Phagocyte activation by Trp-Lys-Tyr-Met-Val-Met, acting through FPRL1/LXA4R, is not affected by lipoxin A4.

Lipoxin A4 (LXA4) has been shown to bind to the leucocyte formyl peptide receptor (FPR) homologue, FPRL1, without triggering the biological activities induced by other FPRL1 agonists. We investigated the direct effect of LXA4 as well as the effect on agonist-induced biological responses using transfected HL-60 cells expressing FPR, FPRL1 or FPRL2. LXA4 neither induced an intracellular rise in calcium in these transfectants nor affected the response induced by the peptide Trp-Lys-Tyr-Met-Val-Met (WKYMVM), an agonist that activates cells through FPRL1 and -2. Both agonists induced Erk-2 activation; however, the eicosanoid-induced activity was independent of FPRL1 and FPRL2. Moreover, LXA4 was unable to trigger neutrophil upregulation of complement receptor 3 and respiratory burst, and it had no effect on the responses induced by triggering with WKYMVM. We conclude that LXA4 is unable to affect the WKYMVM-induced signalling through FPRL1 and suggest that it acts through a receptor different from FPRL1.

The synthetic peptide Trp-Lys-Tyr-Met-Val-Met-NH2 specifically activates neutrophils through FPRL1/lipoxin A4 receptors and is an agonist for the orphan monocyte-expressed chemoattractant receptor FPRL2.

Neutrophils express the G protein-coupled N-formyl peptide receptor (FPR) and its homologue FPRL1, whereas monocytes express FPR, FPRL1, and FPRL2, an orphan receptor sharing 83% amino acid identity with FPRL1. FPRL1 is a promiscuous receptor activated by serum amyloid A and by different synthetic peptides, including the hexapeptide Trp-Lys-Tyr-Met-Val-d-Met-NH(2) (WKYMVm). By measuring calcium flux in HL-60 cells transfected with FPR, FPRL1, or FPRL2, we show that WKYMVm activated all three receptors, whereas the l-conformer WKYMVM activated exclusively FPRL1 and FPRL2. The functionality of FPRL2 was further assessed by the ability of HL-60-FPRL2 cells to migrate toward nanomolar concentrations of hexapeptides. The half-maximal effective concentrations of WKYMVM for calcium mobilization in HL-60-FPRL1 and HL-60-FPRL2 cells were 2 and 80 nm, respectively. Those of WKYMVm were 75 pm and 3 nm. The tritiated peptide WK[3,5-(3)H(2)]YMVM bound to FPRL1 (K(D) approximately 160 nm), but not to FPR. The two conformers similarly inhibited binding of (125)I-labeled WKYMVm to FPRL2-expressing cells (IC(50) approximately 2.5-3 micrometer). Metabolic labeling with orthophosphoric acid revealed that FPRL1 was differentially phosphorylated upon addition of the l- or d-conformer, indicating that it induced different conformational changes. In contrast to FPRL1, FPRL2 was already phosphorylated in the absence of agonist and not evenly distributed in the plasma membrane of unstimulated cells. However, both receptors were internalized upon addition of either of the two conformers. Taken together, the results indicate that neutrophils are activated by WKYMVM through FPRL1 and that FPRL2 is a chemotactic receptor transducing signals in myeloid cells.

The synthetic chemoattractant Trp-Lys-Tyr-Met-Val-DMet activates neutrophils preferentially through the lipoxin A(4) receptor.

