The structure-function relationship of activated protein C. Lessons from natural and engineered mutations

Protein C is the central enzyme of the natural anticoagulant pathway and its activated form APC (activated protein C) is able to proteolyse non-active as well as active coagulation factors V and VIII. Proteolysis renders these cofactors inactive, resulting in an attenuation of thrombin formation and overall down-regulation of coagulation. Presences of the APC cofactor, protein S, thrombomodulin, endothelial protein C receptor and a phospholipid surface are important for the expression of anticoagulant APC activity. Notably, APC also has direct cytoprotective effects on cells: APC is able to protect the endothelial barrier function and expresses anti-inflammatory and anti-apoptotic activities. Exact molecular mechanisms have thus far not been completely described but it has been shown that both the protease activated receptor 1 and EPCR are essential for the cytoprotective activity of APC. Recently it was shown that also other receptors like sphingosine 1 phosphate receptor 1, Cd11b/CD18 and tyrosine kinase with immunoglobulin-like and EGF-like domains 2 are likewise important for APC signalling. Mutagenesis studies are being performed to map the various APC functions and interactions onto its 3D structure and to dissect anticoagulant and cytoprotective properties. The results of these studies have provided a wealth of structure-function information. With this review we describe the state-of-the-art of the intricate structure-function relationships of APC, a protein that harbours several important functions for the maintenance of both humoral and tissue homeostasis.     

The protein C pathway and sepsis

After the discovery of the key components of the protein C (PC) pathway a beneficial effect on survival of the infusion of activated protein C (APC) in animal models of sepsis was demonstrated, leading to the development of recombinant human activated protein C (rh-APC) as a therapeutic agent. It soon became clear that rather than the anticoagulant and profibrinolytic activities of APC, its anti-inflammatory and cytoprotective properties played a major role in the treatment of patients with severe sepsis. Such properties affect the response to inflammation of endothelial cells and leukocytes and are exerted through binding of APC to at least five receptors with intracellular signaling. The main APC protective mechanism involves binding of the Gla-domain to the endothelial protein C receptor (EPCR) and cleavage of protease activated receptor 1 (PAR-1), eliciting suppression of proinflammatory cytokines synthesis and of intracellular proapoptotic pathways and activation of endothelial barrier properties. However, thrombin cleaves PAR-1 with much higher catalytic efficiency, followed by pro-inflammatory, pro-apoptotic and barrier disruptive intracellular signaling, and it is unclear how APC can exert a protective activity through the cleavage of PAR-1 when thrombin is also present in the same environment. Interestingly, in endothelial cell cultures, PAR-1 cleavage by thrombin results in anti-inflammatory and barrier protective signaling provided occupation of EPCR by the PC gla-domain, raising the possibility that the beneficial effects of rh-APC might be recapitulated in vivo by administration of h-PC zymogen to patients with severe sepsis. Recent reports of h-PC infusion in animal models of sepsis support this hypothesis.     

Alpha 2-macroglobulin enhances prothrombin activation and thrombin potential by inhibiting the anticoagulant protein C/protein S system in cord and adult plasma

Protein S (PS) is a vitamin K-dependent plasma protein and serves as a cofactor for the anticoagulant activities of activated protein C (APC). We investigated the effects of different PS concentrations on prothrombin activation and thrombin generation in cord and adult plasma containing APC and different amounts of alpha 2-macroglobulin (a2-M). Prothrombin activation was assessed by monitoring the time-course of prothrombin fragment 1+2 (F1+2) generation. Thrombin generation curves were determined by means of a subsampling technique using the chromogenic substrate S-2238. We demonstrate a dose-dependent inhibition of the anticoagulant action of PS by a2-M: suppression of F1+2 and thrombin generation due to addition of PS was stronger in plasma containing low amounts of a2-M than in plasma with elevated a2-M levels. Since no complex formation between a2-M and PS was observed by means of SDS-PAGE, we attribute decreased anticoagulant action of PS at high a2-M levels to enhanced complex formation between APC and a2-M. Thereby, APC is subtracted from its cofactor PS, resulting in suppressed formation of the anticoagulant APC/PS complex. Thus, our data suggest that a2-M, besides its well-known anticoagulant effects, also acts as a procoagulant by suppressing the formation of the anticoagulant APC/PS complex. Our findings have implications particularly on thrombin generation and inhibition in cord plasma, since a2-M levels in newborns are elevated over adult values and the antithrombotic APC/PS pathway is up-regulated at birth. Therefore, elevated levels of a2-M might restrict the up-regulation of the APC/PS pathway.     

