Development of neutralizing anti-factor (F)VIII antibodies (‘inhibitors’) is a serious clinical problem in hemophilia A. Increased inhibitor risk has been associated with certain FVIII missense substitutions, including R593C in the A2 domain.
The aim of the present study was to identify T-cell epitopes in FVIII and characterize T-cell responses in two unrelated hemophilia A subjects sharing F8-R593C andHLA-DRB1*1101 genotypes. We hypothesized that the hemophilic substitution site coincides with an important T-cell epitope.
The binding affinities of peptides for recombinant HLA-DR proteins were measured and compared with epitope prediction results. CD4+ T cells were stimulated using peptides and stained with fluorescent, peptide-loaded tetramers.
The inhibitor subjects, but not HLA-matched controls, had high-avidity HLA-DRB1* 1101-restricted T-cell responses against FVIII589–608, which contains the hemophilic missense site. Antigen-specific T cells secreted Th1 and Th2 cytokines and proliferated in response to FVIII and FVIII592–603. FVIII589–608 bound with physiologically relevant (micromolar) IC50 values to recombinant DR0101, DR1101 and DR1501 proteins.
Hemophilia A patients with R593C missense substitutions and these HLA haplotypes had an increased incidence of inhibitors in our cohorts, supporting a paradigm in which presentation of FVIII epitopes containing the wild-type R593 influences inhibitor risk in this hemophilia A sub-population.
factor VIII; hemophilia A; HLA; inhibitor; T-cell clones
In recent years, it has become increasingly apparent that the life span of transfused platelets in circulation is regulated, at least in part, by glycan-lectin mediated mechanisms. There is clear evidence that refrigerated platelets are cleared by glycan-lectin mediated clearance mechanisms. Acute platelet cooling clusters glycoprotein (GP) Ibα receptors bearing uncovered N-acetylglucosamine (GlcNAc), and αMβ2 integrins on hepatic macrophages recognise clustered GlcNAc to rapidly clear these platelets from circulation. With prolonged refrigeration GPIbα clustering bearing uncovered galactose increases, which mediates the removal of long-term refrigerated platelets via hepatic Ashwell-Morell receptors (AMR), originally named as asialoglycoprotein receptors. In contrast, little is known about the molecular mechanisms of transfused room temperature platelet clearance. This review examines the role of glycan-lectin mediated clearance of exogenous, i.e. transfused chilled platelet clearance and briefly addresses the current knowledge of stored platelet function, degradation and its relation to platelet clearance.
Increased hypercoagulability has been reported with low doses of direct thrombin inhibitors but not with direct factor Xa inhibitors.
To compare the effects of rivaroxaban with those of melagatran and dabigatran on thrombin generation (TG) and tissue factor-induced hypercoagulability and to explore the possible involvement of the thrombin–thrombomodulin/activated protein C system.
In normal human plasma and in protein C-deficient plasma, TG was investigated in vitro in the presence and absence of recombinant human soluble thrombomodulin (rhs-TM). TG was determined by calibrated automated thrombography and an ELISA for prothrombin fragments 1+2 (F1+2). In an in vivo rat model, hypercoagulability was induced by tissue factor; levels of thrombin–antithrombin (TAT) and fibrinogen and the platelet count were determined.
Rivaroxaban inhibited TG in a concentration-dependent manner. In the absence of rhs-TM, melagatran and dabigatran also inhibited TG concentration dependently. However, in the presence of rhs-TM, lower concentrations of melagatran (119–474 nmol L–1) and dabigatran (68–545 nmol L−1) enhanced endogenous thrombin potential, peak TG, and F1+2 formation in normal plasma but not in protein C-deficient plasma. In vivo, rivaroxaban dose-dependently inhibited TAT generation, whereas melagatran showed a paradoxical effect, with an increase in TAT and a small decrease in fibrinogen and platelet count at lower doses.