A D-methionine-containing peptide, Trp-Lys-Tyr-Met-Val-D-Met-NH(2) (WKYMVm), featuring a unique receptor specificity was investigated with respect to its ability to activate neutrophil effector functions. The peptide was found to be more potent than the N-formylated peptide N-formyl-Met-Leu-Phe (fMLF) at inducing neutrophil chemotaxis, mobilization of neutrophil complement receptor 3 (CR3), and activation of the neutrophil NADPH-oxidase. The fact that binding of fML[(3)H]F was inhibited by both fMLF and WKYMVm suggests that N-formyl peptide receptor (FPR) is shared by these peptides. However, the neutrophil response induced by the WKYMVm peptide was insensitive to the fMLF antagonists, cyclosporin H, and Boc-FLFLF that specifically block the function of the FPR. These results suggest that even though WKYMVm may bind FPR the cells are activated preferentially through a receptor distinct from the FPR. Using transfected HL-60 cells expressing either the FPR or its neutrophil homologue FPRL1, also referred to as LXA(4)R because it has been shown to bind lipoxin A(4), we show that WKYMVm is about 300-fold more active at mobilizing intracellular calcium through FPRL1 than through FPR. The WKYMVm activates FPRL1-expressing cells in a cyclosporin H-independent manner with an EC(50 )of around 75 pmol/L, whereas it activates FPR-expressing cells with an EC(50 )of around 25 nmol/L. The observation that exudated cells are primed in their response to WKYMVm suggests that FPRL1/LXA(4)R like FPR is stored in mobilizable organelles. (Blood. 2000;95:1810-1818)

Human complement 5a (C5a) anaphylatoxin receptor (CD88) phosphorylation sites and their specific role in receptor phosphorylation and attenuation of G protein-mediated responses. Desensitization of C5a receptor controls superoxide production but not receptor sequestration in HL-60 cells.

Upon agonist binding, the anaphylatoxin human complement 5a receptor (C5aR) has previously been found to be phosphorylated on the six serine residues of its carboxyl-terminal tail (Giannini, E., Brouchon, L., and Boulay, F. (1995) J. Biol. Chem. 270, 19166-19172). To evaluate the precise roles that specific phosphorylation sites may play in receptor signaling, a series of mutants were expressed transiently in COS-7 cells and stably in the physiologically relevant myeloid HL-60 cells. Ser(334) was found to be a key residue that controls receptor phosphorylation. Phosphorylation of either of two serine pairs, namely Ser(332) and Ser(334) or Ser(334) and Ser(338), was critical for the phosphorylation of C5aR and its subsequent desensitization. Full phosphorylation and desensitization of C5aR were obtained when these serines were replaced by aspartic acid residues. The mutation S338A had no marked effect on the agonist-mediated phosphorylation of C5aR, but it allowed a sustained C5a-evoked calcium mobilization in HL-60 cells. These findings and the ability of the S314A/S317A/S327A/S332A mutant receptor to undergo desensitization indicate that the phosphorylation of Ser(334) and Ser(338) is critical and sufficient for C5aR desensitization. The lack of phosphorylation was found to result not only in a sustained calcium mobilization and extracellular signal-regulated kinase 2 activity but also in the enhancement of the C5a-mediated respiratory burst in neutrophil-like HL-60 cells. For instance, the nonphosphorylatable S332A/S334A mutant receptor triggered a 1.8-2-fold higher production of superoxide as compared with the wild-type receptor. Interestingly, although the desensitization of this mutant was defective, it was sequestered with the same time course and the same efficiency as the wild-type receptor. Thus, in myeloid HL-60 cells, desensitization and sequestration of C5aR appear to occur through divergent molecular mechanisms.

Overexpression of wild-type and catalytically inactive forms of GRK2 and GRK6 fails to alter the agonist-induced phosphorylation of the C5a receptor (CD88): evidence that GRK6 is autophosphorylated in COS-7 cells.

The G protein-coupled receptor kinase family comprises six members (GRK1 to GRK6) that phosphorylate and desensitize a number of agonist-occupied G protein-coupled receptors. Overexpression of the dominant negative mutant GRK2-K220R is often accompanied by an inhibition of the agonist-mediated phosphorylation of G protein-coupled receptors. In the case of the C5a receptor (C5aR), the overexpression of wild-type GRK2 or GRK6 as well as of catalytically inactive forms of these kinases (GRK2-K220R and GRK6-K215R) failed to increase or to inhibit the agonist-mediated phosphorylation of C5aR, respectively. Replacement of Lys215 by an arginine residue in GRK6 yielded a protein with a relative molecular mass of 63 kDa, whereas wild-type GRK6 had a relative molecular mass of 66 kDa on polyacrylamide gel. The mutations S484D and T485D in the catalytically inactive mutant GRK6-K215R resulted in a protein (GRK6-RDD) with the same electrophoretic mobility as wild-type GRK6. Furthermore, in the absence of phosphatase inhibitors, GRK6 was rapidly converted into the 63 kDa species, whereas GRK6-RDD was not. Overepression of GRK6-RDD failed to alter the agonist-mediated phosphorylation of C5aR. Taken together, the results suggest that C5aR is not a substrate for either GRK2 or GRK6 and that GRK6 is very likely autophosphorylated on Ser484 and Thr485 in vivo.

