Platelet responses to activating agonists are influenced by common population variants within or near G protein-coupled receptor (GPCR) genes that affect receptor activity. However, the impact of rare GPCR gene variants is unknown. We describe the rare single nucleotide variants (SNVs) in the coding and splice regions of 18 GPCR genes in 7,595 exomes from the 1,000-genomes and Exome Sequencing Project databases and in 31 cases with inherited platelet function disorders (IPFDs). In the population databases, the GPCR gene target regions contained 740 SNVs (318 synonymous, 410 missense, 7 stop gain and 6 splice region) of which 70% had global minor allele frequency (MAF) < 0.05%. Functional annotation using six computational algorithms, experimental evidence and structural data identified 156/740 (21%) SNVs as potentially damaging to GPCR function, most commonly in regions encoding the transmembrane and C-terminal intracellular receptor domains. In 31 index cases with IPFDs (Gi-pathway defect n=15; secretion defect n=11; thromboxane pathway defect n=3 and complex defect n=2) there were 256 SNVs in the target regions of 15 stimulatory platelet GPCRs (34 unique; 12 with MAF<1% and 22 with MAF≥1%). These included rare variants predicting R122H, P258T and V207A substitutions in the P2Y12 receptor that were annotated as potentially damaging, but only partially explained the platelet function defects in each case. Our data highlight that potentially damaging variants in platelet GPCR genes have low individual frequencies, but are collectively abundant in the population. Potentially damaging variants are also present in pedigrees with IPFDs and may contribute to complex laboratory phenotypes.
Receptors; G-protein-coupled; genetic variation; blood platelets; blood platelet disorders
Obesity increases the risk for venous thromboembolism (VTE), but whether high-dose thromboprophylaxis is safe and effective in morbidly obese inpatients is unknown.
To quantify the efficacy and safety of high-dose thromboprophylaxis with heparin or enoxaparin in inpatients with weight > 100 kilograms (kg) within the BJC HealthCare system.
In a retrospective cohort study, we analyzed 9241 inpatients with weight > 100 kg discharged from three hospitals in the BJC HealthCare system from 2010 through 2012. We compared the incidence of VTE in patients who received high-dose thromboprophylaxis (heparin 7500 units three times daily or enoxaparin 40 milligrams (mg) twice daily) to those who received standard doses (heparin 5000 units two or three times daily or enoxaparin 40 mg once daily). The primary efficacy outcome was hospital-acquired VTE identified by International Classification of Diseases (ICD)-9 diagnosis codes. The primary safety outcome was bleeding events identified by ICD-9 codes.
Among the 3928 morbidly obese inpatients (weight > 100kg and body mass index (BMI) ≥ 40 kg/m2), high-dose thromboprophylaxis approximately halved the odds of symptomatic VTE (odds ratio (OR) 0.52, 95% CI 0.27-1.00; p-value (p) = 0.050). The rate of VTE was 1.48% (35/2369) in these morbidly obese inpatients who received standard doses of thromboprophylaxis, compared to 0.77% (12/1559) in those who received high doses. High-dose thromboprophylaxis did not increase bleeding (OR 0.84, 95% CI 0.66-1.07, p = 0.15). Independent predictors of VTE include surgery, male, cancer, and BMI.
High-dose thromboprophylaxis nearly halves the rate of VTE in morbidly obese inpatients.
