Placental thromboinflammation impairs embryonic survival by reducing placental thrombomodulin expression.

Excess platelet activation by extracellular vesicles (EVs) results in trophoblast inflammasome activation, interleukin 1ß (IL-1ß) activation, preeclampsia (PE), and partial embryonic lethality. Embryonic thrombomodulin (TM) deficiency, which causes embryonic lethality hallmarked by impaired trophoblast proliferation, has been linked with maternal platelet activation. We hypothesized that placental TM loss, platelet activation, and embryonic lethality are mechanistically linked to trophoblast inflammasome activation. Here, we uncover unidirectional interaction of placental inflammasome activation and reduced placental TM expression: although inflammasome inhibition did not rescue TM-null embryos from lethality, the inflammasome-dependent cytokine IL-1ß reduced trophoblast TM expression and impaired pregnancy outcome. EVs, known to induce placental inflammasome activation, reduced trophoblast TM expression and proliferation. Trophoblast TM expression correlated negatively with IL-1ß expression and positively with platelet numbers and trophoblast proliferation in human PE placentae, implying translational relevance. Soluble TM treatment or placental TM restoration ameliorated the EV-induced PE-like phenotype in mice, preventing placental thromboinflammation and embryonic death. The lethality of TM-null embryos is not a consequence of placental NLRP3 inflammasome activation. Conversely, EV-induced placental inflammasome activation reduces placental TM expression, promoting placental and embryonic demise. These data identify a new function of placental TM in PE and suggest that soluble TM limits thromboinflammatory pregnancy complications.

Membrane type 1-matrix metalloproteinase/Akt signaling axis modulates TNF-α-induced procoagulant activity and apoptosis in endothelial cells.

Membrane type 1-matrix metalloproteinase (MT1-MMP) functions as a signaling molecule in addition to a proteolytic enzyme. Our hypothesis was that MT1-MMP cooperates with protein kinase B (Akt) in tumor necrosis factor (TNF)-α-induced signaling pathways of vascular responses, including tissue factor (TF) procoagulant activity and endothelial apoptosis, in cultured human aortic endothelial cells (ECs). TNF-α (10 ng/mL) induced a decrease in Akt phosphorylation within 60 minutes in ECs. A chemical inhibitor of MMP, TIMP-2 and selective small interfering RNA (siRNA)-mediated suppression of MT1-MMP reversed TNF-α-triggered transient decrease of Akt phosphorylation within 60 minutes, suggesting that MT1-MMP may be a key regulator of Akt phosphorylation in TNF-α-stimulated ECs. In the downstream events, TNF-α increased TF antigen and activity, and suppressed the expression of thrombomodulin (TM) antigen. Inhibition of Akt markedly enhanced TNF-α-induced expression of TF antigen and activity, and further reduced the expression of TM antigen. Silencing of MT1-MMP by siRNA also reversed the changed expression of TF and TM induced by TNF-α. Moreover, TNF-α induced apoptosis of ECs through Akt- and forkhead box protein O1 (FoxO1)-dependent signaling pathway and nuclear factor-kB (NF-kB) activation. Knockdown of MT1-MMP by siRNA reversed apoptosis of ECs by inhibiting TNF-α-induced Akt-dependent regulation of FoxO1 in TNF-α-stimulated ECs. Immunoprecipitation demonstrated that TNF-α induced the changes in the associations between the cytoplasmic fraction of MT1-MMP and Akt in ECs. In conclusion, we show new evidence that MT1-MMP/Akt signaling axis is a key modifier for TNF-α-induced signaling pathways for modulation of procoagulant activity and apoptosis of ECs.

UVB irradiation regulates ERK1/2- and p53-dependent thrombomodulin expression in human keratinocytes.