Isolation and characterization of a variant HL60 cell line defective in the activation of the NADPH oxidase by phorbol myristate acetate.

Promyelocytic human leukemia HL60 cells can be differentiated into neutrophil-like cells that exhibit an NADPH oxidase activity through direct stimulation of protein kinase C (PKC) with PMA or through formyl peptide receptor activation. We have isolated a variant HL60 clone that exhibited a conditional PMA-induced oxidative response depending on the agent used for the differentiation. While cells differentiated with DMSO responded to either PMA or N-formyl peptide (N-formyl-Met-Leu-Phe-Lys or fMLFK), cells differentiated with dibutyryl-cAMP (Bt2cAMP) responded to fMLFK but very poorly to PMA. However, in Bt2cAMP-differentiated cells, the expression of the different PKC isoforms was similar to that observed in DMSO-differentiated cells. Moreover, PMA was able to induce a normal phosphorylation of the cytosolic factor p47phox and to fully activate extracellular signal-regulated kinases (Erk1/2). Interestingly, Bt2cAMP-differentiated cells exhibited a strong and sustained O2- production when costimulated with PMA and suboptimal concentrations of fMLFK which were, per se, ineffective. This sustained response was only slightly reduced by the conjunction of the mitogen-activated protein (MAP) kinase kinase (MEK) inhibitor PD98059 and wortmannin, a phosphatidylinositol-3 kinase (PI3K) inhibitor. Variant HL60 cells that were stably transfected with a constitutively active form of Rac1 were able, when differentiated with Bt2cAMP, to secrete oxidant following PMA stimulation. Altogether, the results suggest that, in addition to the phosphorylation of p47phox, the activation of NADPH oxidase requires the activation of a Rac protein through a pathway that diverges at a point upstream of MEK and that is independent of the activation of wortmannin sensitive PI3K.

Inhibition of platelet endothelial cell adhesion molecule-1 synthesis and leukocyte transmigration in endothelial cells by the combined action of TNF-alpha and IFN-gamma.

Endothelial cell (EC) junctions regulate circulating leukocyte extravasation and infiltration at inflammatory sites. Several lines of evidence show that platelet endothelial cell adhesion molecule-1 (PECAM-1), a specific component of EC junctions, is required for leukocyte transmigration through EC monolayers. In this paper, we examined the effects of two inflammatory cytokines, TNF-alpha and IFN-gamma, on PECAM-1 and vascular endothelial-cadherin/catenin organization. We found that the addition of inflammatory cytokines (TNF-alpha plus IFN-gamma in combination, for > or = 24 h) caused PECAM-1 to disappear from EC intercellular contacts. Confocal microscopy indicated that after treatment with the cytokines, PECAM-1 was rapidly internalized. In addition, a strong inhibition of PECAM-1 synthesis and a decrease in PECAM-1 mRNA were observed. This phenomenon was only found when TNF-alpha plus IFN-gamma were used in combination. Adhesion of polymorphonuclear cells to doubly treated EC was increased compared with control cells or cells incubated with TNF-alpha or IFN-gamma separately. This was correlated with an increased expression of intercellular adhesion molecule-1. However, the disappearance of PECAM-1 from cell junctions after treatment with TNF-alpha plus IFN-gamma was accompanied by a marked reduction of leukocyte migration through EC monolayers. The correlation between PECAM-1 level and leukocyte transmigration was supported by transmigration inhibition assays using blocking anti-PECAM-1 mAb. These data indicate that PECAM-1 is a specific target of inflammatory cytokines and suggest that changes in its synthesis and organization might negatively modulate leukocyte recruitment.

