scholarly journals Protein C, an antithrombotic protein, is reduced in hospitalized patients with intravascular coagulation

Blood ◽  
1982 ◽  
Vol 60 (1) ◽  
pp. 261-264 ◽  
Author(s):  
JH Griffin ◽  
DF Mosher ◽  
TS Zimmerman ◽  
AJ Kleiss
Keyword(s):  
Liver Disease ◽  
Serine Protease ◽  
Vitamin K ◽  
Protein C ◽  
Activated Protein C ◽  
Coagulation System ◽  
Antigen Concentration ◽  
Intravascular Coagulation ◽  
The Mean

Abstract Activated protein C is a potent anticoagulant and profibrinolytic enzyme that can be derived from the vitamin-K-dependent serine protease zymogen, protein C, by the action of thrombin. Protein C antigen concentration was determined in plasmas from normals (n = 40) and from 38 patients with intravascular coagulation as evidenced by positive FDP (greater than micrograms/ml). Plasma protein C was 4 micrograms/ml in normals and was significantly depressed (less than 2 SD below the mean of normals) in 19 of the 38 patients. Of 15 patients with suspected intravascular coagulation but normal FDP, protein C was decreased in 5 individuals; 3 of these 5 patients had liver disease. Based on these results, we suggest that extensive activation of the coagulation system in vivo causes a significant consumption of protein C, presumably due to its activation by thrombin and subsequent clearance.

scholarly journals Protein C, an antithrombotic protein, is reduced in hospitalized patients with intravascular coagulation

Blood ◽  
1982 ◽  
Vol 60 (1) ◽  
pp. 261-264 ◽  
Author(s):  
JH Griffin ◽  
DF Mosher ◽  
TS Zimmerman ◽  
AJ Kleiss
Keyword(s):  
Liver Disease ◽  
Serine Protease ◽  
Vitamin K ◽  
Protein C ◽  
Activated Protein C ◽  
Coagulation System ◽  
Antigen Concentration ◽  
Intravascular Coagulation ◽  
The Mean

Activated protein C is a potent anticoagulant and profibrinolytic enzyme that can be derived from the vitamin-K-dependent serine protease zymogen, protein C, by the action of thrombin. Protein C antigen concentration was determined in plasmas from normals (n = 40) and from 38 patients with intravascular coagulation as evidenced by positive FDP (greater than micrograms/ml). Plasma protein C was 4 micrograms/ml in normals and was significantly depressed (less than 2 SD below the mean of normals) in 19 of the 38 patients. Of 15 patients with suspected intravascular coagulation but normal FDP, protein C was decreased in 5 individuals; 3 of these 5 patients had liver disease. Based on these results, we suggest that extensive activation of the coagulation system in vivo causes a significant consumption of protein C, presumably due to its activation by thrombin and subsequent clearance.

Quantification of Circulating Activated Protein C in Human Plasma by Immunoassays - Enzyme Levels are Proportional to Total Protein C Levels

1996 ◽  
Vol 75 (01) ◽  
pp. 056-061 ◽  
Author(s):  
Francisco España ◽  
Isabel Zuazu ◽  
Vicente Vicente ◽  
Amparo Estellés ◽  
Pascual Marco ◽  
...  
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Limiting Factor ◽  
Healthy Controls ◽  
Blood Samples ◽  
History Of ◽  
The Mean ◽  
The Difference ◽  
Circulating Levels

SummaryWe have developed a simple assay that measures the circulating activated protein C (APC) in plasma. The assay requires collection of duplicate blood samples, one in citrate plus heparin and the other in citrate plus inhibitors of the enzyme. In the heparin tube, APC reacts completely and irreversibly with its major plasma inhibitors, protein C inhibitor (PCI) and α1-antitrypsin (α1AT), and the complexes formed are measured by ELISAs. The amount of circulating APC is calculated from the difference between the total amount of complexed APC (sample in citrate plus heparin) and the amount of APC complexed in vivo (sample in citrate plus inhibitor). Over 95% of the APC added to blood collected with heparin was recovered in the assay. The assay can easily be performed in four hours, and had a detection limit of 0.1 ng/ml APC. The mean APC level in 18 protein C heterozygous members from seven kindreds was significantly lower (0.6 ± 0.3 ng/ml) than in 20 healthy controls (1.1 ± 0.3 ng/ml) (p <0.001), whereas the mean level in 10 non-affected members from the kindreds studied was 1.5 ± 0.3 ng/ml. In the group of 12 nonanticoagulated heterozygous protein C-deficient individuals, the three patients with a history of venous thrombosis had a mean APC level significantly lower than the nine asymptomatic members (p <0.01), both subgroups showing similar protein C levels. There was a significant correlation in all groups between the levels of APC and the levels of protein C antigen (r = 0.758, p <0.0001) and activity (r = 0.745, p <0.0001), which means that APC circulating levels are proportional to protein C levels and suggests that the protein C level is the limiting factor in the rate of protein C activation in vivo.

