The anticoagulant properties of a modified form of protein S

Protein S (PS) is a vitamin K-dependent anticoagulant that acts as a cofactor to activated protein C (APC). To date PS has not been shown to possess anticoagulant activity in the absence of APC. In this study, we have developed monoclonal antibody to protein S and used to purify the protein to homogeneity from plasma. Affinity purified protein S (PSM), although identical to the conventionally purified protein as judged by SDS-PAGE, had significant anticoagulant activity in the absence of APC when measured in a factor Xa recalcification time. Using SDS-PAGE we have demonstrated that prothrombin cleavage by factor Xa was inhibited in the presence of PSM. Kinetic analysis of the reaction revealed that PSM competitively inhibited factor Xa mediated cleavage of prothrombin. PS preincubated with the monoclonal antibody, acquired similar anticoagulant properties. These results suggest that the interaction of the monoclonal antibody with PS results in an alteration in the protein exposing sites that mediate the observed anticoagulant effect. Support that the protein was altered was derived from the observation that PSM was eight fold more sensitive to cleavage by thrombin and human neutrophil elastase than conventionally purified protein S. These observations suggest that PS can be modified in vitro to a protein with APC-independent anticoagulant activity and raise the possibility that a similar alteration could occur in vivo through the binding protein S to a cellular or plasma protein.     

Protein S levels modulate the activated protein C resistance phenotype induced by elevated prothrombin levels

Elevated plasma prothrombin levels, due to the prothrombin 20210 G/A mutation or to acquired causes, are a risk factor for venous thrombosis, partly because of prothrombin-mediated inhibition of the protein C anticoagulant pathway and consequent activated protein C (APC) resistance. We determined the effect of plasma prothrombin concentration on the APC resistance phenotype and evaluated the role of protein S levels as a modulating variable. The effect of prothrombin and protein S levels on APC resistance was investigated in reconstituted plasma systems and in a population of healthy individuals using both the aPTT-based and the thrombin generation-based APC resistance tests. In reconstituted plasma, APC resistance increased at increasing prothrombin concentration in both assays. Enhanced APC resistance was caused by the effect of prothrombin on the clotting time in the absence of APC in the aPTT-based test, and on thrombin formation in the presence of APC in the thrombin generation-based test. In plasma from healthy individuals prothrombin levels were highly correlated to protein S levels. Since prothrombin and protein S had opposite effects on the APC resistance phenotype, the prothrombin/protein S ratio was a better predictor of APC resistance than the levels of either protein alone. Prothrombin titrations in plasmas containing different amounts of protein S confirmed that protein S levels modulate the ability of prothrombin to induce APC resistance. These findings suggest that carriers of the prothrombin 20210 G/A mutation, who have a high prothrombin/protein S ratio, may experience a higher thrombosis risk than non-carriers with comparable prothrombin levels.     

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.     

Circulating levels of inflammatory cytokines (IL-1 beta and TNF-alpha), resistance to activated protein C, thrombin and fibrin generation in uncomplicated pregnancies

We studied 33 women during normal uneventful pregnancies and with no history of previous adverse pregnancy events for markers of activated coagulation and thrombin activity including prothrombin fragment 1.2(PF1.2), thrombin- antithrombin (TAT), and soluble fibrin polymer (SFP). In addition, we measured potential thrombin generation through the addition of thromboplastin to patient plasma in the presence of a thrombin-specific chromogenic substrate determined serially over a period of time--Endogenous Thrombin Potential assay (ETP). This assay was performed with plasma treated and untreated with activated protein C (APC). The fibrinolytic system was assessed by measurement of thrombin activatable fibrinolysis inhibitor (TAFI). These findings were correlated with the levels of pro-inflammatory cytokines, interleukine-1 beta and tumor necrosis factor-alpha. Our data supports previous reports that indicate that resistance to activated protein C and coagulation activation markers are commonly increased in the later 2/3rds of pregnancy. There are no differences in thrombin generation potential, as determined by the ETP assay without the addition of APC, in the three trimesters. However, the thrombin reserve (TR), the ETP result without APC divided by the ETP result with the addition of APC, is increased above the reference range in the 2nd and 3rd trimesters. Patients with increased TR and resistance to APC had increased levels of TNF-alpha. Increased proinflammatory cytokines are reportedly associated with changes in the APC system with a decrease in the ability to generate APC. A sub-group of pregnancies with APC resistance had increased levels of TNF-alpha and may be important in the risk for adverse pregnancy outcomes.     