Obesity; obese inpatient; thromboprophylaxis; venous thromboembolism
Antithrombin (AT) is a heparin-binding serpin in plasma which regulates the proteolytic activity of procoagulant proteases of the clotting cascade. In addition to being an anticoagulant, AT also exhibits antiinflammatory activities when it binds to cell surface heparan sulfate proteoglycans (HSPGs) on the endothelium via its basic residues of D-helix to elicit intracellular signaling responses. By contrast to AT, α1-proteinase inhibitor (α1-PI) is a non-heparin-binding serpin that exhibits very slow reactivity with coagulation proteases and possesses no HSPG-dependent antiinflammatory properties. To determine whether the antiinflammatory signaling specificity of AT can be transferred to α1-PI, we replaced the D-helix of human α1-PI with the corresponding sequence of human AT and expressed the chimeric serpin (α1-PI/D-helix) in a bacterial expression system. High molecular weight heparin bound to α1-PI/D-helix and accelerated the inhibition of thrombin by the serpin mutant by a template mechanism reminiscent of the cofactor effect of heparin on inhibition of thrombin by AT. Like AT, α1-PI/D-helix exhibited antiinflammatory properties in both cellular and animal models. Thus, α1-PI/D-helix inhibited the barrier-disruptive effect of proinflammatory cytokines and inhibited the activation of NF-κB transcription factor in LPS-stimulated endothelial cells by a concentration-dependent manner. Furthermore, the chimeric serpin reduced lipopolysaccharide-mediated lethality, elicited a vascular protective effect and inhibited infiltration of activated leukocytes to the peritoneal cavity of mice in an HMGB1-mediated inflammatory model. These results suggest that grafting the D-helix of AT to α1-PI confers antiinflammatory properties on the serpin and that the chimeric serpin may have therapeutic utility for treating inflammatory disorders.
Coronary artery disease (CAD) is characterised by progressive atherosclerotic plaque leading to flow-limiting stenosis, while myocardial infarction (MI) occurs due to plaque rupture or erosion with abrupt coronary artery occlusion. Multiple inflammatory pathways influence plaque stability, but direct assessment of endothelial inflammation at the site of coronary artery stenosis has largely been limited to pathology samples or animal models of atherosclerosis. We describe a technique for isolating and characterising endothelial cells (ECs) and EC microparticles (EMPs) derived directly from the site of coronary artery plaque during balloon angioplasty and percutaneous coronary intervention. Coronary artery endothelial cells (CAECs) were identified using imaging flow cytometry (IFC), and individual CAEC and EMP expression of the pro-atherogenic adhesion molecule vascular cell adhesion molecule-1 (VCAM-1) was assessed immediately following angioplasty. Patients with MI registered 73 % higher VCAM-1 expression on their CAECs and 79 % higher expression on EMPs compared to patients with stable CAD. In contrast, VCAM-1 expression was absent on ECs in the peripheral circulation from these same subjects. VCAM-1 density was significantly higher on CAECs and EMPs among patients with MI and positively correlated with markers of myocardial infarct size. We conclude that increased VCAM-1 expression on EC and formation of EMP at the site of coronary plaque is positively correlated with the extent of vascular inflammation in patients with myocardial infarction.
Acute myocardial infarction; adhesion molecules; VCAM-1; endothelial cells; inflammation; microparticles
Factor (F)VIIIa, a heterotrimer comprised of A1, A2, and A3C1C2 subunits, is labile due to the tendency of the A2 subunit to dissociate from the A1/A3C1C2 dimer. As dissociation of the A2 subunit inactivates FVIIIa activity, retention of A2 defines FVIIIa stability and thus, FXase activity. Earlier results showed that replacing residues D519, E665, and E1984 at the A2 domain interface with Ala or Val reduced rates of FVIIIa decay, increasing FXa and thrombin generation (Wakabayashi et al., Blood 112: 2761, 2008). We now show the enhanced FVIIIa stability of these variants results from increases in inter-A2 subunit affinity. Using a FVIIIa reconstitution assay to monitor inter-subunit affinity by activity regeneration, the apparent Kd value for the interaction of wild type (WT) A2 subunit with WT A1/A3C1C2 dimer (43 ± 2 nM) was significantly higher than values observed for the A2 point mutants D519A/V, E665A/V, and E1984A/V which ranged from ∼5 to ∼19 nM. Val was determined to be the optimal hydrophobic residue at position 665 (apparent Kd = 5.1 ± 0.7 nM) as substitutions with Ile or Leu at this position increased the apparent Kd value by ∼3- and ∼7-fold, respectively. Furthermore, the double mutant (D519V/E665V) showed an ∼47-fold lower apparent Kd value (0.9 ± 0.6 nM) than WT. Thus these hydrophobic mutations at the A2 subunit interfaces result in high binding affinities for the A2 subunit and correlate well with previously observed reductions in rates in FVIIIa decay.
