Thrombomodulin expression by human keratinocytes. Induction of cofactor activity during epidermal differentiation.

Thrombomodulin is an endothelial cell surface glycoprotein that inhibits the procoagulant activities of thrombin and accelerates activation of the anticoagulant protein C. Because protein C deficiency is associated with cutaneous thrombosis, we investigated the expression of thrombomodulin in human skin. Thrombomodulin was detected by immunohistochemical staining both in dermal endothelial cells and in epidermal keratinocytes. Within the epidermis, thrombomodulin staining was limited to keratinocytes of the spinous layer, suggesting that thrombomodulin is induced when basal keratinocytes begin to terminally differentiate. Thrombomodulin expression also correlated with squamous differentiation in epidermal malignancies; little or no thrombomodulin staining was seen in five basal cell carcinomas, whereas strong thrombomodulin staining was observed in each of five squamous cell carcinomas. Human foreskin keratinocytes cultured in medium containing 0.07 mM calcium chloride synthesized functional thrombomodulin with cofactor activity comparable to thrombomodulin in human umbilical vein endothelial cells. Stimulation of keratinocyte differentiation with 1.4 mM calcium chloride for 48 h produced 3.5-, 3.2-, and 5.6-fold increases in thrombomodulin cofactor activity, antigen, and mRNA, respectively. These observations suggest that thrombin is regulated by keratinocyte thrombomodulin at sites of cutaneous injury, and indicate a potential role for thrombomodulin in epidermal differentiation.     

Effects of the chemotherapeutic agent doxorubicin on the protein C anticoagulant pathway

Although chemotherapy treatment is associated with an increased risk of thrombosis, the pathogenic mechanisms for the thrombogenic effect of chemotherapeutic drugs are poorly understood. We hypothesize that exposure of vascular endothelial cells to chemotherapeutic agents results in the loss of a thromboresistant phenotype. In this study, we examined the effects of the chemotherapeutic agent doxorubicin on the endothelium-based protein C anticoagulant pathway. The endothelial cell protein C receptor (EPCR) and thrombomodulin are two endothelial cell surface receptors required for the conversion of zymogen protein C to the anticoagulant enzyme activated protein C. Exposure of human umbilical vein endothelial cells (HUVEC) to doxorubicin resulted in a dose- and time-dependent decrease in cell surface EPCR levels. This decrease occurred as a result of receptor shedding as well as from a down-regulation in EPCR mRNA levels. In contrast, doxorubicin treatment of HUVECs resulted in a dose- and time-dependent increase in cell surface thrombomodulin attributed to an up-regulation of thrombomodulin mRNA levels. The net effect of the doxorubicin-induced changes in EPCR and thrombomodulin levels was a decrease in the capacity of HUVECs to convert protein C to activated protein C. Preliminary studies suggest that doxorubicin free radical metabolites mediate the doxorubicin-induced changes in EPCR expression but not those of thrombomodulin expression. In summary, these results suggest that doxorubicin alters the hemostatic balance of endothelial cells by down-regulating the endothelium-based protein C anticoagulant pathway.     

Effects of thrombomodulin and coagulation Factor Va-light chain on protein C activation in vitro.

Protein C activation by thrombin is significantly accelerated by the endothelial cell surface protein thrombomodulin, Factor Va, or its light chain. In this study we have compared the activation of protein C in the presence of either cofactor and examined the possibility that thrombomodulin and Factor Va-light chain act together to regulate protein C activation by thrombin. At all concentrations of protein C used, thrombomodulin was 20 times more efficient than Factor Va-light chain in accelerating protein C activation by thrombin. Protein C treated with chymotrypsin to remove the amino-terminal 41 amino acids that contain the gamma-carboxyglutamyl residues was activated by the thrombin-thrombomodulin complex at an identical rate to native protein C, whereas the modified protein C was activated by Factor Va-light chain and thrombin at only 5% of the rate obtained by using native protein C. Increasing concentrations of Factor Va-light chain, greater than or equal to 30 nM, inhibited thrombin-thrombomodulin catalyzed protein C activation with complete inhibition observed at 90 nM Factor Va-light chain. On the other hand, increasing thrombomodulin concentrations did not inhibit protein C activation by Factor Va-light chain and thrombin. These reactions in solution mimic, in part, those obtained on endothelial cells where protein C lacking the gamma-carboxyglutamyl domain is activated poorly and Factor Va-light chain at concentrations greater than 50 nM inhibited the activation of native protein C. The results of this study suggest that thrombomodulin and Factor Va-light chain may act in concert to regulate protein C activation by thrombin.     

