Cocaine and Certain of Its Long-Acting Metabolites Stimulate Endothelial Cell Von Willebrand Factor Secretion

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1148-1148 ◽  
Author(s):  
William E Hobbs ◽  
Rebecca P Penkala ◽  
Emily E Moore ◽  
José A. López
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Intracellular Camp ◽  
Thrombotic Risk ◽  
Cocaine Use ◽  
Cocaine Metabolites ◽  
Increased Risk ◽  
String Formation

Abstract Abstract 1148 Cocaine ingestion is associated with increased risk of myocardial infarction, stroke, and deep vein thrombosis. Elevated risk for these thrombotic events persists for 7–10 days after cocaine ingestion, despite the fact that cocaine is rapidly cleared from the blood (with a half-life in circulation of less than 1 hour) being rapidly converted into numerous metabolites that circulate for 1–2 weeks. Thus, it is likely that these longer-lived cocaine metabolites account for the long-term thrombotic risk with cocaine use. Cocaine ingestion has been associated with increased plasma VWF concentration, early atherosclerosis, and platelet-rich arterial thrombi. VWF is the major adhesive ligand that attaches platelets to the vessel wall, either to the subendothelium at sites of injury where the endothelium has been denuded or, during inflammation, to intact endothelium, from which it is secreted. VWF is secreted by endothelial cells from intracellular storage granules (Weibel-Palade bodies) in a large, hyperadhesive multimeric form (ultra-large VWF, or ULVWF) that either remains tethered to the endothelial surface or is released into bulk flow. Platelet-VWF adhesion, and subsequent thrombus formation, may be augmented further by cocaine-induced vaso-constriction, increasing shear stress. Thus, we hypothesized that cocaine and/or its metabolites would stimulate endothelial VWF secretion as a mechanism of thrombotic risk. To test this possibility, we exposed cultured human endothelial cells from umbilical vein (HUVEC), brain microvasculature (BMVEC), or coronary artery (CAEC) to cocaine or one of its four major metabolites at concentration ranges reported to occur in plasma following cocaine use. The cocaine metabolites we tested were benzoylecgonine (BE), cocaethylene (CE), norcocaine (NC), and ecgonine methyl ester (EME). We assayed VWF release by platelet-VWF string formation in a parallel-plate flow chamber (2.5 dyne/cm2) and by measuring the concentration of VWF released into the supernatant. Cocaine concentrations as low as 0.1 μg/ml induced VWF release from HUVEC; 1–2 μg/ml cocaine was as effective in releasing VWF as 25 μM histamine or 4 mg/ml dDAVP. Of the cocaine metabolites, only BE and CE induced VWF release from endothelial cells. BMVEC were 10-fold more sensitive to cocaine and metabolites BE and CE than HUVEC in both platelet-string formation and VWF antigen assays. In CAEC, VWF release was slightly reduced compared to HUVEC in response to cocaine, BE, or CE. Consistent with this pattern, staining for intracellular VWF revealed the following hierarchy of intracellular VWF: BMVEC > HUVEC >> CAEC. In BMVEC and HUVEC, VWF staining was restricted to Weibel-Palade bodies, with CAEC also demonstrating a diffuse cytoplasmic pattern. We tested whether intracellular cAMP levels increased after cocaine or cocaine metabolite exposure to explore whether VWF release was dependent on Protein Kinase A, as is the case with dDAVP. Intracellular cAMP did not increase following exposure to cocaine or its metabolites in any of the endothelial cell lines. These results suggest that cocaine contributes to thrombosis by activating endothelial cells to secrete ULVWF via a mechanism that does not involve increased intracellular cAMP. The prolonged thrombotic risk after cocaine ingestion likely relates to the continued action of cocaine metabolites BE and/or CE on endothelial ULVWF secretion. VWF secretion is likely to vary between vascular beds, with brain endothelial cells being particularly sensitive. Furthermore, these results suggest that clinical management of cocaine-induced ischemia or vaso-occlusion may benefit from therapies aimed at disrupting the ULVWF–platelet interaction. Disclosures: No relevant conflicts of interest to declare.

