scholarly journals Differential Effects of Anti–β2-Glycoprotein I and Antiprothrombin Antibodies on the Anticoagulant Activity of Activated Protein C

Blood ◽  
1998 ◽  
Vol 91 (6) ◽  
pp. 1999-2004 ◽  
Author(s):  
Monica Galli ◽  
Luisa Ruggeri ◽  
Tiziano Barbui
Keyword(s):  
Protein C ◽  
Time Course ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Limited Information ◽  
Coagulation Profile ◽  
Glycoprotein I ◽  
Increased Risk ◽  
Antiprothrombin Antibodies ◽  
Normal Controls

Abstract Antiprothrombin and anti–β2-glycoprotein I (β2-GPI) antibodies belong to the family of antiphospholipid (APL) antibodies and represent the phospholipid-dependent inhibitors of coagulation. They may be distinguished by analyzing the coagulation profiles generated by the comparison of the ratios of two coagulation tests, the Kaolin Clotting Time (KCT) and the dilute Russell's Viper Venom Time (dRVVT), commonly adopted for their diagnosis. The KCT profile is caused by antiprothrombin antibodies, whereas anti–β2-GPI antibodies are responsible for the dRVVT coagulation profile. The presence of aPL antibodies is frequently associated with acquired resistance to activated Protein C (APC-R), but limited information is available regarding the role of the different antibodies in its development. We studied the time-course of activated Factor V (FVa) generation and inactivation in the plasma of 42 patients with well-defined phospholipid-dependent inhibitors of coagulation: 24 displayed the dRVVT coagulation profile, whereas the other 18 cases showed the KCT profile. In normal pooled plasma, the peak values of FVa (mean ± standard deviation, [SD]: 16.307 ± 4.372 U/mL) were reached in 4 to 5 minutes and an almost complete inactivation (0.088 ± 0.123 U/mL) was obtained within 20 minutes. At this time point, values of residual FVa exceeding 2 SD the mean of controls (0.344 U/mL) were considered abnormal. Patients belonging to the KCT coagulation profile group reached the maximal amount of FVa in plasma (22.740 ± 7.693 U/mL, P = not significant v controls) within 4 to 5 minutes; at 20 minutes, the residual amount of FVa in plasma ranged from 0 to 1.09 U/mL (0.293 ± 0.298; P = .027), but it was found abnormal in only six of the 18 cases. The time-course of FVa in plasma of patients belonging to the dRVVT coagulation profile group differed from that of normal controls in that the peak values (10.955 ± 5.092 U/mL) were reached at 10 minutes and the amount of residual FVa at 20 minutes ranged from 0.320 to 14.450 U/ml (2.544 ± 3.580 U/mL;P = .0191 v normal controls and P = .0114v KCT group patients). Twenty of the 24 patients belonging to the dRVVT profile group had an abnormal inactivation of FVa (χ2 = 0.001 v KCT group patients). History of venous thrombosis was experienced by 15 patients: an abnormal rate of FVa inactivation was found in 11 of them (73%) versus 15 of the 27 cases without thrombosis (56%) (x2= 0.2556). The effect of affinity-purified IgG phospholipid-dependent inhibitors of coagulation on the time-course of FVa generation and inactivation in normal plasma was also investigated. Anti–β2-GPI, but not antiprothrombin antibodies, hampered the inactivation of FVa by endogenous APC, thus reproducing the behavior of the original plasmas. This effect was strictly β2-GPI–dependent. In conclusion, our findings confirm that anti–β2-GPI antibodies identify patients with phospholipid-dependent inhibitors of coagulation at increased risk of thrombosis and suggest acquired APC-R as a possible explanation of the pathogenesis of the thromboembolic events.

scholarly journals Differential Effects of Anti–β2-Glycoprotein I and Antiprothrombin Antibodies on the Anticoagulant Activity of Activated Protein C

Blood ◽  
1998 ◽  
Vol 91 (6) ◽  
pp. 1999-2004 ◽  
Author(s):  
Monica Galli ◽  
Luisa Ruggeri ◽  
Tiziano Barbui
Keyword(s):  
Protein C ◽  
Time Course ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Limited Information ◽  
Coagulation Profile ◽  
Glycoprotein I ◽  
Increased Risk ◽  
Antiprothrombin Antibodies ◽  
Normal Controls

