scholarly journals Specificity of activated human protein C

1985 ◽  
Vol 230 (2) ◽  
pp. 497-502 ◽  
Author(s):  
S R Stone ◽  
J Hofsteenge
Keyword(s):  
Amino Acid ◽  
Rate Constant ◽  
Amino Acid Residue ◽  
Protein C ◽  
Activated Protein C ◽  
Order Rate Constant ◽  
Human Protein ◽  
Functional Protein ◽  
Hydrophobic Residues ◽  
Apolar Residue

Peptide p-nitroanilide substrates and peptidylchloromethane inhibitors were used to examine the specificity of activated human Protein C. Substrates with arginine in the P1 position had the highest activity. The best substrates and inhibitors, as judged by the second-order rate constant for their interaction with the enzyme, had an apolar residue in the P2 position. In contrast with thrombin [Kettner & Shaw (1981) Methods Enzymol. 80, 826-842], activated Protein C was able to accommodate large hydrophobic residues such as phenylalanine and leucine in the P2 position. In the P3 position, the enzyme preferred an apolar D-amino acid residue. The results of the present study have also indicated a suitable substrate and inhibitor to be used in the assay of functional protein C and of thrombomodulin.

scholarly journals Turnover of *I-protein C inhibitor and *I-alpha 1-antitrypsin and their complexes with activated protein C

Blood ◽  
1990 ◽  
Vol 76 (11) ◽  
pp. 2290-2295 ◽  
Author(s):  
M Laurell ◽  
J Stenflo ◽  
TH Carlson
Keyword(s):  
Complex Formation ◽  
Protein C ◽  
Activated Protein C ◽  
Human Protein ◽  
Relative Contribution ◽  
Protein C Inhibitor ◽  
The Difference ◽  
Alpha 1 Antitrypsin

Abstract The rates of clearance and catabolism of human protein C inhibitor (PCI) and human alpha 1-antitrypsin (alpha 1-AT) and their complexes with human activated protein C (APC) were studied in the rabbit. The radioiodinated-free inhibitors had biologic half-lives of 23.4 and 62.1 hours, respectively, while the corresponding *I-labeled activated- protein C complexes were cleared with half-lives of 19.6 +/- 3.1 and 72.2 +/- 6.1 minutes. Complex clearances were linked to their catabolism as shown by a correlation between clearance and the appearance of free radioiodine in the plasma. Thus, the difference in the rates of catabolism would result in a fivefold greater amount of alpha 1-AT-APC complex than PCI-APC complex 1 hour after the formation of equal amounts of these in vivo. These results lead to the conclusion that the relative contribution of PCI and alpha 1-AT to the physiologic inhibition of APC cannot be determined only from the rates of the formation of these complexes in vitro, or from measurement of their levels in plasma. The APC-PCI complex is unstable as compared with the APC-alpha 1-AT complex, compounding the problem of estimating rates of complex formation from their levels in plasma.

DEVELOPMENT OF A PROTEIN C CONCENTRATE

1987 ◽  
Author(s):  
Prabir Bhattacharya ◽  
Carolyn L Orthner ◽  
Dudley K Strickland
Keyword(s):  
Protein C ◽  
Antithrombin Iii ◽  
Activated Protein C ◽  
Protein S ◽  
Exchange Chromatography ◽  
Factor Ix ◽  
Human Protein ◽  
Red Cross ◽  
American Red Cross ◽  
Protein C Concentrate

A Protein C (PC) concentrate may be useful in treating patients with congenital or acquired Protein C deficiencies. A method for preparation of a human Protein C concentrate has been developed using a by-product of American Red Cross Factor IX production as the starting material (Menache et. al. Blood, 64, 1220). Levels of other vitamin K dependent proteins in the Protein C concentrate were measured and found to be <10 units per 100 units of PC, except for Protein S. The level of Protein S as judged by immunological assay was 30 u/100 u PC. Assay of the PC concentrate using chrcmogenic substrates revealed that levels of thrombin, Factor 3�a and Factor IXa were less than 0.006 u/mL. In addition, Antithrombin III and ax -macroglobulin were not detected. The vivo effects of Protein C concentrate and Protein C activated by thrombin have been tested in anesthetized rabbits. Thrombin was removed from the activated Protein C by ion-exchange chromatography; depletion was verified by S-2238 or by a clotting assay (< 0.006 u/mL). Rabbits were injected with Protein C concentrate (400 ug/kg) or activated Protein C 24 - 48 ug/Kg). The activated partial thromboplastin time (APTT), FactorV (FV) and Factor VIII (FVIII) levels were measured in samples collected over the next three hours. Infusion of PC concentrate elevated the level of PC to 150% of the preinfusion level within 30 min. It did not change the levels of FV, FVIII, fibrinogen or platelet count. In contrast, infusion of activated Protein C produced progressive prolongation of the APTT. Levels of FV and FVIII were decreased to 25% and 50% of preinfusion levels, respectivelv, three hours after the infusion. Fibrinogen and platelet levels were unchanged during that period. These data demonstrate that activated human Protein C concentrate induces an anticoagulant effect that can be readily measured in rabbits.

