Proteolytic Activation Of Factor Research Articles

A Single Cleavage Reaction in the Proteolytic Activation of Factor V to the Cofactor Is Rate-Limiting in a Step That Is Selectively Dependent on Anion Binding Exosite 2 of Thrombin

Human factor V (hFV) is a procofactor with a domain organization of A1-A2-B-A3-C1-C2. It requires proteolytic activation before it can assemble within the prothrombinase complex and function as a cofactor. Activation by thrombin results from cleavages following Arg709, Arg1018 and Arg1545 to release the B domain in two fragments and form heterodimeric factor Va (FVa, A1-A2/A3-C1-C2). While A1-A2/A3-C1-C2 is the prototypic active cofactor, variable cofactor function is attributed to partially cleaved species. Several studies have proposed that meizothrombin (mIIa), an intermediate produced during prothrombin activation, is also an efficient activator of membrane-bound hFV and possibly responsible for FVa formation early in the clotting process. However, this is inconsistent with the proposed importance of anion binding exosite 2 (ABE2) for hFV activation by thrombin which is occluded by the covalently attached propiece in mIIa. We have studied the kinetics of hFV cleavage using a quantitative two-color immunoblotting approach to simultaneously and unambiguously identify intermediates and products containing A1-A2 and/or A3-C1-C2. We have also compared hFV cleavage by thrombin by cleavage with a stable mIIa variant and by mIIa lacking both the 4-carboxyglutamic acid and kringle 1 domains (mIIa-desF1) in the presence of membranes. Addition of 0.1 nM thrombin yielded quantitative cleavage of hFV (50 nM) in ~6 minutes, producing products consistent with cleavage at Arg709 and/or Arg1018. However, the C-terminal intermediate beginning at 1019 but uncleaved at Arg1545 persisted for more than 90 minutes. A3-C1-C2, arising from cleavage at Arg1545, appeared at a ~20-fold slower rate than the A1-A2 species. Thus, for the reaction with thrombin, cleavage at Arg1545 represents the rate limiting step in the conversion of hFV to heterodimeric FVa. Similar rates of hFV consumption and appearance of species cleaved at Arg709 and/or Arg1018 were seen with mIIa. However, cleavage at Arg1545 to produce A3-C1-C2 was substantially slower, by a factor of ~100, than A1-A2 formation. Equivalently large differences in the rates of A1-A2 and A3-C1-C2 formation were evident in the absence of membranes except the rate of all cleavages by mIIa was further reduced. Because the results obtained with mIIa were replicated with mIIa-desF1, the data imply that it is covalent occlusion of ABE2 in these mIIa variants that underlies their selectively impaired ability to cleave at Arg1545 and produce heterodimeric Va but does not affect cleavage at Arg709 or Arg1018. The binding of Xa to activated FV, essential for membrane-dependent prothrombinase assembly, is a prerequisite for cofactor function. We correlated hFV cleavage with the development of cofactor function by binding measurements using Xa inactivated with a fluorescent probe covalently tethered to the active site with a peptidyl chloromethylketone. For hFV cleavage by thrombin, Xa binding and the assembly of prothrombinase proceeded unexpectedly slowly and correlated with cleavage at Arg1545, widely separated in time from rapid cleavage at Arg709 and/or Arg1018. Therefore, slow cleavage at Arg1545 dictates both the formation of heterodimeric FVa and the ability of the product to bind Xa with high affinity to form prothrombinase. Only a small fraction of Xa binding developed within an hour in hFV cleavage reactions initiated with mIIa in comparison to the same concentration of thrombin. Our findings indicate that ABE2 in thrombin variants plays an essential role in the rate-limiting cleavage at Arg1545 required for the formation of heterodimeric FVa and for the assembly of prothrombinase. Occlusion of ABE2 by the covalently linked propiece in mIIa variants greatly impairs their ability to activate hFV to the active cofactor by selectively decreasing the rate of cleavage at Arg1545. The surprising and near absolute need for cleavage at Arg1545 to produce cofactor is consistent with new biochemical and structural evidence implicating an essential role played by an acidic sequence immediately N-terminal to Arg1545 in concert with a basic sequence C-terminal to Arg1018 within the B domain in restricting the binding of Xa to the incompletely cleaved procofactor and preventing cofactor function. DisclosuresKrishnaswamy:Novo Nordisk: Consultancy; Janssen: Consultancy, Research Funding; Portola: Research Funding.

