Neutrophil Cathepsin G Proteolysis of Protease Activated Receptor 4 Generates a Novel, Functional Tethered Ligand

Platelet-neutrophil interactions regulate ischemic vascular injury. Platelets are activated by serine proteases that cleave protease activated receptor (PAR) amino-termini, resulting in an activating tethered ligand. Neutrophils release cathepsin G (CatG) at sites of injury and inflammation, which activates PAR4 but not PAR1, although the molecular mechanism of CatG-induced PAR4 activation is unknown. We show that blockade of the canonical PAR4 thrombin cleavage site did not alter CatG-induced platelet aggregation, suggesting CatG cleaves a different site than thrombin. Mass spectrometry analysis using PAR4 N-terminus peptides revealed CatG cleavage at Ser67-Arg68. A synthetic peptide, RALLLGWVPTR, representing the tethered ligand resulting from CatG proteolyzed PAR4, induced PAR4-dependent calcium flux and greater platelet aggregation than the thrombin-generated GYPGQV peptide. Mutating PAR4 Ser67 or Arg68 reduced CatG-induced calcium flux without affecting thrombin-induced calcium flux. Dog platelets, which contain a conserved CatG PAR4 Ser-Arg cleavage site, aggregated in response to human CatG and RALLLGWVPTR, while mouse (Ser-Gln) and rat (Ser-Glu) platelets, were unresponsive. Thus, CatG amputates the PAR4 thrombin cleavage site by cleavage at Ser67-Arg68 and activates PAR4 by generating a new functional tethered ligand. These findings support PAR4 as an important CatG signaling receptor and suggest a novel therapeutic approach for blocking platelet-neutrophil-mediated pathophysiologies.

Thrombin Cleaves Prolactin Into a Potent 5.6-kDa Vasoinhibin: Implication for Tissue Repair

Vasoinhibin is an endogenous prolactin (PRL) fragment with profibrinolytic, antivasopermeability, and antiangiogenic effects. The fact that blood clotting, vascular permeability, and angiogenesis are functionally linked during the wound-healing process led us to investigate whether thrombin, a major protease in tissue repair, generates vasoinhibin. Here, we have incubated human PRL with thrombin and analyzed the resulting proteolytic products by Western blot, mass spectrometry, high-performance liquid chromatography purification, recombinant production, and bioactivity. We unveil a main thrombin cleavage site at R48-G49 that rapidly (< 10 minutes) generates a 5.6-kDa fragment (residues 1-48) with full vasoinhibin activity, that is, it inhibited the proliferation, invasion, and permeability of cultured endothelial cells and promoted the lysis of a fibrin clot in plasma with a similar potency to that of a conventional 14-kDa vasoinhibin (residues 1-123). The R48-G49 cleavage site is highly conserved throughout evolution and precedes the intramolecular disulfide bond (C58-C174), thereby allowing the 5.6-kDa vasoinhibin to be released without a reduction step. Furthermore, the 5.6-kDa vasoinhibin is produced by endogenous thrombin during the clotting process. These findings uncover the smallest vasoinhibin known, add thrombin to the list of PRL-cleaving proteases generating vasoinhibin, and introduce vasoinhibin as a thrombin-activated mechanism for the regulation of hemostasis, vasopermeability, and angiogenesis in response to tissue injury.

Cell-Free DNA Promotes Thrombin Autolysis and Generation of Thrombin-Derived C-Terminal Fragments

Cell-free DNA (cfDNA) is the major structural component of neutrophil extracellular traps (NETs), an innate immune response to infection. Antimicrobial proteins and peptides bound to cfDNA play a critical role in the bactericidal property of NETs. Recent studies have shown that NETs have procoagulant activity, wherein cfDNA triggers thrombin generation through activation of the intrinsic pathway of coagulation. We have recently shown that thrombin binds to NETs in vitro and consequently can alter the proteome of NETs. However, the effect of NETs on thrombin is still unknown. In this study, we report that DNA binding leads to thrombin autolysis and generation of multiple thrombin-derived C-terminal peptides (TCPs) in vitro. Employing a 25-residue prototypic TCP, GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE), we show that TCPs bind NETs, thus conferring mutual protection against nuclease and protease degradation. Together, our results demonstrate the complex interplay between coagulation, NET formation, and thrombin cleavage and identify a previously undisclosed mechanism for formation of TCPs.

