Cofactor Directed Catalysis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1715-1715
Author(s):  
Michael A. Bukys ◽  
Paul Kim ◽  
Melissa A. Blum ◽  
Michael E. Nesheim ◽  
Michael Kalafatis
Keyword(s):  
Heavy Chain ◽  
Serine Proteases ◽  
Membrane Surface ◽  
Limited Proteolysis ◽  
Factor Xa ◽  
Fold Increase ◽  
Cleavage Sites ◽  
Wild Type ◽  
Factor Va ◽  
Initial Cleavage

Abstract Blood coagulation involves specific serine proteases that are activated by limited proteolysis. The process results in the conversion of prothrombin to thrombin which in turn cleaves fibrinogen to produce the insoluble fibrin mesh. Prothrombin is activated physiologically by the prothrombinase complex, which is composed of the non-enzymatic cofactor, factor Va, the enzyme, factor Xa, and the substrate prothrombin associated on a cell membrane-surface in the presence of Ca2+. Membrane-bound factor Xa alone can activate prothrombin by two sequential cleavages at R271 and R320, however the incorporation of Factor Va into prothrombinase results in the reversal of the order of cleavages, different intermediates being generated, and a 300,000-fold increase in the overall rate of catalysis. Initial cleavage at R271 will produce fragment 1•2 and prethrombin-2 while initial cleavage at R320 results in the formation of meizothrombin which has optimum esterase activity and diminished clotting activity. While the existence of these pathways and the kinetics of the rates of the cleavages have long been established, the consequences of the interaction of the cofactor with the components of prothrombinase and the molecular mechanism by which factor Va reverses the order of cleavages and increases the rate of the overall catalysis is unknown. We used recombinant factor Va molecules mutated at specific sites representing the binding domains of factor Va heavy chain for factor Xa (factor Va with the mutations E323 → F, Y324 → F, E330 → M, and V331 → I, factor VaFF/MI) and prothrombin (factor Va with the mutations D695 → K, Y696 → F, D697 → K, and Y698 → F, factor Va2K2F) in combination with plasma-derived prothrombin and mutant prothrombin molecules rMZ-II (prothrombin with the substitution R155 → A, R284 → A, and R271 → A) and rP2-II (prothrombin with the substitutions R155 → A, R284 → A, and R320 → A) to determine the molecular contribution of factor Va to each of the prothrombin-activating cleavage sites separately. The rate of cleavage of plasma-derived prothrombin at R320/R271 by prothrombinase assembled with factor VaFF/MI was 17-fold slower compared to prothrombinase assembled with the wild type cofactor. The incorporation of factor Va2K2F into prothrombinase resulted in an enzymatic complex that was both unable to activate plasma-derived prothrombin following initial cleavages at R320, and impaired in its ability to accelerate prothrombin activation through initial cleavage R271. Similarly, while the rates of cleavage of rMZ-II and rP2-II by prothrombinase assembled with factor VaFF/MI were 18- and 9-fold respectively slower compared to prothrombinase assembled with wild type factor Va, cleavage of both molecules by prothrombinase assembled with factor Va2K2F was considerly impaired. These data demonstrate that while the interaction of factor Va heavy chain with factor Xa is necessary to achieve optimal rates for thrombin formation, the interaction of factor Va with prothrombin is required because it promotes both initial cleavage at R320 and accelerates the rate of the cleavage at R271. The data presented herein dissects the cofactor’s contribution to the rate of each of the two prothrombin-activating cleavage sites, demonstrates that the interaction of factor Va heavy chain with prothrombin is responsible for the reversal of cleavage order, and strongly suggest that factor Va directs catalysis by factor Xa within prothrombinase at two spatially distinct sites.

The Effect of Factor Va on Prothrombinase Function

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1020-1020
Author(s):  
Michael Kalafatis ◽  
Jamila Hirbawi ◽  
Melissa A. Barhoover ◽  
Michael A. Bukys
Keyword(s):  
Membrane Surface ◽  
Factor Xa ◽  
Catalytic Efficiency ◽  
Fold Increase ◽  
Maximum Effect ◽  
Divalent Metal Ions ◽  
Factor Va ◽  
Ping Pong ◽  
Initial Cleavage ◽  
Membrane Bound

