Membrane-bound Factor Xa Research Articles

22 - Lipid oxidation inactivates the anticoagulant function of protein Z-dependent protease inhibitor (ZPI)

Lipid oxidation due to oxidative stress plays an important role in the pathogenesis of inflammatory and thrombotic cardiovascular diseases. Several findings suggest that lipid peroxidation can alter the function of coagulation proteins and contribute to a hypercoagulable state, but the molecular mechanisms are unclear. Here, we report that oxidized phospholipids suppress the anticoagulant function of the serpin, protein Z-dependent protease inhibitor (ZPI), a specific inhibitor of membrane-associated factor Xa (FXa) that requires protein Z (PZ), phospholipid, and calcium as cofactors. We found that this suppression arises from a diminished ability of the oxidized membrane to function as a cofactor to promote ZPI inhibition of membranebound FXa, due fully or in part to the susceptibility of the bound ZPI-PZ complex to oxidative inactivation. Surprisingly, free ZPI was also susceptible to inactivation by oxidized membrane vesicles in the absence of calcium. Oxidized vesicles containing both Phosphatidylserine and polyunsaturated fatty acids were required to promoteinactivation of theZPI-PZcomplexor free ZPI, indicating that binding of thePZ-complexedor freeZPIto peroxide-modified phospholipid vesicles mediates the inactivation. Heparin protected the ZPI-PZ complex and free ZPI from inactivation, suggesting that blocking the heparin-binding site on ZPI interferes with ZPI binding to lipid or to PZ. This was confirmed by direct lipid-binding experiments. Native PAGE indicated that oxidization induced dissociation of the ZPI-PZ complex and increased the negative charge of ZPI. We conclude that compromised ZPI anticoagulant function could contribute to thrombus initiation and growth in oxidative stress-induced cardiovascular diseases.

Lipid oxidation inactivates the anticoagulant function of protein Z-dependent protease inhibitor (ZPI)

Lipid oxidation due to oxidative stress plays an important role in the pathogenesis of inflammatory and thrombotic cardiovascular diseases. Several findings suggest that lipid peroxidation can alter the function of coagulation proteins and contribute to a hypercoagulable state, but the molecular mechanisms are unclear. Here, we report that oxidized phospholipids suppress the anticoagulant function of the serpin, protein Z-dependent protease inhibitor (ZPI), a specific inhibitor of membrane-associated factor Xa (FXa) that requires protein Z (PZ), phospholipid, and calcium as cofactors. We found that this suppression arises from a diminished ability of the oxidized membrane to function as a cofactor to promote ZPI inhibition of membrane-bound FXa, due fully or in part to the susceptibility of the bound ZPI-PZ complex to oxidative inactivation. Surprisingly, free ZPI was also susceptible to inactivation by oxidized membrane vesicles in the absence of calcium. Oxidized vesicles containing both phosphatidylserine and polyunsaturated fatty acids were required to promote inactivation of the ZPI-PZ complex or free ZPI, indicating that binding of the PZ-complexed or free ZPI to peroxide-modified phospholipid vesicles mediates the inactivation. Heparin protected the ZPI-PZ complex and free ZPI from inactivation, suggesting that blocking the heparin-binding site on ZPI interferes with ZPI binding to lipid or to PZ. This was confirmed by direct lipid-binding experiments. Native PAGE indicated that oxidization induced dissociation of the ZPI-PZ complex and increased the negative charge of ZPI. We conclude that compromised ZPI anticoagulant function could contribute to thrombus initiation and growth in oxidative stress-induced cardiovascular diseases.