scholarly journals Hemostatic enzyme generation in the blood of patients with hereditary protein C deficiency

Blood ◽  
1988 ◽  
Vol 71 (5) ◽  
pp. 1418-1426 ◽  
Author(s):  
KA Bauer ◽  
AW Broekmans ◽  
RM Bertina ◽  
J Conard ◽  
MH Horellou ◽  
...  
Keyword(s):  
Protein C ◽  
Factor Xa ◽  
Protein S ◽  
Coagulation System ◽  
Protein C Deficiency ◽  
Clinical Disorder ◽  
Normal Individuals ◽  
The Mean ◽  
Metabolic Behavior

The presence of hereditary protein C deficiency has been shown to predispose patients to the development of venous thrombosis. We used radioimmunoassays for the protein C activation peptide (PCP) and the prothrombin fragment F1 + 2 to quantitate the extent of in vivo activation of protein C by thrombin-thrombomodulin and prothrombin by factor Xa, respectively, in the blood of individuals with this clinical disorder. A total of 46 protein C deficient subjects from 18 kindreds were studied. In 23 nonanticoagulated patients with an isolated deficiency of protein C, the mean level of PCP was substantially reduced while the mean concentration of F1 + 2 was significantly elevated as compared with normal controls (1.10 pmol/L v 1.78 pmol/L, P less than .0005 and 2.54 nmol/L v 1.51 nmol/L, P less than .0005, respectively). The metabolic behavior of 131I-F1 + 2 was found to be similar in protein C deficient patients and normal individuals. However, we were unable to establish a significant correlation between decreased PCP levels and increased F1 + 2 measurements in these 23 patients. This study demonstrates that heterozygous protein C deficient individuals with equivalent plasma levels of the zymogen may have markedly different biochemical profiles when assay techniques are used that quantitate the in vivo activity of the coagulation system. Six individuals from three pedigrees were identified as having combined deficiencies of protein C and either antithrombin III or protein S; the genetic basis for the combined deficiency state was determined in two of the kindreds. Finally we observed that hemostatic system activity as measured by the PCP and F1 + 2 assays is markedly suppressed in protein C deficient patients who are chronically anticoagulated with coumarin derivatives.

scholarly journals Hemostatic enzyme generation in the blood of patients with hereditary protein C deficiency

Blood ◽  
1988 ◽  
Vol 71 (5) ◽  
pp. 1418-1426 ◽  
Author(s):  
KA Bauer ◽  
AW Broekmans ◽  
RM Bertina ◽  
J Conard ◽  
MH Horellou ◽  
...  
Keyword(s):  
Protein C ◽  
Factor Xa ◽  
Protein S ◽  
Coagulation System ◽  
Protein C Deficiency ◽  
Clinical Disorder ◽  
Normal Individuals ◽  
The Mean ◽  
Metabolic Behavior

Abstract The presence of hereditary protein C deficiency has been shown to predispose patients to the development of venous thrombosis. We used radioimmunoassays for the protein C activation peptide (PCP) and the prothrombin fragment F1 + 2 to quantitate the extent of in vivo activation of protein C by thrombin-thrombomodulin and prothrombin by factor Xa, respectively, in the blood of individuals with this clinical disorder. A total of 46 protein C deficient subjects from 18 kindreds were studied. In 23 nonanticoagulated patients with an isolated deficiency of protein C, the mean level of PCP was substantially reduced while the mean concentration of F1 + 2 was significantly elevated as compared with normal controls (1.10 pmol/L v 1.78 pmol/L, P less than .0005 and 2.54 nmol/L v 1.51 nmol/L, P less than .0005, respectively). The metabolic behavior of 131I-F1 + 2 was found to be similar in protein C deficient patients and normal individuals. However, we were unable to establish a significant correlation between decreased PCP levels and increased F1 + 2 measurements in these 23 patients. This study demonstrates that heterozygous protein C deficient individuals with equivalent plasma levels of the zymogen may have markedly different biochemical profiles when assay techniques are used that quantitate the in vivo activity of the coagulation system. Six individuals from three pedigrees were identified as having combined deficiencies of protein C and either antithrombin III or protein S; the genetic basis for the combined deficiency state was determined in two of the kindreds. Finally we observed that hemostatic system activity as measured by the PCP and F1 + 2 assays is markedly suppressed in protein C deficient patients who are chronically anticoagulated with coumarin derivatives.