Human thrombomodulin is not an efficient inhibitor of the procoagulant activity of thrombin.

The effect of human thrombomodulin isolated from placenta on the procoagulant activity of thrombin was studied and compared to that of rabbit thrombomodulin. The isolated protein was proved to be thrombomodulin because a rabbit antibody against the isolated protein blocked protein C activation by thrombomodulin in solution and also blocked the protein-C-activating cofactor activity of human umbilical vein endothelial cells. The affinity of human thrombomodulin for human thrombin in the presence of fibrinogen is 30 times less than that of rabbit thrombomodulin. This value is based on the measurements of the clotting time of human fibrinogen and thrombin in the presence of increasing amounts of thrombomodulin. Human thrombomodulin was also much less effective compared with rabbit thrombomodulin in inhibiting thrombin-induced human coagulation factor V activation. The ability to inhibit release of [3H]serotonin from washed human platelets was at least 10 times less using human thrombomodulin compared with rabbit thrombomodulin. A partially purified preparation of human lung thrombomodulin was also relatively ineffective in inhibiting thrombin-induced serotonin release from platelets, indicating that the difference between rabbit and human thrombomodulin is one of species rather than of tissue. Thus, while human thrombomodulin is a potent cofactor in protein C activation, it is not an efficient inhibitor of the procoagulant actions of thrombin.     

Loss of ATP Diphosphohydrolase Activity with Endothelial Cell Activation

Quiescent endothelial cells (EC) regulate blood flow and prevent intravascular thrombosis. This latter effect is mediated in a number of ways, including expression by EC of thrombomodulin and heparan sulfate, both of which are lost from the EC surface as part of the activation response to proinflammatory cytokines. Loss of these anticoagulant molecules potentiates the procoagulant properties of the injured vasculature. An additional thromboregulatory factor, ATP diphosphohydrolase (ATPDase; designated as EC 3.6.1.5) is also expressed by quiescent EC, and has the capacity to degrade the extracellular inflammatory mediators ATP and ADP to AMP, thereby inhibiting platelet activation and modulating vascular thrombosis. We describe here that the antithrombotic effects of the ATPDase, like heparan sulfate and thrombomodulin, are lost after EC activation, both in vitro and in vivo. Because platelet activation and aggregation are important components of the hemostatic changes that accompany inflammatory diseases, we suggest that the loss of vascular ATPDase may be crucial for the progression of vascular injury.     

Blood coagulation and the risk of atherothrombosis: a complex relationship

The principles of Virchov's triad appear to be operational in atherothrombosis or arterial thrombosis: local flow changes and particularly vacular wall damage are the main pathophysiological elements. Furthermore, alterations in arterial blood composition are also involved although the specific role and importance of blood coagulation is an ongoing matter of debate. In this review we provide support for the hypothesis that activated blood coagulation is an essential determinant of the risk of atherothrombotic complications. We distinguish two phases in atherosclerosis: In the first phase, atherosclerosis develops under influence of "classical" risk factors, i.e. both genetic and acquired forces. While fibrinogen/fibrin molecules participate in early plaque lesions, increased activity of systemic coagulation is of no major influence on the risk of arterial thrombosis, except in rare cases where a number of specific procoagulant forces collide. Despite the presence of tissue factor - factor VII complex it is unlikely that all fibrin in the atherosclerotic plaque is the direct result from local clotting activity. The dominant effect of coagulation in this phase is anticoagulant, i.e. thrombin enhances protein C activation through its binding to endothelial thrombomodulin.The second phase is characterized by advancing atherosclerosis, with greater impact of inflammation as indicated by an elevated level of plasma C-reactive protein, the result of increased production influenced by interleukin-6. Inflammation overwhelms protective anticoagulant forces, which in itself may have become less efficient due to down regulation of thrombomodulin and endothelial cell protein C receptor (EPCR) expression. In this phase, the inflammatory drive leads to recurrent induction of tissue factor and assembly of catalytic complexes on aggregated cells and on microparticles, maintaining a certain level of thrombin production and fibrin formation. In advanced atherosclerosis systemic and vascular wall driven coagulation becomes more important and elevated levels of D-dimer fragments should be interpreted as markers of this hypercoagulability.     

