scholarly journals Kallikrein augments the anticoagulant function of the protein C system in thrombin generation

2021 ◽  
Author(s):  
Jun Wan ◽  
Nadira Vadaq ◽  
Joke Konings ◽  
Martin Jaeger ◽  
Vinod Kumar ◽  
...  
Keyword(s):  
Protein C ◽  
Thrombin Generation ◽  
Anticoagulant Function

scholarly journals Dissociation of Activated Protein C Functions by Elimination of Protein S Cofactor Enhancement

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 21-21
Author(s):  
Roger JS Preston ◽  
Shona Harmon ◽  
Fionnuala B Ni Ainle ◽  
Jennifer A Johnson ◽  
Moya Cunningham ◽  
...  
Keyword(s):  
Protein C ◽  
Thrombin Generation ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Protein S ◽  
Single Amino Acid ◽  
Wild Type ◽  
Maximal Inhibition ◽  
Gla Domain ◽  
Anticoagulant Function

Abstract Activated protein C (APC) plays a critical anticoagulant role by inactivating factor Va (FVa) and factor VIIIa (FVIIIa) and thus down-regulating thrombin generation. In addition, APC bound to the endothelial cell protein C receptor (EPCR) can initiate PAR-1 mediated cytoprotective signalling. Although protein S constitutes a critical cofactor for APC anticoagulant function, the molecular basis through which protein S interacts with APC is not fully understood. In this study, we employed a site-directed mutagenesis strategy to characterise the effects of four single amino acid substitutions (D35T, D36A, L38D and A39V) within a region of the APC Gla domain important for protein S cofactor enhancement. To maintain Gla domain structural integrity, each residue was substituted with the corresponding residue of the human prothrombin Gla domain. Protein C variants were expressed in HEK 293 cells and purified by ion-exchange chromatography. Upon activation, the amidolytic activity of each recombinant APC variant was identical to that of wild type APC. The anticoagulant function of recombinant wild type and variant APC was compared in a tissue factor-initiated thrombin generation assay using protein C-deficient plasma. Wild type APC diminished thrombin generation in a concentration-dependent manner as expected. Variants APC-D35T, APC-D36A and APC-A39V exhibited only mildly impaired (<2-fold) anticoagulant activity compared to wild type APC. The anticoagulant activity of APC-L38D, however, was severely impaired. APC-L38D was unable to achieve half-maximal inhibition of endogenous thrombin potential (ETP) at APC concentrations as high as 150nM, compared to wild type APC, which achieved half-maximal inhibition at 7.2nM APC. To clarify the role of Leu-38 in facilitating APC anticoagulant function, we further studied the ability of APC-L38D to be stimulated in protein S-deficient plasma reconstituted with plasma-purified protein S. Co-incubation of wild type APC with increasing protein S concentration (12.5–200nM) caused a corresponding reduction in ETP (IC50 = 24nM protein S). In contrast, APC-L38D was unresponsive to protein S. In the presence of APC-L38D, ETP was reduced only 22% at 1.5μM protein S (10-fold higher than plasma free protein S). In a phospholipid-dependent FVa proteolysis time course assay, both wild type APC and APC-L38D rapidly reduced FVa cofactor activity, indicating that the observed impaired plasma anticoagulant activity of APC-L38D is not mediated by impaired interaction with anionic phospholipids or FVa. In a modified version of this assay, wild type APC-mediated FVa proteolysis was rapidly enhanced by added protein S, with half-maximal inhibition observed at 5nM protein S. In contrast, APC-L38D exhibited no protein S-enhanced FVa proteolysis. Cumulatively, these data confirm that Leu-38 mediates APC anticoagulant function in plasma by facilitating critical protein S cofactor enhancement of FVa proteolysis. Previous studies have shown that APC Gla domain mutations can influence EPCR binding, a pre-requisite for PAR-1 mediated cytoprotective signalling. Consequently, we assessed APC binding to sEPCR using surface plasmon resonance. Binding affinities of APC-L38D and wild type APC were very similar (KD 112±25nM versus 117±36nM). Furthermore, the ability of APC-L38D to protect EAhy926 cells from staurosporine-induced apoptosis was also investigated using RT-PCR quantification of pro- (bax) and anti- (bcl-2) apoptotic gene expression. Pre-incubation with APC-L38D significantly reduced the bax/bcl-2 ratio to the same extent as wild type APC. The EPCR-dependence of these anti-apoptotic activities was confirmed using RCR-252, (an inhibitory anti-EPCR antibody) which ablated the cytoprotective effect of both APC species. In conclusion, we demonstrate that a single amino acid substitution (L38D) can significantly impair APC anticoagulant activity due to elimination of protein S cofactor enhancement. However, despite the location of Leu-38 in the Gla domain, APC-L38D retains its ability to bind EPCR, and trigger PAR-1 mediated cytoprotective signalling in a manner indistinguishable from that of wild type APC. Consequently, elimination of protein S cofactor enhancement of APC anticoagulant function represents a novel and effective strategy by which to dissociate the anticoagulant and cytoprotective functions of APC for potential therapeutic gain.

