Tissue-type plasminogen activator (t-PA) is a potent and efficacious mitogen for growth-arrested cultured human aortic smooth muscle cells, stimulating an increase in cell number at 0.3-30 nM concentration. Double-chain t-PA is as efficient as single-chain t-PA in stimulating smooth muscle cell mitogenesis, whereas single-chain urokinase-type plasminogen activator (u-PA) or u-PA and plasmin or plasminogen are ineffective. Plasminogen activator inhibitor-1, Pefabloc-TPA, diisopropyl fluorophosphate or alpha 1-anti-trypsin inhibit the mitogenic effect of t-PA for smooth muscle cells in a dose-dependent manner, showing that it is dependent on the enzymatic activity. t-PA activated phosphoinositide turnover in smooth muscle cells through a pertussis toxin-insensitive pathway and stimulated proto-oncogene c-fos and c-jun mRNA levels. These findings indicate that t-PA stimulates vascular human smooth muscle cell proliferation and suggest for the first time that it may contribute to intimal smooth muscle cell proliferation after vascular injury as a result of angioplasty or vascular compromise during atherogenesis.
The synthetic peptide, SFLLRNPNDKYEPF, has been recently described as a peptide mimicking the new amino-terminus created by cleavage of the thrombin receptor, therefore acting as an agonist of the thrombin receptor. This peptide was a potent mitogen for rabbit arterial smooth muscle cells (SMC) and exhibited the same activity as that of native alpha-thrombin. Both compounds stimulated the proliferation of growth-arrested SMCs with half-maximum mitogenic responses at 1 nM. NAPAP, a synthetic inhibitor of the enzymatic activity of thrombin, specifically inhibited thrombin-induced SMC growth (IC50 = 0.35 +/- 0.04 microM) but was without effect on the mitogenic effect of the agonist peptide. These results therefore demonstrate that the mitogenic effect of alpha-thrombin for SMCs is intimately linked to its esterolytic activity. Heparin, which inhibited fetal calf serum-induced SMC growth, was without effect on thrombin-induced SMC growth but strongly reduced the mitogenic effect of the agonist peptide (IC50 = 32 +/- 5 micrograms/ml). This effect was not related to the anti-coagulant activity of heparin but was highly dependent on molecular mass and on the global charge of the molecule and was also observed for other sulphated polysaccharides such as pentosan polysulphate.
Endotoxin (LPS), interleukin‐1β (IL‐1) and tumor necrosis factor‐α (TNF) increased the expression of tissue factor, a membrane‐anchored glycoprotein that initiates blood coagulation on the surface of cultured bovine aortic endothelial cells (ABAE) and human monocytes. These compounds simultaneously reduced the amount of thrombomodulin on the endothelial cell surface, further contributing to the procoagulant activity of the endothelium or monocytes. On endothelial cells and monocytes, interleukin‐4 (IL‐4) and interleukin‐13 (IL‐13), a newly described lymphokine, both strongly inhibited LPS‐induced tissue factor expression, a similar activity also being obtained with regard to the pyrogenic effects of IL‐1 or TNF. When measured in parallel, IL‐4 and IL‐13 counteracted thrombomodulin down‐regulation induced by LPS, IL‐1 or TNF in endothelial cells. These results therefore show that both IL‐4 and IL‐13 protect the endothelial and the monocyte surface against inflammatory mediator‐induced procoagulant changes.
The decay rate of thrombin in plasma is shown to be linearly proportional to the concentration of antithrombin III (AT III), not only in the absence but also in the presence of heparin. This is a consequence of partitioning of heparin between AT III and other plasma proteins. In previous articles were calculated the prothrombin converting activity assuming a fixed concentration of AT III. Since AT III is consumed during the clotting process, prothrombinase activity is more accurately approximated using an algorithm that counts with the decrease of the AT III concentration. It is shown this leads to higher prothrombinase activities. The (absence of) inhibition of prothrombin conversion by prothrombinase in the presence of heparins found with the previous method is also found using the new algorithm. From the results presented it is evident that characteristic parameters of heparin action have to be normalised to the AT III concentration. On this basis we define a Standard Independent Unit of the antithrombin activity of heparin.
Current thrombolytic strategies have a number of important shortcomings including resistance to recanalization and development of acute reocclusion. The purpose of this study was to investigate whether the lysis of venous thrombi by streptokinase could be enhanced by SR 46349, a novel 5-HT2 receptor antagonist, or clopidogrel, an analogue of ticlopidine. The activity of these compounds was evaluated by following the lysis of radiolabelled fibrin under a continuous infusion of streptokinase (4,000 IU kg-1 h-1 over 4 h). Streptokinase alone induced 42% thrombolysis when compared to saline. The i.v. co-administration of SR 46349 or clopidogrel (10 mg/kg) enhanced significantly streptokinase-induced thrombolysis. Thrombolysis measured by [125I]-fibrinogen lysis increased to 65 and 59% respectively. This efficacy was achieved without additional prolongation of the template bleeding time observed with streptokinase alone. Thus, the concomitant use of a 5-HT2 receptor antagonist or an anti-ADP agent during streptokinase therapy may facilitate clot lysis.
The pharmacodynamic profile of standard heparin (SH), a low molecular weight derivative (CY 216) and of dermatan sulfate (DS), a new potential antithrombotic drug, was investigated in the rabbit over a large range of doses. After bolus i.v. injection of low doses, the biological activity of SH disappeared exponentially; however, its half-life was prolonged when the dose injected increased, and over 158 micrograms/kg (100 anti-factor Xa U/kg) the biological activity disappeared as a concave-convex curve. CY 216 disappeared more slowly than SH at low doses but faster than SH at higher doses. More than 90% of the DS biological activity present 1 minute after the i.v. injection disappeared exponentially without dose-dependent effects. Increasing doses of the three drugs were then delivered for 5 h under continuous infusions. Below 500 micrograms/kg/h the DS and CY 216 plateau concentrations were higher than that of SH while above this dose the SH concentration was higher than that of DS and CY 216. These observations may be explained by the results of pharmacokinetics experiments where 125I-labeled compounds were delivered by bolus i.v. injection in association with increasing doses of their unlabeled counterparts. For SH there was a 10-fold difference between the half-life of the lower dose (32 micrograms/kg or 5 anti-factor Xa U/kg) and that of the higher dose (3200 micrograms/kg); it was demonstrated that the half-life of SH continuously shortened as its plasma concentration decreased. In contrast the CY 216 and DS half-lives were very close, independent of the dose delivered, and therefore longer than that of SH at low doses and shorter than that of SH at higher doses. The renal contribution to the clearance of SH, CY 216, and DS was also investigated by injecting a low dose (150-200 micrograms/kg) and a 10 times higher dose to sham-operated or binephrectomized animals: renal function is critical for the elimination of the three compounds from the blood except for SH at low doses. These observations are promising for the development of DS and may have clinical implications.