The value of 64-detector row computed tomography for the exclusion of pulmonary embolism

Identification of patients at high risk of recurrence after a first event of venous thromboembolism (VTE) remains difficult. Resistance to activated protein C (APC) is a known risk factor for VTE, but data on the risk of recurrence is controversial. We wanted to investigate whether APC resistance in the absence of factor V Leiden, determined with global coagulation test such as the thrombin generation assay, could be used as a marker for increased risk of recurrent VTE among women 18-65 years old after a first event of VTE. In a cohort of 243 women with a first event of VTE, plasma was collected after discontinuation of anticoagulant treatment and the patients were followed up for 46 months (median). Thrombin generation was measured via calibrated automated thrombography, at 1 pM and 10 pM of tissue factor (TF). In women without factor V Leiden (n=117), samples were analysed in the absence and in the presence of APC. Increase in ETP (endogenous thrombin potential) and peak height analysed in the presence of APC correlated significantly with higher risk of recurrence. At 1 pM, peak height correlated with increased risk of recurrence. In conclusion, high thrombin generation in the presence of APC, in women after a first event of VTE is indicative for an increased risk of a recurrence. We also found that thrombin generation at low TF (1 pM) is correlated with the risk of recurrence. Our data suggest that APC resistance in the absence of factor V Leiden is a risk factor for recurrent VTE.     

The role of protein S in the activation of thrombin activatable fibrinolysis inhibitor (TAFI) and regulation of fibrinolysis.

Thrombin activatable fibrinolysis inhibitor (TAFI) is a carboxy-peptidase B-like proenzyme that after activation by thrombin downregulates fibrinolysis. Thrombomodulin stimulates the activation of both TAFI and protein C whereas activated protein C inhibits the activation of TAFI by downregulation of thrombin formation, a process in which protein S acts as a cofactor. Here we determined the role of protein S in the activation of TAFI and regulation of fibrinolysis. Depletion of protein S from plasma or inhibition of protein S by specific antibodies resulted in an increased rate of TAFI activation and in an increased maximum of TAFIa activity generated. The effect on the rate of TAFI activation could be attributed to the APC-independent anticoagulant function of protein S whereas the effect on the maximum activity could be attributed to the APC cofactor function of protein S. Therefore it is concluded that protein S inhibits TAFI activation in two ways. On one hand, protein S functions as a cofactor for APC which results in a reduction of the maximum induced TAFI activity and on the other hand protein S inhibits the initial thrombin formation independently of APC which results in a decreased rate of TAFI activation. The effect of the APC-independent anticoagulant activity of protein S on the activation of TAFI provides a new mechanism for the regulation of fibrinolysis in the early stages of clot formation.     

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.     

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.     

Phospholipid-bound tissue factor modulates both thrombin generation and APC-mediated factor Va inactivation

Hemostasis is initiated by tissue factor (TF) exposed on cellular phospholipid (PL) membranes, leading to thrombin generation. The binding of thrombin to thrombomodulin (TM), activates the protein C pathway, resulting in the inactivation of factors Va and VIIIa by activated protein C (APC) and a negative feedback effect on thrombin generation. A new assay system was developed for simultaneous measurement of thrombin and APC generation in defibrinated plasma induced by large unilamellar PL vesicles complexed with full-length recombinant TF (TF:PL). TF:PL preparations with a low TF concentration induced an initial rate of thrombin generation below 100 nM/min, and resulted in less thrombin formation in the presence of TM than in its absence. In contrast, TF:PL preparations with a high concentration of TF induced a higher rate of thrombin generation, and APC-mediated feedback inhibition did not occur, despite maximal APC generation. We used the same TF:PL surfaces to study factor Va inactivation by APC in a non-plasma reaction system, and found an inverse correlation between TF surface density and the rate of factor Va inactivation. This observation suggests a previously unrecognized hemostatic effect of TF, namely a non-enzymatic surface density-based inhibition of the anticoagulant effect of APC. In this model, high concentrations and surface density of TF exert complementary effects by promoting the regular procoagulant cascade and by inhibiting the protein C pathway, thereby maximizing hemostasis after vascular injury.     

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.     

Effect of oral contraceptives on the anticoagulant activity of protein S in plasma

We determined anticoagulant parameters that depend on protein S function in plasma, i.e. the APC-independent anticoagulant activity of protein S (expressed as pSR) and APC resistance determined with thrombin generation-based tests (expressed as APCsr) as well as plasma levels of total and free protein S and prothrombin in men, women not using oral contraceptives (OC), and in women using second or third generation OC. Thrombin generation in the APC resistance assays was initiated either with factor Xa (Xa-APCsr) or tissue factor (TF-APCsr). The APC-independent anticoagulant activity of protein S was highest in men (pSR=1.69) and gradually decreased from women not using OC (pSR=1.49) via women using second generation (pSR=1.35) to women using third generation OC (pSR=1.27). The pSR correlated inversely with nAPCsr determined with the tissue factor-based APC resistance test (TF-APCsr) but not with nAPCsr determined with the factor Xa-based assay (Xa-APCsr). Multiple linear regression analysis in which sex, OC use, and protein S and prothrombin levels were included as independent variables and the pSR, TF-APCsr or Xa-APCsr as dependent variables indicated that plasma protein S levels poorly predict the pSR and the TF-APCsr, but are the main determinant of the Xa-APCsr. This indicates that OC use alters the expression of protein S activity. This phenomenon can be caused by differences in modulation of the activity of protein S by other plasma proteins that change during OC use or by OC-induced changes in the protein S molecule that impair its anticoagulant activity. Functional impairment of protein S as a result of hormonal influence may, at least in part, contribute to the thrombotic risk of OC users.     

