The transcription factor ERG regulates a low shear stress-induced anti-thrombotic pathway in the microvasculature.

Endothelial cells actively maintain an anti-thrombotic environment; loss of this protective function may lead to thrombosis and systemic coagulopathy. The transcription factor ERG is essential to maintain endothelial homeostasis. Here, we show that inducible endothelial ERG deletion (ErgiEC-KO) in mice is associated with spontaneous thrombosis, hemorrhages and systemic coagulopathy. We find that ERG drives transcription of the anticoagulant thrombomodulin (TM), as shown by reporter assays and chromatin immunoprecipitation. TM expression is regulated by shear stress (SS) via Krüppel-like factor 2 (KLF2). In vitro, ERG regulates TM expression under low SS conditions, by facilitating KLF2 binding to the TM promoter. However, ERG is dispensable for TM expression in high SS conditions. In ErgiEC-KO mice, TM expression is decreased in liver and lung microvasculature exposed to low SS but not in blood vessels exposed to high SS. Our study identifies an endogenous, vascular bed-specific anticoagulant pathway in microvasculature exposed to low SS.

The endothelial transcription factor ERG mediates Angiopoietin-1-dependent control of Notch signalling and vascular stability.

Notch and Angiopoietin-1 (Ang1)/Tie2 pathways are crucial for vascular maturation and stability. Here we identify the transcription factor ERG as a key regulator of endothelial Notch signalling. We show that ERG controls the balance between Notch ligands by driving Delta-like ligand 4 (Dll4) while repressing Jagged1 (Jag1) expression. In vivo, this regulation occurs selectively in the maturing plexus of the mouse developing retina, where Ang1/Tie2 signalling is active. We find that ERG mediates Ang1-dependent regulation of Notch ligands and is required for the stabilizing effects of Ang1 in vivo. We show that Ang1 induces ERG phosphorylation in a phosphoinositide 3-kinase (PI3K)/Akt-dependent manner, resulting in ERG enrichment at Dll4 promoter and multiple enhancers. Finally, we demonstrate that ERG directly interacts with Notch intracellular domain (NICD) and ß-catenin and is required for Ang1-dependent ß-catenin recruitment at the Dll4 locus. We propose that ERG coordinates Ang1, ß-catenin and Notch signalling to promote vascular stability.

Von Willebrand factor and angiogenesis: basic and applied issues.

The recent discovery that von Willebrand factor (VWF) regulates blood vessel formation has opened a novel perspective on the function of this complex protein. VWF was discovered as a key component of hemostasis, capturing platelets at sites of endothelial damage and synthesized in megakaryocytes and endothelial cells (EC). In recent years, novel functions and binding partners have been identified for VWF. The finding that loss of VWF in EC results in enhanced, possibly dysfunctional, angiogenesis is consistent with the clinical observations that in some patients with von Willebrand disease (VWD), vascular malformations can cause severe gastrointestinal (GI) bleeding. In vitro and in vivo studies indicate that VWF can regulate angiogenesis through multiple pathways, both intracellular and extracellular, although their relative importance is still unclear. Investigation of these pathways has been greatly facilitated by the ability to isolate EC from progenitors circulating in the peripheral blood of normal controls and patients with VWD. In the next few years, these will yield further evidence on the molecular pathways controlled by VWF and shed light on this novel and fascinating area of vascular biology. In this article, we will review the evidence supporting a role for VWF in blood vessel formation, the link between VWF dysfunction and vascular malformations causing GI bleeding and how they may be causally related. Finally, we will discuss how these findings point to novel therapeutic approaches to bleeding refractory to VWF replacement therapy in VWD.

Methotrexate-mediated activation of an AMPK-CREB-dependent pathway: a novel mechanism for vascular protection in chronic systemic inflammation.

