scholarly journals New functional assays to selectively quantify the activated protein C- and tissue factor pathway inhibitor-cofactor activities of protein S in plasma

2017 ◽  
Vol 15 (5) ◽  
pp. 950-960 ◽  
Author(s):  
N. A. Alshaikh ◽  
J. Rosing ◽  
M. C. L. G. D. Thomassen ◽  
E. Castoldi ◽  
P. Simioni ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Protein C ◽  
Tissue Factor Pathway Inhibitor ◽  
Activated Protein C ◽  
Protein S ◽  
Functional Assays ◽  
Tissue Factor Pathway

Role of protein S and tissue factor pathway inhibitor in the development of activated protein C resistance early in pregnancy in women with a history of preeclampsia

2011 ◽  
Vol 106 (11) ◽  
pp. 914-921 ◽  
Author(s):  
M. Christella Thomassen ◽  
Serban-Dan Costa ◽  
Louis Peeters ◽  
Jan Rosing ◽  
Svetlana Tchaikovski
Keyword(s):  
Tissue Factor ◽  
Protein C ◽  
Thrombin Generation ◽  
Tissue Factor Pathway Inhibitor ◽  
Activated Protein C ◽  
Protein S ◽  
Apc Resistance ◽  
Thrombosis Risk ◽  
History Of ◽  
Tissue Factor Pathway

SummaryPregnancy increases the risk of venous thromboembolism. Particularly in early pregnancy, the thrombosis risk can be attributed to the changes in coagulation. Elevated thrombin generation and resistance to activated protein C (APC) are likely to contribute to the increased thrombosis risk during pregnancy. We studied changes and the determinants of thrombin generation and APC resistance in the first 16 weeks of gestation in women with history of preeclampsia. Additionally, we investigated the influence of pregnancy-induced haemodilution on the coagulation system. We measured thrombin generation, APC resistance and plasma levels of prothrombin, factor V, factor X, protein S and tissue factor pathway inhibitor (TFPI) in 30 non-pregnant and 21 pregnant women at 8, 12 and 16 weeks of gestation. All participants shared a history of a hypertensive complication in the preceding pregnancy. Thrombin generation and APC resistance were higher at eight weeks of pregnancy than in the non-pregnant state, and progressively increased between eight and 16 weeks of gestation. Changes in the TFPI and protein S levels accounted for ~70% of pregnancy-induced APC resistance. Interestingly, a significant correlation (slope 2.23; 95%CI: 1.56 to 2.91; r= 0.58) was observed between protein Stotal or protein Sfree levels and haematocrit. In conclusion, pregnancy induces a decrease of TFPIfree and protein Sfree levels that attenuates the function of the TFPI and protein C systems and results in elevated thrombin generation and increased APC resistance. Besides, our data suggest that pregnancy-dependent haemodilution may contribute to the decreased peripheral protein S levels.

scholarly journals Laminin G1 residues of protein S mediate its TFPI cofactor function and are competitively regulated by C4BP

2021 ◽  
Author(s):  
Adrienn Teraz-Orosz ◽  
Magdalena Gierula ◽  
Anastasis Petri ◽  
David A. Jones ◽  
Renos Keniyopoullos ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein C ◽  
Tissue Factor Pathway Inhibitor ◽  
Activated Protein C ◽  
Protein S ◽  
High Affinity ◽  
Attachment Sites ◽  
Tissue Factor Pathway ◽  
Β Chain ◽  
Variant Protein