Thrombomodulin (TM) is highly expressed in endothelial cells and acts as a natural anticoagulation factor to maintain circulation homeostasis. TM is an interesting molecule with many physiological functions, including anti-inflammation, anti-thrombosis, and carcinogenesis inhibition. TM can also be detected on the spinous layer of epidermal keratinocytes. However, the role of epidermal TM is still under investigation. In this study, we investigated keratinocyte TM expression and regulation in response to sub-cytotoxic ultraviolet B (UVB) irradiation. Oxidative stress was assessed with DCF and the results revealed that UVB irradiation significantly and dose-dependently augmented reactive oxygen species (ROS) production in HaCaT cells. In addition, low-dose UVB irradiation decreased TM mRNA and protein levels. Blocking ROS production and ERK activation prevented UVB-induced TM down-regulation. The nuclear p53 accumulation and TM promoter binding was observed within 3 h after UVB exposure. Small interfering RNA-mediated p53 knockdown disrupted the UVB-mediated TM protein down-regulation. Our study demonstrates that UVB irradiation results in ROS accumulation and ERK activation, which causes the nuclear p53 accumulation and TM promoter binding to inhibit TM expression. This study provides novel evidence demonstrating that p53 serves as a key regulator of keratinocyte TM expression.

Pharmacological targeting of the thrombomodulin-activated protein C pathway mitigates radiation toxicity.

Tissue damage induced by ionizing radiation in the hematopoietic and gastrointestinal systems is the major cause of lethality in radiological emergency scenarios and underlies some deleterious side effects in patients undergoing radiation therapy. The identification of target-specific interventions that confer radiomitigating activity is an unmet challenge. Here we identify the thrombomodulin (Thbd)-activated protein C (aPC) pathway as a new mechanism for the mitigation of total body irradiation (TBI)-induced mortality. Although the effects of the endogenous Thbd-aPC pathway were largely confined to the local microenvironment of Thbd-expressing cells, systemic administration of soluble Thbd or aPC could reproduce and augment the radioprotective effect of the endogenous Thbd-aPC pathway. Therapeutic administration of recombinant, soluble Thbd or aPC to lethally irradiated wild-type mice resulted in an accelerated recovery of hematopoietic progenitor activity in bone marrow and a mitigation of lethal TBI. Starting infusion of aPC as late as 24 h after exposure to radiation was sufficient to mitigate radiation-induced mortality in these mice. These findings suggest that pharmacologic augmentation of the activity of the Thbd-aPC pathway by recombinant Thbd or aPC might offer a rational approach to the mitigation of tissue injury and lethality caused by ionizing radiation.

Functions of rhomboid family protease RHBDL2 and thrombomodulin in wound healing.

The expression of thrombomodulin (TM), a membrane glycoprotein, is upregulated in neoepidermis during cutaneous wound healing. Rhomboid-like-2 (RHBDL2), an intramembrane serine protease, specifically cleaves TM at the transmembrane domain and causes the release of soluble TM (sTM). However, the physiological functions of TM and RHBDL2 in wound healing remain unclear. We demonstrated that both TM and RHBDL2 are upregulated in HaCaT cells stimulated by scratch wounds; furthermore, increased sTM was found in culture medium. Conversely, inhibition of RHBDL2 by 3,4-dichloroisocoumarin (DCI) or short hairpin RNA significantly inhibited wound-induced TM ectodomain shedding and wound healing. Both conditioned media from multiple-scratch-wounded HaCaT and recombinant sTM accelerated wound healing in HaCaT cells; such effects were abrogated by anti-TM antibodies. The RNA released from injured cells is involved in the induction of TM and RHBDL2. RHBDL2 and sTM were upregulated in ex vivo tissue culture of the injured skin. Furthermore, DCI inhibited sTM production and wound healing; this was reversed by recombinant sTM in mice. Thus, RHBDL2 and TM have important roles in wound healing via the release of sTM from keratinocytes; this may function as an autocrine/paracrine signal promoting wound healing.

