Zinc ions bind to and inhibit activated protein C

SummaryZn2+ ions were found to efficiently inhibit activated protein C (APC), suggesting a potential regulatory function for such inhibition. APC activity assays employing a chromogenic peptide substrate demonstrated that the inhibition was reversible and the apparent K I was 13 ± 2 μM. k cat was seven fold decreased whereas K M was unaffected in the presence of 10 μM Zn2+. The inhibitory effect of Zn2+ on APC activity was also observed when factor Va was used as a substrate in an assay coupled to a prothrombinase assay. The interaction of Zn2+ with APC was accompanied by a reversible ~40% decrease in tryptophan fluorescence, consistent with the ion inducing a conformational change in the protein. The apparent K D was 7.4 ± 1.5 μM and thus correlated well with the apparent K I. In the presence of physiological Ca2+ concentration the K I and K D values were three to four fold enhanced, presum-ably due to the Ca2+-induced conformational change affecting the conformation of the Zn2+-binding site. The inhibition mechanism was non-competitive both in the absence and presence of Ca2+. Comparisons of sequences and structures suggested several possible sites for zinc binding. The magnitude of the apparent KI in relation to the blood and platelet concentrations of Zn2+ supports a physiological role for this ion in the regulation of anticoagulant activity of APC. These findings broaden the understanding of this versatile serine protease and enable the future development of potentially more efficient anticoagulant APC variants for treatments of thrombotic diseases.

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Evidence that the Protein C Activation Pathway Amplifies the lnhibition of Thrombin Generation by Recombinant Human Thrombomodulin in Plasma

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649598 ◽

1993 ◽

Vol 70 (03) ◽

pp. 423-426 ◽

Cited By ~ 16

Author(s):

Rika ohishi ◽

Naoko watanabe ◽

Masaharu Aritomi ◽

Komakazu Gomi ◽

Takao Kiyota ◽

...

Keyword(s):

Protein C ◽

Inhibitory Action ◽

Thrombin Generation ◽

Anticoagulant Activity ◽

Activated Protein C ◽

Inhibitory Effect ◽

Phospholipid Vesicles ◽

Activation Pathway ◽

Activated Platelets ◽

Rapid Generation

SummaryThrombomodulin (TM) is a cofactor for the thrombin-catalyzed activation of anticoagulant protein C. However, we have no evidence that thrombomodulin actually activates protein C during blood coagulation processing, nor do we know whether this activated protein C acts as an anticoagulant. We studied the inhibitory action of recombinant human soluble TM (rhs-TM) on thrombin generation in whole plasma. Human plasma was activated with small amounts of tissue factor using phospholipid vesicles in place of activated platelets. Thrombin generation was observed. The addition of only 2 nM of rhs-TM prevented rapid generation of thrombin and reduced the total amount of thrombin generated. In order to study the influence of the protein C activation pathway on this inhibitory action of rhs-TM, protein C-depleted plasma was used. rhs-TM had little inhibitory effect on protein C-depleted plasma. However, the addition of protein C caused a delay in thrombin generation and a reduction of the maximum thrombin concentration. We concluded that the anticoagulant activity of rhs-TM was amplified by the protein C activation pathway.

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

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650220 ◽

1996 ◽

Vol 75 (01) ◽

pp. 049-055 ◽

Cited By ~ 70

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.

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

Clinical Chemistry ◽

10.1373/clinchem.2004.043414 ◽

2005 ◽

Vol 51 (3) ◽

pp. 545-552 ◽

Cited By ~ 42

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.

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Activation and Inactivation of Human Protein C by Plasmin

Thrombosis and Haemostasis ◽

10.1055/s-0038-1642492 ◽

1994 ◽

Vol 71 (05) ◽

pp. 615-621 ◽

Cited By ~ 7

Author(s):

Katalin Váradi ◽

Anton Philapitsch ◽

Thomas Santa ◽

Hans Peter Schwarz

Keyword(s):