Gamma Prime Fibrinogen Does Not Cause Arterial Thrombosis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1092-1092
Author(s):  
Bethany L Walton ◽  
Todd M Getz ◽  
Wolfgang Bergmeier ◽  
Shirley Uitte de Willige ◽  
Alisa S. Wolberg
Keyword(s):  
Platelet Aggregation ◽  
Arterial Thrombosis ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Formation Rate ◽  
Thrombotic Risk ◽  
Fibrin Formation ◽  
Increased Risk ◽  
Human Thrombin

Abstract Elevated levels of the acute phase plasma protein fibrinogen are associated with increased risk of thrombosis. Using a mouse model of arterial thrombosis, we have previously shown that elevated total fibrinogen levels increase the rate of fibrin formation, increase thrombus fibrin content, and directly promote arterial thrombosis (Machlus et al. 2011 Blood 117:4953-63). Fibrinogen is composed of two copies each of three polypeptide chains: Aα, Bβ, and γ. The fibrinogen γ chain undergoes alternative splicing, resulting in a dominant form (γA/γA) that comprises ∼90% of circulating fibrinogen and a minor species (γA/γ’) that comprises 8-15% of circulating fibrinogen. Epidemiologic studies have detected elevated levels of γA/γ’ fibrinogen in patients with a history of arterial thrombosis, indicating that this isoform associates with thrombotic risk. However, in vitro data show that γA/γ’ fibrinogen has anticoagulant properties due to its ability to bind and sequester thrombin, and suggest that its expression is upregulated in response to a proinflammatory/procoagulant process. To determine whether γA/γ’ fibrinogen is pro- or antithrombotic in vivo, we purified γA/γA and γA/γ’ fibrinogen from human plasma by anion exchange chromatography, raised circulating levels in mice by intravenous infusion, and determined the effects of increased levels of these proteins on arterial thrombus formation in vivo. We also determined the effects of γA/γA and γA/γ’ fibrinogen on whole plasma clot formation, platelet aggregation, and endogenous procoagulant activity. Compared to buffer controls (7.3 [4.6-40.0] minutes, median [range]), γA/γA fibrinogen shortened the time to carotid artery occlusion (5.5 [4.0-8.0] minutes, P<0.05), whereas γA/γ’ fibrinogen did not (7.6 [4.9-40.0] minutes, P=0.9). Both γA/γA and γA/γ’ fibrinogen could be cleaved by murine and human thrombin, were incorporated into murine and human clots, and supported murine and human thrombin-induced platelet aggregation. When γA/γA or γA/γ’ fibrinogen was spiked into normal human or murine plasma, both γA/γA and γA/γ’ fibrinogen accelerated the fibrin formation rate (P<0.02). However, γA/γA fibrinogen increased the rate to a greater extent than γA/γ’ fibrinogen (P<0.02). Interestingly, compared to buffer controls (18.9 [9.8-126.2] ng/mL), mice infused with γA/γ’ fibrinogen had lower levels of circulating plasma thrombin-antithrombin complexes (6.2 [0.9-89.4] ng/mL, P<0.01) following arterial injury, whereas mice infused with γA/γA fibrinogen did not (13.2 [10.7-80.1] ng/mL, P=0.7). These data suggest γA/γ’ fibrinogen exhibits “antithrombin I” activity in vivo by binding thrombin and decreasing circulating prothrombotic activity. Together, these findings indicate that elevated levels of the γA/γA fibrinogen isoform promote arterial thrombosis in vivo, whereas the γA/γ’ isoform does not. Disclosures: No relevant conflicts of interest to declare.

An Electron Microscopic Study of Platelet Thrombus Formation in the Rabbit with Particular Regard to 5-Hydroxytryptamine Release

1967 ◽  
Vol 18 (03/04) ◽  
pp. 592-604 ◽  
Author(s):  
H. R Baumgartner ◽  
J. P Tranzer ◽  
A Studer
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Vessel Wall ◽  
Thrombus Formation ◽  
Endothelial Damage ◽  
Electron Microscopic ◽  
Rabbit Ear ◽  
Basement Lamina ◽  
Platelet Thrombus

SummaryElectron microscopic and histologic examination of rabbit ear vein segments 4 and 30 min after slight endothelial damage have yielded the following findings :1. Platelets do not adhere to damaged endothelial cells.2. If the vessel wall is denuded of the whole endothelial cell, platelets adhere to the intimai basement lamina as do endothelial cells.3. The distance between adherent platelets as well as endothelial cells and intimai basement lamina measures 10 to 20 mµ, whereas the distance between aggregated platelets is 30 to 60 mµ.4. 5-hydroxytryptamine (5-HT) is released from platelets during viscous metamorphosis at least in part as 5-HT organelles.It should be noted that the presence of collagen fibers is not necessary for platelet thrombus formation in vivo.

