Diagnostic potential of NETosis-derived products for disease activity, atherosclerosis and therapeutic effectiveness in Rheumatoid Arthritis patients.

OBJECTIVES: 1) To assess the association of NETosis and NETosis-derived products with the activity of the disease and the development of cardiovascular disease in RA; 2) To evaluate the involvement of NETosis on the effects of biologic therapies such as anti-TNF alpha (Infliximab) and anti-IL6R drugs (Tocilizumab). METHODS: One hundred and six RA patients and 40 healthy donors were evaluated for the occurrence of NETosis. Carotid-intimae media thickness was analyzed as early atherosclerosis marker. Inflammatory and oxidative stress mediators were quantified in plasma and neutrophils. Two additional cohorts of 75 RA patients, treated either with Infliximab (n = 55) or Tocilizumab (n = 20) for six months, were evaluated. RESULTS: NETosis was found increased in RA patients, beside myeloperoxidase and neutrophil elastase protein levels. Cell-free nucleosomes plasma levels were elevated, and strongly correlated with the activity of the disease and the positivity for autoantibodies, alongside inflammatory and oxidative profiles in plasma and neutrophils. Moreover, ROC analyses showed that cell-free nucleosomes levels could identify RA patients showing early atherosclerosis with high specificity. RA patients treated either with IFX or TCZ for six months exhibited decreased generation of NETs. Concomitantly, clinical parameters and serum markers of inflammation were found reduced. Mechanistic in vitro analyses showed that inhibition of NETs extrusion by either DNase, IFX or TCZ, further abridged the endothelial dysfunction and the activation of immune cells, thus influencing the global activity of the vascular system. CONCLUSIONS: NETosis-derived products may have diagnostic potential for disease activity and atherosclerosis, as well as for the assessment of therapeutic effectiveness in RA.

miR-146a rs2431697 identifies myeloproliferative neoplasm patients with higher secondary myelofibrosis progression risk.

Myelofibrosis (MF) occurs as part of the natural history of polycythemia vera (PV) and essential thrombocythemia (ET), and remarkably shortens survival. Although JAK2V617F and CALR allele burden are the main transformation risk factors, inflammation plays a critical role by driving clonal expansion toward end-stage disease. NF-κB is a key mediator of inflammation-induced carcinogenesis. Here, we explored the involvement of miR-146a, a brake in NF-κB signaling, in MPN susceptibility and progression. rs2910164 and rs2431697, that affect miR-146a expression, were analyzed in 967 MPN (320 PV/333 ET/314 MF) patients and 600 controls. We found that rs2431697 TT genotype was associated with MF, particularly with post-PV/ET MF (HR = 1.5; p < 0.05). Among 232 PV/ET patients (follow-up time=8.5 years), 18 (7.8%) progressed to MF, being MF-free-survival shorter for rs2431697 TT than CC + CT patients (p = 0.01). Multivariate analysis identified TT genotype as independent predictor of MF progression. In addition, TT (vs. CC + CT) patients showed increased plasma inflammatory cytokines. Finally, miR-146a-/- mice showed significantly higher Stat3 activity with aging, parallel to the development of the MF-like phenotype. In conclusion, we demonstrated that rs2431697 TT genotype is an early predictor of MF progression independent of the JAK2V617F allele burden. Low levels of miR-146a contribute to the MF phenotype by increasing Stat3 signaling.

The genetic interaction between VKORC1 c1173t and calumenin a29809g modulates the anticoagulant response of acenocoumarol.