Factor V is an immune inhibitor that is expressed at increased levels in leukocytes of patients with severe Covid-19

Severe Covid-19 is associated with elevated plasma Factor V (FV) and increased risk of thromboembolism. We report that neutrophils, T regulatory cells (Tregs), and monocytes from patients with severe Covid-19 express FV, and expression correlates with T cell lymphopenia. In vitro full length FV, but not FV activated by thrombin cleavage, suppresses T cell proliferation. Increased and prolonged FV expression by cells of the innate and adaptive immune systems may contribute to lymphopenia in severe Covid-19. Activation by thrombin destroys the immunosuppressive properties of FV. Anticoagulation in Covid-19 patients may have the unintended consequence of suppressing the adaptive immune system.

Big Defensin ApBD1 from the scallop Argopecten purpuratus is an antimicrobial peptide which entraps bacteria through nanonets formation

Antimicrobial peptides (AMPs) are ancient innate immune components. Big defensins is a family of AMPs found in a restricted number of animal phyla, in particular mollusks where they have highly diversified. Big defensins are composed of a highly hydrophobic N-terminal region and a C-terminal β-defensin-like region, stabilized by three disulfide bridges. They have been shown to be active against both Gram-positive, Gram-negative bacteria and fungi. Antimicrobial aggregates called nanonets entrapping bacteria have been recently described as the mechanism of action of the Cg-BigDef1 from the oyster Crassostrea gigas. Specifically, the N-terminal domain of Cg-BigDef1 was identified as responsible of nanonet formation. In order to determine whether nanonets are specific to oyster Cg-BigDef1 or common to other big defensins outside this species, we assessed the potential entrapping of bacteria through nanonets of the big defensin from the scallop Argopecten purpuratus, namely ApBD1. Recombinant ApBD1 was produced as a fusion polypeptide which carried a N-terminal His6 tag, with a thrombin cleavage site before the mature peptide sequence and an unfolded C-terminal domain by mutating the last Cys to Arg. Activity of rApBD1 was assessed against the gram-positive bacteria Staphylococcus aureus SG511. rApBD1 inhibited bacterial growth. Moreover, strong immune staining of rApBD1 in numerous areas surrounding bacteria was observed. Overall, results suggest that rApBD1 entrap bacteria in peptide aggregates similar to those reported to Cg-BigDef1. This study demonstrates the conservation of nanonet formation across big defensins and supports further a role for the N-terminal domain in this conserved process.

Characterization of the Interaction between Tissue Factor Pathway Inhibitor α (TFPIα) and Factor V Species