Downregulation of thrombomodulin, a novel target of Snail, induces tumorigenesis through epithelial-mesenchymal transition.

The expression of thrombomodulin (TM), a calcium-dependent adhesion molecule, is frequently downregulated in various cancer types. However, the mechanism responsible for the low expression level of TM in tumorigenesis is unknown. Here, an inverse expression of TM and Snail was detected in different cancer cell lines. We further confirmed this inverse relation using the epithelial-mesenchymal transition cell model in HaCaT and A431 cells. We demonstrated that Snail suppressed TM expression by binding to E-box (CACCTG) in TM promoter. Moreover, TM knockdown by short hairpin RNA disrupted E-cadherin-mediated cell junctions and contributed to tumorigenesis. In the calcium switch assay, E-cadherin lost the ability to associate with ß-catenin and accumulated in cytoplasm in TM knockdown cells. Meanwhile, wound healing and invasive assays showed that TM knockdown promoted cell motility. A subcutaneous injection of TM knockdown transfectants into immunocompromised mice induced squamous cell carcinoma-like tumors. Besides, forced expression of murine TM in TM knockdown cells made the cells reassume epithelium-like morphology and increased calcium-dependent association of E-cadherin and ß-catenin. In conclusion, TM, a novel downstream target of Snail in epithelial-mesenchymal transition, is required for maintaining epithelial morphology and functions as a tumor suppressor.

Knockdown of thrombomodulin enhances HCC cell migration through increase of ZEB1 and decrease of E-cadherin gene expression.

BACKGROUND: Thrombomodulin (TM) is a key molecule mediating circulation homeostasis through its binding to thrombin. The TM-thrombin complex can activate protein C and thrombin-activatable fibrinolysis inhibitor to form a tight clot. In many cancer tissues, decrease of TM expression may correlate with cancer metastasis. However, the role of TM in hepatocellular carcinoma (HCC) progression is still unclear. METHODS: We characterized TM expression in HCC cells (HepJ5 and skHep-1 cells) using real-time polymerase chain reaction (PCR) and Western blotting. We then manipulated TM expression using both TM-specific short hairpin RNA (shRNA) and overexpressing it in HCC cells. Transwell migration assay was performed to monitor the migratory ability of HCC cells under different levels of TM expression. RESULTS: We found that TM was ectopically highly expressed in skHep-1 at both transcriptional and translational levels. After silencing TM expression in skHep-1 cells, we found that metastatic capability was dramatically increased. Conversely, overexpression of TM in HepJ5 cells decreased metastatic ability. We investigated the possible mechanism and found that decreased TM-mediated enhancement of cell migration was dependent on upregulation of ZEB1, a repressor of E-cadherin. CONCLUSIONS: TM may be a modulator of cancer metastasis in HCC. Downregulation of TM expression may increase ZEB1 and decrease E-cadherin levels.

Endothelial cell activity in chronic obstructive pulmonary disease without severe pulmonary hypertension.

Pulmonary hypertension is common in patients with chronic obstructive pulmonary disease (COPD), but the precise mechanism of vascular impairment in these patients is unknown. We, therefore, decided to investigate whether endothelial cell dysfunction is present in patients with COPD with a wide range of chronic airflow obstruction before the development of severe pulmonary hypertension. Selected plasma markers of endothelial cell activity were studied: nitrate+nitrite (NO-(2)/NO-(3)), thrombomodulin (TM), tissue factor pathway inhibitor (TFPI), soluble selectins (endothelium sES, leukocyte sLS, platelet sPS), soluble intercellular adhesion molecule-1 (sICAM-1), and soluble platelet endothelial cell adhesion molecule-1 (sPECAM-1). Twenty-five patients with COPD (forced expiratory volume in one second/vital capacity [FEV(1)/VC] < 88% predicted) and 29 healthy control subjects were recruited to the study. Among patients nine had a pulmonary artery systolic pressure (PASP) between 15 and 30 mmHg, 13 between 32 and 38 mmHg, 2 had a PASP of 41 and 42 mmHg, respectively. One patient had severe pulmonary hypertension with a PASP of 70 mmHg. The average FEV(1) of patients with COPD was 46 +/- 4% predicted. As compared to control subjects, patients with COPD showed a significant increase in plasma levels of TM and TFPI, indicating that their endothelial cells are still able to produce potent coagulation inhibitors. Levels of NO-(2)/NO-(3) were similar in the two groups of subjects examined, further suggesting preserved endothelial function in patients with COPD. In regard to adhesion molecules, patients with COPD showed a reduction in sLS, sPS, and sPECAM-1, and an increase in sICAM-1. This study shows that endothelial cell activity is largely preserved in patients with COPD without severe pulmonary hypertension, suggesting that these patients, despite quite severe airway obstruction, retain reasonably normal endothelial function until they develop severe pulmonary hypertension.