Low concentrations of the direct thrombin inhibitors melagatran and dabigatran enhanced TG and hypercoagulability, possibly via inhibition of the protein C system. In contrast, rivaroxaban reduced TG and hypercoagulability under all conditions studied, suggesting that it does not suppress this negative-feedback system.
dabigatran; hypercoagulability; melagatran; rivaroxaban; thrombin
Knowledge of independent, baseline risk factors of catheter-related thrombosis (CRT) may help select adult cancer patients at high risk to receive thromboprophylaxis.
We conducted a meta-analysis of individual patient-level data to identify these baseline risk factors.
MEDLINE, EMBASE, CINAHL, CENTRAL, DARE, Grey literature databases were searched in all languages from 1995-2008.
Prospective studies and randomized controlled trials (RCTs) were eligible. Studies were included if original patient-level data were provided by the investigators and if CRT was objectively confirmed with valid imaging.
Multivariate logistic regression analysis of 17 prespecified baseline characteristics was conducted. Adjusted odds ratios (OR) and 95% confidence intervals (CI) were estimated.
A total sample of 5636 subjects from 5 RCTs and 7 prospective studies was included in the analysis. Among these subjects, 425 CRT events were observed. In multivariate logistic regression, the use of implanted ports as compared with peripherally implanted central venous catheters (PICC), decreased CRT risk (OR = 0.43; 95% CI, 0.23-0.80), whereas past history of deep vein thrombosis (DVT) (OR = 2.03; 95% CI, 1.05-3.92), subclavian venipuncture insertion technique (OR = 2.16; 95% CI, 1.07-4.34), and improper catheter tip location (OR = 1.92; 95% CI, 1.22-3.02), increased CRT risk.
CRT risk is increased with using PICC catheters, previous history of DVT, subclavian venipuncture insertion technique and improper positioning of the catheter tip. These factors may be useful for risk stratifying patients to select those for thromboprophylaxis. Prospective studies are needed to validate these findings.
Catheter-related thrombosis; risk factors; meta-analysis; cancer; thromboprophylaxis; adults
Thrombus resolution is a complex process that involves thrombosis, leukocyte-mediated thrombolysis, and the final resolution of inflammation. Activated Protein C (APC) is an anticoagulant that also possesses immunoregulatory activities.
In this study, we sought to examine the effects of APC administration on thrombus resolution using a mouse model of deep vein thrombosis by ligating the inferior vena cava (IVC).
The IVCs of C57BL/6 mice were ligated. Beginning on Day 4 post-IVC ligation, mice were injected i.p. daily with APC, APC plus a heme oxygenase-1 (HO-1) inhibitor Sn-protoporphyrin IX (SnPP), SnPP alone, or vehicle control. At different time points following surgery, the thrombus-containing IVCs were weighed and then analyzed by biochemical assays and histology.
Venous thrombi reached maximum size on Day 4 post ligation. The APC-treated group exhibited a significant reduction in thrombus weights on Day 12 but not on Day 7 as compared to control mice. The enhanced thrombus resolution in APC-treated mice correlated with an increased HO-1 expression and a reduced interleukin-6 production. No significant difference was found in urokinase-type plasminogen activator, plasminogen activator inhibitor-1, matrix metalloproteinase-2 and -9 between APC-treated and control mice. Co-injection of an HO-1 inhibitor SnPP abolished the ability of APC to enhance thrombus resolution.
Our data show that APC enhances the resolution of existing venous thrombi via a mechanism that is in part dependent on HO-1, suggesting that APC could be used as a potential treatment for patients with deep vein thrombosis to accelerate thrombus resolution.
Protein C; Venous Thrombosis; Heme oxygenase-1; Inflammation; Thrombolytic Therapy
Tumor cell tissue factor (TF)-initiated coagulation supports hematogenous metastasis by fibrin formation, platelet activation, and monocyte/macrophage recruitment. Recent studies identified host anticoagulant mechanisms as a major impediment for successful hematogenous tumor cell metastasis.
