Biosynthesis and intracellular post-translational processing of normal and mutant platelet glycoprotein GPIb-IX.

The multisubunit leucine-rich glycoprotein (GP) Ib-IX-V complex mediates von Willebrand factor-dependent platelet adhesion at sites of blood-vessel injury. Molecular defects of this receptor are reported to cause the Bernard-Soulier haemorrhagic disorder. To gain insight into the mechanisms controlling expression of normal and defective receptors, we performed pulse-chase metabolic studies and detailed analysis of intracellular processing in GPIb-IX-transfected Chinese-hamster ovary cells. In the native complex, after early subunit association, sugars N-linked to the three subunits are trimmed and sialylated in the Golgi compartment and GPIbalpha undergoes extensive O-glycosylation. Surface biotinylation during chase demonstrated that only fully processed complexes reach the cell surface. Tunicamycin treatment revealed that early N-glycosylation is not required for O-glycosylation of GPIbalpha and surface expression of the complex. Biosynthetic studies were then performed on a Bernard-Soulier variant based on previous description of abnormal GPIbalpha size and decreased surface expression. The mutant complex associated normally, but displayed defective processing of its N-linked sugars and abnormal O-glycosylation of GPIbalpha. Confocal immunofluorescence microscopy revealed that the mutant complexes could reach the cell surface but also accumulated intracellularly, while use of compartment specific markers showed strong co-localization in the endoplasmic reticulum (ER) and ER-to-Golgi intermediate compartments ('ERGIC') and only slight labelling of the cis-Golgi. Blockade before the Golgi was confirmed by brefeldin A treatment, which restored O-glycosylation and processing of N-linked sugars. The present study has shown that transfer from the ER to the Golgi represents an important step for controlling post-translational processing and surface expression of normal GPIb-IX-V complex.

A novel monoclonal antibody against the extracellular domain of GPIbbeta modulates vWF mediated platelet adhesion.

GPIbbeta is disulfide-linked to GPIbalpha to form GPIb, a platelet receptor for von Willebrand factor (vWF). GPIb is in turn non covalently linked to GPIX and GPV to form the GPIb/V/IX complex. Apart from its contribution to controlling surface expression of the complex, the exact function of GPIbbeta is not well established due to a lack of suitable ligands or antibodies. The present report describes a monoclonal antibody (RAM.1) that labeled the 26 kDa GPIbbeta subunit on western blots and coprecipitated the three subunits of the GPIb/IX complex from lysates of platelets and transfected CHO and K562 cells. RAM.1 bound to GPIbbeta deleted of its intracellular domain whereas Gi27, directed against intracellular GPIbbeta, did not. Using synthetic peptides, the RAM.1 epitope was mapped to a putative cysteine loop within the COOH-terminal leucine-rich flanking region. In functional assays, RAM.1 had no effect on platelet aggregation induced by ADP, collagen or thrombin, but inhibited ristocetin induced platelet agglutination and botrocetin induced vWF binding. RAM.1 inhibited adhesion of GPIb/V/IX transfected K562 cells to a vWF matrix under flow, increased their rolling velocity and decreased the resistance of cells to detachment at high shear. This study suggests a role of GPIbbeta in modulating the adhesive properties of GPIb/V/IX and describes a useful tool to analyze the exact functions of GPIbbeta.

The alpha(IIb)beta(3) integrin and GPIb-V-IX complex identify distinct stages in the maturation of CD34(+) cord blood cells to megakaryocytes.