scholarly journals Activation and complexation of protein C and cleavage and decrease of protein S in plasma of patients with intravascular coagulation

Blood ◽  
1989 ◽  
Vol 73 (2) ◽  
pp. 455-461 ◽  
Author(s):  
MJ Heeb ◽  
D Mosher ◽  
JH Griffin
Keyword(s):  
Disseminated Intravascular Coagulation ◽  
Protein C ◽  
Activated Protein C ◽  
Protein S ◽  
Coagulation Test ◽  
Major Band ◽  
Intravascular Coagulation ◽  
Protein C Inhibitor ◽  
Dependent Protein

Abstract Activated protein C (APC) is inhibited by two major plasma inhibitors (PCIs). To find evidence for in vivo complexation of APC, immunoblotting studies were performed on plasmas of 85 patients with suspected disseminated intravascular coagulation (DIC). Samples from 62 of these patients contained 5% to 35% of protein C antigen in APC:inhibitor complexes, indicating that protein C activation and inhibition had occurred. In 24 normal plasmas, no detectable APC:PCI complexes were observed (less than 5%). Patients with higher levels of complexes had more abnormal coagulation test data for DIC. The major band of APC complexes detected by anti-protein C antibodies did not react with antibodies to the heparin-dependent protein C inhibitor (PCI- 1) previously described. Rather, APC was complexed with another recently described plasma protein C inhibitor, PCI-2. Immunoblotting studies for protein S, the cofactor for APC, revealed that the majority of the DIC patient plasmas contained a higher than normal proportion of protein S in cleaved form, suggesting that protein S may have been proteolytically inactivated. Protein S total antigen levels were also found to be low in DIC patients, excluding those with malignancy. These studies support the hypothesis that the protein C pathway is activated during DIC.

Low Level of Circulating Activated Protein C Is a Risk Factor for Venous Thromboembolism

2001 ◽  
Vol 86 (12) ◽  
pp. 1368-1373 ◽  
Author(s):  
Amparo Vayá ◽  
Yolanda Mira ◽  
Pilar Medina ◽  
Amparo Estellés ◽  
Piedad Villa ◽  
...  
Keyword(s):  
Risk Factor ◽  
Venous Thromboembolism ◽  
Protein C ◽  
Independent Risk Factor ◽  
Activated Protein C ◽  
Control Group ◽  
Low Level ◽  
History Of ◽  
The Mean

SummaryThe levels of circulating activated protein C (APC) reflect in vivo protein C activation. The aim of this study was to determine whether a low APC level is an independent risk factor for venous thromboembolism (VTE). We measured APC in 160 patients with a history of VTE and without recognized thrombophilic defects, and in 199 healthy individuals. The mean (±SD) APC level was lower in patients (0.99 ± 0.44 ng/ml) than in controls (1.19 ± 0.41 ng/ml) (p <0.0001), and showed a different distribution in the two groups. Thirty-eight patients (23.7%) had APC levels below the 5th percentile of the control group (<0.69 ng/ml) and 57 patients (35.6%) had APC levels below the 10th percentile (<0.77 ng/ml). APC levels <0.69 ng/ml increased the risk of a single or recurrent episode of VTE 4.2-fold (95% confidence interval, 2.0-9.0) or 6.9-fold (2.6-17.9), respectively, and APC levels <0.77 ng/ml increased these risks 3.4-fold (1.9-6.2) or 5.1-fold (2.3-11.2), respectively, compared with controls. Familial studies revealed that in some cases the low APC phenotype seems to be hereditary. We conclude that a low level of circulating APC in individuals without any of the most recognized thrombophilic defects is a prevalent, independent risk factor for VTE, and that it predisposes to recurrent VTE.

scholarly journals Activation and complexation of protein C and cleavage and decrease of protein S in plasma of patients with intravascular coagulation

Blood ◽  
1989 ◽  
Vol 73 (2) ◽  
pp. 455-461
Author(s):  
MJ Heeb ◽  
D Mosher ◽  
JH Griffin
Keyword(s):  
Disseminated Intravascular Coagulation ◽  
Protein C ◽  
Activated Protein C ◽  
Protein S ◽  
Coagulation Test ◽  
Major Band ◽  
Intravascular Coagulation ◽  
Protein C Inhibitor ◽  
Dependent Protein