Here we address mechanisms that contribute to enhanced metastasis in hyperthrombotic mice with functional thrombomodulin deficiency (TMPro mice).
Pharmacological and genetic approaches were combined to characterize relevant thrombin targets in a mouse model of experimental hematogenous metastasis.
TF-dependent, but contact pathway-independent syngeneic breast cancer metastasis was associated with marked platelet hyper-reactivity and formation of leukocyte-platelet aggregates in immune-competent TMPro mice. Blockade of CD11b or genetic deletion of platelet glycoprotein Ibα excluded contributions of these receptors to enhanced platelet-dependent metastasis in hyperthrombotic mice. Mice with very low levels of the endothelial protein C receptor (EPCR) did not phenocopy the enhanced metastasis seen in TMPro mice. Genetic deletion of the thrombin receptor PAR1 or endothelial thrombin signaling targets alone did not diminish enhanced metastasis in TMPro mice. Combined deficiency of PAR1 on tumor cells and the host reduced metastasis in TMPro mice.
Metastasis in the hyperthrombotic TMPro mouse model is mediated by platelet hyper-reactivity and contributions of PAR1 signaling on tumor and host cells.
Tissue Factor; Thrombin; Hypercoagulability; Metastasis; Platelets
The glycoprotein (GP)Ib-IX complex is critical to hemostasis and thrombosis. Its proper assembly is closely correlated with its surface expression level and requires cooperative interactions among extracellular and transmembrane domains of Ibα, Ibβ and IX subunits. Two interfaces have been previously identified between the extracellular domains of Ibβ and IX.
To understand how extracellular domains interact in GPIb-IX.
The Ibβ extracellular domain (IbβE) or the IX counterpart (IXE) in GPIb-IX was replaced with a well-folded IbβE/IXE chimera called IbβEabc, and the effect of domain replacement on assembly and expression of the receptor complex in transiently transfected Chinese hamster ovary cells was analyzed.
Replacing IXE with IbβEabc in GPIb-IX retained interface 1 but not interface 2 between the extracellular domains. While this domain replacement preserved complex integrity, the expression levels of Ibβ and Ibα were significantly reduced. Additional domain replacement with IbβEabc or IbβE in GPIb-IX produced the complex at disparate expression levels that cannot be simply explained by two separate interfaces. In particular, when IbβE in GPIb-IX was replaced by IbβEabc, Ibα and IX were expressed at approximately 70% of the wild-type level. Their levels were not reduced when IXE was changed further to IbβE.
Our results demonstrate the importance of the association between Ibβ and IX extracellular domains for complex assembly and efficient expression, and provide evidence for the structural malleability of these domains that may accommodate and propagate conformational changes therein.
Bernard-Soulier syndrome; leucine-rich repeat proteins; platelet glycoprotein GPIb-IX complex; protein-protein interaction domains; von Willebrand factor receptors
Background and objectives
Src family kinases (SFKs) play a critical role in initiating and propagating signals in platelets. The aims of this study were to quantitate SFK members present in platelets and to analyze their contribution to platelet regulation using glycoprotein VI (GPVI) and intregrin αIIbβ3, and in vivo.
Methods and Results
Mouse platelets express four SFKs, Fgr, Fyn, Lyn and Src, with Lyn expressed at a considerably higher level than the others. Using mutant mouse models, we demonstrate that platelet activation by collagen-related peptide (CRP) is delayed and then potentiated in the absence of Lyn, but only marginally reduced in the absence of Fyn or Fgr, and unaltered in the absence of Src. Compound deletions of Lyn/Src or Fyn/Lyn, but not of Fyn/Src or Fgr/Lyn, exhibit a greater delay in activation relative to Lyn-deficient platelets. Fibrinogen-adherent platelets show reduced spreading in the absence of Src, potentiation in the absence of Lyn, but no change in the absence of Fyn or Fgr. In mice double-deficient in Lyn/Src or Fgr/Lyn, the inhibitory role of Lyn on spreading on fibrinogen is lost. Lyn is the major SFK-mediating platelet aggregation on collagen at arterial shear and its absence leads to a reduction in thrombus size in a laser injury model.
