Ursolic Acid Promotes the Sensitization of rhTRAIL-resistant Triple-negative Breast Cancer.

BACKGROUND/AIM: Triple-negative breast cancer (TNBC) can be characterized as the deadliest breast cancer type considering the lack of efficacious therapeutics. Recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL) is an encouraging anti-cancer therapeutic with the capacity to induce apoptosis in cancer cells but there are TNBCs less susceptible to rhTRAIL. The aim of this study was to assess the potential of the natural product ursolic acid (UA) to sensitize of rhTRAIL-resistant TNBCs. MATERIALS AND METHODS: In order to evaluate apoptosis induction in rhTRAIL and UA-treated TNBC BT-20 and HCC1937 cells that are resistant to rhTRAIL, western blot analysis and Annexin V/PI assays were executed. RESULTS: UA increased the expression of death receptors 4 and 5 and decreased the expression of c-FLIPL transcriptionally sensitizing rhTRAIL-resistant TNBC cells to apoptosis induced by rhTRAIL. CONCLUSION: UA is a possible potent sensitizer of rhTRAIL-resistant TNBCs to rhTRAIL-induced apoptosis.

Sensitization of recombinant human tumor necrosis factor-related apoptosis-inducing ligand-resistant malignant melanomas by quercetin.

Malignant melanoma is the most commonly diagnosed skin cancer associated with a high rate of metastasis. Low-stage melanoma is easily treated, but metastatic malignant melanoma is an extremely treatment-resistant malignancy with low survival rates. The application of recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL) for the treatment of metastatic malignant melanoma holds considerable promise because of its selective proapoptotic activity towards cancer cells and not nontransformed cells. Unfortunately, the clinical utilization of rhTRAIL has been terminated due to the resistance of many cancer cells to undergo apoptosis in response to rhTRAIL. However, rhTRAIL-resistance can be abrogated through the cotreatment with compounds derived from 'Mother Nature' such as quercetin that can modulate cellular components responsible for rhTRAIL-resistance. Here, we show that rhTRAIL-resistant malignant melanomas are sensitized by quercetin. Quercetin action is manifested by the upregulation of rhTRAIL-binding receptors DR4 and DR5 on the surface of cancer cells and by increased rate of the proteasome-mediated degradation of the antiapoptotic protein FLIP. Our data provide for a new efficient and nontoxic treatment of malignant melanoma.

TRAIL-Induced Apoptosis in TRAIL-Resistant Breast Carcinoma Through Quercetin Cotreatment.

Breast cancer is the most commonly diagnosed cancer in women. There is a continued interest for the development of more efficacious treatment regimens for breast carcinoma. Recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL) shows potential as a potent anticancer therapeutic for the treatment of breast cancer, whereas displaying minimal toxicity to normal cells. However, the promise of rhTRAIL for the treatment of breast cancer is dismissed by the resistance to rhTRAIL-induced apoptosis exhibited by many breast cancers. Thus, a cotreatment strategy was examined by applying the natural compound quercetin (Q) as a sensitizing agent for rhTRAIL-resistant breast cancer BT-20 and MCF-7 cells. Quercetin was able to sensitize rhTRAIL-resistant breast cancers to rhTRAIL-induced apoptosis as detected by Western blotting through the proteasome-mediated degradation of c-FLIPL and through the upregulation of DR5 expression transcriptionally. Overall, these in vitro findings establish that Q is an effective sensitizing agent for rhTRAIL-resistant breast cancers.

Spellbinding Effects of the Acidic COOH-Terminus of Factor Va Heavy Chain on Prothrombinase Activity and Function.