Activation of factor V (FV) involves removal of its central B-domain following proteolysis at R709, R1018 and R1545. Two evolutionary conserved regions (basic region; BR; residues 964-1008 and acidic region 2; AR2; residues 1493-1538) of the B-domain play an essential role in keeping FV inactive. FV derivatives lacking the BR but retaining AR2 (FVAR) have cofactor-like properties while the BR fragment added in trans blocks their procoagulant function. Physiological important forms of FVAR include: platelet FV, FXa activated FV and FV-short. The latter is a splice variant lacking most of the B-domain, including the BR, yet retains AR2. In normal plasma, FV-short represents <2% of the total FV but is overexpressed in patients affected by the East-Texas bleeding disorder due to a single point mutation or deletion in exon 13. In plasma, FV-short forms a complex with tissue factor pathway inhibitor α (TFPIα) through a high affinity interaction between AR2 and the basic C-terminal region of TFPIα (TFPIα-BR; residues 249-264) which is homologous to FV-BR. It has also been found that FV interacts with TFPIα via its BR, albeit with reduced affinity compared to FVAR. Furthermore, TFPIα and FV levels in plasma appear linked suggesting FV may act as carrier for TFPIα. Collectively these results are puzzling considering the mechanism by which these proteins are thought to interact. How can FV, with its endogenous BR engaged in interactions with AR2, simultaneously interact with TFPIα? To gain more insight into this question, we characterized the binding of TFPIα to different physiologic FV species including full-length (fl) FV, FVa, FV-short and other FVAR species. In direct binding measurements, we found that fluorescently labelled TFPIα-BR (OG488-TFPIα-BR) bound FV-short with high affinity (Kd = 0.66 nM). Unlabeled TFPIα and TFPIα-BR displaced OG488-TFPIα-BR from FVshort equivalently indicating specific binding of the BR region of TFPIα to FV-short. No detectable binding was observed to FVa and the OG488-TFPIα-BR also failed to bind fl-FV. These data indicated that AR2 is required for binding to TFPIα-BR and that the endogenous BR in fl-FV is associated with AR2 and precludes binding to TFPIα-BR. In support of this, thrombin cleavage of FV-short over time during binding measurements showed a gradual and marked decreased in fluorescence which correlated with cleavage at R1545 and release of AR2 as observed by western blotting. Cleavage of fl-FV by thrombin during the binding assay transiently increased fluorescence, indicating that TFPIα-BR binds to cleaved FV which correlated with removal of the endogenous BR (cleavage at R709 and R1018) as shown by western blotting. Subsequent cleavage at R1545 resulted in a decrease in fluorescence and hence binding. Using a FV-derivative that cannot be cleaved at R1018 (R1018Q), no binding of TFPIα-BR could be detected upon thrombin incubation, despite cleavage at R709. Together these data indicate that 1) cleavage of FV at R709 has little, if any influence on disrupting the BR-AR2 interaction; 2) cleavage at R1018 releases endogenous FV BR allowing TFPIα to engage via AR2; and 3) cleavage at R1545 removes AR2 eliminating TFPIα binding. Our data suggests that intramolecular binding of FV BR to AR2 has high affinity. To further assess the difference in apparent affinity of the intramolecular BR for AR2 compared to TFPIα-BR, we compared rates of FV-short activation (± TFPIα-BR) by thrombin to fl-FV and monitored cleavage at R1545. Based on the data, we estimate that intramolecular FV BR binds at least 25-50-fold tighter compared to TFPIα-BR binding to FV-short. Overall, we conclude that TFPIα via its BR binds to FV-short and cleaved forms of FV which retain AR2 but have its BR removed. TFPIa binding to these FV species not only blocks procoagulant function but also delays further cleavage at R1545. FVa and fl-FV do not bind TFPIα and are not regulated by this anticoagulant. Fl-FV must first be cleaved at R709 and R1018 prior to any possible TFPIα binding/regulation. Our data support the findings that TFPIα regulates the procoagulant function of FV-short and dampens thrombin generation by delaying the generation of FVa by tuning the activity of FVAR during the initiation of coagulation. This is especially evident when the coagulation stimulus is weak (e.g. low tissue factor), and much less important with a strong stimulus (e.g. high tissue factor) where other anticoagulant mechanisms dominate. Disclosures Camire: Pfizer: Research Funding.

Residues 1680-1684 in the A3 Domain of Factor VIII Contain a Novel Thrombin-Interactive Site Responsible for Proteolytic Cleavage at Arg1689