These results demonstrate that SFKs share individual and overlapping roles in regulating platelet activation, with Lyn having a dual role in regulating GPVI signaling and an inhibitory role downstream of αIIbβ3, which requires prior signaling through Src.
GPVI; integrin αIIbβ3; Lyn; platelets; Src family kinases
Intravascular hemolysis occurs after blood transfusion, in hemolytic anemias and other conditions, and is associated with hypercoagulable states. Hemolysis has been shown to potently activate platelets in vitro and in vivo and several mechanisms have been suggested to account for this including (1) direct activation by hemoglobin, (2) increase in reactive oxygen species (ROS), (3) scavenging of nitric oxide by released hemoglobin, and (4) release of intraerythrocytic ADP.
The aim of the current study is to elucidate the mechanism of hemolysis-mediated platelet activation.
We used flow cytometry to detect PAC-1 binding to activated platelets for in vitro experiments and a Siemens’ Advia 120 hematology system to assess platelet aggregation using platelet counts from in vivo experiments in a rodent model.
We show that Hb does not directly activate platelets. However, ADP bound to Hb can cause platelet activation. Furthermore, platelet activation due to shearing of RBCs is reduced in the presence of apyrase which metabolizes ADP to AMP. Use of ROS scavengers did not affect platelet activation. We also show that cell free Hb does enhance platelet activation by abrogating the inhibitory effect of NO on platelet activation. In vivo infusions of ADP and purified (ADP-free) Hb as well as hemolysate result in platelet aggregation as evidenced by decreased platelet counts.
Two primary mechanisms account for red blood cell hemolysis-associated platelet activation: ADP release which activates platelets and cell-free hemoglobin release which enhances platelet activation by lowering NO bioavailability.
hemoglobin; hemolysis; nitric oxide; platelets; red blood cells
The development of anti-factor VIII (fVIII) antibodies (inhibitors) is a significant complication in the management of patients with hemophilia A, leading to significant increases in morbidity and treatment cost. Using a panel of anti-fVIII monoclonal antibodies (MAbs) to different epitopes on fVIII, we recently have shown that epitope specificity, inhibitor kinetics, and time to maximum inhibition are more important than inhibitor titer in predicting response to fVIII and the combination of fVIII and recombinant factor VIIa. In particular, a subset of high-titer inhibitors responded to high dose fVIII, which would not be predicted based on their inhibitor titer alone. Thus the ability to quickly map the epitope spectrum of patient plasma using a clinically feasible assay may fundamentally change how clinicians approach the treatment of high-titer inhibitor patients.
To map the epitopes of anti-fVIII MAbs, of which 3 are classical inhibitors and one non-classical, using hydrogen-deuterium exchange coupled with liquid chromatography-mass spectrometry (HDX-MS).
Binding epitopes of 4 MAbs targeting fVIII C2 domain were mapped using HDX-MS.
The epitopes determined by HDX-MS are consistent with those obtained earlier through structural characterization and antibody competition assays. In addition classical and non-classical inhibitor epitopes could be distinguished using a limited subset of C2-derived peptic fragments.
Our results demonstrate the effectiveness and robustness of the HDX-MS method for epitope mapping and suggest a potential role of rapid mapping of fVIII inhibitor epitopes in facilitating individualized treatment of inhibitor patients.
Antigen-antibody complex; epitope mapping; factor VIII; hemophilia A; hydrogen deuterium exchange measurement
Ectodomain shedding of GPIbα, a proteolytic event in which metalloprotease ADAM17 cleaves the Gly464-Val465 bond and releases glycocalicin to the plasma, is considered a critical step in mediating clearance of stored platelets. Supporting evidence has largely come from studies using ADAM17 inhibitors. However, the definitive proof is lacking due to the broad substrate specificity of ADAM17.