Human factor Va (hfVa) is the important regulatory subunit of prothrombinase. Recent modeling data have suggested a critical role for amino acid Arg701 of hfVa for human prothrombin (hPro) activation by prothrombinase. Furthermore, it has also been demonstrated that hfVa has a different effect than that of bovine fVa on prethrombin-1 activation by prothrombinase. The difference between the two cofactor molecules was also found within the Asn700-Arg701 dipeptide in the human factor V (hfV) molecule, which is replaced by the Asp-Glu sequence in bfV. As a consequence, we produced a recombinant hfV (rhfV) molecule with the substitution 700NR701→DE. rhfVNR→DE together with the wild-type molecule (rhfVWT) were expressed in COS7 cells, purified, and tested for their capability to function within prothrombinase. Kinetic studies showed that the Kd of rhfVaNR→DE for human fXa as well as the kcat and Km of prothrombinase made with rhfVaNR→DE for hPro activation were similar to the values obtained following hPro activation by prothrombinase made with rhfVaWT. Remarkably, sodium dodecyl sulfate polyacrylamide gel electrophoresis analyses of hPro activation time courses demonstrated that the rate of cleavage of hPro by prothrombinase reconstituted with rhfVaNR→DE was significantly delayed with substantial accumulation of meizothrombin, and delayed thrombin generation, when compared to activation of hPro by prothrombinase made with rhfVaWT. These unanticipated results provide significant insights on the role of the carboxyl-terminal end of the heavy chain of hfVa for hPro cleavage and activation by prothrombinase and show that residues 700NR701 regulate at least in part the enzyme-substrate/product interaction during fibrin clot formation.

Sensitization of rhTRAIL-resistant Triple-negative Breast Carcinoma Through Silibinin Co-Treatment.

BACKGROUND/AIM: Triple-negative breast cancer (TNBC) is the most fatal form of breast cancer due to the shortcomings of therapies. However, recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL) is a promising anticancer therapeutic that possesses the capability to promote the induction of apoptosis in cancer cells, but some TNBCs are resistant to rhTRAIL's pro-apoptotic effects. Therefore, a combinatorial treatment approach with silibinin and rhTRAIL was considered in order to sensitize rhTRAIL-resistant TNBCs. MATERIALS AND METHODS: The co-treatment of rhTRAIL and silibinin's impact on apoptosis induction in rhTRAIL-resistant TNBC BT-20 and HCC1937 cells was inspected via application of Annexin V/PI assays and western blot analysis. RESULTS: Silibinin possessed the ability to sensitize the examined rhTRAIL-resistant TNBC cells to rhTRAIL-induced apoptosis through the up-regulation of death receptors 4 and 5 and the down-regulation of survivin transcriptionally. CONCLUSION: Silibinin is a good sensitizing agent for rhTRAIL-resistant TNBCs.

The Case Back on the TRAIL: Death Receptors as Markers for rhTRAIL Sensitivity.

BACKGROUND: Personalized cancer treatments can be applied to the clinical use of recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL). rhTRAIL holds great promise because of its selectivity for cancer cells. However, rhTRAIL clinical trials were conducted without the screening of patients' tumors for rhTRAIL-binding death receptor (DR)4 and DR5, and the unselected treatment resulted in a lack of clinical benefit. Here we propose an in vitro test to analyze tumor cells isolated from patients for the membrane expression of DRs to determine patient suitability for rhTRAIL treatment. METHODS: Using a panel of malignant melanoma cell lines, the correlation between DR membrane expression and rhTRAIL sensitivity was evaluated. The membrane expression of DR4 and DR5 was examined through staining with anti-DR4 and -DR5 antibodies followed by fluorescence-activated cell sorting. rhTRAIL sensitivity was determined through Annexin-V and propidium iodide staining and Western blotting after rhTRAIL treatment. RESULTS: Here we show a direct correlation between the membrane expression of DRs and rhTRAIL sensitivity. rhTRAIL-sensitive melanoma lines, on average, had nearly 4-fold more DR4 and >2-fold more DR5 than rhTRAIL-resistant lines. For a cancer cell to display rhTRAIL sensitivity, the optimum expression of DRs is essential. To overcome the apoptotic threshold, cancer cells must express DRs >2-fold higher compared with their benign counterpart. CONCLUSION: These data show the potential of this flow cytometry-based assay for the analysis of isolated tumor cells for DR membrane expression. By first determining a patient's susceptibility to rhTRAIL-based treatments, they can be more appropriately placed in rhTRAIL clinical trials and improve rhTRAIL as an anticancer therapeutic.