Activated protein C (APC) is inhibited by two major plasma inhibitors (PCIs). To find evidence for in vivo complexation of APC, immunoblotting studies were performed on plasmas of 85 patients with suspected disseminated intravascular coagulation (DIC). Samples from 62 of these patients contained 5% to 35% of protein C antigen in APC:inhibitor complexes, indicating that protein C activation and inhibition had occurred. In 24 normal plasmas, no detectable APC:PCI complexes were observed (less than 5%). Patients with higher levels of complexes had more abnormal coagulation test data for DIC. The major band of APC complexes detected by anti-protein C antibodies did not react with antibodies to the heparin-dependent protein C inhibitor (PCI- 1) previously described. Rather, APC was complexed with another recently described plasma protein C inhibitor, PCI-2. Immunoblotting studies for protein S, the cofactor for APC, revealed that the majority of the DIC patient plasmas contained a higher than normal proportion of protein S in cleaved form, suggesting that protein S may have been proteolytically inactivated. Protein S total antigen levels were also found to be low in DIC patients, excluding those with malignancy. These studies support the hypothesis that the protein C pathway is activated during DIC.

A Sensitive and Facile Assay for the Measurement of Activated Protein C Activity Levels in Vivo

1993 ◽  
Vol 69 (05) ◽  
pp. 441-447 ◽  
Author(s):  
Carolyn L Orthner ◽  
Billy Kolen ◽  
William N Drohan
Keyword(s):  
Protein C ◽  
Plasma Concentrations ◽  
Limited Proteolysis ◽  
Activated Protein C ◽  
Microtiter Plate ◽  
Reversible Inhibitor ◽  
Activity Levels ◽  
Standard Curve ◽  
Agarose Beads

SummaryActivated protein C (APC) is a serine protease which plays an important role as a naturally occurring antithrombotic enzyme. APC, which is formed by thrombin-catalyzed limited proteolysis of the zymogen protein C, functions as an anticoagulant by proteolytic inactivation of the coagulation cofactors VIIIa and Va. APC is inhibited by several members of the serpin family as well a by α2-macroglobulin. APC is being developed as a therapeutic for the prevention and treatment of thrombosis. We have developed an assay to quantify circulating levels of enzymatically active APC during its administration to patients, in healthy individuals, and in various disease states. This assay utilizes an EDTA-dependent anti-protein C monoclonal antibody (Mab) 7D7B10 to capture both APC and protein C from plasma, prepared from blood collected in an anticoagulant supplemented with the reversible inhibitor p-aminobenzamidine. Mab 7D7B10-derivatized agarose beads are added to the wells of a 96-well filtration plate, equilibrated with Tris-buffered saline, and incubated for 10 min with 200 μl of plasma. After washing, APC and protein C are eluted from the immunosorbent beads with a calcium-containing buffer into the wells of a 96-well microtiter plate containing antithrombin III (ATIII) and heparin. The amidolytic activity of APC is then measured on a kinetic plate reader following the addition of L-pyroglutamyl-L-prolyl-L-arginine-p-nitroanilide (S-2366) substrate.The rate of substrate hydrolysis was proportional to APC concentration over a 200-fold concentration range (5.0 to 1,000 ng/ml) when measured continuously over a 15 to 30 min time period. The coefficient of variation was 5.9% at 35 ng/ml and 8.8% at 350 ng/ml APC. The sensitivity of the assay could be increased by measuring the amount of color produced after longer incubation times in the endpoint mode. The measured APC activity levels were little affected by varying protein C or prothrombin over the extremes of 0 to 150% of normal plasma concentrations. By constructing the standard curve in protein C-deficient plasma, the concentration of APC activity in normal pooled plasma was determined to be 2.8 ng/ml (45 pM), which represents 0.08% of the protein C concentration. The assay was approximately 50-fold more sensitive than the identical assay, but using Mab-coated microtiter wells rather than immunosorbent beads as the capture step.