High Levels of Circulating Thrombomodulin in Human Foetuses and Children

1999 ◽  
Vol 81 (06) ◽  
pp. 906-909 ◽  
Author(s):  
Marie-Hélène Aurousseau ◽  
Danielle Gozin ◽  
Fernand Daffos ◽  
Armando D’Angelo ◽  
François Forestier ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cell ◽  
Protein C ◽  
Endothelial Cell Surface ◽  
A Value ◽  
Median Level ◽  
Group A ◽  
Anticoagulant Function ◽  
Cell Surface Proteoglycan ◽  
Human Foetuses

SummaryThrombomodulin (TM) is an endothelial cell surface proteoglycan with anticoagulant functions, also implicated in cell proliferation, cell-cell adhesion and differentiation. In this study we determined circulating plasma TM (pTM) levels in human foetuses at different stages of pregnancy, at birth and in childhood. TM levels increased with gestational age, the median level reaching a peak of approximately 165 ng/ml between the 23rd and 26th week, thereafter decreasing gradually, reaching a value of 108 ng/ml at birth. pTM continues to decrease progressively during childhood, reaching in the 5-15 years group a median of 56 ng/ml which approaches the adult value. The pTM peak was statistically significant and represents a specific foetal phenomenon as it was independent of the corresponding maternal values. As a whole, the pTM pattern during foetal maturation appears totally different from that of protein C, prothrombin and other coagulation activators and inhibitors and thus, TM may play in the foetus another role in addition to its well-known anticoagulant function.

Plasma Factor V Activation Is Prevented by Activated Protein C in the Presence of Phospholipid Vesicles, Not Platelets

1993 ◽  
Vol 69 (02) ◽  
pp. 124-129 ◽  
Author(s):  
Susan Solymoss ◽  
Kim Thi Phu Nguyen
Keyword(s):  
Western Blotting ◽  
Protein C ◽  
Thrombin Generation ◽  
Blood Platelets ◽  
Activated Protein C ◽  
Phospholipid Vesicles ◽  
Factor V ◽  
Two Stage ◽  
One Stage ◽  
Plasma Factor

SummaryActivated protein C (APC) is a vitamin K dependent anticoagulant which catalyzes the inactivation of factor Va and VIIIa, in a reaction modulated by phospholipid membrane surface, or blood platelets. APC prevents thrombin generation at a much lower concentration when added to recalcified plasma and phospholipid vesicles, than recalcified plasma and platelets. This observation was attributed to a platelet associated APC inhibitor. We have performed serial thrombin, factor V one stage and two stage assays and Western blotting of dilute recalcified plasma containing either phospholipid vesicles or platelets and APC. More thrombin was formed at a given APC concentration with platelets than phospholipid. One stage factor V values increased to higher levels with platelets and APC than phospholipid and APC. Two stage factor V values decreased substantially with platelets and 5 nM APC but remained unchanged with phospholipid and 5 nM APC. Western blotting of plasma factor V confirmed factor V activation in the presence of platelets and APC, but lack of factor V activation with phospholipid and APC. Inclusion of platelets or platelet membrane with phospholipid enhanced rather than inhibited APC catalyzed plasma factor V inactivation. Platelet activation further enhanced factor V activation and inactivation at any given APC concentration.Plasma thrombin generation in the presence of platelets and APC is related to ongoing factor V activation. No inhibition of APC inactivation of FVa occurs in the presence of platelets.

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.

scholarly journals Intensity of thrombin formation and myocardial contractility in patients with ischemic heart disease after coronary stenting

2017 ◽  
Vol 16 (2) ◽  
pp. 63-69
Author(s):  
G. A. Berezovskaya ◽  
E. S. Klokova
Keyword(s):  
Myocardial Contractility ◽  
Protein C ◽  
Stress Echocardiography ◽  
Thrombin Generation ◽  
Wall Motion ◽  
Primary Pci ◽  
Before And After ◽  
Wall Motion Abnormalities ◽  
Artery Disease ◽  
Thrombin Formation

