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.

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.

Modulation of Protein C Inhibitor Activity by Histidine-Rich Glycoprotein and Platelet Factor 4: Role of Zinc and Calcium Ions in the Heparin-Neutralizing Ability of Histidine-Rich Glycoprotein

1992 ◽  
Vol 67 (01) ◽  
pp. 050-055 ◽  
Author(s):  
Yoshiaki Kazama ◽  
Takehiko Koide
Keyword(s):  
Calcium Ions ◽  
Protein C ◽  
Activated Protein C ◽  
Platelet Factor 4 ◽  
Dependent Manner ◽  
Platelet Factor ◽  
Thrombin Inhibition ◽  
Significant Difference ◽  
Protein C Inhibitor

SummaryEffects of zinc and calcium ions on the heparin-neutralizing abilities of histidine-rich glycoprotein (HRG) and platelet factor 4 (PF4) were examined. Both HRG and PF4 effectively neutralized the ability of heparin to accelerate the activated protein C (APC) and the thrombin inhibitions by protein C inhibitor (PCI). The heparin-neutralizing ability of HRG in the APC inhibition by PCI, however, was decreased in a Ca2+-dependent manner and apparently lost at 1 mM Ca2+, while it was enhanced by Zn2+ regardless of the presence or absence of Ca2+. The heparinneutralizing ability of HRG in the thrombin inhibition by PCI was not affected by Ca2+.In contrast to HRG, there was no significant difference in the heparin-neutralizing ability of PF4 in the presence or absence of 1 mM Ca2+. These results strongly suggest additional physiological functions of HRG and PF4 as modulators of PCI.

Interaction of Plasma Kallikrein with Protein C Inhibitor in Purified Mixtures and in Plasma

1991 ◽  
Vol 65 (01) ◽  
pp. 046-051 ◽  
Author(s):  
Francisco España ◽  
Amparo Estelles ◽  
John H Griffin ◽  
Justo Aznar
Keyword(s):  
Complex Formation ◽  
Protein C ◽  
Room Temperature ◽  
Activated Protein C ◽  
Physiological Role ◽  
Plasma Kallikrein ◽  
Amidolytic Activity ◽  
Normal Plasma ◽  
Protein C Inhibitor ◽  
Subsequent Addition

SummaryThe interaction between plasma kallikrein (KK) and protein C inhibitor (PCI) and the influence of KK on the complex formation between activated protein C (APC) and PCI was studied in purified systems as well as in plasma in order to assess the significance of these reactions in the plasma milieu. PCI complexed to KK (KK: PCI) or to APC (APC: PCI) was measured by sandwich ELISA’s using antibodies directed against each protein in the complexes. The formation of KK: PCI complexes assayed by this method paralleled the inhibition of KK amidolytic activity by PCI in purified system. Incubation of normal plasma (NHP) at 4 °C, which can induce prekallikrein activation due to cold activation, resulted in PCI inactivation and appearance of KK: PCI complexes. PCI activity fell to 35% of the NHp and 1.2 μ/ml of KK: PCI complex was formed. However, incubation of NHP at room temperature or of prekallikrein deficient plasma at 4 °C did not result in significant decrease of PCI activity. Thus the PCI inactivation was associated with prekallikrein activation and complexation to PCI following cold activation. Incubation of exogenous purified KK with NHP resulted in PCI inactivation and complexation with KK in a temperaturedependent manner. Addition of 2.8 μ/ml KK to plasma at 4 °C resulted in the inactivation of 55% of plasma PCI and the formation of 0.9 μ/ml KK: PCI which represents 21% of the KK added, whereas at 37 °C PCI was inactivated to 30% and only 0.30 μg/ml KK: PCI complexes were measured. These results indicate that PCI is a major KK inhibitor at 4 °C. At 37 °C, PCI accounted for aborfi 7% of the inhibition of the KK added. In separate experiments, following addition of 2.5 μg/ml APC to NHR more than 1 μg/ml of APC: PCI complex was formed in 3 h. When NHP was prior incubated with KK, PCI activity decreased to 10% of that of the normal plasma. Subsequent addition of APC to the plasma treated with KK resulted in formation of only 35 μg/ml of APC: PCI complex compared to 1,350 μg/ml when plasma was not previously incubated with KK. These results indicate that PCI could play a physiological role in the inhibition of plasma KK, and that, in turn, plasma KK can either complex to or inactivate plasma PCI. Thus, KK could modulate the PCI inhibition of APC in plasma.

