Activated protein C generation is greatly decreased in plasma from newborns compared to adults in the presence or absence of endothelium

2004 ◽  
Vol 91 (02) ◽  
pp. 238-247 ◽  
Author(s):  
Sanjay Patel ◽  
Christoph Male ◽  
Leslie Berry ◽  
Lesley Mitchell ◽  
Anthony Chan
Keyword(s):  
Cell Surface ◽  
Tissue Factor ◽  
Umbilical Cord ◽  
Protein C ◽  
Thrombin Generation ◽  
Activated Protein C ◽  
Age Related ◽  
Adult Plasma ◽  
The Difference ◽  
Inhibition Of Thrombin

SummaryActivated protein C (APC) generation strongly affects sepsis and thrombosis by inhibition of thrombin generation. However, it is unclear if there are age-related differences in effectiveness of protein C (PC). We studied age effects on plasma APC generation ± endothelium. Defibrinated (Ancrod) plasma (from adults or newborns (umbilical cord)) was recalcified with buffer containing tissue factor ± thrombomodulin (TM) on either plastic or endothelium (HUVEC) at 37oC. Timed subsamples of reaction mixture were taken into either heparin-EDTA or FFRCMK-EDTA solutions and analyzed for APC-PC inhibitor (APC-PCI) or APC-α1antitrypsin (APC-α1AT) by ELISAs. Since heparin converts free APC to APC-PCI, the difference in APCPCI measured in heparin-EDTA and FFRCMK-EDTA samples was equal to free active APC. APC-α2macroglobulin (APC-α2M) was measured as remaining chromogenic activity in heparin-EDTA. Free APC, APC-PCI and APC-α1AT were decreased in newborn compared to adult plasma on plastic. However, APC-α2M made up a larger fraction of inhibitor complexes in newborn plasma. On endothelium, significantly more APC, APC-PCI and APC-α1AT were generated in either plasma compared to that on plastic with excess added TM. APC, APC-PCI and APC-α1AT were also reduced and total APC-α2M increased in newborn plasma on HUVEC. Addition of PC to newborn plasma gave APC generation similar to adult plasma. Thus, free APC, APC-PCI and APC-α1AT generation is reduced in newborn compared to adult plasma with or without endothelium, likely due to reduced plasma PC levels. Endothelium enhances APC generation, regardless of plasma type, possibly because of cell surface factors such as TM, phospholipid and endothelial PC receptor.

Additive effects of anticoagulants: recombinant human activated protein C and heparin or melagatran, in tissue factor-activated umbilical-cord plasma

2005 ◽  
Vol 94 (07) ◽  
pp. 69-74 ◽  
Author(s):  
Siegfried Gallistl ◽  
Wolfgang Muntean ◽  
Bettina Leschnik ◽  
Peter Fritsch ◽  
Gerhard Cvirn ◽  
...  
Keyword(s):  
Severe Sepsis ◽  
Tissue Factor ◽  
Umbilical Cord ◽  
Protein C ◽  
Thrombin Generation ◽  
Activated Protein C ◽  
Thrombin Inhibitor ◽  
Human Umbilical Cord ◽  
Cord Plasma

SummarySevere sepsis in children or adults may cause a life-threatening coagulopathy, with widespread consumption of activated protein C (APC); recombinant human APC (rhAPC) is a promising candidate anticoagulant treatment. We investigated the effects of rhAPC and other anticoagulants on coagulation triggered by adding small quantities of lipidated tissue factor to human umbilical-cord plasma in vitro. rhAPC, unfractionated heparin (UH),and melagatran (a direct thrombin inhibitor) were studied individually, and in combinations of rhAPC with either UH or melagatran. rhAPC alone dose-dependently prolonged the activated partial-thromboplastin time (aPTT) but not the prothrombin time (PT), and dose-dependently suppressed two indices of thrombin generation, namely prothrombin fragment F 1.2 (F 1.2) generation and thrombin–antithrombin (TAT) complex formation. UH alone dose-dependently prolonged the aPTT but not the PT, while melagatran alone dose-dependently prolonged both the aPTT and the PT. Adding either UH or melagatran dose-dependently augmented the capacity of rhAPC to suppress F 1.2 generation (with addition of UH showing a greater effect) and TAT formation (with addition of melagatran showing a greater effect). Both the capacity of UH to prolong the aPTT and the capacity of melagatran to prolong the aPTT and the PT were augmented by adding rhAPC. In our in-vitro study, adding either UH or melagatran augmented the capacity of rhAPC to suppress thrombin generation in human umbilical-cord plasma, with the anticoagulant effect of melagatran being more predictable than that of UH. Hence, combining rhAPC with melagatran might be a valuable therapeutic option in patients with severe sepsis.