Thrombin-catalyzed activation of factor (F)VIII by proteolytic cleavages at Arg372, Arg740, and Arg1689 is essential for the propagation phase of blood coagulation cascade. Activated FVIII (FVIIIa) forms the tenase complex and markedly amplifies FX activation as a cofactor of FIXa. We previously reported that thrombin interacts with FVIII through the A2 domain (residues 392-394 and 484-509) and C2 domain, and these interactions governed the cleavages at Arg740, Arg372 and Arg1689, respectively (Nogami, JBC 2000, 2005, BJH 2008). A previous report suggested, however, that FVIII lacking the C2 domain retained >50% cofactor activity (Wakabayashi, JBC 2010), supportive of the presence of other thrombin-binding site responsible for cleavage at Arg1689 within the A3-C1 domain. Recently, we focused on similar sequence in A3 acidic region to hirugen residues 54-65, and demonstrated that two region of residues 1659-1669 and 1675-1685 might contained the thrombin-binding site(s) responsible for cleavage at Arg1689 by functional and binding experiments using synthetic peptide (Minami. ASH 2015). In this study, to identify the crucial residues, seven acidic clustered residues and two sulfated Tyr residues in these regions as a series of rFVIII mutants were converted to Ala, and 9 single mutants for D1663A, Y1664A, D1665A, D1666A, Y1680A, D1681A, E1682A, D1683A, E1684A and 2 double mutants for D1665A/D1666A, D1683A/E1684A, were prepared by using a BHK system. Specific activities of FVIII in all of 5 mutants (D1663A, Y1664A, D1665A, D1666A, D1665/D1664) in the former region and 4 mutants (Y1680A, D1683A, E1684A, D1683A/E1684A) in the latter region, assessed by a one-stage clotting assay were 40-70% and 50-70% of wild type (WT), respectively. These mutants exhibited the assay discrepancy for FVIII activity between one-stage clotting assay and FVIIIa-dependent FXa generation assay (one-stage < FXa generation assay), guessing possible association with these mutated residues for thrombin reaction. Next, FVIII mutants (10 nM) were examined for activation by thrombin (0.4 nM) in a one-stage clotting assay. Regards FVIII mutants in the 1663-1666 region, FVIII activation by thrombin in all mutants were not significant difference from that in WT. We further examined thrombin-catalyzed cleavage at Arg1689 of these mutants by using SDS-PAGE and Western blot using an anti-FVIII monoclonal antibody recognizing the A3 acidic region for detection. The initial velocity of thrombin cleavage at Arg1689 in these mutants showed no significant difference from that in WT. In addition, the rate of thrombin cleavage at Arg372 in them was also almost similar to that in WT. These results indicated that the 1663-1666 region appeared unlikely to participate in the functional association between FVIII and thrombin. On the other hand, regards the 1680-1684 region, peak activity in FVIII activation by thrombin for D1683A, E1684A and D1683A/E1684A mutants were modestly reduced with an ~60% level of WT, and that for Y1680A mutant was significantly diminished with peak activity of ~30% of WT. FVIII activation by thrombin in D1681A and E1682A mutants was not significant difference from WT, however. Evaluated by SDS-PAGE, the initial velocities of cleavage at Arg1689 of D1681A and E1682A were comparable to WT, whilst those of D1683A, E1684A and D1683A/E1684A mutants were ~60% level of WT. It was note that Y1680A mutant showed a ~10% of velocity rates of WT on the cleavage at Arg1689, supportive of the results obtained from thrombin-catalyzed activation of FVIII. However, the rate of thrombin cleavage at Arg372 in these mutants was almost similar to that in WT. These results suggested that three residues were involved with the cleavage at Arg1689. In conclusion, we for the first time identified that that 1680-1684 residues in A3 acidic region, in particular sulfated Tyr1680, played a key role in thrombin-interactive sites responsible for cleavage at Arg1689. Disclosures No relevant conflicts of interest to declare.

Evaluating the Effects of Fibrinogen αC Mutations on the Ability of Factor XIII to Crosslink the Reactive αC Glutamines (Q237, Q328, Q366)

Fibrinogen (Fbg) levels and extent of fibrin polymerization have been associated with various pathological conditions such as cardiovascular disease, arteriosclerosis, and coagulation disorders. Activated factor XIII (FXIIIa) introduces γ-glutamyl-ε-lysinyl isopeptide bonds between reactive glutamines and lysines in the fibrin network to form a blood clot resistant to fibrinolysis. FXIIIa crosslinks the γ-chains and at multiple sites in the αC region of Fbg. Fbg αC contains a FXIII binding site involving αC (389–402) that is located near three glutamines whose reactivities rank Q237 >> Q366 ≈ Q328. Mass spectrometry and two-dimensional heteronuclear single-quantum correlation nuclear magnetic resonance assays were used to probe the anchoring role that αC E396 may play in controlling FXIII function and characterize the effects of Q237 on the reactivities of Q328 and Q366. Studies with αC (233–425) revealed that the E396A mutation does not prevent the transglutaminase function of FXIII A2 or A2B2. Other residues must play a compensatory role in targeting FXIII to αC. Unlike full Fbg, Fbg αC (233–425) did not promote thrombin cleavage of FXIII, an event contributing to activation. With the αC (233–425) E396A mutant, Q237 exhibited slower reactivities compared with αC wild-type (WT) consistent with difficulties in directing this N-terminal segment toward an anchored FXIII interacting at a weaker binding region. Q328 and Q366 became less reactive when Q237 was replaced with inactive N237. Q237 crosslinking is proposed to promote targeting of Q328 and Q366 to the FXIII active site. FXIII thus uses Fbg αC anchoring sites and distinct Q environments to regulate substrate specificity.