Megakaryocytopoiesis is a complex multistep process involving cell division, endoreplication, and maturation and resulting in the release of platelets into the blood circulation. Megakaryocytes (MK) progressively express lineage-restricted proteins, some of which play essential roles in platelet physiology. Glycoprotein (GP)Ib-V-IX (CD42) and GPIIb (CD41) are examples of MK-specific proteins having receptor properties essential for platelet adhesion and aggregation. This study defined the progressive expression of the GPIb-V-IX complex during in vitro MK maturation and compared it to that of GPIIb, an early MK marker. Human cord blood CD34(+) progenitor cells were cultured in the presence of cytokines inducing megakaryocytic differentiation. GPIb-V-IX expression appeared at day 3 of culture and was strictly dependent on MK cytokine induction, whereas GPIIb was already present in immature CD34(+) cells. Analysis by flow cytometry and of the messenger RNA level both showed that GPV appeared 1 day later than GPIb-IX. Microscopy studies confirmed the late appearance of GPV, which was principally localized in the cytoplasm when GPIb-IX was found on the cell surface, suggesting a delayed program of GPV synthesis and trafficking. Cell sorting studies revealed that the CD41(+)GPV(+) population contained 4N and 8N cells at day 7, and was less effective than CD41(+)GPV(-) cells in generating burst-forming units of erythrocytes or MK colonies. This study shows that the subunits of the GPIb-V-IX complex represent unique surface markers of MK maturation. The genes coding for GPIb-IX and GPV are useful tools to study megakaryocytopoiesis and for tissue-specific or conditional expression in mature MK and platelets. (Blood. 2000;96:4169-4177)

Role of the leucine-rich domain of platelet GPIbalpha in correct post-translational processing--the Nancy I Bernard-Soulier mutation expressed on CHO cells.

The mechanisms governing the biosynthesis and surface expression of platelet adhesive receptors on parent megakaryocytes are as yet poorly understood. In particular, the assembly and processing of the multisubunit glycoprotein (GP) Ib-IX-V complex, a receptor for von Willebrand factor (vWf) is not fully understood. In the present work, these questions were addressed by reproducing a natural mutation of GPIbalpha found in a variant case of Bernard-Soulier syndrome (Nancy I), due to the deletion of leucine 179 in the seventh leucine-rich repeat of the polypeptide. Wild type and mutated GPIbalpha were transfected into CHO cells expressing GPlbbeta and GPIX. Flow cytometry showed surface expression of the three subunits of both GPIb-IX complexes, but GPlbalphadeltaLeu was present at lower levels (20-40%) and was recognized only by a sub class of monoclonal antibodies which epitopes were not modified by the mutation. These properties reproduce the defect found in the patient's platelets, demonstrating the causative nature of the mutation and validate the use of the CHO cells model. Biochemical studies were performed in an attempt to elucidate the mechanism of the conformational change of GPIbalphadeltaLeu. They unexpectedly revealed a major glycosylation deficiency of the mutated GPIbalpha leading to a 40% decrease in molecular weight. The other two subunits of the complex were however normal and present at the plasma membrane. The deletion led to complete functional deficiency with lack of vWf binding of CHOalphadeltaLeu transfected cells in the presence of botrocetin and defective adhesion to a vWf coated surface under static conditions. Finally, in contrast to normal CHOalphabetaIX cells, which displayed rolling and deceleration when perfused over a vWf surface, CHOalphadeltaLeubetaIX cells were unable to roll over or attach to a vWf substratum. These results show that the integrity of the leucine-rich region of GPIbalpha is essential for normal processing and function of the GPIb-IX complex. In addition, these results obtained in a cellular system supported the suspected role of the macroglycopeptide region of GPIbalpha in maintaining a suitable conformation of this multisubunit receptor to perform its adhesive function.

The von Willebrand factor-glycoprotein Ib/V/IX interaction induces actin polymerization and cytoskeletal reorganization in rolling platelets and glycoprotein Ib/V/IX-transfected cells.