Thrombomodulin enhances the invasive activity of mouse mammary tumor cells.

Thrombomodulin (TM) is a thrombin receptor on the surface of endothelial cells that converts thrombin from a procoagulant to an anticoagulant. Thrombin promotes invasion by various tumor cells, and positive or negative correlations are found between the expression of TM and tumorigenesis in some patients. In this study, we used an invasion assay to investigate the effect of TM on the invasive activity of a mouse mammary tumor cell line, MMT cells, and the effects of TM were compared with those of thrombin as a positive control. In the presence of 1% fetal calf serum (FCS), TM significantly stimulated MMT cell invasion in a dose-dependent manner, resulting in an approximately 3-fold increase at 1-10 pg/ml over the untreated control. Thrombin also caused a similar degree of stimulation at 50 ng/ml. Since thrombin activity was detected in the components of the assay system, an invasion assay was also performed in a thrombin-activity-depleted assay system constructed to eliminate the effect of thrombin activity; TM (10 pg/ml) plus thrombin (1 pg/ml) stimulated invasion by approximately 3.5-fold in this assay system. Hirudin, a specific thrombin inhibitor, inhibited stimulation by TM as well as by thrombin in both the presence and absence of 1% FCS. Investigations of the effects of TM on proliferation, adhesion and chemotaxis to clarify the mechanism of stimulation by TM revealed that TM does not affect proliferation or adhesion in the presence of 1% FCS, but stimulates chemotaxis by approximately 2.3-fold. Similar results were obtained in experiments using thrombin. TM (10 pg/ml) plus thrombin (1 pg/ml), on the other hand, stimulated chemotaxis by approximately 2.3-fold in the thrombin-activity-depleted assay system. Binding studies using [125I]-thrombin revealed that the cells have specific saturable binding sites for thrombin. These results show that TM stimulates the invasive activity of MMT cells, probably by acting as a cofactor for the thrombin-stimulated invasion of the cells via its receptor and lowering the effective concentration of thrombin. The findings also indicate that the stimulation of invasive activity in the presence of 1% FCS and in the thrombin-activity-depleted assay system may mainly be mediated by the stimulation of chemotaxis.

NMR structures reveal how oxidation inactivates thrombomodulin.

Oxidation of Met 388, one of the three linker residues connecting the fourth and fifth EGF-like domains of thrombomodulin (TM), is deleterious for TM activity. An NMR structure of the smallest active fragment of TM (TMEGF45) and a crystal structure of a larger fragment (TMEGF456) bound to thrombin both show that Met 388 is packed into the fifth domain. Using multidimensional NMR, we have solved the structure of TMEGF45 in which Met 388 is oxidized (TMEGF45ox) and the structure of TMEGF45 in which Met 388 is mutated to Leu (TMEGF45ML). Comparison of the structures shows that the fifth domain has a somewhat different structure depending on the residue at position 388, and several of the thrombin-binding residues are packed into the fifth domain in the oxidized protein while they are exposed and free to interact with thrombin in the native structure and the Met-Leu mutant. This observation is consistent with kinetic measurements showing that the K(m) for TMEGF45ox binding to thrombin is 3.3-fold higher than for the native protein. Most importantly, the connection between the two domains, as indicated by interdomain NOEs, appears to be essential for activity. In the TMEGF45ox structure which has a reduced k(cat) for protein C activation by the thrombin-TMEGF45ox complex, interaction between the two domains is lost. Conversely, a tighter connection is observed between the two domains in TMEGF45ML, which has a higher k(cat) for protein C activation by the thrombin-TMEGF45ML complex.

