thromboplastin and phenylalanyl-prolyl-arginine-chloromethyl-ketone

Proteolytic cleavage of the peptide bond between Arg(152) and Ile(153) converts the procoagulant protein Factor VII (FVII) to an activated two-chain form (FVIIa). The formation of a salt bridge between Ile(153) and Asp(343) drives the conversion of FVIIa from being zymogen-like to the active form. In the present paper, we describe the novel FVII mutant V154G (Val(154)-->Gly mutation; residue 17 in the chymotrypsin numbering system), found in three FVII-deficient patients, which models a zymogen-like form of FVIIa. Recombinant V154G FVIIa, although normally cleaved, shows markedly reduced activity towards peptidyl substrate and undetectable activity towards macromolecular substrates. Susceptibility of Ile(153) to chemical modification, in either the presence or the absence of tissue factor (TF), suggests that the reduced V154G FVIIa activity is caused by impaired salt-bridge formation, thus resulting in a zymogen-like FVIIa form. The TF-mediated protection from chemical modification of V154A indicated that Gly(154) is responsible for this peculiar feature, and suggests that this region, proximal to the heavy chain N-terminus, is directly involved in the conversion of FVII into FVIIa. V154G FVII was exploited to study the FVII-TF interaction, together with three additional FVII variants that were expressed to serve as models for different FVII forms. The comparison of binding affinities of full-length TF after relipidation in L-alpha-phosphatidylcholine for the zymogen FVII (Arg(152)-->Gln, K (d)=1.04+/-0.27 nM), inactive FVIIa (Ser(344)-->Ala, K (d)=0.27+/-0.06 nM) and a zymogen-like FVIIa (V154G, K (d)=1.15+/-0.16 nM) supports the hypothesis that preferential binding of TF to active FVIIa is insufficient to drive the 10(5)-fold enhancement of FVIIa activity. In addition, the inability of V154G FVIIa to accommodate an inhibitor in the active site, indicating an improperly shaped specificity pocket, would explain the low activity of the zymogen-like form of FVIIa, which is predominant in the absence of TF.

Topics: Alanine; Amino Acid Chloromethyl Ketones; Binding Sites; Enzyme Precursors; Factor VII; Factor VIIa; Humans; Isoleucine; Point Mutation; Protein Conformation; Serine Proteinase Inhibitors; Thromboplastin; Valine

Tissue factor-induced coagulation leads to the generation of a small amount of thrombin, resulting in the formation of a fibrin clot. After clot formation, thrombin generation continues resulting in the activation of thrombin activatable fibrinolysis inhibitor, leading to downregulation of fibrinolysis. In this study, the effect of anticoagulant drugs targeting different steps in the coagulation cascade on clot formation and subsequent breakdown was investigated using a plasma-based clot lysis assay. All drugs tested significantly delayed clot formation; only those drugs targeting activated factor X (FXa) (tissue factor pathway inhibitor, fondaparinux, and low molecular weight heparin) accelerated fibrinolysis. Anticoagulant drugs targeting tissue factor (active site-inactivated recombinant activated factor VII) or thrombin (hirudin and d-phenylalanyl-l-prolyl-l-arginyl chloromethyl ketone) did not affect clot lysis time. In accordance with these findings, it was shown that total thrombin generation, as quantified by the endogenous thrombin potential, was only affected by anticoagulant drugs targeting FXa when all drugs were used in a concentration resulting in doubling of clotting time. Induction of hyperfibrinolysis by anticoagulant drugs directed against FXa might be beneficial as increased clot breakdown might facilitate thrombolysis or prevent re-occlusion. On the other hand, the induction of hyperfibrinolysis by these compounds might increase the risk of bleeding complications.

Topics: Amino Acid Chloromethyl Ketones; Anticoagulants; Blood Coagulation Tests; Dose-Response Relationship, Drug; Factor VII; Factor VIIa; Factor Xa Inhibitors; Fibrinolysis; Heparin, Low-Molecular-Weight; Humans; Nephelometry and Turbidimetry; Oligosaccharides; Recombinant Proteins; Thrombin; Thromboplastin

Protein modification with poly(ethylene glycol) (PEG) can prolong circulatory lifetime and lower protein antigenicity in an animal. These benefits may arise from the proposed mechanism of PEG action, molecular shielding of the protein surface, and lowered interaction with other macromolecules. Proteins that depend on macromolecule association for their function would not seem good targets for PEG modification as the benefits may be mitigated by loss of function. Indeed, high loss of function applied to PEG-modified factor VIIa and to active site-blocked blood clotting factors Xa or IXa was studied. A surprising finding was that PEG-modified, active site-blocked factor VIIa (PEG-VIIai, PEG-40 000) retained 40% of its function despite an 18-fold increase in circulatory lifetime. The discrepancy between functional loss and increased circulatory lifetime was consistent with a process that was limited by the diffusion step of assembly rather than the chemical binding step. The impact of PEG-40 000 on diffusion of VIIai is small (about 3-fold) relative to its potential impact on molecular shielding during the chemical binding step of association. These properties extended to a mutant of VIIai (P10Q/K32E, QE-VIIai) that has 25-fold higher function than wild-type factor VIIai. Overall, properties of PEG-modified proteins can suggest features of the kinetic mechanism and may provide enhanced proteins for anticoagulation therapy.