AIMS: Premature cardiovascular events complicate chronic inflammatory conditions. Low-dose weekly methotrexate (MTX), the most widely used disease-modifying drug for rheumatoid arthritis (RA), reduces disease-associated cardiovascular mortality. MTX increases intracellular accumulation of adenosine monophosphate (AMP) and 5-aminoimidazole-4-carboxamide ribonucleotide which activates AMP-activated protein kinase (AMPK). We hypothesised that MTX specifically protects the vascular endothelium against inflammatory injury via induction of AMPK-regulated protective genes. METHODS/RESULTS: In the (NZW×BXSB)F1 murine model of inflammatory vasculopathy, MTX 1 mg/kg/week significantly reduced intramyocardial vasculopathy and attenuated end-organ damage. Studies of human umbilical vein endothelial cells (HUVEC) and arterial endothelial cells (HAEC) showed that therapeutically relevant concentrations of MTX phosphorylate AMPKα(Thr172), and induce cytoprotective genes including manganese superoxide dismutase (MnSOD) and haem oxygenase-1 (HO-1). These responses were preserved when HUVECs were pretreated with tumour necrosis factor-α to mimic dysfunctional endothelium. Furthermore, MTX protected against glucose deprivation-induced endothelial apoptosis. Mechanistically, MTX treatment led to cyclic AMP response element-binding protein (CREB)(Ser133) phosphorylation, while AMPK depletion attenuated this response and the induction of MnSOD and HO-1. CREB siRNA inhibited upregulation of both cytoprotective genes by MTX, while chromatin immunoprecipitation demonstrated CREB binding to the MnSOD promoter in MTX-treated EC. Likewise, treatment of (NZW×BXSB)F1 mice with MTX enhanced AMPKα(Thr172) phosphorylation and MnSOD, and reduced aortic intercellular adhesion molecule-1 expression. CONCLUSIONS: These data suggest that MTX therapeutically conditions vascular endothelium via activation of AMPK-CREB. We propose that this mechanism contributes to the protection against cardiovascular events seen in patients with RA treated with MTX.

Statin-mediated cytoprotection of human vascular endothelial cells: a role for Kruppel-like factor 2-dependent induction of heme oxygenase-1.

BACKGROUND: Heme oxygenase-1 (HO-1), by exerting anti-inflammatory, antiproliferative, antiapoptotic and antioxidant effects in the vasculature, protects against atherosclerosis and post-transplant vasculopathy. We noted the overlap between the effects of HO-1 and those attributed to 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins). This led to an investigation of the role of HO-1 in statin-mediated cytoprotection in primary human endothelial cells (ECs), and the ability of Kruppel-like factor 2 (KLF2) to regulate HO-1 function. METHODS/RESULTS: Treatment of human umbilical vein and aortic ECs with atorvastatin significantly upregulated HO-1 promoter activity, mRNA expression and protein expression, increasing HO-1 enzymatic activity as shown by raised intracellular bilirubin IXalpha. This effect was indirect, dependent upon inhibition of HMG-CoA reductase and geranylgeranylation, and independent of nitric oxide or changes in mRNA stability. Atorvastatin protected ECs against the generation of reactive oxygen species and H(2)O(2)-induced injury. HO-1 inhibition, with small interfering RNA (siRNA) or zinc protoporphyrin IX, abrogated atorvastatin-mediated cytoprotection. Atorvastatin upregulated KLF2 expression, whereas KLF2 siRNA attenuated statin-induced HO-1 and its associated antioxidant cytoprotective effects. Iron chelation, adenoviral-mediated overexpression of ferritin or supplementation of culture media with biliverdin reversed the inhibitory effects of HO-1 and KLF2 siRNA, suggesting that bile pigments and ferritin mediate the antioxidant actions of statin-induced HO-1. CONCLUSIONS: We have identified a novel link between KLF2 and HO-1 in human vascular ECs, demonstrating that atorvastatin-mediated HO-1 upregulation, and its associated antioxidant effect, is KLF2-dependent. The relationship between KLF2 and HO-1 is likely to represent an important component of the vasculoprotective profile of statins.

Atorvastatin affects leukocyte gene expression in dyslipidemia patients: in vivo regulation of hemostasis, inflammation and apoptosis.