Endothelium and regulation of coagulation

Endothelial cells form the luminal vascular surface and thus have a central role in the regulation of coagulation. One important way in which endothelial cells control the clotting system is by regulating the expression of binding sites for anticoagulant and procoagulant factors on the cell surface. In the quiescent state, endothelial cells maintain blood fluidity by promoting the activity of numerous anticoagulant pathways, including the protein C/protein S pathway. After activation, as can be brought about by cytokines, the balance of endothelial properties can be tipped to favor clot formation through coordinated induction of procoagulant and suppression of anticoagulant mechanisms. Tumor necrosis factor suppresses the endothelial anticoagulant cofactor thrombomodulin and induces expression of the procoagulant cofactor tissue factor. Working in concert, these changes can allow fibrin formation to proceed in an inflamed focus but maintain blood fluidity in the surrounding area of normal vasculature. Recent studies suggest that similar changes in endothelial coagulant properties can be induced by advanced glycosylation end products, proteins modified by glucose that accumulate in the vasculature at a rapid rate in diabetic subjects, indicating the potential relevance of these mechanisms in diabetic vascular disease.     

The normal role of Activated Protein C in maintaining homeostasis and its relevance to critical illness

Thrombin is a multifunctional protein, with procoagulant, inflammatory and anticoagulant effects. Binding of thrombin to thrombomodulin results in activation of Protein C and initiation of the Activated Protein C anticoagulant pathway, a process that is augmented by the endothelial cell Protein C receptor (EPCR). Activated Protein C has demonstrated antithrombotic, anti-inflammatory, and profibrinolytic properties. Its antithrombotic activity is particularly important in the microcirculation, and Protein C deficiency is associated with microvascular thrombosis. Activated Protein C has also been shown to modulate inflammation. When the level of thrombomodulin or Protein C is reduced in sepsis there is a vicious cycle of coagulation and inflammation, with potentially lethal consequences. In vitro studies and animal models have shown that Activated Protein C blunts the inflammatory and coagulant response to sepsis through a variety of mechanisms.     

Endothelial cells and vascular hemostasis]

Procoagulant, anticoagulant, and fibrinolytic activities are associated with endothelial cells and involve the production, secretion, and receptor mediated binding of proteins involved in these processes. The procoagulant aspect of endothelial cells function involves the production and release of von Willebrand Factor(vWF), the production of tissue factor, and the presence of Factor IX/IXa receptors on the cell surface. Secretion of vWf will promote the initial steps in thrombus formation by supporting platelet-platelet interaction and platelet-subendothelial matrix adhesion. Tissue factor which is undetectable in resting cells appears after exposure to various cytokines and initiates factor VIIa activation of factors IX and X. Receptors of Factor IX/IXa are also present and mediate the assembly of the prothrombinase complex on the endothelial cell surface. The anticoagulant pathway involves the cell surface protein thrombomodulin, protein C and its cofactor protein S. Thrombomodulin binds thrombin which activates protein C which in the presence of protein S cleaves and inactivates Factors V and VIII. Inactivation of these two coagulation cofactors halts the coagulation. Finally, endothelial cells also play a pivotal role in the fibrinolytic system. Production and regulated secretion of tissue plasminogen activator creates a profibrinolytic state in the endothelial cell environment. In addition, receptors for plasminogen and urokinase are also present, constituting a cell surface mediated fibrinolytic pathway. Plasminogen activator inhibitor type I, the primary inhibitor of tPA, is also produced by endothelial cells. Thus endothelial cells can promote and inhibit fibrinolysis, depending on the prevailing environmental conditions.     