The Dual Regulatory Role of Amino Acids Leu480 and Gln481 of Prothrombin.

Prothrombin (FII) is activated to α-thrombin (IIa) by prothrombinase. Prothrombinase is composed of a catalytic subunit, factor Xa (fXa), and a regulatory subunit, factor Va (fVa), assembled on a membrane surface in the presence of divalent metal ions. We constructed, expressed, and purified several mutated recombinant FII (rFII) molecules within the previously determined fVa-dependent binding site for fXa (amino acid region 473-487 of FII). rFII molecules bearing overlapping deletions within this significant region first established the minimal stretch of amino acids required for the fVa-dependent recognition exosite for fXa in prothrombinase within the amino acid sequence Ser(478)-Val(479)-Leu(480)-Gln(481)-Val(482). Single, double, and triple point mutations within this stretch of rFII allowed for the identification of Leu(480) and Gln(481) as the two essential amino acids responsible for the enhanced activation of FII by prothrombinase. Unanticipated results demonstrated that although recombinant wild type α-thrombin and rIIa(S478A) were able to induce clotting and activate factor V and factor VIII with rates similar to the plasma-derived molecule, rIIa(SLQ→AAA) with mutations S478A/L480A/Q481A was deficient in clotting activity and unable to efficiently activate the pro-cofactors. This molecule was also impaired in protein C activation. Similar results were obtained with rIIa(ΔSLQ) (where rIIa(ΔSLQ) is recombinant human α-thrombin with amino acids Ser(478)/Leu(480)/Gln(481) deleted). These data provide new evidence demonstrating that amino acid sequence Leu(480)-Gln(481): 1) is crucial for proper recognition of the fVa-dependent site(s) for fXa within prothrombinase on FII, required for efficient initial cleavage of FII at Arg(320); and 2) is compulsory for appropriate tethering of fV, fVIII, and protein C required for their timely activation by IIa.

Amino acid region 1000-1008 of factor V is a dynamic regulator for the emergence of procoagulant activity.

Single chain factor V (fV) circulates as an Mr 330,000 quiescent pro-cofactor. Removal of the B domain and generation of factor Va (fVa) are vital for procoagulant activity. We investigated the role of the basic amino acid region 1000-1008 within the B domain of fV by constructing a recombinant mutant fV molecule with all activation cleavage sites (Arg(709)/Arg(1018)/Arg(1545)) mutated to glutamine (fV(Q3)), a mutant fV molecule with region 1000-1008 deleted (fV(ΔB9)), and a mutant fV molecule containing the same deletion with activation cleavage sites changed to glutamine (fV(ΔB9/Q3)). The recombinant molecules along with wild type fV (fV(WT)) were transiently expressed in COS-7L cells, purified, and assessed for their ability to bind factor Xa (fXa) prior to and following incubation with thrombin. The data showed that fV(Q3) was severely impaired in its interaction with fXa before and after incubation with thrombin. In contrast, KD(app) values for fV(ΔB9) (0.9 nM), fVa(ΔB9) (0.4 nM), and fV(ΔB9/Q3) (0.7 nM) were similar to the affinity of fVa(WT) for fXa (0.3 nM). Two-stage clotting assays revealed that although fV(Q3) was deficient in its clotting activity, fV(ΔB9/Q3) had clotting activity comparable with fVa(WT). The kcat value of prothrombinase assembled with fV(ΔB9/Q3) was minimally affected, whereas the Km value of the reaction was increased 57-fold compared with the Km value obtained with prothrombinase assembled with fVa(WT). These findings strongly suggest that amino acid region 1000-1008 of fV is a regulatory sequence protecting the organisms from spontaneous binding to fXa and unnecessary prothrombinase complex formation, which in turn results in catastrophic physiological consequences.

Cleavage at both Arg306 and Arg506 is required and sufficient for timely and efficient inactivation of factor Va by activated protein C.