Naturally Produced Extracellular Matrix Is an Excellent Substrate for Canine Endothelial Cell Proliferation and Resistance to Shear Stress on PTFE Vascular Grafts

1997 ◽  
Vol 78 (05) ◽  
pp. 1392-1398 ◽  
Author(s):  
A Schneider ◽  
M Chandra ◽  
G Lazarovici ◽  
I Vlodavsky ◽  
G Merin ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Shear Stress ◽  
Endothelial Cell ◽  
Cell Attachment ◽  
Thrombus Formation ◽  
Endothelial Cell Proliferation ◽  
Ptfe Graft ◽  
Vascular Prostheses ◽  
Endothelial Cell Attachment

SummaryPurpose: Successful development of a vascular prosthesis lined with endothelial cells (EC) may depend on the ability of the attached cells to resist shear forces after implantation. The present study was designed to investigate EC detachment from extracellular matrix (ECM) precoated vascular prostheses, caused by shear stress in vitro and to test the performance of these grafts in vivo. Methods: Bovine aortic endothelial cells were seeded inside untreated polytetrafluoro-ethylene (PTFE) vascular graft (10 X 0.6 cm), PTFE graft precoated with fibronectin (FN), or PTFE precoated with FN and a naturally produced ECM (106 cells/graft). Sixteen hours after seeding the medium was replaced and unattached cells counted. The strength of endothelial cell attachment was evaluated by subjecting the grafts to a physiologic shear stress of 15 dynes/cm2 for 1 h. The detached cells were collected and quantitated. PTFE or EC preseeded ECM coated grafts were implanted in the common carotid arteries of dogs. Results: While little or no differences were found in the extent of endothelial cell attachment to the various grafts (79%, 87% and 94% of the cells attached to PTFE, FN precoated PTFE, or FN+ECM precoated PTFE, respectively), the number of cells retained after a shear stress was significanly increased on ECM coated PTFE (20%, 54% and 85% on PTFE, FN coated PTFE, and FN+ECM coated PTFE, respectively, p <0.01). Implantation experiments in dogs revealed a significant increase in EC coverage and a reduced incidence of thrombus formation on ECM coated grafts that were seeded with autologous saphenous vein endothelial cells prior to implantation. Conclusion: ECM coating significantly increased the strength of endothelial cell attachment to vascular prostheses subjected to shear stress. The presence of adhesive macromolecules and potent endothelial cell growth promoting factors may render the ECM a promising substrate for vascular prostheses.

Abstract 531: Tie-2/Cre--Mediated Conditional Deletion of Lipid Phosphate Phosphatase-3 Reveals Its Role in Endothelial Cell Apoptosis

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Ishita Chatterjee ◽  
Kishore K Wary
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Genome Wide Association Study ◽  
Vascular Endothelial Cell ◽  
Protein Levels ◽  
Conditional Deletion ◽  
Transmission Electron ◽  
Increased Risk

Rationale: A recent genome-wide association study (GWAS) has linked a frequently occurring variation in the LPP3 (also known as PPAP2b) loci to increased risk of coronary heart disease (CAD). However, the in vivo function of LPP3 in vascular endothelial cell is incompletely understood. Goal: To address the endothelial cell (EC) specific function of Lpp3 in mice. Results: Tie-2/Cre mediated Lpp3 deletion did not affect normal vasculogenesis in early embryonic development, in contrast, in late embryonic stages it led to impaired angiogenesis associated with hemorrhage, edema and late embryonic lethal phenotype. Immunohistochemical staining followed by microscopic analyses of mutant embryos revealed reduced fibronectin and VE-cadherin expression throughout different vascular bed, and increased apoptosis in CD31+ vascular structures. Transmission electron microscopy (TEM) showed the presence of apoptotic endothelial cells and disruption of adherens junctions in mutant embryos. LPP3-knockdown in vitro showed an increase in p53 and p21 protein levels, with concomitant decrease in cell proliferation. LPP3-knockdown also decreased transendothelial electrical resistance (TER), interestingly re-expression of ß-catenin cDNA into LPP3-depleted endothelial cells partially restored the effect of loss of LPP3. Conclusion: These results suggest the ability of LPP3 to regulate survival and apoptotic activities of endothelial cells during patho/physiological angiogenesis.