BACKGROUND: The efficacy of oral anticoagulant therapy is largely conditioned by both environmental and genetic factors. OBJECTIVES: To attempt to define the genetic profile involved in the response to this treatment. PATIENTS AND METHODS: We selected 100 men younger than 75 years, with non-valvular atrial fibrillation, who started anticoagulation with acenocoumarol following the same protocol: 3 mg for three consecutive days. Then, doses were individually adjusted to achieve a steady International Normalized Ratio (INR). The basal plasma level and the level after 3 days were obtained, and the INR was determined. We studied five functional polymorphisms: FVII -323 Del/Ins, CYP2C*9, VKORC1 c1173t, calumenin (CALU) R4Q and CALU a29809g. The dose required for a steady INR was also recorded. RESULTS: Only the VKORC1 genotype had significant impact on the efficacy of therapy. Carriers of the 1173t allele were significantly more sensitive to therapy for 3 days [INR 2.07 (1.59-2.87) vs. 1.74 (1.30-2.09); P = 0.015] and they needed lower acenocoumarol doses to stabilize their INR (15.8 +/- 5.6 vs. 19.5 +/- 6.0 mg week(-1); P = 0.004). Its effect was exacerbated by combination with the CALU a29809g polymorphism. Carriers of both variants (27% of the sample) achieved the highest INR [2.26 (1.70-3.32)] and required the lowest dose (14.1 +/- 5.1 mg week(-1)). This genetic profile was particularly relevant in patients with INR >or= 3.5 at the start of therapy (P = 0.005; odds ratio = 6.67, 95% confidence interval = 1.32-37.43). CONCLUSIONS: Our results suggest that CALU a29809g might be a new genetic factor involved in the pharmacogenetics of anticoagulant therapy, and confirm that specific genetic profiles defined by different polymorphisms will determine the initial response and dose required to achieve a stable and safe INR.

The role of microRNA-27a/b and microRNA-494 in estrogen-mediated downregulation of tissue factor pathway inhibitor α.

UNLABELLED: Essentials Estrogens are known to influence the expression of microRNAs in breast cancer cells. We looked at microRNAs in estrogenic regulation of tissue factor pathway inhibitor α (TFPIα). Estrogen upregulated microRNA-27a/b and microRNA-494 through the estrogen receptor α. MicroRNA-27a/b and microRNA-494 are partly involved in estrogenic downregulation of TFPIα. SUMMARY: Background Tissue factor pathway inhibitor (TFPI) has been linked to breast cancer pathogenesis. We have recently reported TFPI mRNA levels to be downregulated by estrogens in a breast cancer cell line (MCF7) through the estrogen receptor α (ERα). Accumulating evidence also indicates that activation of ERα signaling by estrogens may modulate the expression of target genes indirectly through microRNAs (miRNAs). Objectives To examine if miRNAs are involved in the estrogenic downregulation of TFPIα. Methods Computational analysis of the TFPI 3'-untranslated region (UTR) identified potential binding sites for miR-19a/b, miR-27a/b, miR-494, and miR-24. Transient overexpression or inhibition of the respective miRNAs was achieved by transfection of miRNA mimics or inhibitors. Direct targeting of TFPI 3'-UTR by miR-27a/b and miR-494 was determined by luciferase reporter assay in HEK293T cells. Effects of 17α-ethinylestradiol (EE2) and fulvestrant on relative miR-27a/b, miR-494, and TFPI mRNA levels in MCF7 cells were determined by qRT-PCR and secreted TFPIα protein by ELISA. Transient knockdown of ERα was achieved by siRNA transfection. Results EE2 treatment lead to a significant increase in miR-19a, miR-27a/b, miR-494, and miR-24 mRNA levels in MCF7 cells through ERα. miR-27a/b and miR-494 mimics lead to reduced TFPI mRNA and protein levels. Luciferase assay showed direct targeting of miR-27a/b and miR-494 on TFPI mRNA. Impaired estrogen-mediated downregulation of TFPI mRNA was detected in anti-miR-27a/b and anti-miR-494 transfected cells. Conclusions Our results provide evidence that miR-27a/b and miR-494 regulate TFPIα expression and suggest a possible role of these miRNAs in the estrogen-mediated downregulation of TFPIα.

Uniparental disomy causes deficiencies of vitamin K-dependent proteins.

Essentials Vitamin K-dependent coagulant factor deficiency (VKCFD) is a rare autosomal recessive disorder. We describe a case of inherited VKCFD due to uniparental disomy. The homozygous mutation caused the absence of GGCX isoform 1 and overexpression of Δ2GGCX. Hepatic and non-hepatic vitamin K-dependent proteins must be assayed to monitor VKCFD treatment. SUMMARY: Background Inherited deficiency of all vitamin K-dependent coagulant factors (VKCFD) is a rare autosomal recessive disorder caused by mutations in the γ-glutamyl carboxylase gene (GGCX) or the vitamin K epoxide reductase gene (VKORC1), with great heterogeneity in terms of both clinical presentation and response to treatment. Objective To characterize the molecular basis of VKCFD in a Spanish family. Methods and Results Sequencing of candidate genes, comparative genomic hybridization and massive sequencing identified a new mechanism causing VKCFD in the proband. Uniparental disomy (UPD) of chromosome 2 caused homozygosity of a mutation (c.44-1G>A) resulting in aberrant GGCX splicing. This change contributed to absent expression of the mRNA coding for the full-length protein, and to four-fold overexpression of the smaller mRNA isoform lacking exon 2 (Δ2GGCX). Δ2GGCX might be responsible for two unexpected clinical observations in the patient: (i) increased plasma osteocalcin levels following vitamin K1 supplementation; and (ii) a mild non-bleeding phenotype. Conclusions Our study identifies a new autosomal disease, VKCFD1, caused by UPD. These data suggest that the Δ2GGCX isoform may retain enzymatic activity, and strongly encourage the evaluation of both hepatic and non-hepatic vitamin K-dependent proteins to assess differing responses to vitamin K supplementation in VKCFD patients.