Activated protein C (APC) inactivates membrane-bound factor Va following cleavages of the heavy chain at Arg, Arg, and Arg. The objective of this study is to examine which cleavage is most important for inactivation. The recombinant factor V molecules were constructed as follows: factor V (mutations R→Q), factor V (mutations R→Q), and factor V (mutations R→Q and R→Q). The recombinant molecules were expressed in mammalian cells, purified, and assayed prior and after incubation with APC and lipids for 30 min (factor Vai) in clotting assays and in an assay using purified reagents and saturating concentrations of factor Va. Clotting assays demonstrated that wild-type factor Vai (Vai), factor Vai, and factor Vai were devoid of activity, whereas factor Vai maintained approximately 70% activity following a 30 min incubation with APC. Prothrombinase assembled with all mutant cofactor molecules before and after treatment with APC had kinetic constant (Km) values similar to values found with prothrombinase assembled with factor Va. Prothrombinase assembled with factor Vai demonstrated a 20-fold reduction in kcat, whereas prothrombinase assembled with factor Vai had a two-fold reduction in kcat as compared with prothrombinase assembled with factor Va. In contrast, factor Vai and factor Vai did not show any loss in kcat under similar experimental conditions. In conclusion, our data demonstrate that the activity of an APC-treated factor Va molecule bearing a single mutation at Arg or Arg depends on the assay used; and regardless of the assay employed, in the absence of the APC-cleavage sites at Arg and Arg, the active cofactor is unable to be significantly inactivated by APC in the presence of a membrane surface.

Contribution of amino acid region 659-663 of Factor Va heavy chain to the activity of factor Xa within prothrombinase .

Factor Va, the cofactor of prothrombinase, is composed of heavy and light chains associated noncovalently in the presence of divalent metal ions. The COOH-terminal region of the heavy chain contains acidic amino acid clusters that are important for cofactor activity. In this work, we have investigated the role of amino acid region 659-663, which contains five consecutive acidic amino acid residues, by site-directed mutagenesis. We have generated factor V molecules in which all residues were mutated to either lysine (factor V(5K)) or alanine (factor V(5A)). We have also constructed a mutant molecule with this region deleted (factor V(Δ659-663)). The recombinant molecules along with wild-type factor V (factor V(WT)) were transiently expressed in mammalian cells, purified, and assessed for cofactor activity. Two-stage clotting assays revealed that the mutant molecules had reduced clotting activities compared to that of factor Va(WT). Kinetic analyses of prothrombinase assembled with the mutant molecules demonstrated diminished k(cat) values, while the affinity of all mutant molecules for factor Xa was similar to that for factor Va(WT). Gel electrophoresis analyses of plasma-derived and recombinant mutant prothrombin activation demonstrated delayed cleavage of prothrombin at both Arg(320) and Arg(271) by prothrombinase assembled with the mutant molecules, resulting in meizothrombin lingering throughout the activation process. These results were confirmed after analysis of the cleavage of FPR-meizothrombin. Our findings provide new insights into the structural contribution of the acidic COOH-terminal region of factor Va heavy chain to factor Xa activity within prothrombinase and demonstrate that amino acid region 659-663 from the heavy chain of the cofactor contributes to the regulation of the rate of cleavage of prothrombin by prothrombinase.

Cooperative regulation of the activity of factor Xa within prothrombinase by discrete amino acid regions from factor Va heavy chain.

The prothrombinase complex catalyzes the activation of prothrombin to alpha-thrombin. We have repetitively shown that amino acid region (695)DYDY(698) from the COOH terminus of the heavy chain of factor Va regulates the rate of cleavage of prothrombin at Arg(271) by prothrombinase. We have also recently demonstrated that amino acid region (334)DY(335) is required for the optimal activity of prothrombinase. To assess the effect of these six amino acid residues on cofactor activity, we created recombinant factor Va molecules combining mutations at amino acid regions 334-335 and 695-698 as follows: factor V(3K) ((334)DY(335) --> KF and (695)DYDY(698) --> KFKF), factor V(KF/4A) ((334)DY(335) --> KF and (695)DYDY(698) --> AAAA), and factor V(6A) ((334)DY(335) --> AA and (695)DYDY(698) --> AAAA). The recombinant factor V molecules were expressed and purified to homogeneity. Factor Va(3K), factor Va(K4/4A), and factor Va(6A) had reduced affinity for factor Xa, when compared to the affinity of the wild-type molecule (factor Va(Wt)) for the enzyme. Prothrombinase assembled with saturating concentrations of factor Va(3K) had a 6-fold reduced second-order rate constant for prothrombin activation compared to the value obtained with prothrombinase assembled with factor Va(Wt), while prothrombinase assembled with saturating concentrations of factor Va(KF/4A) and factor Va(6A) had approximately 1.5-fold reduced second-order rate constants. Overall, the data demonstrate that amino acid region 334-335 together with amino acid region 695-698 from factor Va heavy chain are part of a cooperative mechanism within prothrombinase regulating cleavage and activation of prothrombin by factor Xa.