A randomized cross-over study on the effects of levonorgestrel- and desogestrel-containing oral contraceptives on the anticoagulant pathways

The use of oral contraceptives (OC) causes disturbances of the procoagulant, anticoagulant and fibrinolytic pathways of blood coagulation which may contribute to the increased risk of venous thrombosis associated with OC therapy. Here we report the results of a cycle-controlled randomized cross-over study, in which we determined the effects of so-called second and third generation OC's on a number of anticoagulant parameters. In this study, 28 non-OC using women were randomly prescribed either a second generation (150 microg levonorgestrel/30 microg ethinylestradiol) or a third generation OC (150 microg desogestrel/30 microg ethinylestradiol) and who switched to the other OC after a two month wash out period. The anticoagulant parameters determined were: antithrombin (AT), alpha2-macroglobulin (alpha2-M), alpha1-antitrypsin, protein C inhibitor (PCI), protein C, total and free protein S and activated protein C sensitivity ratios (APC-sr) measured with two functional APC resistance tests which quantify the effect of APC on either the activated partial thromboplastin time (aPTT) or on the endogenous thrombin potential (ETP). During the use of desogestrel-containing OC the plasma levels of alpha2-M, alpha1-antitrypsin, PCI and protein C significantly increased, whereas AT and protein S significantly decreased. Similar trends were observed with levonorgestrel-containing OC, although on this kind of OC the changes in AT, PCI and protein S (which was even slightly increased) did not reach significance. Compared with levonorgestrel, desogestrel-containing OC caused a significant decrease of total (p <0.005) as well as free protein S (p <0.0001) and more pronounced APC resistance in both the aPTT (p = 0.02) and ETP-based (p <0.0001) APC resistance tests. These observations indicate that the activity of the anticoagulant pathways in plasma from users of desogestrel-containing OC is more extensively impaired than in plasma from users of levonorgestrel-containing OC.     

Control of thrombin mediated cleavage of protein S

Thrombin has been shown to cleave the vitamin K dependent cofactor protein S with subsequent loss of its cofactor activity. This study examines the control mechanisms for thrombin cleavage of protein S. The anticoagulant activity of activated protein C (APC) is enhanced fourteen fold by the addition of protein S. Thrombin cleaved protein S is seven fold less efficient than the native protein, and this loss of activity is due to reduced affinity of cleaved protein S for APC or the lipid surface compared to the intact protein. In the absence of Ca++, protein S is very sensitive to minimal concentrations of thrombin. As little as 1.5 nM thrombin results in complete cleavage of 20 nM protein S in 10 min and loss of cofactor activity. Ca++, in concentrations greater than 0.5 mM, will inhibit this cleavage and in the presence of physiological Ca++ concentrations, no cleavage of protein S could be demonstrated in spite of high concentrations of thrombin (up to 1 microM) and prolonged incubations (up to two hours). The endothelial surface protein thrombomodulin is very efficient in inhibiting the cleavage of protein S by thrombin suggesting that any thrombin formed on the endothelial cell surface is unlikely to cleave protein S, thus allowing the intact protein to act as a cofactor to APC. We conclude that the inhibitory effects of Ca++ and thrombomodulin on thrombin mediated cleavage of protein S imply that this event, by itself, is unlikely to represent a physiological control of the activity of protein S.     

Activated protein C induction of MCP-1 in human endothelial cells: a possible role for endothelial cell nitric oxide synthase.