Antiprothrombin and anti–β2-glycoprotein I (β2-GPI) antibodies belong to the family of antiphospholipid (APL) antibodies and represent the phospholipid-dependent inhibitors of coagulation. They may be distinguished by analyzing the coagulation profiles generated by the comparison of the ratios of two coagulation tests, the Kaolin Clotting Time (KCT) and the dilute Russell's Viper Venom Time (dRVVT), commonly adopted for their diagnosis. The KCT profile is caused by antiprothrombin antibodies, whereas anti–β2-GPI antibodies are responsible for the dRVVT coagulation profile. The presence of aPL antibodies is frequently associated with acquired resistance to activated Protein C (APC-R), but limited information is available regarding the role of the different antibodies in its development. We studied the time-course of activated Factor V (FVa) generation and inactivation in the plasma of 42 patients with well-defined phospholipid-dependent inhibitors of coagulation: 24 displayed the dRVVT coagulation profile, whereas the other 18 cases showed the KCT profile. In normal pooled plasma, the peak values of FVa (mean ± standard deviation, [SD]: 16.307 ± 4.372 U/mL) were reached in 4 to 5 minutes and an almost complete inactivation (0.088 ± 0.123 U/mL) was obtained within 20 minutes. At this time point, values of residual FVa exceeding 2 SD the mean of controls (0.344 U/mL) were considered abnormal. Patients belonging to the KCT coagulation profile group reached the maximal amount of FVa in plasma (22.740 ± 7.693 U/mL, P = not significant v controls) within 4 to 5 minutes; at 20 minutes, the residual amount of FVa in plasma ranged from 0 to 1.09 U/mL (0.293 ± 0.298; P = .027), but it was found abnormal in only six of the 18 cases. The time-course of FVa in plasma of patients belonging to the dRVVT coagulation profile group differed from that of normal controls in that the peak values (10.955 ± 5.092 U/mL) were reached at 10 minutes and the amount of residual FVa at 20 minutes ranged from 0.320 to 14.450 U/ml (2.544 ± 3.580 U/mL;P = .0191 v normal controls and P = .0114v KCT group patients). Twenty of the 24 patients belonging to the dRVVT profile group had an abnormal inactivation of FVa (χ2 = 0.001 v KCT group patients). History of venous thrombosis was experienced by 15 patients: an abnormal rate of FVa inactivation was found in 11 of them (73%) versus 15 of the 27 cases without thrombosis (56%) (x2= 0.2556). The effect of affinity-purified IgG phospholipid-dependent inhibitors of coagulation on the time-course of FVa generation and inactivation in normal plasma was also investigated. Anti–β2-GPI, but not antiprothrombin antibodies, hampered the inactivation of FVa by endogenous APC, thus reproducing the behavior of the original plasmas. This effect was strictly β2-GPI–dependent. In conclusion, our findings confirm that anti–β2-GPI antibodies identify patients with phospholipid-dependent inhibitors of coagulation at increased risk of thrombosis and suggest acquired APC-R as a possible explanation of the pathogenesis of the thromboembolic events.

Coagulation, inflammation, and apoptosis: different roles for protein S and the protein S–C4b binding protein complex

Blood ◽  
2004 ◽  
Vol 103 (4) ◽  
pp. 1192-1201 ◽  
Author(s):  
Suely Meireles Rezende ◽  
Rachel Elizabeth Simmonds ◽  
David Anthony Lane
Keyword(s):  
Venous Thromboembolism ◽  
Complement Activation ◽  
Protein C ◽  
Binding Protein ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Protein S ◽  
Increased Risk ◽  
Established Role ◽  
Structure And Functions

AbstractProtein S (PS) has an established role as an important cofactor to activated protein C (APC) in the degradation of coagulation cofactors Va and VIIIa. This anticoagulant role is evident from the consequences of its deficiency, when there is an increased risk of venous thromboembolism. In human plasma, PS circulates approximately 40% as free PS (FPS) and 60% in complex with C4b-binding protein (C4BP). Formation of this complex results in loss of PS cofactor function, and C4BP can then modulate the anticoagulant activity of APC. It had long been predicted that the complex could act as a bridge between coagulation and inflammation due to the involvement of C4BP in regulating complement activation. This prediction was recently supported by the demonstration of binding of the PS-C4BP complex to apoptotic cells. This review aims to summarize recent findings on the structure and functions of PS, the basis and importance of its deficiency, its interaction with C4BP, and the possible physiologic and pathologic importance of the PS-C4BP interaction.

scholarly journals Coagulation and fibrinolytic activities in 2 siblings with β2-glycoprotein I deficiency

