated that some of these research lines will yield improved thrombolytic agents or therapeutic regimens

Revascularization of ischemic tissues by PlGF treatment, and inhibition of tumor angiogenesis, arthritis and atherosclerosis by anti-Flt1

The therapeutic potential of placental growth factor (PlGF) and its receptor Flt1 in angiogenesis is poorly understood. Here, we report that PlGF stimulated angiogenesis and collateral growth in ischemic heart and limb with at least a comparable efficiency to vascular endothelial growth factor (VEGF). An antibody against Flt1 suppressed neovascularization in tumors and ischemic retina, and angiogenesis and inflammatory joint destruction in autoimmune arthritis. Anti-Flt1 also reduced atherosclerotic plaque growth and vulnerability, but the atheroprotective effect was not attributable to reduced plaque neovascularization. Inhibition of VEGF receptor Flk1 did not affect arthritis or atherosclerosis, indicating that inhibition of Flk1-driven angiogenesis alone was not sufficient to halt disease progression. The anti-inflammatory effects of anti-Flt1 were attributable to reduced mobilization of bone marrow-derived myeloid progenitors into the peripheral blood; impaired infiltration of Flt1-expressing leukocytes in inflamed tissues; and defective activation of myeloid cells. Thus, PlGF and Flt1 constitute potential candidates for therapeutic modulation of angiogenesis and inflammation.status: publishe

Mouse models of angiogenesis, arterial stenosis, atherosclerosis and hemostasis

The development of efficient transgenic technologies in mice has allowed the study of the consequences of genetic alterations on cardiovascular (patho)physiology, although the development of such models have been hampered by size limitation of species resulting in time-consuming, labor-intensive and costly analyses. This overview summarizes the murine models currently available for studying or manipulating angiogenesis, arterial stenosis, atherosclerosis, transplant arteriopathy, thrombosis, thrombolysis and bleeding and addresses techniques to evaluate vascular development during embryogenesis.status: publishe

Reduced atherosclerotic plaque but enhanced aneurysm formation in mice with inactivation of the tissue inhibitor of metalloproteinase-1 (TIMP-1) gene

Development and progression of atherosclerotic lesions and aneurysm formation were investigated in mice with single or combined deficiency of apolipoprotein E (ApoE) and tissue inhibitor of metalloproteinase-1 (TIMP-1) kept on a cholesterol-rich diet for 30 weeks. Atherosclerotic lesions throughout the thoracic aorta were significantly (P<0.001) larger in mice wild-type for TIMP-1 (ApoE-/-:TIMP-1+/+) than in mice deficient in TIMP-1 (ApoE-/-:TIMP-1-/-). Aneurysms in the thoracic and abdominal aortas were less frequent in ApoE-/-:TIMP-1+/+ mice than in ApoE-/-:TIMP-1-/- mice (11+/-3.0 versus 23+/-5.1 aneurysms per 100 sections analyzed, mean+/-SD, P<0.001). Immunocytochemistry revealed enhanced accumulation of Oil red O-stained lipids, colocalizing with macrophages in atherosclerotic lesions of ApoE-/-:TIMP-1-/- mice (P<0.05). In situ zymography using a casein substrate showed enhanced lysis in plaques of ApoE-/-:TIMP-1-/- mice as compared with ApoE-/-:TIMP-1+/+ mice (P<0.01). MMP activity was most pronounced at sites where degradation of the elastic lamina occurred. These data suggest that enhanced MMP activity, as a result of TIMP-1 deficiency, contributes to a reduction of atherosclerotic plaque size but promotes aneurysm formation.status: publishe

Humoral immune response in mice against a circulating antigen induced by adenoviral transfer is strictly dependent on expression in antigen-presenting cells

Adenoviral transfer of human apo A-I in Balb/c mice induces a strong humoral immune response against the transgene product when expression is driven from the ubiquitously active CMV promoter but induces no immune response when driven by the hepatocyte-specific 256-base pair apo A-I promoter. Here the hypothesis was tested, which is that the humoral immune response against the circulating transgene product correlates with its expression in antigen-presenting cells. No humoral immune response was observed after adenoviral transfer of vectors with human apo A-I expression driven by the hepatocyte-specific apo C-II or 1.5-kilobase (kb) human alpha(1)-antitrypsin promoter, but antibodies were induced after transfer with vectors driven by the ubiquitously active U1b promoter and the murine MHCII E beta promoter. A strict correlation was observed between antigen expression in the spleen and the occurrence of an immune response. Coinjection of the 1.5-kb human alpha(1)-antitrypsin and the murine MHCII E beta promoter-driven vectors resulted in a very short-lived humoral immune response against human apo A-I, suggesting that the time course of human apo A-I expression is a critical determinant of the development of tolerance for human apo A-I. High titers of antibodies against human apo A-I after subcutaneous gene transfer with the MHCII E beta promoter-driven vector underscore the potential of this promoter for vaccination purposes. In conclusion, humoral immune response in mice against a circulating antigen induced by adenoviral transfer is strictly dependent on expression in antigen-presenting cells.status: publishe

Measurement of urokinase-type plasminogen-activator (u-pa) with an enzyme-linked-immunosorbent-assay (elisa) based on 3 murine monoclonal-antibodies

An enzyme-linked immunosorbent assay (ELISA) for the measurement of urokinase-type plasminogen activator (u-PA) was developed. Three murine monoclonal antibodies to single chain urokinase-type plasminogen activator (scu-PA) were isolated and shown to react with non-overlapping epitopes in scu-PA. Two of the three antibodies were coated onto microtiter plates and bound u-PA was quantitated with the third antibody conjugated to horseradish peroxidase. The assay was equally sensitive to Mr 54,000 u-PA in the single chain or two-chain form but did not respond to Mr 33,000 urokinase. The lower limit of sensitivity of the assay was 0.1 ng/ml in buffer and 1 ng/ml in plasma. Coefficients of variation of the assay at physiological levels of u-PA were 6.5 percent within assays and 13 percent between assays. The level of u-PA in normal resting plasma was 1.9 +/- 0.66 ng/ml (mean +/- SD, n = 54). The assay can be performed within one working day and provides an efficient, reproducible, and stable means for the measurement of u-PA in biological fluids. As such it may facilitate physiological and pharmacological studies of urokinase-type plasminogen activators in man.status: publishe