scholarly journals Prevalence of Non‐Specific Cross‐Reactive Epitopes to Bovine Factor Va Light Chain Fragments in Normal and Patient Plasma

2007 ◽  
Vol 21 (6) ◽  
Author(s):  
Debra Hoppensteadt ◽  
Vinod Bansal ◽  
Josephine Cunanan ◽  
Kuldeep Patel ◽  
Rakesh Wahi ◽  
...  
Keyword(s):  
Light Chain ◽  
Factor Va ◽  
Bovine Factor

Prevalence of Cross Reactive Epitopes to Bovine Factor Va Light Chain Fragments in Normal and Patients Plasma.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4022-4022
Author(s):  
Debra A. Hoppensteadt ◽  
Vinod Bansal ◽  
Josephine Cunanan ◽  
Kuldeep Patel ◽  
Rakesh Wahi ◽  
...  
Keyword(s):  
Western Blotting ◽  
Light Chain ◽  
Coagulation Factors ◽  
Factor V ◽  
Plasma Samples ◽  
Bovine Thrombin ◽  
Coronary Syndrome ◽  
Factor Va ◽  
V Antigen ◽  
Bovine Factor

Abstract Several reports have described the presence of antibodies to bovine coagulation factors, such as factor V, prothrombin and factor X in plasma samples obtained from patients exposed to topical bovine thrombin. Other reports have also demonstrated the presence of anti-bovine coagulation factors in patients who have not been exposed to bovine thrombins, suggesting that anti-bovine protein antibodies can be generated in normal individuals. It has been suggested that surgical patients treated with topical bovine thrombin develop specific antibodies to bovine factor V which may be responsible for the bleeding and thrombotic complications. However, there is no definitive clinical study demonstrating a relationship between the apparent hemostatic defects and the presence of bovine factor Va antibodies. It was hypothesized that bovine factor Va antibodies are usually present in patients plasma because of the exposure to dietary bovine products. To test this hypothesis plasma samples from patients with end state renal disease (ESRD)(n=80), acute coronary syndrome (ACS)(n=160), burns (n=40) and healthy normal volunteers (n=140) were profiled for the presence of human factor V antigen (HFVA), bovine FVa antigen as measured by using a modified Elisa method and western blotting methods where bovine factor Va light chain fragment is used as a probe. In contrast to the normals (89±12%), the factor V antigen levels were found to be increased in the ESRD (148±30%), ACS (164±41%) and burn (145±27%) patients. Thus, there appears to be an up regulation of factor V antigen in these patients. All of the groups tested for the presence of immunoreactive material to the bovine factor Va light chain exhibited 2–3 ug/ml levels which were not significantly different. However, in the western blotting studies all groups exhibited cross reactivity with the factor Va light epitopes. There were bands present in the molecular weight range of 22, 36, 45 and 97 Kda in both the ESRD and burn patients. In the ACS patients there was an additional band observed at 166 Kda. These observations underscore the notion that bovine antifactor Va antibodies are non-specific and highly prevalent in both the surgical/interventional patients and normal population. A possible explanation for the presence of these antibodies is that most normal individual and patients problem are exposed to bovine proteins. Moreover, the higher prevalence of these antibodies in the ESRD and ACS patients may be due to additional exposure to heparin and aprotonin.

scholarly journals Platelet coagulation factor Va: the major secretory platelet phosphoprotein

Blood ◽  
1994 ◽  
Vol 83 (8) ◽  
pp. 2180-2190
Author(s):  
MD Rand ◽  
M Kalafatis ◽  
KG Mann
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Activated Protein C ◽  
Coagulation Factor ◽  
Casein Kinase Ii ◽  
Casein Kinase ◽  
Factor V ◽  
Platelet Factor ◽  
Factor Va ◽  
Plasma Factor

