The Activities of Recombinant .gamma.-Carboxyglutamic-Acid-Deficient Mutants of Activated Human Protein C toward Human Coagulation Factor Va and Factor VIII in Purified Systems and in Plasma

Biochemistry ◽  
1994 ◽  
Vol 33 (7) ◽  
pp. 1869-1875 ◽  
Author(s):  
Ashish Jhingan ◽  
Li Zhang ◽  
William T. Christiansen ◽  
Francis J. Castellino
Keyword(s):  
Factor Viii ◽  
Protein C ◽  
Coagulation Factor ◽  
Human Protein ◽  
Factor Va ◽  
Carboxyglutamic Acid

scholarly journals Role of individual gamma-carboxyglutamic acid residues of activated human protein C in defining its in vitro anticoagulant activity

Blood ◽  
1992 ◽  
Vol 80 (4) ◽  
pp. 942-952 ◽  
Author(s):  
L Zhang ◽  
A Jhingan ◽  
FJ Castellino
Keyword(s):  
Protein C ◽  
Anticoagulant Activity ◽  
Coagulation Factor ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Factor Ix ◽  
Human Protein ◽  
Complete Disappearance ◽  
Carboxyglutamic Acid

Abstract To evaluate the contributions of individual gamma-carboxyglutamic acid (gla) residues to the overall Ca(2+)-dependent anticoagulant activity of activated human protein C (APC), we used recombinant (r) DNA technology to generate protein C (PC) variants in which each of the gla precursor glutamic acid (E) residues (positions 6, 7, 14, 16, 19, 20, 25, 26, and 29) was separately altered to aspartic acid (D). In one case, a gla26V mutation ([gla26V]r-PC) was constructed because a patient with this particular substitution in coagulation factor IX had been previously identified. Two additional r-PC mutants were generated, viz, an r-PC variant containing a substitution at arginine (R) 15 ([R15]r-PC), because this particular R residue is conserved in all gla- containing blood coagulation proteins, as well as a variant r-PC with substitution of an E at position 32 ([F31L, Q32E]r-PC), because gla residues are found in other proteins at this sequence location. This latter protein did undergo gamma-carboxylation at the newly inserted E32 position. For each of the 11 recombinant variants, a subpopulation of PC molecules that were gamma-carboxylated at all nonmutated gla- precursor E residues has been purified by anion exchange chromatography and, where necessary, affinity chromatography on an antihuman PC column. The r-PC muteins were converted to their respective r-APC forms and assayed for their amidolytic activities and Ca(2+)-dependent anticoagulant properties. While no significant differences were found between wild-type (wt) r-APC and r-APC mutants in the amidolytic assays, lack of a single gla residue at any of the following locations, viz, 7, 16, 20, or 26, led to virtual complete disappearance of the Ca(2+)-dependent anticoagulant activity of the relevant r-APC mutant, as compared with its wt counterpart. On the other hand, single eliminations of any of the gla residues located at positions 6, 14, or 19 of r-APC resulted in variant recombinant molecules with substantial anticoagulant activity (80% to 92%), relative to wtr-APC. Mutation of gla residues at positions 25 and 29 resulted in r-APC variants with significant but low (24% and 9% of wtr-APC, respectively) levels of anticoagulant activity. The variant, [R15L]r-APC, possessed only 19% of the anticoagulant activity of wrt-APC, while inclusion of gla at position 32 in the variant, [F31L, Q32gla]r-APC, resulted in a recombinant enzyme with an anticoagulant activity equivalent to that of wtr-APC.

scholarly journals Role of individual gamma-carboxyglutamic acid residues of activated human protein C in defining its in vitro anticoagulant activity

Blood ◽  
1992 ◽  
Vol 80 (4) ◽  
pp. 942-952
Author(s):  
L Zhang ◽  
A Jhingan ◽  
FJ Castellino
Keyword(s):  
Protein C ◽  
Anticoagulant Activity ◽  
Coagulation Factor ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Factor Ix ◽  
Human Protein ◽  
Complete Disappearance ◽  
Carboxyglutamic Acid