Role of hydrophobic mutations on the binding affinity and stability of blood coagulation factor VIIIa: A computational molecular dynamics and free-energy analysis

Factor VIIIa is a non-covalently bound hetero-trimer among A1, A2 and A3–C1–C2 domains and an essential co-factor for factor IXa enzyme during proteolytic activation of factor X zymogen. The relatively weak interactions between A2 and the interface A1/A3 domains dampen the functional stability of FVIIIa in plasma and results in rapid degradation. We studied the mutational effect of three charged residues (Asp519, Glu665 and Asp666) to several hydrophobic residues by molecular dynamics simulations. Analysis of the binding free energy by MM-PBSA and MM-GBSA methods shows that the mutation of Asp519 and Glu665 residues to either Val or Ala enhance the A2 domain binding affinity in agreement with the experimental site-specific mutagenesis data. Mutation of Asp666 to Val, Tyr, Met and Phe showed largest improvement in the A2-domain binding among the eight hydrophobic mutants studied. Our studies suggest that the enrichment of hydrophobic interactions in the buried surface regions of A2 domain plays crucial role in improving the overall stability of FVIIIa.

A cell adhesion molecule (Ig‐SF/hemolin) from caterpillar regulates cell survival and induces extracellular matrix components in human osteoarthritic chondrocytes (LB528)

Background: Losac is a hemolin from Lonomia obliqua caterpillar that promotes blood coagulation through proteolytic activation of factor X. In HUVECs, Losac increased cell proliferation and inhibited the apoptosis induced by starvation. Additionally, we have observed the cytoprotective effect of rLosac in other cell types, such as neurons and fibroblasts. Recombinant Losac (rLosac) was obtained from bacteria system. This report aims to investigate the potential of rLosac as a chondroprotective agent in human articular chondrocytes and to analyze its capacity to stimulate the production of extracellular matrix molecules and to reduce senescence. Methods: Primary cultures of human chondrocytes were prepared from cartilage of osteoarthritic knee joints obtained from 4 donors (age range: 68‐83) undergoing total knee replacement surgery. The study was carried out in full accordance with local ethics guidelines, and cartilage samples were collected after obtaining written informed consent of the donors. Chondrocytes were treated with rLosac and several parameters were measured: specific senescence‐associated beta‐galactosidase activity, cell viability by the MTT assay, production of extracellular matrix molecules by western blot and immunofluorescence microscopy, and cell cycle by flow cytometry. Results: rLosac was found to be an effective anti‐apoptotic agent, as evidenced by the strong inhibition of cellular death. The cytoprotective effect was observed specially starting from 48h and 72h of treatment, even in the presence of IL‐1β (50 ng/ml) to induce an inflammatory environment. Moreover, rLosac was able to reduce the expression of β‐galactosidade after 6 days of treatment. In addition, rLosac stimulated the production of collagen type I, laminin and fibronectin, and to increase the expression of the receptor of collagen type I, and to decrease the expression of IL‐1β. Conclusion: rLosac has the potential to become a therapeutic agent for protection of articular cartilage against the progression of osteoarthritic diseases.Grant Funding Source: CETICs/FAPESP (Processo No. 2013/07467‐1), CAPES, INCTTox‐CNPq

Functional Regulation of TFPI in Membrane Lipid Rafts.