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 Protein C Inhibitor Acts as a Procoagulant by Inhibiting the Thrombomodulin-Induced Activation of Protein C in Human Plasma

Blood ◽  
1998 ◽  
Vol 91 (5) ◽  
pp. 1542-1547 ◽  
Author(s):  
Marc G.L.M. Elisen ◽  
Peter A.Kr. von dem Borne ◽  
Bonno N. Bouma ◽  
Joost C.M. Meijers
Keyword(s):  
Tissue Factor ◽  
Protein C ◽  
Activated Protein C ◽  
Anticoagulant Effect ◽  
Clotting Time ◽  
Thrombin Inhibition ◽  
Normal Plasma ◽  
Protein C Inhibitor ◽  
Inhibition Of Thrombin

AbstractProtein C inhibitor (PCI), which was originally identified as an inhibitor of activated protein C, also efficiently inhibits coagulation factors such as factor Xa and thrombin. Recently it was found, using purified proteins, that the anticoagulant thrombin-thrombomodulin complex was also inhibited by PCI. The paradoxical inhibitory effect of PCI on both coagulant and anticoagulant proteases raised questions about the role of PCI in plasma. We studied the role of thrombomodulin (TM)-dependent inhibition of thrombin by PCI in a plasma system. Clotting was induced by addition of tissue factor to recalcified plasma in the absence or presence of TM, and clot formation was monitored using turbidimetry. In the absence of TM, PCI-deficient plasma showed a slightly shorter coagulation time compared with normal plasma. Reconstitution with a physiologic amount of PCI gave normal clotting times. Addition of PCI to normal plasma and protein C–deficient plasma resulted in a minor prolongation of the clotting time. This suggested that PCI can act as a weak coagulation inhibitor in the absence of TM. TM caused a strong anticoagulant effect in normal plasma due to thrombin scavenging and activation of the protein C anticoagulant pathway. This effect was less pronounced when protein C–deficient plasma was used, but could be restored by reconstitution with protein C. When PCI was added to protein C–deficient plasma in the presence of TM, a strong anticoagulant effect of PCI was observed. This anticoagulant effect was most likely caused by the TM-dependent thrombin inhibition by PCI. However, when PCI was added to normal plasma containing TM, a strong procoagulant effect of PCI was observed, due to the inhibition of protein C activation. PCI-deficient plasma was less coagulant in the presence of TM. A concentration-dependent increase in clotting time was observed when PCI-deficient plasma was reconstituted with PCI. The combination of these results suggest that the major function of PCI in plasma during coagulation is the inhibition of thrombin. A decreased generation of activated protein C is a procoagulant consequence of the TM-dependent thrombin inhibition by PCI. We conclude that TM alters PCI from an anticoagulant into a procoagulant during tissue factor-induced coagulation.

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.

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.

Recombinant human activated protein C, heparin and melagatran in umbilical cord versus adult plasma

2005 ◽  
Vol 94 (7) ◽  
pp. 884-889 ◽  
Author(s):  
Martin Koestenberger ◽  
Gerhard Cvirn ◽  
Siegfried Gallistl ◽  
Joerg Kutschera ◽  
Wolfgang Muntean
Keyword(s):  
Umbilical Cord ◽  
Protein C ◽  
Activated Protein C ◽  
Adult Plasma

A New Assay Based on Thrombin Generation Inhibition to Detect Both Protein C and Protein S Deficiencies in Plasma

1994 ◽  
Vol 71 (03) ◽  
pp. 331-338 ◽  
Author(s):  
Jérôme Duchemin ◽  
Jean-Louis Pittet ◽  
Michel Tartary ◽  
Suzette Béguin ◽  
Pascale Gaussem ◽  
...  
Keyword(s):  
Oral Contraceptives ◽  
Oral Anticoagulant ◽  
Protein C ◽  
Thrombin Generation ◽  
Normal Values ◽  
Oral Anticoagulant Therapy ◽  
Activated Protein C ◽  
Protein S ◽  
Heparin Therapy ◽  
Inhibition Of Thrombin

SummaryActivated protein C reduces thrombin generation by inactivating factors V and VIII in the presence of protein S. This prompted us to develop an assay which would allow specific exploration of this reaction. The total amount of thrombin formed in plasma after activation by tissue factor and phospholipids was reduced by adding thrombomodulin. This addition allowed protein C to be activated by endogenous thrombin. The inhibition of thrombin generation (ITG) due to protein C activation could be measured by comparing thrombin formation in the presence and in the absence of thrombomodulin. ITG increased with both protein C and protein S concentrations. Normal values of ITG expressed as a percentage were between 40 and 65% and were not influenced by age or sex. ITG increased in patients under heparin therapy, decreased in patients under oral anticoagulant therapy and was decreased in women using oral contraceptives. This method could be used for screening patients for protein C and protein S deficiencies.

