Reducing agents induce thrombomodulin shedding in human endothelial cells

The level of thrombomodulin (TM) on cell surfaces reflects its biosynthesis, intracellular turnover, proteolytic cleavage, and release in soluble form (sTM). In the present study we examined the mechanisms mediating and regulating sTM release. Inducers of endothelial protein C receptor (EPCR) shedding, such as proinflammatory cytokines, phorbol ester, and ionomycin did not affect sTM release from human umbilical endothelial cells (HUVECs). In contrast, several natural and synthetic reducing compounds (i.e., glutathione, dihydrolipoic acid, homocysteine, N-acetyl-L-cysteine, dithiothreitol, and non-thiol cell-impermeable reductant, tris-(2-carboxyethyl)phosphine), but not oxidized glutathione or α-lipoic acid effectively up-regulated the release of sTM in endothelial cells. In addition, the direct activator of metalloproteases, 4-aminophenylmercuric acetate (APMA), was an effective inducer of TM shedding. Considerable inhibition of protein C activation was found with APMA, which is consistent with the effects of this agent on TM shedding. In addition to metalloproteases, serine proteases were shown by pharmacological inhibition studies to be involved in a similar degree in basal sTM release; however, serine proteases seem preferentially to be involved in thiol-induced TM proteolytic processing. From comparisons of non-thiol containing synthetic substrate with human recombinant TM it was demonstrated that disulfide bonds within TM are most likely modified by thiols making TM more susceptible to serine protease-mediated cleavage. In summary, the study shows that the extracellular redox state plays a crucial role in the regulation of TM shedding in HUVECs thereby offering new strategies to interfere with diminished activation of protein C during inflammatory diseases.     

Thrombomodulin-dependent effect of factor V Leiden mutation on the cross-linking of α(2)-plasmin inhibitor to fibrin and its consequences on fibrinolysis

Introduction

It has been shown that thrombomodulin (TM) considerably delays factor XIII (FXIII) activation and this effect is abrogated by Factor V Leiden (FV_Leiden) mutation. The aim of the study was to explore the effect of TM on the cross-linking of α₂-plasmin inhibitor (α₂-PI) to fibrin in plasma samples of different FV genotypes and how this effect is related to the impaired fibrinolysis of FV_Leiden carriers.

Methods

In the plasma samples of fifteen individuals with different FV genotypes and in FV deficient plasma supplemented with wild type FV or FV_Leiden coagulation was initiated by recombinant human tissue factor and phospholipids with or without recombinant human TM (rhTM). In the recovered clots the extent of α₂-PI-fibrin cross-linking was evaluated by Western blotting and quantitative densitometry. The effect of rhTM on tissue plasminogen activator (tPA) induced clot lysis was measured by turbidimetric method.

Results

rhTM significantly delayed the formation of α₂-PI-fibrin α-chain heterodimers/oligomers in plasma samples containing wild type FV. This effect of rhTM was impaired in the presence of FV_Leiden. rhTM delayed tPA-induced clot lysis and this effect of rhTM was more pronounced in plasma containing FV_Leiden. When TAFIa was inhibited by potato carboxypeptidase inhibitor, rhTM accelerated clot lysis in the presence of wild type FV, which is explained by the delayed α₂-PI-fibrin cross-linking. This effect of rhTM did not prevail in the presence of FV_Leiden.

Conclusion

FV_Leiden abrogates the delaying effect of rhTM on α₂-PI-fibrin cross-linking, which contributes to the impaired fibrinolysis observed in FV_Leiden carriers.     

Thrombomodulin-dependent effect of factor VLeiden mutation on factor XIII activation

Introduction

Factor V Leiden mutation (FV_Leiden) is associated with impaired down-regulation of activated FV procoagulant activity and loss of FV anticoagulant function that result in an increased risk of venous thromboembolism. As the downstream effects of FV_Leiden on clot formation and fibrinolyis have only partially been revealed, we investigated its effect on the activation of factor XIII (FXIII) and the cross-linking of fibrin.

Methods

In the plasma samples of fifteen healthy individuals with known FV genotypes coagulation was initiated by recombinant human tissue factor and phospholipids with or without recombinant human thrombomodulin (rhTM). FV deficient plasma supplemented with purified wild type FV or FV_Leiden were also investigated. Clots were recovered and analyzed by SDS-PAGE and quantitative densitometric evaluation of Western blots.

Results

rhTM considerably delayed the activation of FXIII in the plasma from FV wild type individuals. This effect of rhTM was significantly impaired in the plasma from FV_Leiden carriers. The results were confirmed in experiments with FV deficient plasma supplemented by FV prepared from wild type individuals or FV_Leiden homozygotes. Fibrin γ-chain dimerization was also considerably delayed by rhTM in plasma samples from individuals without Leiden mutation, but not in plasma samples from FV_Leiden heterozygotes or homozygotes. The difference between heterozygotes and homozygotes was not statistically significant.

Conclusion

The highly diminished delaying effect of TM on FXIII activation and on the cross-linking of fibrin in FV_Leiden carriers might represent a novel mechanism contributing to the increased thrombosis risk of these individuals.     

Calibrated automated thrombography demonstrates hypercoagulability in patients with idiopathic pulmonary arterial hypertension

Introduction

Pathogenesis of idiopathic pulmonary arterial hypertension (iPAH) includes endothelial dysfunction and in situ thrombosis. A hypercoagulable state has also been postulated but never demonstrated. Our objective was to determine whether patients with iPAH had a hypercoagulable state using calibrated automated thrombography (CAT), a new tool to phenotype coagulation in vitro.

Patients and methods

16 patients with iPAH and 29 controls were studied. In vitro platelet dependent coagulation phenotyping by CAT monitored the activity of thrombin generation over time. Plasma levels of soluble thrombomodulin, tissue factor pathway inhibitor (TFPI) and von Willebrand factor (VWF) were measured as endothelial biomarkers.

Results

Endogenous thrombin potential (ETP) in the absence of activated protein C (APC) tended to be increased in patients compared to controls (1769 versus 1656 nM.min; p = 0.053). ETP was higher in the presence of APC 25 nM (ETP-APC) in patients (781 versus 494 nM.min; p = 0.005). Five patients had ETP-APC higher than the 95th centile of controls. Other CAT parameters (lag time, peak thrombin and time to peak) were all consistent with some degree of hypercoagulability in patients. Regarding endothelial plasma biomarkers sTM was lower (28.4 versus 40.6 μg/l, p = 0.0108) in patients; TFPI antigen and activity (respectively: 14.3 versus 10.5 μg/l, p = 0.0167; 1.155 versus 1.070, p = 0.0021) and VWF (1300 versus 976%, p = 0.0108) were higher in patients.

Conclusion

We have demonstrated that at least some patients with iPAH have a hypercoagulable phenotype.