Protein C ◽

Anticoagulant Activity ◽

Activated Protein C ◽

Order Rate Constant ◽

Inhibitory Effect ◽

Fibrinolytic Therapy ◽

Second Order ◽

Amidolytic Activity ◽

Order Rate

SummaryTransient procoagulant states resulting in failure of recanalization or rethrombosis of the reperfused artery during thrombolytic therapy might be due to an inhibitory effect of plasmin on the anticoagulant properties of protein C. We therefore studied the effect of plasmin on protein C (PC) and activated protein C (APC) using purified human proteins.Incubation of 70 nM purified human PC with 40-400 nM human plasmin resulted in rapid activation and subsequent inactivation of PC as measured by amidolytic and anticoagulant assays. The rates of activation and inactivation were dependent on the concentration of plasmin. Lower concentrations of plasmin resulted in higher peaks of generated APC and more sustained activity, while at higher concentrations, both activation and inactivation were more rapid. Anticoagulant activity appeared more sensitive to inactivation by plasmin than amidolytic activity; e. g., while amidolytic activity reached a maximum of 13.8 nM in 6 min and declined to approximately 6 nM after 30 min, anticoagulant activity reached its maximum of only 1.4 nM within 30 s and completely disappeared within 90 s.Plasmin rapidly destroyed both the anticoagulant and amidolytic activity of purified APC, with second order rate constants of 2.8 × 105 M−1 s−1 and 1.2 × 104 M−1 −1, respectively, for 70 nM APC. The rates of activation and subsequent inactivation were slowed by the presence of CaCl2. The second order rate constant of inactivation of APC amidolytic activity decreased to 6.6 × 103 M−1 s−1 in the presence of 5 mM CaCl2. Proteolytic degradation of both PC and APC corresponding to the loss of amidolytic activity was demonstrated on SDS-PAGE using 125I-labelled proteins. When normal human plasma was incubated with plasmin or streptokinase a substantial loss of PC anticoagulant activity was observed.These results in vitro suggest that plasmin modulates the anticoagulant properties of protein C in a way that might be of relevance for the success of fibrinolytic therapy.

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Low Levels of the Anticoagulant Activity of Protein C in Patients with Chronic Renal Insufficiency: an Inhibitor of Protein C Is Present in Uremic Plasma

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646431 ◽

1991 ◽

Vol 66 (04) ◽

pp. 420-425 ◽

Cited By ~ 21

Author(s):

Elena M Faioni ◽

Franca Franchi ◽

Alessandro Krachmalnicoff ◽

Carla Valsecchi ◽

Gian Luigi Viganò ◽

...

Keyword(s):

Protein C ◽

Anticoagulant Activity ◽

Activated Protein C ◽

Inhibitory Effect ◽

Freezing And Thawing ◽

Repeated Freezing ◽

Low Levels ◽

Positively Charged

SummaryTwenty-nine of 54 uremic patients had low levels of protein C measured as anticoagulant activity, contrasting with normal levels measured as amidolytic activity or antigenic concentration. We demonstrate that this discrepancy is due to the presence of a soluble plasma inhibitor that interferes specifically with the anticoagulant activity of activated protein C. The inhibitor does not interfere with other coagulation assays. It is resistent to diisopropylfluorophosphate, high temperatures and repeated freezing and thawing. It can be dissociated from protein C by anti-protein C antibodies or by dialysis in vitro and in vivo. It binds to positively charged resins and can be eluted with high salt concentrations without losing its inhibitory capacity. The inhibitory effect is correlated with plasma creatinine levels and fluctuates with time.

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Immunoglobulin Fractions Isolated from Patients with Antiphospholipid Antibodies Prevent the Inactivation of Factor Va by Activated Protein C on Human Endothelial Cells

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656363 ◽

1992 ◽

Vol 68 (03) ◽

pp. 268-272 ◽

Cited By ~ 62

Author(s):

Montserrat Borrell ◽

Nuria Sala ◽

Conxita de Castellarnau ◽

Silvia Lopez ◽

Merce Gari ◽

...

Keyword(s):

Endothelial Cells ◽

Antiphospholipid Antibodies ◽

Protein C ◽

Umbilical Vein ◽

Anticoagulant Activity ◽

Activated Protein C ◽

Reaction Times ◽

Protein S ◽

Inhibitory Effect ◽

Factor Va

SummaryWe studied the effect of purified immunoglobulins (Ig) from 21 patients with antiphospholipid antibodies (aPL) on factor Va degradation by activated protein C (aPC) on cultured human umbilical vein endothelial cells (HUVEC). Sera from patients were tested on an ELISA aPL assay to determine the isotype with aPL activity. HUVEC were incubated with purified IgG or IgM fraction from controls or patients. Activated PC and factor Va were then added and factor Va degradation was measured after several reaction times. 13 of 14 IgM and 8 of 10 IgG from patients showed an inhibitory effect on factor Va degradation by aPC when compared with control Ig. We also observed the same inhibitory effect with patients’ Ig on studying the degradation of factor Va by aPC in a purified system containing aPC, protein S and phospholipids. These results suggest that aPL antibodies disturb the anticoagulant activity of aPC, which may contribute to the thrombotic tendency of these patients.

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Gly74Ser mutation in protein C causes thrombosis due to a defect in protein S-dependent anticoagulant function

Thrombosis and Haemostasis ◽

10.1160/th17-01-0043 ◽

2017 ◽

Vol 117 (07) ◽

pp. 1358-1369 ◽

Cited By ~ 3

Author(s):

Changming Chen ◽

Likui Yang ◽

Bruno O. Villoutreix ◽

Xuefeng Wang ◽

Qiulan Ding ◽

...

