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 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.

Modulation of Prothrombinse Assembly and Activity by Phosphotidylethanolamine.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4207-4207
Author(s):  
Rinku Majumder ◽  
Xiaoe Liang ◽  
Mary Ann Quinn-Allen ◽  
Barry R. Lentz ◽  
William H. Kane
Keyword(s):  
Binding Sites ◽  
Conflicts Of Interest ◽  
Membrane Surface ◽  
Factor Xa ◽  
Catalytic Efficiency ◽  
Prothrombinase Complex ◽  
Factor Va ◽  
Naturally Occurring ◽  
Early Phases

Abstract Abstract 4207 Constituents of naturally occurring phospholipid membranes regulate the activity of the prothrombinase complex. In the present study we demonstrate that membranes containing phosphatidylcholine and phosphatidylethanolamine (PC:PE) bind factor Va with high affinity (Kd ∼10 nM) in the absence of phosphatidylserine (PS). These membranes support formation of a functional prothrombinase complex though thrombin generation at saturating factor Va concentrations is reduced approximately 60-70% compared to membranes containing 5% or more PS. The presence of PE markedly enhances the catalytic efficiency of the prothrombinase complex on membranes containing 1% PS with only modest effects on membranes containing 5% or more PS. The effect of PE on factor Va membrane binding appears to be due to direct interactions between PE and factor Va rather than to changes in membrane surface packing. Finally, we find that soluble C6PE is able to bind to factor Va (Kd ∼6.5 uM) and factor Xa (Kd ∼ 91 uM). We also show that soluble C6PE is able to stimulate formation of a partially active factor Va-factor Xa complex capable of catalyzing conversion of prothrombin to thrombin in the absence of a membrane surface. We further demonstrate that C6PE and C6PS binding sites in factor Xa are linked, as binding of one lipid enhances the binding and activity of the other. These findings provide important new insights into the role of PE in assembly of the prothrombinase complex that are relevant to understanding the activity of factor Xa on the surface of platelets particularly in the early phases of hemostasis when the concentration of PS may be limiting. Disclosures: No relevant conflicts of interest to declare.

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.

Factor Va Cofactor Activity Is Dependent on the Interaction of Prothrombin with the Membrane Surface.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2702-2702
Author(s):  
Michael A. Bukys ◽  
Tivadar Orban ◽  
Paul Y. Kim ◽  
Michael E. Nesheim ◽  
Michael Kalafatis
Keyword(s):  
Membrane Surface ◽  
Enzymatic Reaction ◽  
Specific Inhibitor ◽  
Catalytic Efficiency ◽  
Inhibitory Effect ◽  
Factor Va ◽  
Initial Cleavage ◽  
Membrane Bound ◽  
Cofactor Activity ◽  
Thrombin Formation

Abstract Activation of human prothrombin (Pro) occurs because of two sequential cleavages in prothrombin by membrane-bound factor Xa (fXa) (Arg271 followed by Arg320, prethrombin 2 pathway). However, the catalytic efficiency of membrane-bound fXa is poor in the absence of factor Va (fVa), and the overall reaction is incompatible with efficient thrombin formation required for the timely arrest of bleeding. Binding of fVa to fXa on a membrane surface in the presence of divalent metal ions results in the formation of prothrombinase. This complex catalyzes the activation of Pro following the opposite pathway (cleavage at Arg320 followed by Arg271, meizothrombin pathway). This pathway results in a dramatic increase in the catalytic efficiency of fXa. It is well established that initial cleavage of prothrombin at Arg320 is strictly dependent on the incorporation of fVa into prothrombinase and that the increase in the overall efficiency of the enzymatic reaction is solely credited to the interaction of the cofactor molecule with discrete amino acid residues from both membrane-bound enzyme and membrane-bound substrate. Thus, the activity of fXa within prothrombinase is controlled by the presence of the soluble, non-enzymatic cofactor, fVa. We have shown that a pentapeptide with the sequence DYDYQ specifically inhibits this pathway. It has been also established that Hir54–65(SO3−) is a specific inhibitor of prothrombinase. To understand the role of fVa within prothrombinase at the molecular level, we have studied thrombin formation by prothrombinase in the presence of various Pro-derived fragments alone or in combination. Activation of prethrombin 1 is slow with cleavages at Arg320 and Arg271 occurring with similar rates. Addition of purified fragment 1 to prethrombin 1 accelerates both the rate of cleavage at Arg320 and thrombin formation. Both reactions were inhibited by Hir54–65(SO3−) while DYDYQ had no significant inhibitory effect on prethrombin 1 cleavage in the absence or presence of fragment Similarly, activation of prethrombin 2 by prothrombinase, which is notably slow and inhibited by Hir54–65(SO3−), is not affected by DYDYQ. Addition of purified fragment 1·2 to prethrombin 2 accelerates the rate of cleavage at Arg320 by prothrombinase resulting in a rate of thrombin formation comparable to the rate of Pro activation by prothrombinase. This addition results in a significant inhibition of thrombin formation by DYDYQ and is concurrent with the elimination of the inhibitory effect of Hir54–65(SO3−) on the same reaction. Finally, a membrane-bound ternary complex composed of prethrombin 2/fragment 1·2/Hir54–65(SO3−) is inhibited by DYDYQ. These data demonstrate that DYDYQ and Hir54–65(SO3−) inhibit prothrombinase activity through different mechanisms because the peptides most likely interact with distinct portions of prethrombin Altogether, the data demonstrate that membrane-bound fragment 1 is required to promote optimum fVa cofactor activity which in turn is translated by efficient initial cleavage of Pro by prothrombinase at Arg320. Therefore, our findings put in the context of the literature suggest that fVa incorporation into prothrombinase equalizes the rate of both Pro activating cleavages, and it is the interaction of membrane-bound fragment 1 with fVa and/or fXa within prothrombinase that promotes a further accelerating effect of the rate of cleavage at Arg320, resulting in meizothrombin generation.