To achieve substrate-specific inhibition of GPIbα shedding.
Development of monoclonal antibodies that directly bind the sequence around the GPIbα shedding cleavage site and inhibit GPIbα shedding by blocking ADAM17 access to the cleavage site.
Six anti-GPIbα monoclonal antibodies with varying binding affinities were obtained. The prototypic clone, designated 5G6, and its monomeric Fab fragment, bind specifically purified GPIb-IX complex, human platelets, and transgenic murine platelets expressing human GPIbα. 5G6 showed similar inhibitory potency as a widely used shedding inhibitor GM6001 in both constitutive and induced GPIbα shedding in human platelets. It does not recognize mouse GPIbα. Nor does it inhibit shedding of other platelet receptors. Finally, 5G6 binding displays no detectable effect on platelet activation and aggregation.
5G6 specifically inhibits GPIbα shedding with no detectable effect on platelet functions. The method of substrate-specific shedding inhibition by macromolecular binding of the shedding cleavage site can be applicable to many other transmembrane receptors undergoing ectodomain shedding.
Glycoprotein Ib; Glycocalicin; Platelet transfusion; ADAM proteins; Antibodies, Monoclonal
Protein disulfide isomerase (PDI) catalyzes disulfide bond oxidation, reduction and isomerization during protein synthesis in the endoplasmic reticulum (ER). In addition to its critical role in the ER, in vitro and in vivo studies with blocking antibodies and conditional knockout mice have demonstrated that cell surface PDI is required for thrombosis, hemostasis and vascular inflammation in a manner dependent on its isomerase activity. This review will focus on our current understanding of the pathophysiologic role of PDI in regulating integrin-mediated platelet and neutrophil functions during vascular disease.
inflammation; integrins; neutrophils; platelet; protein disulfide isomerase; thrombosis
Worldwide, streptokinase remains the most used thrombolytic agent for the treatment of myocardial infarction. Recombinant streptokinase, from E. coli, is increasingly used in developing countries as a biosimilar of native streptokinase; however, potency assignments relative to the WHO International Standard (IS) are highly variable with potentially dangerous consequences. A proportion of recombinant streptokinase appears to be incompletely processed, retaining the amino-terminal methionine engineered for intracellular expression.
To investigate and quantify the impact of an amino-terminal methionine on streptokinase activity.
Mature native streptokinase (rSK) was cloned and a novel variant constructed to include an amino-terminal methionine (rSK-Met) that is not susceptible to processing during expression. Potencies of rSK and rSK-Met were determined relative to the WHO IS using a chromogenic solution (European Pharmacopoeia) assay, and fibrin-based assays.
In the chromogenic solution assay there was no measurable difference between rSK and rSK-Met activities. In the fibrin-based methods, however, potency estimates for rSK-Met were greatly reduced compared with rSK, and fibrinolytic activity for rSK-Met was shown to increase over time with methionine aminopeptidase treatment. This apparent difference in activity and fibrin selectivity was consistent with potency estimates for several different batches of commercial recombinant streptokinase products also tested; consequently, different potencies would be assigned to therapeutic recombinant streptokinase products depending on the degree of amino-terminal methionine processing, and on the pharmacopoeial assay method used, affecting the dosage patients receive. This has serious health implications and provides an example of the danger in the unregulated clinical use of biosimilars.
biosimilar harmaceuticals; DNA; recombinant; fibrinolytic agents; reference standards; thrombolytic therapy
The ISTH bleeding assessment tool (ISTH-BAT) was developed to record bleeding symptoms and to aid diagnosis in patients with a possible bleeding disorder.
To investigate the utility of the ISTH-BAT in predicting functional defects in platelet activation in participants with suspected inherited platelet function disorders.
Participants with clinical evidence of excessive bleeding and suspected inherited platelet function disorders and healthy volunteers were recruited to the Genotyping and Phenotyping of Platelets study (GAPP; ISRCTN 77951167). The ISTH-BAT questionnaire was applied by a trained investigator prior to lumiaggregometry.