Abstract Following injury to the vasculature, timely activation of prothrombin (Pro) to thrombin (IIa) induces fibrin formation and platelet activation. Activation of human Pro occurs because of two cleavages by membrane-bound factor Xa (fXa) (Arg271 followed by Arg320, pathway I, pre2 pathway). However, the catalytic efficiency of membrane-bound fXa is poor in the absence of the non-enzymatic cofactor, factor Va (fVa), and the overall reaction is incompatible with efficient IIa formation. Binding of fVa to fXa on a membrane surface in the presence of divalent metal ions results in the formation of prothrombinase (IIase). This enzymatic complex catalyzes the activation of Pro following the opposite pathway characterized by the formation of meizothrombin (Mz)(cleavage at Arg320 followed by Arg271, pathway II, meizo pathway). This pathway results in a dramatic increase in the catalytic efficiency of fXa. Thus, the activity of fXa within IIase is controlled by the presence of the non-enzymatic cofactor, fVa. Earlier data suggested that IIase activation of Pro may be best described as an ordered ping-pong reaction and, more recently, data using recombinant Pro established the existence of two forms of IIase. It has been also shown that IIase is a 1:1 stoichiometric association between fVa and fXa on a lipid surface. If IIase is one enzyme, theoretically, reversing the concentrations of reactants will have no effect on the pathway and catalytic efficiency of fXa in the presence of fVa for cleavage of Pro. We have performed such experiments and gel electrophoresis data showed that in the absence of fVa, membrane-bound fXa (10nM) activates Pro slowly through pathway I with production of prethrombin 2. Very little IIa is formed under these conditions (as indicated by the small amounts of B chain of IIa). In the presence of 10nM factor Xa and 100pM factor Va, there is a significant increase in the concentration of IIa as demonstrated by the increase in the appearance of B chain of IIa. However, IIa is still activated through pathway I as indicated by the presence of prethrombin 2 and the absence of fragment 1•2-A both of which are an indication of the activation of Pro via pathway I. In the presence of 100pM fXa with 10nM fVa, Pro is activated exclusively through the meizo pathway as demonstrated by the presence of fragment 1•2-A and the absence of prethrombin 2. These data demonstrate the existence of two enzymes: one form of IIase that exists at low, limiting concentrations of fVa (with respect to fXa) and activates Pro following pathway I and prethrombin 2 formation as intermediate (E271), and one form of IIase that exists at saturating concentrations of fVa (with respect to fXa) and activates Pro via the meizo pathway (E320). In order to distinguish between activation of Pro by fXa alone and activation of Pro by fXa in the presence of low concentrations of fVa (E271) we used mutant fVa molecules. Our data show that the Vmax of IIase assembled in the presence of 5 nM fXa and 100 pM fVa is 28 nM IIa/min, while fXa alone (5 nM) activates Pro with a Vmax of ~6 nM IIa/min. This value is similar to the Vmax for the activation of Pro by fXa (5 nM) in the presence of either fVa2M (100 pM, 7.3 nM IIa/min) or fVa680–709 (100 pM, 9.5 nM IIa/min). fVa2M is a fVa molecule that is impaired in the interaction with fXa, while fVa680–709 is a cofactor molecule that is impaired in its ability to promote acceleration of cleavage of at Arg271. The data demonstrate a ~4.7-fold increase in the activation of Pro by fXa in the presence of limiting concentrations of wild type fVa through the prethrombin 2 pathway (initial cleavage a Arg271) compared to the rate of Pro cleavage by fXa alone. Earlier data established that the maximum effect of fVa on fXa for cleavage at Arg271 is 4–5 fold. In addition, we have recently demonstrated that the acidic COOH-terminal region of fVa heavy chain is responsible for this enhancing effect. Overall, the data demonstrate that E271 activates Pro through initial cleavage at Arg271 with a rate that is ~8-fold slower than activation of Pro through the meizo pathway by E320. These data are in complete agrement with recent findings demonstrating that activation of Pro by IIase is governed by a ping-pong mechanism involving two enzymes that have ~10-fold difference in catalytic efficiency. Our data strongly suggest that the mechanism and pathway of prothrombin activation by fXa within IIase is dictated by the amount of active cofactor present during the initiation phase of blood coagulation locally at the site of vascular injury.

Controlling the Pathway for Prothrombin Activation by Prothrombinase.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1695-1695
Author(s):  
Michael A. Bukys ◽  
Paul Y. Kim ◽  
Michael E. Nesheim ◽  
Michael Kalafatis
Keyword(s):  
Amino Acid ◽  
Heavy Chain ◽  
Factor Xa ◽  
Competitive Inhibitor ◽  
Amino Acid Region ◽  
Factor Va ◽  
Initial Cleavage ◽  
Membrane Bound ◽  
Prothrombin Activation ◽  
Thrombin Formation