Platelet adhesion to sites of vascular injury is initiated by the binding of the platelet glycoprotein (GP) Ib-V-IX complex to matrix-bound von Willebrand factor (vWf). This receptor-ligand interaction is characterized by a rapid on-off rate that enables efficient platelet tethering and rolling under conditions of rapid blood flow. We demonstrate here that platelets adhering to immobilized vWf under flow conditions undergo rapid morphological conversion from flat discs to spiny spheres during surface translocation. Studies of Glanzmann thrombasthenic platelets (lacking integrin alpha(IIb)beta(3)) and Chinese hamster ovary (CHO) cells transfected with GPIb/IX (CHO-Ib/IX) confirmed that vWf binding to GPIb/IX was sufficient to induce actin polymerization and cytoskeletal reorganization independent of integrin alpha(IIb)beta(3). vWf-induced cytoskeletal reorganization occurred independently of several well characterized signaling processes linked to platelet activation, including calcium influx, prostaglandin metabolism, protein tyrosine phosphorylation, activation of protein kinase C or phosphatidylinositol 3-kinase but was critically dependent on the mobilization of intracellular calcium. Studies of Oregon Green 488 1, 2-bis(o-amino-5-fluorophenoxy)ethane-N,N,N',N-tetraacetic acid tetraacetoxymethyl ester-loaded platelets and CHO-Ib/IX cells demonstrated that these cells mobilize intracellular calcium in a shear-dependent manner during surface translocation on vWf. Taken together, these studies suggest that the vWf-GPIb interaction stimulates actin polymerization and cytoskeletal reorganization in rolling platelets via a shear-sensitive signaling pathway linked to intracellular calcium mobilization.

Functional characterization of the human platelet glycoprotein V gene promoter: A specific marker of late megakaryocytic differentiation.

Glycoprotein V (GPV), a subunit of the platelet GPIb-V-IX receptor for von Willebrand factor and thrombin, is specifically found in platelets and mature megakaryocytes. Studies of the GPV gene can therefore provide insight into the mechanisms governing megakaryocyte differentiation. The human GPV promoter was isolated, and elements important for its tissue specific transcriptional activity were localized using systematic DNase I protection and reporter deletion assays. A -1413/+25 fragment inserted into a luciferase reporter construct displayed promoter activity in Dami and HEL but not in K562, HL60, or HeLa cells. Progressive 5' to 3' deletion showed a putative enhancer region in the -1413/-903 segment that contained closely spaced GATA and Ets sites protected from DNase I digestion in Dami extracts. Regions similar to a GPIIb gene repressor were found at -816 and -610, with the first exhibiting repressor activity in Dami and HEL cells and the second protected from DNAse I. Deletions from -362 to -103, an area containing protected sites for Sp1, STAT, and GATA, induced a progressive decrease in activity. The -103/+1 fragment, bearing a proximal Ets footprinted site and a GATA/Ets tandem footprint, displayed 75% activity relative to the full-length promoter and retained cell specificity. In summary, this work defines several regions of the GPV gene promoter important for its activity. It contains megakaryocyte-specific signals, including erythro-megakaryocytic GATA, and Ets cis-acting elements, GPIIb-like repressor domains, and binding sites for ubiquitous factors such as Sp1, ETF, and STAT.

Glycoprotein (GP) Ib-IX-transfected cells roll on a von Willebrand factor matrix under flow. Importance of the GPib/actin-binding protein (ABP-280) interaction in maintaining adhesion under high shear.