Protein S Is a Cofactor for Platelet TFPIα, but Not Cell Surface-Associated TFPI

Abstract 494 Background:Tissue factor pathway inhibitor (TFPI), the primary physiologic inhibitor of the tissue factor (TF)/factor VIIa (FVIIa) enzymatic complex, is found in plasma, on endothelial cells, and within platelets. There are two alternatively-spliced isoforms of TFPI, TFPIα and TFPIβ. TFPIα is secreted from endothelial cells, present in plasma and stored in circulating platelets, while TFPIβ is the primary cell surface-expressed isoform of TFPI. TFPIα is composed of three Kunitz-type inhibitor domains: K1 and K2 bind to and inhibit the serine proteases FVIIa and factor Xa (FXa), respectively, and K3 binds to protein S (PS). TFPIβ contains the same K1 and K2 domains and a glycosylphosphatidylinositol (GPI)-anchored C-terminus. PS serves as a cofactor for plasma TFPIα in the inhibition of factor Xa (FXa), as measured by amidolytic activity assays. However, TFPIα is a poor inhibitor of thrombin generation by the prothrombinase complex (FXa and its cofactor, factor Va (FVa)) regardless of whether PS is present. Thus, the physiologic function of FXa inhibition by the TFPI/PS anticoagulant system is not entirely clear, particularly on the platelet surface where prothrombinase assembly occurs. In addition, it is not known if PS cofactor activity is mediated by localizing TFPI to the membrane surface, thereby enhancing its interaction with membrane-bound FXa, or by directly enhancing the interaction between TFPI and FXa once both are on the membrane surface. Methods/Results:The cofactor activity of PS toward recombinant TFPIα (rTFPIα), platelet TFPIα, and cell surface-associated forms of TFPI was measured. As previously published, low concentrations of PS (5 nM) enhanced the inhibitory activity of rTFPIα in a FXa amidolytic activity assay. PS also substantially delayed thrombin generation in assays where rTFPIα, FXa, and PS were incubated prior to addition of FVa, but not when FVa was first incubated with FXa (before rTFPIα and PS addition). PS was also found to enhance the inhibition of FXa by platelet TFPIα (measured in platelet lysates), in a dose-dependent manner, with enhancement apparent at concentrations as low as 5 nM. Unlike with experiments performed using recombinant or platelet TFPIα, PS (at concentrations up to 80 nM) had no impact on the inhibition of either FXa amidolytic activity or thrombin generation when assays were performed using CHO cells expressing TFPIβ or a GPI-anchored TFPIα (K1K2K3-GPI). Additionally, PS did not act as a cofactor for endogenous TFPI on the surface of EA.hy926 cells or primary HUVECs. However, TFPIα released from EA.hy926 cells by treatment with heparin was enhanced by addition of PS, to a similar extent as observed with rTFPIα. TFPIβ, released by treatment with phosphoinositol-specific phospholipase C, displayed little inhibitory activity either in the presence or absence of PS. Conclusions:(1) PS is a cofactor for the inhibition of FXa by soluble TFPIα, including platelet TFPIα, but not for surface-associated forms of TFPI, regardless of the presence of the K3 domain. (2) PS functions by localizing and retaining TFPIα at the membrane surface, rather than through a direct enhancement of the interaction between TFPIα and FXa. (3) PS stabilizes the TFPIα/FXa inhibitory complex. In this manner, PS may act physiologically to sequester FXa away from FVa and slow assembly of prothrombinase on the surface of activated platelets. Disclosures:Mast:Novo Nordisk: Research Funding.

P2.05 Contribution of Platelets To Tumor Aggressivity

ABSTRACT Background: The interaction between cancer and coagulation process has been shown since many years. The aim of our study is to better understand the mechanisms implicated and to propose new therapeutic approaches. Methods: Two breast cancer cell lines were used: a very aggressive (MDA-MB231) and a much less aggressive (MCF-7) cell lines. Platelets aggregation tests were done with washed platelets, normal or fibrin removed plasma and cancer cells (20 to 2·105 cells per ml). Procoagulant activity of cancer cells was also studied. Aspirin, Apyrase (ADPase), a direct thrombin inhibitor (Hirudin) and two Xa inhibitors (Fondaparinux and Rivaroxaban) were the different inhibitors tested at therapeutic blood levels. Interaction between platelets and cancer cells was visualized using confocal immunofluorescence microscopy. Angiogenic effect of supernatants from platelets-cancer cells co-incubation was investigated. Results: The data obtained show that a platelet aggregation is induced by cancer cells in the presence of a small amount of plasma. This aggregation depends on both the type and number of cancer cells. Aggressive MDA-MB231 cells have a more potent pro-aggregating activity than MCF-7. This aggregation appears to be due to thrombin generation since it is inhibited by Hirudin. Rivaroxaban, a direct inhibitor of factor Xa, inhibits platelets aggregation but not Fondaparinux, an indirect anti Xa inhibitor which binds to antithrombin III. Aspirin and Apyrase have no effect. Moreover, the procoagulant activity of cancer cells with plasma is inhibited by Hirudin and Rivaroxaban but not by Fondaparinux. This suggests that the Fondaparinux-Antithrombin III complex, cannot access to cell membrane-bound factor Xa. It may be due to its steric hindrance since thromboplastin induced coagulation is inhibited by Rivaroxaban but not by Fondaparinux. The confocal microscopic study shows that platelets protect tumor cells from immune attack, probably via the formation from a protective envelope around cancer cells. Angiogenesis assays show that activation of platelets by cancer cells releases angiogenesis stimulating factors and cytokines. Conclusion: The present work confirms the crucial role of platelets in cancer aggressiveness and reveals that Rivaroxaban or thrombin inhibitors could be efficient drugs to reduce cancer progression, metastasis and thrombosis.