'Atherothrombosis-associated microRNAs in Antiphospholipid syndrome and Systemic Lupus Erythematosus patients'.

MicroRNAs markedly affect the immune system, and have a relevant role in CVD and autoimmune diseases. Yet, no study has analyzed their involvement in atherothrombosis related to APS and SLE patients. This study intended to: 1) identify and characterize microRNAs linked to CVD in APS and SLE; 2) assess the effects of specific autoantibodies. Six microRNAs, involved in atherothrombosis development, were quantified in purified leukocytes from 23 APS and 64 SLE patients, and 56 healthy donors. Levels of microRNAs in neutrophils were lower in APS and SLE than in healthy donors. Gene and protein expression of miRNA biogenesis-related molecules were also reduced. Accordingly, more than 75% of identified miRNAs by miRNA profiling were underexpressed. In monocytes, miR124a and -125a were low, while miR-146a and miR-155 appeared elevated. Altered microRNAs' expression was linked to autoimmunity, thrombosis, early atherosclerosis, and oxidative stress in both pathologies. In vitro treatment of neutrophils, monocytes, and ECs with aPL-IgG or anti-dsDNA-IgG antibodies deregulated microRNAs expression, and decreased miRNA biogenesis-related proteins. Monocyte transfections with pre-miR-124a and/or -125a caused reduction in atherothrombosis-related target molecules. In conclusion, microRNA biogenesis, significantly altered in neutrophils of APS and SLE patients, is associated to their atherothrombotic status, further modulated by specific autoantibodies.

Mutations in the shutter region of antithrombin result in formation of disulfide-linked dimers and severe venous thrombosis.

BACKGROUND: Missense mutations causing conformational alterations in serpins can be responsible for protein deficiency associated with human diseases. However, there are few data about conformational consequences of mutations affecting antithrombin, the main hemostatic serpin. OBJECTIVES: To investigate the conformational and clinical effect of mutations affecting the shutter region of antithrombin. PATIENTS AND METHODS: We identified two families with significant reduction of circulating antithrombin displaying early and severe venous thrombosis, frequently associated with pregnancy or infection. Mutations were determined by standard molecular methods. Biochemical studies were performed on plasma samples. One variant (P80S) was purified by heparin-affinity chromatography and gel filtration, and evaluated by proteomic analysis. Finally, we modelled the structure of the mutant dimer. RESULTS: We identified two missense mutations affecting the shutter region of antithrombin: P80S and G424R. Carriers of both mutations presented traces of a similar abnormal antithrombin, supporting inefficiently expressed rather than non-expressed variants. The abnormal antithrombin purified from P80S carriers is an inactive disulfide-linked dimer of mutant antithrombin whose properties are consistent with head-to-head insertion of the reactive loop. CONCLUSIONS: Our data support the conclusion that missense mutations affecting the shutter region of serpins have specific conformational effects resulting in the formation of mutant oligomers. The consequent inefficiency of secretion explains the accompanying deficiency and loss of function, but the severity of thrombosis associated with these mutations suggests that the oligomers also have new and undefined pathological properties that could be exacerbated by pregnancy or infection.

Detection of factor V Leiden from a drop of blood by PCR-SSCP.