Thrombin-induced increase in endothelial permeability is associated with changes in cell-to-cell junction organization.

Thrombin increases endothelial permeability in a rapid and reversible way. This effect requires the catalytic activity of the enzyme and thrombin receptor engagement. Endothelial cell permeability is mostly regulated by intercellular junction organization. In the present study, we investigated whether opening of intercellular gaps after thrombin treatment could be related to changes in adherence-junction molecular organization. By immunofluorescence analysis, we found that thrombin stimulation of endothelial cells caused a marked alteration of the distribution of vascular endothelial (VE)-cadherin and of the associated catenins. These molecules, which are strictly localized at intercellular boundaries in confluent resting cells, were absent in the areas of intercellular retraction. Immunoprecipitation analysis indicated that thrombin disrupted the VE-cadherin/catenin complex. This effect was reversible and correlated with the increase in endothelial permeability. The use of a protein kinase C inhibitor (calphostin C) blocked both thrombin-induced permeability and disassembly of adherence-junction components. We propose that thrombin's effect on endothelial cell junction organization is an important determinant in the increase in endothelial permeability induced by this agent.

Role of the thrombin insertion loop 144-155. Study of thrombin mutations W148G, K154E and a thrombin-based synthetic peptide.

Thrombin is a multifunctional serine protease that plays a critical role in hemostasis. Crystallographic studies revealed that the insertion loop, residues 144-155 (human thrombin B chain numbering) located on the surface of thrombin, might be involved in the access of substrates to the active-site of the enzyme. This loop has also been proposed as a potential candidate for a binding site for thrombomodulin and selected thrombin substrates. In order to examine this hypothesis, we have introduced single amino acid substitutions into the loop 144-155 (W148G, K154E). These point mutations did not result in major changes in thrombin specificity. However, the mutant thrombins presented slight modifications in their catalytic activity on the tripeptidic substrate H-D-Lys-(epsilon-benzyloxycarbonyl)-Pro-Arg-NH-nitroanilide ([K154E]thrombin) or tosyl-Gly-Pro-Arg-NH-nitroanilide ([W148G]thrombin), and in the second-order rate constants of inhibition by antithrombin III ([K154E]thrombin) and ([W148G]thrombin) compared to recombinant wild-type thrombin. Kinetics of fibrinogen hydrolysis were minimally affected by the K154E mutation and were not affected by the W148G mutation. Neither of the mutations affected thrombin interaction with hirudin or its C-terminal tail, protein C activation by thrombin or thrombin-thrombomodulin, or platelet activation. We also examined the properties of a synthetic peptide corresponding to the sequence T147-S158. The synthetic peptide T147-S158 did not inhibit thrombin interaction with fibrin, thrombomodulin or protein C. Together, our results indicate that the thrombin loop 144-155 is indirectly involved in the catalytic function of the enzyme, most probably by limiting the access of the substrates to the catalytic site, and argue against the presence of a recognition exosite for fibrin(ogen), thrombomodulin or platelets within the loop.

Thrombin-induced endothelial cell dysfunction.

Endothelium is a multifunctional organ which can directly influence circulating blood components as well as other cells within the vessel wall. The clotting enzyme thrombin, generated at the surface of damaged endothelium, induces blood coagulation but also exerts a variety of functional effects on the endothelium itself. Thrombin acts on endothelial cells to stimulate synthesis and release of various agents, such as inflammatory mediators, vasoactive substances and growth factors. It causes leukocyte adhesion to the endothelium by triggering expression of adhesion molecules on the cell surface and causes disruption of endothelial permeability properties. The majority of thrombin effects on endothelial cells are mediated by its receptor and require its lytic activity. Differences have been observed among the response to thrombin of endothelial cells of different origin. In general microvascular endothelial cells appear to be particularly sensitive to this enzyme. Thrombin induced microvascular dysfunction can have pathological consequences and contribute to organ reactions to inflammation and ischaemia.

Evidence for a selective inhibitory effect of thrombin on megakaryocyte progenitor growth mediated by the thrombin receptor.