The assembly of tissue factor-factor VIIa (TF-FVIIa) complex results in the proteolytic activation of factors X (FX) and IX and ultimate thrombin generation. The inhibition of this pathway by the Kunitz-type inhibitor, tissue factor pathway inhibitor (TFPI), involves the formation of a stable TF-FVIIa-FXa-TFPI complex. TFPI in endothelial cells (EC) locates primarily in rafts and caveolae, which are membrane microdomains enriched in cholesterol, glycosphingolipids (GSL) and caveolins, and which regulate the function of TFPI. Since caveolin-1 supports the TFPI-dependent inhibition of TF-FVIIa, we aimed to decipher the role played by the individual components of rafts in the anticoagulant function of cell surface TFPI. To this end, we studied the distribution of TFPI, TF and caveolin-1 by immunofluorescence microscopy, and we assayed the functional activity of TFPI after cholesterol-complexation on EC (EA. hy926 and HUVEC) and HEK293 expressing TFPI or TFPI+caveolin-1, or we used GSL-deficient CHO mutant cell lines. In EC, cholesterol complexation with filipin led to patching of TFPI over the cell surface and reduced inhibition of TF-FVIIa. Extraction of cholesterol from the external leaflet of the membrane with methyl-β-cyclodextrin (M-β-CD) shifted the partition of TFPI from predominantly raft-associated to the non-raft cellular fractions isolated through temperature-induced phase separation of Triton X-114 lysates. Although activation of FX by TF-FVIIa was significantly enhanced by M-β-CD and reversed after cholesterol replenishment, the effect was only modestly affected by the TFPI activity reduction. By immunofluorescence we observed that M-β-CD produced redistribution of both TFPI and TF over the EC and 293 cell surface with apparent segregation into separate domains and complete lack of co-localization. Such accumulations of TF will likely promote strong procoagulant activity when not inhibited by TFPI. Since M-β-CD selectively disrupts the glycerophospholipid-rich regions of the membrane while leaving the caveolar cholesterol virtually intact, we also tested progesterone, which extracts cholesterol specifically from caveolae. Treatment of HEK293 cells with progesterone for 2 hrs reduced significantly the inhibition of TF-FVIIa-dependent activation of FX by TFPI for TFPI+Cav+ cells but not for TFPI+ cells, suggesting that the process was specific for cells that have caveolae. To study the role of GSL for the activity of TFPI, we used Ly-B cells, a GSL-deficient mutant derived from CHO-K1, which have a defect in the LCB1 subunit of serine palmitoyltransferase. Characterization of endogenous TFPI in CHO-K1, Ly-B and its genetically corrected revertant Ly-B/cLCB1 (cLCB) revealed strong similarities between CHOs and EC with regard to the expression and function of TFPI. Whereas not affecting cLCB cells, incubation of Ly-B for 2 days in sphingolipid-deficient medium shifted the partition of cellular TFPI from the detergent-soluble (rafts) to the water-soluble (non-raft) fraction, which suggests that GSL play a major role in the distribution and function of the membrane TFPI. The fundamental knowledge developed by these studies will improve our understanding of the mechanisms by which TFPI functions against TF-FVIIa procoagulant activity on cell surfaces. In the long term, they may guide novel therapeutic approaches to prevent inflammation and thrombosis.

Mapping Factor VIIIa Inter-Subunit Interactions Using Chemical Modification and Zero-Length Cross-Linking.

Activated factor VIII (FVIIIa) is a complex of three subunits, designated A1, A2, and A3C1C2, that serves as a cofactor for factor IXa in the proteolytic activation of factor X. The structure and surface interactions between factor VIIIa subunits have yet to be fully defined, but a homology-based model is available as a predictive tool (Stoylova-McPhie et al, Blood, 2002). To investigate FVIIIa inter-subunit interactions we used a chemical modification approach to determine surface-exposed and potentially interactive residues. The protein modification reagents acetic anhydride and diethylpyrocarbonate (DEPC), that modify lysine and histidine residues, respectively, were used to identify residues that are modified in the free subunits but protected in the bound complex. The modified samples were digested with various proteases and the resulting digests were analyzed by MALDI-TOF mass spectrometry to determine sites of peptide modification (Figure 1). The majority of data observed was in agreement with the A1/A3C1C2 interface in the predicted model. Protection was observed at Lys-89, Lys-142, and Lys-230 in the A1 subunit, all of which are near the A3C1C2 interface. While most His residues were completely modified by DEPC, partial protection was seen at His-1954, His-1957, and His-1961 in A3C1C2, which we have previously shown to be an A1-interactive region (Ansong and Fay, Biochemistry, 2005). In addition to this data, we attempted to define inter-subunit contacts by covalently cross-linking the FVIIIa complex. Using the zero-length cross-linker 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC), which cross-links Glu/Asp residues to Lys, four distinct cross-linked bands were identified by SDS-PAGE (Figure 2). The composition of the cross-linked products was determined using proteolytic digestion and MALDI analysis. The four products correspond to each possible combination of the three subunits. Interestingly, there are no candidate cross-link sites between A1 and A2 in the model, yet this cross-link is the most predominant of the four. One alternative is that the A1–A2 cross-link may involve the A1 337–372 region that is not represented in the model. Taken together, the data are directed toward physically defining interactive regions between FVIIIa subunits and serve to test and supplement current structure models. [Display omitted]

Removal of B-domain Sequences from Factor V Rather than Specific Proteolysis Underlies the Mechanism by Which Cofactor Function Is Realized