Factor VIII; Factor VIIIa; inter-subunit binding affinity; protein stability
FVIIIa is labile due to the dissociation of A2 subunit. Previously, we introduced hydrophobic mutations at select A1/A2/A3 subunit interfaces yielding more stable FVIII(a) variants. Separately we showed that altering the sequence flanking the primary FXa cleavage site in FVIIIa (Arg336) yielded reduced rates of proteolytic inactivation of FVIIIa. In this study we prepared the FXa-cleavage resistant mutant (336(P4-P3’)562) combined with mutations of Ala108Ile, Asp519Val/Glu665Val or Ala108Ile/Asp519Val/Glu665Val and examined the effects of these combinations relative to FVIII thermal stability, rates of FVIIIa decay and proteolytic inactivation of FVIIIa by FXa. Thermal decay rates for 336(P4-P3’)562/Ala108Ile, 336(P4-P3’)562/Asp519Val/Glu665Val, and 336(P4-P3’)562/Ala108Ile/Asp519Val/Glu665Val variants were reduced by ~2–5-fold as compared with WT primarily reflecting the effects of the A domain interface mutations. FVIIIa decay rates for 336(P4-P3’)562/Asp519Val/Glu665Val and 336(P4-P3’)562/Ala108Ile/Asp519Val/Glu665Val variants were reduced by ~25 fold, indicating greater stability than the control Asp519Val/Glu665Val variant (~14-fold). Interestingly, 336(P4-P3’)562/Asp519Val/Glu665Val and 336(P4-P3’)562/Ala108Ile/Asp519Val/Glu665Val variants showed reduced FXa-inactivation rates compared with the 336(P4-P3’)562 control (~4-fold), suggesting A2 subunit destabilization is a component of proteolytic inactivation. Thrombin generation assays using the combination variants were similar to the Asp519Val/Glu665Val control. These results indicate that combining multiple gain-of-function FVIII mutations yields FVIII variants with increased stability relative to a single type of mutation.
factor VIII; factor VIIIa; factor Xa; protein stability; thrombin generation assay
Monocytes/macrophages respond to external stimuli with rapid changes in the expression of numerous inflammation-related genes to undergo polarisation towards the M1 (pro-inflammatory) or M2 (anti-inflammatory) phenotype. We have previously shown that, independently of Toll-like receptor activation, extracellular RNA (eRNA) could exert pro-thrombotic and pro-inflammatory properties in the cardiovascular system and provoked cytokine mobilisation. Here, mouse bone marrow-derived-macrophages (BMDM) differentiated with mouse macrophage-colony-stimulating factor (M-CSF) were found to be skewed towards the M1 phenotype when exposed to eRNA. This resulted in up-regulated expression of inflammatory markers such as Tnf-α and Il-6, together with Il-12 and iNOS, whereas anti-inflammatory genes such as chitinase-like proteins (Ym1/2) and macrophage mannose receptor-2 (Cd206) were significantly down-regulated. Human peripheral blood monocytes were treated with eRNA and analysed by micro-array analysis of the whole human genome, revealing an up-regulation of 79 genes by at least four-fold; 27 of which are related to signal transduction and 15 genes associated with inflammatory response. In accordance with the proposed actions of eRNA as a pro-inflammatory “alarm signal”, these data shed light on the role of eRNA in the context of chronic inflammatory diseases such as atherosclerosis.