An efficient method for the culture of human megakaryocyte precursors in serum-free medium has been developed facilitating study of the effect of regulators of megakaryocyte growth and maturation without interference by serum-derived factors. We have investigated how megakaryocytes and their precursors respond to the procoagulant enzyme, thrombin. In addition to its already documented agonist effect on mature megakaryocytes, thrombin was found to have a marked inhibitory effect on the growth of megakaryocyte colonies from CD34+ bone marrow cells stimulated by IL3. This inhibitory effect, not previously reported, was selective for megakaryocytic cells. The growth of granulomonocytic and erythroid colonies was not affected. A monoclonal antibody which neutralized the effect of exogenous transforming growth factor beta (TGF beta) was unable to fully neutralize the inhibitory effect of thrombin. With the use of a synthetic peptide, corresponding to the tethered thrombin receptor ligand, and of a recombinant inactive form of thrombin, we provide direct evidence that both the inhibitory effect of thrombin on megakaryocyte proliferation and its agonist effect on mature megakaryocytes are mediated by a receptor analogous to the recently cloned platelet thrombin receptor.

Expression of monocyte chemotactic protein-1 by monocytes and endothelial cells exposed to thrombin.

Thrombin, in addition to being a key enzyme in hemostasis, affects a series of endothelial and leukocyte functions and thus may be involved in the regulation of inflammatory reactions. Because leukocyte recruitment and activation are important events in inflammatory and thrombotic processes, in this study we have examined the possibility that thrombin induces the production of a cytokine chemotactic for mononuclear phagocytes. Human peripheral blood mononuclear cells (PBMC) exposed in vitro to thrombin expressed transcripts of monocyte chemotactic protein-1 (MCP-1; alternative acronyms: JE, monocyte chemotactic and activating factor, tumor-derived chemotactic factor). Thrombin was two- to threefold less effective than endotoxin in inducing MCP-1 transcripts in PBMC. Among circulating mononuclear cells, monocytes were identified as the cells expressing MCP-1 in response to thrombin. Monocytes expressed thrombin receptor transcripts. Boiling, hirudin, antithrombin III, and mutation of the catalytic site serine 205 into alanine) blocked the capacity of thrombin to induce MCP-1 expression. The thrombin receptor-activating peptide mimicked the effect of thrombin in inducing MCP-1 expression. Induction of MCP-1 transcript by thrombin was not reduced by blocking interleukin-1 and tumor necrosis factor, suggesting that these mediators are not involved in thrombin-induced expression of MCP-1. In addition to monocytes, endothelial cells (EC) also expressed MCP-1 in response to thrombin, although at lower levels compared with monocytes. Actinomycin D experiments indicated that induction of MCP-1 by thrombin in PBMC and EC was gene transcription dependent. The inhibition of protein synthesis blocked thrombin-induced MCP-1 expression in PBMC, whereas it superinduced both constitutive and thrombin-inducible expression of MCP-1 in EC, indicating different mechanisms of regulation of this gene in mononuclear phagocytes versus endothelial cells. Thrombin stimulated mononuclear cells and EC to release chemotactic activity for monocytes that could be inhibited by absorption with anti-MCP-1 antibodies. Induction of a chemotactic cytokine for monocytes by thrombin points to the importance of this enzyme in regulating inflammatory processes and further indicates that hemostasis, inflammation, and immunity are strictly interconnected processes.

Ca(2+)-binding properties of the platelet glycoprotein IIb ligand-interacting domain.