Contribution of platelets to tumor aggressivity.

e21156 Background: The interaction between cancer and coagulation process has been shown since many years. The aim of our study is to understand the mechanisms implicated and to propose new therapeutic approaches. Methods: Two breast cancer cell lines were used: a very aggressive (MDA-MB231) and a much less aggressive (MCF-7). Platelet aggregation tests were done with washed platelets, normal or fibrin removed plasma and cancer cells (20 to 2.105 cells/ml). Procoagulant activity of cancer cells was studied. Aspirin, Apyrase (ADPase), a direct thrombin inhibitor (Hirudin), two Xa inhibitors (Fondaparinux, Rivaroxaban) were the inhibitors tested. Interaction between platelets and cancer cells was visualized by confocal microscopy. Angiogenic effect of supernatants from platelets-cancer cells co-incubation was investigated. Results: The data obtained show that a platelet aggregation is induced by cancer cells in the presence of a small amount of plasma. This aggregation depends on both the type and number of cancer cells. Aggressive MDA-MB231 cells have a more potent pro-aggregating activity than MCF-7. This aggregation appears to be due to thrombin generation since it is inhibited by Hirudin. Rivaroxaban, a direct inhibitor of factor Xa, inhibits platelets aggregation but not Fondaparinux, an indirect anti Xa inhibitor which binds to Antithrombin III. Aspirin and Apyrase have no effect. Moreover, the procoagulant activity of cancer cells with plasma is inhibited by Hirudin and Rivaroxaban but not by Fondaparinux. This suggests that the Fondaparinux-Antithrombin III complex cannot access to cell membrane-bound factor Xa. It may be due to its steric hindrance since thromboplastin induced coagulation is inhibited by Rivaroxaban but not by Fondaparinux. The confocal microscopic study shows that platelets protect tumor cells from immune attack via the formation from a protective envelope around cancer cells. Angiogenesis assays show that activation of platelets by cancer cells releases angiogenesis stimulating factors and cytokines. Conclusions: The present work confirms the crucial role of platelets in cancer aggressiveness and reveals that Rivaroxaban or Thrombin inhibitors could be efficient drugs to reduce cancer progression, metastasis and thrombosis.

The Effect of Factor Va on Prothrombinase Function

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.

Kinetic Characterization of the Protein Z-dependent Protease Inhibitor Reaction with Blood Coagulation Factor Xa

Protein Z-dependent protease inhibitor (ZPI) is a recently identified member of the serpin superfamily that functions as a cofactor-dependent regulator of blood coagulation factors Xa (FXa) and XIa. Here we show that ZPI and its cofactor, protein Z (PZ), inhibit procoagulant membrane-bound factor Xa by the branched pathway acyl-intermediate trapping mechanism used by other serpins, but with significant variations of this mechanism that are unique to ZPI. Rapid kinetic analyses showed that the reaction proceeded by the initial assembly of a membrane-associated PZ-ZPI-FXa Michaelis complex (K(M) 53+/-5 nM) followed by conversion to a stable ZPI-FXa complex (k(lim) 1.2+/-0.1 s(-1)). Cofactor premixing experiments together with independent kinetic analyses of ZPI-PZ and factor Xa-PZ-membrane complex formation suggested that assembly of the Michaelis complex through either ZPI-PZ-lipid or factor Xa-PZ-lipid intermediates was rate-limiting. Reaction stoichiometry analyses and native PAGE showed that for every factor Xa molecule inhibited by ZPI, two serpin molecules were cleaved. Native PAGE and immunoblotting showed that PZ dissociated from ZPI once ZPI forms a stable complex with FXa, and kinetic analyses confirmed that PZ acted catalytically to accelerate the membrane-dependent ZPI-factor Xa reaction. The ZPI-FXa complex was only transiently stable and dissociated with a rate constant that showed a bell-shaped pH dependence indicative of participation of factor Xa active-site residues. The complex was detectable by SDS-PAGE when denatured at low pH, consistent with it being a kinetically trapped covalent acyl-intermediate. Together our findings show that ZPI functions like other serpins to regulate the activity of FXa but in a manner uniquely dependent on protein Z, procoagulant membranes, and pH.