Protein S is a cofactor in the tissue factor pathway inhibitor (TFPI) anticoagulant pathway. It enhances TFPIα-mediated inhibition of factor (F)Xa activity and generation. The enhancement is dependent on a TFPIα-protein S interaction, involving TFPIα Kunitz 3 and protein S laminin G-type (LG)-1. C4b binding protein (C4BP), which binds to protein S LG1, almost completely abolishes its TFPI cofactor function. However, neither the amino acids involved in TFPIα enhancement, nor the mechanisms underlying the reduced TFPI cofactor function of C4BP-bound protein S, are known. To screen for functionally important regions within protein S LG1 we generated seven variants with inserted N-linked glycosylation attachment sites. Protein S D253T and Q427N/K429T, displayed severely reduced TFPI cofactor function while showing normal activated protein C (APC) cofactor function and C4BP binding. Based on these results, we designed four protein S variants in which 4-6 surface exposed charged residues were substituted for alanine. One variant, protein S K255A/E257A/D287A/R410A/K423A/E424A, exhibited either abolished or severely reduced TFPI cofactor function in plasma and FXa inhibition assays, both in the presence or absence of FV-short, but retained normal APC cofactor function and high affinity C4BP-binding. The C4BP β-chain was expressed to determine the mechanisms behind the reduced TFPI cofactor function of C4BP-bound protein S. Like C4BP-bound protein S, C4BP β-chain-bound protein S had severely reduced TFPI cofactor function. These results show that protein S Lys255, Glu257, Asp287, Arg410, Lys423 and Glu424 are critical for protein S-mediated enhancement of TFPIα and that binding of the C4BP β-chain blocks this function.

The anticoagulant action of recombinant human activated protein C (rhAPC, Drotrecogin α activated): comparison between cord and adult plasma

2004 ◽  
Vol 91 (05) ◽  
pp. 912-918 ◽  
Author(s):  
Siegfried Gallistl ◽  
Martin Koestenberger ◽  
Katrin Baier ◽  
Peter Fritsch ◽  
Joachim Greilberger ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Protein C ◽  
Tissue Factor Pathway Inhibitor ◽  
Activated Protein C ◽  
Clotting Time ◽  
Promising Candidate ◽  
Anticoagulant Action ◽  
Adult Plasma ◽  
Tissue Factor Pathway

SummaryThe present study was performed to compare the anticoagulant efficiency of recombinant human activated protein C (rhAPC) in cord with that in adult plasma. RhAPC is a promising candidate to improve the outcome of severe sepsis. However, different anticoagulant efficiency of rhAPC in cord compared with adult plasma has to be expected due to physiological low plasma levels of tissue factor pathway inhibitor (TFPI) and antithrombin (AT) present in neonates, two inhibitors known to markedly influence the anticoagulant action of APC. Clot formation was induced in our experiments by addition of high (30 µM) or low (20 pM) amounts of lipidated tissue factor (TF). High amounts of TF are conventionally applied in standard clotting assays, whereas plasma activation with low amounts of TF probably better matches the conditions in vivo. We demonstrate that under low coagulant challenge increasing amounts of rhAPC (0.1 – 0.5 µg/ml final plasma concentration) dose-dependently prolonged clotting time and suppressed thrombin potential and prothrombin fragment 1 + 2 generation in both cord and adult plasma. The same was true for experiments performed under high coagulant challenge when 4 – 16 µg/ml of rhAPC were added. Whereby, cord plasma was significantly more susceptible to addition of rhAPC in the presence of high amounts of TF and adult plasma was significantly more susceptible to addition of rhAPC in the presence of low amounts of TF. We demonstrate that increased anticoagulant efficiency of rhAPC in adult plasma under low coagulant challenge is attributable to the physiological high levels of TFPI and AT present in adults.

Synergistic Anticoagulant Effect of Activated Protein C and Tissue Factor Pathway Inhibitor As a Mechanism for Acute Traumatic Coagulopathy

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1487-1487
Author(s):  
Michael A Meledeo ◽  
Melanie V Valenciana ◽  
Armando C Rodriguez ◽  
Andrew P Cap
Keyword(s):  
Synergistic Effect ◽  
Tissue Factor ◽  
Protein C ◽  
Thrombin Generation ◽  
Tissue Factor Pathway Inhibitor ◽  
Activated Protein C ◽  
Lag Time ◽  
Acute Traumatic Coagulopathy ◽  
Tissue Factor Pathway ◽  
Traumatic Coagulopathy