Adhesion of platelets to sites of vascular injury is critical for hemostasis and thrombosis and is dependent on the binding of the vascular adhesive protein von Willebrand factor (vWf) to the glycoprotein (GP) Ib-V-IX complex on the platelet surface. A unique but poorly defined characteristic of this receptor/ligand interaction is its ability to support platelet adhesion under conditions of high shear stress. To examine the structural domains of the GPIb-V-IX complex involved in mediating cell adhesion under flow, we have expressed partial (GPIb-IX), complete (GPIb-V-IX), and mutant (GPIbalpha cytoplasmic tail mutants) receptor complexes on the surface of Chinese hamster ovary (CHO) cells and examined their ability to adhere to a vWf matrix in flow-based adhesion assays. Our studies demonstrate that the partial receptor complex (GPIb-IX) supports CHO cell tethering and rolling on a bovine or human vWf matrix under flow. The adhesion was specifically inhibited by an anti-GPIbalpha blocking antibody (AK2) and was not observed with CHO cells expressing GPIbbeta and GPIX alone. The velocity of rolling was dependent on the level of shear stress, receptor density, and matrix concentration and was not altered by the presence of GPV. In contrast to selectins, which mediate cell rolling under conditions of low shear (20-200 s-1), GPIb-IX was able to support cell rolling at both venous (150 s-1) and arterial (1500-10,500 s-1) shear rates. Studies with a mutant GPIbalpha receptor subunit lacking the binding domain for actin-binding protein demonstrated that the association of the receptor complex with the membrane skeleton is not essential for cell tethering or rolling under low shear conditions, but is critical for maintaining adhesion at high shear rates (3000-6000 s-1). These studies demonstrate that the GPIb-IX complex is sufficient to mediate cell rolling on a vWf matrix at both venous and arterial levels of shear independent of other platelet adhesion receptors. Furthermore, our results suggest that the association between GPIbalpha and actin-binding protein plays an important role in enabling cells to remain tethered to a vWf matrix under conditions of high shear stress.

Angiogenesis inhibitor SR 25989 upregulates thrombospondin-1 expression in human vascular endothelial cells and foreskin fibroblasts.

The aim of this work was to evaluate the effects of SR 25989, a member of the thienopyridine family devoid of antiplatelet activity but possessing anti-angiogenic properties, on the regulation of proteins involved in matrix remodeling during wound healing or tumor progression. Human endothelial cells grown in the presence of SR 25989 showed moderate increases in the production of activators (tissue plasminogen activator and urokinase) and one inhibitor (plasminogen activator inhibitor type 1) of fibrinolysis, together with a significant rise in intracellular thrombospondin-1. SR 25989 induced a similar increase in thrombospondin-1 in human foreskin fibroblasts. This over-expression of thrombospondin-1 was correlated to a decrease in cell density. A concomitant increase in the tumor suppressor gene protein p53 was observed in endothelial cells and in fibroblasts, in which the slowing down of proliferation could be related to an accumulation of cells in the S and G2/M phases of the cell cycle. Northern blot analysis revealed a temporary rise in thrombospondin-1 transcripts, followed by a decrease along with a moderate increase in p53 transcripts. Thus the anti-angiogenic properties of SR 25989 appear to result from an upregulation of thrombospondin-1 which is possibly mediated by p53. The thienopyridine SR 25989 could therefore be a good candidate for adjuvant anti-angiogenic therapy in cancer.

Membrane and soluble Fc gamma RII/III modulate the antigen-presenting capacity of murine dendritic epidermal Langerhans cells for IgG-complexed antigens.

Murine dendritic epidermal Langerhans cells (LC) are APC. This implies that LC take up, process, and present Ag to T cells. One way of doing so that could allow Ag internalization is provided by the low affinity receptors for the Fc region of IgG (Fc gamma R), which murine LC are known to express, although their isoform(s) and function(s) have not been defined. By using molecular biology and biochemical approaches, we demonstrated that LC expressed Fc gamma RIIb2 and Fc gamma RIII. Furthermore, LC internalized Fc gamma R by receptor-mediated endocytosis, as observed with gold-labeled anti-Fc gamma RII/III mAb or immune complexes. We demonstrated the biologic relevance of this process by observing that Fc gamma R-mediated Ag internalization improved by approximately 300-fold the Ag-presenting capacity of LC to T cells. Moreover, analysis of cell culture supernatants showed that two forms of soluble Fc gamma R (sFc gamma R) were released by LC: the first most probably was the secreted transmembrane-deleted Fc gamma RII isoform, Fc gamma RIIb3, and the second was a soluble receptor probably derived from the membrane-associated Fc gamma RII/III. The ability of two recombinant forms, corresponding to the two sFc gamma R released by LC, to inhibit Fc gamma R-mediated presentation enhancement was assayed. Preincubation of IgG-complexed Ag with either rsFc gamma R led to a dose-dependent decrease in the Ag-presenting capacity of LC. Taken together, our results suggest that, in vivo, LC express membrane Fc gamma R, which increase their Ag-presenting capacity for IgG-complexed Ag, and release sFc gamma R, which might be able to modulate this Ag presentation.