Understanding the Factor Va Cofactor Effect within Prothrombinase.

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.

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

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 fragment1.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 prethrombin2.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 that1.fVa incorporation into prothrombinase equalizes the rate of both Pro activating cleavages, and2.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.

Exosites in the substrate specificity of blood coagulation reactions.

The specificity of blood coagulation proteinases for substrate, inhibitor, and effector recognition is mediated by exosites on the surfaces of the catalytic domains, physically separated from the catalytic site. Some thrombin ligands bind specifically to either exosite I or II, while others engage both exosites. The involvement of different, overlapping constellations of exosite residues enables binding of structurally diverse ligands. The flexibility of the thrombin structure is central to the mechanism of complex formation and the specificity of exosite interactions. Encounter complex formation is driven by electrostatic ligand-exosite interactions, followed by conformational rearrangement to a stable complex. Exosites on some zymogens are in low affinity proexosite states and are expressed concomitant with catalytic site activation. The requirement for exosite expression controls the specificity of assembly of catalytic complexes on the coagulation pathway, such as the membrane-bound factor Xa*factor Va (prothrombinase) complex, and prevents premature assembly. Substrate recognition by prothrombinase involves a two-step mechanism with initial docking of prothrombin to exosites, followed by a conformational change to engage the FXa catalytic site. Prothrombin and its activation intermediates bind prothrombinase in two alternative conformations determined by the zymogen to proteinase transition that are hypothesized to involve prothrombin (pro)exosite I interactions with FVa, which underpin the sequential activation pathway. The role of exosites as the major source of substrate specificity has stimulated development of exosite-targeted anticoagulants for treatment of thrombosis.

Expression of Allosteric Linkage between the Sodium Ion Binding Site and Exosite I of Thrombin during Prothrombin Activation

The specificity of thrombin for procoagulant and anticoagulant substrates is regulated allosterically by Na+. Ordered cleavage of prothrombin (ProT) at Arg320 by the prothrombinase complex generates proteolytically active, meizothrombin (MzT), followed by cleavage at Arg271 to produce thrombin and fragment 1.2. The alternative pathway of initial cleavage at Arg271 produces the inactive zymogen form, the prethrombin 2 (Pre 2).fragment 1.2 complex, which is cleaved subsequently at Arg320. Cleavage at Arg320 of ProT or prethrombin 1 (Pre 1) activates the catalytic site and the precursor form of exosite I (proexosite I). To determine the pathway of expression of Na+-(pro)exosite I linkage during ProT activation, the effects of Na+ on the affinity of fluorescein-labeled hirudin-(54-65) ([5F]Hir-(54-65)(SO-3)) for the zymogens, ProT, Pre 1, and Pre 2, and for the proteinases, MzT and MzT-desfragment 1 (MzT(-F1)) were quantitated. The zymogens showed no significant linkage between proexosite I and Na+, whereas cleavage at Arg320 caused the affinities of MzT and MzT(-F1) for [5F]Hir-(54-65)(SO-3) to be enhanced by Na+ 8- to 10-fold and 5- to 6-fold, respectively. MzT and MzT(-F1) showed kinetically different mechanisms of Na+ enhancement of chromogenic substrate hydrolysis. The results demonstrate for the first time that MzT is regulated allosterically by Na+. The results suggest that the distinctive procoagulant substrate specificity of MzT, in activating factor V and factor VIII on membranes, and the anticoagulant, membrane-modulated activation of protein C by MzT bound to thrombomodulin are regulated by Na+-induced allosteric transition. Further, the Na+ enhancement in MzT activity and exosite I affinity may function in directing the sequential ProT activation pathway by accelerating thrombin formation from the MzT fast form.