Classically, activated protein C (APC) of the protein C/protein S anticoagulant pathway has functioned not only to inactivate the procoagulant factors Va and VIIIa but also to inhibit the activity of plasminogen activator inhibitor-1 (PAI-1). More recent data have suggested that the protein C/protein S pathway may serve as a physiological link between coagulation and inflammation. This APC pathway link was proposed because of observations showing that APC could both modulate the effects of cytokines and block neutrophil activation. As a further extension of the effect(s) of APC on cytokines, we found that APC, at the equivalent physiological protein C concentration of 4 microg/ml, significantly upregulated monocyte chemotactic protein-1 (MCP-1) RNA in human umbilical vein endothelial cells (HUVECs), as indicated by a ribonuclease protection assay (RPA) at 3 and 6 h with a return to near basal levels by 24 h. ELISA determinations demonstrated that 4 microg/ml of APC induced a significant (P=.0001) increase in MCP-1 protein production over basal levels within a 24-h period. At the same concentration, APC downregulated endothelial cell nitric oxide synthase (eNOS) RNA. Downregulation first became apparent at 6 h and continued through 48 h of culture. This downregulation was concentration dependent over a range of 1.3-12 microg/ml, and there was no effect on cell viability within this range. In support of other studies, we also found that exogenously added nitric oxide (NO) inhibited MCP-1 production. These data suggest that APC may induce MCP-1 through the inhibition of eNOS.     

The effects of supra-normal protein C levels on markers of coagulation, fibrinolysis and inflammation in a human model of endotoxemia

The protein C pathway serves as a modulating system with both anti-inflammatory and anticoagulant properties and is intimately involved in the pathophysiology of inflammation and sepsis. Treatment with recombinant human activated protein C (rhAPC) can reduce the mortality of severe sepsis.We investigated whether an elevation of plasma protein C levels to supra-normal levels by infusion of a protein C zymogen concentrate has an effect on coagulation, protein C activation or inflammation in a human endotoxemia model. Eleven healthy male volunteers were enrolled in a double-blind, placebo-controlled two-way cross-over trial. Ten minutes after infusion of 2ng/kg endotoxin each volunteer received either placebo or a plasma-derived protein C zymogen concentrate (Ceprotin, Baxter) (150 U/kg as a slow bolus infusion followed by 30 U/kg/h continuous infusion until 4 hours after LPS-infusion). Protein C antigen and activity increased 4- to 5-fold after infusion of the concentrate. APC was generated during endotoxin-induced inflammation in the placebo (1.6 fold increase) and the protein C period (4.0-fold increase). The increase of APC levels correlated with the TNF-alpha and IL-6 release in both periods (r = 0.65-0.68; p < 0.05) and paralleled the protein C antigen and activity levels in the period with supranormal protein C levels. Supra normal protein C levels resulted in slightly, although non-significant, lower tissue factor mRNA expression and thrombin generation (TAT, F1+2). Systemic inflammation (TNF-alpha, IL-6) was not influenced by protein C zymogen concentrate administration. Infusion of protein C zymogen was safe and no adverse effects occurred. The increase of protein C levels several fold above the normal range resulted in a proportional increase of the APC levels, but had no major anticoagulant, anti-inflammatory or profibrinolytic effects. Low grade endotoxemia itself induces significant protein C activation, which correlates with the TNF release.     

Dermatan sulfate and LMW heparin enhance the anticoagulant action of activated protein C

Unfractionated heparin potentiates the anticoagulant action of activated protein C (APC) through several mechanisms, including the recently described enhancement of proteolytic inactivation of factor V. Possible anticoagulant synergism between APC and physiologic glycosaminoglycans, pharmacologic low molecular weight heparins (LMWHs), and other heparin derivatives was studied. Dermatan sulfate showed potent APC-enhancing effect. Commercial LMWHs showed differing abilities to promote APC activity, and the molecular weight of LMWHs correlated with enhancement of APC activity. Degree of sulfation of the glycosaminoglycans influenced APC enhancement. However, because dextran sulfates did not potentiate APC action, the presence of sulfate groups per se on a polysaccharide is not sufficient for APC enhancement. As previously for unfractionated heparin, APC anticoagulant activity was enhanced by glycosaminoglycans when factor V but not factor Va was the substrate. Thus, dermatan sulfate and LMWHs exhibit APC enhancing activity in vitro that could be of physiologic and pharmacologic significance.     