Abstract Prothrombinase is the enzymatic complex responsible for timely thrombin formation. Activation of human prothrombin is the consequence of two cleavages at Arg271 and Arg320 in prothrombin by factor Xa. Membrane-bound factor Xa alone catalyzes prothrombin activation following initial cleavage at Arg271 and prethrombin 2 formation (pre2 pathway). Factor Va directs prothrombin activation by factor Xa through the meizothrombin pathway, characterized by initial cleavage at Arg320 (meizo pathway). We have previously shown that a pentapeptide encompassing amino acid sequence 695–699 from the COOH-terminus of the heavy chain of factor Va (Asp-Tyr-Asp-Tyr-Gln, DYDYQ) interacts with anion binding exosite I (ABE-I) of thrombin and inhibits prothrombin activation by prothrombinase. The peptide was found to be a competitive inhibitor of prothrombinase with respect to substrate. According to the mode of inhibition, we postulated that the peptide binds prothrombin in competition with the binding of the substrate to the enzyme, and inhibits prothrombinase activity by substrate depletion. This mode of DYDYQ inhibition of prothrombin activation by the factor Va-factor Xa complex is similar to that previously demonstrated for sulfated hirugen. To understand the mechanism of inhibition of thrombin formation by DYDYQ we have studied prothrombin activation by gel electrophoresis. Titration of plasma-derived prothrombin activation by fully assembled prothrombinase, with increasing concentrations of peptide, resulted in complete inhibition of the meizo pathway. However, thrombin formation still occurred through the pre2 pathway. Higher peptide concentrations were required to impair thrombin formation through the latter pathway. These data demonstrate that the peptide preferentially inhibits initial cleavage of prothrombin by prothrombinase at Arg320. These findings were corroborated by studying the kinetics of activation of recombinant mutant prothrombin molecules rMZ-II (R155A/R284A/R271A) and rP2-II (R155A/R284A/R320A) which can be only cleaved at Arg320 and Arg271 respectively. Cleavage of rMZ-II by prothrombinase was completely inhibited by low concentrations of DYDYQ while high concentrations of pentapeptide were required to inhibit cleavage of rP2-II. The pentapeptide also interfered with thrombin formation by membrane-bound factor Xa alone in the absence of factor Va. Nonetheless, while the rate for cleavage at Arg271 of plasma-derived prothrombin or rP2-II by membrane-bound factor Xa alone was significantly accelerated in the presence of DYDYQ, resulting in accumulation of prethrombin 2, the rate for cleavage at Arg320 of plasma-derived prothrombin or rMZ-II by membrane-bound factor Xa alone was only moderately affected by the pentapeptide. Our data demonstrate that a pentapeptide mimicking amino acids 695–699 of the heavy chain of factor Va has opposing effects on membrane-bound factor Xa for prothrombin activation, depending on the incorporation of factor Va in prothrombinase. In the presence of the cofactor the peptide inhibits the rate of thrombin generation by specifically interfering with initial cleavage of prothrombin at Arg320, while in the absence of factor Va the pentapeptide accelerates cleavage of prothrombin by factor Xa at Arg271. Thus, the amino acid region spatially surrounding proexosite I in prothrombin most likely has two interactive sites for the components of prothrombinase, a factor Va interactive site and a factor Xa binding site.

A Peptide Region from Factor Va Increases Factor Xa Efficiency for Cleavage of Prothrombin.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3977-3977
Author(s):  
Melissa A. Blum ◽  
Daniel O. Beck ◽  
Michael Kalafatis
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Heavy Chain ◽  
Membrane Surface ◽  
Factor Xa ◽  
Amino Acid Region ◽  
Factor Va ◽  
Prothrombinase Activity ◽  
Prothrombin Activation ◽  
Acid Region

Abstract The procoagulant enzymatic complex, prothrombinase, which is required for normal hemostasis, is composed of the enzyme, factor Xa, the protein cofactor, factor Va, associated on a cell surface in the presence of divalent metal ions. Incorporation of factor Va into prothrombinase and its interaction with factor Xa increases the catalytic efficiency of the enzyme by five orders of magnitude as compared to factor Xa alone. While the importance of the contribution of factor Va to the activity of factor Xa for rapid thrombin formation by prothrombinase at the place of vascular injury has been long established, the consequence of the interaction of the cofactor with the members of prothrombinase and the molecular mechanism by which factor Va accelerates prothrombin activation remains an enigma. Prothrombin is activated following two cleavages (Arg271/Arg320). Depending on the order of peptide bond cleavage different intermediates are formed. Factor Xa alone cleaves prothrombin sequentially, first at Arg271 to produce fragment 1•2 and prethrombin-2, followed by cleavage at Arg320 to produce fragment 1•2 and thrombin. The prothrombinase complex catalyzes the activation of prothrombin following the opposite pathway (Arg320 followed by Arg271), resulting in a formation of an active intermediate (meizothrombin) and a 300,000-fold increase in the rate of the overall reaction compared with the rate of prothrombin activation observed with factor Xa alone. We have shown that amino acid region 307–348 of factor Va heavy chain is critical for cofactor activity. A peptide containing this amino acid sequence (42 amino acids, N42R) was found to interact with fluorescently labeled factor Xa and to inhibit prothrombinase activity. Our present data show that N42R can be cross-linked to the heavy chain of membrane-bound factor Xa in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). We have also demonstrated that amino acid region 323–331 from N42R (AP4′) contains a binding site for factor Xa of factor Va heavy chain. Our present data show that a peptide containing amino acid residues 317–326 (AP3) inhibited both prothrombinase activity and the high affinity interaction of factor Va with factor Xa on the membrane surface. Moreover, we have found using site directed mutagenesis and recombinant factor Va that amino acids at the NH2-terminal end of AP4′ (i.e. residues 323–325, Glu-Tyr-Phe) are responsible for the inhibitory effect of AP3 and AP4′ and are crucial for the interaction of factor Va with factor Xa. A tripeptide with this sequence inhibited prothrombinase activity in an assay using a fluorescent thrombin inhibitor. To identify the effect of these peptides on factor Xa’s ability to cleave and activate prothrombin, we studied prothrombin activation by gel electrophoresis. The data demonstrated that several peptides that inhibited both the factor Va-factor Xa interaction on the membrane surface and prothrombinase activity, had the ability to accelerate cleavage of prothrombin by factor Xa alone, in the absence of factor Va. Specifically, N42R and AP3 were found to increase the rate of prothrombin consumption by factor Xa by approximately four-fold when compared to factor Xa acting alone. Both peptides induced acceleration in prethrombin-2 formation suggesting an increased in the rate of cleavage of prothrombin at Arg271. These data suggest that the binding of factor Va to factor Xa through amino acid region 323–331 alone produces an effect on factor Xa that increases its potency for cleavage at Arg271.