Release of Fc gamma RIIa2 by activated platelets and inhibition of anti-CD9-mediated platelet aggregation by recombinant Fc gamma RIIa2.

Thrombin-activated human platelets and megakaryocyte cell lines release soluble Fc gamma RII (Fc gamma RIIa2) containing the extracellular and intracellular regions of Fc gamma RIIa1, but lacking the transmembrane domain. Use of polyclonal antibodies directed either against the entire intracytoplasmic tail, or against a peptide located near the C-terminal part of the intracellular region of Fc gamma RIIa2, showed the presence of both a complete form of Fc gamma RIIa2 and a C-terminal truncated form in supernatants of platelets after release of their alpha granule contents and in culture supernatants of megakaryocyte cell lines. Furthermore, recombinant Fc gamma RIIa2 inhibited in a dose-dependent manner Fc-dependent anti-CD9 antibody-induced platelet aggregation. Thus, release of Fc gamma RIIa2 by activated platelets could play an important role in the regulation of platelet activation by immune complexes.

A three-base deletion removing a leucine residue in a leucine-rich repeat of platelet glycoprotein Ib alpha associated with a variant of Bernard-Soulier syndrome (Nancy I).

Leucine-rich repeats are conserved structural motifs present in the four components of the human platelet glycoprotein Ib/IX/V complex receptor for the adhesive protein von Willebrand factor. The absence or abnormality of this complex is responsible for Bernard-Soulier disease, an autosomal recessive bleeding disorder. We report a deletion of leucine 179, located in a highly conserved position of the seventh leucine-rich repeat of GPIb alpha, found in a variant form of Bernard-Soulier disease (Bernard-Soulier Nancy I). Three affected siblings of a family were characterized by absence of ristocetin-induced platelet agglutination, although ADP aggregation was normal. Flow cytometry studies showed detectable amounts of all four members of the GPIb/IX/V complex on the surface of the patients' platelets. Western blotting revealed normal levels of GPIX, decreased levels of GPIb beta and GPV, and < 1% of GPIb alpha. RT-PCR studies showed the presence of mRNA coding for GPIb alpha, GPIb beta, GPIX and GPV. Sequencing showed a three-base deletion which results in the absence of a leucine residue, highly conserved across the seven leucine-rich repeats of GPIb alpha and also within the other members of the leucine-rich glycoprotein family. The absence of the leucine 179 in a patient's GPIb alpha is believed to cause a conformational change in the protein which would account for the lack of binding of most of the MoAbs tested and would be responsible for the absence of von Willebrand factor binding. These results point to the leucine-rich region of GPIb alpha as being required for the correct exposure of the von Willebrand binding site as well as for the correct assembly and stability of the GPIb/IX/V complex on the platelet surface.

Biochemical and molecular basis of Bernard-Soulier syndrome: a review.

Bernard-Soulier syndrome (BSS) is a rare hereditary recessive autosomal bleeding disorder characterized by a prolonged bleeding time, giant platelets, thrombocytopenia, normal platelet aggregation in response to ADP and no agglutination in response to ristocetin. This disease is due to absence or abnormality of the platelet membrane glycoprotein GPIb-IX-V, the receptor for von Willebrand factor. All four genes encoding the complex have been cloned and 17 forms of BSS have to date been characterized at the functional, immunological and molecular levels. The mutations can be divided into two main groups. Firstly, mutations located in leucine rich repeats (LRR), responsible for conformational modifications of the molecule, in some cases higher sensitivity to proteases and loss of adhesive function of the receptor, which is expressed at lower than normal levels at the platelet membrane. When mutations affect the LRR of GPIbalpha, the presence of the other chains varies from normal to residual amounts. When mutations affect the LRR of GPIX, expression of the other chains is strongly diminished, suggesting that GPIX plays a major role in the stability of the complex. A second type of mutations leads to synthesis of a truncated molecule lacking the transmembrane domain and absence of its expression at the platelet surface, while the other chains are present in residual amounts. Expression of recombinant proteins in eukaryotic cells has recently confirmed the results derived from studies of natural mutations. Separate expression of each chain can be obtained, although the presence of all subunits is required for full expression.