Species specificity of the fibrinolytic effects of activated protein C.

Activated protein C (APC) has been shown to stimulate fibrinolysis in both in vitro and in vivo experimental systems. In order to test the importance of protein S in the fibrinolytic activity of APC we have compared the activity of APC, prepared from rabbit, bovine and human plasma, in the stimulation of whole blood clot lysis, the inactivation of plasminogen activator inhibitors and anticoagulant activity. When whole blood clot lysis was performed using tissue plasminogen activator in either human or rabbit blood, APC was found to enhance clot lysis in a species specific manner that paralleled the pattern observed for its anticoagulant activity. Bovine APC, was the poorest stimulator of fibrinolysis in human plasma. However, if bovine protein S was also added to human plasma, bovine APC was as effective in promoting fibrinolysis as human APC. In contrast, no species specific effects on the inactivation of plasminogen activator inhibitor activity was observed. Though substantial effects of APC on plasminogen activator inhibitor levels were made by rabbit, human and bovine activated protein C in human plasma, there was no effect of activated protein C on the rate of clot lysis of human plasma. These results suggest that protein S is important for the expression of the fibrinolytic activity of activated protein C and that the effect of protein S may be useful for the differentiation of fibrinolytic effects of activated protein C that may be related to the inactivation of plasminogen activator inhibitors and those that are not.     

Cytoprotective protein C pathways and implications for stroke and neurological disorders

Recent studies indicate that single-action-single-target agents are unlikely to cure CNS disorders sharing a pathogenic triad consisting of vascular damage, neuronal injury/neurodegeneration and neuroinflammation. Here we focus on a recent example of a multiple-action-multiple-target approach for CNS disorders based on newly discovered biological properties of activated protein C (APC), an endogenous plasma protease with antithrombotic, cytoprotective and anti-inflammatory activities in the CNS. We propose that APC-mediated signaling through the protease activated receptor-1 (PAR1) can favorably regulate multiple pathways within the neurovascular unit in non-neuronal cells and neurons during acute or chronic CNS insults, leading to stabilization of the blood-brain barrier (BBB), neuroprotection and control of neuroinflammation. Although much remains to be understood regarding the biology of APC, preclinical studies suggest that APC has promising applications as disease-modifying therapy for ischemic stroke and other neuropathologies whose underlying pathology involves deficits in the vasculo-neuronal-inflammatory triad.     

Inflammation and coagulation. An overview

Inflammation and coagulation are two main host-defence systems that interact with each other. Inflammation activates coagulation and coagulation modulates the inflammatory activity in many ways. The contributing molecular pathways are reviewed. Thrombin and activated protein C (APC) and its receptor EPCR constitute a major physiological regulatory system to control vascular wall permeability during sepsis. Pro-inflammatory cellular effects of coagulation proteases as well as the anti-inflammatory effects of APC/EPCR are mediated by signaling via protease activated receptors PAR on mononuclear cells, endothelial cells, platelets, fibroblast, and smooth muscle cells. The beneficial effects of APC in sepsis are mainly dependent on the PAR-mediated cell-protective properties rather than the anticoagulant protease function on coagulation cofactors FV/Va and FVIII/VIIIa. Animal experiments with signaling selective APC-variants show promise in improving the therapeutic efficacy and safety of APC in sepsis.     