The Specific Contribution of Amino Acids 334 and 335 from Factor Va Heavy Chain to the Catalytic Efficiency of Prothrombinase.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1027-1027
Author(s):  
Melissa A. Blum ◽  
Tivadar Orban ◽  
Daniel O. Beck ◽  
Michael Kalafatis
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Heavy Chain ◽  
Factor Xa ◽  
Catalytic Efficiency ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Factor Va ◽  
Cofactor Activity ◽  
Type Factor

Abstract The prothrombinase complex, composed of the enzyme factor Xa, the cofactor factor Va, and the substrate prothrombin associated on a cell surface in the presence of divalent metal ions, catalyzes the activation of prothrombin to thrombin 300,000-fold more effectively than the enzyme, factor Xa, alone. We have demonstrated that amino acids E323, Y324 and E330, V331 are binding sites for factor Xa on the factor Va heavy chain and are required for coordinating the spatial arrangement of enzyme and substrate directing prothrombin cleavage at two spatially distinct sites. We have also demonstrated that amino acid region 332–336 contains residues that are involved in cofactor function. Peptide studies have identified amino acid residues 334DY335 as major participants in factor Va cofactor activity. We have employed site-directed mutagenesis to study the effect of these amino acids on the catalytic efficiency of prothrombinase. Recombinant factor V molecules with the mutations D334K and Y335F, designated factor VKF, and D334A and Y335A, designated factor VAA were produced, transiently transfected, expressed in COS7L cells, and purified. Kinetic studies demonstrate that while factor VaKF has a KD for factor Xa similar to the KD observed for wild type factor Va, the kcat of prothrombinase assembled with factor VaKF has approximately a 1.5-fold decreased value compared to kcat of prothrombinase assembled with the wild type cofactor molecule. On the contrary, prothrombinase assembled with factor VaAA was found to have a nearly 10-fold decrease kcat, compared to prothrombinase assembled with wild type factor Va. This data suggest that not all amino acid substitutions are well tolerated at positions 334–335. Analysis of the sequence 323–340 using the recently published completed model of coagulation factor Va (pdb entry 1Y61) revealed that amino acids 334–335 are located at the end of a beta-sheet. To ascertain the importance of these mutants and their contribution to cofactor activity we have combined the mutations of amino acids 334–335 with mutations at amino acids 323–324 (E323F, Y324F) and 330–331 (E330M, V331I). We thus created quadruple mutants resulting in recombinant factor VFF/KF, factor VFF/AA, factor VMI/KF and factor VMI/AA. These molecules were transiently expressed in COS-7L cells and studied for their ability to be incorporated into prothrombinase. Free energies associated with the catalytic efficiencies of prothrombinase assembled with each mutant were also calculated (ΔΔGint). The ΔΔGint of interaction for the double mutants, factor VaFF/KF and factor VaMI/KF, had positive values indicating that the side chains of amino acids 330EV331, 323EY324 and 334DY335 located in and around the factor Xa binding site interact in a synergistic manner resulting in the destabilization of the transition state complex and a decelerated rate of catalysis. Conversely, combining the factor Xa binding site mutants with recombinant factor VaAA result in ΔΔGint values of approximately zero. In conclusion, the data demonstrate that replacement of amino acids 334–335 by two hydrophilic residues results in decreased cofactor function. In contrast, replacement of these amino acids by two small hydrophobic residues do not appear to be well tolerated by the cofactor resulting in severely impaired cofactor activity. Altogether, these data demonstrate the importance of amino acid residues D334 and Y335 for the rearrangement of enzyme and substrate required for efficient catalysis.