Glycoprotein (GP) IIb is the alpha subunit of platelet integrin GPIIb-IIIa. Analysis of the primary structure of this subunit has indicated the presence of four stretches of amino acid residues that are highly conserved among various integrin alpha subunits and that have been suggested to be putative calcium-binding sites. To verify the Ca(2+)-binding capacity of these conserved domains and their implication in integrin adhesive functions, a fragment corresponding to the amino acid sequence of GPIIb from positions 171 to 464 was expressed. The nucleotide sequence coding for this GPIIb domain was generated by polymerase chain reaction, cloned into the pTG1924 expression vector, and expressed in Escherichia coli strain TGE901. The recombinant protein was purified by gel exclusion chromatography and used in equilibrium dialysis experiments. The results demonstrate that the four binding sites can be occupied by Ca2+. Two classes of binding sites can be detected, including two sites with a Kd of 30 microns and two sites of lower affinity with a Kd of 120 microns. Interaction of Ca2+ with these two classes of sites was inhibited by a large excess of Mg2+ or Mn2+, suggesting that these cations are competitive for the same sites on GPIIb. Thus, the four Ca(2+)-binding sites of GPIIb are not similar and exhibit different affinities for divalent ions. To verify the functional implication of these Ca(2+)-binding sites, the effect of Ca2+ on the binding of fibrinogen to the recombinant protein was analyzed using a solid-phase assay. The results indicate that optimal fibrinogen binding occurs when the four calcium-binding sites are occupied and establish the functional importance of this Ca(2+)-binding domain in the ligand-binding activity of GPIIb.

A highly conserved sequence of the Arg-Gly-Asp-binding domain of the integrin beta 3 subunit is sensitive to stimulation.

The Arg-Gly-Asp (RGD)-binding domain of GPIIb-IIIa has been localized in a fragment of the GPIIIa subunit that includes the sequence between amino acids 109 and 171. To examine, in a platelet membrane environment, the activated versus nonactivated status of this domain, we have produced a monoclonal antibody against a synthetic peptide (residues 109-128) located within the RGD-binding region on GPIIIa. This kappa-IgM, named AC7, was specific for GPIIIa peptide 109-128 and interacted only with activated platelets. Fibrinogen, RGDF peptide, and the fibrinogen phi chain decapeptide LGGAKQAGDV inhibited the binding of AC7 to ADP-stimulated platelets. AC7 IgM and "small fragments" inhibited fibrinogen binding and platelet aggregation in a dose-dependent fashion. Induction of AC7 binding by D33C, a monoclonal antibody recognizing the GPIIb 426-437 sequence and stimulating fibrinogen binding, indicated that the GPIIb 426-437 and the GPIIIa 109-128 sequences were both involved in a stimulation-dependent conformational modification of the receptor. AC7 was able to recognize beta subunits other than GPIIIa on leucocyte surfaces but only after cell fixation with glutaraldehyde. The results are consistent with the implication of the RGD-binding domain in receptor ligand interaction on the platelet surface and its conformational modification and exposure upon receptor induction.

Identification of a monoclonal antibody against platelet GPIIb that interacts with a calcium-binding site and induces aggregation.

We have characterized a monoclonal antibody named D33C, specific for platelet glycoprotein (GP) IIb, which induces fibrinogen binding and platelet aggregation. D33C Fab fragments interact with an average of 44,000 +/- 20,000 sites on resting platelet with a Kd value of 0.8 microM. This value decreased to 0.17 microM in the presence of 1 mM EDTA suggesting that Ca2+ chelation increases the antibody affinity. Purified IgGs and Fab fragments exhibit a similar potency and induce binding of fibrinogen and aggregation at levels comparable to those obtained with ADP. D33C-induced platelet aggregation, however, was not inhibited by 1 microM PGE1 and was not associated with a significant [14C]serotonin release, suggesting differences with ADP in the mechanism of activation. Among a large series of synthetic peptides corresponding to potential antigenic sequences within the structure of GPIIb, one peptide with the sequence DIDDNGYPDLIV was found to inhibit D33C activity. This peptide corresponds to a putative calcium-binding site whose sequence is highly homologous to similar sequences present in the alpha subunits of the fibronectin and the vitronectin receptors. Despite this homology, D33C interacts only with platelet GPIIb suggesting that the identified epitope may be differently exposed at the surface of the cells. This antibody may prove to be a valuable tool to study the induction reaction on recombinant GPIIbIIIa expressed in cells that lack the appropriate signal transduction reactions.

Factor IX San Dimas. Substitution of glutamine for Arg-4 in the propeptide leads to incomplete gamma-carboxylation and altered phospholipid binding properties.

