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The antithrombotic action of thrombomodulin was studied in mice. Rat and mouse thrombomodulin were isolated from lung acetone powders, and anti-rat thrombomodulin antibodies were prepared in rabbits. The antibodies neutralized both mouse (Kd approximately 150 nM) and rat thrombomodulin (Kd approximately 50 nM). A role for thrombomodulin in vivo was shown in mice injected intravenously (IV) with thrombin. All mice injected with 15 U thrombin (bolus) died of thromboembolism (mean survival 55 minutes), whereas those injected with a lower dosage survived. Prior injection with anti-rat thrombomodulin (1.8 mg IgG/mouse) potentiated the lethal effects of subsequent thrombin, whereas injection of thrombomodulin (isolated from mouse lung) prior to thrombin prolonged survival in a thrombomodulin concentration-dependent manner. The protective effect of thrombomodulin persisted for 30 minutes but after one hour thrombin injection was as toxic as in control animals. The half life (t1/2) for plasma clearance of 125I- mouse lung thrombomodulin was nine minutes. The major site of clearance was the liver, although thrombomodulin accumulated in several organs ten minutes after injection. The mechanism by which antithrombomodulin antibodies potentiated the lethal effects of thrombin was studied by measuring the protein C activating cofactor activity on vena cava removed from animals injected with antibodies. Protein C activation was inhibited by antibodies, suggesting a role for activated protein C in prevention of lethal thromboembolism. We found no effect of antibodies on the clearance of thrombin from mouse plasma, suggesting that blockade of endothelial endocytosis of thrombin does not play a significant role in the effects of antibodies. These results indicate that thrombomodulin participates in the defense against thrombosis in vivo.

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The effect of covalent bonding of fibronectin on the patency and graft healing of high-porosity expanded polytetrafluoroethylene (ePTFE) grafts was evaluated. Bilateral carotid grafting was performed in ten mongrel dogs using high-porosity (60 microm) ePTFE grafts, 4 cm in length and 4 mm in internal diameter, that either had been pretreated by the covalent bonding of fibronectin (fibronectin grafts) or were untreated (control grafts). The grafts were harvested 4 to 6 weeks after surgery and subjected to macroscopic and light-microscopic observations. There was no significant difference in patency between the fibronectin grafts and the control grafts with rates of 80% and 70%, respectively. The thrombus-free area score was significantly greater in the fibronectin grafts than in the control grafts, at 86.9% vs 34.0%. Furthermore, the pseudointima was better replaced by fibrous tissue in the fibronectin grafts than in the control grafts, being lined with a layer of endothelial-like cells. More transmural tissue ingrowth was evident in the fibronectin grafts than in the control grafts. The covalent bonding of fibronectin improves graft healing by stimulating transmural tissue ingrowth in high-porosity ePTFE grafts.

We have measured mRNA levels for thrombomodulin, an endothelial membrane cofactor for the activation ofprotein C by thrombin, in a mouse hemangioma cell line. Cycloheximide, an inhibitor of protein synthesis, increased levels of thrombomodulin mRNA, as measured in an S1 nuclease protection assay, to 2.5-4.0 times control levels. Thrombomodulin transcription in response to cycloheximide treatment, as determined by nuclear run-on analysis, was 3.9 ± 1.3 (mean ± SD) times that found in untreated cells. Thrombin also increased thrombomodulin mRNA levels to 151 ± 21% (mean ± SD) of control levels after 2 hr. Transcription increased in response to thrombin by 2.1-to 7.3-fold. The combination of thrombin and cycloheximide had no additive effect on thrombomodulin mRNA levels. Thrombin treatment of hemangioma cells also caused an increase in thrombomodulin protein synthesis to 142 ± 17% (mean ± SD) of control levels as determined by immunoprecipitation of [32S]methionine-labeled thrombomodulin. We conclude that thrombomodulin expression is determined in part by the rate of transcription and that thrombomodulin mRNA levels in hemangioma cells are increased by treatment with cycloheximide or thrombin. The increased transcription in response to cycloheximiide suggests the existence of a labile protein repressor of thrombomodulin transcription.