Understanding the Factor Va Cofactor Effect within Prothrombinase.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2700-2700
Author(s):  
Michael Kalafatis ◽  
Michael A. Bukys ◽  
Jamila Hirbawi ◽  
Paul Y. Kim ◽  
Melissa A. Barhoover ◽  
...  
Keyword(s):  
Thrombin Generation ◽  
Membrane Surface ◽  
Factor Xa ◽  
Catalytic Efficiency ◽  
Divalent Metal Ions ◽  
Experimental Conditions ◽  
Factor Va ◽  
Low Concentrations ◽  
Initial Cleavage ◽  
Lipid Surface

Abstract Activation of human prothrombin (Pro) occurs following two cleavages by membrane-bound factor Xa (fXa) (Arg271 followed by Arg320, generating prethrombin 2 as intermediate). Binding of factor Va (fVa) to fXa on a membrane surface in the presence of divalent metal ions results in the formation of prothrombinase (IIase). It has been established that IIase is a 1:1 stochiometric complex between fVa and fXa on a lipid surface. This complex catalyzes the activation of Pro following the opposite pathway (cleavage at Arg320 followed by Arg271, generating meizothrombin as intermediate). Thus, the activity of factor Xa within IIase is controlled by fVa. Most of the data published in the literature on IIase function was obtained using limiting amounts of fXa in the presence of saturating concentrations of fVa. It is always assumed that IIase acts as one enzyme and that fXa needs to be saturated with fVa. However, if IIase is one enzyme, theoretically reversing the concentrations of reactants will have no effect on the pathway and catalytic efficiency of the enzyme for cleavage of Pro. We have assessed the pathway to thrombin formation in the presence of high or low concentrations of fVa (with respect to fXa). In the absence of fVa, fXa (10nM) activates Pro slowly through initial cleavage at Arg271. Very little thrombin is formed under these conditions. In the presence of 10nM fXa and 100pM fVa there is a significant increase in the concentration of thrombin as demonstrated by the increase in the appearance of the B chain of thrombin. Under these experimental conditions Pro is activated through the pathway characterized by the appearance of prethrombin 2 as intermediate. No fragment 1·2-A was apparent. In the presence of 100pM fXa with 10nM fVa, Pro is activated exclusively through the pathway characterized by the appearance of meizothrombin as demonstrated by the presence of fragment 1·2-A and the absence of prethrombin 2. Kinetics assessments of thrombin generation using both conditions revealed that the kcat for thrombin generation by IIase assembled in the presence of low concentrations of fVa is ∼240 min−1 while the the kcat of IIase assembled with saturating concentrations of fVa is ∼2100 min−1. These data were verified by studying the activation of recombinant Pro that can only be cleaved at Arg320 (rMZ). In the presence of limiting concentrations of fVa, rMZ was cleaved with a rate that is approximately 7-10-fold slower than the rate of rMZ cleavage in the presence of saturating concentrations of fVa. Overall our data support the following hypothesis: in the presence of low concentrations of fVa that are generated during initiation of clotting, IIase activates Pro through initial cleavage at Arg271 (E271). As clotting occurs and more fVa is generated, a new form of IIase is formed following the binding of fXa to fVa and the interaction of the complex with Pro (E320). Under these conditions, while both forms of IIase exist, E320 that has higher catalytic efficiency activates Pro via initial cleavage at Arg320. Our data are in agreement with recent findings that have suggested that IIase activation of Pro may be best described as an ordered ping-pong reaction and imply that the concentration of fVa generated during clotting, dictates which form of IIase will first cleave Pro. In conclusion, our findings suggest that fVa shuttling between E271 and E320 directs Pro activation by factor Xa locally at the place of vascular injury.

The Acidic COOH-Terminal Region of Factor Va Heavy Chain Controls the Rate of Prothrombin Activation by Prothrombinase.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1745-1745
Author(s):  
Jamila Hirbawi ◽  
Michael A. Bukys ◽  
Melissa A. Barhoover ◽  
Michael Kalafatis
Keyword(s):  
Amino Acids ◽  
Heavy Chain ◽  
Alternative Pathway ◽  
Factor Xa ◽  
Coagulation Cascade ◽  
Factor V ◽  
Divalent Metal Ions ◽  
Factor Va ◽  
Initial Cleavage ◽  
Acidic Region