Topics: Amino Acid Chloromethyl Ketones; Animals; Anticoagulants; Binding Sites; Diffusion; Factor IXa; Factor VIIa; Factor Xa; Factor Xa Inhibitors; Female; Humans; Injections, Intravenous; Isoantibodies; Macromolecular Substances; Mice; Mice, Inbred BALB C; Partial Thromboplastin Time; Polyethylene Glycols; Recombinant Proteins; Substrate Specificity; Thromboplastin

Tissue factor (TF), the primary initiator of blood coagulation in vivo, is expressed in vitro by a variety of cells. Previous efforts to localize TF in tissue and cells have been limited principally to the use of immunological techniques. In the present study, we describe a novel functional probe for TF expression, which can be utilized to localize functional TF in situ in human cells and tissues. This probe, a biotinylated phe-pro-arg-chloro-methyl-ketone-labeled rVIIa (FPR-ck-VIIa), interacts with TF via high-affinity binding sites. The binding of FPR-ck-VIIa, therefore, can be correlated with the ability of TF to activate clotting. In the described studies, TF antigen (TF:Ag) expression was examined immunohistochemically with various TF-specific monoclonal antibodies (MAbs) and was correlated with functional TF expression using our novel TF-binding probe (eg, FPR-ck-VIIa). Initial results indicate that TF:Ag expression correlates with the expression of functional TF (TF:VIIa), and the specificity of both types of probes was confirmed. Parallel antigenic and functional TF expression in situ was demonstrated in various human tumors. We believe this to be the first demonstration of functional TF in situ in human cells and tissues. We suggest that FPR-ck-VIIa should prove a useful reagent for studying the role of TF in the pathogenesis of clotting complications of human disease.

Topics: Amino Acid Chloromethyl Ketones; Antibodies, Monoclonal; Antigens; Factor VIIa; Humans; Neoplasms; Recombinant Proteins; Thromboplastin; Tumor Cells, Cultured

The effects of thrombin, D-phenylalanyl-L-propyl-L-arginine chloromethyl ketone (PPACK)-inhibited thrombin, and thrombin receptor agonist peptide, SFLLRNPNDKYEPF (SFLL, a portion of the receptor unmasked after thrombin cleavage), on the expression of tissue factor (TF) and thrombomodulin by human saphenous vein endothelial cells (HSVECs) in culture were studied. Unstimulated cells contained very low amounts of TF mRNA as measured by the reverse transcriptase-PCR method. Thrombin treatment increased TF mRNA to 8.0 +/- 1.9 (n = 3) times the control level. The increase was detectable within 2 h and declined to near basal level by 6 h. Induction of TF mRNA was not blocked by cycloheximide, treatment with cycloheximide alone also increased TF mRNA levels, and thrombin in combination with cycloheximide further enhanced the accumulation of TF mRNA. Thrombin caused a 14.5 +/- 1.5-fold (n = 5) increase in TF activity on the surface of HSVECs and a 20.5 +/- 1.4-fold (mean +/- S.D., n = 2) increase in the extracellular matrix. The thrombin-induced effects on TF synthesis could be fully reproduced by the thrombin receptor agonist peptide, SFLL, whereas PPACK-inhibited thrombin did not influence TF expression. Thrombin increased thrombomodulin mRNA to 190 +/- 39% (n = 5) of control levels, whereas PPACK-inhibited thrombin or SFLL did not influence thrombomodulin mRNA levels. In contrast, surface-bound thrombomodulin cofactor activity and thrombomodulin antigen in the cell lysates did not change over 24 h of incubation with thrombin. However, thrombin caused a 2-fold increase in thrombomodulin antigen released into the conditioned medium, and immunoelectron microscopy of HSVECs also demonstrated the presence of thrombomodulin vesicles close to the luminal cell surface in thrombin-treated cultures. The Western blot pattern thrombomodulin in the conditioned medium of untreated and thrombin-treated cells was found to be similar, and soluble thrombomodulin occurred mainly as fragments of the cell-associated form. We conclude that the transcriptional control by thrombin causes an increase in both TF and thrombomodulin mRNA. The increase in TF mRNA levels is also paralleled by an increase in surface expression, is dependent on the proteolytic activity of thrombin, and is mediated by the same receptor as the recently cloned thrombin receptor in platelets. Up-regulation of thrombomodulin mRNA levels by thrombin is distinct from this pathway and is associated with unchanged exp

Topics: Amino Acid Chloromethyl Ketones; Amino Acid Sequence; Base Sequence; Cells, Cultured; Cycloheximide; Endothelium, Vascular; Humans; Immunoenzyme Techniques; Kinetics; Molecular Sequence Data; Oligodeoxyribonucleotides; Peptides; Polymerase Chain Reaction; Receptors, Cell Surface; Receptors, Thrombin; RNA; RNA, Messenger; Saphenous Vein; Thrombin; Thromboplastin