A reactive center loop–based prediction platform to enhance the design of therapeutic SERPINs

2021 ◽  
Vol 118 (45) ◽  
pp. e2108458118
Author(s):  
Wariya Sanrattana ◽  
Thibaud Sefiane ◽  
Simone Smits ◽  
Nadine D. van Kleef ◽  
Marcel H. Fens ◽  
...  
Keyword(s):  
Cleavage Site ◽  
Serine Proteases ◽  
Protein C ◽  
Activated Protein C ◽  
Reactive Center Loop ◽  
Physiological Processes ◽  
Naturally Occurring ◽  
Reactive Center ◽  
Insight Into

Serine proteases are essential for many physiological processes and require tight regulation by serine protease inhibitors (SERPINs). A disturbed SERPIN–protease balance may result in disease. The reactive center loop (RCL) contains an enzymatic cleavage site between the P1 through P1’ residues that controls SERPIN specificity. This RCL can be modified to improve SERPIN function; however, a lack of insight into sequence–function relationships limits SERPIN development. This is complicated by more than 25 billion mutants needed to screen the entire P4 to P4’ region. Here, we developed a platform to predict the effects of RCL mutagenesis by using α1-antitrypsin as a model SERPIN. We generated variants for each of the residues in P4 to P4’ region, mutating them into each of the 20 naturally occurring amino acids. Subsequently, we profiled the reactivity of the resulting 160 variants against seven proteases involved in coagulation. These profiles formed the basis of an in silico prediction platform for SERPIN inhibitory behavior with combined P4 to P4’ RCL mutations, which were validated experimentally. This prediction platform accurately predicted SERPIN behavior against five out of the seven screened proteases, one of which was activated protein C (APC). Using these findings, a next-generation APC-inhibiting α1-antitrypsin variant was designed (KMPR/RIRA; / indicates the cleavage site). This variant attenuates blood loss in an in vivo hemophilia A model at a lower dosage than the previously developed variant AIKR/KIPP because of improved potency and specificity. We propose that this SERPIN-based RCL mutagenesis approach improves our understanding of SERPIN behavior and will facilitate the design of therapeutic SERPINs.

Abstract 34: Endogenous Superoxide Dismutase Protects From Impaired Generation of Activated Protein C and Enhanced Susceptibility to Experimental Thrombosis in Mice

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Sanjana Dayal ◽  
Sean X Gu ◽  
Katinan M Wilson ◽  
Ryan Hutchins ◽  
Steven R Lentz
Keyword(s):  
Superoxide Dismutase ◽  
Protein C ◽  
Activated Protein C ◽  
Vena Cava ◽  
Oxidative Modification ◽  
Mrna Levels ◽  
Endothelial Protein C Receptor ◽  
Experimental Thrombosis

In vitro studies have suggested that reactive oxygen species such as superoxide can produce prothrombotic effects, including enhanced platelet activation, increased tissue factor (TF) expression, and an oxidative modification in thrombomodulin impairing its capacity to enhance the generation of activated protein C (APC) by thrombin. It is not known, however, if elevated levels of superoxide accelerate susceptibility to experimental thrombosis in vivo . We used mice genetically deficient in superoxide dismutase-1 (SOD1, an antioxidant enzyme that dismutates superoxide to hydrogen peroxide), to test the hypothesis that lack of SOD1 enhances susceptibility to thrombosis. Susceptibility to carotid artery thrombosis in a photochemical injury model demonstrated that Sod1-/- mice formed stable occlusions significantly faster than Sod1+/+ mice (P<0.05). In an inferior vena cava (IVC) stasis model Sod1- /- mice developed significantly larger thrombi 48 hours after IVC ligation (P<0.05 vs. Sod1+/+ mice). After activation with thrombin (0.5 U/ml) or convulxin (200 ng/ml), no differences in surface expression of P-selectin or binding of fibrinogen were observed between platelets from Sod1-/- and Sod1+/+ mice. The expression of TF mRNA in lung measured by real time qPCR showed similar levels in Sod1-/- and Sod1 +/+ mice. However, the activation of exogenous protein C by thrombin in lung homogenates was decreased in Sod1 -/- mice (P<0.05 vs. Sod1 +/+ mice). Further, in vivo generation of activated protein C in response to thrombin (40 U/Kg) infusion was significantly lower in Sod1-/- mice (P<0.05 vs. Sod1+/+ mice). No differences in mRNA levels for thrombomodulin or endothelial protein C receptor were detected in Sod1 -/- mice vs. Sod1 +/+ mice, suggesting that altered generation of activated protein C in Sod1-/- mice may be related to a direct oxidative effect on thrombomodulin. In accordance, thrombomodulin treated with xanthine/hypoxanthine showed 40% loss of ability to activate protein C that was overcome by addition of SOD and catalase (P<0.05). We conclude that endogenous SOD1 in mice protects from impaired generation of activated protein C and accelerated thrombosis.

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