Role of the acidic hirudin-like COOH-terminal amino acid region of factor Va heavy chain in the enhanced function of prothrombinase.

Prothrombinase activates prothrombin through initial cleavage at Arg(320) followed by cleavage at Arg(271). This pathway is characterized by the generation of an enzymatically active, transient intermediate, meizothrombin, that has increased chromogenic substrate activity but poor clotting activity. The heavy chain of factor Va contains an acidic region at the COOH terminus (residues 680-709). We have shown that a pentapeptide from this region (DYDYQ) inhibits prothrombin activation by prothrombinase by inhibiting meizothrombin generation. To ascertain the function of these regions, we have created a mutant recombinant factor V molecule that is missing the last 30 amino acids from the heavy chain (factor V(Delta680-709)) and a mutant molecule with the (695)DYDY (698) --> AAAA substitutions (factor V(4A)). The clotting activities of both recombinant mutant factor Va molecules were impaired compared to the clotting activity of wild-type factor Va (factor Va (Wt)). Using an assay employing purified reagents, we found that prothrombinase assembled with factor Va(Delta680-709) displayed an approximately 39% increase in k cat, while prothrombinase assembled with factor Va(4A) exhibited an approximately 20% increase in k cat for the activation of prothrombin as compared to prothrombinase assembled with factor Va(Wt). Gel electrophoresis analyzing prothrombin activation by prothrombinase assembled with the mutant molecules revealed a delay in prothrombin activation with persistence of meizothrombin. Our data demonstrate that the COOH-terminal region of factor Va heavy chain is indeed crucial for coordinated prothrombin activation by prothrombinase because it regulates meizothrombin cleavage at Arg(271) and suggest that this portion of factor Va is partially responsible for the enhanced procoagulant function of prothrombinase.

Contribution of amino acid region 334-335 from factor Va heavy chain to the catalytic efficiency of prothrombinase.

We have demonstrated that amino acids E (323), Y (324), E (330), and V (331) from the factor Va heavy chain are required for the interaction of the cofactor with factor Xa and optimum rates of prothrombin cleavage. We have also shown that amino acid region 332-336 contains residues that are important for cofactor function. Using overlapping peptides, we identified amino acids D (334) and Y (335) as contributors to cofactor activity. We constructed recombinant factor V molecules with the mutations D (334) --> K and Y (335) --> F (factor V (KF)) and D (334) --> A and Y (335) --> A (factor V (AA)). Kinetic studies showed that while factor Va (KF) and factor Va (AA) had a K D for factor Xa similar to the K D observed for wild-type factor Va (factor Va (WT)), the clotting activities of the mutant molecules were impaired and the k cat of prothrombinase assembled with factor Va (KF) and factor Va (AA) was reduced. The second-order rate constant of prothrombinase assembled with factor Va (KF) or factor Va (AA) for prothrombin activation was approximately 10-fold lower than the second-order rate constant for the same reaction catalyzed by prothrombinase assembled with factor Va (WT). We also created quadruple mutants combining mutations in the amino acid region 334-335 with mutations at the previously identified amino acids that are important for factor Xa binding (i.e., E (323)Y (324) and E (330)V (331)). Prothrombinase assembled with the quadruple mutant molecules displayed a second-order rate constant up to 400-fold lower than the values obtained with prothrombinase assembled with factor Va (WT). The data demonstrate that amino acid region 334-335 is required for the rearrangement of enzyme and substrate necessary for efficient catalysis of prothrombin by prothrombinase.