Objective. To study the relationship between the intensity of thrombin formation, estimated by thrombin generation test (TGT) in platelet poor plasma, and myocardial contractility in patients with coronary artery disease (CAD) before and after percutaneous coronary intervention (PCI). Methods. The study included 75 patients with coronary artery disease aged between 40 to 75 years, who underwent primary PCI (10 patients) or elective (65 patients) procedure, as well as 35 individuals matched for age and sex with no clinical signs of CAD. We investigated the venous blood obtained before and after 6 and 12 months following PCI. In the same period, stress echocardiography was performed. The intensity of thrombin formation was assessed using a TGT, formed in platelet poor plasma and the modified reaction mixture by adding human recombinant thrombomodulin (rh-TM) to assess the degree of activation of the protein C system. Results. The association between stress echocardiography parameters characterizing myocardial contractile capacity (ejection fraction (EF) of the left ventricle and a wall motion abnormalities (WMAs)) and TGT parameters, reflecting the intensity (ETP and the Peak) of the thrombin formation rate (V), was identified to be more expressed in patients undergoing primary PCI. The presence of the reverse correlation between EF and WMAs and the percentage reduction of V, ETP and Peak after the addition of rh-TM, as well as a significant association of the EF and WMAs with TGT indicators staged with rh-TM demonstrates the role of protein C system in the changes of myocardial contractility. The intensity of thrombin generation was also associated with hypertension. Conclusion. It was determined that TGT parameters were strongly associated with stress echocardiography parameters. The changes in thrombin generation rate were most closely associated with left ventricular ejection fraction, index of wall motion abnormalities and arterial hypertension, including hypertensive reaction to physical activity.

scholarly journals Protein C deficiency resulting from possible double heterozygosity and its response to danazol

Blood ◽  
1988 ◽  
Vol 71 (2) ◽  
pp. 370-374
Author(s):  
RA Gruppo ◽  
P Leimer ◽  
RB Francis ◽  
RA Marlar ◽  
E Silberstein
Keyword(s):  
Functional Activity ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Anticoagulation Therapy ◽  
Renal Vein Thrombosis ◽  
Type I ◽  
Amidolytic Activity ◽  
Protein C Deficiency ◽  
Protein Levels ◽  
Anticoagulant Function

A unique family with protein C (PC) deficiency is described. The proband had a history of renal vein thrombosis as a newborn and iliofemoral thrombosis at the age of 6 years. After 6 months of heparin treatment, discontinuation of anticoagulation therapy was accompanied by persistent hypofibrinogenemia with increased fibrinogen consumption. With continuous infusion of heparin, fibrinogen turnover normalized, and the child has remained free of thrombosis. Both the immunologic level of PC and the functional activity measured by amidolytic assay were moderately reduced (47% and 34%, respectively). Functional activity of PC measured by its anticoagulant activity was disproportionately lower (14%). A 3-year-old asymptomatic sibling had a similar disproportionate reduction of PC anticoagulant activity compared with the amidolytic activity or immunologic level. The mother demonstrated type I PC deficiency with a proportionate reduction in immunologic protein levels (59%), anticoagulant activity (52%), and amidolytic activity (46%), whereas the father had type II PC deficiency with normal immunologic protein levels (102%), normal amidolytic function (98%), but a low anticoagulant function (50%). An abnormal PC molecule was detected by two-dimensional immunoelectrophoresis in the father and two children. These data are consistent with the hypothesis that the children are doubly heterozygous for two different types of PC deficiency inherited from each of the parents. A 14-day trial of danazol in the proband resulted in a rise in the PC antigen concentration from 66% to 98% but no change in PC anticoagulant function.

scholarly journals Increased prothrombin activation in protein S-deficient plasma under flow conditions on endothelial cell matrix: an independent anticoagulant function of protein S in plasma

Blood ◽  
1995 ◽  
Vol 85 (7) ◽  
pp. 1815-1821 ◽  
Author(s):  
C van't Veer ◽  
TM Hackeng ◽  
D Biesbroeck ◽  
JJ Sixma ◽  
BN Bouma
Keyword(s):  
Endothelial Cell ◽  
Tissue Factor ◽  
Protein C ◽  
Activated Protein C ◽  
Coagulation Factor ◽  
Protein S ◽  
Flow Conditions ◽  
Cell Matrix ◽  
Anticoagulant Function ◽  
Prothrombin Activation

Protein S is a vitamin K-dependent nonenzymatic coagulation factor involved in the regulation of activated protein C (aPC). In this study, we report an aPC-independent anticoagulant function of protein S in plasma under flow conditions. Plasma, anticoagulated with low-molecular-weight heparin allowing tissue factor-dependent prothrombin activation, was perfused at a wall shear rate of 100 s-1 over tissue factor containing matrices of stimulated endothelial cells placed in a perfusion chamber. Fractions were collected in time at the outlet and prothrombin activation was determined by measuring the activation fragment F1+2 of prothrombin. In normal plasma, a time-dependent prothrombin activation was detected by the generation of fragment1+2. Prothrombin activation had ceased after 12 minutes perfusion, independent of the amount of tissue factor present in the matrix. Depletion of protein S from plasma or inhibition of protein S in plasma by monoclonal antibodies induced a 5- to 25-fold increase of prothrombin activation on the procoagulant endothelial cell matrix. A prolonged prothrombin activation was detected in protein S-depleted plasma up to 20 minutes after onset of the thrombin generation. The increased prothrombin activation in protein S-depleted plasma could not be explained by the absence of the cofactor function of protein S for aPC because depletion of protein C from plasma did not result in increased prothrombin activation. These data provide further evidence for a strong anticoagulant function of protein S in plasma independent from activated protein C.

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