Abstract The blood coagulation cascade is initiated at the site of vascular injury and results in the activation of prothrombin (Pro) to thrombin by the prothrombinase (IIase) complex. IIase is composed of the enzyme, factor Xa (fXa) bound to its cofactor, factor Va (fVa) on a phospholipid surface in the presence of Ca2+ ions. Two pathways for Pro activation are possible: membrane-bound fXa alone activates Pro following initial cleavage at Arg271 followed by cleavage at Arg320, while the fully assembled IIase activates Pro following the opposite pathway, initial cleavage at Arg320 followed by cleavage at Arg271. Activation of Pro via this latter pathway is characterized by the generation of a transient intermediate, meizothrombin (MzT) that has proteolytic activity. Initial cleavage of prothrombin at Arg320 resulting in MzT generation is absolutely fVa-dependent. Human factor V (fV) is activated by thrombin to produce a heterodimer consisting of a heavy chain and a light chain associated through divalent metal ions. The heavy chain of fVa contains an acidic hirudin-like region at the COOH-terminus (amino acids 680–709). We have shown using overlapping peptides from this region that a pentapeptide with the sequence DYDYQ inhibits Pro activation by IIase by inhibiting MzT generation. In has been reported that various proteases can cleave the acidic region of fVa heavy chain to produce a cofactor with a truncated heavy chain. All the studies revealed that removal of the acidic COOH-terminal portion of fVa heavy chain results in a cofactor molecule that is deficient in its clotting activity in a clotting assay using fV-deficient plasma, however IIase assembled with cofactor molecules missing the acidic COOH-terminus have significant higher kcat for Pro activation as assessed in an assay using purified reagents and a chromogenic substrate specific for thrombin. A molecular explanation for these paradoxical observations has not yet been provided. We have created a mutant recombinant fV molecule that is missing the last 30 amino acids from the heavy chain (fVΔ680-709). The clotting activity of the mutant molecule was impaired as compared to wild type fVa. IIase assembled with fVaΔ680-709 demonstrated a 30–40% increase in the kcat for the activation of Pro. Interstingly, gel electrophoresis revealed a delay in Pro activation with persistence of MzT during activation. Further experiments demonstrated that peptide DYDYQ inhibited MzT formation by IIase assembled with fVaΔ680-709. It has been well established that while MzT has poor clotting activity, its amidolytic activity is considerable increased towards small chromogenic substrates compared to thrombin. A logical explanation that will reconcile all the findings described above is that, the acidic COOH-terminus of fVa heavy chain regulates MzT concentration within IIase during the factor Xa catalyzed Pro activation. Thus, activation of Pro by IIase assembled with a cofactor that is missing the acidic region will result in increased MzT production. This result will be translated by a fVa molecule that is deficient in its clotting activity and produces an increase in kcat when introduced into IIase. In contrast, in the presence of an excess of DYDYQ no MzT is made by IIase resulting in the slow generation of thrombin through the alternative pathway. Our data are consistent with the interpretation that the acidic COOH-terminus of fVa heavy chain contributes a major productive interactive site for Pro within IIase.

scholarly journals Activities of Thrombin and Factor Xa Are Essential for Replication of Hepatitis E Virus and Are Possibly Implicated in ORF1 Polyprotein Processing

2018 ◽  
Vol 92 (6) ◽  
Author(s):  
Gayatri D. Kanade ◽  
Kunal D. Pingale ◽  
Yogesh A. Karpe
Keyword(s):  
Cell Culture ◽  
Cell Line ◽  
Liver Cell ◽  
Serine Proteases ◽  
Hepatitis E Virus ◽  
Factor Xa ◽  
Hepatitis E ◽  
Cleavage Sites ◽  
Polyprotein Processing ◽  
Liver Cell Line

ABSTRACTHepatitis E virus (HEV) is a clinically important positive-sense RNA virus. The ORF1 of HEV encodes a nonstructural polyprotein of 1,693 amino acids. It is not clear whether the ORF1 polyprotein (pORF1) is processed into distinct enzymatic domains. Many researchers have attempted to understand the mechanisms of pORF1 processing. However, these studies gave various results and could never convincingly establish the mechanism of pORF1 processing. In this study, we demonstrated the possible role of thrombin and factor Xa in pORF1 processing. We observed that the HEV pORF1 polyprotein bears conserved cleavage sites of thrombin and factor Xa. Using a reverse genetics approach, we demonstrated that an HEV replicon having mutations in the cleavage sites of either thrombin or factor Xa could not replicate efficiently in cell culture. Further, we demonstratedin vitroprocessing when we incubated recombinant pORF1 fragments with thrombin, and we observed the processing of pORF1 polyprotein. The treatment of a liver cell line with a serine protease inhibitor as well as small interfering RNA (siRNA) knockdown of thrombin and factor Xa resulted in significant reduction in the replication of HEV. Thrombin and factor Xa have been well studied for their roles in blood clotting. Both of these proteins are believed to be present in the active form in the blood plasma. Interestingly, in this report, we demonstrated the presence of biologically active thrombin and factor Xa in a liver cell line. The results suggest that factor Xa and thrombin are essential for the replication of HEV and may be involved in pORF1 polyprotein processing of HEV.IMPORTANCEHepatitis E virus (HEV) causes a liver disorder called hepatitis in humans, which is mostly an acute and self-limiting infection in adults. A high mortality rate of about 30% is observed in HEV-infected pregnant women in developing countries. There is no convincing opinion about HEV ORF1 polyprotein processing owing to the variability of study results obtained so far. HEV pORF1 has cleavage sites for two host cellular serine proteases, thrombin and factor Xa, that are conserved among HEV genotypes. For the first time, this study demonstrated that thrombin and factor Xa cleavage sites on HEV pORF1 are obligatory for HEV replication. Intracellular biochemical activities of the said serine proteases are also essential for efficient HEV replication in cell culture and must be involved in pORF1 processing. This study sheds light on the presence and roles of clotting factors with respect to virus replication in the cells.