Extracellular RNA; inflammation; microarray technology; gene expression; monocyte/macrophage polarisation
Recent studies have shown that ultra-large complexes (ULCs) of platelet factor 4 (PF4) and heparin (H) play an essential role in the pathogenesis of Heparin-Induced Thrombocytopenia (HIT), an immune-mediated disorder caused by PF4/H antibodies. Because antigenic PF4/H ULCs assemble through non-specific electrostatic interactions, we reasoned that disruption of charge-based interactions can modulate the immune response to antigen. We tested a minimally anticoagulant compound (2-O, 3-O desulfated heparin or ODSH) with preserved charge to disrupt PF4/H complex formation and immunogenicity. We show that ODSH disrupts complexes when added to pre-formed PF4/H ULCs and prevents ULC formation when incubated simultaneously with PF4 and UFH. In other studies, we show that excess ODSH reduces HIT antibody (Ab) binding in immunoassays and that PF4/ODSH complexes do not cross-react with HIT Abs. When ODSH and UFH are mixed at equimolar concentrations, we show that there is a negligible effect on amount of protamine required for heparin neutralization and reduced immunogenicity of PF4/UFH in the presence of ODSH. Taken together, these studies suggest that ODSH can be used concurrently with UFH to disrupt PF4/H charge interactions and provides a novel strategy to reduce antibody mediated complications in HIT.
Platelet Factor 4; PF4; ODSH; heparin and HIT; immunogenicity
The pathogenesis of atherosclerosis involves the interplay of hematopoietic, stromal and endothelial cells. Platelet interactions with endothelium and leukocytes are pivotal for atherosclerosis promotion. Glycoprotein (GP) Ibα is the ligand-binding subunit of the platelet GPIb-IX-V receptor complex; its deficiency causes the Bernard-Soulier syndrome (BSS), characterized by absent platelet GPIb-IX-V, macrothrombocytopenia and bleeding. We designed this study to determine the role of platelet GPIbα in the pathogenesis of atherosclerosis using two unique knockout models. Ldlr−/− mice were reconstituted with wild type (wt), GPIbα−/− (lacks GPIbα) or chimeric IL-4R/GPIbα-Tg (lacks GPIbα extracellular domain) bone marrow and assayed for atherosclerosis development after feeding with pro-atherogenic “western diet”.
Here, we report that Ldlr−/− mice reconstituted with GPIbα−/− bone marrow developed less atherosclerosis compared to wt controls; accompanied by augmented accumulation of pro-inflammatory CD11b+ and CD11c+ myeloid cells, reduced oxLDL uptake and decreased TNF and IL12p35 gene expression in the aortas. Flow cytometry and live cell imaging in whole blood-perfused microfluidic chambers revealed reduced platelet-monocyte aggregates in GPIbα−/− mice, which resulted in decreased monocyte activation. Interestingly, Ldlr−/− mice reconstituted with IL-4R/GPIbα-Tg bone marrow, producing less abnormal platelets, showed atherosclerotic lesions similar to wt mice.Platelet interaction with blood monocytes and accumulation of myeloid cells in the aortas was also essentially unaltered. Moreover, only complete GPIbα ablation altered platelet microparticles and CCL5 chemokine production.
Thus, atherosclerosis reduction in mice lacking GPIbα may not result from the defective GPIbα-ligand binding, but more likely is a consequence of functional defects of GPIbα−/− platelets and reduced blood platelet counts.
aorta; atherosclerosis; inflammation; myeloid cells; platelets
Del-1; Mac-1; iC3b; phagocytosis
Upon conversion of fibrinogen into fibrin, fibrinogen αC-domains containing the RGD recognition motif form ordered αC polymers. Our previous study revealed that polymerization of these domains promotes integrin-dependent adhesion and spreading of endothelial cells, as well as integrin-mediated activation of the FAK and ERK1/2 signaling pathways. The major goal of this study was to test the impact of αC-domain polymerization on endothelial cell migration and proliferation during wound healing, and to clarify the mechanism underlying superior activity of αC polymers toward endothelial cells. In an in vitro wound healing assay, confluent endothelial cell monolayers on tissue culture plates coated with the αC monomer or αC polymers were wounded by scratching and wound closure was monitored by time-lapse videomicroscopy. Although the plates were coated with equal amounts of αC species, as confirmed by ELISA, wound closure by the cells occurred much faster on αC polymers, indicating that αC-domain polymerization promotes cell migration and proliferation. In agreement, endothelial cell proliferation was also more efficient on αC polymers, as revealed by cell proliferation assay. Wound closure on both types of substrates was equally inhibited by the integrin-blocking GRGDSP peptide and a specific antagonist of the ERK1/2 signaling pathway. In contrast, blocking the FAK signaling pathway by a specific antagonist decreased wound closure only on αC polymers. These results indicate that polymerization of the αC-domains enhances integrin-dependent endothelial cell migration and proliferation mainly through the FAK signaling pathway. Furthermore, clustering of integrin-binding RGD motifs in αC polymers is the major mechanism triggering these events.
