Blood Coagulation; Blood Coagulation Tests; Blood Platelets; metabolism; pathology; Cell-Derived Microparticles; metabolism; pathology; Centrifugation; methods; Factor Xa; metabolism; Flow Cytometry; Fluorometry; Humans; Particle Size; Phospholipids; blood; Platelet Count; Thrombin; metabolism
Although factor (F) VIIIa is inactivated by activated protein C (APC) through cleavages in the FVIII heavy chain-derived A1 (Arg336) and A2 subunits (Arg562), the FVIII light chain (LC) contributes to catalysis by binding the enzyme. ELISA-based binding assays showed that FVIII and FVIII LC bound to immobilized active site-modified activated protein C (DEGR-APC) (apparent Kd = 273 nM and 1.0 μM, respectively). Furthermore, FVIII LC effectively competed with FVIIIa in blocking APC-catalyzed cleavage at Arg336 (Ki = 709 nM). A binding site previously identified near the C-terminal end of the A3 domain (residues 2007–2016) of FVIII LC was subjected to Ala-scanning mutagenesis. FXa generation assays and Western and dot blotting were employed to assess the contribution of these residues to FVIIIa interactions with APC. Virtually all variants tested showed some reductions in the rates of APC-catalyzed inactivation of the cofactor and cleavage at the primary inactivation site (Arg336), with maximal reductions in inactivation rates (~3-fold relative to WT) and cleavage rates (~3 to ~9-fold relative to WT) observed for the Met2010Ala, Ser2011Ala, and Leu2013Ala variants. Titration of FVIIIa substrate concentration monitoring cleavage by a dot blot assay indicated that these variants also showed ~3-fold increases relative to WT while a double mutant (Met2010Ala/Ser2011Ala) showed a >4-fold increase in Km. These results show a contribution of a number of residues within the 2007–2016 sequence, and in particular residues Met2010, Ser2011, and Leu2013 to an APC-interactive site.
factor VIIIa; activated protein C; factor VIII mutants; proteolysis
Anticoagulant plasma protein S (PS) is essential for maintaining hemostatic balance. About 2.5% of PS is stored in platelets and released upon platelet stimulation. So far, little is known about the functionality and importance of platelet (plt)PS. A platelet-associated protease cleaves plasma-derived (pd)PS and pltPS in the “thrombin-sensitive region”, abolishing activated protein C (APC) cofactor activity. However we showed that cleaved PS retains APC-independent anticoagulant activities (“PS-direct”). To investigate whether pltPS or pdPS exert PS-direct on platelets or platelet-shed microparticles, thrombin and factor (F)Xa generation on unstimulated or stimulated washed platelets and microparticles were measured. Western blotting revealed that pltPS and pdPS bound to washed, stimulated platelets and microparticles, and that pltPS had slower electrophoretic mobility than pdPS. Platelet stimulation in the presence of inhibitory anti-PS antibodies resulted in 2.6 ± 1.6-fold (p<0.0004, n=20) more thrombin generation upon addition of FXa and prothrombin. PltPS exerted PS-direct that was similar to or greater than that of Zn2+-containing pdPS and much greater than that of Zn2+-deficient pdPS. Findings were confirmed using purified pltPS. Platelet-bound pltPS and microparticle-bound pltPS had similar PS-direct. Finally, platelet stimulation in the presence of inhibitory anti-PS antibodies resulted in 1.5 ± 0.2-fold (p<0.0001, n=11) more FXa generation upon addition of TF/FVIIa and FX. Thus, pltPS inhibits both prothrombinase and extrinsic FXase activities. Neutralizing antibodies against APC and TFPI had no effect on the PS-direct of pltPS or pdPS on platelets. This study indicates that pltPS may be an essential pool of PS that counterbalances procoagulant activities on platelets.