Role of the A+ Helix in Heparin Binding to Protein C Inhibitor

1996 ◽  
Vol 75 (05) ◽  
pp. 760-766 ◽  
Author(s):  
Marc G L M Elisen ◽  
Machiel H H Maseland ◽  
Frank C Church ◽  
Bonno N Bouma ◽  
Joost C M Meijers
Keyword(s):  
Protein C ◽  
Electrostatic Interactions ◽  
Deletion Mutant ◽  
Activated Protein C ◽  
Structural Features ◽  
Heparin Binding ◽  
Wild Type ◽  
Protein C Inhibitor ◽  
Positively Charged

SummaryInteractions between proteins and heparin(-like) structures involve electrostatic forces and structural features. Based on charge distributions in the linear sequence of protein C inhibitor (PCI), two positively charged regions of PCI were proposed as possible candidates for this interaction. The first region, the A+ helix, is located at the N-terminus (residues 1-11), whereas the second region, the H helix, is positioned between residues 264 and 280 of PCI. Competition experiments with synthetic peptides based on the sequence of these regions demonstrated that the H helix has the highest affinity for heparin. In contrast to previous observations we found that the A+ helix peptide competed for the interaction of PCI with heparin, but its affinity was much lower than that of the H helix peptide.Recombinant PCI was also used to investigate the role of the A+ helix in heparin binding. Full-length (wild-type) rPCI as well as an A+ helix deletion mutant of PCI (rPCI-Δ2-l 1) were expressed in baby hamster kidney cells and both had normal inhibition activity with activated protein C and thrombin. The interaction of the recombinant PCIs with heparin was investigated and compared to plasma PCI. The A+ helix deletion mutant showed a decreased affinity for heparin in inhibition reactions with activated protein C and thrombin, but had similar association constants compared to wild-type rPCI. The synthetic A+ helix peptide competed with rPCI-Δ2-11 for binding to heparin. This indicated that the interaction between PCI and heparin is fairly non-specific and that the interaction is primarily based on electrostatic interactions.In summary, our data suggest that the H helix of PCI is the main heparin binding region of PCI, but the A+ helix increases the overall affinity for the PCI-heparin interaction by contributing a second positively charged region to the surface of PCI.

scholarly journals Normal titer of functional and immunoreactive protein-C inhibitor in plasma of patients with congenital combined deficiency of factor V and factor VIII

Blood ◽  
1983 ◽  
Vol 62 (6) ◽  
pp. 1266-1270 ◽  
Author(s):  
K Suzuki ◽  
J Nishioka ◽  
S Hashimoto ◽  
T Kamiya ◽  
H Saito
Keyword(s):  
Factor Viii ◽  
Protein C ◽  
Activated Protein C ◽  
Factor V ◽  
Amidolytic Activity ◽  
Normal Plasma ◽  
Coagulant Activity ◽  
Protein C Inhibitor ◽  
Combined Deficiency ◽  
Good Agreement

Protein-C inhibitor (PCI) is a newly described plasma inhibitor directed against a vitamin-K-dependent serine protease, activated protein-C, which is involved in the inactivation of factor V and factor VIII. Marlar and Griffin have reported that PCI activity is absent in the plasma of patients with congenital combined factor V/VIII deficiency. We have measured the levels of PCI in the plasma of seven unrelated patients with this disorder using both functional and immunologic methods. The rate at which the amidolytic activity of activated protein-C was neutralized in the patients' plasma was essentially identical to that observed in normal plasma. The titer of PCI antigen, as measured by an electroimmunoassay using a monospecific anti-PCI serum, was 5.3 +/- 1.6 micrograms/ml in the patients' plasma and was not significantly different from that of normal plasma (5.3 +/- 2.7 micrograms/ml, n = 30). The levels of factor-V-related antigen, factor V coagulant antigen, and factor VIII coagulant antigen were low in all patient plasma and were in good agreement with their respective coagulant activity. Our results do not appear to support the hypothesis that combined factor V/VIII defect is due to a lack of PCI.

