Madhu S. Bajaj

Priming of human neutrophil functions by tumor necrosis factor : enhancement of superoxide anion generation, degranulation, and chemotaxis to chemoattractants C5a and F-Met-Leu-Phe

Recombinant human tumor necrosis factor-α (rTNF) stimulated increased generation of Superoxide anion (O2−) by human neutrophils in a concentration-dependent fashion. Preincubation of human neutrophils with rTNF (2.2–2200 units/ml) for 10 min enhanced the subsequent generation of O2− in response to C5a and f-MetLeu-Phe(FMLP). Recombinant TNF did not enhance O2− generation by neutrophils stimulated with phorbol myristate acetate (PMA). Recombinant TNF alone failed to induce release of myeloperoxidase (MPO) and lysozyme by neutrophils. However, it did enhance the release of MPO and lysozyme by neutrophils stimulated with C5a and FMLP, but not with PMA. Although rTNF alone (0.001–50,000 units/ml) was not chemotactic for neutrophils, preincubation of neutrophils with rTNF (0.001–0.1 units/ml) enhanced the chemotactic activity of suboptimal concentrations of C5a (0.1 nM) and FMLP (5 nM). Neutrophils treated with high concentrations of rTNF (100–10,000 units/ml) showed inhibition of random movement and of chemotaxis induced by C5a or FMLP. We conclude from these studies that rTNF primes neutrophils for enhanced responses to subsequent stimuli and thus may augment the inflammatory response by increased oxidant production and lysosomal enzyme release and promote down-regulation of chemotactic movement.

High Resolution Structures of p-Aminobenzamidine- and Benzamidine-VIIa/Soluble Tissue Factor UNPREDICTED CONFORMATION OF THE 192-193 PEPTIDE BOND AND MAPPING OF Ca2+, Mg2+, Na+, AND Zn2+ SITES IN FACTOR VIIa

Factor VIIa (FVIIa) consists of a γ-carboxyglutamic acid (Gla) domain, two epidermal growth factor-like domains, and a protease domain. FVIIa binds seven Ca2+ ions in the Gla, one in the EGF1, and one in the protease domain. However, blood contains both Ca2+ and Mg2+, and the Ca2+ sites in FVIIa that could be specifically occupied by Mg2+ are unknown. Furthermore, FVIIa contains a Na+ and two Zn2+ sites, but ligands for these cations are undefined. We obtained p-aminobenzamidine-VIIa/soluble tissue factor (sTF) crystals under conditions containing Ca2+, Mg2+, Na+, and Zn2+. The crystal diffracted to 1.8Å resolution, and the final structure has an R-factor of 19.8%. In this structure, the Gla domain has four Ca2+ and three bound Mg2+. The EGF1 domain contains one Ca2+ site, and the protease domain contains one Ca2+, one Na+, and two Zn2+ sites. 45Ca2+ binding in the presence/absence of Mg2+ to FVIIa, Gla-domainless FVIIa, and prothrombin fragment 1 supports the crystal data. Furthermore, unlike in other serine proteases, the amide N of Gly193 in FVIIa points away from the oxyanion hole in this structure. Importantly, the oxyanion hole is also absent in the benzamidine-FVIIa/sTF structure at 1.87Å resolution. However, soaking benzamidine-FVIIa/sTF crystals with d-Phe-Pro-Arg-chloromethyl ketone results in benzamidine displacement, d-Phe-Pro-Arg incorporation, and oxyanion hole formation by a flip of the 192-193 peptide bond in FVIIa. Thus, it is the substrate and not the TF binding that induces oxyanion hole formation and functional active site geometry in FVIIa. Absence of oxyanion hole is unusual and has biologic implications for FVIIa macromolecular substrate specificity and catalysis.