Bacterial lipopolysaccharide decreases thrombomodulin expression in the sinusoidal endothelial cells of rats -- a possible mechanism of intrasinusoidal microthrombus formation and liver dysfunction.

BACKGROUND/AIMS: To elucidate the mechanism of liver dysfunction occurring in patients with sepsis, we evaluated the effect of bacterial lipopolysaccharide (LPS) on the expression of thrombomodulin (TM) in rat sinusoidal endothelial cells (SECs) and the therapeutic efficacy of exogenous recombinant TM. METHODS: We induced endotoxemia in rats by bolus intraperitoneal injection of LPS. TM antigen levels within tissues were assessed by immunohistochemistry. We measured TM in cultured SECs by enzyme immunoassay, functional analysis and real-time polymerase chain reaction (PCR). RESULTS: TM antigen and activity levels were significantly decreased in SECs isolated from LPS-treated rats after 3 and 6 h treatment, and recovered after 12 h treatment, correlating with immunohistochemical observations. In contrast, TM messenger RNA was decreased after 6 and 12 h treatment, and slightly recovered after 24 h treatment. TM expression in cultured SECs isolated from normal rats was also reduced after treatment with LPS and tumor necrosis factor (TNF)-alpha in vitro. The increased levels of serum fibrin degradation products (FDP), fibrin deposition within liver sinusoids, injury of SECs and liver dysfunction induced by LPS in our rat model was improved by recombinant TM treatment. CONCLUSIONS: Decreased TM expression in SECs of LPS-treated rats may result in intrasinusoidal microthrombus formation and subsequent liver dysfunction during sepsis.

Thrombomodulin prolongs thrombin-induced extracellular signal-regulated kinase phosphorylation and nuclear retention in endothelial cells.

On endothelial cells, thrombin binds to thrombomodulin (TM), an integral membrane-bound glycoprotein, and to protease-activated receptors (PARs). Thrombin binding to TM modulates endothelial cell and smooth muscle cell proliferation mediated through PAR1. We studied the phosphorylation and nuclear translocation of extracellular signal-regulated kinases (ERKs) 1 and 2 in human umbilical vein endothelial cells activated by thrombin. Thrombin and thrombin receptor-activating peptide (TRAP)-induced DNA synthesis were significantly inhibited by PD98059, an inhibitor of ERK phosphorylation. Immunoblots of phosphorylated ERKs (pERKs) and immunocytochemical studies of pERK localization revealed differences in the signal generated by thrombin and TRAP. After a short activation (15 minutes), the phosphorylation and the intracellular localization of pERKs were the same with the 2 agonists. After 4 hours, however, pERKs were visualized in the nuclei of thrombin-activated cells but barely detectable in TRAP-activated cells. Moreover, after 4 hours, the pERKs were visualized in the nuclei of cells stimulated by TRAP in the presence of a thrombin mutant that bound to TM, whereas they were around the nuclei in cells stimulated by thrombin in the presence of a monoclonal antibody preventing thrombin binding to TM. The results demonstrate that ERKs are involved in human umbilical vein endothelial cell DNA synthesis mediated by PAR agonists, that the duration of pERK nuclear retention is in inverse ratio to the mitogenic response, and that in addition to its role in the regulation of blood coagulation, TM acts as a thrombin receptor that modulates the duration of pERK nuclear retention and cell proliferation in response to thrombin.