One hundred participants were included (79 with suspected inherited platelet function disorders, and 21 healthy volunteers). The ISTH-BAT score in participants with suspected inherited platelet function disorders (median 12; interquartile range [IQR] 8–16) was significantly higher than in healthy volunteers (median 0; IQR 0–0). There was no difference between participants with suspected inherited platelet function disorders with a platelet defect detected by lumiaggregometry (median 11; IQR 8–16) and those with normal platelet function (median 12; IQR 8–14) (P > 0.05). The ISTH-BAT score was not associated with a demonstrable platelet defect on platelet function testing (area under the receiver operating characteristic curve = 0.501 [95% confidence interval 0.372–0.630, P = 0.98] and odds ratio 1.01 [95% confidence interval 0.93–1.09, P = 0.91]).
The ISTH-BAT is a powerful tool for documenting lifelong bleeding history. However, the score obtained is not predictive of the presence of a platelet defect on lumiaggregometry in patients with suspected inherited platelet function disorders.
Blood platelet disorders; Coagulation disorders, inherited; Decision support techniques; Platelet aggregation; Platelet function tests
The clinical impact of a tumor thrombus in renal cell carcinoma (RCC) patients awaiting radical nephrectomy and thrombectomy is unknown.
To determine the incidence of venous thromboembolism (VTE) in RCC patients with tumor thrombus prior to nephrectomy.
Patients and methods
We conducted a retrospective cohort study including all late-stage (stage 3–4 excluding T1–2 N0M0) RCC patients who underwent radical nephrectomy at our institution between 1 January 2005 and 1 July 2012. Tumor thrombus was defined as the presence of an intraluminal filling defect in the renal vein, hepatic vein, portal vein, or inferior vena cava, directly extending from a renal mass detected on computed tomography.
A total of 176 patients were included in the study. Fifty-three (30.1%) patients had tumor thrombus diagnosed on imaging Three patients with tumor thrombus (5.7%; 95% confidence interval [CI] 1.4–16.8) developed a VTE while awaiting radical nephrectomy, whereas none (0%; 95% CI 0–2.9) of the patients without a tumor thrombus had an event (P = 0.026). All three events were deep vein thrombosis. Times from tumor thrombus diagnosis to VTE were 5, 15 and 21 days.
Tumor thrombus on imaging is a frequent finding among RCC patients awaiting nephrectomy. The presence of tumor thrombus in these patients increases the incidence of preoperative VTE.
neoplasm; nephrectomy; renal cell carcinoma; thrombosis; venous thromboembolism
G protein-coupled receptors (GPCRs) are a major family of signaling molecules, central to the regulation of inflammatory responses. Their activation upon agonist binding is attenuated by GPCR kinases (GRKs), which desensitize the receptors through phosphorylation. G protein-coupled receptor kinase 2(GRK2) down-regulation in leukocytes has been closely linked to the progression of chronic inflammatory disorders such as rheumatoid arthritis and multiple sclerosis. Because leukocytes must interact with the endothelium to infiltrate inflamed tissues, we hypothesized that GRK2 down-regulation in endothelial cells would also be pro-inflammatory.
To determine whether GRK2 down-regulation in endothelial cells is pro-inflammatory.
siRNA-mediated ablation of GRK2 in human umbilical vein endothelial cells (HUVECs) was used in analyses of the role of this kinase. Microscopic and biochemical analyses of Weibel-Palade body (WPB) formation and functioning, live cell imaging of calcium concentrations and video analyses of adhesion of monocyte-like THP-1 cells provide clear evidence of GRK2 function in histamine activation of endothelial cells.
G protein-coupled receptor kinase 2 depletion in HUVECs increases WPB exocytosis and P-selectin-dependent adhesion of THP-1 cells to the endothelial surface upon histamine stimulation, relative to controls. Further, live imaging of intracellular calcium concentrations reveals amplified histamine receptor signaling in GRK2-depleted cells, suggesting GRK2 moderates WPB exocytosis through receptor desensitization.