Engineering Kunitz Domain 1 (KD1) of Human Tissue Factor Pathway Inhibitor-2 to Selectively Inhibit Fibrinolysis PROPERTIES OF KD1-L17R VARIANT

Tissue factor pathway inhibitor-2 (TFPI-2) inhibits factor XIa, plasma kallikrein, and factor VIIa/tissue factor; accordingly, it has been proposed for use as an anticoagulant. Full-length TFPI-2 or its isolated first Kunitz domain (KD1) also inhibits plasmin; therefore, it has been proposed for use as an antifibrinolytic agent. However, the anticoagulant properties of TFPI-2 or KD1 would diminish its antifibrinolytic function. In this study, structure-based investigations and analysis of the serine protease profiles revealed that coagulation enzymes prefer a hydrophobic residue at the P2′ position in their substrates/inhibitors, whereas plasmin prefers a positively charged arginine residue at the corresponding position in its substrates/inhibitors. Based upon this observation, we changed the P2′ residue Leu-17 in KD1 to Arg (KD1-L17R) and compared its inhibitory properties with wild-type KD1 (KD1-WT). Both WT and KD1-L17R were expressed in Escherichia coli, folded, and purified to homogeneity. N-terminal sequences and mass spectra confirmed proper expression of KD1-WT and KD1-L17R. Compared with KD1-WT, the KD1-L17R did not inhibit factor XIa, plasma kallikrein, or factor VIIa/tissue factor. Furthermore, KD1-L17R inhibited plasmin with ∼6-fold increased affinity and effectively prevented plasma clot fibrinolysis induced by tissue plasminogen activator. Similarly, in a mouse liver laceration bleeding model, KD1-L17R was ∼8-fold more effective than KD1-WT in preventing blood loss. Importantly, in this bleeding model, KD1-L17R was equally or more effective than aprotinin or tranexamic acid, which have been used as antifibrinolytic agents to prevent blood loss during major surgery/trauma. Furthermore, as compared with aprotinin, renal toxicity was not observed with KD1-L17R.

Fatal Tumor Thrombosis Due to an Inferior Vena Cava Leiomyosarcoma in a Patient With Antiphospholipid Antibody Syndrome

We describe a patient with antiphospholipid antibody syndrome (APS) who died because of relentless inferior vena cava (IVC) tumor thrombosis due to an unsuspected leiomyosarcoma. Laboratory confirmation for APS was provided by functional identification of a lupus anticoagulant and anticardiolipin IgG and anti-beta2-glycoprotein I IgM antibodies. Although sensitive for detecting vascular obstruction, radiocontrast venography and magnetic resonance imaging and angiography detected the IVC thrombosis but failed to distinguish its malignant nature. Concomitant refractory thrombocytopenia prevented further invasive diagnostic and therapeutic maneuvers for progressive, severe IVC thrombosis unresponsive to aggressive treatment of APS. Deep venous thrombosis refractory to anticoagulant and immunomodulatory therapies in a patient with APS may be due to a concomitant underlying malignancy, such as a leiomyosarcoma, causing vascular obstruction.

Platelets Contain Tissue Factor Pathway Inhibitor-2 Derived from Megakaryocytes and Inhibits Fibrinolysis

Background: TFPI-2 inhibits plasma kallikrein, FXIa, and plasmin, but its concentration in normal plasma is insufficient to inhibit clotting or fibrinolysis. Results: Platelets contain TFPI-2 derived from megakaryocytes and binds to platelet FV/Va and circulating FV in late pregnancy when plasma TFPI-2 is ∼7 nm. Conclusion: Platelet-derived TFPI-2 regulates intrinsic coagulation and tPA-induced fibrinolysis. Significance: Platelet-derived TFPI-2 promotes clot stabilization while attenuating intrinsic clotting. Tissue factor pathway inhibitor-2 (TFPI-2) is a homologue of TFPI-1 and contains three Kunitz-type domains and a basic C terminus region. The N-terminal domain of TFPI-2 is the only inhibitory domain, and it inhibits plasma kallikrein, factor XIa, and plasmin. However, plasma TFPI-2 levels are negligible (≤20 pm) in the context of influencing clotting or fibrinolysis. Here, we report that platelets contain significant amounts of TFPI-2 derived from megakaryocytes. We employed RT-PCR, Western blotting, immunohistochemistry, and confocal microscopy to determine that platelets, MEG-01 megakaryoblastic cells, and bone marrow megakaryocytes contain TFPI-2. ELISA data reveal that TFPI-2 binds factor V (FV) and partially B-domain-deleted FV (FV-1033) with Kd ∼9 nm and binds FVa with Kd ∼100 nm. Steady state analysis of surface plasmon resonance data reveal that TFPI-2 and TFPI-1 bind FV-1033 with Kd ∼36–48 nm and bind FVa with Kd ∼252–456 nm. Further, TFPI-1 (but not TFPI-1161) competes with TFPI-2 in binding to FV. These data indicate that the C-terminal basic region of TFPI-2 is similar to that of TFPI-1 and plays a role in binding to the FV B-domain acidic region. Using pull-down assays and Western blots, we show that TFPI-2 is associated with platelet FV/FVa. TFPI-2 (∼7 nm) in plasma of women at the onset of labor is also, in part, associated with FV. Importantly, TFPI-2 in platelets and in plasma of pregnant women inhibits FXIa and tissue-type plasminogen activator-induced clot fibrinolysis. In conclusion, TFPI-2 in platelets from normal or pregnant subjects and in plasma from pregnant women binds FV/Va and regulates intrinsic coagulation and fibrinolysis.