The interaction of fragment 1 of prothrombin with the membrane surface is a prerequisite for optimum expression of factor Va cofactor activity within prothrombinase.

Incorporation of factor (F) Va into prothrombinase directs prothrombin activation by FXa through the meizothrombin pathway, characterized by initial cleavage at Arg(320). We have shown that a pentapeptide with the sequence DYDYQ specifically inhibits this pathway. It has been also established that Hir(54-65)(SO(3)(-)) 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 prothrombin-derived fragments alone or in combination. Activation of prethrombin 1 is slow with cleavages at Arg(320) and Arg(271) occurring with similar rates. Addition of purified fragment 1 to prethrombin 1 accelerates both the rate of cleavage at Arg(320) and thrombin formation. Both reactions were inhibited by Hir(54-65)(SO(3)(-)) while DYDYQ had no significant inhibitory effect on prethrombin 1 cleavage in the absence or presence of fragment 1. Similarly, activation of prethrombin 2 by prothrombinase, is inhibited by Hir(54-65)(SO(3)(-)), but is not affected by DYDYQ. Addition of purified fragment 1*2 to prethrombin 2 accelerates the rate of cleavage at Arg(320) by prothrombinase. This addition also results in a significant inhibition of thrombin formation by DYDYQ and is concurrent with the elimination of the inhibitory effect of Hir(54-65)(SO(3)(-)) on the same reaction. Finally, a membrane-bound ternary complex composed of prethrombin 2/fragment 1*2/Hir(54-65)(SO(3)(-)) is inhibited by DYDYQ. 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 prothrombin by prothrombinase at Arg(320).

Identification of an inactivating cleavage site for alpha-thrombin on the heavy chain of factor Va.

Previous studies of factor (F)Va inactivation on human umbilical vein endothelial cells have shown that alpha-thrombin cleaves the heavy chain near the COOH-terminus to produce a M(r) 97,000 fragment containing the NH(2)-terminal portion of the heavy chain and a M(r) 8,000 peptide containing the rest of the molecule. The alpha-thrombin cleavage appeared to occur between amino acid residues 586 and 654 of FV. This region contains a consensus sequence for alpha-thrombin cleavage located at residues 640-644 (S-S-P-R-S). To test the hypothesis that alpha-thrombin cleaves the FVa heavy chain at Arg(643) and to evaluate the functional importance of this cleavage for FVa inactivation, site-directed mutagenesis was used to create recombinant FV molecules with mutations R(643) --> Q (FV(R643Q)) and R(643) --> A (FV(R643A)). All recombinant molecules were purified to homogeneity and assayed for activity following extended activation with alpha-thrombin. Under similar experimental conditions, appearance of the M(r) 97,000 heavy chain fragment in the plasma and wild-type FVa molecules correlated with partial loss of cofactor activity, while following extended incubation of FV(R643Q) and FV(R643A) with alpha-thrombin no cleavage of the heavy chain at Arg(643) was detected and no presence of the M(r) 97,000 heavy-chain fragment was noticed. Further, no loss in cofactor activity was observed using these mutant recombinant FVa molecules. Our data demonstrate that cleavage of FVa at Arg(643) by alpha-thrombin results in a partially inactive cofactor molecule and provides for an activated protein C (APC)-independent anticoagulant effect of alpha-thrombin.

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.

The structural integrity of anion binding exosite I of thrombin is required and sufficient for timely cleavage and activation of factor V and factor VIII.