Fibrinogen/fibrin; endothelial cells; cell migration; cell proliferation; wound healing
3-phosphoinositide-dependent protein kinase 1 (PDK1), a member of the protein A,G and C (AGC) family of proteins, is a Ser/Thr protein kinase that can phosphorylate and activate other protein kinases from the AGC family, including Akt at Thr308, all of which play important roles in mediating cellular responses. The functional role of PDK1 or the importance of phosphorylation of Akt on Thr308 for its activity has not been investigated in human platelets. In this study, we tested two pharmacological inhibitors of PDK1, BX795 and BX912, to assess the role of Thr308 phosphorylation on Akt. PAR4-induced phosphorylation of Akt onThr308 was inhibited by BX795 without affecting phosphorylation of Akt on Ser473. The lack of Thr308 phosphorylation on Akt also led to the inhibition of PAR4-induced phosphorylation of two downstream substrates of Akt, viz. GSK3β and PRAS40. In vitro kinase activity of Akt was completely abolished if Thr308 on Akt was not phosphorylated. BX795 caused inhibition of 2-MeSADP-induced or collagen-induced aggregation, ATP secretion and thromboxane generation. Primary aggregation induced by 2-MeSADP was also inhibited in the presence of BX795. PDK1 inhibition also resulted in reduced clot retraction indicating its role in outside-in signalling. These results demonstrate that PDK1 selectively phosphorylates Thr308 on Akt thereby regulating its activity and plays a positive regulatory role in platelet physiological responses.
Kinases; platelet pharmacology; signal transduction
There is a significant recruitment of leucocytes into aortas during atherogenesis. L-selectin regulates leucocyte migration into secondary lymphoid and peripheral tissues and was proposed to play a role in leucocyte homing into aortas. Here, we determine the role of L-selectin in atherosclerosis. L-selectin-deficient Apoe−/− (Sell−/−Apoe−/−) mice had a 74% increase in plaque burden compared to Apoe−/− mice fed a chow diet for 50 weeks. Elevated atherosclerosis was accompanied by increased aortic leucocyte content, but a 50% reduction in aortic B cells despite elevated B cell counts in the blood. Follicular B cells represented 65%, whereas B1a and regulatory B cells (Breg) comprised 5% of aortic B cells. B1a and Breg cell subsets were reduced in Sell−/−Apoe−/− aortas with accompanied 2-fold decrease in aortic T15 antibody and 1.2-fold decrease of IL-10 levels. L-selectin was required for B1 cell homing to the atherosclerotic aorta, as demonstrated by a 1.5-fold decrease in the migration of Sell−/−Apoe−/− versus Apoe−/− cells. Notably, we found a 1.6-fold increase in CD68hi macrophages in Sell−/−Apoe−/− compared to Apoe−/− aortas, despite comparable blood monocyte numbers and L-selectin-dependent aortic homing. L-selectin had no effect on neutrophil migration into aorta, but led to elevated blood neutrophil numbers, suggesting a potential involvement of neutrophils in atherogenesis of Sell−/−Apoe−/− mice. Thus, L-selectin deficiency increases peripheral blood neutrophil and lymphocyte numbers, decreases aortic B1a and Breg populations, T15 antibody and IL-10 levels, and increases aortic macrophage content of Sell−/−Apoe−/− mice. Altogether, these data provide evidence for an overall athero-protective role of L-selectin.
