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

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.

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Molecular Events in the Activation of Bovine Factor V

10.1055/s-0039-1684693 ◽

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.

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Relative Immunologic Potential of Bovine Prothrombin and Purified Thrombins

Blood ◽

10.1182/blood.v112.11.5463.5463 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5463-5463

Author(s):

He Zhu ◽

Debra Ann Hoppensteadt ◽

Rodger L. Bick ◽

Cafer Adiguzel ◽

Rakesh Wahi ◽

...

Keyword(s):

Western Blotting ◽

Membrane Filtration ◽

Specific Activity ◽

Factor Viia ◽

Rank Order ◽

Coagulation Factors ◽

Bovine Thrombin ◽

Factor Va ◽

Bovine Factor ◽

Time Point

Abstract Topical bovine thrombin preparations have been used successfully in the vast majority of patients however, isolated reports of adverse events including deranged hemostasis resulting in severe or refractory bleeding and/or thrombosis exist. It has been assumed that a severe coagulopathy following exposure to topical bovine thrombin may be attributed to the impurities in bovine thrombin preparations such as factor Va. Obtained through the activation of bovine prothrombin by thromboplastin, crude thrombin preparation was further purified using ion-exchange chromatography and membrane filtration steps yielding thrombin 4A and 4B preparations which exhibit a higher specific activity and are devoid of some of the protein contaminants seen in earlier generation products. Consequently, a purer preparation of bovine thrombin might prove to be less immunogenic. The aims of this study are to evaluate the immunogenic potential of bovine prothrombin; to compare the purities of crude thrombin, 4A and 4B preparations by virtue of the detection of prothrombin related antigens. Bovine prothrombin was administered intravenously to 3 individual rabbits on days 0, 21, 42, 91, 123 and 151 using standard immunologic methods. Blood was drawn from each rabbit on days 30, 50, 105, 137 and 165 and the pooled antisera from 3 rabbits were purified to obtain the immunoglobulinG (IgG) using protein G affinity columns. Utilizing western blotting, the specificity of bovine prothrombin IgG collected on each time point (day 30, 50, 105, 137 and 165) was determined by using specific human and bovine coagulation factors such as prothrombin, thrombin, factor Xa, factor VIIa and factor Va fragment. In addition, serial diluted bovine crude thrombin, 4A and 4B preparations were also probed using the prothrombin IgG from day 30 and day 165 to explore prothrombin related-antigens in these samples. Under the experimental conditions used, neither cross-reactivity with human coagulation factors nor the recognition of bovine factor Va antigen was observed with the prothrombin IgG collected on any time point. The results of Western Blotting using the prothrombin IgG collected on day 30 and day 165 revealed that the lowest amount of crude thrombin, 4A and 4B preparations which prothrombin IgG could detect was 0.25U, 10U and 20U, respectively. The rank order of the number of detectable immunoreactive bands in each preparation by prothrombin IgG was: crude thrombin > thrombin 4A > thrombin 4B. Compared with the IgG collected on day 30, the 165 day’s IgG showed a littler stronger detecting ability for the prothrombin antigens in bovine thrombin samples. The results suggest that despite of the presence of trace amounts of bovine factor Va and other coagulation factors-related antigens in bovine prothrombin preparation, these contaminants failed to elicit the generation of relevant antibodies in rabbit. The results also indicate that among the three thrombin preparations tested, thrombin 4B preparation contains the least antigens which could be found in bovine prothrombin preparation. The suggested relationship between factor Va contaminants and the development of corresponding neutralizing human antibodies that could result in a coagulopathy in humans needs further study.