DNA sequence analysis of the Factor IX gene from a hemophilia B patient (98% Factor IX antigen; less than 0.01 unit/ml clotting activity) has identified a point mutation in exon II. A guanine to adenine transition causes the substitution of a glutamine codon for an arginine codon at -4 in the propeptide of Factor IX. This variant, termed Factor IX San Dimas, circulates in the plasma as proFactor IX with a mutant 18-amino acid propeptide still attached. Like Factor IX Cambridge (Arg-1----Ser), Factor IX San Dimas is unable to express metal-induced epitopes recognized by conformation-specific polyclonal antibodies. Amino acid analysis of the alkaline hydrolysate indicates that purified Factor IX San Dimas contains a reduced number of gamma-carboxyglutamyl residues compared to Factor IX. However, this protein undergoes metal-induced quenching of the intrinsic fluorescence. In addition, Factor IX San Dimas is unable to interact with phospholipid vesicles. The absence of coagulant activity in Factor IX San Dimas can be attributed to impaired calcium-induced conformational changes and loss in the ability to bind phospholipid vesicles in the presence of calcium ions.

Molecular defects of factor IX Chicago-2 (Arg 145----His) and prothrombin Madrid (Arg 271----cys): arginine mutations that preclude zymogen activation.

Factor IX Chicago-2 and prothrombin Madrid were purified from patients with hemophilia B and congenital dysprothrombinemia, respectively. Each protein displays defects in zymogen activation secondary to the failure to cleave one of the sessile bonds whose cleavage is necessary for full coagulant activity. These proteins were isolated by immunoaffinity chromatography using conformation-specific antibodies directed at either factor IX or prothrombin. Factor IX Chicago-2 is cleaved abnormally by factor XIa, yielding a pattern consistent with the failure to cleave the sessile bond between Arg 145 and Ala 146. Prothrombin Madrid is cleaved abnormally by factor Xa, yielding a pattern consistent with the failure to cleave the sessile bond between Arg 271 and Thr 272. Peptide mapping was performed on reduced and alkylated factor IX, factor IX Chicago-2, prothrombin, and prothrombin Madrid, and the hydrolysates were separated by high-performance liquid chromatography. The mutant peptide in factor IX Chicago-2 was identified by automated Edman degradation as residues 143 through 188 of factor IX, and had a histidine substituted for arginine at residue 145. The mutant peptide identified in prothrombin Madrid corresponds to residues 267 through 285 of prothrombin and has the substitution of cysteine for arginine at residue 271. These mutations, each occurring at arginines, are identical to those in factor IX Chapel Hill and prothrombin Barcelona. These results suggest that a limited repertoire of point mutations, many affecting arginine residues, may be responsible for hereditary defects of the vitamin K-dependent proteins in patients with normal antigen levels.

Effect of propeptide mutations on post-translational processing of factor IX. Evidence that beta-hydroxylation and gamma-carboxylation are independent events.

Post-translational processing of Factor IX includes glycosylation, cleavage of the signal peptide and propeptide, vitamin K-dependent carboxylation of specific glutamic acid residues to form gamma-carboxyglutamic acid, and beta-hydroxylation of aspartic acid at residue 64 to form beta-hydroxyaspartic acid. The human Factor IX cDNA coding sequence was modified in the propeptide region (residue -18 to -1) using oligonucleotide-directed site-specific mutagenesis, and the altered Factor IX cDNA was expressed in Chinese hamster ovary cells. The effects of the mutations on proteolytic processing, gamma-carboxylation, and beta-hydroxylation were assessed by direct structural analysis. After purification, the molecular weight of each of the recombinant Factor IX species and its NH2-terminal amino acid sequence were shown to be identical to those of plasma Factor IX. gamma-Carboxyglutamic acid and beta-hydroxyaspartic acid analyses revealed that recombinant wild-type Factor IX contained 9.2 gamma-carboxyglutamic acid and 0.3 beta-hydroxyaspartic acid residues/molecule compared with 11.4 gamma-carboxyglutamic acid and 0.39 beta-hydroxyaspartic acid residues in plasma Factor IX. When the 18-residue propeptide was deleted or when the cells were grown in the presence of sodium warfarin, secreted Factor IX contained no detectable gamma-carboxyglutamic acid but 0.36 and 0.40 residues of beta-hydroxyaspartic acid, respectively. Point mutations leading to substitution of alanine for phenylalanine at residue -16 or glutamic acid for alanine at residue -10 contained 0.2 and 1.7 gamma-carboxyglutamic acid residues, respectively, and 0.2 residues of beta-hydroxyaspartic acid. These data confirm that the propeptide mutations made do not interfere with proteolytic processing and that the Factor IX propeptide contains a recognition site that designates the adjacent glutamic acid-rich domain for gamma-carboxylation. In contrast, beta-hydroxylation of aspartic acid 64 is an independent process which does not require vitamin K and is mediated through a hydroxylation recognition site in the mature Factor IX, not in the propeptide.

