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.

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.

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.

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 Arg 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.

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.

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.

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).

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)).

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).

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.

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.

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).

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.

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.

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).

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.

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.

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.

Incorporation of factor Va into prothrombinase is required for coordinated cleavage of prothrombin by factor Xa.

Prothrombin is activated to thrombin by two sequential factor Xa-catalyzed cleavages, at Arg271 followed by cleavage at Arg320. Factor Va, along with phospholipid and Ca2+, enhances the rate of the process by 300,000-fold, reverses the order of cleavages, and directs the process through the meizothrombin pathway, characterized by initial cleavage at Arg320. Previous work indicated reduced rates of prothrombin activation with recombinant mutant factor Va defective in factor Xa binding (E323F/Y324F and E330M/V331I, designated factor VaFF/MI).

Coagulation factor V: a plethora of anticoagulant molecules.

Purpose Of Review: Thrombin is necessary for survival and is produced after activation of prothrombin by prothrombinase at the site of a vascular injury. While the enzyme component of prothrombinase alone, factor Xa, bound to a membrane surface can activate prothrombin, incorporation of the cofactor molecule, factor Va, into prothrombinase results in a five orders of magnitude increase in the catalytic efficiency of factor Xa that provides the physiologic pathway for thrombin generation. While the kinetic constants and the identity of peptide bonds cleaved in prothrombin to generate alpha-thrombin have been long established, the peptidyl portions of the factor Va molecule responsible for its interactions with factor Xa, prothrombin, and the lipid surface are still the subject of intense investigation.

The contribution of amino acid region ASP695-TYR698 of factor V to procofactor activation and factor Va function.

There is strong evidence that a functionally important cluster of amino acids is located on the COOH-terminal portion of the heavy chain of factor Va, between amino acid residues 680 and 709. To ascertain the importance of this region for cofactor activity, we have synthesized five overlapping peptides representing this amino acid stretch (10 amino acids each, HC1-HC5) and tested them for inhibition of prothrombinase assembly and function. Two peptides, HC3 (spanning amino acid region 690-699) and HC4 (containing amino acid residues 695-704), were found to be potent inhibitors of prothrombinase activity with IC(50) values of approximately 12 and approximately 10 microm, respectively.

Structural requirements for expression of factor Va activity.

Thrombin activated factor Va (factor VIIa, residues 1-709 and 1546-2196) has an apparent dissociation constant (Kd,app) for factor Xa within prothrombinase of approximately 0.5 nM. A protease (NN) purified from the venom of the snake Naja nigricollis nigricollis, cleaves human factor V at Asp697, Asp1509, and Asp1514 to produce a molecule (factor VNN) that is composed of a Mr 100,000 heavy chain (amino acid residues 1-696) and a Mr 80,000 light chain (amino acid residues 1509/1514-2196).

Amino acids Glu323, Tyr324, Glu330, and Val331 of factor Va heavy chain are essential for expression of cofactor activity.

We have recently demonstrated that amino acid region 323-331 of factor Va heavy chain (9 amino acids, AP4') contains a binding site for factor Xa (Kalafatis, M., and Beck, D. O.