Abstract Introduction Severe trauma with tissue damage and shock can rapidly (<30 min) result in abnormal coagulation function which is independent of consumption or dilution effects; this acute traumatic coagulopathy (ATC) has been linked to increased transfusion requirements, morbidity and mortality. It has been suggested that ATC is caused by an increase in activated protein C (aPC; from 40 pM in normal to 175 pM) which has been identified in trauma patients [Cohen MJ, Ann Surg 255:379 (2012)]. aPC inactivates factor Va (FVa) as part of normal hemostasis, but our previous results indicate that aPC at trauma levels is not sufficient to prevent coagulation, particularly when platelets are present, as platelet fVa is resistant to aPC [Campbell JE, PLoS ONE 9:e99181 (2014); Camire RM, Blood 91:2818 (1998)]. Tissue Factor Pathway Inhibitor (TFPI) is an endothelial-bound protein which inhibits coagulation through effects on factor Xa and the factor VIIa-tissue factor complex. It is released in the plasma (normal level 2.5nM, pathophysiologic states including trauma up to 10nM) and is typically cleared by the liver or kidneys. TFPIα accounts for 20% of circulating TFPI and binds FV and FVa [Ndonwi M, J Thromb Haemost 10:1944 (2012)]. Recently, a truncated FV (termed FV-short) produced by an autosomal dominant mutation was found to form complexes with TFPIα, resulting in retention of TFPIα in a 10-fold increase over normal levels in affected individuals [Vincent LM, J Clin Invest 123:3777 (2013)]. FV-short has significantly reduced thrombogenic potential and, by concomitantly raising TFPIα, causes a bleeding disorder. We hypothesize that the activation of PC in acute trauma may result in the production of FV-degradation products which could stabilize TFPIα similarly to the effect of FV-short. The combination of reduced FVa and increased TFPIα may contribute to ATC. Methods Whole blood from healthy volunteers was drawn by phlebotomy into ACD-containing tubes. Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were collected by centrifugation (200g for 10 min and 1000g for 15 min, respectively). Calibrated automated thrombogram (CAT) assays and thromboelastography (TEG) were conducted using PRP and PPP with exogenously delivered aPC (0, 100pM, or 1nM) and TFPI (0, 2.5nM, or 10nM). An immunodepleted FV-deficient (<1% normal) plasma (FVdp) was used as a reference. Results CAT assays verified that aPC and TFPI each delay and suppress thrombin generation in PPP in a dose-dependent manner, but the combination of aPC and TFPI had a synergistic effect at the highest doses tested. The endogenous thrombin potential (ETP) was eliminated in PPP (control: 1663 nM-min; 1nM aPC + 10nM TFPI: 0 nM-min; P<0.001); lag time was similarly affected (control: 2.5 min; 1nM aPC + 10nM TFPI: >60 min; P<0.001). FVdp also featured a delayed lag time (11.17 min, P<0.001 versus control), but the delay induced by the addition of 10nM TFPI was not nearly as severe in FVdp (19.67 min) as in the PPP + 10nM aPC sample (>60 min, P<0.001). For PRP, there was no statistical difference between control and the highest doses of aPC and TFPI in ETP (1636 nM-min versus 1388 nM-min) or lag time (8.72 min versus 9.13 min). Conclusions In vitro studies suggest that PC activation is not the sole cause of ATC; the concentrations of aPC measured in trauma patient blood have little effect on the coagulation potential of plasma with or without platelets when aPC is delivered exogenously. The results here demonstrate that there is a synergistic effect between aPC and TFPI. When TFPI is added to FV deficient plasma, the thrombin generation is delayed and suppressed; however, in normal plasma digested with aPC (containing fragmented FVa), the addition of TFPI is sufficient to suppress the generation of thrombin beyond the window of measurement (>60 min). The addition of platelets to the milieu eliminated the effects of aPC, TFPI, and the combination on thrombin generation, highlighting the central role of platelets in maintaining hemostatic function. Disclosures No relevant conflicts of interest to declare.

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