Loxosceles spider venom induces the release of thrombomodulin and endothelial protein C receptor: implications for the pathogenesis of intravascular coagulation as observed in loxoscelism

BACKGROUND: The venom of the spider Loxosceles can cause both local and systemic effects including disseminated intravascular coagulation. AIM: The aim of this study was to investigate the effects of the venom of Loxosceles intermedia (L. intermedia) and the purified Sphingomyelinase D (SMaseD) toxin upon the Protein C (PC) natural anticoagulant pathway. RESULTS: Both the venom and e purified SMaseD reduced the cell surface expression of thrombomodulin (TM) and Endothelial PC Receptor on endothelial cells in culture. The reduction of cell surface expression was caused by cleavage from the cell surface mediated by activation of an endogenous metalloproteinase. Reduction of TM and Endothelial PC Receptor on the surface of these cells resulted in an impaired ability of the cells to assist in the thrombin-induced activation of PC. CONCLUSION: This novel observation gives further insight into the mechanisms of the pathology induced by venom from Loxosceles spiders and may aid the development of a suitable therapy.     

Homocysteine, a thrombogenic agent, suppresses anticoagulant heparan sulfate expression in cultured porcine aortic endothelial cells.

Previous studies showed that homocysteine, a thrombo-atherogenic and atherogenic agent, inhibits an endothelial thrombomodulin-protein C anticoagulant pathway. We examined whether homocysteine might affect another endothelial anticoagulant mechanism; i.e., heparin-like glycosaminoglycan-antithrombin III interactions. Incubations of porcine aortic endothelial cell cultures with homocysteine reduced the amount of antithrombin III bound to the cell surface in a dose- and time-dependent fashion. The inhibitory effect was observed at a homocysteine concentration as low as 0.1 mM, and the maximal suppression occurred at 1 mM of homocysteine after 24 h. In contrast with a marked reduction in the maximal antithrombin III binding capacity (approximately 30% of control), the radioactivity of [35S]sulfate incorporated into heparan sulfate on the cell surface was minimally (< 15%) reduced. The cells remained viable after homocysteine treatment. Although neither net negative charge nor proportion in total glycosaminoglycans of cell surface heparan sulfate was altered by homocysteine treatment, a substantial reduction in antithrombin III binding capacity of heparan sulfate isolated from homocysteine-treated endothelial cells was found using both affinity chromatography and dot blot assay techniques. The antithrombin III binding activity of endothelial cells decreased after preincubation with 1 mM homocysteine, cysteine, or 2-mercaptoethanol; no reduction in binding activity was observed after preincubation with the same concentration of methionine, alanine, or valine. This sulfhydryl effect may be caused by generation of hydrogen peroxide, as incubation of catalase, but not superoxide dismutase, with homocysteine-treated endothelial cells prevented this reduction, whereas copper augmented the inhibitory effects of the metabolite. Thus, our data suggest that the inhibited expression of anticoagulant heparan sulfate may contribute to the thrombogenic property resulting from the homocysteine-induced endothelial cell perturbation, mediated by generation of hydrogen peroxide through alteration of the redox potential.     