CONCENTRATION DEPENDENCE OF ACTIVATION OF ACARBOXYPROTEIN C BY THE CONTORTRIX ACTIVATOR

1987 ◽  
Author(s):  
T Exner
Keyword(s):  
Protein C ◽  
Solid Phase ◽  
Oral Anticoagulant Therapy ◽  
Activated Protein C ◽  
Apparent Molecular Weight ◽  
Single Chain ◽  
Chromogenic Substrates ◽  
Functional Protein ◽  
High Concentrations ◽  
Agkistrodon Contortrix Contortrix

The protein C activator in Southern Copperhead (Agkistrodon Contortrix Contortrix) venom was isolated by sequential chromatographies on SP�Sephadex, Con A Sepharose and hydroxylapatite. It was found to be a single chain glycoprotein with an apparent molecular weight of 36,000 and an enzymatic specificity on chromogenic substrates resembling kallikein.This "contortrix activator" was used in a solid-phase immunochromometric assay (ICMA) for functional protein C in which heterologous antibody against protein C was passively coated onto microtitre wells and used to immoblize protein C. This was then activated, easily freed of excess activator by washing and assessed by its subsequent overnight cleavage of chromogenic substrates sensitive to activated protein C.Correlation between protein C results obtained by ICMA and immunoradiometric assay (IRMA) on a variety of patient samples was excellent when relatively high concentrations of the venom activator was used. However with lower concentration of activator plasmas from patients deficient in vitamin K gave lower protein C values by ICMA then obtained by IRMA.Normal protein C and "acarboxy" protein C from a patient on oral anticoagulant therapy were immuno-immobilized and studied by the ICMA technique using varying concentrations of the venom activator. The acarboxy-protein C, although completely activatable by high concentrationa of activator, was found to activate much more slowly than normal protein C at low concentrations of the contortrix activator. Thus by reducing the intensity of the activation step, the ICMA protein C results were increased in their sensitivity for functional protein C.

scholarly journals The structure and evolution of a 461 amino acid human protein C precursor and its messenger RNA, based upon the DNA sequence of cloned human liver cDNAs

1985 ◽  
Vol 13 (14) ◽  
pp. 5233-5247 ◽  
Author(s):  
Robert J. Beckmann ◽  
Robert J. Schmidt ◽  
Robert F. Santerre ◽  
Jorge Plutzky ◽  
Gerald R. Crabtree ◽  
...  
Keyword(s):  
Amino Acid ◽  
Dna Sequence ◽  
Messenger Rna ◽  
Human Liver ◽  
Protein C ◽  
Human Protein

The Effect of Enzymatic Removal of Sialic Acids on the Functional Properties of Protein C

1988 ◽  
Vol 60 (02) ◽  
pp. 267-270 ◽  
Author(s):  
Lena Hau ◽  
Hatem H Salem
Keyword(s):  
Sialic Acid ◽  
Functional Properties ◽  
Snake Venom ◽  
Protein C ◽  
Activated Protein C ◽  
Sialic Acids ◽  
Human Protein ◽  
Native Protein ◽  
Chromogenic Substrates ◽  
Acid Component

SummaryThe effect of enzymatic removal of sialic acid residues on the functional properties of human protein C was examined. The rates of activation of native and asialo protein C were identical using either the thrombin thrombomodulin complex or the snake venom activator, protac. Desialylated activated protein C (APC) was, two-three-fold more active than native protein C, both as an anticoagulant and in its ability to hydrolyze chromogenic substrates. The potentiating effect of the desialylation was confirmed by initially fully activating protein C followed by the desialylation process. In these studies, a two-three-fold enhancement in the activity of the protein was observed which paralleled the extent of the desialylation. The activity of asialo-protein C was completely abolished by monoclonal and polyclonal antiprotein C antibodies, confirming that the effect was mediated by APC. Our studies suggest that the sialic acid component of protein C is not essential for the expression of APC activity but may act to modulate the function of the protein.

scholarly journals Functional and immunologic protein S levels are decreased during pregnancy

Blood ◽  
1986 ◽  
Vol 68 (4) ◽  
pp. 881-885 ◽  
Author(s):  
PC Comp ◽  
GR Thurnau ◽  
J Welsh ◽  
CT Esmon
Keyword(s):  
Pregnant Women ◽  
Total Protein ◽  
Postpartum Period ◽  
Protein C ◽  
Binding Protein ◽  
Activated Protein C ◽  
Protein S ◽  
Functional Protein ◽  
Protein Levels ◽  
Protein S Activity

Abstract Protein S, is a natural anticoagulant protein which serves as a cofactor for activated protein C. During pregnancy and in the postpartum period, functional protein S levels are significantly reduced (38% +/- 17.3%, mean +/- 1 SD) when compared to nonpregnant females (97% +/- 31.6%) (P less than 0.001). In plasma an equilibrium exists between functionally active free protein S and protein S complexed with C4b-binding protein, which is functionally inactive. As a result of this equilibrium either a decreased level of total protein S antigen or an elevation of C4b-binding protein could lead to reduced protein S activity. C4b-binding protein levels measured by enzyme- linked immunoassay are not significantly different in pregnant women versus nonpregnant controls (103.5% +/- 21.2% v 100% +/- 16.9%). However, during pregnancy and in the postpartum period, total protein S levels are reduced (68% +/- 10.7%) compared to nonpregnant controls (100% +/- 17.0%). This difference is significant at P less than 0.001. These data demonstrated that the reduction in protein S activity observed during pregnancy is a result of reduced total protein S antigen.

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