Blood ◽  
2000 ◽  
Vol 96 (4) ◽  
pp. 1594-1595
Author(s):  
Rie Takeuchi ◽  
Tatsuya Atsumi ◽  
Masahiro Ieko ◽  
Hiroyuki Takeya ◽  
Shinsuke Yasuda ◽  
...  
Keyword(s):  
Protein C ◽  
Thrombin Generation ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Healthy Woman ◽  
Contact Activation ◽  
Glycoprotein I ◽  
Normal Ranges

β2-Glycoprotein I (β2GPI) is a major antigen for antiphospholipid antibodies, and its multiple in vitro functions have been reported. This glycoprotein not only down-regulates thrombin formation by inhibiting contact activation or prothrombinase activity, but also up-regulates coagulation by reducing protein C anticoagulant activity. However, the in vivo roles of β2GPI remain obscure. Coagulation and fibrinolytic characteristics were investigated in individuals with β2GPI deficiency. An apparently healthy woman and her brother are homozygotes for β2GPI deficiency. In these patients, Russell viper venom time was shortened (40.4 seconds; normal range, 47.8 ± 4.95 seconds), but all markers of thrombin generation and fibrin turnover were within normal ranges. Exogenous activated protein C adequately prolonged the clotting time of the β2GPI-deficient plasma, and euglobulin lysis time was also normal. Thus, elevated thrombin generation, enhancement of activated protein C response, and an altered fibrinolytic system were not found in congenitally β2GPI-deficient plasma.

Abstract 33: Activated Protein C Light Chain Provides an Extended Binding Surface for Anticoagulant Cofactor Protein S

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Jose A Fernandez ◽  
Xiao Xu ◽  
Ranjeet K Sinha ◽  
Laurent O Mosnier ◽  
John H Griffin
Keyword(s):  
Light Chain ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Protein S ◽  
Crystallographic Structure ◽  
Binding Surface ◽  
Increased Risk ◽  
Extended Surface ◽  
Cofactor Activity

Plasma protein S whose deficiency is linked to increased risk for thrombosis provides anticoagulant cofactor activity for activated protein C (APC) by enhancing rates of inactivation of factors Va and VIIIa. Previous APC mutagenesis studies showed that residues 35-39 in the gamma-carboxyglutamic acid domain are required for normal interactions with protein S, indicating the APC Gla domain binds protein S. Here we used mutagenesis of APC to interrogate the surface of APC’s light chain to identify the extended binding surface for protein S. We characterized the ability of protein S to enhance the anticoagulant activity of multiple recombinant APC variants using factor Xa-1-stage clotting assays using normal pooled plasma and protein S-depleted plasma. Mutations of residues L38, K43, I73, F95, and W115 in APC significantly reduced protein S’s cofactor activity. An APC variant carrying all of these five mutations lost all of protein S cofactor activity. On the crystallographic structure of APC, these five residues delineate an extended surface on only one side of the APC light chain that identifies the putative protein S binding site which is found on a face that is opposite APC’s catalytic triad site. Each of the APC variants with single or multiple L38, K43, I73, F95, and W115 mutations showed a normal ability to cleave SEAP-labeled PAR1 at Arg 41 and Arg 46, implying that the protein S-binding surface does not bind EPCR or PAR1. In summary, mutagenesis studies identify an extended surface on a single face of APC’s light chain for binding protein S. This knowledge will enable design and interpretation of new APC biologics with enhanced translational value.

Protein S Regulates Factor IXa in the Absence and Presence of Factor VIIIa Independently of Activated Protein C

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1197-1197
Author(s):  
Rinku Majumder ◽  
Rima Chattopadhyay ◽  
Tanusree Sengupta
Keyword(s):  
Protein C ◽  
Thrombin Generation ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Protein S ◽  
Activity Measurement ◽  
Clotting Time ◽  
Thrombotic Risk ◽  
Increased Risk