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.

scholarly journals Protein C Inhibitor Acts as a Procoagulant by Inhibiting the Thrombomodulin-Induced Activation of Protein C in Human Plasma

Blood ◽  
1998 ◽  
Vol 91 (5) ◽  
pp. 1542-1547 ◽  
Author(s):  
Marc G.L.M. Elisen ◽  
Peter A.Kr. von dem Borne ◽  
Bonno N. Bouma ◽  
Joost C.M. Meijers
Keyword(s):  
Tissue Factor ◽  
Protein C ◽  
Activated Protein C ◽  
Anticoagulant Effect ◽  
Clotting Time ◽  
Thrombin Inhibition ◽  
Normal Plasma ◽  
Protein C Inhibitor ◽  
Inhibition Of Thrombin

Protein C inhibitor (PCI), which was originally identified as an inhibitor of activated protein C, also efficiently inhibits coagulation factors such as factor Xa and thrombin. Recently it was found, using purified proteins, that the anticoagulant thrombin-thrombomodulin complex was also inhibited by PCI. The paradoxical inhibitory effect of PCI on both coagulant and anticoagulant proteases raised questions about the role of PCI in plasma. We studied the role of thrombomodulin (TM)-dependent inhibition of thrombin by PCI in a plasma system. Clotting was induced by addition of tissue factor to recalcified plasma in the absence or presence of TM, and clot formation was monitored using turbidimetry. In the absence of TM, PCI-deficient plasma showed a slightly shorter coagulation time compared with normal plasma. Reconstitution with a physiologic amount of PCI gave normal clotting times. Addition of PCI to normal plasma and protein C–deficient plasma resulted in a minor prolongation of the clotting time. This suggested that PCI can act as a weak coagulation inhibitor in the absence of TM. TM caused a strong anticoagulant effect in normal plasma due to thrombin scavenging and activation of the protein C anticoagulant pathway. This effect was less pronounced when protein C–deficient plasma was used, but could be restored by reconstitution with protein C. When PCI was added to protein C–deficient plasma in the presence of TM, a strong anticoagulant effect of PCI was observed. This anticoagulant effect was most likely caused by the TM-dependent thrombin inhibition by PCI. However, when PCI was added to normal plasma containing TM, a strong procoagulant effect of PCI was observed, due to the inhibition of protein C activation. PCI-deficient plasma was less coagulant in the presence of TM. A concentration-dependent increase in clotting time was observed when PCI-deficient plasma was reconstituted with PCI. The combination of these results suggest that the major function of PCI in plasma during coagulation is the inhibition of thrombin. A decreased generation of activated protein C is a procoagulant consequence of the TM-dependent thrombin inhibition by PCI. We conclude that TM alters PCI from an anticoagulant into a procoagulant during tissue factor-induced coagulation.

Age-Related Differences in the Glycosylation of Anticoagulant Protein C.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2031-2031
Author(s):  
Leslie R. Berry ◽  
Anthony K. C. Chan
Keyword(s):  
Alkaline Phosphatase ◽  
Protein C ◽  
Thrombin Generation ◽  
Pediatric Population ◽  
Plasma Concentrations ◽  
Coagulation Factor ◽  
Ethical Guidelines ◽  
Factor Va ◽  
Age Related ◽  
Adult Plasma

Abstract Thromboembolic events can result from procoagulant stimuli and the inability of native anticoagulant mechanisms to pacify thrombin generation. In the pediatric population, although thrombosis occurs in the presence of major coagulant insults, children are relatively resistant to onset of thrombotic complications. A key aspect contributing to developmental differences in the young is that coagulation factors and inhibitors have been shown to exist in different plasma concentrations and developmental forms compared to adults. Increasing evidence indicates that protein C (PC) is a major factor that, upon activation, controls coagulation by shutting down thrombin generation. In adults, PC exists as either 4 N-linked glycan (alpha-PC) or 3 N-linked glycan (beta- PC) proteins that differ in the rate at which their corresponding activated forms react with coagulation factor Va. Our previous work has shown that although plasma PC concentrations are vastly reduced in newborn plasma, inactivation of factor Va is similar to adults, suggesting a potential difference in PC molecules during development. To assess the potential for age-related PC molecule variation, we performed carbohydrate analyses to determine the relative glycoform composition of PC in newborns compared to adults. Venous plasma from either adults or umbilical cords of healthy full-term babies was collected following the ethical guidelines of our institution. Samples of plasma were run on non-reducing SDS polyacrylamide gel (7.5%) electrophoresis, followed by transfer onto PVDF blotting membranes. Blots were probed with polyclonal sheep anti-human PC, followed by anti-sheep IgG-alkaline phosphatase conjugate and then alkaline phosphatase substrate to visualize the protein bands. Relative intensity of alpha-PC and beta-PC bands was determined by laser densitometry. Analyses revealed that significant age differences existed in PC glycoform composition between adults and newborns. While the mass ratio of alpha-PC to the lower molecular weight band of beta PC was 2.3 to 1 for the adult plasma protein, newborns were observed to have an alpha-PC to beta-PC ratio of 8.8 to 1. Thus, an almost 4-fold increase in the circulating alpha-PC to beta-PC ratio was observed in newborns compared to adults. As a confirmation that the difference in migration between the alpha-PC and beta-PC bands was due to glycosylation, removal of N-glycans by treatment of newborn and adult plasma with N-glycosidase F prior to electrophoresis resulted in single bands that now migrated at the same rate for both age groups. We have demonstrated that relative expression of the 2 major PC glycoforms varies widely during development. Given that alpha-PC in its activated form has a much faster rate for factor Va inactivation compared to beta-PC, its increased appearance in newborn plasma may play a role in protecting neonates from thrombosis, despite reduced plasma inhibitor concentrations.

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