Detection of protein C activation in humans.

Bauer, Kenneth A.; Kass, BL; Beeler, David; Rosenberg, R D

doi:10.1172/jci111626

Cited by 96 publications

(39 citation statements)

References 38 publications

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“…The answer would appear to be yes because it is now well established that even a 2-fold reduction in protein C concentration can result in an increased risk of venous thrombosis (36). This propensity toward thrombosis is almost certainly the result of a reduction in protein C activation rates associated with the decrease in circulating protein C concentration rather than consumption because very little protein C is activated tonically (37). Therefore, increases in protein C activation rates of 9-fold or greater as observed here are likely to play an important role in regulating the blood coagulation process in large vessels.…”

Section: Discussionmentioning

confidence: 99%

Reconstitution of the Human Endothelial Cell Protein C Receptor with Thrombomodulin in Phosphatidylcholine Vesicles Enhances Protein C Activation

Esmon

1999

Journal of Biological Chemistry

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Blocking protein C binding to the endothelial cell protein C receptor (EPCR) on the endothelium is known to reduce protein C activation rates. Now we isolate human EPCR and thrombomodulin (TM) and reconstitute them into phosphatidylcholine vesicles. The EPCR increases protein C activation rates in a concentration-dependent fashion that does not saturate at 14 EPCR molecules/TM. Without EPCR, the protein C concentration dependence fits a single class of sites (K m ‫؍‬ 2.17 ؎ 0.13 M). With EPCR, two classes of sites are apparent (K m ‫؍‬ 20 ؎ 15 nM and K m ‫؍‬ 3.2 ؎ 1.7 M). Increasing the EPCR concentration at a constant TM concentration increases the percentage of high affinity sites. Holding the TM: EPCR ratio constant while decreasing the density of these proteins results in a decrease in the EPCR enhancement of protein C activation, suggesting that there is little affinity of the EPCR for TM. Negatively charged phospholipids also enhance protein C activation. EPCR acceleration of protein C activation is blocked by anti-EPCR antibodies, but not by annexin V, whereas the reverse is true with negatively charged phospholipids. Human umbilical cord endothelium expresses approximately 7 times more EPCR than TM. Anti-EPCR antibody reduces protein C activation rates 7-fold over these cells, whereas annexin V is ineffective, indicating that EPCR rather than negatively charged phospholipid provide the surface for protein C activation. EPCR expression varies dramatically among vascular beds. The present results indicate that the EPCR concentration will determine the effectiveness of the protein C activation complex.

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Section: Discussionmentioning

confidence: 99%

Reconstitution of the Human Endothelial Cell Protein C Receptor with Thrombomodulin in Phosphatidylcholine Vesicles Enhances Protein C Activation

Esmon

1999

Journal of Biological Chemistry

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“…Unfortunately, these molecular species are present in large excess within the blood. Only a small percentage of the zymogens are converted to active enzymes under in vivo conditions (6)(7)(8). This level of activated intermediate is responsible for only minimal depletion of inhibitors and negli-gible conversion of substrates to final products.…”

Section: Introductionmentioning

confidence: 99%

“…Based upon this information, a series of highly sensitive and specific RIAs have been developed which can quantify the activities of various steps of the hemostatic mechanism in vivo at the subnanomolar level. We have established assays for the F1+2 fragment and the protein C activation peptide (PCP)' that measure the cleavage of the prothrombin molecule by Factor Xa (9,10) and the scission of protein C by the thrombin-thrombomodulin complex (8), respectively. Nossel and co-workers (1 l1-13) have developed a similar assay for fibrinopeptide A (FPA) that monitors the cleavage of fibrinogen by thrombin.…”

Section: Introductionmentioning

confidence: 99%

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Aging-associated changes in indices of thrombin generation and protein C activation in humans. Normative Aging Study.