BACKGROUND: The beneficial effect of HMG-CoA reductase inhibitors (statins) on coronary artery disease has been linked to mechanisms beyond their lipid-lowering effect. However the existence of direct, lipid-independent effects of statin in humans is still controversial. OBJECTIVE: To investigate the early effect of atorvastatin on peripheral blood mononuclear cells (PBMC) in dyslipidemic patients using gene arrays. PATIENTS AND METHODS: Eleven male patients with primary hyperlipidemia received 20 mg atorvastatin daily for 4 weeks. Blood was collected at baseline, 12 h, 36 h, 1 and 4 weeks after the start of treatment. RESULTS: Human microarrays containing 12 650 genes were used to study the effect of atorvastatin on PBMC gene expression at all time-points. Two hundred and forty genes were significantly regulated by atorvastatin treatment, several of which are involved in hemostasis, inflammation and other processes critical to atherosclerosis. Different patterns of response over time suggested both lipid-dependent and independent effects of atorvastatin on gene expression. CONCLUSIONS: This study demonstrates for the first time that atorvastatin regulates gene expression in PBMC in man before changes in the lipid profile are detectable in serum. Using blood leukocytes as a pharmacogenomic surrogate, we have identified new in vivo targets of atorvastatin treatment.

Clinical inhibition of the seven-transmembrane thrombin receptor (PAR1) by intravenous aprotinin during cardiothoracic surgery.

BACKGROUND: Protease-activated receptor-1 (PAR1) is the principal thrombin receptor in the vasculature, and antagonists against this receptor are in preclinical trials. Aprotinin, already approved for clinical use to reduce transfusion requirements in cardiopulmonary bypass (CPB) surgery, has been shown to inhibit PAR1 activation in vitro. Here, we exploit CPB as a model for thrombin generation in humans to examine whether aprotinin can inhibit platelet PAR1 activation clinically. METHODS AND RESULTS: PAR1 expression and function on platelets was examined in coronary artery bypass grafting (CABG) patients randomized into 2 groups: (1) those receiving saline infusion during CPB (n=17) and (2) those receiving aprotinin (2x10(6) kallikrein inhibitor units [KIU] in pump prime, 2x10(6) KIU loading dose, followed by 0.5x10(6) KIU/h [n=13]). Platelets in the saline group showed loss of PAR1-specific function at 2 hours after CPB, but this was preserved in the aprotinin group (P<0.001). These effects were most likely targeted at PAR1 receptor cleavage, because (1) the level of thrombin generated during CPB did not vary significantly between groups, (2) expression of SPAN12, which detects only uncleaved PAR1 receptors, was preserved in the aprotinin but not the placebo group (P<0.05), and (3) supporting evidence in vitro showed reduced thrombin-induced PAR1 cleavage (P<0.001) and platelet aggregation (P<0.001) in the presence of aprotinin. CONCLUSIONS: This study demonstrates that platelet PAR1 activation by thrombin can be inhibited by aprotinin. Our results extend the clinical mechanism of action of aprotinin and provide the first proof of principle that PAR1 can be inhibited clinically. This has implications beyond cardiac surgery for the development of therapeutic PAR1 blockade.

Identification of differentially expressed genes in coronary atherosclerotic plaques from patients with stable or unstable angina by cDNA array analysis.

The composition of atherosclerotic plaques is a crucial factor in determining rupture, thrombosis and clinical events. In this study, we analyzed gene expression in coronary plaques from patients with stable or unstable angina using gene arrays. Total RNA was extracted from eight plaques collected by therapeutic directional coronary atherectomy. cDNA probes, generated by amplification, were hybridized to nylon arrays containing 482 genes. Here we report the results for the inflammation, adhesion and hemostasis subsets. Many genes not previously associated with atherosclerosis, such as the lymphocyte adhesion molecule MadCAM, were expressed in the plaques. anova analysis showed higher tissue factor (TF) expression in unstable angina samples. Five genes were expressed at lower levels in unstable angina samples: anticoagulant protein S, cyclooxygenase (COX)-1, interleukin (IL)-7 and chemokines monocyte chemotactic protein (MCP)-1 and -2. Gene arrays provide a new approach to study plaque composition and identify candidate markers of plaque instability.