Abstract Abstract 1197 Coagulation is a finely tuned process. During thrombin formation, several anticoagulant reactions are initiated to prevent systematic activation of coagulation, and impairment of anticoagulant activity causes an increased risk of venous thrombosis. One such anticoagulant factor is protein S, deficiencies of which have been linked to venous and arterial thrombosis. While protein S has been studied for over three decades, the precise role this protein plays in attenuating the hemostatic response is far from clear. Protein S is a vitamin K-dependent plasma protein that functions in feedback regulation of thrombin generation. Protein S was initially identified as a cofactor for activated protein C (APC) but later it was observed that there is only a 3–10 fold increase in APC activity in the presence of protein S. Plasma coagulation assays in the absence of APC suggest that protein S may have other anticoagulant role(s). We report here an anticoagulant activity of Protein S mediated by inhibition of fIXa in the absence and presence of fVIIIa independent of APC. Although an APC-independent anticoagulant activity has been reported for protein S interacting with fVIIIa, no study has shown that the inhibitory effect of protein S is mediated through its interaction with fIXa, thus making our observations novel and significant. Moreover, previous studies that reported an interaction between fVIIIa and protein S were performed with low amounts of phospholipid, a condition that produces activity measurement artifacts due to the presence of active protein S multimers. We used both ex vivo (plasma studies) and in vitro methods at high phospholipid (100–200 micro molar) concentration to determine whether and how the intrinsic pathway of blood coagulation is regulated by protein S. We obtained the following results: 1) activated partial thromboplastin time (aPTT) assays with protein S-supplemented plasma confirmed that protein S prolongs clotting time, and a normal clotting time was restored with addition of anti-protein S antibody, 2) a modified aPPT assay with fIX-deficient plasma confirmed that protein S affects fIX-initiated clotting time, 3) thrombin generation assay through fIXa/fVIIIa pathway, initiated with a limiting amount of tissue factor (TF), was regulated by protein S, 4) in vitro studies with fIXa/fVIIIa and protein S in the presence of phosphatidylserine (PS) vesicles showed ∼40% and ∼65% inhibition in the activity of fIXa in the absence and presence of fVIIIa, respectively, and 5) protein S altered only the KM for fX activation by fIXa but altered both kcat and KM for fX activation by fIXa and fVIIIa. Our findings underscore the central role of protein S in regulation of coagulation. We anticipate these results will unravel important implications for the evaluation of thrombotic risk associated with protein S-deficiency. Disclosures: No relevant conflicts of interest to declare.

β2-Glycoprotein I Modulates the Anticoagulant Activity of Activated Protein C on the Phospholipid Surface

1996 ◽  
Vol 75 (01) ◽  
pp. 049-055 ◽  
Author(s):  
Tatsuyuki Mori ◽  
Hiroyuki Takeya ◽  
Junji Nishioka ◽  
Esteban C Gabazza ◽  
Koji Suzuki
Keyword(s):  
Protein C ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Protein S ◽  
Inhibitory Effect ◽  
Factor V ◽  
Clotting Time ◽  
Factor Va ◽  
Glycoprotein I ◽  
Prothrombinase Activity

SummaryThe objective of this study was to determine whether (β2-glycoprotein I (β2GPI) has procoagulant activity by inhibiting the anticoagulant activity of activated protein C (APC). β2GPI inhibited significantly the APC-catalyzed inactivation of factor Va in an assay using factor V-deficient plasma and physiological levels of protein S and factor Va. This inhibitory effect was diminished by the addition of increasing concentrations of phospholipids, suggesting that β2GPI competitively inhibits the binding of APC to the phospholipid surface. β2GPI inhibited weakly factor Va- and phospholipid-dependent prothrombinase activity at concentrations similar to those to inhibit APC activity. The depletion of β2GPI from plasma led to only a slight shortening of the diluted Russell’s viper venom-dependent clotting time, but to a strong and significant potentiation of the anticoagulant activity of APC. These results suggest that under certain physiological conditions β2GPI has procoagulant property by inhibiting the phospholipid-dependent APC anticoagulant activity.

scholarly journals Coagulation and fibrinolytic activities in 2 siblings with β2-glycoprotein I deficiency

Blood ◽  
2000 ◽  
Vol 96 (4) ◽  
pp. 1594-1595 ◽  
Author(s):  
Rie Takeuchi ◽  
Tatsuya Atsumi ◽  
Masahiro Ieko ◽  
Hiroyuki Takeya ◽  
Shinsuke Yasuda ◽  
...  
Keyword(s):  
Protein C ◽  
Thrombin Generation ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Healthy Woman ◽  
Contact Activation ◽  
Glycoprotein I ◽  
Normal Ranges