Human epidermal Langerhans cells secrete a soluble receptor for IgG (Fc gamma RII/CD32) that inhibits the binding of immune complexes to Fc gamma R+ cells.

Langerhans cells (LC) express Fc gamma RII on their cell surface. In this paper, we demonstrate that these cells also release soluble Fc gamma RII (sFc gamma RII) molecules. LC express transcripts encoding a membrane-associated receptor and a transmembrane-deleted Fc gamma RIIA. The latter form was identified in LC culture supernatants using specific antibodies. CHO cells, transfected with LC-derived cDNA encoding the transmembrane-deleted Fc gamma RIIA, secrete sFc gamma RIIA that include the intracellular domain and exhibit the same backbone as the protein identified in LC supernatants. Secreted sFc gamma RIIA exhibits the same pattern of binding to human and mouse IgG subclasses as do membrane Fc gamma RII and inhibits the binding of immune complexes to Fc gamma RII+ cells. In addition, CHO cells expressing the membrane-associated Fc gamma RIIA release truncated and unstable Fc gamma RIIA molecules that lack the intracellular domain. Thus, sFc gamma RII can result from shedding of membrane molecules and/or from secretion of soluble receptors lacking the transmembrane domain.

Cloning and characterization of the gene encoding the human platelet glycoprotein V. A member of the leucine-rich glycoprotein family cleaved during thrombin-induced platelet activation.

Glycoprotein V (GPV) is a major platelet membrane 82-kDa glycoprotein, missing in the Bernard-Soulier syndrome, that is cleaved when platelets are treated with thrombin. We report the cloning and sequencing of the GPV cDNA and gene obtained by a combination of polymerase chain reaction amplification of platelet mRNA and genomic library screening. The single-copy gene for GPV is contained within 6.5 kilobase pairs (kb) of genomic sequence and has a simple structure with a single intron of 958 base pairs in the 5'-untranslated sequence; the coding sequence is contained within a single exon. The promoter region contains a canonical TATA box, and putative GATA, Ets-1, and Sp1 cis-acting elements. Reverse transcription-polymerase chain reaction analysis on RNAs from cells of different hematopoietic origins revealed that GPV was specifically transcribed from platelets and from cells of the megakaryocytic lineage (megakaryocytes, HEL cells). A single transcript of 4.5 kb for GPV was detected in human platelets by Northern blot analysis. The entire amino acid sequence of GPV was deduced from the cDNA and genomic sequences. Mature GPV was composed of 544 amino acids which contained a single transmembrane domain, a short cytoplasmic domain (16 residues), and a large extracellular domain with 8 potential N-glycosylation sites. Analysis of the extracellular domain revealed the presence of 15 tandem Leu-rich repeats of 24 amino acids with homology to GPIb alpha and identified a cleavage site for thrombin near the COOH terminus with similarity to the A alpha chain of fibrinogen, but no hirudin-like sequence was found.

Identification, in mouse macrophages and in serum, of a soluble receptor for the Fc portion of IgG (Fc gamma R) encoded by an alternatively spliced transcript of the Fc gamma RII gene.