Platelet-derived coagulation factor Va is the primary secreted substrate for a thrombin-stimulation-dependent platelet kinase. Human platelet factor Va, consisting of a molecular weight (M(r)) 105,000 heavy chain and an M(r) 74,000 light chain, incorporates phosphate in at least two sites on the light chain. Phosphorylated factor Va represents 50% of the secreted protein-associated phosphate. This modification occurs exclusively at serine residues and is inhibited by H-7 and staurosporine, which suggests a protein kinase C (PKC)-mediated event. Purified plasma factor V and Va are phosphorylated in the light chain region by rat brain PKC. The activity of platelet factor Va in prothrombinase on platelets is not altered when phosphorylation is inhibited by staurosporine. Plasma-derived factor Va in the presence of thrombin stimulated platelets is phosphorylated on both the heavy chain and the light chain. Plasma factor V and factor Va heavy chain phosphorylation occurs without light chain phosphorylation in the presence of added 32P gamma-ATP and non-stimulated or collagen- stimulated platelets or casein kinase II. This differential phosphorylation of factor Va heavy and light chain shows two independent platelet kinase activities that act on factor Va. The heavy chain factor V/Va kinase activity is similar to casein kinase II, which we have demonstrated previously to act on factor Va and accelerate activated protein C inactivation of the cofactor. Our data show platelet-dependent phosphorylation of platelet and plasma factor V and Va resulting in significant covalent modifications of the cofactor. These modifications may play a role in directing the extracellular distribution of factor V and factor Va.

scholarly journals Insights into the complex association of bovine factor Va with acidic-lipid-containing synthetic membranes

1996 ◽  
Vol 70 (6) ◽  
pp. 2938-2949 ◽  
Author(s):  
G.A. Cutsforth ◽  
V. Koppaka ◽  
S. Krishnaswamy ◽  
J.R. Wu ◽  
K.G. Mann ◽  
...  
Keyword(s):  
Synthetic Membranes ◽  
Acidic Lipid ◽  
Factor Va ◽  
Bovine Factor

Phenotypic Comparison of Platelet- and Plasma-Derived Factor Va: Tyrosine Phosphorylation Differentiates the Two Cofactor Pools.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1696-1696
Author(s):  
Jeremy P. Wood ◽  
Paula B. Tracy
Keyword(s):  
Monoclonal Antibody ◽  
Tyrosine Phosphorylation ◽  
Light Chain ◽  
Whole Blood ◽  
Membrane Surface ◽  
Tyrosine Phosphatase ◽  
Light Chains ◽  
Factor V ◽  
Phosphatase Inhibitors ◽  
Factor Va

Abstract Factor Va, the essential cofactor for thrombin formation via the Prothrombinase complex, exists in two pools in whole blood: 75–80% is found in the plasma, while platelet alpha-granules contain the remaining 20–25%. Platelet-derived factor Va possesses several modifications not present in its plasma counterpart, such as an O-glycosylation on Thr402, and is the more procoagulant molecule, exhibiting resistance to inactivation by activated protein C (APC) and plasmin when bound to the activated platelet membrane surface. As the platelet-derived cofactor pool originates from endocytosis of plasma-derived factor V by megakaryocytes, it can be hypothesized that, subsequent to its endocytosis, factor V is post-translationally modified, proteolytically processed, and packaged into alpha-granules to form the unique platelet-derived molecule. Platelet- and plasma-derived factor Va were purified from three individual donors. Platelets were isolated from whole blood and lysed with Triton X-100 in the presence of the tyrosine phosphatase inhibitors sodium orthovanadate and okadaic acid to prevent dephosphorylation by any released tyrosine phosphatases. Factor V in both the platelet lysates and the platelet-poor plasma was activated with thrombin, adsorbed onto a resin-coupled monoclonal antibody against human factor V, washed extensively, and eluted using 10% SDS. Western blotting analyses, using a monoclonal antibody against phosphotyrosine residues, revealed the presence of tyrosine phosphorylation on the light chain of plasma-derived, but not platelet-derived, factor Va. No such modification was observed on the heavy chain of either cofactor. Monoclonal antibodies against the heavy and light chains of factor V were used to verify that similar levels of factor V were present in all samples. This differential modification was observed in all three donors. Subsequent MALDI-TOF analyses of trypsin-digested plasma- and platelet-derived factor Va light chains were performed to identify the site(s) of tyrosine phosphorylation. These data identified phosphorylation of the C-terminal tyrosine residue (Tyr2196) in plasma-derived, but not platelet-derived, factor Va. The phosphorylated fragment (residues 2188–2196, m/z = 1152.5) is consistently present in spectra of plasma-derived factor Va, while only the non-phosphorylated fragment (m/z = 1072.5) has been observed in the platelet-derived cofactor. To demonstrate that the phosphatase inhibitors used were effective, the platelets were lysed in the presence of added plasma-derived factor Va. Tyrosine phosphorylation, as determined by immunoblotting, was still present on the light chain, indicating that the dephosphorylation was occurring prior to platelet lysis. Tyrosine phosphorylation of other proteins has been implicated in regulation of endocytosis. Further experiments will need to be performed to determine if the modification has a similar role in megakaryocyte endocytosis of factor V.