activated protein C; protein S; platelets; microparticles; anticoagulant activity
The mechanism underlying a hyperreactive platelet phenotype remains unknown. Since serotonin has been shown to influence platelet biology and atherothrombosis, we sought to investigate the association of platelet serotonin transporter number, binding affinity, and uptake kinetics to platelet aggregation. A total of 542 healthy volunteers had light transmittance platelet aggregometry measured in response to varying concentrations of epinephrine, serotonin, epinephrine plus serotonin, ADP and collagen. Transporter-dependent serotonin uptake rate was determined (Vmax), as were serotonin transporter number (Bmax) and binding affinity (Kd) using 3H paroxetine binding in a homologous displacement assay, nonlinear regression and validated algorithms for kinetic modeling. Stimulation with submaximal (2 μM) epinephrine concentration elicited a distinct, bimodal pattern of platelet aggregation in this population. In contrast, subjects exhibited minimal aggregation in response to serotonin alone. Co-stimulation with submaximal epinephrine and serotonin induced platelet aggregation to a level beyond that observed with either agonist alone and maintained a bimodal response distribution. Subjects with heightened (>60%) platelet aggregation to both epinephrine alone and epinephrine plus serotonin exhibited increased platelet serotonin uptake, and transporter number and affinity. In a population of healthy subjects, co-stimulation with submaximal concentrations of epinephrine and serotonin identifies a subset of individuals with a hyperreactive platelet aggregation profile that is associated with changes in platelet serotonin function.
Platelets; platelet activity; serotonin; epinephrine; transporter
Recent in vitro studies have shown that the zymogen and activated form of FVII bind to endothelial cell protein C receptor (EPCR). At present, there is no evidence that FVIIa binds to EPCR on vascular endothelium in vivo in the presence of circulating protein C, a primary ligand for EPCR. The present study was carried out to investigate the interaction of murine and human ligands with murine EPCR both in vivo and in vitro. Measurement of endogenous plasma levels of FVII in wild-type, EPCR-deficient and EPCR-over expressing mice showed slightly lower levels of FVII in EPCR-over expressing mice. However, infusion of high concentrations of competing ligands, either human APCi or FVIIai, to EPCR-over expressing mice failed to increase plasma levels of mouse FVII whereas they increased the plasma levels of protein C by 2 to 3-fold. Examining the association of exogenously administered mouse FVIIa or human FVIIa by immunohistochemistry revealed that human, but not murine FVIIa, binds to the murine endothelium in an EPCR-dependent manner. In vitro binding studies performed using surface plasmon resonance and endothelial cells revealed that murine FVIIa binds murine EPCR negligibly. Human FVIIa binding to EPCR, particularly to mouse EPCR, is markedly enhanced by availability of Mg2+ ions. In summary, our data show that murine FVIIa binds poorly to murine EPCR, whereas human FVIIa binds efficiently to both murine and human EPCR. Our data suggest that one should consider the use of human FVIIa in mouse models to investigate the significance of FVIIa and EPCR interaction.
Endothelial cell protein C receptor; factor VIIa; protein C
Although protein kinases and phosphatases participate in integrin αIIbβ3 signaling, whether integrin functions are regulated by the catalytic subunit of protein phosphatase 1 (PP1c) isoforms are unclear. We show that siRNA mediated knockdown of all PP1c isoforms (α, β and γ1) in 293 αIIbβ3 cells decreased adhesion to immobilized fibrinogen and fibrin clot retraction. Selective knockdown of only PP1cγ1 did not alter adhesion or clot retraction, while depletion of PP1cβ decreased both functions. Unexpectedly, knockdown of PP1cα enhanced αIIbβ3 adhesion to fibrinogen and clot retraction. Protein interaction studies revealed that all PP1c isoforms can interact with the integrin αIIb subunit. Phosphoprofiling studies revealed an enhanced activation of mitogen-activated protein kinase (MAPK) p38 in the PP1cα depleted cells. Enhanced adhesive phenotype displayed by the PP1cα depleted 293 αIIbβ3 cells was blocked by pharmacological inhibition of p38. Conversely, the decreased adhesion of PP1cα overexpressing cells was rescued by the expression of constitutively active p38α or p38γ. Thus, PP1c isoforms have distinct contribution to the outside-in αIIbβ3 signaling-dependent functions in 293 αIIbβ3 cells. Moreover, PP1cα negatively regulates integrin function by suppressing the p38 pathway.