Keyword(s):

Mammalian Cells ◽

Protein C ◽

Anticoagulant Activity ◽

Activated Protein C ◽

Protein S ◽

Inhibition Mechanism ◽

Heterozygous Mutation ◽

Thrombin Generation Assay ◽

Clotting Cascade ◽

Anticoagulant Function

SummaryProtein C is a vitamin K–dependent serine protease zymogen in plasma which upon activation by thrombin in complex with thrombomodulin (TM) down-regulates the clotting cascade by a feedback loop inhibition mechanism. Activated protein C (APC) exerts its anticoagulant function through protein S-dependent degradation of factors Va and VIIIa. We recently identified a venous thrombosis patient whose plasma level of protein C antigen is normal, but its anticoagulant activity is only 34% of the normal level. Genetic analysis revealed that the proband and her younger brother carry a novel heterozygous mutation c.346G>A, p.Gly74Ser (G74S) in PROC. Thrombin generation assay indicated that the TM-dependent anticoagulant activity of the proband’s plasma has been significantly impaired. We expressed protein C-G74S in mammalian cells and characterised its properties in established coagulation assays. We demonstrate that the protein C variant can be normally activated by the thrombin-TM complex and the resulting APC mutant also exhibits normal amidolytic and proteolytic activities toward both FVa and FVIIIa. However, it was discovered the protein S-dependent catalytic activity of APC variant toward both procoagulant cofactors has been significantly impaired. Protein S concentration-dependence of FVa degradation revealed that the capacity of APC variant to interact with the cofactor has been markedly impaired. The same results were obtained for inactivation of FVa-Leiden suggesting that the protein S-dependent activity of APC variant toward cleavage of Arg-306 site has been adversely affected. These results provide insight into the mechanism through which G74S substitution in APC causes thrombosis in the proband carrying this mutation.

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Dermatan Sulfate and LMW Heparin Enhance the Anticoagulant Action of Activated Protein C

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614856 ◽

1999 ◽

Vol 82 (11) ◽

pp. 1462-1468 ◽

Cited By ~ 22

Author(s):

José Fernández ◽

Jari Petäjä ◽

John Griffin

Keyword(s):

Molecular Weight ◽

Unfractionated Heparin ◽

Protein C ◽

Dermatan Sulfate ◽

Anticoagulant Activity ◽

Activated Protein C ◽

Factor V ◽

Factor Va ◽

Anticoagulant Action

SummaryUnfractionated 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.

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Platelet-mediated proteolytic down regulation of the anticoagulant activity of protein S in individuals with haematological malignancies

Thrombosis and Haemostasis ◽

10.1160/th11-07-0457 ◽

2012 ◽

Vol 107 (03) ◽

pp. 468-476 ◽

Cited By ~ 12

Author(s):

Ilze Dienava-Verdoold ◽

Marina R. Marchetti ◽

Liane C. J. te Boome ◽

Laura Russo ◽

Anna Falanga ◽

...

Keyword(s):

Protein C ◽

Normal Values ◽

Anticoagulant Activity ◽

Activated Protein C ◽

Protein S ◽

Intact Protein ◽

The Impact ◽

Independent Protein

SummaryThe natural anticoagulant protein S contains a so-called thrombin-sensitive region (TSR), which is susceptible to proteolytic cleavage. We have previously shown that a platelet-associated protease is able to cleave protein S under physiological plasma conditions in vitro. The aim of the present study was to investigate the relation between platelet-associated protein S cleaving activity and in vivo protein S cleavage, and to evaluate the impact of in vivo protein S cleavage on its anticoagulant activity. Protein S cleavage in healthy subjects and in thrombocytopenic and thrombocythaemic patients was evaluated by immunological techniques. Concentration of cleaved and intact protein S was correlated to levels of activated protein C (APC)-dependent and APC-independent protein S anticoagulant activity. In plasma from healthy volunteers 25% of protein S is cleaved in the TSR. While in plasma there was a clear positive correlation between levels of intact protein S and both APC-dependent and APC-independent protein S anticoagulant activities, these correlations were absent for cleaved protein S. Protein S cleavage was significantly increased in patients with essential thrombocythaemia (ET) and significantly reduced in patients with chemotherapy-induced thrombocytopenia. In ET patients on cytoreductive therapy, both platelet count and protein S cleavage returned to normal values. Accordingly, platelet transfusion restored cleavage of protein S to normal values in patients with chemotherapy-induced thrombocytopenia. In conclusion, proteases from platelets seem to contribute to the presence of cleaved protein S in the circulation and may enhance the coagulation response in vivo by down regulating the anticoagulant activity of protein S.

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