Complex Interaction between Factor Va-Light Chain and Thrombomodulin in the Regulation of Protein C Activation

1986 ◽  
Vol 485 (1 Bioregulatory) ◽  
pp. 221-227 ◽  
Author(s):  
HATEM H. SALEM ◽  
NAOMI L. ESMON ◽  
CHARLES T. ESMON ◽  
PHILIP W. MAJERUS
Keyword(s):  
Light Chain ◽  
Protein C ◽  
Complex Interaction ◽  
Factor Va

scholarly journals Binding of bovine factor Va to phosphatidylcholine membranes

1996 ◽  
Vol 70 (6) ◽  
pp. 2930-2937 ◽  
Author(s):  
V. Koppaka ◽  
B.R. Lentz
Keyword(s):  
Factor Va ◽  
Bovine Factor

scholarly journals Activation of Bovine Blood Coagulation Factor V, A Prerequisite for it of Bind Both Prothrombin and Factor Xa.

1977 ◽  
Author(s):  
M.C. Guillin ◽  
A. Bezeaud ◽  
J.P. Freeman ◽  
C.M. Jackson
Keyword(s):  
Factor Xa ◽  
Coagulation Factor ◽  
Factor V ◽  
Factor Va ◽  
Bovine Plasma ◽  
Coagulation Factor V ◽  
Russell’S Viper ◽  
Blood Coagulation Factor V ◽  
Bovine Factor ◽  
Plasma Heparin

It is known that prior to bind bovine prothrombin and to become fully functional, bovine Factor V must itself be “activated” by either thrombin or an enzyme isolable from Russell’s viper venom. The purpose of this work was to determine if Factor V activation is also required in order for it to bind bovine Factor Xa.This has been investigated by measuring the binding of both “native” (unactivated) Factor V and Factor V activated by the Russell’s viper venom activating enzyme, to a column of agarose-bound Factor Xa. The experiments were also performed using diisopropylfluorophosphate (DFP) inhibited Factor Xa covalently bound to agarose. Both purified bovine Factor V (Va) and bovine plasma were used and gave the same results. In order to prevent initiation of clotting in bovine plasma, heparin wad added to the plasma to promote inactivation of Factor Xa by antithrombin III.The results indicate that Factor V activation is a prerequisite for it to bind Factor Xa ; Factor Va binds both Factor Xa and DFP inhibited Factor Xa, unmodified Factor V does not.These experiments suggest that Factor V may not participate in prothrombin activation at all, until after some thrombin has been formed. If this is so, an alternate pathway by which the first thrombin is generated must be considered and may be proposed to be simply that involving Factor Xa, phospholipid and Ca2+ alone.

Molecular Events in the Activation of Bovine Factor V

1979 ◽  
Author(s):  
C.T. Esmon ◽  
S.F. Gray
Keyword(s):  
The Other ◽  
Antigenic Determinants ◽  
Factor V ◽  
Factor Va ◽  
Molecular Events ◽  
Activation Products ◽  
Activation Peptide ◽  
Peptide Region ◽  
Bovine Factor