Multicenter Evaluation of a Kit for Activated Protein C Resistance on Various Coagulation Instruments Using Plasmas from Healthy Individuals

1994 ◽  
Vol 72 (02) ◽  
pp. 255-60 ◽  
Author(s):  
S Rosén ◽  
K Johansson ◽  
K Lindberg ◽  
B Dahlbäck ◽  
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Factor V ◽  
Clotting Time ◽  
Apc Resistance ◽  
Normal Plasma ◽  
The Individual ◽  
Anticoagulant Response ◽  
Hemostatic Disorder

SummaryRecently a new hemostatic disorder has been described which appears to be an important risk factor for familial thromboembolism. The disorder is characterized by a poor anticoagulant response to activated Protein C (APC) and has been shown to be due to lack of an APC cofactor activity which is a property of factor V.A kit for determining the response of plasma samples towards addition of APC in an APTT-based assay - COATEST APC Resistance -has been evaluated on 35 coagulation instruments in a multicenter study involving 32 laboratories. A lyophilized normal plasma and identical plasma aliquots from 20 individuals, one of whom had a borderline resistance to APC, were analysed in each laboratory and the sensitivity of each plasma to APC was determined as the ratio between the clotting times obtained in the presence and absence of APC (APC ratio).The plasma from the individual with a borderline resistance to APC activity was correctly classified as the lowest responder in each laboratory, with an APC ratio in the range 1.6-2.4. In comparison, plasmas from individuals with a pronounced response to APC activity resulted in APC ratios above 3.4 in most cases. Interestingly, although the actual APT time for a plasma from a given individual showed a more than 10 s difference due to the type of instrumentation used, the variation in the APC ratio was limited. A similar discrimination was also obtained from evaluation of the actual prolongation of the clotting time in the presence of APC.The intra-laboratory coefficient of variation for the clotting times were on average 2.0% and 3.9% in the absence and presence of APC, respectively, indicating that the precision for the prolonged clotting times obtained in the presence of APC is sufficient to allow a safe assignment of the APC response. The APC ratio for the lyophilized normal plasma was 2.7 ± 0.2 (2 S.D.) illustrating a narrow distribution between instruments which shows the feasibility of including such plasma for assay validation. Altogether, the results indicate that all the coagulation instruments included in the study can be used for detection of individuals with resistance to APC activity through determination of the APC ratio or the prolongation time.

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.

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.

Role of Protein C Inhibitor and Tissue Factor in Fertilization

2007 ◽  
Vol 33 (1) ◽  
pp. 013-020 ◽  
Author(s):  
José Fernández ◽  
Mary Heeb
Keyword(s):  
Tissue Factor ◽  
Protein C ◽  
Protein C Inhibitor

scholarly journals Identification of monoclonal antibodies that inhibit the function of protein C inhibitor. Evidence for heparin-independent inhibition of activated protein C in plasma

Blood ◽  
1988 ◽  
Vol 72 (4) ◽  
pp. 1401-1403 ◽  
Author(s):  
JC Meijers ◽  
RA Vlooswijk ◽  
DH Kanters ◽  
M Hessing ◽  
BN Bouma
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Protein C ◽  
Activated Protein C ◽  
Rapid Phase ◽  
Plasma Environment ◽  
Normal Plasma ◽  
Protein C Inhibitor

Abstract Monoclonal antibodies specific for protein C inhibitor (PCI) partially blocked the inactivation of activated protein C (APC) in plasma, whereas in a purified system, the PCI activity could be completely blocked. The inactivation of APC in normal and in PCI-depleted plasma was similar in the absence of heparin. The addition of heparin did not change the rate of inactivation of APC in PCI-depleted plasma, whereas in normal plasma a rapid phase of inhibition of APC was followed by a slower phase of inhibition. The slower phase was identical to the rate of inhibition of APC in the absence of heparin. After incubation of normal plasma with a monoclonal antibody specific for PCI that blocked its activity, there was no difference in heparin-dependent or heparin- independent inhibition of APC. These results indicate that in the absence of heparin PCI is unable to inactivate APC in a plasma environment.

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