To evaluate the contributions of individual gamma-carboxyglutamic acid (gla) residues to the overall Ca(2+)-dependent anticoagulant activity of activated human protein C (APC), we used recombinant (r) DNA technology to generate protein C (PC) variants in which each of the gla precursor glutamic acid (E) residues (positions 6, 7, 14, 16, 19, 20, 25, 26, and 29) was separately altered to aspartic acid (D). In one case, a gla26V mutation ([gla26V]r-PC) was constructed because a patient with this particular substitution in coagulation factor IX had been previously identified. Two additional r-PC mutants were generated, viz, an r-PC variant containing a substitution at arginine (R) 15 ([R15]r-PC), because this particular R residue is conserved in all gla- containing blood coagulation proteins, as well as a variant r-PC with substitution of an E at position 32 ([F31L, Q32E]r-PC), because gla residues are found in other proteins at this sequence location. This latter protein did undergo gamma-carboxylation at the newly inserted E32 position. For each of the 11 recombinant variants, a subpopulation of PC molecules that were gamma-carboxylated at all nonmutated gla- precursor E residues has been purified by anion exchange chromatography and, where necessary, affinity chromatography on an antihuman PC column. The r-PC muteins were converted to their respective r-APC forms and assayed for their amidolytic activities and Ca(2+)-dependent anticoagulant properties. While no significant differences were found between wild-type (wt) r-APC and r-APC mutants in the amidolytic assays, lack of a single gla residue at any of the following locations, viz, 7, 16, 20, or 26, led to virtual complete disappearance of the Ca(2+)-dependent anticoagulant activity of the relevant r-APC mutant, as compared with its wt counterpart. On the other hand, single eliminations of any of the gla residues located at positions 6, 14, or 19 of r-APC resulted in variant recombinant molecules with substantial anticoagulant activity (80% to 92%), relative to wtr-APC. Mutation of gla residues at positions 25 and 29 resulted in r-APC variants with significant but low (24% and 9% of wtr-APC, respectively) levels of anticoagulant activity. The variant, [R15L]r-APC, possessed only 19% of the anticoagulant activity of wrt-APC, while inclusion of gla at position 32 in the variant, [F31L, Q32gla]r-APC, resulted in a recombinant enzyme with an anticoagulant activity equivalent to that of wtr-APC.

scholarly journals Blood coagulation factor Va abnormality associated with resistance to activated protein C in venous thrombophilia

Blood ◽  
1994 ◽  
Vol 83 (11) ◽  
pp. 3120-3125 ◽  
Author(s):  
X Sun ◽  
B Evatt ◽  
JH Griffin
Keyword(s):  
Factor Viii ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Laboratory Finding ◽  
Activated Protein C ◽  
Coagulation Factor ◽  
Factor V ◽  
Apc Resistance ◽  
Factor Va ◽  
Common Laboratory

Abstract A coagulation test abnormality, termed activated protein C (APC) resistance, involving poor anticoagulant response to APC is currently the most common laboratory finding among venous thrombophilic patients. Because the anticoagulant activity of APC involves inactivation of factors Va and VIIIa, studies were made to assess the presence of abnormal factors V or VIII. Diluted aliquots of plasma from two unrelated patients with APC resistance and thrombosis were added to either factor VIII-deficient or factor V-deficient plasma and APC resistance assays were performed. The results suggested that patients' factor V but not factor VIII rendered the substrate plasma APC resistant. When factor V that had been partially purified from normal or APC resistant patients' plasmas using immunoaffinity chromatography was added to factor V-deficient plasma, APC resistance assays showed that patients' factor V or factor Va, but not normal factor V, rendered the substrate plasma resistant to APC. Studies of the inactivation of each partially purified thrombin-activated factor Va by APC suggested that half of the patients' factor Va was resistant to APC. These results support the hypothesis that the APC resistance of some venous thrombophilic plasmas is caused by abnormal factor Va.

scholarly journals Blood coagulation factor Va abnormality associated with resistance to activated protein C in venous thrombophilia

Blood ◽  
1994 ◽  
Vol 83 (11) ◽  
pp. 3120-3125 ◽  
Author(s):  
X Sun ◽  
B Evatt ◽  
JH Griffin
Keyword(s):  
Factor Viii ◽  
Protein C ◽  
Anticoagulant Activity ◽  
Laboratory Finding ◽  
Activated Protein C ◽  
Coagulation Factor ◽  
Factor V ◽  
Apc Resistance ◽  
Factor Va ◽  
Common Laboratory

A coagulation test abnormality, termed activated protein C (APC) resistance, involving poor anticoagulant response to APC is currently the most common laboratory finding among venous thrombophilic patients. Because the anticoagulant activity of APC involves inactivation of factors Va and VIIIa, studies were made to assess the presence of abnormal factors V or VIII. Diluted aliquots of plasma from two unrelated patients with APC resistance and thrombosis were added to either factor VIII-deficient or factor V-deficient plasma and APC resistance assays were performed. The results suggested that patients' factor V but not factor VIII rendered the substrate plasma APC resistant. When factor V that had been partially purified from normal or APC resistant patients' plasmas using immunoaffinity chromatography was added to factor V-deficient plasma, APC resistance assays showed that patients' factor V or factor Va, but not normal factor V, rendered the substrate plasma resistant to APC. Studies of the inactivation of each partially purified thrombin-activated factor Va by APC suggested that half of the patients' factor Va was resistant to APC. These results support the hypothesis that the APC resistance of some venous thrombophilic plasmas is caused by abnormal factor Va.