Combined genomic and antisense analysis reveals that the transcription factor Erg is implicated in endothelial cell differentiation.

It has recently been shown that the transcription factor Erg, an Ets family member, drives constitutive expression of the intercellular adhesion molecule 2 (ICAM-2) in human umbilical vein endothelial cells (HUVECs) and that its expression is down-regulated by the pleiotropic cytokine tumor necrosis factor alpha (TNF-alpha). To identify other Erg target genes and to define its function in the endothelium, a combined approach of antisense oligonucleotides (GeneBloc) and differential gene expression was used. Treatment of HUVECs with Erg-specific GeneBloc for 24, 48, and 72 hours suppressed Erg mRNA and protein levels at all time points. Total RNA extracted from HUVECs treated with Erg-specific or control GeneBloc was analyzed for differences in gene expression using high-density, sequence-verified cDNA arrays containing 482 relevant genes. Inhibition of Erg expression resulted in decreased expression of ICAM-2, as predicted. Four more genes decreased in Erg-deficient HUVECs were the extracellular matrix proteins SPARC and thrombospondin, the adhesive glycoprotein von Willebrand factor, and the small GTPase RhoA. Each of these molecules has been directly or indirectly linked to angiogenesis because of its role in vascular remodeling, adhesion, or shape change. Therefore, the role of Erg in vascular remodeling was tested in an in vitro model, and the results showed that HUVECs treated with Erg GeneBloc had a decreased ability to form tubulelike structures when grown on Matrigel. These results suggest that Erg may be a mediator of the TNF-alpha effects on angiogenesis in vivo.

Cryopreservation of spores of Dicksonia sellowiana: an endangered tree fern indigenous to South and Central America.

Spores of Dicksonia sellowiana (Presl.) Hook., an endangered tree fern, were stored in liquid nitrogen. Surface sterilized spores were placed in 1 ml sterile polypropylene cryotubes and were plunged into liquid nitrogen cryo-cans for 15 minutes, 15 days, 1 month and 3 months. In all, of the treatments the percentage of germination was higher than the control (fresh spores). Germination in Dyer and MS media supplement with 10 (-7) M and 5 x 10(-7) M BA was also promoted as comparing to control. There was no difference between the germination of spores thawed rapidly in a water bath at 45 degree C during 5 minutes or slowly at room temperature. Cryopreservation seems to promote germination of some dormant spores of D. sellowiana. The pre-treatment in cryoprotective solution of dimethyl sulphoxide 15%(v/v) in 1 M glycerol inhibited the germination of cryopreserved spores

Characterisation of the tumour necrosis factor (TNF)-(alpha) response elements in the human ICAM-2 promoter.

ICAM-2 is a cell surface adhesion molecule constitutively expressed on the endothelium, involved in leukocyte recruitment into tissues. We recently showed that pro-inflammatory cytokines tumour necrosis factor (TNF)-(alpha) and interleukin (IL)-1(beta) down-regulate ICAM-2 expression at the transcriptional level. Here we investigate the elements in the ICAM-2 promoter required for the TNF-(alpha)-mediated down-regulation. Site directed mutagenesis of the ICAM-2 promoter implicated three consensus sites for Ets transcription factors in basal activity; two of these sites were also involved in the TNF-(alpha)-induced down-regulation. Electrophoretic mobility shift assays (EMSA) performed in human umbilical vein endothelial cells (HUVEC) showed that all three Ets binding sites (EBS) bind nuclear proteins. TNF-(alpha) treatment (10 ng/ml for 24 hours) decreased binding to the double -135/-127EBS, but not to the -44EBS. The Ets family member Erg was found to be constitutively expressed in HUVEC, and TNF-(alpha) down-regulated Erg protein levels. Furthermore, an Erg cDNA transactivated the ICAM-2 promoter when transiently transfected into both HeLa cells and HUVEC. Protein expression of ICAM-2 and Erg was found to be similarly regulated by TNF-(alpha) in an ex vivo artery model. These data suggest that constitutive endothelial genes ICAM-2 and Erg are on the same pathway of cytokine-dependent regulation of gene expression.