Low affinity Fc gamma R are a heterogeneous group of glycoproteins which exist in transmembrane (TM) as well as in soluble forms. Two membrane isoforms of the murine type II Fc gamma R, Fc gamma RIIb1 and Fc gamma RIIb2, have been described. They result from the translation of alternatively spliced pre-mRNA, Fc gamma RIIb2 lacking sequences of the first intracytoplasmic domain (IC1). Soluble forms of Fc gamma R (sFc gamma R) have previously been shown to result from proteolysis of membrane receptors. We report here the identification, in macrophages, of a mRNA derived from the Fc gamma RII gene by splicing exons encoding the TM and IC1 domains, i.e. corresponding to a TM-deleted Fc gamma RIIb2 mRNA. A soluble protein possibly encoded by this mRNA was identified in macrophage supernatants. In accordance with Fc gamma R nomenclature, we propose to name this new Fc gamma RII isoform Fc gamma RIIb3. It is the most abundant sFc gamma R present in serum, as compared with sFc gamma R resulting from cleavage of membrane Fc gamma R.

Role of cyclic AMP in promoting the thromboresistance of human endothelial cells by enhancing thrombomodulin and decreasing tissue factor activities.

1. The effects of forskolin, prostaglandin E1 (PGE1), dibutyryl cyclic AMP (db cyclic AMP), dibutyryl cyclic GMP (db cyclic GMP) and 3-isobutyl-l-methyl-xanthine (IBMX) were investigated on the expression of tissue factor and thrombomodulin activities on the surface of human saphenous vein endothelial cells (HSVEC) in culture. 2. Forskolin (10(-6) to 10(-4) M), PGE1 (10(-7) to 10(-5) M) and db cyclic AMP (10(-4) to 10(-3) M) caused a concentration-dependent decrease of cytokine-induced tissue factor activity. 3. Similar concentrations of forskolin, PGE1 and db cyclic AMP enhanced significantly constitutive thrombomodulin activity and reversed the decrease of this activity caused by interleukin-1 (IL-1). 4. IBMX (10(-4) M) decreased tissue factor activity and enhanced the effect of forskolin on tissue factor and thrombomodulin activities. 5. Forskolin (10(-4) M) decreased the IL-1-induced tissue factor mRNA and increased the thrombomodulin mRNA level. IL-1 did not change the thrombomodulin mRNA level after 2 h of incubation with HSVEC in culture. 6. Dibutyryl cyclic GMP (10(-4) M to 10(-3) M) did not influence tissue factor or thrombomodulin activity. 7. Our data suggest that elevation of intracellular cyclic AMP levels may participate in the regulation of tissue factor and thrombomodulin expression, thus contributing to promote or restore antithrombotic properties of the endothelium.

The Arg-4 mutant factor IX Strasbourg 2 shows a delayed activation by factor XIa.

We have characterized at the DNA and protein levels a mutant factor IX, factor IX Strasbourg 2, which is responsible for a severe form (< 0.01 U/ml) of haemophilia B. Factor IX Strasbourg 2 has a higher molecular weight than normal factor IX. A mutation G-->A at position 6365 of the gene was demonstrated by DNA sequencing and confirmed by restriction mapping which showed absence of a Hae III site. This leads to the substitution of glutamine for arginine at position -4 of the propeptide. Factor IX Strasbourg 2 was purified from plasma by DEAE Sepharose chromatography and immunoaffinity and relative to normal factor IX, binding of calcium to the mutant protein was clearly reduced in calcium lactate agarose gel. Quantification of gamma-carboxyglutamic acid residues gave about 50% carboxylation as compared to normal factor IX. Microsequencing of the NH2-terminal part of factor IX Strasbourg 2 confirmed the attachment of the propeptide and the mutation Arg-->Gln. Activation of factor IX Strasbourg 2 by purified factor XIa was found to be retarded as compared to normal factor IX, but after activation the mutant factor IXa was able to activate factor X. In conclusion, factor IX Strasbourg 2 circulates with the attached propeptide and shows reduced gamma-carboxylation and delayed activation by factor XIa but a normal capacity to activate factor X after total cleavage by factor XIa.

Thrombin regulates tissue factor and thrombomodulin mRNA levels and activities in human saphenous vein endothelial cells by distinct mechanisms.

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 expression on the cell surface.