A Control Switch for Prothrombinase: CHARACTERIZATION OF A HIRUDIN-LIKE PENTAPEPTIDE FROM THE COOH TERMINUS OF FACTOR Va HEAVY CHAIN THAT REGULATES THE RATE AND PATHWAY FOR PROTHROMBIN ACTIVATION

Membrane-bound factor Xa alone catalyzes prothrombin activation following initial cleavage at Arg(271) and prethrombin 2 formation (pre2 pathway). Factor Va directs prothrombin activation by factor Xa through the meizothrombin pathway, characterized by initial cleavage at Arg(320) (meizo pathway). We have shown previously 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) inhibits prothrombin activation by prothrombinase in a competitive manner with respect to substrate. 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 prothrombinase, with increasing concentrations of peptide, resulted in complete inhibition of the meizo pathway. However, thrombin formation still occurred through the pre2 pathway. These data demonstrate that the peptide preferentially inhibits initial cleavage of prothrombin by prothrombinase at Arg(320). These findings were corroborated by studying the activation of recombinant mutant prothrombin molecules rMZ-II (R155A/R284A/R271A) and rP2-II (R155A/R284A/R320A) which can be only cleaved at Arg(320) and Arg(271), respectively. Cleavage of rMZ-II by prothrombinase was completely inhibited by low concentrations of DYDYQ, whereas high concentrations of pentapeptide were required to inhibit cleavage of rP2-II. The pentapeptide also interfered with prothrombin cleavage by membrane-bound factor Xa alone in the absence of factor Va increasing the rate for cleavage at Arg(271) of plasma-derived prothrombin or rP2-II. Our data demonstrate that pentapeptide DYDYQ has opposing effects on membrane-bound factor Xa for prothrombin cleavage, depending on the incorporation of factor Va in prothrombinase.

Controlling the Pathway for Prothrombin Activation by Prothrombinase.

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.

Cofactor Directed Catalysis.

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.

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

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.

Fibroblasts, glial, and neuronal cells are involved in extravascular prothrombin activation.

A membrane-associated prothrombin activator (MAPA) was found on various cultured cells derived from non-hematopoietic cells [Sekiya, F. et al. (1994) J. Biol. Chem. 269, 32441-32445]. In this study, we investigated the enzymatic properties of this enzyme using protease inhibitors. While the metalloproteinase inhibitor, o-phenanthroline, had no effect, some Kunitz type serine protease inhibitors attenuated MAPA activity. Recombinant tissue factor pathway inhibitor (rTFPI) also markedly reduced the activity (IC(50), 1. 3+/-0.6 x 10(-10) M). MAPA activity is, therefore, most likely to be due to factor Xa. We evaluated the effect of exogenous factor Xa on MAPA activity. Factor Xa-dependent prothrombin activation was observed on fibroblast cells (apparent K(d), 1.47+/-0.72 nM). Activation was also observed on glial and neuronal cells, which expressed MAPA activity. These results imply that membrane-bound factor Xa results in MAPA activity on these cells. Therefore, we considered the involvement of factor Va, a component of prothrombinase, in this activity. We examined whether or not the prothrombinase complex is assembled on these cells. Prothrombin was activated in a manner dependent on both exogenous factor Xa and factor Va (apparent K(d) of 0.51-1.81 nM for factor Va). These results indicate that the prothrombinase complex forms specifically on various extravascular cells. Although the prothrombinase complex can be assembled on monocytes and lymphocytes, it is not known why these cells can activate prothrombin specifically. These cells which have the capacity for prothrombin activator activity could also activate factor X; i.e. cells with factor X activation activity were able to convert prothrombin. These observations suggest that thrombin was generated via two procoagulant activities; factor X activation and subsequent prothrombinase complex formation on the surface of these cells. This mechanism may explain the various pathological states involving or resulting from extravascular thrombin and fibrin formation.

Proteolysis of factor V by cathepsin G and elastase indicates that cleavage at Arg1545 optimizes cofactor function by facilitating factor Xa binding.