Bauer

Weiss²,

Sparrow³

et al. 1987

J. Clin. Invest.

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210

100

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In view of the known association of vascular disease with increasing age, we have conducted an analysis of hemostatic system activity with respect to perturbations induced by aging phenomena. We have utilized an immunochemical assay for prothrombin fragment F1+2 to quantify Factor Xa activity upon prothrombin in the plasma of 199 healthy males between the ages of 42 and 80. The levels of F1+2 in this population generally increased as a function of age (P < 0.0001). The metabolic behavior of this marker was determined in 10 individuals > 65 yr of age with varying levels of F1+2, which ranged from 1.28 to 5.85 nM. The elevations in the concentration of this component were not due to diminished clearance of the fragment. Radioimmunoassays for fibrinopeptide A (FPA) and the protein C activation peptide (PCP) were subsequently employed to measure thrombin activity upon fibrinogen and thrombin-thrombomodulin activity upon protein C, respectively, in 82 members of this population ranging in age from 42 to 80. Significant positive correlations were again observed between increasing age and the level of F1+2 (P < 0.0001) as well as FPA (P < 0.01) and PCP (P < 0.002). The results of this cross-sectional study indicate that many apparently normal males of increasing age with normal immunologic levels of antithrombin III and protein C exhibit a biochemical defect that denotes the presence of an acquired prethrombotic state.

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“…1 The zymogen protein C is converted to the active protease, activated protein C (APC), through proteolytic cleavage by thrombin bound to its endothelial membrane receptor thrombomodulin. 1 The demonstration that APC is a normal plasma component, 2,3 whose enzymatic activity can be detected with specific and sensitive methods, 4,5 indicates that the protein C anticoagulant pathway is continuously activated in vivo. Measurement of APC plasma levels might therefore be helpful in determining the in vivo integrity of the protein C anticoagulant pathway.…”

mentioning

confidence: 99%

Plasma Levels of Activated Protein C in Healthy Subjects and Patients With Previous Venous Thromboembolism

Cattaneo

Franchi

Zighetti

et al. 1998

ATVB

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Abstract-The proteolytic enzyme activated protein C (APC) is a normal plasma component, indicating that protein C (PC) is continuously activated in vivo. High concentrations of homocysteine (Hcy) inhibit the activation of PC in vitro; this effect may account for the high risk for thrombosis in patients with hyperhomocysteinemia (HyperHcy). We measured the plasma levels of APC in 128 patients with previous venous thromboembolism (VTE) and in 98 age-and sex-matched healthy controls and correlated them with the plasma levels of total Hcy (tHcy) measured before and after an oral methionine loading (PML). Forty-eight patients had HyperHcy and 80 had normal levels of tHcy. No subject was known to have any of the congenital or acquired thrombophilic states at the time of the study. Because the plasma levels of APC and PC were correlated in healthy controls, the APC/PC ratios were also analyzed. Plasma APC levels and APC/PC ratios were significantly higher in VTE patients than in controls (Pϭ0.03 and 0.0004, respectively). Most of the increase in APC levels and APC/PC ratios were attributable to patients with HyperHcy. Patients with normal tHcy had intermediate values, which did not differ significantly from those of healthy controls. There was no correlation between the plasma levels of tHcy or its PML increments and APC or APC/PC ratios in controls. The fasting plasma levels of APC and APC/PC ratios of 10 controls did not increase 4 hours PML, despite a 2-fold increase in tHcy. This study indicates that APC plasma levels are sensitive markers of activation of the hemostatic system in vivo and that Hcy does not interfere with the activation of PC in vivo. (Arterioscler Thromb Vasc Biol. 1998;18:1371-1375.)

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Detection of protein C activation in humans.

Abstract: Abstract. We have developed a radioimmunoassay (RIA) for the dodecapeptide that is liberated from protein C when this zymogen is activated by thrombin bound to thrombomodulin present on the vascular endothelium. The

Cited by 96 publications

References 38 publications

Reconstitution of the Human Endothelial Cell Protein C Receptor with Thrombomodulin in Phosphatidylcholine Vesicles Enhances Protein C Activation

Reconstitution of the Human Endothelial Cell Protein C Receptor with Thrombomodulin in Phosphatidylcholine Vesicles Enhances Protein C Activation

Aging-associated changes in indices of thrombin generation and protein C activation in humans. Normative Aging Study.

Plasma Levels of Activated Protein C in Healthy Subjects and Patients With Previous Venous Thromboembolism

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