Molecular defect of prothrombin Barcelona. Substitution of cysteine for arginine at residue 273.

Prothrombin Barcelona has been isolated from a patient with a normal prothrombin antigen level but low prothrombin coagulant activity. The activation of this protein is impaired by the absence of one of the two factor Xa-catalyzed cleavages that normally lead to the formation of thrombin. Prothrombin Barcelona and prothrombin were isolated from patient plasma and normal plasma, respectively, in a single-step, high-yield immunoaffinity purification using conformation-specific antibodies immobilized on Sepharose. After reduction and alkylation, the purified proteins were subjected to trypsin hydrolysis. The resulting peptides were separated by reverse-phase high performance liquid chromatography. Comparison of the peptide maps of prothrombin Barcelona and prothrombin demonstrated that a peptide, identified as fragment 274-287 in prothrombin by automated Edman degradation, was missing in the prothrombin Barcelona digest. In the chromatogram derived from prothrombin Barcelona, an additional peptide was observed. The amino acid sequence of this peptide was Ala-Ile-Glu-Gly-Cys-Thr-Ala-Thr-Ser-Glu-Tyr-Gln-Thr-Phe-Phe-Asn-Pro-Arg, corresponding to residues 269-287 in prothrombin except for the substitution of cysteine for arginine at residue 273. The substitution of cysteine for arginine was confirmed by tryptic digestion of 14C-carboxymethylated prothrombin Barcelona. Edman degradation of fragment 269-287 indicated the association of 14C with the cysteine at residue 273. The replacement of arginine by cysteine at residue 273, adjacent to the known factor Xa cleavage site, precludes normal activation of prothrombin Barcelona by factor Xa and the generation of thrombin.

Prothrombin fragment 1 X 2 X 3, a major product of prothrombin activation in human plasma.

The conversion of the blood coagulation zymogen prothrombin to thrombin is associated with the production of several cleavage intermediates and products. In contrast to earlier studies of prothrombin cleavage in chemically defined systems, the current investigation examines the fragmentation of human prothrombin in normal plasma. Radiolabeled prothrombin was added to platelet-poor relipidated normal human plasma, and clotting was initiated with the addition of Ca(II) and kaolin. Analysis of the radiolabeled prothrombin cleavage products by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate and beta-mercaptoethanol identified a heretofore unobserved product of prothrombin activation with an apparent molecular weight of 45,000. This product was identified as fragment 1 X 2 X 3, the NH2-terminal 286 amino acids of prothrombin. The product was isolated from a prothrombin digest by immunoaffinity chromatography using anti-prothrombin:Ca(II) antibodies and by preparative gel electrophoresis. Its amino-terminal sequence is identical to that of prothrombin. Digestion of this product with either Factor Xa or thrombin yields, at a minimum, fragment 1 X 2 and fragment 1. Amino-terminal sequence analysis of the products obtained by digestion with Factor Xa of the unknown activation product indicated 3 amino acid residues at each cycle consistent with the presence of fragment 1, fragment 2, and fragment 3. To unambiguously identify the COOH-terminal amino acid sequence of the product, its factor Xa digestion products were separated by reverse-phase high performance liquid chromatography. Edman degradation of one peptide revealed the complete sequence of fragment 3. On this basis, we identify the Mr 45,000 polypeptide as fragment 1 X 2 X 3 and indicate that it is a prominent product of prothrombin conversion to thrombin when activation occurs in plasma.