Factor V, the precursor of factor Va, circulates in plasma with little or no procoagulant activity. Activity is generated following limited proteolysis indicating that the conversion of factor V to factor Va results in appropriate structural changes, which impart cofactor function. We have produced recombinant partial B-domain-truncated derivatives of factor V (FV(des811-1491) and FV(des811-1491) with Arg(709) and Arg(1545) mutated to Gln) to investigate whether discrete proteolysis within the B-domain followed by a conformational transition is responsible for activation. Direct binding fluorescence measurements as well as steady-state kinetic assays were employed to assess the ability of these factor V derivatives to assemble and function in prothrombinase. In contrast to human factor V, single-chain B-domain-truncated factor V bound to FXa membranes with an affinity that was identical to factor Va. Additionally, it was found that, once this modified derivative was assembled in prothrombinase, it functioned in an equivalent manner to factor Va. Taken together these data support the hypothesis that proteolysis within the B-domain of factor V, although necessary, is incidental to the mechanism by which cofactor function is realized. Instead, our results are more consistent with the interpretation that proteolytic activation of factor V simply eliminates steric and/or conformational constraints contributed by the B-domain that otherwise interfere with discrete binding interactions that govern the eventual function of factor Va.

A serine protease zymogen functions as a pattern-recognition receptor for lipopolysaccharides.

Bacterial lipopolysaccharide (LPS)-induced exocytosis of granular hemocytes is a key component of the horseshoe crab's innate immunity to infectious microorganisms; stimulation by LPS induces the secretion of various defense molecules from the granular hemocytes. Using a previously uncharacterized assay for exocytosis, we clearly show that hemocytes respond only to LPS and not to other pathogen-associated molecular patterns, such as beta-1,3-glucans and peptidoglycans. Furthermore, we show that a granular protein called factor C, an LPS-recognizing serine protease zymogen that initiates the hemolymph coagulation cascade, also exists on the hemocyte surface as a biosensor for LPS. Our data demonstrate that the proteolytic activity of factor C is both necessary and sufficient to trigger exocytosis through a heterotrimeric GTP-binding protein-mediating signaling pathway. Exocytosis of hemocytes was not induced by thrombin, but it was induced by hexapeptides corresponding to the tethered ligands of protease-activated G protein-coupled receptors (PARs). This finding suggested the presence of a PAR-like receptor on the hemocyte surface. We conclude that the serine protease zymogen on the hemocyte surface functions as a pattern-recognition protein for LPS.

Optimization of factor VIII replacement therapy: can structural studies help in evading antibody inhibitors?

Optimization of factor VIII replacement therapy: can structural studies help in evading antibody inhibitors?

Hepsin, a Putative Membrane-associated Serine Protease, Activates Human Factor VII and Initiates a Pathway of Blood Coagulation on the Cell Surface Leading to Thrombin Formation

Previous studies have shown that hepsin is a putative membrane-associated serine protease that is required for cell growth (Torres-Rosado, A., O'Shea, K. S., Tsuji, A., Chou, S.-H., and Kurachi, K. (1993) Proc. Natl. Acad. Sci. U.S. A. 90, 7181 7185). In the present study, we have transfected baby hamster kidney (BHK) cells with a plasmid containing the cDNA for human hepsin and examined these cells for their ability to activate several blood coagulation factors including factors X, IX, VII, prothrombin, and protein C. Little, if any, proteolytic activation of factors X, IX, prothrombin, or protein C was observed when these clotting factors were incubated with hepsin-transfected cells. On the other hand, hepsin-transfected cells proteolytically activated significant concentrations of human factor VII in a time- and calcium-dependent manner, whereas essentially no activation of factor VII was observed in BHK cells transfected with plasmid lacking the cDNA for hepsin. The factor VII activating activity in the hepsin-transfected BHK cell line was confined exclusively to the total membrane fraction and was inhibited > 95% by antibody raised against a fusion protein consisting of maltose-binding protein and the extracellular domain of human hepsin. An active site factor VII mutant, S344A factor VII, was cleaved as readily as plasma-derived factor VII by hepsin-transfected cells, indicating that factor VII was not converted to factor VIIa autocatalytically on the cell surface. In contrast, an activation cleavage site factor VII mutant, R152E factor VII, was not cleaved by hepsin-transfected cells, suggesting that factor VII and S344A factor VII were activated on these cells by cleavage of the Arg152-Ile153 peptide bond. In the copresence of factor VII and factor X, hepsin-transfected BHK cells supported the formation of factor Xa. In addition, in the copresence of factor VII, factor X, and prothrombin, hepsin-transfected BHK cells supported the formation of thrombin. These results strongly suggest that membrane-associated hepsin converts zymogen factor VII to factor VIIa, which in turn, is capable of initiating a coagulation pathway on the cell surface that ultimately leads to thrombin formation.

