Regulation of Prothrombinase Activity by Protein S

1999 ◽  
Vol 82 (07) ◽  
pp. 80-87 ◽  
Author(s):  
Saulius Butenas ◽  
Neal Golden ◽  
Kenneth Mann ◽  
Cornelis van't Veer
Keyword(s):  
Tissue Factor ◽  
Protein C ◽  
Thrombin Generation ◽  
Plasma Concentrations ◽  
Factor Xa ◽  
Protein S ◽  
Coagulation Factors ◽  
Independent Effect ◽  
Factor Va ◽  
Prothrombinase Activity

SummaryThe independent effect of protein S as prothrombinase inhibitor has been proposed to depend on binding to both coagulation factors Va and factor Xa or on the binding to phospholipid thereby limiting the phospholipid available for prothrombinase activity. In this study we show that plasma concentrations of protein S (300 nM) equilibrated with the prothrombinase components (factor Va, factor Xa, phospholipid) cause a profound inhibition at low phospholipid concentrations (~0.2 μM). This inhibition by protein S of prothrombinase activity is abrogated with increasing phospholipid concentrations. Modeling of the effect of protein S on prothrombinase based only on the reported affinity of protein S for phospholipids (Kd ~ 10-8 M) in an equilibrium model (Clotspeed), predicted the experimentally obtained thrombin generation rates at low phospholipid in the presence of protein S based on the diminished available phospholipid binding sites for the prothrombinase components. Consistently, initial rates of prothrombinase activity are already maximally inhibited when protein S is preincubated with the phospholipid prior to the addition of factor Xa, factor Va and pro-thrombin. The results indicate that the order of addition of prothrombinase components and the availability of phospholipid may have a profound influence on observed effects of protein S on prothrombinase activity. All prothrombinase components (factor Xa, factor Va, phospholipid) become available during the course of the physiological thrombin generation. The effect of protein S was therefore studied on tissue factor-induced, platelet-dependent thrombin generation. Protein S delayed and inhibited the rate of thrombin generation of tissue factor-induced thrombin formation when surface is provided at physiologic concentrations using isolated platelets (2 × 108/ml). In contrast, protein S hardly affected thrombin generation in this model when platelets were pre-activated with collagen. Furthermore, the observed effects of addition of protein C and thrombomodulin in the absence or presence of protein S on tissue factor-induced, platelet-dependent thrombin generation, indicate that protein S and protein C may cooperate in the regulation of prothrombinase activity through independent mechanisms.

Activated protein C cleaves factor Va more efficiently on endothelium than on platelet surfaces

Blood ◽  
2002 ◽  
Vol 100 (2) ◽  
pp. 539-546 ◽  
Author(s):  
Julie A. Oliver ◽  
Dougald M. Monroe ◽  
Frank C. Church ◽  
Harold R. Roberts ◽  
Maureane Hoffman
Keyword(s):  
Endothelial Cells ◽  
Tissue Factor ◽  
Protein C ◽  
Thrombin Generation ◽  
Activated Protein C ◽  
Protein S ◽  
Lipid Vesicles ◽  
Factor Va ◽  
Lipid Surface

Abstract The protein C/protein S system is known to regulate thrombin generation in vivo by cleaving factors Va and VIIIa. We have examined the activity of activated protein C in several tissue factor–initiated models of coagulation. We used 4 models: monocytes as the tissue factor source with platelets as the thrombin-generating surface; endothelial cells as the tissue factor source with platelets as the thrombin-generating surface; endothelial cells as both the tissue factor source and the thrombin-generating surface; and relipidated tissue factor with lipid vesicles providing the surface for thrombin generation. With the lipid surface, activated protein C dose-dependently reduced thrombin generation. Similarly, when endothelial cells provided the only surface for thrombin generation, activated protein C dose-dependently decreased thrombin generation significantly. By contrast, whenever platelets were present, activated protein C only minimally affected the amount of thrombin generated. When endothelial cells were the tissue factor source with platelets providing the surface for thrombin generation, activated protein C did increase the time until the burst of thrombin generation but had minimal effects on the total amount of thrombin generated. Activated protein C had essentially no effect on thrombin generation when monocytes were the tissue factor source with platelets providing the surface for thrombin generation. From the studies reported here, we conclude that in vivo, despite the important role of the protein C system in regulating thrombosis, activated protein C does not serve as a primary regulator of platelet-dependent thrombin generation.

Phospholipid-bound Tissue Factor Modulates both Thrombin Generation and APC-mediated Factor Va Inactivation

1999 ◽  
Vol 82 (12) ◽  
pp. 1673-1679 ◽  
Author(s):  
Katalin Váradi ◽  
Jürgen Siekmann ◽  
Peter Turecek ◽  
H. Peter Schwarz ◽  
Victor Marder
Keyword(s):  
Tissue Factor ◽  
Surface Density ◽  
Protein C ◽  
Thrombin Generation ◽  
Feedback Inhibition ◽  
Activated Protein C ◽  
Reaction System ◽  
Inverse Correlation ◽  
High Concentration ◽  
Factor Va

SummaryHemostasis 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.