Protein phosphatase 1; Adhesion; Clot retraction; Fibrinogen
Factor IXa (FIXa) is a vitamin K-dependent coagulation serine protease which binds to factor VIIIa (FVIIIa) on negatively charged phospholipid vesicles (PCPS) to catalyze the activation of factor X (FX) to factor Xa (FXa) in the intrinsic pathway. Fluorescence resonance energy transfer (FRET) studies have indicated that the Gla-domain-dependent interaction of FIXa and FX with PCPS in the presence of FVIIIa positions the active-site of the protease at an appropriate height above the membrane surface to optimize the catalytic reaction. In this study, we investigated the contribution of the NH2-terminal EGF-domain (EGF1) of FIXa to the recognition specificity of intrinsic Tenase by constructing an EGF1 deletion mutant of FIXa (FIXa-desEGF1) and characterizing the properties of the mutant in kinetic, direct binding and FRET assays. The results of direct binding and kinetic studies demonstrated that the binding affinity of the mutant for interaction with FVIIIa on PCPS has been impaired greater than 10- fold and the catalytic efficiency of the mutant protease-FVIIIa-PCPS complex in the activation of FX has been decreased ~100-fold. By contrast, the mutant protease exhibited a normal activity toward FX in the absence of the protein cofactor. FRET measurements revealed that the distance of the active-site of the mutant FIXa relative to PCPS vesicles has been decreased 10 Å from 75 ±2 Å for FIXa to 65 ±2 Å for FIXa-desEGF1 independent of FVIIIa. These results suggest that the NH2-terminal EGF-domain of FIXa provides a binding-site for FVIIIa and plays an essential spacer function in the intrinsic Tenase complex.
Factor IXa; factor VIIIa; intrinsic Tenase; FRET; Gla-domain; EGF-domains
Advances in imaging technology have provided powerful tools for dissecting the angiogenic and inflammatory aspects of atherosclerosis. Improved technology along with multi-modal approaches has expanded the utilisation of imaging. Recent advances provide the ability to better define structure and development of angiogenic vessels, identify relationships between inflammatory mediators and the vessel wall, validate biological effects of anti-inflammatory and anti-angiogenic drugs, delivery and/or targeting specific molecules to inflammatory regions of atherosclerotic plaques.
Atherosclerosis; inflammation; angiogenesis; imaging
Vascular hyperpermeability contributes to morbidity in inflammation. Current methodologies for in vivo assessment of permeability based on extravasation of Evans Blue (EB)-bound albumin are cumbersome and often lack sensitivity. We developed a novel infrared fluorescence (IRF) methodology for measurement of EB-albumin extravasation to quantify vascular permeability in murine models. Vascular permeability induced by endotoxemia was examined for all solid organs, brain, skin and peritoneum by IRF and the traditional absorbance-based measurement of EB in tissue extracts. Organ IRF increased linearly with increasing concentrations of i.v. EB (2.5-25 mg/kg). Tissue IRF was more sensitive for EB accumulation compared to the absorbance-based method. Accordingly, differences in vascular permeability and organ EB accumulation between lipopolysaccharide-treated and saline-treated mice were often significant when analyzed by IRF-based detection but not by absorbance-based detection. EB was detected in all 353 organs analyzed with IRF but only in 67% (239/353) of organs analyzed by absorbance-based methodology, demonstrating improved sensitivity of EB detection in organs with IRF. In contrast, EB in plasma after EB administration was readily measured by both methods with high correlation between the two methods (n=116, r2=0.86). Quantitation of organ-specific EB-IRF differences due to endotoxin was optimal when IRF was compared between mice matched for weight, gender, and age, and with appropriate corrections for organ weight and EB plasma concentrations. Notably, EB-IRF methodology leaves organs intact for subsequent histopathology. In summary, EB-IRF is a novel, highly sensitive, rapid, and convenient method for the relative quantification of EB in intact organs of treatment versus control mice.