The I domain of integrin leukocyte function-associated antigen-1 is involved in a conformational change leading to high affinity binding to ligand intercellular adhesion molecule 1 (ICAM-1).

On T cells the leukocyte integrin leukocyte function-associated antigen-1 (LFA-1) (CD11a/CD18) can be induced to bind its ligand intercellular adhesion molecule 1 (ICAM-1) (CD54) either by increasing the affinity of the receptor with Mg2+ and EGTA or by receptor clustering following activation with phorbol ester. The existence of these two adhesion-inducing pathways implies that alternative mechanisms might exist by which LFA-1 engages ICAM-1. The LFA-1 alpha subunit I domain contains a major binding site for ICAM-1. In this study we show that soluble LFA-1 I domain blocks ICAM-1 binding of the high affinity Mg2+-induced form of LFA-1 but not the phorbol ester-induced form. Under conditions of Mg2+-activation, the soluble I domain also prevents expression of an activation dependent epitope on LFA-1, implying that it inhibits a conformational change necessary for conversion to the high affinity form of this integrin. In addition, the binding of Mg2+-activated LFA-1 to ICAM-1 is blocked by peptides covering the alpha4-beta3 loop, the beta3-alpha5 loop, and the alpha5 helix of the I domain, whereas none of the peptides tested blocks phorbol ester-mediated adhesion. The blocking peptides localize to the same face of the crystal structure of the LFA-1 I domain and define an area that, during activation, may be involved in association of the I domain with another region of LFA-1, potentially the beta-propeller domain. This is the first evidence linking a structural domain of an integrin, in this case the I domain, with a particular activation mechanism.

Tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta down-regulate intercellular adhesion molecule (ICAM)-2 expression on the endothelium.

Leukocyte recruitment is a crucial step in inflammation. Inflammatory stimuli upregulate the expression of some endothelial adhesion molecules, such as E-selectin or ICAM-1, but not of others such as ICAM-2. ICAM-2, a constitutively expressed endothelial ligand for beta2 integrins LFA-1 and Mac-1, is involved in leukocyte adhesion to resting endothelium and in transmigration in vitro, however its role in inflammation is unclear. We have studied the effect of TNF-alpha and IL-1beta on ICAM-2 expression on human umbilical vein endothelial cells (HUVECs). Prolonged treatment (24 h) of HUVECs with TNF-alpha (10 ng/ml) or IL-1beta (34 ng/ml) reduced ICAM-2 surface expression to 50% of control, while interferon (IFN)-gamma had no effect. The loss in ICAM-2 surface expression correlated with a reduction of ICAM-2 mRNA to approximately 40% of control after 24 h of cytokine treatment. The activity of an ICAM-2 promoter reporter plasmid transfected into HUVECs was down-regulated by TNF-alpha and IL-1beta to similar values. Thus inflammatory cytokines inhibit ICAM-2 transcription, despite the absence of known cytokine-responsive elements in the promoter. Immunocytochemistry on HUVEC monolayers showed that ICAM-2 expression, mainly at the cell junctions in resting cells, was markedly decreased by cytokine treatment. This data suggest that ICAM-2 expression on the endothelium may be regulated during inflammation.

The sticking point: How integrins bind to their ligands.

The integrin adhesion receptors are alpha beta heterodimers that exist in different ligand-binding states. Because of their large size and conformational lability, it has been difficult to determine how they interact with their ligands. Ligand-binding sites have been identified in the beta subunit, and now more recently in the 'I' domain and EF-hand-like domains V and VI of the alpha subunit. We speculate here about how these various sites might operate together to bind ligand in a stable manner.

I domain of beta 2 integrin lymphocyte function-associated antigen-1 contains a binding site for ligand intercellular adhesion molecule-1.