Abstract β2-Glycoprotein I (β2GPI) is a major antigen for antiphospholipid antibodies, and its multiple in vitro functions have been reported. This glycoprotein not only down-regulates thrombin formation by inhibiting contact activation or prothrombinase activity, but also up-regulates coagulation by reducing protein C anticoagulant activity. However, the in vivo roles of β2GPI remain obscure. Coagulation and fibrinolytic characteristics were investigated in individuals with β2GPI deficiency. An apparently healthy woman and her brother are homozygotes for β2GPI deficiency. In these patients, Russell viper venom time was shortened (40.4 seconds; normal range, 47.8 ± 4.95 seconds), but all markers of thrombin generation and fibrin turnover were within normal ranges. Exogenous activated protein C adequately prolonged the clotting time of the β2GPI-deficient plasma, and euglobulin lysis time was also normal. Thus, elevated thrombin generation, enhancement of activated protein C response, and an altered fibrinolytic system were not found in congenitally β2GPI-deficient plasma.

scholarly journals Acquired Activated Protein C Resistance Associated with IgG Antibodies against β2-Glycoprotein I and Prothrombin as a Strong Risk Factor for Venous Thromboembolism

2005 ◽  
Vol 51 (3) ◽  
pp. 545-552 ◽  
Author(s):  
Junzo Nojima ◽  
Hirohiko Kuratsune ◽  
Etsuji Suehisa ◽  
Yoshinori Iwatani ◽  
Yuzuru Kanakura
Keyword(s):  
Venous Thromboembolism ◽  
Lupus Erythematosus ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Complete Removal ◽  
Inhibitory Effect ◽  
Glycoprotein I ◽  
Venous Thromboembolic Events

Abstract Background: Venous thromboembolic events such as deep vein thrombosis and pulmonary embolism are common manifestations of antiphospholipid syndrome. Our aim was to clarify the roles of anti-phospholipid (aPL) antibodies in the pathogenesis of venous thromboembolism (VTE) in patients with systemic lupus erythematosus (SLE). Methods and Results: We examined anti-cardiolipin/β2-glycoprotein I (anti-CL/β2-GPI) antibody concentrations, anti-phosphatidylserine/prothrombin (anti-PS/PT) antibody concentrations, and lupus anticoagulant (LA) activity in 87 patients with SLE (21 with VTE and 66 without thrombosis). Both anti-CL/β2-GPI and anti-PS/PT antibodies strongly correlated with LA activity. Multivariate logistic analysis confirmed that both anti-CL/β2-GPI and anti-PS/PT antibodies were significant independent risk factors for VTE (odds ratios = 4.98 and 7.54, respectively; 95% confidence intervals, 1.51–16.4 and 2.30–24.7, respectively). We therefore studied the in vitro effects of IgG fractions containing anti-CL/β2-GPI or anti-PS/PT antibodies on the anticoagulant activity of activated protein C (APC) and found that purified IgG containing anti-CL/β2-GPI or anti-PS/PT antibodies significantly hampered the anticoagulant activity of APC. We also studied the ability of IgG fractions to impede the anticoagulant activity of APC before and after complete removal of anti-CL/β2-GPI or anti-PS/PT antibodies by adsorption. Removal of anti-CL/β2-GPI or anti-PS/PT antibodies from all positive IgG samples clearly decreased the inhibitory effect of those samples on APC anticoagulant activity. Conclusions: Anti-CL/β2-GPI and anti-PS/PT antibodies independently cause APC resistance, which may contribute to risk of VTE in patients with SLE.

Activated protein C variants with normal cytoprotective but reduced anticoagulant activity

Blood ◽  
2004 ◽  
Vol 104 (6) ◽  
pp. 1740-1744 ◽  
Author(s):  
Laurent O. Mosnier ◽  
Andrew J. Gale ◽  
Subramanian Yegneswaran ◽  
John H. Griffin
Keyword(s):  
Endothelial Cell ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Site Directed Mutagenesis ◽  
Bleeding Complications ◽  
Cell Protein ◽  
Cytoprotective Activity ◽  
Increased Risk ◽  
Antiapoptotic Activity

Abstract Recombinant activated protein C (APC), a well-defined anticoagulant enzyme, reduced mortality in severe sepsis patients in a phase 3 trial. However, 2 potent anticoagulants, antithrombin III and recombinant tissue factor pathway inhibitor, failed to do so, implying the physiologic relevance of APC's less well-defined anti-inflammatory and antiapoptotic activities. Recombinant APC therapy conveys an increased risk of serious bleeding complications due to APC anticoagulant activity. To generate recombinant APC variants with reduced risk of bleeding due to reduced anticoagulant activity, we dissected APC's anticoagulant activity from its cytoprotective activity by site-directed mutagenesis. Using staurosporine-induced endothelial cell apoptosis assays, we show here that Ala mutations (RR229/230AA and KKK191_ 193AAA) in 2 APC surface loops that severely reduce anticoagulant activity result in 2 APC variants that retain normal antiapoptotic activity that requires protease activated receptor-1 and endothelial cell protein C receptor. Thus, it is possible to reduce anticoagulant activity while preserving antiapoptotic activity of recombinant APC variants. We suggest that therapeutic use of such APC variants may reduce serious bleeding risks while providing the beneficial effects of APC acting directly on cells. (Blood. 2004;104: 1740-1744)