G protein-coupled receptor kinase 2 deficiency in endothelial cells results in increased pro-inflammatory signaling and enhanced leukocyte recruitment to activated endothelial cells. The ability of GRK2 to modulate initiation of inflammatory responses in endothelial cells as well as leukocytes now places GRK2 at the apex of control of this finely balanced process.
G protein coupled receptor kinase 2; human umbilical vein endothelial cells; P-selectin; von Willebrand factor; Weibel-Palade bodies
Vatreptacog alfa, a recombinant factor VIIa (rFVIIa) analog with three amino acid substitutions and 99% identity to native FVIIa, was developed to improve the treatment of hemophilic patients with inhibitors.
To confirm the safety and assess the efficacy of vatreptacog alfa in treating bleeding episodes in hemophilic patients with inhibitors.
Patients and methods
In this international, multicenter, randomized, double-blind, active-controlled, crossover, confirmatory phase III trial (adept™2) in patients with hemophilia A or B and inhibitors, bleeds were randomized 3 : 2 to treatment with vatreptacog alfa (one to three doses at 80 μg kg−1) or rFVIIa (one to three doses at 90 μg kg−1). Treatment failures after three doses of trial product (TP) were managed according to the local standard of care.
In the 72 patients enrolled, 567 bleeds were treated with TP. Both vatreptacog alfa and rFVIIa gave 93% effective bleeding control at 12 h. Vatreptacog alfa was superior to rFVIIa in secondary efficacy outcomes, including the number of doses used to treat a bleed and sustained bleeding control 24–48 h after the first dose. Eight patients (11%) developed antibodies against vatreptacog alfa, including four with cross-reactivity against rFVIIa and one with an in vitro neutralizing effect to vatreptacog alfa.
This large randomized controlled trial confirmed the well-established efficacy and safety profile of rFVIIa, and showed that vatreptacog alfa had similar or better efficacy than rFVIIa. However, because of the development of anti-drug antibodies, a positive benefit–risk profile is unlikely to be achieved with vatreptacog alfa.
antibodies; clinical trial, phase III; hemophilia; inhibitors; recombinant factor VIIa
Pannexin-1 (Panx1) forms an anion-selective channel with a permeability up to ∼1 kDa and represents a non-lytic, non-vesicular ATP release pathway in erythrocytes, leukocytes and neurons. Related connexin gap junction proteins have been reported in platelets; however, the expression and function of the pannexins remain unknown.
To determine the expression and function of pannexins in human plate-lets, using molecular, cellular and functional techniques.
Panx1 expression in human platelets was det-ermined using qPCR and antibody-based techniques. Contributions of Panx1 to agonist-evoked efflux of cytoplasmic calcein, Ca2+ influx, ATP release and aggregation were assessed in washed platelets under conditions where the P2X1 receptor response was preserved (0.32 U mL−1 apyrase). Thrombus formation in whole blood was assessed in vitro using a shear chamber assay. Two structurally unrelated and widely used Panx1 inhibitors, probenecid and carbenoxolone, were used throughout this study, at concentrations that do not affect connexin channels.
PANX1, but not PANX2 or PANX3, mRNA was detected in human platelets. Furthermore, Panx1 protein is glycosylated and present on the plasma membrane of platelets, and displays weak physical association with P2X1 receptors. Panx1 inhibition blocked thrombin-evoked efflux of calcein, and reduced Ca2+ influx, ATP release, platelet aggregation and thrombus formation under arterial shear rates in vitro. The Panx1-dependent contribution was not additive to that of P2X1 receptors.
Panx1 is expressed on human platelets and amplifies Ca2+ influx, ATP release and aggregation through the secondary activation of P2X1 receptors. We propose that Panx1 represents a novel target for the management of arterial thrombosis.
ATP; calcium; collagen; P2X1 receptor; pannexin 1, human