Factor VIIa Residue Arg290 Is Required for Efficient Activation of the Macromolecular Substrate Factor X

The serine protease factor VIIa (VIIa) in complex with tissue factor is responsible for initiating proteolytic events in the coagulation pathways. Efficient proteolysis by the extrinsic activation complex appears to depend on structural determinants in the cofactor as well as the light and heavy chain domains of VIIa. This study characterizes the functional defect resulting from alanine replacement for R290 in the VIIa protease domain. VIIa R290-->A bound both full-length and soluble tissue factor with affinities indistinguishable from wild-type VIIa, consistent with overall unaltered folding of the mutant protein. The catalytic function of VIIa R290-->A was further demonstrated to be unperturbed when analyzed with three different peptidyl p-nitroanilide substrates, indicating that the function of the catalytic triad is not affected by the mutation. However, proteolytic activation of factor X was diminished due to a 4-5-fold decreased kcat in the presence and a > 10-fold decreased rate in the absence of a negatively charged phospholipid surface. The functional defect resulting from the R290-->A replacement was observed in the presence and absence of cofactor. Within the structural framework of serine protease domains, R290 is predicted to be localized in a surface-exposed loop suggested to contribute to substrate selectivity in other serine proteases, consistent with the proposed functional role of R290 in the proteolytic activation of the natural substrate factor X.

Evaluation of the proteolytic activity of factor D accumulated as an active serine protease in patients with chronic renal failure.

Complement factor D, a complement system serine protease, circulating in vivo as its active form, accumulates in patients with chronic renal failure. The pathophysiological role of this active protease in these patients was examined by studies on activities of excess factor D on 10 synthetic peptide substrates for some usual serine proteases. The most sensitive of these substrates to factor D was Boc-Gln-Ala-Arg-MCA, which is used as a substrate for trypsin. The proteolytic activity of factor D (2.17 unit/mg/h) on this substrate was estimated to be 10(-5)-fold that of trypsin (2.18 x 10(5) unit/mg/h). The activities of factor D on other synthetic substrates were lower. Thus the proteolytic activity of factor D is considered to be very specific for its natural substrate, complement factor B bound with C3b, even when it is highly accumulated in vivo. The inhibitory effects of some serine protease inhibitors used clinically (nafamostat mesilate, sepinostat mesilate, camostat mesilate and gabexate mesilate) on the proteolytic activity of factor D on its natural substrate, factor B, were also investigated. Of these synthetic compounds, nafamostat mesilate was the most effective inhibitor (ID50:25 microM) of the activity of factor D on factor B.

Factor IX Deventer-Evidence for the Heterogeneity of Hemophilia BM

Factor IX Deventer was isolated from the plasma of a patient with severe hemophilia B. The patient was classified as BM because of an abnormal prolongation (2.1 times) of the ox-brain prothrombin time, that could be corrected by addition of antifactor IX serum. Experiments with the isolated factor IX Deventer showed that one of the two peptide bonds involved in the proteolytic activation of factor IX cannot be cleaved by physiological or non-physiological activators (XIa and RVV-X, respectively). Such a defect can explain why the molecule has no procoagulant activity. At present it is not clear why this defect makes factor IX Deventer such an effective inhibitor of the ox-brain prothrombin time. It is proposed that hemophilia BM is a heterogeneous disorder.

Coagulation factor III (tissue factor) interaction with phospholipid vesicles induced by cadmium: characterization of the reconstituted protein-membrane complex.

Coagulation factor III (tissue factor) is a membrane glycoprotein which serves as a cofactor in the proteolytic activation of factor X and factor IX by factor VIIa. Mixing of human placental factor III apoprotein with vesicles of bovine brain phospholipids does not produce significant reconstitution of factor III activity, but, when the mixture of apoprotein and vesicles is made 5 mM with CdCl2, the apoprotein is incorporated into the vesicles. Ultracentrifugation on sucrose density gradients demonstrated that the active factor III-lipid complex formed by reconstitution with vesicles had a density indistinguishable from that of the complex formed by detergent dialysis. Vesicles isolated after centrifugation were shown to range in diameter from 20 nm to over 100 nm using the electron microscope. Gel filtration showed that factor-III activity was associated with all size-classes of vesicles. The presence of factor III activity in the smallest vesicles argues for a specific cadmium-mediated reconstitution of the apoprotein with phospholipid vesicles.