vascular permeability; endotoxemia; method; lipopolysaccharide; Evans Blue
Noninvasive imaging plays an emerging role in preclinical and clinical cancer research and has high potential to improve clinical translation of new drugs. This article summarizes and discusses tools and methods to image tumor angiogenesis and monitor anti-angiogenic therapy effects. In this context, micro-computed tomography (μCT) is recommended to visualize and quantify the micro-architecture of functional tumor vessels. Contrast-enhanced ultrasound (US) and magnetic resonance imaging (MRI) are favorable tools to assess functional vascular parameters, such as perfusion and relative blood volume. These functional parameters have been shown to indicate anti-angiogenic therapy response at an early stage, before changes in tumor size appear. For tumor characterization, the imaging of the molecular characteristics of tumor blood vessels, such as receptor expression, might have an even higher diagnostic potential and has been shown to be highly suitable for therapy monitoring as well. In this context, US using targeted microbubbles is currently evaluated in clinical trials as an important tool for the molecular characterization of the angiogenic endothelium. Other modalities, being preferably used for molecular imaging of vessels and their surrounding stroma, are photoacoustic imaging (PAI), near-infrared fluorescence optical imaging (OI), MRI, positron emission tomography (PET) and single photon emission computed tomography (SPECT). The latter two are particularly useful if very high sensitivity is needed, and/or if the molecular target is difficult to access. Carefully considering the pros and cons of different imaging modalities in a multimodal imaging setup enables a comprehensive longitudinal assessment of the (micro)morphology, function and molecular regulation of tumor vessels.
angiogenesis; functional imaging; molecular imaging; vessel normalization; cancer therapy
Pharmacogenetic dosing algorithms help predict warfarin maintenance doses, but their predictive performance differs in different populations, possibly due to unsuspected population-specific genetic variants. The objectives of this study were to quantify the effect of the VKORC1 D36Y variant (a marker of warfarin resistance previously described in 4% of Ashkenazi Jews) on warfarin maintenance doses and to examine how this variant affects the performance of the International Warfarin Pharmacogenetic Consortium (IWPC) dose prediction model. In 210 Israeli patients on chronic warfarin therapy recruited at a tertiary care center, we applied the IWPC model and then added D36Y genotype as covariate to the model (IWPC+D36Y) and compared predicted with actual doses. Median weekly warfarin dose was 35 mg (interquartile range [IQR], 24.5 to 52.5 mg). Among 16 heterozygous D36Y carriers (minor allele frequency = 3.8%), warfarin weekly dose was increased by a median of 43.7 mg (IQR, 40.5 to 47.2 mg) compared to non-carriers after adjustment for all IWPC parameters, a greater than 2-fold dose increase. The IWPC model performed suboptimally (coefficient of determination R2=27.0%; mean absolute error (MAE), 14.4 ± 16.2 mg/week). Accounting for D36Y genotype using the IWPC+D36Y model resulted in a significantly better model performance (R2=47.2%, MAE=12.6±12.4 mg/week). In conclusion, even at low frequencies, variants with a strong impact on warfarin dose may greatly decrease the performance of a commonly used dose prediction model. Unexpected discrepancies of the performance of universal prediction models in subpopulations should prompt searching for unsuspected confounders, including rare genetic variants.
Warfarin; pharmacogenetics; VKORC1; dose prediction; ethnicity
Apart from obesity, it remains controversial whether atherosclerosis and its cardiovascular risk disease (CVD) factors are associated with risk of venous thromboembolism (VTE). Using data from the Atherosclerosis Risk in Communities study (ARIC), we evaluated associations between CVD risk factors and incident VTE in a cohort of 15,340 participants who were free a history of VTE and/or anticoagulant use on enrolment. The CVD risk factors were updated during the follow-up period. Over a mean follow-up time of 15.5 years (237,375 person-years), 468 participants had VTE events. Adjusting for demographic variables and body mass index (BMI), current smokers were at greater risk [HR of 1.44 (95% CI: 1.12–1.86)] compared to non-smokers. There was a positive monotonic association between BMI and VTE risk. Individuals with a BMI ≥35 kg/m2 had a HR for VTE of 3.09 (95%CI: 2.26–4.23) compared to those with normal BMI (<25 kg/m2). Greater physical activity was associated with lower VTE risk in a demographic adjusted model; however, this association became non-significant following adjustment for BMI. Alcohol intake, diabetes, hypertension, high-density lipoprotein and low-density lipoprotein cholesterol, and triglycerides were not associated with VTE risk. In conclusion, among the well-established CVD risk factors, only current smoking and obesity were independently associated with VTE risk in this large cohort where risk factors were updated serially during follow-up. This finding corroborates that the pathogenesis of venous disease differs from that of atherosclerotic disease.