scholarly journals Enhanced Rate of Cleavage at Arg-306 and Arg-506 in Coagulation Factor Va by Gla Domain-mutated Human-activated Protein C

2004 ◽  
Vol 279 (46) ◽  
pp. 47528-47535 ◽  
Author(s):  
Yong-Hui Sun ◽  
Sinh Tran ◽  
Eva A. Norstrøm ◽  
Björn Dahlbäck
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Coagulation Factor ◽  
Factor Va ◽  
Gla Domain

Increased Risk for Thromboembolism Associated with High Coagulation Factor VIII (F VIII) Levels in Patients with Malignant Lymphoma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1380-1380
Author(s):  
Martin Mohren ◽  
Ilka Markmann ◽  
Astrid Franke ◽  
Kathleen Jentsch-Ullrich ◽  
Michael Koenigsmann ◽  
...  
Keyword(s):  
Factor Viii ◽  
Malignant Lymphoma ◽  
Solid Tumors ◽  
Protein C ◽  
Factor V Leiden ◽  
Coagulation Factor ◽  
Hematologic Malignancies ◽  
Low Grade ◽  
Factor V Leiden Mutation ◽  
Increased Risk

Abstract An increased annual incidence of thromboembolic events (TE) of up to 11% has been observed in patients with solid tumors, whereas there exists little data on TE in hematologic malignancies. A previous study found a 6,6% incidence of TE in patients with high grade Non Hodgkin’s lymphoma (HG-NHL) mostly occuring during the first three months of therapy. Little is known about pathogenesis and risk factors in this patient group. We retrospectively evaluated the medical records of all patients with malignant lymphoma treated at our institution between 1991 and 2004. In a seperate effort laboratory analysis for detection of acquired and hereditary thrombophilia was performed at diagnosis and during treatment in 44 patients with various hematologic malignancies: HG-NHL (n = 22), Low grade- NHL (LG-NHL) (n = 7), Hodgkin’s disease (HD) (n = 6), CNS-lymphoma (n = 1) and acute myeloid leukemia (AML) (n = 8). A total of 96 TE occurred in 80 of 1048 patients (7,6%) with malignant lymphoma: DVT (n = 51), pulmonary embolism (n = 19), central venous catheter thrombosis (n = 11), upper extremity thrombosis due to tumor compression (n = 9), central nervous system thrombosis (n = 3), arterial thrombosis (n = 2) and portal vein thrombosis (n = 1). 69 TE (72%) occurred during treatment, whereas 27 (28%) were diagnosed prior to (n = 16) (17%) or after completion of therapy (n = 11) (11%). 9 patients (9%) had comorbid solid tumors. In 12 patients (15%) results of thrombophilia screening were available and FVIII levels were high (> 150%) in 7 (58%). In the prospectively analyzed patient cohort 30 (68%) had high FVIII levels, 22 (50%) showing very high levels (> 180%). High FVIII was associated with high von Willebrand factor (vWF) and increased collagen binding activity, but not with elevated IL 6 or TNF-a. 4 patients (9%) had heterozygous factor V Leiden mutation, one had the G20210A mutation of the prothrombin gene. Fibrinolysis was normal in all patients as were protein C, S and AT-III. No anticardiolipin antibodies or lupus anticoagulants were detected. However only 2 patients (4,4%) in this cohort developed TE, one of whom also had heterozygous protein C resistance. Patients with malignant lymphoma are at substantial risk for TE, especially during treatment, thus prophylactic anticoagulation seems warranted. Our study shows sustained strikingly high factor VIII levels in patients with malignant lymphoma even months or years after a TE as well as in a prospectively analyzed, yet mostly asymptomatic cohort with lymphoma and acute leukemia. Infection as a cause of secondary F VIII elevation in these patients was ruled out by absence of fever and normal IL 6 and TNF-a. Increased FVIII activity (> 150%) has been recognized as an independent risk factor for TE, however the pathogenesis is unclear so far and high FVIII and vWF levels have previously also been found in Multiple Myeloma patients. Ongoing investigations will focus on the implication of these findings in the pathophysiology of hematologic disease.

Sign in / Sign up

Export Citation Format

Share Document