Advanced Strategies for Food-Grade Protein Production: A New E. coli/Lactic Acid Bacteria Shuttle Vector for Improved Cloning and Food-Grade Expression

Food-grade production of recombinant proteins in Gram-positive bacteria, especially in LAB (i.e., Lactococcus, Lactobacillus, and Streptococcus), is of great interest in the areas of recombinant enzyme production, industrial food fermentation, gene and metabolic engineering, as well as antigen delivery for oral vaccination. Food-grade expression relies on hosts generally considered as safe organisms and on clone selection not dependent on antibiotic markers, which limit the overall DNA manipulation workflow, as it can be carried out only in the expression host and not in E. coli. Moreover, many commercial expression vectors lack useful elements for protein purification. We constructed a “shuttle” vector containing a removable selective marker, which allows feasible cloning steps in E. coli and subsequent protein expression in LAB. In fact, the cassette can be easily excised from the selected recombinant plasmid, and the resulting marker-free vector transformed into the final LAB host. Further useful elements, as improved MCS, 6xHis-Tag, and thrombin cleavage site sequences were introduced. The resulting vector allows easy cloning in E. coli, can be quickly converted in a food-grade expression vector and harbors additional elements for improved recombinant protein purification. Overall, such features make the new vector an improved tool for food-grade expression.

Characterization of Interaction of Factor VIII with Engineered Variants of a Single-Chain Variable Antibody Fragment

Introduction Replacement therapy for Hemophilia A requires frequent infusions of Factor VIII (FVIII) due to its relatively short half-life of ~12 h in plasma. Previous attempts to extend this half-life by genetic and chemical modification of FVIII met the barrier of ~20 h, which is a half-life of von Willebrand factor (VWF), a carrier of FVIII in plasma. A single-chain variable antibody fragment (scFv) KM33 was shown to inhibit FVIII activity, and interactions with VWF and the low-density lipoprotein receptor-related protein 1 (LRP), the major clearance receptor of FVIII (Bovenschen et al, 2005, Blood, 106:906-12). A study indicated that scFv KM33 may prolong the half-life of FVIII in mice to the level exceeding that of VWF half-life (Mertens et al, US Patent 2008, 20080219983A1). This would make scFv KM33 a promising tool for new designs of the longer-acting FVIII products. Study objective We aimed to generate a scFv KM33 variant that can delay FVIII clearance but can be removed from FVIII during its activation by thrombin. Such antibody fragment may extend the half-life of FVIII above that of VWF. Experimental design We generated three scFv KM33 variants with different linkers connecting the subunits VL and VH of the antibody fragment. The linkers contained variants of thrombin cleavage sites identical to those on FVIII. The proteins were expressed using a baculovirus system, purified by Ni-affinity and size exclusion chromatography (SEC), and tested for their properties. Results The engineered scFv variants, along with the unmodified KM33, were tested for binding to FVIII by surface plasmon resonance (SPR). All scFv versions demonstrated similar affinity for FVIII (~1 nM). In addition, a selected variant of scFv inhibited FVIII binding to LRP. These showed that the modifications of scFv did not affect its binding to FVIII. Thrombin treatment of the engineered scFv variants resulted in dissociation of their VL and VH domains, verified by SEC. However, the respective rates of thrombin cleavage were slower than that of FVIII. The preparation of a thrombin-cleaved scFv still inhibited the interaction of FVIII with LRP by SPR, similarly to that observed for the unmodified KM33. All variants of scFv inhibited FVIII activity in a thrombin generation assay suggesting that their moiety remained in complex with FVIII upon its activation. Conclusions The rate of thrombin cleavage of sites within FVIII is higher than that of the identical sites within the scFv. This suggests that additional determinants of FVIII (e. g. sulfated tyrosines adjacent to the sites) contribute to the higher rate of cleavage. The cleavage of the linker between the VL and VH subunits of scFv KM33 results in dissociation of the subunits and breakdown of the antibody fragment. This mechanism is likely applicable to any scFv, and may be useful in a broad range of applications involving such ligands. Both subunits of thrombin-cleaved scFv KM33, most likely, re-assemble on FVIII and form a tertiary complex FVIII/VL/VH. In turn, thrombin cleavage of the scFv, complexed with FVIII, does not result in its dissociation from FVIII. These indicate that in such design, an scFv should have lower affinity for FVIII to ensure its release from the complex. Disclosures No relevant conflicts of interest to declare.