Preservation of Beneficial TAFI Functions by an Activated Protein C Variant with Normal Cytoprotective Functions but Minimal Anticoagulant Activity.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 268-268
Author(s):  
Laurent O. Mosnier ◽  
Xia V. Yang ◽  
John H. Griffin
Keyword(s):  
Protein C ◽  
Time Course ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Significant Inhibition ◽  
Clot Lysis ◽  
Proteolytic Activation ◽  
Carboxypeptidase N ◽  
Anti Inflammatory

Abstract Activated Thrombin Activatable Fibrinolysis Inhibitor (TAFIa), a carboxypeptidase B-like enzyme, both inhibits fibrinolysis and exerts anti-inflammatory effects via removal of C-terminal Arg residues of bradykinin (BK) and the complement anaphylatoxins, C3a and C5a. Proteolytic activation of TAFI is caused by thrombin or thrombin-thrombomodulin; thus, TAFI activation can be down-regulated by the anticoagulant, activated protein C (APC). Recombinant APC reduces mortality in severe sepsis patients (PROWESS trial), but it increases risk of serious bleeding. This risk is presumably due to APC’s anticoagulant activity and possibly exacerbated by profibrinolytic effects derived from down-regulation of thrombin-mediated activation of TAFI. In addition, APC therapy might ironically aggravate sepsis by reducing TAFIa generation because TAFIa inactivates BK, C3a and C5a, well known pathological mediators in sepsis. Recently we made a non-anticoagulant APC variant, 5A-APC (RR229/230AA and KKK191–193AAA), that retains normal in vitro cytoprotective effects and an ability to reduce mortality in murine sepsis models (Kerschen et al, J Exp Med, 2007). This current study compares the effects of this variant to wild type (wt) APC on profibrinolytic and TAFIa’s anti-inflammatory activities in plasma-based assays. To determine the effects of APC on TAFIa-mediated inhibition of fibrinolysis, clot lysis was studied in a plasma system of thrombin-induced clot formation and tPA-mediated fibrinolysis. Compared to wt-APC (IC50=0.36 nM APC), almost 10-times more 5A-APC was required for 50% inhibition of clot lysis (IC50=3.1 nM 5A-APC) and 20-times more 5A-APC was needed to inhibit completely TAFI activation under the conditions employed. The effects of wt-APC and 5A-APC on TAFIa’s anti-inflammatory activities were determined by following proteolytic inactivation of BK in plasma during tissue factor-induced clotting. In the absence of added thrombomodulin, angiotensin-converting enzyme, carboxypeptidase N, and TAFIa each inactivated approximately 10–15% of BK under the study conditions. But in the presence of thrombomodulin, BK was fully inactivated in plasma and converted to des-Arg9-BK (> 98%), an effect mainly attributable to TAFIa. Analysis of the time course of BK inactivation in the presence of thrombomodulin indicated that TAFIa rapidly converted BK to des-Arg9-BK with only a minor contribution from carboxypeptidase N. Under conditions where TAFIa fully converted BK to des-Arg9-BK, wt-APC, but not 5A-APC, dose-dependently diminished BK inactivation and des-Arg9-BK generation. At the highest tested concentrations of APC (40 nM), wt-APC completely inhibited TAFIa-dependent BK inactivation whereas non-anticoagulant 5A-APC permitted normal TAFI activation and showed no significant inhibition of TAFIa-dependent BK inactivation. Thus, 5A-APC left intact the TAFIa-mediated anti-inflammatory mechanism for inactivation of BK in plasma and, presumably, TAFIa’s ability to inactivate the C3a and C5a anaphylatoxins. These studies show that genetic engineering can selectively alter APC’s multiple activities to provide an APC variant that retains this enzyme’s beneficial anti-inflammatory and cytoprotective effects but that diminishes bleeding risk due to reduction in APC’s anticoagulant and profibrinolytic activities. Thus, these data support the hypothesis that preservation of TAFI activation in plasma by normally cytoprotective 5A-APC variant would preserve TAFIa-dependent anti-inflammatory effects that are lost with the use of wt-APC.

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