Protein C pathway in sepsis

The goals of this chapter are to provide a brief review of the biology of the protein C pathway and some of the features of the pathway that make it uniquely positioned to control microvascular coagulation and control the acute inflammatory response. Activated protein C works as an antithrombotic agent by inactivating factors Va and VIIIa. It is particularly effective at preventing microvascular thrombosis. Platelets may provide a margin of safety for activated protein C as an antithrombotic. Approximately 25% of the factor V/Va in plasma is contained within the platelet and hence resistant to time dependent inactivation by activated protein C. In addition, factor Va bound to the platelet surface is relatively resistant to inactivation by activated protein C. Activated protein C also facilitates clot lysis by inhibiting plasminogen activator inhibitor 1, a process that is accelerated markedly by vitronectin. Inflammatory cytokines like tumor necrosis factor alpha (TNFalpha) and interleukin-1beta (IL-1beta) downregulate two key components of the protein C activation complex, thrombomodulin and the endothelial cell protein C receptor resulting in decreased protein C activation. Activated protein C in turn has been shown in several animal models and in vitro to inhibit TNF elaboration in response to endotoxin. This inhibition appears to be due to diminished nuclear factor kappaB (NF kappaB) expression and nuclear translocation. Activated protein C has been shown to reduce the rate of death due to severe sepsis. This reduction may be due to both the anticoagulant effects as demonstrated by a reduction in D-dimer and inflammatory effects as demonstrated by a reduction in interleukin 6.     

Activation Mechanism of Anticoagulant Protein C in Large Blood Vessels Involving the Endothelial Cell Protein C Receptor

Protein C is an important regulatory mechanism of blood coagulation. Protein C functions as an anticoagulant when converted to the active serine protease form on the endothelial cell surface. Thrombomodulin (TM), an endothelial cell surface receptor specific for thrombin, has been identified as an essential component for protein C activation. Although protein C can be activated directly by the thrombin–TM complex, the conversion is known as a relatively low-affinity reaction. Therefore, protein C activation has been believed to occur only in microcirculation. On the other hand, we have identified and cloned a novel endothelial cell surface receptor (EPCR) that is capable of high-affinity binding of protein C and activated protein C. In this study, we demonstrate the constitutive, endothelial cell–specific expression of EPCR in vivo. Abundant expression was particularly detected in the aorta and large arteries. In vitro cultured, arterial endothelial cells were also found to express abundant EPCR and were capable of promoting significant levels of protein C activation. EPCR was found to greatly accelerate protein C activation by examining functional activity in transfected cell lines expressing EPCR and/or TM. EPCR decreased the dissociation constant and increased the maximum velocity for protein C activation mediated by the thrombin–TM complex. By these mechanisms, EPCR appears to enable significant levels of protein C activation in large vessels. These results suggest that the protein C anticoagulation pathway is important for the regulation of blood coagulation not only in microvessels but also in large vessels.     

Endothelium-specific loss of murine thrombomodulin disrupts the protein C anticoagulant pathway and causes juvenile-onset thrombosis

The thrombomodulin (TM) gene was ablated in mice in a cell type–restricted manner from vascular endothelium by Cre-recombinase–mediated excision controlled by the endothelial cell lineage–specific Tie2 promoter. Forty percent of mutant (TMLox-) mice display a distinct lethal embryonic phenotype not observed in completely TM-deficient embryos. The remaining 60% of TMLox mice survive beyond birth, but invariably succumb to a severe hypercoagulable state and massive thrombosis after 3 weeks, terminating in a lethal consumptive coagulopathy. The progression of thrombosis was age- and sex-dependent. Disruption of the TM/protein C pathway was not associated with a latent proinflammatory state. Disease onset and progression could be prevented by warfarin anticoagulation. These results show that in mice, loss of endothelial cell TM function causes spontaneous and fatal thrombosis in the arterial and venous circulation, resulting from unfettered activation of the coagulation system. The combination of complete disease penetrance, uniform disease onset at young age, large vessel thrombosis of the extremities and multiple organ systems, and consumptive coagulopathy as the disease end-point provides a unique mouse model of human thrombotic disease.     

Homocysteine and markers of coagulation and endothelial cell activation.