Deep-vein thrombosis; pulmonary embolism; risk factors
Identifying coagulation abnormalities in patients with combined bleeding and thrombosis history is clinically challenging. Our goal was to probe the complexity of dysregulated coagulation in humans by characterizing pathophysiologic mechanisms in a patient with both bleeding and thrombosis. The patient is a 56-year old female with a history of hematomas, poor wound healing, and thrombosis (retinal artery occlusion and transient cerebral ischemia). She had a normal activated partial thromboplastin time, prolonged thrombin and reptilase times, and decreased functional and antigenic fibrinogen levels, and was initially diagnosed with hypodysfibrinogenemia. This diagnosis was supported by DNA analysis revealing a novel FGB mutation (c.656A>G) predicting a Q189R mutation in the mature Bβ chain that was present in the heterozygote state. However, turbidity analysis showed that purified fibrinogen polymerization and degradation were indistinguishable from normal, and Bβ chain subpopulations appeared normal by two-dimensional difference in-gel electrophoresis, indicating the mutated chain was not secreted. Interestingly, plasma thrombin generation testing revealed the patient’s thrombin generation was higher than normal and could be attributed to elevated levels of factor VIII (FVIII, 163-225%). Accordingly, in an arterial injury model, hypofibrinogenemic mice (Fgn+/−) infused with FVIII demonstrated significantly shorter vessel occlusion times than saline-infused Fgn+/− mice. Together, these data associate the complex bleeding and thrombotic presentation with combined hypofibrinogenemia plus plasma hypercoagulability. These findings suggest previous cases in which fibrinogen abnormalities have been associated with thrombosis may also be complicated by co-existing plasma hypercoagulability and illustrate the importance of “global” coagulation testing in patients with compound presentations.
thrombin generation; factor VIII; hypofibrinogenemia; thrombosis; bleeding
DZ-697b is a new orally active antiplatelet agent that inhibits collagen and ristocetin-mediated platelet activation. It does not require metabolisation to generate its active compound and has a safer profile than clopidogrel in pre-clinical studies. We compared the antithrombotic effects and bleeding time prolongations of three DZ-697b doses with clopidogrel 300 mg. In a four-treatment, three-period, crossover-design, 20 healthy subjects (31 ± 7 years, 85% males) were randomised to single oral doses of DZ-697b (60, 120 and 360 mg), and clopidogrel (300 mg) (n=15 in each treatment with crossing-over). Antithrombotic effects were assessed by measuring six-hour post-dose changes from baseline in thrombus size in the Badimon chamber and platelet adhesion using Diamed Impact-R platelet function assay. Bleeding times were also measured pre-dose and at six hours post-dose. DZ-697b caused dose-dependent reductions in thrombus size at both high- and low-shear rates (mean reductions at 60, 120 and 360 mg doses of: 13.0%, 18.7%, 26.4% and 11.4%, 12.7%, 22.1% respectively, p<0.05 for all). Effect of clopidogrel (reductions of 18.7% and 11.0% respectively, p<0.05 for both) was closest to DZ-697b 120 mg. Reductions in platelet adhesion were also dose-dependent. Bleeding time ratio from baseline were shorter with DZ-697b versus clopidogrel (1.3, 1.4 and 1.5 versus 1.9, p<0.05 for all). The oral agent DZ-697b shows potent, dose-dependent, antithrombotic effects that are comparable to 300 mg clopidogrel at the 120 mg dose. Despite having equal or greater anti-platelet potency than 300 mg clopidogrel, bleeding time prolongations are significantly shorter with DZ-697b.