The single-chain procofactor factor V is cleaved by thrombin (FVaIIa) at Arg709, Arg1018, and Arg1545 and by a variety of other proteases to generate a cofactor species with various levels of cofactor function. Having demonstrated previously that monocyte-bound forms of cathepsin G and elastase cleave and activate factor V, studies were initiated here using purified proteins to probe factor V structure/function. Electrophoretic, Western blotting, and amino-terminal sequence analyses revealed that cathepsin G cleaves factor V at several sites (Phe1031, Leu1447, Tyr1518, and potentially Tyr696), ultimately generating an amino-terminal 103 kDa heavy chain and a carboxy-terminal 80 kDa light chain (FVaCG). Elastase also cleaves factor V at several sites (Ile708, Ile819, Ile1484, and potentially Thr678), generating a cofactor species, FVaHNE, with an amino-terminal 102 kDa heavy chain and a carboxy-terminal 90 kDa light chain. Incubation of FVaIIa with either cathepsin G or elastase resulted in cleavage within the heavy chain, releasing peptides of approximately 2000 and approximately 3000 Da, respectively, generating FVaIIa/CG and FVaIIa/HNE. The functional activity of each cofactor species was assessed either by clotting assay or by employing a purified prothrombinase assay using saturating amounts of factor Xa. Significant differences in cofactor function were observed between the two assay systems. Whereas FVaIIa, FVaCG, FVaIIa/CG, FVaHNE, and FVaIIa/HNE all had similar cofactor activities in the purified prothrombinase assay, FVaCG and FVaHNE had no cofactor activity in the clotting-based assay, and FVaIIa/CG and FVaIIa/HNE had approximately 30-35% clotting activity relative to FVaIIa. These disparate results led us to examine the binding interactions of these cofactors with the various prothrombinase components. Kinetic analyses indicated that FVaIIa (Kd(app) = 0.096 nM), FVaIIa/CG (Kd(app) = 0.244 nM), and FVaIIa/HNE (Kd(app) = 0.137 nM) bound to membrane-bound factor Xa much more effectively than FVaCG (Kd(app) = 1.46 nM) and FVaHNE (Kd(app) = 0.818 nM). In contrast, studies of the activated protein C (APC)-catalyzed inactivation of each of the factor V(a) species indicated that they were all equivalent substrates for APC with no differences observed in the rate of inactivation or the cleavage mechanism, suggesting that APC interacts with the light chain at a site distinct from factor Xa. The Km values for prothrombin, as well as the kcat values for each of the FV(a) species, were all similar (approximately 0.25 microM and approximately 1900 min-1). In addition, kinetic analyses indicated that whereas FVaCG and FVaHNE exhibited a slightly reduced ability to interact with phospholipid vesicles (approximately 2-3-fold), the remaining FV(a) species assembled equally well on this surface. Collectively, these data indicate that FVaCG and FVaHNE have a diminished capacity to support factor Xa binding; however, cleavage at Arg1545 and removal of the extended B-domain in these cofactors restore near-total factor Xa binding. Thus, cleavage at Arg1545 optimizes cofactor function within prothrombinase by facilitating factor Xa binding to membrane-bound FVa.

Transient high affinity binding of tissue factor pathway inhibitor-factor Xa complexes to negatively charged phospholipid membranes.

The interaction of tissue factor pathway inhibitor (TFPI), factor Xa, and TFPI-factor Xa complexes with negatively charged phospholipid membranes composed of 25 mol % phosphatidylserine and 75 mol % phosphatidylcholine was studied by ellipsometry. The binding of TFPI alone was negligible; factor Xa bound with moderate affinity, with a dissociation constant Kd = 42 nM. Formation of the TFPI-factor Xa complex drastically enhanced the affinity for phospholipid membranes, Kd = 5 nM, compared to that of either protein alone. TFPI1-161, a TFPI variant lacking the third Kunitz domain and the positively charged C-terminus did not enhance binding affinity of the factor Xa. Analysis of the kinetics of adsorption and desorption confirmed the equilibrium binding data, although upon longer residence at the lipid membrane the desorption rate of TFPI-factor Xa complexes became slower, indicating an increase in affinity with longer residence of the TFPI-factor Xa complexes at the membrane. In contrast, binding of TFPI-factor Xa complexes in the presence of an excess factor Xa was transient; maximal binding is followed by a slow desorption of the complex. Immunoblot analysis revealed that this desorption was accompanied with cleavage of TFPI by membrane-bound factor Xa. Collectively, our results show that phosphatidylserine containing membranes will accumulate tightly bound TFPI-factor Xa complexes, and that uncomplexed, phospholipid-bound, factor Xa, will cause limited proteolysis of TFPI accompanied by simultaneous release of these complexes from the phospholipid membrane.