Recently, a point mutation in the coagulation Factor V gene (G to A in position 1691) has been identified which makes the mutant Factor V (called Factor V Leiden) resistant to activated protein C. This defect is a well established genetic risk factor in venous thrombosis. Because of the high prevalence of Factor V Leiden in normal population (2-7%), it would be reasonable to perform a rapid and simple method for screening the genetic abnormality in population at risk. We have developed a simple, reproducible, rapid and cheap procedure that, using PCR and SSCP, allows the identification of the mutation responsible for Factor V Leiden. Specificity of this method has been tested in 319 samples: 304 normal, 14 heterozygous and 1 homozygous for Factor V Leiden. This assay allows non-isotopic Factor V Leiden identification by using frozen whole blood in 3 h. All these features make this test adequate for routine screening of this mutation in a large number of samples.

Role of the 807 C/T polymorphism of the alpha2 gene in platelet GP Ia collagen receptor expression and function--effect in thromboembolic diseases.

The variability of the platelet GP Ia/IIa density has been associated with the 807 C/T polymorphism (Phe 224) of the GP Ia gene in American Caucasian population. We have investigated the genotype and allelic frequencies of this polymorphism in Spanish Caucasians. The T allele was found in 35% of the 284 blood donors analyzed. We confirmed in 159 healthy subjects a significant association between the 807 C/T polymorphism and the platelet GP Ia density. The T allele correlated with high number of GP Ia molecules on platelet surface. In addition, we observed a similar association of this polymorphism with the expression of this protein in other blood cell types. The platelet responsiveness to collagen was determined by "in vitro" analysis of the platelet activation and aggregation response. We found no significant differences in these functional platelet parameters according to the 807 C/T genotype. Finally, results from 3 case/control studies involving 302 consecutive patients (101 with coronary heart disease, 104 with cerebrovascular disease and 97 with deep venous thrombosis) determined that the 807 C/T polymorphism of the GP Ia gene does not represent a risk factor for arterial or venous thrombosis.

A common polymorphism flanking the ATG initiator codon of GPIb alpha does not affect expression and is not a major risk factor for arterial thrombosis.

The platelet membrane glycoprotein (GP) Ib alpha plays a key role in the initial formation of thrombi. Polymorphisms (VNTR and HPA-2) in this receptor are associated with increased risk of coronary heart disease (CHD) and cerebral vascular disease (CVD). We investigated whether a recently described polymorphism (S/R), due to a single base change (T-->C) five nucleotides upstream the initiator codon of GPIb alpha, might influence the expression of the protein, and be implicated in the development of arterial thrombosis. One hundred and thirty nine healthy individuals provided blood samples for DNA analysis of platelet GPIb alpha polymorphisms, and for flow cytometric analysis of the surface expression of the receptor. A group of 20 S/R normal individuals and an identical number of S/S participants, age and sex matched, was investigated for the analysis of the density of various platelet receptors. The distribution of the S/R polymorphism was also analyzed in two case/control studies including 104 CVD patients, 101 CHD patients, and one control age, sex, and environmental risk factors matched for each case patient. Surface density of GPIb alpha showed no wide variations between individuals, was not influenced by the presence of S or R alleles, nor associated with the VNTR or HPA-2 polymorphisms. The prevalence of the S/R genotype among CVD and CHD patients was not distinct from that in the control groups. We conclude that the S/R polymorphism of GPIb alpha, flanking the initiator codon of the receptor, does not seem to be associated with surface levels of the protein, and is not an independent risk factor for arterial thrombosis.

Binding of recombinant apolipoprotein(a) to human platelets and effect on platelet aggregation.

The interaction of lipoprotein(a) [Lp(a)] with platelets is not well defined, particularly with regards to the individual contribution of the protein components of Lp(a), the apo B-100 and the apolipoprotein apo(a). This study investigated the binding of different recombinant apo(a) [r-apo(a)] isoforms, to human platelets and its effect on platelet aggregation. Scatchard analysis of saturation binding experiments demonstrated that human platelets display a single class of high affinity r-apo(a) binding sites (71 +/- 46 molec./platelet, Kd = 5.6 +/- 2.0 nmol/L). Platelet activation with strong agonists (thrombin, arachidonic acid) increased 2- to 10-fold the r-apo(a) binding, without affecting the affinity. Competition assays showed that the binding sites are highly specific for r-apo(a) and Lp(a). At high concentration t-PA could also bind to the r-apo(a) binding sites. By contrast, neither fibrinogen nor plasminogen inhibited to the r-apo(a) binding. The lysine analogue EACA inhibits the binding of r-apo(a) to platelets, thus suggesting the involvement of lysine residues in that interaction. Moreover, the r-apo(a) binding to platelets is unlikely mediated by GPIIb/IIIa-attached fibrin since it is not affected by platelet treatment with either LJ-CP8, a monoclonal antibody that specifically blocks fibrinogen binding to GPIIb/IIIa, nor GPRP, an inhibitor of fibrin polymerisation. Finally, we show that the distinct recombinant apo(a) proteins, as well as native Lp(a), promote an aggregation response of platelets to otherwise subaggregant doses of arachidonic acid. This proaggregant effect of r-apo(a) is dependent on its binding to platelets since it requires a minimum incubation time, and it is prevented by EACA at concentration inhibiting the r-apo(a)-platelet interaction. These results suggest that the prothrombotic action of Lp(a) may be in part mediated by modulating the platelet function through the interaction of its apo(a) subunit with a specific receptor at the platelet surface.