Atherosclerosis; L-selectin; B cell subsets
Factor VIII (FVIII), a procoagulant cofactor, plays a crucial role in the intrinsic coagulation cascade. A causal association between elevated FVIII levels and venous thrombosis incidence has been established; no such association has been confirmed with arterial thrombosis.
The independent role of elevated FVIII levels in arteriolar thrombosis was evaluated in a mouse model to determine the thrombogenic potential of elevated levels of FVIII.
The in vitro thrombogenic effect of elevated FVIII levels was examined using thrombin-antithrombin (TAT) complex generation and thromboelastography (TEG) assays. The thrombogenic potential of acute and extended elevation of circulating FVIII levels was assessed using ferric chloride induced injury of the cremaster arterioles.
The rate of TAT complex formation, and the final concentration of TAT complexes, significantly increased as FVIII levels were elevated from 100% to 400% FVIII activity. TEG analysis of fibrin and clot formation showed that as FVIII levels were elevated, the time to initial fibrin formation decreased and the rate of fibrin formation increased. The acute elevation of circulating FVIII to 400% FVIII activity resulted in significantly decreased times to vessel occlusion. Prolonged elevation of FVIII activity did not significantly affect time to vessel occlusion.
Acute elevations in FVIII levels result in a non-linear thrombogenic effect, with non-significant increases in thrombogenic risk within the physiological range (FVIII levels up to 200%). Prolonged elevation of plasma FVIII did not further increase the thrombogenic potential of elevated FVIII levels.
Animal Models; Arterial Thrombosis; Factor VIII; Intravital Microscopy
Tissue factor (TF), the cellular receptor and cofactor for factor VII/VIIa, initiates haemostasis and thrombosis. Initial tissue distribution studies suggested that TF was sequestered from the circulation and only present at perivascular sites. However, there is now clear evidence that TF also exists as a blood-borne form with critical contributions not only to arterial thrombosis following plaque rupture and to venous thrombosis following endothelial perturbation, but also to various other clotting abnormalities associated with trauma, infection, or cancer. Because thrombin generation, fibrin deposition, and platelet aggregation in the contexts of haemostasis, thrombosis, and pathogen defence frequently occur without TF de novo synthesis, considerable efforts are still directed to understanding the molecular events underlying the conversion of predominantly non-coagulant or cryptic TF on the surface of haematopoietic cells to a highly procoagulant molecule following cellular injury or stimulation. This article will review some of the still controversial mechanisms implicated in cellular TF activation or decryption with particular focus on the coordinated effects of outer leaflet phosphatidylserine exposure and thiol-disulfide exchange pathways involving protein disulfide isomerase (PDI). In this regard, our recent findings of ATP-triggered stimulation of the purinergic P2X7 receptor on myeloid and smooth muscle cells resulting in potent TF activation and shedding of procoagulant microparticles as well as of rapid monocyte TF decryption following antithymocyte globulin-dependent membrane complement fixation have delineated specific PDI-dependent pathways of cellular TF activation and thus illustrated additional and novel links in the coupling of inflammation and coagulation.
Tissue factor; phosphatidylserine; protein disulfide isomerase; thiol-disulfide exchange; thrombosis
To identify a physiological agonist of PAR3, we used PAR4 null murine platelets, which were known to express only PAR3. In this study, we tested several proteases and found that trypsin, but not heat-inactivated trypsin, activated PAR4 null murine platelets. Even at high concentrations, trypsin caused shape change without increasing intracellular calcium levels in PAR4 null murine platelets. Consistent with this result, the Gq inhibitor YM-254890 had no effect on trypsin-induced shape change. However, trypsin-induced platelet shape change was abolished by either p160ROCK inhibitor, Y27632 or H1152. Furthermore, trypsin caused phosphorylation of myosin light chain (Thr18), but not Akt or Erk. Surprisingly, trypsin caused a similar shape change in PAR4-desensitized PAR3 null murine platelets as in PAR4 null murine platelets, indicating that trypsin did not activate PAR3 to cause shape change. More interestingly, the Src family kinase (SFK) inhibitor PP2 abolished trypsin-induced, but not AYPGKF-induced, shape change. Hence, trypsin activated a novel signaling pathway through RhoA/p160ROCK and was regulated by SFKs. In conclusion, our study demonstrates a novel protease signaling pathway in platelets that is independent of PARs. This protease-induced novel signaling pathway regulates platelet shape change through SFKs and p160ROCK.