OBJECTIVES: In vitro studies suggest an influence of hyperhomocysteinemia on the coagulation system, but the influence of mild hyperhomocysteinemia in vivo is unclear. METHODS AND RESULTS: We studied the relation between homocysteine and markers of coagulation activation and endothelial cell activation in 279 patients with established atherosclerotic disease. In addition, we performed an investigator-blinded placebo-controlled cross-over study to investigate the influence of acute hyperhomocysteinemia by oral methionine load on these markers in 20 healthy volunteers. In the atherosclerotic patients prothrombin fragment F1+2 and soluble thrombomodulin (sTM) were associated with homocysteine in univariate analyses (P = 0.003 and P = 0.001, respectively), but not in multivariate analyses. Age, creatinine and MTHFR C677T polymorphism were major determinants of homocysteine concentration. MTHFR C677T polymorphism status was not associated with F1+2 and sTM. Median homocysteine concentrations increased in the healthy volunteers after methione load. However, after methionine load or after placebo, we did not observe different plasma concentrations of F1+2 (0.9 nmol L-1 vs. 0.9 nmol L-1, P = 0.39), d-dimer (153 micro g L-1 vs. 151 micro g L-1, P = 0.63) and von Willebrand factor (103% vs. 107%, P = 1.00). CONCLUSIONS: These results provide evidence against a major effect of mild hyperhomocysteinemia on activation of the coagulation system and endothelial cell activation in vivo.     

Stimulatory effect of homocysteine on interleukin-8 expression in human endothelial cells.

Elevated plasma homocysteine is an independent risk factor for atherosclerosis. An important initial step of atherosclerosis is the adhesion and infiltration of monocytes to the lesion site. It has been shown that the pro-inflammatory cytokine interleukin-8 can rapidly cause rolling monocytes to adhere firmly onto monolayers expressing E-selectin. The objective of the present study was to investigate the effect of homocysteine on interleukin-8 production in human endothelial cells. Cells were incubated with various concentrations of homocysteine for 20 h. The gene expression was determined by real-time PCR and the interleukin-8 protein was measured by immunoassay analysis. Homocysteine enhanced the expression of interleukin-8 in a dose-dependent manner (181% of controls at 2.5 mmol/l homocysteine). Stimulation of gene expression was associated with a parallel increase in interleukin-8 protein synthesis (160% of controls at 5.0 mmol/l homocysteine). By co-incubation of endothelial cells with homocysteine and copper sulfate, a further elevation of interleukin-8 expression (251% of controls) was observed, whereas copper sulfate alone had no stimulatory effect. In conclusion, the present study demonstrated that homocysteine altered endothelial cell function by stimulating interleukin-8 expression, suggesting a contribution of homocysteine to the initiation and progression of atherosclerosis. The formation of homocysteine-induced oxidation products might serve as one of the underlying mechanisms of this effect.     

Crucial role of the protein C pathway in governing microvascular inflammation in inflammatory bowel disease

Endothelial protein C receptor (EPCR) and thrombomodulin (TM) are expressed at high levels in the resting microvasculature and convert protein C (PC) into its activated form, which is a potent anticoagulant and antiinflammatory molecule. Here we provide evidence that in Crohn disease (CD) and ulcerative colitis (UC), the 2 major forms of inflammatory bowel disease (IBD), there was loss of expression of endothelial EPCR and TM, which in turns caused impairment of PC activation by the inflamed mucosal microvasculature. In isolated human intestinal endothelial cells, administration of recombinant activated PC had a potent antiinflammatory effect, as demonstrated by downregulated cytokine-dependent cell adhesion molecule expression and chemokine production as well as inhibited leukocyte adhesion. In vivo, administration of activated PC was therapeutically effective in ameliorating experimental colitis as evidenced by reduced weight loss, disease activity index, and histological colitis scores as well as inhibited leukocyte adhesion to the inflamed intestinal vessels. The results suggest that the PC pathway represents a new system crucially involved in governing intestinal homeostasis mediated by the mucosal microvasculature. Restoring the PC pathway may represent a new therapeutic approach to suppress intestinal inflammation in IBD.     