Factor XIII Val34Leu polymorphism in primary intracerebral haemorrhage.

INTRODUCTION: Recently, the common Val34Leu polymorphism of the A-chain factor XIII gene, associated with high factor XIII activity, has been identified as a protective genetic factor against occlusive arterial and venous diseases. Moreover, this polymorphism has been suggested to be the first one to increase the risk of cerebral haemorrhage in a small number of Caucasian patients. The aim of our study was to investigate the role of this polymorphism in patients with primary intracerebral haemorrhage from a distinct population. MATERIAL AND METHODS: Patients with non-traumatic primary intracerebral haemorrhage (n=116), age-, race-, sex- and risk factor-matched controls (n=116), and individuals from the general population (n=465) were genotyped for the factor XIII Val34Leu polymorphism by polymerase chain reaction and allele specific restriction assay. The relationships of the Val/Leu genotype with distinct intracerebral haemorrhagic risk factors and with early mortality associated with the haemorrhagic episode were also analysed. RESULTS: No statistical difference in terms of prevalence was detected between patients (P=0.190) and controls (P=0.181). The frequency of the FXIII Leu34 allele was similar in the general population (P=0.191). CONCLUSION: The results suggest that the Leu 34 allele of the A-chain factor XIII gene has a minor role in the development of non-traumatic primary intracerebral haemorrhage. Moreover, the simultaneous presence of the Leu 34 allele with selected risk factors for this disease does not increase the risk of developing this disease.

Autoaggression syndrome resembling acute graft-versus-host disease grade IV after autologous peripheral blood stem cell transplantation for breast cancer.

Acute graft-versus-host disease (aGVHD) after autologous progenitor cell transplantation has been associated with blood transfusion or cyclosporine. Mild aGVHD grades I-II, identified as autoaggression or engraftment syndrome, has recently been described in autologous progenitor transplantation. Here, we report the first case of pathologically documented grade IV aGVHD after autologous peripheral blood progenitor cell transplantation in a patient with breast cancer. The allogeneic origin was excluded by molecular techniques, and no cyclosporine or cytokines were administered.

Genetic determinants of platelet function in thromboembolic diseases.

Within the past decade our understanding of thromboembolic disorders has become even more sophisticated as recent discoveries have suggested the influence of gene variants on the development of atherosclerotic disease and arterial thrombosis. Candidate genes encode proteins involved in processes relevant to atherosclerosis, ranging from cholesterol metabolism to arterial thrombosis. Platelets are key elements in primary hemostasis, but also in arterial thrombosis. Moreover, a number of genetic polymorphisms of platelet proteins may also induce gain or loss of function, supporting a role predisposing some individuals to thrombotic events. However, after thousands of studies, much controversy remains whether individual platelet polymorphisms contribute to an increased likelihood of thromboembolic disorders. Although platelet polymorphisms are a promising addition to more established cardiovascular risk factors, identifying genetic variants as a single cause of cardiovascular disease would be an oversimplification; instead, the contribution of these polymorphisms should also be considered in the context of a multifactorial disease. Gene-gene and gene-environment studies would identify specific combinations associated with a high risk to suffer from these diseases. The platelet's genetic heterogeneity should also be considered in every aspect of clinical medicine, ranging from susceptibility to diseases, pathogenesis, and clinical outcome to diversity in responses to drug treatment (pharmacogenomics), and bleeding.

HPA-1 genotype in arterial thrombosis--role of HPA-1b polymorphism in platelet function.