Thrombinography shows acquired resistance to activated protein C in patients with lupus anticoagulants

In patients with lupus anticoagulants (LA), acquired resistance to activated protein C (APC) is difficult to demonstrate with clot-based assays due to the presence of the anticoagulant. Via the conversion of a fluorogenic substrate (thrombinography), we monitored the complete process of thrombin formation and decay and its delimitation by the protein C system in eight consecutive LA-patients without anticoagulant therapy and non-carriers of the V Leiden polymorphism. Thrombin generation was triggered in platelet-poor and platelet-rich plasma by recalcification in the presence of a low concentration of tissue factor. In 7 out of 8 patients we observed a long lag-time before the thrombin burst (LA effect) together with a marked inability of APC to diminish the thrombin activity. The lag-phase was however prolonged to some degree by APC. The effects were more outspoken in the presence of phospholipids from patients' platelets than with added phospholipids. Thrombinography thus demonstrates APC resistance in LA-patients despite the occurrence of long lag-times (clotting times). The amount of thrombin activity generated in the presence of APC could be a better indicator of the thrombotic risk than the moment at which the thrombin burst starts.     

Honokiol protects rat brain from focal cerebral ischemia-reperfusion injury by inhibiting neutrophil infiltration and reactive oxygen species production

We have previously shown that honokiol, an active component of Magnolia officinalis, displayed protective effect against focal cerebral ischemia-reperfusion (FCI/R) injury in rats. Production of reactive oxygen species (ROS) and infiltration of neutrophils to injured tissue play deleterious roles during cerebral ischemia. To study the mechanism(s) in mediating neuroprotective effect of honokiol, FCI/R-induced neutrophil infiltration and lipid peroxidation in brain tissue, and activation of neutrophils in-vitro were examined. Intravenous administration of honokiol (0.01-1.0 microg/kg) 15 min before (pretreatment) or 60 min after (post-treatment) middle cerebral artery occlusion reduced the total infarcted volume by 20-70% in dose-dependent manner. Pretreatment or post-treatment of honokiol at concentration of 0.1 and 1.0 microg/kg significantly decreased the neutrophil infiltration in the infarcted brain. Time course of neutrophil infiltration was performed in parallel with the lipid peroxidation in infracted brain tissue during FCI/R injury. The results indicate that honokiol can protect brain tissue against lipid peroxidation and neutrophil infiltration during FCI/R injury and cerebral infarction induced by FCI/R is accompanied with a prominent neutrophil infiltration to the infarcted area during FCI/R course. In-vitro, honokiol (0.1-10 microM) significantly diminished fMLP (N-formyl-methionyl-leucyl-phenylalanine)- or PMA (phorbol-12-myristate-13-acetate)-induced neutrophil firm adhesion, a prerequisite step behind neutrophil infiltration, and ROS production in neutrophils. Intracellular calcium overloading activates calcium-stimulated enzymes and further exaggerates FCI/R injury. Honokiol (0.1-10 microM) impeded the calcium influx induced by fMLP (a receptor agonist), AlF(4)(-) (a G-protein activator) or thapsigargin (an intracellular calcium pool releaser). Therefore, we conclude that the amelioration of FCI/R injury by honokiol can be attributed to its anti-oxidative and anti-inflammatory actions through, at least in part, limiting lipid peroxidation and reducing neutrophil activation/infiltration by interfering firm adhesion, ROS production, and calcium overloading that may be primed/activated during FCI/R injury.     

Hyperprothrombinaemia-induced APC resistance: differential influence on fibrin formation and fibrinolysis