Antiplatelet agents; thrombosis; clinical studies; antiplatelet drugs
A large number of individuals are at risk for deep venous thrombosis (DVT) due to alterations in multiple coagulation factors and inhibitors secondary to malignancy, drug interactions, or other general medical conditions. Traditional metrics of haemostasis such as prothrombin time, partial thromboplastin time, and bleeding time, generally estimate anticoagulation status and bleeding risk rather than thrombosis risk.
The objective of this study was to correlate a novel, systems-based metric of clotting potential to risk of DVT from a database derived from the Leiden Thrombophilia Study (LETS).
We utilized a computational model of blood coagulation, which addresses the interplay between biochemical factors, blood flow, and physiologic surface initiation of coagulation, to calculate an individualized, systems-based metric of clotting potential, termed the flow simulated thrombin generation (FSTG), for 210 pre-menopausal women in LETS.
Both DVT and oral contraceptive (OC) use were associated with higher values of FSTG. We demonstrated a nearly 3-fold increased risk of DVT for each standard deviation increase above the mean in FSTG determined under venous flow conditions, which remained highly predictive after adjustment for age and OC status (adjusted OR 2.66; 95% CI 1.69–4.19; P<0.0001).
A systems-based screening approach that integrates biochemical factors and flow haemodynamics identifies small subgroups of patients at risk of thrombosis that may benefit from oral anticoagulants.
thrombosis; blood coagulation; computer simulation; thromboembolism
The transient receptor potential (melastatin) 2 (TRPM2), is an oxidant-activated nonselective cation channel, that is widely expressed in mammalian tissues including the vascular endothelium. Oxidative stress, through the generation of oxygen metabolites including H2O2, stimulates intracellular ADP-ribose formation which, in turn, opens TRPM2 channels. These channels act as an endogenous redox sensor for mediating oxidative stress/ROS-induced Ca2+ entry and the subsequent specific Ca2+-dependent cellular reactions such as endothelial hyper-permeability and apoptosis. This review summarizes recent findings on the mechanism by which oxidants induce TRPM2 activation, the role of these channels in the signaling vascular endothelial dysfunctions, and the modulation of oxidant-induced TRPM2 activation by PKCα and phospho-tyrosine phosphates L1.
TRPM2; Oxidative stress; Endothelial cells
Farnesyl pyrophosphate (FPP) is an intermediate in cholesterol biosynthesis, and it has also been reported to activate platelet LPA (lysophosphatidic acid) receptors. The aim of this study was to investigate the role of extracellular FPP in platelet aggregation. Human platelets were studied with light transmission aggregometry, flow cytometry and [35S]GTPγS binding assays. As shown previously, FPP could potentiate LPA-stimulated shape change. Surprisingly, FPP also acted as a selective insurmountable antagonist to ADP-induced platelet aggregation. FPP inhibited ADP-induced expression of P-selectin and the activated glycoprotein (Gp)llb/llla receptor. FPP blocked ADP-induced inhibition of cAMP accumulation and [35S]GTPγS binding in platelets. In Chinese hamster ovary cells expressing the P2Y12 receptor, FPP caused a right-ward shift of the [35S]GTPγS binding curve. In Sf9 insect cells expressing the human P2Y12 receptor, FPP showed a concentration-dependent, although incomplete inhibition of [3H]PSB-0413 binding. Docking of FPP in a P2Y12 receptor model revealed molecular similarities with ADP and a good fit into the binding pocket for ADP. In conclusion, FPP is an insurmountable antagonist of ADP-induced platelet aggregation mediated by the P2Y12 receptor. It could be an endogenous antithrombotic factor modulating the strong platelet aggregatory effects of ADP in a manner similar to the use of clopidogrel, prasugrel or ticagrelor in the treatment of ischaemic heart disease.