Inhibition of thrombin generation by the zymogen factor VII: implications for the treatment of hemophilia A by factor VIIa

Blood ◽  
2000 ◽  
Vol 95 (4) ◽  
pp. 1330-1335 ◽  
Author(s):  
Cornelis van 't Veer ◽  
Neal J. Golden ◽  
Kenneth G. Mann
Keyword(s):  
Factor Viii ◽  
Tissue Factor ◽  
Thrombin Generation ◽  
Hemophilia A ◽  
Factor Viia ◽  
Plasma Concentrations ◽  
Factor Xa ◽  
Factor Vii ◽  
Lag Phase ◽  
Single Chain

Factor VII circulates as a single chain inactive zymogen (10 nmol/L) and a trace (∼10-100 pmol/L) circulates as the 2-chain form, factor VIIa. Factor VII and factor VIIa were studied in a coagulation model using plasma concentrations of purified coagulation factors with reactions initiated with relipidated tissue factor (TF). Factor VII (10 nmol/L) extended the lag phase of thrombin generation initiated by 100 pmol/L factor VIIa and low TF. With the coagulation inhibitors TFPI and AT-III present, factor VII both extended the lag phase of the reaction and depressed the rate of thrombin generation. The inhibition of factor Xa generation by factor VII is consistent with its competition with factor VIIa for TF. Thrombin generation with TF concentrations >100 pmol/L was not inhibited by factor VII. At low tissue factor concentrations (<25 pmol/L) thrombin generation becomes sensitive to the absence of factor VIII. In the absence of factor VIII, factor VII significantly inhibits TF-initiated thrombin generation by 100 pmol/L factor VIIa. In this hemophilia A model, approximately 2 nmol/L factor VIIa is needed to overcome the inhibition of physiologic (10 nmol/L) factor VII. At 10 nmol/L, factor VIIa provided a thrombin generation response in the hemophilia model (0% factor VIII, 10 nmol/L factor VII) equivalent to that observed with normal plasma, (100% factor VIII, 10 nmol/L factor VII, 100 pmol/L factor VIIa). These results suggest that the therapeutic efficacy of factor VIIa in the medical treatment of hemophiliacs with inhibitors is, in part, based on overcoming the factor VII inhibitory effect.

Haemostatic Studies in Carbohydrate-deficient Glycoprotein Syndrome Type I

1996 ◽  
Vol 76 (04) ◽  
pp. 502-504 ◽  
Author(s):  
A Fiumara ◽  
R Barone ◽  
P Buttitta ◽  
R Musso ◽  
L Pavone ◽  
...  
Keyword(s):  
Plasma Levels ◽  
Protein C ◽  
Antithrombin Iii ◽  
Plasma Concentrations ◽  
Protein S ◽  
Coagulation Factors ◽  
Type I ◽  
Clotting Factors ◽  
Blood Coagulation Factors ◽  
D Dimer

SummaryCDG syndrome (CDGS) type I is the most frequent form of a group of metabolic disorders characterised by a defect of the carbohydrate moiety of glycoproteins. A large number of plasma glycoproteins, including clotting factors and inhibitors, are decreased and stroke-like episodes have been described in about half of the reported patients. We studied blood coagulation factors, inhibitors and D-dimer plasma levels in four subjects, aged 12-23 years, with CDGS type I. Factors VIII, XI, antithrombin III activity, antigen plasma levels of antithrombin III, free protein S and protein C were decreased whereas protein C as activity was normal. In addition two patients had reduction of factors II, V, VII, IX, X reflecting the phenotypic heterogeneity associated with CDGS type I. D-dimer plasma concentrations were elevated in all subjects. The hypercoagulable state as consequence of the combined deficiencies of coagulation inhibitors could contribute to the stroke-like phenomena in CDGS type I.