Transforming growth factor-beta mediates astrocyte-specific regulation of brain endothelial anticoagulant factors.

BACKGROUND AND PURPOSE: Astrocytes are potent regulators of brain capillary endothelial cell function. Recently, astrocytes were shown to regulate brain capillary endothelial expression of the fibrinolytic enzyme tissue plasminogen activator (tPA) and the anticoagulant thrombomodulin (TM). To study the mechanism of this process, we examined the hypothesis that astrocyte regulation of endothelial tPA and TM is mediated by transforming growth factor-beta (TGF-beta). METHODS: Brain capillary endothelial cells were grown in blood-brain barrier models. We examined astrocyte-endothelial cocultures, endothelial monocultures, and astrocyte-conditioned media (ACM) for the expression of TGF-beta. We also incubated endothelial cells with ACM to determine the role of TGF-beta. Following 24 hours of incubation, we assayed for tPA and TM mRNA, as well as tPA and TM activity. RESULTS: Astrocyte-endothelial cocultures and ACM exhibited significantly higher levels of active TGF-beta than brain endothelial monocultures and endothelial cells grown in nonconditioned media, respectively. Brain endothelial cells incubated with ACM exhibited reduced tPA and TM mRNA and activity. Treatment with exogenous TGF-beta produced dose-dependent reductions in tPA and TM. The effects of ACM on both tPA and TM were blocked by TGF-beta neutralizing antibody. CONCLUSIONS: These data indicate that TGF-beta mediates astrocyte regulation of brain capillary endothelial expression of tPA and TM.

Thrombomodulin modulates the mitogenic response to thrombin of human umbilical vein endothelial cells.

Thrombin interacts with its receptor and thrombomodulin on endothelial cells. We evaluated the respective roles of these two proteins on human umbilical vein endothelial cell (HUVEC) growth by comparing thrombin, S195A (a mutant thrombin in which the serine of the charge stabilizing system had been replaced by alanine), and the receptor activating peptide (TRAP). Thrombin and TRAP induced DNA synthesis (half maximal cell proliferation with 5 nM and 25 microM, respectively), whereas S195A thrombin was inactive, inferring that growth is mediated through the thrombin receptor. Surprisingly, cells stimulated by TRAP exhibited a maximal proliferation twice greater than that obtained with thrombin. Combination of thrombin and TRAP resulted in a mitogenic response higher than by thrombin alone, but lower than by TRAP alone. The role of thrombomodulin was evaluated by adding an anti-thrombomodulin antibody, which prevents formation of the thrombin-thrombomodulin complex. Antibody did not interfere with cell proliferation induced by TRAP, but enhanced that induced by thrombin. We conclude that formation of the thrombin-thrombomodulin complex restrains HUVEC proliferation mediated through the thrombin receptor.

Major basic protein binding to thrombomodulin potentially contributes to the thrombosis in patients with eosinophilia.

The contribution of an eosinophil granule protein, major basic protein (MBP), to the pathogenesis of thrombosis seen in patients with eosinophilia was investigated. The sera from eosinophilic patients containing elevated levels of MBP inhibited thrombomodulin (TM) function as a cofactor for the thrombin-catalysed activation of protein C more significantly than those from normal individuals (means 48.5% v 17.4%, respectively). It was suggested that the binding of mature MBP in the sera to TM was electrostatic, because mature MBP (pI 10.9) bound to TM, whereas pro-MBP (pI 6.2) did not. The inhibition of TM cofactor activity by eosinophil granule proteins was mainly attributed to the mature MBP, because MBP-depleted eosinophil granule proteins did not inhibit TM cofactor activity significantly. This inhibition seemed to be due to the specific thrombin-binding to TM being blocked. We concluded that eosinophil granule proteins, particularly MBP, potentially contribute to the hypercoagulation seen in some conditions of eosinophilia, at least because of the inhibition of TM function as a cofactor of the anticoagulation system.