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Pseutarin C, a Prothrombin Activator from Pseudonaja textilis Venom: Its Structural and Functional Similarity to Mammalian Coagulation Factor Xa-Va Complex

Thrombosis and Haemostasis ◽

10.1055/s-0037-1613264 ◽

2002 ◽

Vol 88 (10) ◽

pp. 611-619 ◽

Cited By ~ 64

Author(s):

Veena Rao ◽

R. Kini

Keyword(s):

Structural Information ◽

Sequence Data ◽

Domain Architecture ◽

Factor Xa ◽

Coagulation Factor ◽

Peptide Bonds ◽

Amino Terminal ◽

Factor Va ◽

Group D ◽

Bovine Factor

SummarySeveral snake venoms contain procoagulant proteins that can activate prothrombin. We have purified pseutarin C, a prothrombin activator from the venom of the Australian brown snake (Pseudonaja textilis). It converts prothrombin to thrombin by cleaving both the peptide bonds Arg274 – Thr275 and Arg323 – Ile324, similar to mammalian factor Xa. It is a protein complex (∼250 Kd) consisting of an enzymatic and a nonenzymatic subunit. These subunits were separated by reverse phase HPLC and their interactions with bovine factor Xa and factor Va were studied. The enzymatic subunit of pseutarin C has a ∼13 fold higher affinity for bovine factor Va (K d of 11.4 nM for pseutarin C enzymatic subunit – bovine factor Va interaction as compared to a K d of 147.4 nM for the bovine factor Xa-Va interaction). The non-enzymatic component, however, was unable to activate bovine factor Xa. N-terminal sequence analysis of the catalytic subunit of pseutarin C showed ∼ 60% homology to mammalian factor Xa and ∼78% homology to trocarin, a group D prothrombin activator from Tropidechis carinatus venom. Structural information for the non-enzymatic subunit of pseutarin C was obtained by amino terminal sequencing of several internal peptides. The sequence data obtained indicates that the non-enzymatic subunit of pseutarin C has similar domain architecture like the mammalian factor Va and the overall homology is ∼55%. Thus pseutarin C is the first venom procoagulant protein that is structurally and functionally similar to mammalian factor Xa-Va complex.

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Phosphatidylserine Binding Alters the Conformation and Specifically Enhances the Cofactor Activity of Bovine Factor Va†

Biochemistry ◽

10.1021/bi011844d ◽

2002 ◽

Vol 41 (17) ◽

pp. 5675-5684 ◽

Cited By ~ 27

Author(s):

Xin Zhai ◽

Arvind Srivastava ◽

Daryl C. Drummond ◽

David Daleke ◽

Barry R. Lentz

Keyword(s):

Factor Va ◽

Cofactor Activity ◽

Bovine Factor

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Roles of factor Va heavy and light chains in protein and lipid rearrangements associated with the formation of a bovine factor Va-membrane complex

Biophysical Journal ◽

10.1016/s0006-3495(97)78293-6 ◽

1997 ◽

Vol 73 (5) ◽

pp. 2638-2652 ◽

Cited By ~ 10

Author(s):

V. Koppaka ◽

W.F. Talbot ◽

X. Zhai ◽

B.R. Lentz

Keyword(s):

Light Chains ◽

Membrane Complex ◽

Factor Va ◽

Bovine Factor

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The crystal structure of activated protein C-inactivated bovine factor Va: Implications for cofactor function

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0403072101 ◽

2004 ◽

Vol 101 (24) ◽

pp. 8918-8923 ◽

Cited By ~ 92

Author(s):

Ty E. Adams ◽

Matthew F. Hockin ◽

Kenneth G. Mann ◽

Stephen J. Everse

Keyword(s):

Crystal Structure ◽

Protein C ◽

Activated Protein C ◽

Factor Va ◽

Bovine Factor

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Differences in prethrombin-1 activation with human or bovine factor Va can be attributed to the heavy chain

Thrombosis and Haemostasis ◽

10.1160/th09-04-0238 ◽

2009 ◽

Vol 102 (10) ◽

pp. 623-633 ◽

Cited By ~ 2

Author(s):

Paul Y. Kim ◽

Reginald Manuel ◽

Michael E. Nesheim

Keyword(s):