Protein C pathway in sepsis

The goals of this chapter are to provide a brief review of the biology of the protein C pathway and some of the features of the pathway that make it uniquely positioned to control microvascular coagulation and control the acute inflammatory response. Activated protein C works as an antithrombotic agent by inactivating factors Va and VIIIa. It is particularly effective at preventing microvascular thrombosis. Platelets may provide a margin of safety for activated protein C as an antithrombotic. Approximately 25% of the factor V/Va in plasma is contained within the platelet and hence resistant to time dependent inactivation by activated protein C. In addition, factor Va bound to the platelet surface is relatively resistant to inactivation by activated protein C. Activated protein C also facilitates clot lysis by inhibiting plasminogen activator inhibitor 1, a process that is accelerated markedly by vitronectin. Inflammatory cytokines like tumor necrosis factor &#102 (TNF &#102 ) and interleukin1 &#103 (IL-1 &#103 ) downregulate two key components of the protein C activation complex, thrombomodulin and the endothelial cell protein C receptor resulting in decreased protein C activation. Activated protein C in turn has been shown in several animal models and in vitro to inhibit TNF elaboration in response to endotoxin. This inhibition appears to be due to diminished nuclear factor &#115 B (NF &#115 B) expression and nuclear translocation. Activated protein C has been shown to reduce the rate of death due to severe sepsis. This reduction may be due to both the anticoagulant effects as demonstrated by a reduction in D-dimer and inflammatory effects as demonstrated by a reduction in interleukin 6.     

Specificity of coagulation factor signaling

Summary.  Coagulation serine proteases signal through protease-activated receptors (PARs). Thrombin-dependent PAR signaling on platelets is essential for the hemostatic response and vascular thrombosis, but regulation of inflammation by PAR signaling is now recognized as an important aspect of the pro- and anti-coagulant pathways. In tissue factor (TF)-dependent initiation of coagulation, factor (F) Xa is the PAR-1 or PAR-2-activating protease when associated with the transient TF–FVIIa–FXa complex. In the anticoagulant protein C (PC) pathway, the thrombin–thrombomodulin complex activates PC bound to the endothelial cell PC receptor (EPCR), which functions as a required coreceptor for activated PC-mediated signaling through endothelial cell PAR-1. Thus, the pro- and anti-inflammatory receptor cascades are mechanistically coupled to immediate cell signaling, which precedes systemic coagulant or anticoagulant effects. In contrast to the substrate-like recognition of PARs by thrombin, TF- or EPCR-targeted activation of PARs generates cell-type specificity, PAR selectivity and protease receptor cosignaling with the G-protein-coupled PAR response. Protease receptors are thus major determinants of the biological outcome of coagulation factor signaling on vascular cells.     

Thalidomide protects endothelial cells from doxorubicin-induced apoptosis but alters cell morphology

Summary.  Antiangiogenesis agents are now being used in clinical trials to reduce the risk of recurrence of cancer. Several of these agents, however, are associated with thrombosis, especially when used in combination with chemotherapy. Antiangiogenesis and thrombosis are both endothelial-related activities, and we therefore evaluated one presumed antiangiogenesis agent (thalidomide) on intact cultured endothelial cells, and on cultured endothelial cells injured by preincubation with doxorubicin. We evaluated cell viability, caspase-3 activation, morphology of cells using light microscopy, and protease activated receptor-1 (PAR-l) expression. In our experiments, doxorubicin induced a dose- and incubation time-dependent and caspase-3-mediated apoptosis of endothelial cells. Thalidomide alone caused no changes in intact endothelial cells in terms of morphology, cell viability or activation of caspase-3. In contrast, when thalidomide was added to doxorubicin-injured endothelial cells, there was protection from cell death, increase in viability of endothelial cells, induction of differentiation and formation of neotubules. Doxorubicin reduced the expression of thrombin receptor, PAR-1, as evaluated by immunostaining and flow cytometry. Thalidomide did not alter PAR-1 expression in untreated cells but restored its expression reduced by doxorubicin. These findings suggest that thalidomide may be procoagulant, not by enhancing doxorubicin-mediated endothelial cell injury, but by altering the expression of PAR-1 on injured endothelium and resulting in endothelial dysfunction, which may explain hypercoagulability in patients treated with chemotherapy followed by thalidomide.