ALBERT

Description: Thrombomodulin (TM) is a widely expressed glycoprotein receptor that plays a physiologically important role in maintaining normal hemostatic balance postnatally. Inactivation of the TM gene in mice results in embryonic lethality without thrombosis, suggesting that structures of TM not recognized to be involved in coagulation might be critical for normal fetal development. Therefore, the in vivo role of the cytoplasmic domain of TM was studied by using homologous recombination in ES cells to create mice that lack this region of TM (TMcyt/cyt). Cross-breeding of F1 TMwt/cyt mice (1 wild-type and 1 mutant allele) resulted in more than 300 healthy offspring with a normal Mendelian inheritance pattern of 25.7% TMwt/wt, 46.6% TMwt/cyt, and 27.7% TMcyt/cyt mice, indicating that the tail of TM is not necessary for normal fetal development. Phenotypic analyses showed that the TMcyt/cyt mice responded identically to their wild-type littermates after procoagulant, proinflammatory, and skin wound challenges. Plasma levels of plasminogen, plasminogen activator inhibitor 1 (PAI-1), and 2-antiplasmin were unaltered, but plasmin:2-antiplasmin (PAP) levels were significantly lower in TMcyt/cyt mice than in TMwt/wt mice (0.46 ± 0.2 and 1.99 ± 0.1 ng/mL, respectively; P 〈 .001). Tissue levels of TM antigen were also unaffected. However, functional levels of plasma TM in the TMcyt/cyt mice, as measured by thrombin-dependent activation of protein C, were significantly increased (P 〈 .001). This supported the hypothesis that suppression in PAP levels may be due to augmented activation of thrombin-activatable fibrinolysis inhibitor (TAFI), with resultant inhibition of plasmin generation. In conclusion, these studies exclude the cytoplasmic domain of TM from playing a role in the early embryonic lethality of TM-null mice and support its function in regulating plasmin generation in plasma.

Description: Impaired fibrinolysis, resulting from increased plasminogen activator inhibitor-1 (PAI-1) or reduced tissue-type plasminogen activator (t-PA) plasma levels, may predispose the individual to subacute thrombosis in sepsis and inflammation. The objective of these studies was to show that adenovirus-mediated gene transfer could increase systemic plasma t-PA levels and thrombolytic capacity in animal model systems. Recombinant adenovirus vectors were constructed that express either human wild type or PAI-1–resistant t-PA from the cytomegalovirus (CMV) promoter. Both t-PA-deficient (t-PA−/−) and PAI-1–overexpressing transgenic mice were infected by intravenous injection of these viruses. Intravenous injection of recombinant adenovirus resulted in liver gene transfer, t-PA synthesis, and secretion into the plasma. Virus dose, human t-PA antigen, and activity concentrations in plasma and extent of lysis of a 125I-fibrin–labeled pulmonary embolism were all closely correlated. Plasma t-PA antigen and activity were increased approximately 1,000-fold above normal levels. Clot lysis was significantly increased in mice injected with a t-PA–expressing virus, but not in mice injected with saline or an irrelevant adenovirus. Comparable levels of enzyme activity and clot lysis were obtained with wild type and inhibitor-resistant t-PA viruses. Adenovirus-mediated t-PA gene transfer was found to augment clot lysis as early as 4 hours after infection, but expression levels subsided within 7 days. Adenovirus-mediated transfer of a t-PA gene can effectively increase plasma fibrinolytic activity and either restore (in t-PA–deficient mice) or augment (in PAI-1–overexpressing mice) the thrombolytic capacity in simple animal models of defective fibrinolysis.

Description: Thrombomodulin (TM) is a multidomain protein that serves as a cofactor in a major natural anticoagulant system. To further characterize the structure-function of TM, we have transfected COS cells with different truncated forms of TM. In the first form, COS cells expressing TM that lacks the putative signal peptide (17 residues); the lectin-like, hydrophobic N-terminal domain (226 residues); and 12 residues of the first epidermal growth factor (EGF )-like repeat (COSdel.238 cells) were found to function normally with respect to TM transport to the cell surface and thrombin-dependent protein C activation. However, in contrast to wild-type TM, as visually studied by immunofluorescence and immunogold electron microscopy, the COSdel.238 cells did not constitutively internalize anti-TM–TM or thrombin-TM complexes. To identify the region responsible for mediating the endocytic process, deletant forms of TM lacking either the lectin-like region (residues 2-155) or the hydrophobic region of the N-terminal domain (residues 161-202) were expressed in COS cells (COSdel.2-155 and COSdel.161-202, respectively). Protein C cofactor activity was maintained in both cells. Although the COSdel.161-202 cells behaved similarly to wild-type TM-transfected cells, visual studies showed a lack of constitutive internalization of thrombin-TM or anti-TM–TM complexes in the COSdel.2-155 cells. We conclude that the lectin-like domain of human TM serves to regulate cell surface expression of TM via the endocytic route and therefore may also play a major physiologic role in controlling intracellular and extracellular accumulation of thrombin in a variety of biologic systems.