Role of GATA Motifs in Tissue Factor Pathway Inhibitor Gene Expression in Malignant Cells

Tissue factor pathway inhibitor (TFPI) is the primary physiologic inhibitor of tissue factor-induced clotting. The TFPI gene contains three GATA motifs in the region flanking its transcription initiation sites. GATA motifs present in promoters of other genes bind GATA-2 transcription factor and thereby regulate their transcriptional expression. Both TFPI and GATA-2 transcription factor are synthesized by a variety of normal as well as malignant cells including hepatocellular carcinoma HepG2 and bladder carcinoma ECV304. Here, we studied whether the three GATA motifs flanking the transcription initiation sites regulate TFPI gene expression in HepG2 and ECV304 cells by binding to the GATA-2 transcription factor. Synthetic oligonucleotides containing GATA sequences from the TFPI regulatory region formed DNA-protein complexes with HepG2 and ECV304 nuclear extracts in an electrophoretic mobility shift assay. Using a 740-bp fragment (-496/+244) from TFPI regulatory region, the effect of base substitutions at each of the three GATA motifs was studied in a luciferase reporter gene system. TFPI promoter activity in HepG2 cells was increased 3-fold with mutation in one of the three GATA motifs and in ECV304 cells was essentially unchanged with mutations in all three GATA motifs. Thus, GATA motifs appear to serve a tissue-specific regulatory role in TFPI gene expression in malignant cells.

Decoy plasminogen receptor containing a selective Kunitz-inhibitory domain.

Kunitz domain 1 (KD1) of tissue factor pathway inhibitor-2 in which P2′ residue Leu17 (bovine pancreatic trypsin inhibitor numbering) is mutated to Arg selectively inhibits the active site of plasmin with ∼5-fold improved affinity. Thrombin cleavage (24 h extended incubation at a 1:50 enzyme-to-substrate ratio) of the KD1 mutant (Leu17Arg) yielded a smaller molecule containing the intact Kunitz domain with no detectable change in the active-site inhibitory function. The N-terminal sequencing and MALDI-TOF/ESI data revealed that the starting molecule has a C-terminal valine (KD1L17R-VT), whereas the smaller molecule has a C-terminal lysine (KD1L17R-KT). Because KD1L17R-KT has C-terminal lysine, we examined whether it could serve as a decoy receptor for plasminogen/plasmin. Such a molecule might inhibit plasminogen activation as well as the active site of generated plasmin. In surface plasmon resonance experiments, tissue plasminogen activator (tPA) and Glu-plasminogen bound to KD1L17R-KT (Kd ∼ 0.2 to 0.3 μM) but not to KD1L17R-VT. Furthermore, KD1L17R-KT inhibited tPA-induced plasma clot fibrinolysis more efficiently than KD1L17R-VT. Additionally, compared to ε-aminocaproic acid KD1L17R-KT was more effective in reducing blood loss in a mouse liver-laceration injury model, where the fibrinolytic system is activated. In further experiments, the micro(μ)-plasmin–KD1L17R-KT complex inhibited urokinase-induced plasminogen activation on phorbol-12-myristate-13-acetate-stimulated U937 monocyte-like cells, whereas the μ-plasmin–KD1L17R-VT complex failed to inhibit this process. In conclusion, KD1L17R-KT inhibits the active site of plasmin as well as acts as a decoy receptor for the kringle domain(s) of plasminogen/plasmin; hence, it limits both plasmin generation and activity. With its dual function, KD1L17R-KT could serve as a preferred agent for controlling plasminogen activation in pathological processes.