Recently, the HPA-1b (PlA2) polymorphism of the platelet glycoprotein IIIa has been suggested as a genetic risk factor for coronary artery disease. We conducted two case-control studies of 103 patients with ischaemic cerebrovascular disease (CVD) and 101 patients with ischaemic heart disease (IHD). The groups were matched for age, race and sex. No significant differences regarding selected risk factors (hypertension, diabetes mellitus, hypercholesterolaemia and smoking) were found between case patients and controls. Moreover, we investigated 286 normal individuals from the Mediterranean area. Genotyping of HPA-1 was performed by PCR-allelic specific restriction and single-strand conformation polymorphism analysis. The prevalence of HPA-1b was similar among case patients and controls (29.2% vs. 25.3% and 26.7% vs. 34.6% for CVD and IHD case-control studies, respectively). The HPA-1b allele was found in 36.4% of the normal population. Finally, the analysis of platelet function in nine controls with the three possible HPA-1 genotypes (three a/a, three a/b and three b/b) indicates that HPA-1b genotype does not modify either the in vitro platelet aggregation and activation profile, nor the GP IIb/IIIa interaction with fibrinogen or von Willebrand factor. Our results do not support the role of HPA-1b polymorphism as an inherited risk factor for arterial thrombotic disease.

Genetic polymorphisms of factor VII are not associated with arterial thrombosis.

A high plasma concentration of factor VII procoagulant activity is considered an independent risk factor for coronary heart disease. Recently, two polymorphisms of factor VII gene (insertion of a decanucleotide at -323 [A2], and the variant Q353) have been associated with 20-25% lower levels of this protein in plasma. In this study, the prevalence of these two factor VII polymorphisms were evaluated in relation to the development of acute thromboembolic events. Thus, we conducted three case-control studies of patients diagnosed with acute coronary syndromes, acute cerebrovascular events and deep venous thrombosis (101, 104 and 97 cases, respectively). No significant differences were detected in the prevalence of these polymorphisms between patients and controls, suggesting that the A1/A2 or R/Q alleles do not play an important role in the development of thromboembolic episodes.

Platelet aggregation through prothrombinase activation induced by non-aggregant doses of platelet agonists.

Activation of the prothrombinase complex, which catalyzes the formation of thrombin from prothrombin, is crucial for the (patho)physiological processes of hemostasis and thrombosis. We here report that washed platelets supplemented with prothrombin can be irreversibly aggregated with otherwise non-aggregant doses of adenosine diphosphate (10 micromol/l), thrombin (0.06 U/ml), or collagen (1 microg/ml). Prothrombinase-catalyzed prothrombin to thrombin conversion most probably supports this aggregation response, since inhibitors of thrombin (hirudin or heparin) and an inhibitor of activated factor X (DX-9065a) impair the response. A certain degree of agonist-induced platelet activation seems to be required for this prothrombin-supported aggregation response, since prothrombin alone does not induce aggregation, and blockade of glycoprotein Ia/IIa with a specific antibody inhibits the platelet aggregation response to collagen and prothrombin. These results may suggest that activation of the prothrombinase complex could be a common step of the platelet response to distinct agonists, which may be achieved at low levels of platelet stimulation.

Platelet GP Ib/IX/V complex: physiological role.

Platelets play an essential role in primary hemostasis and in thrombotic events, particularly in arterial vessels, as rheological conditions originate closer interactions between platelets and endothelium than lower shear rates. In response to vascular injury, platelets adhere to the subendothelial matrix by membrane receptors potentiating the generation of thrombin, become activated, and a series of biochemical processes induce platelet aggregation and liberation of intracellular metabolic products to the extracelular medium. Among platelet receptors, glycoprotein (GP) Ib/IX/V complex is peculiar, as it binds adhesive proteins, mainly von Willebrand factor (vWF), and thrombin, the main platelet agonist. Platelet adhesion and subsequent aggregation under conditions of high shear flow, essentially relies upon this receptor's capacity of binding to the subendothelial matrix, initiating signal transduction. Two proteins associated to GP Ib/IX/V, actin-binding protein (ABP) 280 and 14-3-3zeta, are potential mediators of signal transduction by the complex, but their specific contribution in this process is not yet fully understood. Additionally, two proteins implicated in signal transduction by immune stimuli, FcgammaRIIA and FcR gamma-chain, associate with GPIb/IX/V complex, and increasing data indicate a potential role in GPIbalpha mediated signal transduction.