Heavy Chain ◽

Primary Structure ◽

Species Difference ◽

Light Chains ◽

Heavy Chains ◽

Factor Va ◽

Interspecies Hybrids ◽

Sds Page ◽

Km Value ◽

Bovine Factor

SummaryHuman and bovine factor Va (FVa) function similarly in the activation of prothrombin but differently in the activation of prethrombin-1 (Pre-1). Pre-1 activation with human FVa proceeds at about 22 percent of the rate with bovine FVa. The dependencies of initial rates on the FVa and Pre-1 concentrations indicate that the differential activity is expressed in kcat differences, rather than differences in the assembly of prothrombinase or the Km value of the substrate. The heavy and light chains of both species of FVa were separated and interspecies hybrids were constructed in the presence of Ca++. Studies of the activation of Pre-1 with these hybrids indicate that the species difference can be attributed specifically to the heavy chain of FVa. Analyses of the reactions by SDS-PAGE indicated that cleavage at Arg271 occurs at about the same rate with both species of FVa, but cleavage at Arg320 with human FVa is specifically retarded. A major difference in primary structure between the human and bovine FVa heavy chains comprises 10 residues at COOH-terminus, adjacent to the negatively charged hirudin-like DYDYQ sequence. These residues have pI values of 12.5 and 4.26 in human and bovine FVa, respectively. The lower value would complement the negatively charged DYDYQ sequence but the higher value would counteract it. Thus, we suggest that the differences in the COOH-terminus of the heavy chain are responsible for the differences in Pre-1 activation, and that it specifically influences cleavage at Arg320 in Pre-1.

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Isolation And Properties Of Intermediates Involved In Thrombin-Catalyzed Conversion Of Bovine Factor V To Factor Va

10.1055/s-0038-1652212 ◽

1981 ◽

Author(s):

M E Nesheim ◽

K G Mann

Keyword(s):

Electrophoretic Analysis ◽

Complete Loss ◽

The Other ◽

Factor V ◽

The Third ◽

Factor Va ◽

Specific Activities ◽

Cofactor Activity ◽

The Individual ◽

Bovine Factor

Bovine Factor V was incubated briefly with bovine β- thrombin, causing partial conversion to Factor Va. QAE- cellulose chromatography in Ca++ of the partially activated material yielded 3 fractions. Electrophoresis in DodSO4 indicated primarily starting material (Mr=330,000) in the first fraction; two peptides of Mr=150,000 and 200,000 in the second; and two peptides of Mr=200,000 and 90,000 in the third. The first of these latter fractions was active only after further incubation with thrombin, while the other was active without further thrombin catalyzed activation. After thrombin treatment both fractions had specific activities equal to that of fully activated, unfractionated Factor V. Incubation of either of these fractions with EDTA caused complete loss of activity and allowed isolation of the constituent polypeptides by further chromatography on QAE- cellulose in EDTA. The NH2-termini of the 150,000 and 90.000 apparent Mr peptides had sequences identical to that of Factor V. The 200,000 apparent Mr peptides of the two fractions obtained by chromatography in Ca++ had NH2- terminal sequences identical to each other but different from that of Factor V. The summed amino acid compositions of the Mr=200,000 and Mr=150,000 were equivalent to that of Factor V. Electrophoretic analysis indicated that thrombin converts the Mr=150,000 peptide to endproducts of Mr=90,000 and 71,000, and the Mr=200,000 peptide to endproducts of 83.000 and 31,000 apparent Mr. Recalcification of the individual isolated intermediates, followed by incubation with thrombin did not yield cofactor activity. Recombina- ion of the recalcified intermediates followed by incubation with thrombin, however, generated samples with activities approaching that of unfractionated, activated Factor V. These data indicate that the intermediates of Mr=150,000 and Mr=200,000 arise by a single cleavage of Factor V, and that Factor Va comprises peptides derived from both of these intermediates.

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