Regulation Of Bovine Activated Protein C By Protein S: The Role Of The Cofactor Protein In Species Specificity

1981 ◽  
Author(s):  
F J Walker
Keyword(s):  
Protein C ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Factor Xa ◽  
Protein S ◽  
Species Specificity ◽  
Rabbit Plasma ◽  
Factor Va ◽  
Bovine Plasma ◽  
Species Specific

The anticoagulant activity of activated Protein C has been observed to be species specific. This could be due either to the inability of the bovine enzyme to recognize its substrate, Factor Va, in non-bovine plasmas, or the absence of cofactor-Protein S, a protein that has been shown to be necessary for the maximum expression of the anticoagulant activity of activated Protein C. Activated Protein C was found to be an effective inhibitor of Factor Xa-initiated clotting of bovine plasma, but without activity in either human or rabbit plasma. Human and rabbit plasma supplemented with bovine Protein S was sensitive to the anticoagulant activity of activated Protein C. Neither rabbit nor human plasma contained bovine activated Protein C cofactor activity as measured by the enhancement of bovine activated Protein C-catalyzed inactivation of Factor Va. However, bovine activated Protein C was able to inactivate both human and rabbit Factor Va. The inactivation of both of these proteins could be stimulated by the addition of bovine Protein S. These results indicate that the species specificity of bovine activated Protein C is due to the absence of a cofactor protein in non-bovine plasma that will interact with the bovine enzyme. Secondly, these findings further confirm that Protein S is required for the maximal expression of the anticoagulant activity of activated Protein C.

Oestradiol-responsive miR-365a-3p interacts with tissue factor 3’-UTR to modulate tissue factor-initiated thrombin generation

2021 ◽  
Author(s):  
Jiayin Tian ◽  
Murray J Adams ◽  
Jasmine Wee Ting Tay ◽  
Ian James ◽  
Suzanne Powell ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Direct Interaction ◽  
Protein S ◽  
Coagulation Factors ◽  
Luciferase Reporter ◽  
Protein Levels ◽  
Thrombin Generation Assay ◽  
Increased Risk ◽  
Reporter Assays

Background: High oestradiol (E2) levels are linked to an increased risk of venous thromboembolism, however, the underlying molecular mechanism(s) remain poorly understood. We previously identified an E2-responsive microRNA (miR), miR-494-3p that downregulates protein S expression, and posited additional coagulation factors, such as tissue factor, may be regulated in a similar manner via miRs. Objectives: To evaluate the coagulation capacity of cohorts with high physiological E2, and to further characterise novel E2-responsive miR and miR regulation on tissue factor in E2-related hypercoagulability. Methods: Ceveron® Alpha thrombin generation assay (TGA) was used to assess plasma coagulation profile of three cohorts. The effect of physiological levels of E2, 10 nM on miR expression in HuH-7 cells was compared using NanoString nCounter® and validated with independent assays. The effect of tissue factor interacting miR was confirmed by dual-luciferase reporter assays, immunoblotting, flow cytometry, biochemistry assays and TGA. Results: Plasma samples from pregnant women and women on the contraceptive pill were confirmed to be hypercoagulable (compared with sex-matched controls). At equivalent and high physiological levels of E2, miR-365a-3p displayed concordant E2-down-regulation in two independent miR quantification platforms, and tissue factor mRNA (F3) was up-regulated by E2 treatment. Direct interaction between miR-365a-3p and F3-3’UTR was confirmed and overexpression of miR-365a-3p led to a decrease of 1) tissue factor mRNA transcripts, 2) protein levels, 3) activity and 4) tissue factor-initiated thrombin generation. Conclusion: miR-365a-3p is a novel tissue factor regulator. High E2 concentrations induces a hypercoagulable state via a miR-network specific for coagulation factors.

scholarly journals Coagulative Disorders in Critically Ill COVID-19 Patients with Acute Distress Respiratory Syndrome: A Critical Review

2021 ◽  
Vol 10 (1) ◽  
pp. 140
Author(s):  
Chiara Robba ◽  
Denise Battaglini ◽  
Lorenzo Ball ◽  
Alberto Valbusa ◽  
Italo Porto ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Critically Ill ◽  
Clinical Manifestations ◽  
Factor Xa ◽  
Protein S ◽  
Coagulation Factors ◽  
Coagulation Cascade ◽  
Acute Distress ◽  
Estimated Risk ◽  
Tissue Factor Pathway

In critically ill patients with acute respiratory distress syndrome (ARDS) coronavirus disease 2019 (COVID-19), a high incidence of thromboembolic and hemorrhagic events is reported. COVID-19 may lead to impairment of the coagulation cascade, with an imbalance in platelet function and the regulatory mechanisms of coagulation and fibrinolysis. Clinical manifestations vary from a rise in laboratory markers and subclinical microthrombi to thromboembolic events, bleeding, and disseminated intravascular coagulation. After an inflammatory trigger, the mechanism for activation of the coagulation cascade in COVID-19 is the tissue factor pathway, which causes endotoxin and tumor necrosis factor-mediated production of interleukins and platelet activation. The consequent massive infiltration of activated platelets may be responsible for inflammatory infiltrates in the endothelial space, as well as thrombocytopenia. The variety of clinical presentations of the coagulopathy confronts the clinician with the difficult questions of whether and how to provide optimal supportive care. In addition to coagulation tests, advanced laboratory tests such as protein C, protein S, antithrombin, tissue factor pathway inhibitors, D-dimers, activated factor Xa, and quantification of specific coagulation factors can be useful, as can thromboelastography or thromboelastometry. Treatment should be tailored, focusing on the estimated risk of bleeding and thrombosis. The aim of this review is to explore the pathophysiology and clinical evidence of coagulation disorders in severe ARDS-related COVID-19 patients.