The conversion of bovine Factor V (FV) to Factor Va (FVa) by thrombin appears to proceed in two steps as shown below. FV(300,000) → thrombin Factor V intermediate (FVi) 2 sub-units; 220,000 & 100,000)→FVa (2 subunits; FVa-H.C., 100,000 & FVa-L.C., 73,000)+activation peptide/s. The 220,000 Mol. wt. subunit of FVi gives rise to the FVa-L.C. EDTA dissociates both FVi and FVa into subunits, Mn2+ facilitates reassociation. Even following dissociation with EDTA, 1 mole of Ca2+ remains bound to each of the FVa subunits.Support of the above model of FV activation comes from an immunological characterization of the FV. Antibodies to FV cross react with FVa, FVi and both subunits of Fva. Antibodies to FVa cross react with FVi both subunits of FVi and both subunits of FVa. Antibodies to the FVa-L.C. cross react with FV, FVj and FVa, but do not with the FVa-H.L. Antibodies to the FVa-H.C. cross react with FV, the FVi, FVa but not with the FVa-L. C.A protease from Russell’s viper venom activated FV by making a single proteolytic cleavage. This results in the formation of two subunits of Mol. wt., 250,000 & 73,000. The 73,000 Mol. wt. subunit is functionally and immunologically identical to the FVa-L.C. the 250,000 Mol. wt. subunit contains the antigenic determinants to the FVa-H.C. and can be converted into this subunit by incubation with thrombin. The appearance of two disdinct high Mol. wt. activation products from FV; one containing the FVa-L.C.; the other containing the FVa-H.C., is compatible with placing the activation peptide region between the two subunits of Fva.

Identification of the Multimerin Binding Region within the C2 Domain of Human Factor V Using Constructs Generated by Alanine-Scanning and Site-Directed Mutagenesis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 124-124
Author(s):  
Samira B. Jeimy ◽  
Rachael A. Woram ◽  
Nola Fuller ◽  
Mary Anne Quinn-Allen ◽  
Gerard Nicolaes ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Light Chain ◽  
Coagulation Factor ◽  
Procoagulant Activity ◽  
Site Directed Mutagenesis ◽  
Factor V ◽  
C2 Domain ◽  
Wild Type ◽  
Factor Va ◽  
Covalently Linked

Abstract Activated coagulation factor V is a key non-enzymatic cofactor that is an essential component of the prothrombinase complex. In blood, much of the procoagulant factor V is stored in platelets, as a complex with the α-granule protein multimerin, for activation-induced release during clot formation. Presently, the molecular nature of multimerin - factor V binding has not been determined, although multimerin is known to interact with the light chain of factor V and Va. Using modified enzyme-linked immunoassays and recombinant factor V constructs, we previously found that discontinuous regions in the C2 domain of factor V were important for binding multimerin, and that these regions overlapped with areas in factor V important for its procoagulant function. Specifically, four (S2183T, W2063A/W2064A, K2060Q/K2061Q, K2060Q/K2061Q/W2063A/ W2064A) full-length, site-directed C2 mutants, and 12 (W2063A, W2064A (W2063, W2064)A, R2074A (R2072, R2074)A (K2101, K2103, K2104)A, L2116A (K2157, H2159, K2161)A, R2171A, R2174A, E2189A (R2187, E2189)A) B domain deleted, charge to alanine constructs had significantly reduced multimerin binding (p< 0.01), relative to the corresponding wild-type. In the present study, we evaluated multimerin-factor V binding with a new assay that used affinity purified, recombinant multimerin immobilized onto microtitre wells to test the binding of recombinant factor V constructs. Because results from the new binding assays were in agreement on the regions of the C2 domain important for multimerin binding, the new assay was used to examine the effect of thrombin on factor V-multimerin binding. Thrombin exposure led to significant dissociation of preformed multimerin-factor V complexes (p<0.01). In addition, thrombin cleaved factor Va had significantly reduced multimerin-binding in assays using antibodies against the factor Va heavy chain and light chain (p<0.01). Recently, our lab identified that platelets contain forms of factor V covalently linked to multimerin via cysteine 1085 in the factor V B-domain. After recombinant factor V was activated by thrombin, there was no detectable binding of the liberated B-domain to multimerin (p<0.001). Nonetheless, the B domain of factor V appeared to enhance factor V binding to multimerin, as factor V constructs synthesized without the B-domain had reduced multimerin binding even after conversion to factor Va, compared to wild-type factor V. Based on the overlap between multimerin-binding and procoagulant, PS binding regions in the C2 domain of factor V, we assessed the effect of multimerin on factor V procoagulant activity in one stage and two stage prothrombinase assays. However, multimerin did not neutralize factor V procoagulant activity when tested in molar excess. Our study indicates that multimerin binding of factor V is modulated by conformational changes in factor V upon activation, and that the factor V B-domain may function to enhance binding to multimerin. The dissociation of multimerin-factor V complexes by thrombin suggests multimerin might be important for delivering and localizing factor V onto platelets, prior to prothrombinase assembly.

Sign in / Sign up

Export Citation Format

Share Document