The inhibitory effect of recombinant human soluble thrombomodulin on initiation and extension of coagulation--a comparison with other anticoagulants

Recombinant human soluble thrombomodulin (rhsTM) was compared with various anticoagulants for in vitro anticoagulant effects on thrombin generation, clotting time, and thromboelastography. rhsTM as well as APC reduced the level of the peak of the thrombin generation curve, but we did not observe any time-delay to reach the peak. This effect of rhsTM was diminished in PC-deficient plasma and was closely associated with the inhibitory effect on prothrombinase and factor Va. On the other hand, hirudin and argatroban delayed the time to reach the level of the peak, without reducing it. rhsTM and other anticoagulants except for activated protein C (APC) were found to have concentration-dependent anticoagulant activity by conventional clotting tests. However, the concentration of rhsTM for clotting time was slightly affected by anti-protein C antibody. Moreover, the concentration of rhsTM required to inhibit thrombin activity directly was 50 times higher than that needed to inhibit thrombin generation. The effect of rhsTM on clot development was compared with that of other anticoagulants by thromboelastography; rhsTM reduced the growth of the clot but had little effect on the time to activate clotting, while the other anticoagulants had the opposite effect. This effect of rhsTM was completely abolished by the addition of anti-protein C or anti-protein S antibody. These findings suggest that rhsTM attenuates blood clotting by reducing the level of generated thrombin through protein C activation and subsequent factor Va inactivation and prothrombinase inhibition.     

Effects of hereditary and acquired risk factors of venous thrombosis on a thrombin generation-based APC resistance test

BACKGROUND: Several hereditary and acquired risk factors for venous thromboembolism (VTE) are associated with impaired down-regulation of thrombin formation via the protein C pathway. To identify individuals at risk, functional tests are needed that estimate the risk to develop venous thrombosis. METHOD: We determined the effects of hereditary and acquired risk factors of venous thrombosis on an APC resistance test that quantifies the influence of APC on the time integral of thrombin formation (the endogenous thrombin potential, ETP) initiated in plasma via the extrinsic coagulation pathway. APC sensitivity ratios (APCsr) were determined in plasma from carriers of factor V(Leiden) (n = 56) or prothrombin G20210A (n = 18), of individuals deficient in antithrombin (n = 9), protein C (n = 7) or protein S (n = 14) and of women exposed to acquired risk factors such as hormone replacement therapy (n = 49), oral contraceptive use (n = 126) or pregnancy (n = 35). We also analysed combinations of risk factors (n = 60). RESULTS: The thrombin generation-based APC resistance test was sensitive for the factor V(Leiden) and prothrombin G20210A mutation, to protein S deficiency, hormone replacement therapy, oral contraceptive use and pregnancy. The assay was not influenced by antithrombin or protein C deficiency. The presence of more than one risk factor of venous thrombosis resulted in more pronounced APC resistance. The APCsr of individuals with a single or combined risk factors of VTE correlated well with reported risk increases. INTERPRETATION: The thrombin generation-based APC resistance test identifies individuals at risk for venous thrombosis due to acquired risk factors and/or hereditary thrombophilic disorders that affect the protein C pathway.     

Activated protein C, a natural anticoagulant protein, has antioxidant properties and inhibits lipid peroxidation and advanced glycation end products formation

INTRODUCTION: Activated protein C (APC) is an important natural anticoagulant that is proteolytically generated from protein C (PC) by the modulation of thrombin activity in the presence of thrombomodulin on an endothelial surface. Recent studies have demonstrated that, beyond its anticoagulant acitivities, APC had anti-inflammatory and cytoprotective properties. The mechanisms underlying APC's anti-inflammatory effects remain unknown. Our goal was to elucidate and confirm these mechanisms. METHODS: We first examined the effect of APC on reactive oxygen species (ROS) and inflammatory cytokine production in murine macrophage-like RAW264.7 cells. We further examined the effect of APC on chemically induced lipid peroxidation and advanced glycation end-products (AGE) formation. RESULTS AND CONCLUSIONS: APC in the range of 10-50 microg/mL could reduce lipopolysaccharide (LPS)-induced ROS generation, nuclear factor kappaB (NF-kappaB) activation and resultant proinflammatory cytokine production. Additional cell-free experiments revealed that APC (10-50 microg/mL) had inhibitory effects on lipid peroxidation and AGE formation. These findings suggest that APC, via its intrinsic anti-oxidant properties, may, in settings of oxidant stress, exert important cytoprotective and anti-inflammatory effects that are distinct from its anticoagulant activity as an antioxidant protein. If that is true, APC may contribute to ROS-related chronic disorders including atherosclerosis and diabetes as well as acute shock conditions.     