Trypsin; Protease-activated receptor; Platelet shape change
In Haemophilia B (HB) (factor IX (FIX) deficiency), F9 genotype largely determines clinical phenotype. Aimed to characterise Argentine families with HB, this study presents F9 genotype frequencies and their specific FIX inhibitor risk and 10 novel F9 mutations. Ninety-one DNA samples from HB patients and relatives were subjected to a new scheme: a primary screen for large deletions, a secondary screen for point mutations using conformation sensitive gel electrophoresis, DNA-sequencing and bioinformatic analysis.
Our unbiased HB population (N=52)(77% with severe, 11.5% moderate and 11.5% mild HB) showed 32 missense (61.5%) including three novel mutations predicting specific structural/functional defects in silico, 7 nonsense (13.5%)(one novel), 5 large deletions, 4 splice including three novel mutations affecting predicted splicing scores, 3 indels (two novel) and one Leiden mutation.
Our comprehensive HB population included five patients with long-lasting FIX inhibitors: three nonsense (p.E35* (novel), p.R75*, p.W240*) and two entire-F9 deletions. A further patient with an indel (p.A26Rfs*14) developed transient inhibitors.
A case-control analysis, based on our global prevalence of 3.05% for developing inhibitors in HB revealed that missense mutations were associated with a low risk odds ratio (OR) of 0.05 and a prevalence of 0.39%, whereas nonsense and entire-F9 deletions had significantly higher risks (OR 11.0 and 32.7) and prevalence (14.3% and 44.5%, respectively).
Our cost-effective practical approach enabled identification of the causative mutation in all 55 Argentine families with HB, analysis of the molecular pathology of novel F9 defects and determination of mutation-associated FIX inhibitor risks.
F9; haemophilia B; mutation; FIX inhibitors; CSGE
Several cohort studies have investigated the molecular basis of von Willebrand disease (VWD); however these have mostly focused on European and North American populations. This study aimed to investigate mutation spectrum in 26 index cases (IC) from Turkey diagnosed with all three VWD types, the majority (73%) with parents who were knowingly related. IC were screened for mutations using multiplex ligation-dependent probe amplification and analysis of all von Willebrand factor gene (VWF) exons and exon/intron boundaries. Selected missense mutations were expressed in vitro. Candidate VWF mutations were identified in 25 of 26 IC and included propeptide missense mutations in four IC (two resulting in type 1 and two in recessive 2A), all influencing VWF expression in vitro. Four missense mutations, a nonsense mutation and a small in-frame insertion resulting in type 2A were also identified. Of 15 type 3 VWD IC, 13 were homozygous and two compound heterozygous for 14 candidate mutations predicted to result in lack of expression and two propeptide missense changes. Identification of intronic breakpoints of an exon 17–18 deletion suggested that the mutation resulted from non-homologous end joining. This study provides further insight into the pathogenesis of VWD in a population with a high degree of consanguineous partnerships.
large-scale deletion; multiplex ligation-dependent probe amplification; mutation analysis; recessive 2A; von Willebrand disease
Background and Objective
The miR-143/145 cluster regulates VSMC specific gene expression, thus controlling differentiation, plasticity and contractile function, and promoting the VSMC phenotypic switch from a contractile/non-proliferative to a migrating/proliferative state. More recently increased miR-145 expression was observed in human carotid atherosclerotic plaques from symptomatic patients. The goal of this study was to investigate the contribution of miR-143/145 during atherogenesis by generating mice lacking miR-143/145 on an Ldlr-deficient background.