The prothrombin gene mutation G20210A is a common risk factor for thrombosis and has been reported to cause APC resistance. However, the inhibition of thrombin formation by APC not only limits fibrin formation but also stimulates fibrinolysis by reducing TAFI activation. We evaluated the influence of prothrombin G20210A mutation on the anticoagulant and fibrinolytic activities of APC (1 microg/ml). Thirty-two heterozygous carriers and 32 non carriers were studied. APC anticoagulant activity was assessed by aPTT prolongation whereas APC fibrinolytic activity was determined by a microplate clot lysis assay. APC-induced aPTT prolongation was markedly less pronounced in carriers than in non carriers. On the contrary, fibrinolysis time was shortened by APC to a comparable extent in both groups. Accordingly, prothrombin levels were strongly correlated with APC-induced aPTT prolongation but not with APC-induced shortening of lysis time. The addition of purified prothrombin to normal plasma (final concentration 150%) caused APC resistance in the clotting assay over the whole range of tested APC concentrations (0.125-1.5 microg/ml). In the fibrinolytic assay, instead, prothrombin supplementation made the sample resistant to low but not to high concentrations of APC (>0.5 microg/ml). Thrombin and TAFIa determination in the presence of 1 microg/ml APC revealed that hyperprothrombinemia, although capable of enhancing thrombin generation, was unable to induce detectable TAFIa formation. It is suggested that APC resistance caused by hyperprothrombinaemia does not translate in impaired fibrinolysis, at least in the presence of high APC levels, because the increase in thrombin formation is insufficient to activate the amount of TAFI required to inhibit plasminogen conversion. These data might help to better understand the relationship between thrombin formation and fibrinolysis down-regulation.     

Recombinant activated protein C attenuates coagulopathy and inflammation when administered early in murine pneumococcal pneumonia

Recombinant human activated protein C (APC), which has both anticoagulant and anti-inflammatory properties, improves survival of patients with severe sepsis. This beneficial effect is especially apparent in patients with pneumococcal pneumonia. Earlier treatment with APC in sepsis has been associated with a better therapeutic response as compared to later treatment. In a mouse model it was recently confirmed that recombinant murine (rm-)APC decreases coagulation activation and improves survival in pneumococcal pneumonia; however, APC did not impact on the inflammatory response. The aim of this study was to determine the effect of APC treatment instigated early in infection on activation of coagulation and inflammation after induction of pneumococcal pneumonia. Mice were infected intranasally with viable S. pneumoniae . Mice were treated with rm-APC (125 μg) or vehicle intraperitoneally 12 hours after infection and were sacrificed after 20 hours, after which blood and organs were harvested for determination of bacterial outgrowth, coagulation activation and inflammatory markers. In this early treatment model, rm-APC treatment inhibited pulmonary and systemic activation of coagulation as reflected by lower levels of thrombin-antithrombin complexes and D-dimer. Moreover, rm-APC reduced the levels of a large number of cytokines and chemokines in the lung. When administered early in pneumococcal pneumonia, rm-APC inhibits systemic and pulmonary activation of coagulation and moreover exerts various anti-inflammatory effects in the lung.     

Investigations on oxidative stress and therapeutical implications in dementia

Alzheimer’s disease (AD) is a progressive dementia affecting a large proportion of the aging population. The histopathological changes in AD include neuronal cell death and formation of amyloid plaques and neurofibrillary tangles (NFTs) NFTs are composed of hyperphosphorylated tau protein, and senile plaques contain aggregates of the β-peptide. There is also evidence that brain tissue in patients with AD is exposed to oxidative stress during the course of the disease. Advanced glycation endproducts (AGEs), which are formed by a non-enzymatic reaction of glucose with long-lived protein deposits, are potentially toxic to the cell, are present in brain plaques in AD, and its extracellular accumulation in AD may be caused by an accelerated oxidation of glycated proteins. The microtubuli-associated protein tau is also subject to intracellular AGE formation. AGEs participate in neuronal death causing direct (chemical) radical production: Glycated proteins produce nearly 50-fold more radicals than non-glycated proteins, and indirect (cellular) radical production: Interaction of AGEs with cells increases oxidative stress. During aging cellular defence mechanisms weaken and the damages to cell constituents accumulate leading to loss of function and finally cell death. The development of drugs for the treatment of AD remains at a very unsatisfying state. However, pharmacological approaches which break the vicious cycles of oxidative stress and neurodegeneration offer new opportunities for the treatment of AD. Theses approaches include AGE-inhibitors, antioxidants, and anti-inflammatory substances, which prevent radical production. AGE inhibitors might be able to stop formation of AGE-modified β-amyloid deposits, antioxidants are likely to scavenge intracellular and extracellular superoxide radicals and hydrogen peroxide before these radicals damage cell constituents or activate microglia, and anti-inflammatory drugs attenuating microglial radical and cytokine production.     