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 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.

Kinetic Description Of Conversion Of Bovine Prethrombin 2 To Thrombin

1981 ◽  
Author(s):  
Thomas L Carlisle ◽  
Craig M Jackson
Keyword(s):  
Calcium Ion ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Factor Xa ◽  
Maximum Velocity ◽  
Catalytic Efficiency ◽  
Kinetic Description ◽  
Factor Va ◽  
Carboxyglutamic Acid ◽  
Thrombin Formation

Bovine Factor Xa slowly converts Prethrombin 1 to thrombin plus Fragment 2. Inclusion of Ca2+ increased the rates of Prethrombin 1 consumption, Prethrombin 2 production and thrombin formation detected by SDS polyacrylamide gel electrophoresis or by thrombin assay. Ca2+ also increased the rate of thrombin formation from equimolar mixtures of Prethrombin 2 and Fragment 2 (Prethrombin 2/Fragment 2) by approximately 1.8 fold. Calcium ion thus increases the rate of both proteolyses required to generate thrombin from Prethrombin 1. Studies using Factor Xa (des light chain residues 1-44) indicated that this effect of Ca2+ required the region of Factor Xa containing gamma-carboxyglutamic acid.Factor Va markedly lowered the apparent Km of Factor Xa for Prethrombin 2/Fragment 2, with decreases greater than 20 fold observed under some conditions. The apparent maximum velocity also increased by up to 50 fold. The extent of increase was greater at higher concentrations of Factor Va, and was about 6 fold greater in the presence of Ca2+ than in its absence. Factor Va binding to Factor Xa (forming XaVa with enhanced substrate binding and/or catalytic efficiency), and Factor Va binding to Prethrombin 2/Fragment 2 (forming a substrate more readily bound and/or cleaved) must be considered among the possible explanations for these effects. Previous qualitative observations suggest that these effects of Factor Va on activation of Prethrombin 2/ Fragment 2 are important in understanding the activation of prothrombin.

Serotoninergic Mechanisms Play a Major Role on Platelet-Mediated Thrombogenicity: Studies under Flow Conditions and Effects of Inhibitory Strategies.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2636-2636
Author(s):  
Ana M. Galan ◽  
Irene Lopez-Vilches ◽  
Maribel Diaz-ricart ◽  
Fulgencio Navalon ◽  
Marcos Pino ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Platelet Aggregation ◽  
Serotonin Reuptake ◽  
Selective Serotonin Reuptake Inhibitors ◽  
Annexin V ◽  
Selective Serotonin ◽  
Experimental Conditions ◽  
Factor Va ◽  
Low Concentrations ◽  
Platelet Aggregates