Kinetics of human factor VII activation.

In this study the activation of human factor VII by a variety of potential activators in the presence and absence of mixed phospholipid vesicles [25% phosphatidylserine (PS), 75% phosphatidylcholine (PC)] is evaluated. At the plasma concentration of factor VII, 10 nM, the activation rate of the zymogen by 0.05 nM factor Xa is anionic phospholipid (PCPS) dependent and achieves a maximum value of 18 pM/s at 5-20 microM PCPS; further increases in the levels of PCPS decrease the activation rate of factor VII. The maximum activation rate of factor VII (10 nM) by the factor VIIa-tissue factor complex (0.1 nM), 0.76 pM/s, is achieved at 200 microM PCPS. No detectable activation of 10 nM factor VII is observed under similar conditions when either thrombin (0.1 nM) or factor IXa (0.1 nM) is used as an activator. Factor VIIa (10 nM) and factor XIa (1 nM) are not observed to activate factor VII at detectable rates. The observed Michaelis-Menten constants (KM) for factor VII activation in the presence of PCPS at optimal concentrations vary from 1.2 microM for factor Xa to 3.2 microM for the factor VIIa-tissue factor complex. The highest catalytic constant (kcat) value (15.2 s-1) is observed for factor Xa-PCPS. The factor VIIa-tissue factor complex, factor IXa, and thrombin kcat values are 1.4, 0.32, and 0.061 s-1, respectively. Tissue factor does not increase the factor VII activation rate by factor Xa, factor IXa, or thrombin. Factor VIIIa in the presence of PCPS has no effect on factor VII activation by factor IXa. In contrast, factor Va decreases the factor VII activation rate by factor Xa, reaching saturation at concentrations consistent with complete prothrombinase complex formation. The formed prothrombinase complex activates factor VII at approximately 30% the rate of factor Xa bound to phospholipids. These data allow us to conclude that the predominant physiological factor VII activator is, most likely, membrane-bound factor Xa.

A membrane-mediated catalytic event in prothrombin activation.

Prothrombinase assembly takes place on the surface of unsaturated phosphatidylcholine (PC), phosphatidylserine (PS) membranes in the presence of Ca2+, through the rapid association of membrane-bound factor Va and factor Xa. The present study uses saturated PCPS (75:25, w/w) vesicles to study prothrombinase assembly and catalytic properties in order to differentiate the influences of the membrane upon catalyst assembly, substrate delivery, and peptide bond cleavage. In contrast to studies using unsaturated phospholipid, prothrombin activation studies using saturated PCPS (75:25, w/w) (C14:0, C16:0, and C18:0) revealed up to a 20-fold decrease in prothrombinase activity. C18:0 membranes support at least 50% of the prothrombinase binding capacity (KdVa-Xa = 1 nM and nVa-Xa = 1.1) of C18:1 PCPS (75:25, w/w). Thus, the 95% loss in activity cannot be explained by gross alterations in catalyst concentration or assembly. Stopped-flow studies with saturated lipids demonstrate that factor Va, factor Xa, and prothrombin have decreased kon values. Compensatory changes in koff leave the Kd values for these protein-lipid interactions almost unchanged relative to unsaturated PCPS. The profoundly decreased activation rate on saturated phospholipid membranes as compared to unsaturated phospholipids is in part due to slowed substrate/enzyme delivery caused by the saturated lipids. However, studies using prethrombin-1 and C18:0 PCPS (75:25, w/w) also revealed a 15-fold decrease in activity for preassembled prothrombinase. Although there was a slight change in Km (+2-fold), the major cause of the decrease is an 18-fold decrease in kcat. Similar differences for Km and kcat values were obtained for prothrombin. Substrate delivery is thus only partially responsible for the diminished prothrombinase activity observed with saturated phospholipids. Since the activity of prothrombinase is decreased for both prothrombin and prethrombin-1 principally by reducing kcat, it appears that catalyst formation on saturated phospholipids somehow compromises the proteolytic activity of the enzyme complex. This implies that the phospholipid bilayer serves not merely as a surface for condensing the proteins but also as a functional element of the prothrombinase enzyme.