scholarly journals Factor Xa interacts with two sites on monocytes with different functional activities

Blood ◽  
1992 ◽  
Vol 80 (8) ◽  
pp. 1989-1997 ◽  
Author(s):  
LA Worfolk ◽  
RA Robinson ◽  
PB Tracy
Keyword(s):  
Substrate Specificity ◽  
Membrane Surface ◽  
Factor Xa ◽  
Factor Ix ◽  
Phospholipid Vesicles ◽  
Limiting Factor ◽  
Functional Activities ◽  
Functional Sites ◽  
Factor Va ◽  
Combined Data

Abstract Studies were performed to elucidate the functional significance of factor Xa interactions at the monocyte membrane in the presence and absence of factor Va, with respect to prothrombin and factor IX cleavage. Factor Xa-catalyzed prothrombin activation at the monocyte surface was absolutely dependent on the addition of factor Va, indicating that thrombin was generated solely by a membrane-bound complex of factors Va and Xa. In contrast, in the absence of added factor Va, factor Xa bound to monocytes catalyzed the cleavage of factor IX to the nonenzymatic intermediate factor IX alpha through a reaction that was dependent on both monocyte and factor Xa concentration. At limiting factor Xa concentration, added factor Va inhibited the factor Xa-catalyzed cleavage of factor IX, suggesting that a monocyte-bound complex of factors Va and Xa did not recognize factor IX as a substrate. These combined data suggest that factor Xa interacts with the monocyte through two sites which can be distinguished by their requirement for added factor Va and their expression of different functional activities. Both functional sites could be distinguished also by their differential susceptibility to inhibition by a monoclonal antibody directed against the light chain of factor Va (alpha-HFV1). At the monocyte surface, the factor Va/Xa- catalyzed activation of prothrombin was maximally inhibited with 0.25 mumol/L alpha-HFV1, whereas 1.0 mumol/L alpha-HFV1 was required to effect 50% inhibition of the factor Xa-catalyzed cleavage of factor IX. The ability of factor Va to modulate factor Xa substrate specificity was investigated further. Factor Xa bound to thrombin-activated platelets either through platelet-released factor Va or added factor Va did not cleave factor IX. Consistent with this result, a plasma concentration of factor IX had no effect on thrombin generation catalyzed by a platelet-bound complex of factors Va and Xa. In marked contrast, factor Xa bound to phospholipid vesicles either independently or in complex with factor Va catalyzed factor IX cleavage with equal efficiency. These combined data indicate that factor Va bound to cell surfaces modulates factor Xa substrate specificity, whereas no discriminatory effect is conferred by factor Va bound to phospholipid vesicles. Thus, by providing two distinct sites at its membrane surface, the monocyte modulates factor Xa binding and the functional activity expressed by the bound enzyme, depending on the availability of factor Va.

Mechanisms of Plasmin-Catalyzed Inactivation of the Factor VIII.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1017-1017
Author(s):  
Keiji Nogami ◽  
Midori Shima ◽  
Tomoko Matsumoto ◽  
Katsumi Nishiya ◽  
Masahiro Takeyama ◽  
...  
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Activated Protein C ◽  
Factor Xa ◽  
Protein S ◽  
Fold Increase ◽  
Clotting Factors ◽  
Fviii Activity ◽  
A1 Domain ◽  
Von Willebrand

Abstract Factor VIII (FVIII) functions as a cofactor for factor IXa in the intrinsic tenase complex. This tenase activity is down-regulated by activated protein C (APC) or factor Xa (FXa). Plasmin, the most potent fibrinolytic protease, inactivates FVIII as well as other clotting factors. However, the mechanism of FVIII inactivation by plasmin is poorly understood. FVIII activity reached to the peak value of ~2-fold increase at 3 min after the addition of plasmin in a one-stage clotting assay. Then, the activity was decreased rapidly and was undetectable within 30 min. This time-dependent reaction was not affected in the presence of von Willebrand factor and phospholipid. The activation of FVIII by plasmin was an ~50% level of that by FXa. The rate constant (min-1) of inactivation of FVIIIa by plasmin possessed ~11.3- and ~2.5-folds greater than those by FXa and APC in the presence of protein S, respectively. SDS-PAGE analysis showed that plasmin cleaved the 90~210-kDa heavy chain of FVIII to 50, 48,45, 40, and 38-kDa fragments via 90-kDa fragment. Using western blot and N-terminal sequence analyses, these fragments derived from the heavy chain were identified as A11-372, A1337-372-A2, A11-336, A2, and A137-336, respectively, by cleavages at Arg372, Arg740, Lys36 and Arg336 in the A1 domain. On the other hand, the 80-kDa light chain was cleaved to 67-kDa fragment via 70-kDa fragment by cleavages at Arg1721 and Arg1689, respectively, consistent with the pattern of cleavage by FXa. However, the cleavage at Arg336 by plasmin was much quicker than that at Arg372, contrast with that by FXa. Furthermore, this cleavage was faster than that by APC, consistent with rapid inactivation of FVIII. In addition, the cleavage at Arg336 of FVIIIa by plasmin was faster than that of isolated A1 or A1/A3-C1–C2 dimer, different with that by FXa. These results demonstrate the importance of cleavage at Arg336 for the mechanism of plasmin-catalyzed FVIII inactivation. Furthermore, this cleavage appears to be selectively modulated by the A2 domain that may interact with plasmin.