ADP receptors; platelet pharmacology; platelet physiology
Cells of both the innate and adaptive immune system participate in the development of atherosclerosis, a chronic inflammatory disorder of medium and large arteries. Natural killer T (NKT) cells express surface markers characteristic of natural killer cells and conventional T cells and bridge the innate and adaptive immune systems. The development and activation of NKT cells is dependent upon CD1d, a MHC-class I-type molecule that presents lipids, especially glycolipids to the TCR on NKT cells. There are two classes of NKT cells; invariant NKT cells that express a semi-invariant T cell receptor and variant NKT cells. This review summarizes studies in murine models in which the effect of the activation, overexpression or deletion of NKT cells or only invariant NKT cells on atherosclerosis has been examined.
atherosclerosis; CD1d; NKT cells
CLEC-2 is a C-type lectin receptor which is highly expressed on platelets but also found at low levels on different immune cells. CLEC-2 elicits powerful platelet activation upon engagement by its endogenous ligand, the mucin-type glycoprotein podoplanin. Podoplanin is expressed in a variety of tissues including lymphatic endothelial cells, kidney podocytes, type I lung epithelial cells, lymph node stromal cells and the choroid plexus epithelium. Animal models have shown that the correct separation of the lymphatic and blood vasculatures during embryonic development is dependent on CLEC-2-mediated platelet activation. Additionally, podoplanin deficient mice show abnormalities in heart, lungs, and lymphoid tissues, whereas absence of CLEC-2 affects brain development. This review summarizes the current understanding of the molecular pathways regulating CLEC-2 and podoplanin function and suggests other physiological and pathological processes where this molecular interaction might exert crucial roles.
Platelets; CLEC-2; podoplanin; lymphangiogenesis
The relationship between protein Z levels and thrombosis is controversial. We performed a systematic review and meta-analysis of the available studies to assess the association between protein Z and vascular thrombotic diseases. We conducted an electronic literature search through MedLine, Embase, Google Scholar, Web of Science, The Cochrane Library, bibliographies of retrieved articles and abstracts of congresses up to October, 2009. Studies were included if they analysed protein Z levels in patients with vascular thrombotic diseases. After the review process, 28 case-control studies (33 patient cohorts), including 4,218 patients with thrombotic diseases and 4,778 controls, were selected for analysis. The overall analysis using a random-effects model showed that low protein Z levels were associated with an increased risk of thrombosis (odds ratio [OR] 2.90, 95% confidence interval [CI] 2.05–4.12; p<0.00001). On subgroup analysis, a significant association was found between low protein Z levels and arterial vascular diseases (OR 2.67, 95%CI 1.60–4.48; p=0.0002), pregnancy complications (OR 4.17, 95%CI 2.31–7.52; p<0.00001), and venous thromboembolic diseases (OR 2.18, 95%CI 1.19–4.00; p=0.01). The results of this meta-analysis are consistent with a role for protein Z deficiency in thrombotic diseases, including arterial thrombosis, pregnancy complications and venous thromboembolism.
Protein Z; thrombosis; coagulation; meta-analysis
Desmopressin (DDAVP) is the treatment of choice in those with mild von Willebrand disease (VWD), yet 20% are unresponsive to DDAVP, and among the 80% who respond, the response is transient, as endothelial stores are depleted after 3 days. We, therefore, conducted a single-center Phase II clinical trial to determine safety and biologic efficacy of recombinant interleukin-11 (rhIL-11, Neumega®) in patients with VWD unresponsive or allergic to DDAVP, or mild or moderate hemophilia A (HA). Increases in VWF:RCo were observed by 48 hours after rhIL-11, with a 1.54-fold increase by day 4, 1.30-fold in VWD and 1.73-fold in HA. Similarly, by 48 hours, increases in VIII:C were observed, with a 1.65-fold increase by day 4, 1.86-fold in VWD and 1.48-fold in HA. Platelet VWFmRNA expression by qPCR increased 0.81-fold but did not correlate with plasma VWF:Ag responses. rhIL-11 was well tolerated, with grade 1 or less fluid retention, flushing, conjunctival erythema, except for transient grade 3 hyponatremia in one subject after excess fluid intake for diabetic hyperglycemia, which resolved with fluid restriction. In summary, rhIL-11 increases VWF levels in 2 of 4 DDAVP-unresponsive or allergic VWD and F.VIII levels in 4 of 5 mild or moderate hemophilia A subjects, suggesting its potential use in treatment of these disorders.
clinical trial; recombinant interleukin-11; von Willebrand disease; hemophilia A