Lymphocyte function-associated antigen-1 (LFA-1) is a beta 2 integrin that participates in a broad range of leukocyte functions through binding to its ligand intercellular adhesion molecule-1 (ICAM-1). The location of the ICAM-1 binding site on LFA-1 is not known. A approximately 200-amino acid "inserted" or "I" domain, which is part of the beta 2 integrin alpha subunit, is homologous to the "A" domains found in the adhesive protein von Willebrand factor and in a number of other proteins. In von Willebrand factor, the A domains are involved in ligand binding, but their function in the other proteins is still unclear. In this report, we show that the LFA-1 I domain contains a binding site for ICAM-1, which can be expressed as an isolated functional unit. The I domain contains the epitopes for 18 out of 20 anti-LFA-1 monoclonal antibodies, many of which interfere with the interaction between LFA-1 and ICAM-1. The I domain binds directly to purified recombinant ICAM-1 and also inhibits LFA-1-dependent T cell adhesion to ICAM-1. This report establishes the I domain as an ICAM-1 binding region in LFA-1 and the first ligand binding site to be identified in a beta 2 integrin.

Effect of type IIB von Willebrand disease mutation Arg(545)Cys on platelet glycoprotein Ib binding--studies with recombinant von Willebrand factor.

Type IIB von Willebrand disease (vWD) is characterized by a selective loss of high molecular weight von Willebrand factor (vWF) multimers in plasma due to their abnormally enhanced reactivity with platelets. Several missense mutations in the platelet glycoprotein Ib (GPIb) binding domain of vWF were recently characterized that cause type IIB vWD. The effect of type IIB mutation Arg(545)Cys on vWF binding to platelet GPIb was studied using recombinant wild type (rvWFWT) and mutant rvWFR545C expressed in COS-7 cells. In the absence of ristocetin, 50% of rvWFR545C bound spontaneously to platelet GPIb and the binding increased to 70% in the presence of 0.2 mg/ml ristocetin; rvWFWT did not bind significantly under either condition. Botrocetin-induced binding of rvWFR545C was only slightly increased compared to rvWFWT. These data demonstrate that the Arg(545)Cys mutation increases the affinity of vWF for GPIb, resulting in the characteristics gain-of-function type IIB vWD phenotype.

Type IIB mutation His-505-->Asp implicates a new segment in the control of von Willebrand factor binding to platelet glycoprotein Ib.

Type IIB von Willebrand disease is characterized by increased affinity of mutant von Willebrand factor (vWF) for platelet glycoprotein Ib. Eight different missense mutations that cause this phenotype have been reported within the disulfide loop defined by Cys-509 and Cys-695 of the mature vWF subunit; this disulfide loop is required for normal binding of vWF to platelet glycoprotein Ib. A new mutation was identified in a patient with type IIB von Willebrand disease (vWD) characterized by a lifelong bleeding disorder, mild thrombocytopenia, normal levels of factor VIII, vWF antigen, and ristocetin cofactor activity but increased ristocetin-induced platelet agglutination at low concentrations of ristocetin. Exon 28 of the patient's vWF gene was amplified, cloned, and sequenced. At nucleotide 3802 (numbering the cDNA from translation initiation), a C to G transversion was identified, which changes the encoded amino acid sequence from His-505 to Asp. The corresponding mutant recombinant vWF was expressed in transiently transfected COS cells. Relative to wild type vWF, the mutant vWF exhibited markedly increased binding to platelets at low concentrations of ristocetin, confirming the association between the His-505-->Asp substitution and the type IIB vWD phenotype. The His-505-->Asp mutation lies outside the disulfide loop affected by other type IIB vWD mutations and implicates a new segment of vWF in the regulation of platelet glycoprotein Ib binding.

Identification of three candidate mutations causing type IIA von Willebrand disease using a rapid, nonradioactive, allele-specific hybridization method.