Methods and Results
Ldlr−/− and Ldlr−/−-miR-143/145−/− (DKO) were fed a Western diet (WD) for 16 weeks. At the end of the treatment, the lipid profile and the atherosclerotic lesions were assessed in both groups of mice. Absence of miR-143/145 significantly reduced atherosclerotic plaque size and macrophage infiltration. Plasma total cholesterol levels were lower in DKO and FLPC analysis showed decreased cholesterol content in VLDL and LDL fractions. Interestingly miR-143/145 deficiency per se resulted in increased hepatic and vascular ABCA1 expression. Experiments with the luciferase coding sequence fused to the ABCA1 3’UTR, Western blotting, qRT-PCR and mimicMiR confirmed the direct regulation of ABCA1 expression by miR-145.
miR-143/145 deficiency significantly reduces atherosclerosis in mice. Therapeutic inhibition of miR-145 might be useful for treating atherosclerotic vascular disease.
miR-143/145; atherosclerosis; ABCA1; smooth muscle cells
Plasma kallikrein (PK) is a serine protease generated from plasma prekallikrein, an abundant circulating zymogen expressed by the Klkb1 gene. The physiological actions of PK have been primarily attributed to its production of bradykinin and activation of coagulation factor XII, which promotes inflammation and the intrinsic coagulation pathway. Recent genetic, molecular, and pharmacological studies of PK have provided further insight into its role in physiology and disease. Genetic analyses have revealed common Klkb1 variants that are association with blood metabolite levels, hypertension, and coagulation. Characterization of animal models with Klkb1 deficiency and PK inhibition have demonstrated effects on inflammation, vascular function, blood pressure regulation, thrombosis, hemostasis, and metabolism. These reports have also identified a host of PK substrates and interactions, which suggest an expanded physiological role for this protease beyond the bradykinin system and coagulation. The review summarizes the mechanisms that contribute to PK activation and its emerging role in diabetes and metabolism.
Plasma kallikrein; diabetes; Klkb1; contact activation pathway
Following initial platelet activation, arachidonic acid is metabolised by cyclooxygenase-1 and 12-lipoxygenase (12-LOX). While the role of 12-LOX in the platelet is not well defined, recent evidence suggests that it may be important for regulation of platelet activity and is agonist-specific in the manner in which it regulates platelet function. Using small molecule inhibitors selective for 12-LOX and 12-LOX-deficient mice, the role of 12-LOX in regulation of human platelet activation and thrombosis was investigated. Pharmacologically inhibiting 12-LOX resulted in attenuation of platelet aggregation, selective in hibition of dense versus alpha granule secretion, and inhibition of platelet adhesion under flow for PAR4 and collagen. Additionally, 12-LOX-deficient mice showed attenuated integrin activity to PAR4-AP and convulxin compared to wild-type mice. Finally, platelet activation by PARs was shown to be differentially dependent on COX-1 and 12-LOX with PAR1 relying on COX-1 oxidation of arachi donic acid while PAR4 being more dependent on 12-LOX for normal platelet function. These studies demonstrate an important role for 12-LOX in regulating platelet activation and thrombosis. Furthermore, the data presented here provide a basis for potentially targeting 12-LOX as a means to attenuate unwanted platelet activation and clot formation.
Platelet activation; 12-lipoxygenase; eicosanoids; aggregation; thrombosis
The kallikreins are a family of serine proteases with a range of tissue-specific and essential proteolytic functions. Among the most well-studied are the prostate tissue specific KLK2 and KLK3 genes and their secreted protease products, hk2 and PSA. Members of the so-called classic kallikreins, these highly active trypsin-like serine proteases play established roles in human reproduction. Both hK2 and PSA expression is regulated by the androgen receptor, whose activity has a fundamental role in prostate tissue development and progression of disease. This feature, combined with the ability to sensitively detect different forms of these proteins in blood and biopsies, result in a crucially important biomarker for the presence and recurrence of cancer. Emerging evidence has begun to suggest a role for these kallikreins in critical vascular events. This review discusses the established and developing biological roles of hK2 and PSA, as well as the historical and advanced use of their detection to accurately and non-invasively detect and guide treatment of prostatic disease.