Description: Circulating complexes of leukocytes and activated platelets are markers for atherosclerosis, but their interaction with the arterial endothelial lining has not been studied. Therefore, the effect of activated platelets on rolling and adhesion of labeled human THP-1 monocytoid cells to human umbilical vein endothelial cell (HUVEC) monolayers was studied by epifluorescence microscopy in a parallel plate flow chamber. In the absence of activated platelets, THP-1 rolling on resting HUVEC was negligible at shear rates greater than 300 s−1. Activation of HUVEC with 100 nmol/L phorbol myristate acetate (PMA) increased THP-1 cell adhesion at shear rates less than 400 s−1. Therefore, a shear rate of 400 s−1 was identified as a threshold for THP-1 adhesion. THP-1 rolling on activated HUVEC was reduced by 64% after L-selectin inhibition but was not affected by P-selectin inhibition. The addition of 1 to 50 thrombin receptor-activating peptide (TRAP)-activated platelets per THP-1 cell enhanced interactions between THP-1 cells and HUVEC, resulting in a steep bell-shaped dose-response curve, with a peak of 10 ± 3 rolling cells/50 seconds at 3 platelets per THP-1 cell (P 〈 .01v control) with a concomitant 2- to 3-fold increase of firmly adhering cells (P 〈 .01 v control). In reconstituted blood, low numbers of activated platelets had the same effect on THP-1 rolling and adhesion. P-selectin inhibition reduced platelet/THP-1 cell interaction in suspension and deposition of the complexes on the endothelial monolayer. Inhibition of both P- and L-selectin reduced THP-1/HUVEC interactions to 14% (P 〈 .01, n = 4). Sialidase digestion and removal of terminal sialic acid residues from HUVEC or THP-1 cells but not from platelets abolished the platelet mediated augmentation of THP-1 cell adhesion. Thus, THP-1 rolling on HUVEC is shear-dependent and largely mediated by L-selectin. P-selectin expressed on activated platelets increases monocytoid cell adhesion to endothelial cells at shear rates found in coronary arteries through interactions with both endothelial and monocytoid cells and may facilitate macrophage accumulation in the vessel wall.

Description: It is widely held that the plasminogen (Plg) system plays a role in inflammation through plasmin-mediated directional cell migration. However, substantial evidence for its involvement in the inflammatory response has been obtained from indirect studies and lacks firm biological confirmation. To directly characterize plasminogen's involvement in the inflammatory response, we used thioglycollate to induce a peritoneal inflammatory reaction in Plg(+/+),Plg(+/−), and Plg(−/−) mice. At 6 hours poststimulation, neutrophil recruitment into the peritoneum was maximal and similar between Plg(+/+), Plg(+/−), andPlg(−/−) mice. In contrast, monocyte recruitment was significantly diminished after 24 hours poststimulation inPlg(−/−) mice relative to Plg(+/+) mice. Lymphocyte recruitment also was blunted. Blood monocyte levels in these mice indicated that diminished recruitment into the peritoneum was not the result of a diminished source of cells in the circulation. Macrophage phagocytic function was similar between Plg(+/+) and Plg(−/−) mice. This study establishes a direct involvement of plasminogen in monocyte recruitment during a representative inflammatory response.

Description: It is widely held that the plasminogen (Plg) system plays a role in inflammation through plasmin-mediated directional cell migration. However, substantial evidence for its involvement in the inflammatory response has been obtained from indirect studies and lacks firm biological confirmation. To directly characterize plasminogen's involvement in the inflammatory response, we used thioglycollate to induce a peritoneal inflammatory reaction in Plg(+/+),Plg(+/−), and Plg(−/−) mice. At 6 hours poststimulation, neutrophil recruitment into the peritoneum was maximal and similar between Plg(+/+), Plg(+/−), andPlg(−/−) mice. In contrast, monocyte recruitment was significantly diminished after 24 hours poststimulation inPlg(−/−) mice relative to Plg(+/+) mice. Lymphocyte recruitment also was blunted. Blood monocyte levels in these mice indicated that diminished recruitment into the peritoneum was not the result of a diminished source of cells in the circulation. Macrophage phagocytic function was similar between Plg(+/+) and Plg(−/−) mice. This study establishes a direct involvement of plasminogen in monocyte recruitment during a representative inflammatory response.

Description: Impaired fibrinolysis, resulting from increased plasminogen activator inhibitor-1 (PAI-1) or reduced tissue-type plasminogen activator (t-PA) plasma levels, may predispose the individual to subacute thrombosis in sepsis and inflammation. The objective of these studies was to show that adenovirus-mediated gene transfer could increase systemic plasma t-PA levels and thrombolytic capacity in animal model systems. Recombinant adenovirus vectors were constructed that express either human wild type or PAI-1–resistant t-PA from the cytomegalovirus (CMV) promoter. Both t-PA-deficient (t-PA−/−) and PAI-1–overexpressing transgenic mice were infected by intravenous injection of these viruses. Intravenous injection of recombinant adenovirus resulted in liver gene transfer, t-PA synthesis, and secretion into the plasma. Virus dose, human t-PA antigen, and activity concentrations in plasma and extent of lysis of a 125I-fibrin–labeled pulmonary embolism were all closely correlated. Plasma t-PA antigen and activity were increased approximately 1,000-fold above normal levels. Clot lysis was significantly increased in mice injected with a t-PA–expressing virus, but not in mice injected with saline or an irrelevant adenovirus. Comparable levels of enzyme activity and clot lysis were obtained with wild type and inhibitor-resistant t-PA viruses. Adenovirus-mediated t-PA gene transfer was found to augment clot lysis as early as 4 hours after infection, but expression levels subsided within 7 days. Adenovirus-mediated transfer of a t-PA gene can effectively increase plasma fibrinolytic activity and either restore (in t-PA–deficient mice) or augment (in PAI-1–overexpressing mice) the thrombolytic capacity in simple animal models of defective fibrinolysis.