Type IIA von Willebrand disease (vWD), the most common type II vWD variant, is characterized by decreased binding of von Willebrand factor (vWF) to platelet glycoprotein Ib (Gplb) and by a decrease in large and intermediate vWF multimers. Mutations reported to cause vWD type IIA are clustered within the A2 domain of vWF, which is encoded by exon 28. Genomic DNA from affected members of 12 unrelated families with type IIA vWD were screened for these mutations by a rapid, nonradioactive, allele-specific oligonucleotide (ASO) hybridization method. Oligonucleotides containing each of eight mutations were cross-linked onto a nylon membrane by UV irradiation. A fragment of vWF exon 28 was amplified from peripheral blood leukocyte DNA using biotinylated primers and hybridized to the immobilized oligonucleotides. Positive signals were detected with an avidin-alkaline phosphatase conjugate and chemiluminescent substrate. Thus, in a single hybridization reaction, a patient sample could be analyzed for a large number of mutations simultaneously. Polymerase chain reaction (PCR) products from four patients did not contain any of the tested mutations and therefore were sequenced. Three additional candidate missense mutations, two of them novel, were identified: Arg(834)-->Gln in one patient, Gly(846)-->Arg in one patient, and Val(902)-->Glu in three ostensibly unrelated patients. By ASO hybridization, the mutations were confirmed in the affected patients and excluded in unaffected relatives and 50 normal controls. In one family, the Val(902)-->Glu mutation was shown to be a de novo mutation. This rapid screening method is applicable to other subtypes of vWD for which mutations have been identified.

The genetic defect of type I von Willebrand disease "Vicenza" is linked to the von Willebrand factor gene.

Type I von Willebrand disease (vWD) Vicenza is a rare variant with autosomal dominant transmission, characterized by the presence of supranormal von Willebrand factor (vWF) multimers in plasma, similar to those normally found in endothelial cells and megakaryocytes. The patients have very low levels of plasma vWF contrasting with a mild bleeding tendency. The pathophysiology of this subtype is still unknown. The presence of supranormal multimers in the patients' plasma could be due to a mutation in the vWF molecule which affects post-translational processing, or to a defect in the cells' processing machinery, independent of the vWF molecule. In order to determine if type I vWD Vicenza is linked to the vWF gene, we studied six polymorphic systems identified within the vWF gene in two apparently unrelated families with type I vWD Vicenza. The results of this study indicate a linkage between vWF gene and the type I vWD Vicenza trait. This strongly suggests that type I vWD Vicenza is due to a mutation in one of the vWF alleles, which results in an abnormal vWF molecule that is processed to a lesser extent than normal vWF.

Identification of a new congenital defect of platelet function characterized by severe impairment of platelet responses to adenosine diphosphate.

This study characterizes a congenital hemorrhagic disorder caused by a platelet function defect with the following features: (1) severely impaired platelet aggregation and fibrinogen or von Willebrand factor (vWF) binding induced by adenosine diphosphate (ADP); (2) defective aggregation, release reaction, and fibrinogen or vWF binding induced by other agonists; (3) normal aggregation and release reaction induced by high concentrations of thrombin or collagen; (4) no further inhibition by ADP scavengers of aggregation, release reaction, and fibrinogen or vWF binding, comparable with those observed for normal platelets in the presence of ADP scavengers; (5) normal membrane glycoprotein (GP) composition and normal binding of the anti-GP IIb/IIIa monoclonal antibody 10E5; (6) no acceleration by ADP of binding of the anti-GP IIb/IIIa monoclonal antibody 7E3; (7) normal platelet-fibrin clot retraction if induced by thrombin or reptilase plus epinephrine, absent if induced by reptilase plus ADP; (8) no inhibition by ADP of the prostaglandin E1-induced increase in platelet cyclic adenosine monophosphate, but normal inhibition by epinephrine; (9) defective mobilization of cytoplasmic Ca2+ by ADP; (10) normal binding of 14C-ADP to fresh platelets, but defective binding of [2-3H]-ADP to formalin-fixed platelets. This congenital platelet function defect is characterized by selective impairment of platelet responses to ADP, caused by either decreased number of platelet ADP receptors or abnormalities of the signal-transduction pathway of platelet activation by ADP.