Anticoagulant activity of beta 2-glycoprotein I is potentiated by a distinct subgroup of anticardiolipin antibodies.

Plasmas of 16 patients positive for both IgG anticardiolipin (aCL) antibodies and lupus anticoagulant (LA) antibodies were subjected to adsorption with liposomes containing cardiolipin. In 5 of these plasmas both the anticardiolipin and the anticoagulant activities were co-sedimented with the liposomes in a dose-dependent manner, whereas in the remaining cases only the anticardiolipin activity could be removed by the liposomes, leaving the anticoagulant activity (LA) in the supernatant plasma. aCL antibodies purified from the first 5 plasmas were defined as aCL-type A, while the term aCL-type B was used for antibodies in the other 11 plasmas, from which 2 were selected for this study. Prolongation of the dRVVT was produced by affinity-purified aCL-type A antibodies in plasma of human as well as animal (bovine, rat and goat) origin. aCL-type B antibodies were found to be devoid of anticoagulant activity, while the corresponding supernatants containing LA IgG produced prolongation of the dRVVT only in human plasma. These anticoagulant activities of aCL-type A and of LA IgG's were subsequently evaluated in human plasma depleted of beta 2-glycoprotein I (beta 2-GPI), a protein which was previously shown to be essential in the binding of aCL antibodies to anionic phospholipids. Prolongation of the dRVVT by aCL-type A antibodies was abolished using beta 2-GPI deficient plasma, but could be restored upon addition of beta 2-GPI. In contrast, LA IgG caused prolongation of the dRVVT irrespective of the presence or absence of beta 2-GPI.(ABSTRACT TRUNCATED AT 250 WORDS)     

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 influence of direct and antithrombin-dependent thrombin inhibitors on the procoagulant and anticoagulant effects of thrombin

INTRODUCTION: Clinical trials evaluating direct thrombin inhibitors in unstable coronary artery disease (CAD) have been disappointing. The hypothesis tested in the present study was that these agents may inhibit the anticoagulant effect of thrombin to a further extent than the procoagulant effect of thrombin. MATERIALS AND METHODS: We studied both reversible and irreversible thrombin inhibitors and compared the effects of each inhibitor on activated protein C (APC) generation vs. the effect on fibrinopeptide A (FPA) generation. A mixture of protein C, thrombin inhibitor, fibrinogen, fibrin polymerisation blocker and thrombin was incubated with thrombomodulin (TM)-expressing human saphenous vein endothelial cells (HSVECs). The inhibitors investigated were melagatran, inogatran, hirudin, hirugen, D-Phe-D-Pro-D-arginyl chloromethyl ketone (PPACK), and antithrombin (AT) alone or in combination with unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH). RESULTS: All agents, except hirugen, inhibited APC and FPA generation in a dose-dependent manner. FPA inhibition/APC inhibition ratios, based on IC50 for inogatran, melagatran, hirudin, PPACK, AT, AT-UFH and AT-LMWH were 1.73, 0.85, 0.55, 2.1, 0.5, 0.65 and 3.1 respectively. CONCLUSIONS: All agents, except hirugen, inhibited APC and FPA generation approximately to a similar extent. Thus, it can be inferred that the poor efficacy of thrombin inhibitors in recent clinical trials in patients with unstable CAD is unlikely to be a consequence of their effects on the protein C system.     

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.     

The effect of enzymatic removal of sialic acids on the functional properties of protein C

The effect of enzymatic removal of sialic acid residues on the functional properties of human protein C was examined. The rates of activation of native and asialo protein C were identical using either the thrombin thrombomodulin complex or the snake venom activator, protac. Desiaylated activated protein C (APC) was, two-three-fold more active than native protein C, both as an anticoagulant and in its ability to hydrolyze chromogenic substrates. The potentiating effect of the desialylation was confirmed by initially fully activating protein C followed by the desialylation process. In these studies, a two-three-fold enhancement in the activity of the protein was observed which paralleled the extent of the desialylation. The activity of asialo-protein C was completely abolished by monoclonal and polyclonal antiprotein C antibodies, confirming that the effect was mediated by APC. Our studies suggest that the sialic acid component of protein C is not essential for the expression of APC activity but may act to modulate the function of the protein.