Identification of Amino Acid Residues in the C1 Domain of Human Factor Va2 That Affect Phosphatidylserine-Triggered Cofactor Activity.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1711-1711
Author(s):  
Rinku Majumder ◽  
Mary Ann Quinn-Allen ◽  
Barry R. Lentz ◽  
William H. Kane
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Factor Xa ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Hydrophobic Residues ◽  
Factor Va ◽  
C1 Domain ◽  
Cofactor Activity ◽  
Prothrombin Activation

Abstract Tightly associated factors Va and Xa serve as the essential prothrombin-activating complex whose assembly is triggered by occupancy of phosphatidylserine (PS) regulatory sites on both proteins. Factor Va C2 domain contains a binding site for soluble, short chain PS (C6PS) that includes the indole moieties of Trp2063/Trp2064 at the apex of a loop (“spike-1”) (Srivastava A, Quinn-Allen MA, Kim SW, Kane WH, Lentz BR. Biochemistry, 2001, 40(28): 8246–55). Our recent data show that there is a C6PS site in the factor Va2 C1 domain that serves as a regulatory site for assembly and/or activity of the FVa2-FXa complex (Majumder R, Quinn-Allen MA, Kane WH & Lentz BR. Manuscript in Preparation). This C6PS-binding site also involves aromatic and hydrophobic residues (Tyr1956/Tyr1957) located in a homologous loop whose apex is termed “spike 3”. In order to identify the amino acid residues in the C1 domain that contribute to the PS-mediated cofactor activity of factor Va2, charged and hydrophobic residues predicted to be exposed in FVa2-C1 domain were mutated to alanine in clusters of 1–3 mutations per construct. The resultant 20 mutants (R1880A, D1892A, (K1896,E1899)A, (F1900,L1901,Y1903)A, (E1905,R1907)A, Y1917A, (E1923,K1924)A, (K1941,E1942)A, (K1954,H1955)A, (Y1956,L1957)A, Y1956A, L1957A, K1958A, E1964A, K1980A, D1995A, R2019A, (R2023,R2027)A, R2023A, R2027A,) and factor V wild type were expressed in Cos-7 cells followed by activation with thrombin, partial purification and concentration using HiTrap SP HP columns. The specific activities of all factor Va mutants were greater than 70% of wild type, with concentrations in the 1.5-7μM range. Recently it has been shown that two mutants (Y1956, L1957)A and (R2023,R2027)A showed decreased binding to immobilized PS and a selective decrease in prothrombinase activity on membranes containing 5% PS (Saleh M, Peng W, Quinn-Allen MA, Macedo-Ribeiro S, Fuentes-Prior P, Bode W & Kane WH. Thromb. Haemost.2004, 91:16–27). Here we report the rate of prothrombin activation in the presence of 1 nM factor Xa, 5 nM factorVa2 (mutants and wild type) and 400 mM C6PS. Enhancement of cofactor activity (E) of factor Va-C1 wild type and mutants by C6PS was measured using the following equation ( Zhai X, Srivastava A, Drummond DC, Daleke D and Lentz BR. Biochemistry. 2002, 41: 5675–84): \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \[E=\ (r_{Xa.Va.PL}/r_{Xa.PL})/(r_{Xa.Va}/r_{Xa})\] \end{document} Here, rXa·Va·PL is the rate of prothrombin activation measured as the initial slope of the rate of change of normalized DAPA fluorescence with time by enzyme in the presence of factor Va and lipid, and other terms are defined analogously. The cofactor activities of (Y1956, L1957) A, Y1956A and L1957A were drastically reduced (values are 1.1, 4.2 and 5.1 respectively) relative to the cofactor activity of the wild type factor Va2 (15). The cofactor activities of (R2023, R2027) A, E1964A and (K1954, H1955) A were also reduced but to a lesser extent (values are 8, 10.6 and 12 respectively). We plan to monitor the binding of these mutants to C6PS and to factor Xa in the presence of C6PS in order to determine the role of these mutations on the assembly and activity of prothrombinase. Supported by grants from the NHLBI (HL43106 to W. Kane and HL 072827 to BRL).

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