scholarly journals The Action of a Synthetic Pentasaccharide on Thrombin Generation in Whole Plasma

1989 ◽  
Vol 61 (03) ◽  
pp. 397-401 ◽  
Author(s):  
S Béguin ◽  
J Choay ◽  
H C Hemker
Keyword(s):  
Binding Site ◽  
Blood Coagulation ◽  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
Factor Xa ◽  
Heparin Binding ◽  
Extrinsic Pathway ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Prothrombinase Activity

SummaryWe investigated the effect on thrombin generation in plasma of the pentasaccharide that represent the AT II/binding site in heparin. This compound has no effect on the breakdown of thrombin in plasma. It dose-dependently inhibits the formation of thrombin in both the intrinsic and the extrinsic pathway. If coagulation is triggered by the complete prothrombinase complex (phospholipid – factor Va – factor Xa) under conditions in which the large majority of factor Xa is bound to the complex, the inhibition of prothrombinase activity is only minor. If no factor Va is present or if the prothrombinase activity is triggered by adding complete tenase (PL-FVIIIa-FIXa) or incomplete tenase (PLFIXa) to the plasma the inhibition by pentasaccharide is of the same magnitude as that in the intrinsic or extrinsic system.We conclude that the pentasaccharide inhibits blood coagulation by katalysing the inactivation of free factor Xa. In contrast to classical heparin it does inhibit the peak of thrombin formation in platelet rich plasma, probably because it is less subject to inactivation by heparin binding proteins from platelets than classical heparin is.

Heritability of Inter-Related Clotting Factors.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2585-2585
Author(s):  
Carla Vossen ◽  
Peter Callas ◽  
Frits Rosendaal ◽  
Sandra Hasstedt ◽  
Bruce Scott ◽  
...  
Keyword(s):  
Protein C ◽  
Plasma Concentrations ◽  
Principal Component ◽  
Protein S ◽  
Coagulation Factors ◽  
Pleiotropic Effect ◽  
Factor Ix ◽  
Factor V ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The identification of genes affecting plasma concentrations of biological traits remains difficult, as the loci affecting such traits (termed quantitative trait loci) tend to explain only a fraction of the phenotypic variation. Evidence on inter-relation (i.e. clustering) of coagulation factors in the literature (Van Hylckama Vlieg 2003) suggests the existence of quantitative trait loci, which influence plasma concentrations of several quantitative traits (i.e.have a pleiotropic effect) outside the genes coding for these factors. The aim of the present study was to identify clusters of pro- and anticoagulant factors within a large protein C deficient kindred using principal components analysis. In addition, we wanted to determine how much of the variance within these clusters could be attributed to the genetic variation within a single large pedigree. Levels of the following analytes were measured in family members: prothrombin, factor V, VII, VIII, IX, X, fibrinogen, von Willebrand factor, antithrombin, protein C and protein S. Subjects with the 3363C protein C mutation, a personal history of thrombosis or those using oral anticoagulants, and women pregnant at the time of the blood draw were excluded from the analyses. To identify clusters of haemostatic factors, the principal component method with orthogonal varimax rotation was performed using SPSS. We used a factor loading of >0.40 as a marginal value to include coagulation factors in a cluster. Heritability, the proportion of the phenotypic variance attributed to polygenes, and common household effect, the proportion of the variance attributed to environmental factors shared within a household, were estimated for each principal component score with an eigenvalue (the variance attributable to a particular principal component) greater than or equal to 1 using the variance component method in SOLAR (Almasy & Blangero 1998). The distribution of each score was assumed to be multivariate normal with a variance-covariance matrix following the formula: covariance (one person to another person)=h2K + c2H + e2I, with K derived from the kinship matrix, H from the household matrix and I from the identity matrix. The additive genetic and household components of variance were estimated using maximum likelihood analysis. A total of 87 family members met the inclusion criteria. The principal components analysis identified 3 components which explained 60% of the variance: component 1 included all vitamin K dependent factors (prothrombin, factor VII, factor IX and factor X, protein C and protein S), component 2 consisted of factor V, factor IX, fibrinogen and antithrombin, which all can interact directly with thrombin, and component 3 consisted of factor VIII and its carrier protein von Willebrand factor. The heritability estimates for these 3 components were, respectively, 96% (p=0.002), 87% (p<0.001) and 12% (p=0.33). These findings appear to provide evidence for the existence of genes that regulate the levels of distinct groups of proteins in the coagulation system, thus leading to clustering of levels suggestive of a pleiotropic effect.

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