Alpha-thrombin has two separate electropositive binding exosites (anion binding exosite I, ABE-I and anion binding exosite II, ABE-II) that are involved in substrate tethering necessary for efficient catalysis. Alpha-thrombin catalyzes the activation of factor V and factor VIII following discrete proteolytic cleavages. Requirement for both anion binding exosites of the enzyme has been suggested for the activation of both procofactors by alpha-thrombin. We have used plasma-derived alpha-thrombin, beta-thrombin (a thrombin molecule that has only ABE-II available), and a recombinant prothrombin molecule rMZ-II (R155A/R284A/R271A) that can only be cleaved at Arg(320) (resulting in an enzymatically active molecule that has only ABE-I exposed, rMZ-IIa) to ascertain the role of each exosite for procofactor activation. We have also employed a synthetic sulfated pentapeptide (DY(SO(3)(-))DY(SO(3)(-))Q, designated D5Q1,2) as an exosite-directed inhibitor of thrombin. The clotting time obtained with beta-thrombin was increased by approximately 8-fold, whereas rMZ-IIa was 4-fold less efficient in promoting clotting than alpha-thrombin under similar experimental conditions. Alpha-thrombin readily activated factor V following cleavages at Arg(709), Arg(1018), and Arg(1545) and factor VIII following proteolysis at Arg(372), Arg(740), and Arg(1689). Cleavage of both procofactors by alpha-thrombin was significantly inhibited by D5Q1,2. In contrast, beta-thrombin was unable to cleave factor V at Arg(1545) and factor VIII at both Arg(372) and Arg(1689). The former is required for light chain formation and expression of optimum factor Va cofactor activity, whereas the latter two cleavages are a prerequisite for expression of factor VIIIa cofactor activity. Beta-thrombin was found to cleave factor V at Arg(709) and factor VIII at Arg(740), albeit less efficiently than alpha-thrombin. The sulfated pentapeptide inhibited moderately both cleavages by beta-thrombin. Under similar experimental conditions, membrane-bound rMZ-IIa cleaved and activated both procofactor molecules. Activation of the two procofactors by membrane-bound rMZ-IIa was severely impaired by D5Q1,2. Overall the data demonstrate that ABE-I alone of alpha-thrombin can account for the interaction of both procofactors with alpha-thrombin resulting in their timely and efficient activation. Because formation of meizothrombin precedes that of alpha-thrombin, our findings also imply that meizothrombin may be the physiological activator of both procofactors in vivo in the presence of a procoagulant membrane surface during the early stages of coagulation.

Completed three-dimensional model of human coagulation factor va. Molecular dynamics simulations and structural analyses.

Factor Va is the critical cofactor for prothrombinase assembly required for timely and efficient prothrombin activation. In the absence of a complete crystal structure for the cofactor, Pellequer et al. [(2000) Thromb. Haemostasis 84, 849-857] proposed an incomplete homology model of factor Va (it lacks 46 amino acids from the carboxyl terminus of the heavy chain), which is a static model in a vacuum. A recently published X-ray structure of activated protein C (APC) inactivated bovine factor Va(i) (without the A2 domain) suggests a completely new arrangement of the C1 and C2 domains as compared with the previously published structure of the recombinant C1 and C2 domains. Our aims were (a) to exchange the C1 and C2 domains of the homology model with the modified bovine C1 and C2 domains using the X-ray structure as a template, (b) to determine by computation the three-dimensional model for the carboxyl-terminal peptide of the factor Va heavy chain (Ser(664)-Arg(709)) and incorporate it into the incomplete model, (c) to obtain a complete model of the cofactor folded in solution that might account for its physiological functions and interactions with other components of prothrombinase, and (d) to use the model in order to understand the mechanism of factor Va inactivation by APC. In the first step a sequence alignment of the human and bovine C1 and C2 domains was performed followed by amino acid changes in the three-dimensional structure where the sequences were not identical. The new model of the C1 and C2 domains was then attached to the homology model. The analysis of the MD simulation data revealed that several domains of the cofactor were significantly displaced during simulation. Using our completed model of human factor Va, we are also demonstrating for the first time that cleavage of membrane-bound normal factor Va as well as membrane-bound factor V(LEIDEN) by APC at Arg(306) is required for the dissociation of the A2 domain from the rest of the molecule. Thus, differences in the inactivation rates of the two cofactor molecules are due to differences in the rate of cleavage at Arg(306). The data demonstrate that our model represents the foundation for the establishment of a complete prothrombinase complex model, which might be successful in describing accurately the ternary protein-protein interaction and thus accounts for experimental observations.