Protective effects of activated protein C in sepsis

Sepsis remains a complex syndrome associated with significant morbidity and mortality. It is now widely accepted that the pathways of inflammation, coagulation, apoptosis, and endothelial permeability are intimately linked in sepsis pathophysiology. The clinical success of activated protein C (APC), a natural anticoagulant, in reducing mortality in patients with severe sepsis has fuelled basic and preclinical research on the protective effects of this molecule. Over the past 15 years, impressive research advances have provided novel insights into the multifunctional activities of APC. APC is now viewed not only as an anticoagulant, but also as a cell signaling molecule that dampens the excessive or insufficiently controlled host response during sepsis. This review attempts to summarize the pleiotropic activities of APC with focus on its ability to inhibit coagulation, inflammation, apoptosis, and endothelial barrier breakdown. A comprehensive PUBMED literature review up to May 2008 was conducted.     

Cytotoxicity of advanced glycation endproducts in human micro- and astroglial cell lines depends on the degree of protein glycation

Advanced glycation endproducts (AGEs) arise from the reaction of sugars with side chains and the N-terminus of proteins and are thought to be involved in the pathogenesis of several diseases by inducing oxidative stress, inflammation and cell death presumably mediated through activation of the receptor of AGE (RAGE). To address the question whether the cell damaging effect of AGE depends on the degree of its protein glycation, differential modified AGEs derived from incubating human serum albumin with increasing concentrations of methyl glyoxal were tested on cell viability, reactive oxygen species (ROS) formation, intracellular ATP levels, and activation of caspases 3/7 in two human glial cell lines, which were used as a model for human glia cells. All AGEs tested, regardless of their degree of modification, were found to induce ROS formation in both microglial (CHME-5) and astroglial cells (U373 MG), while only highly modified AGEs were able to decrease the cell viability and to induce apoptosis. This indicates that apoptotic events may be involved in the change of physiological parameters.     

Polymorphisms in the endothelial protein C receptor gene and thrombophilia

The protein C anticoagulant pathway plays a crucial role as a regulator of the blood clotting cascade. Protein C is activated on the vascular endothelial cell membrane by the thrombin-thrombomodulin complex. Once formed, activated protein C (APC) down-regulates thrombin formation by inactivating factors (F)Va and FVIIIa. Endothelial protein C receptor (EPCR) is able to bind protein C and increase the rate of protein C activation. Normal APC generation depends on the precise assemblage, on the surface of endothelial cells, of thrombin, thrombomodulin, protein C and EPCR. Therefore, any change in the efficiency of this assemblage may cause reduced/increased APC generation and modify the risk of thrombosis. This review highlights the different mutations/polymorphisms reported in the EPCR gene and their association with the risk of thrombosis.     

Interspecies loop grafting in the protease domain of human protein C yielding enhanced catalytic and anticoagulant activity.

Human anticoagulant activated protein C (hAPC) is less potent than the bovine APC (bAPC) molecule and our aims were to elucidate the molecular background for this difference and to create an APC with enhanced anticoagulant activity. In the protease domain of human protein C (hPC), the loop 148 (GWGYHSSREKEAKRN) is four residues longer than the corresponding loop in bovine APC (GWGY RDETKRN). To investigate whether this caused the species difference, the loop in hPC was replaced by the shorter bovine loop, whereas the longer human loop was introduced in bovine protein C. The mutation in hAPC yielded enhanced catalytic activity against chromogenic (4-fold) as well as natural (factors Va and VIIIa) substrates and 2-3-fold increased anticoagulant activity. The opposite effects were obtained with the bovine mutant. As compared to wild-type hAPC, the mutant hAPC was inhibited slightly faster by the protein C inhibitor, whereas the inhibition by alpha1-antitrypsin was unaffected by the mutation. A computer model of bAPC was developed in order to analyse further our data. Collectively, our results demonstrate enhanced catalytic efficiency to result from mutagenesis in the loop 148 and show that APC mutant with increased anticoagulant activity can be created.