Abstract Serotoninergic mechanisms are reciprocally implicated in depression and cardiovascular disorders. Serotonin (5-HT) may facilitate the development of a subpopulation of platelets with increased procoagulant activity. We have investigated the involvement of serotoninergic mechanisms in adhesive, cohesive and procoagulant function of platelets. Furthermore, we evaluated the antithrombotic properties of selective serotonin reuptake inhibitors (SSRI). For these purposes we used a series of experimental strategies including standard aggregometry, flow cytometry, perfusion techniques and determination of coagulation parameters. Serotonin concentrations ranging 0.5–5 μM were used in these studies. Citalopram, a selective serotonin reuptake inhibitor (SSRI), was used at concentrations equivalent to those reached in clinical practice. Aggregation studies indicate that 5-HT is a weak agonist for platelets in comparison with standard activating agents. Only the highest concentrations of 5-HT tested (5 μM) caused minimal and reversible platelet aggregation. Despite this modest effect on aggregation, 5-HT potentiated the aggregation induced by low concentrations of ADP (0.5 μM) in PRP samples (16.3±5.9 % vs. 28.1±5.5 %). Potentiation of aggegation was more evident when PRP was obtained from blood anticoagulated with LMWH (p<0.05), where some thrombin could be generated. Citalopram at 0.6 μM caused statistical reductions in % of maximal aggregation induced by ADP (2 or 0.5 μM) and COL (2.5 μg/ml) (p<0.05). Platelet aggregation was maximally inhibited with citalopram at concentrations exceeding clinical doses (6 μM). Flow cytometry studies revealed a mild increase of P-selectin expression in washed platelets in the first minute after activation with 5-HT. In isolated platelets, 5-HT produced irreversible aggregation when studies were performed in the presence of human-TF enriched microvesicles. A significant increase in Annexin V binding and presence of factor Va was detected by flow cytometry in the latter studies. Further studies performed in perfusion models using human blood flowing though chambers exposing a surface rich in collagen and TF demonstrated that presence of 5-HT in the perfusates increased thrombin generation (p<0.05) as demonstrated by elevations of F1+2 levels. Presence of citalopram in the perfusates significantly inhibited the formation of large platelet aggregates on the perfused thrombogenic surfaces (20.2±3.4 % vs. 11.5±1.7 %; p<0.05) and prevented elevations of F1+2 levels observed in previous studies. Our studies confirm that 5-HT is a weak agonist for platelets, though it possesses a marked ability to enhance the activation induced by other agonists. Moreover, 5-HT showed a powerful capacity to potentiate procoagulant responses of platelets under different experimental conditions and to enhance the thrombogenesis on damaged vascular surfaces. The fact that SSRI inhibited the responses elicited by 5-HT in the previous situations further confirm the implications of serotoninergic mechanisms in platelet activation. Our data reinforce the opinion that serotoninergic mechanisms play an important role in platelet-mediated thrombogenicity, suggesting that modulation of 5-HT mediated responses may offer a new potential target for the development of more powerful antithrombotic drugs.

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.

scholarly journals Factor XI contributes to thrombin generation in the absence of factor XII

Blood ◽  
2009 ◽  
Vol 114 (2) ◽  
pp. 452-458 ◽  
Author(s):  
Dmitri V. Kravtsov ◽  
Anton Matafonov ◽  
Erik I. Tucker ◽  
Mao-fu Sun ◽  
Peter N. Walsh ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Factor Ix ◽  
Factor Xii ◽  
Factor Xi ◽  
Factor Xii Deficiency ◽  
Low Concentrations

Abstract During surface-initiated blood coagulation in vitro, activated factor XII (fXIIa) converts factor XI (fXI) to fXIa. Whereas fXI deficiency is associated with a hemorrhagic disorder, factor XII deficiency is not, suggesting that fXI can be activated by other mechanisms in vivo. Thrombin activates fXI, and several studies suggest that fXI promotes coagulation independent of fXII. However, a recent study failed to find evidence for fXII-independent activation of fXI in plasma. Using plasma in which fXII is either inhibited or absent, we show that fXI contributes to plasma thrombin generation when coagulation is initiated with low concentrations of tissue factor, factor Xa, or α-thrombin. The results could not be accounted for by fXIa contamination of the plasma systems. Replacing fXI with recombinant fXI that activates factor IX poorly, or fXI that is activated poorly by thrombin, reduced thrombin generation. An antibody that blocks fXIa activation of factor IX reduced thrombin generation; however, an antibody that specifically interferes with fXI activation by fXIIa did not. The results support a model in which fXI is activated by thrombin or another protease generated early in coagulation, with the resulting fXIa contributing to sustained thrombin generation through activation of factor IX.

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