Acquired protein C dysfunction but not decreased activity of thrombomodulin is a possible marker of thrombophilia in patients with lupus anticoagulant.

Lupus anticoagulants (LAs) are acquired antiphospholipid antibodies, and the occurrence of LA is associated with an increased risk of developing thrombosis. In a population of 46 patients with LA with or without LA-associated thrombophilia, it was analyzed whether the concentration of LA could be correlated to the individual thrombotic risk in patients with LA. No significant difference was found in the concentrations of LA measured by routinely used functional and immunologic assays in patients with LA with thrombophilia when compared with patients with LA without thrombophilia. Inhibition of thrombomodulin (TM) activity by LA has been postulated to be one of the major pathogenic mechanisms causing thrombophilia in LA. Therefore the inhibition of endothelial cell-dependent TM activity by LA was analyzed by using a protein C (PC) activation assay. Reduced rates of PC activation were found in only 2 out of the 46 cases, ruling out that inhibition of TM activity is a common phenomenon in patients with LA. However, anionic phospholipids are necessary to ascertain the anticoagulant activity of activated PC (APC). To prove the hypothesis that the anticoagulant activity of APC is inhibited by LA, the anticoagulant response of purified APC added to LA-containing plasma was measured through the amount of factor VIII inactivation. Thirteen out of 14 patients with recurrent thrombotic events and 10 out of 19 patients with one single episode of thrombosis showed an APC response outside the mean--2 SD range of normal human controls. In contrast, among 13 patients with LA without symptoms, only one showed an abnormal APC response. From these data it is concluded that LA inhibits the APC anticoagulant activity and that this type of acquired APC dysfunction may contribute to the pathogenesis of LA-associated thrombophilia. Moreover, the APC anticoagulant response assay may prove to be a useful marker to identify patients with LA with a high thrombotic risk.

Antithrombotic effects of recombinant human soluble thrombomodulin in a rat model of vascular shunt thrombosis.

Thrombomodulin on endothelial cells is a cofactor for thrombin-catalyzed activation of protein C. We have investigated the anticoagulant function of recombinant human soluble thombomodulin (rsTM) in a rat model of arterio-venous (AV)-shunt thrombosis. A bolus injection of rsTM 30 s before the induction of AV-shunt thrombosis inhibited the thrombus formation in a dose-dependent manner. The dose of anticoagulant that inhibited thrombus formation by 50% was 0.4 mg/kg rsTM alpha, 0.15 mg/kg rsTM beta, and 13 U/kg heparin. Recently, we characterized three monoclonal antibodies (moAbs) against human TM whose epitopes are located in the TM epidermal growth factor-like domain (Nawa et al., 1994). moAb 2A2 inhibited thrombin binding to rsTM, and abolished both TM functions as a cofactor in thrombin-catalyzed activation of protein C and as an anticoagulant by modifying thrombin-induced fibrinogen clotting and platelet aggregation. moAb 1F2 preserved the latter activities as an anticoagulant, but inhibited cofactor activity. moAb 10A3 had no inhibitory effect on either activity. Analysis of the in vivo anticoagulant mechanism of rsTM was facilitated by the availability of these moAbs. After incubation at rsTM/moAb molar ratios of 1:1.25, the effect of the mixtures were examined in the AV-shunt thrombosis model. An injection of 0.8 mg/kg rsTM alpha or 0.4 mg/kg rsTM beta resulted in a significant reduction on thrombus formation, as expected. moAb 10A3 had no effect on rsTM activity. However, co-injection of rsTM with moAb 1F2 resulted in a significant decrease of the inhibitory activity on thrombus formation. moAb 2A2 essentially abolished the inhibitory effect of rsTM.(ABSTRACT TRUNCATED AT 250 WORDS)