Inflammation and thrombosis in diabetes

2011 ◽  
Vol 105 (S 06) ◽  
pp. S43-S54 ◽  
Author(s):  
Katharina Hess ◽  
Peter Grant
Keyword(s):  
Plaque Rupture ◽  
Thrombus Formation ◽  
Atherosclerotic Lesion ◽  
Coagulation Factors ◽  
Fibrin Clot ◽  
Thrombotic Risk ◽  
Cardiovascular Morbidity And Mortality ◽  
Increased Risk ◽  
Patients With Diabetes ◽  
Inflammatory State

SummaryPatients with diabetes mellitus are at increased risk of cardiovascular morbidity and mortality. Atherothrombosis, defined as atherosclerotic lesion disruption with superimposed thrombus formation, is the most common cause of death among these patients. Following plaque rupture, adherence of platelets is followed by local activation of coagulation, the formation of a cross-linked fibrin clot and the development of an occlusive platelet rich fibrin mesh. Patients with diabetes exhibit a thrombotic risk clustering which is composed of hyper-reactive platelets, up regulation of pro-thrombotic markers and suppression of fibrinolysis. These changes are mainly mediated by the presence of insulin resistance and dysglycaemia and an increased inflammatory state which directly affects platelet function, coagulation factors and clot structure. This prothrombotic state is related to increased cardiovascular risk and may account for the reduced response to antithrombotic therapeutic approaches, underpinning the need for adequate antithrombotic therapy in patients with diabetes to reduce their cardiovascular mortality.

Daunorubicin Induces Procoagulant Activity of Cultured Endothelial Cells through Phosphatidylserine Exposure and Microparticles Release

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 5185-5185
Author(s):  
Yueyue Fu ◽  
Huibo Li ◽  
Wen Li ◽  
Xiushuai Dong ◽  
Jinxiao Hou ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Conflicts Of Interest ◽  
Umbilical Vein ◽  
Chemotherapeutic Agents ◽  
Significant Inhibition ◽  
Procoagulant Activity ◽  
Clotting Time ◽  
Increased Risk ◽  
Umbilical Vein Endothelial Cells ◽  
The Impact

Abstract Abstract 5185 Administration of various chemotherapeutic agents is associated with an increased risk of thrombotic events. Although vascular endothelium plays a predominant role in blood coagulation and fibrinolysis, the effect of cytotoxic drugs on the procoagulant activity (PCA) of endothelial cells has not been well evaluated. Our study aims to investigate the possibility that daunorubicin, a chemotherapeutic agent, exerts prothrombotic effect on endothelial cells. We tested the impact of daunorubicin on phosphatidylserine (PS) exposure, endothelial microparticles (EMPs) release and consequent PCA. Human umbilical vein endothelial cells (HUVECs) were treated with daunorubicin (0.1, 0.2, 0.5, 1, 2 μ M) for 24 h. PCA of HUVECs was measured using clotting time and purified coagulation complex assays. Counts and PCA of EMPs were evaluated by flow cytometry and clotting time assay, respectively. Lactadherin was used as a novel probe for detection of PS exposure and EMPs release. We found that daunorubicin dose-dependently increased the PS exposure and consequent PCA of HUVECs. Moreover, daunorubicin treatment also enhanced the release of EMPs which were highly procoagulant. This increment was especially significant at 0.2 μ M of daunorubicin or over. Blockade of PS with lactadherin inhibited over 90% of HUVECs and EMPs PCA. However, anti-TF antibody had no significant inhibition effect. Our results demonstrate that daunorubicin treatment enhanced PCA of HUVECs through PS exposure and shedding of procoagulant EMPs. Lactadherin acts as an efficient anticoagulant in this process. Disclosures: No relevant conflicts of interest to declare.

Markers of Endothelial Angiogenic Dysfunction in Patients with Antiphospholipid Antibodies

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2300-2300
Author(s):  
Karen A Breen ◽  
Kiran Parmar ◽  
Beverley J Hunt
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Endothelial Dysfunction ◽  
Antiphospholipid Antibodies ◽  
Conflicts Of Interest ◽  
Control Group ◽  
Vegf Receptor ◽  
Healthy Controls ◽  
Related Complication ◽  
Obstetric Aps

Abstract Abstract 2300 Background: The antiphospholipid syndrome (APS) is characterised by presence of persistent antiphospholipid antibodies in association with thrombosis and/or pregnancy morbidity and mortality. Activation of endothelial cells by aPL has been proposed to pay a role in the pathogenesis of APS;antiphospholipid antibodies(aPL) activate endothelial cells in vitro and some evidence of endothelial cell perturbation has been found in patients with aPL. Vascular endothelial growth factor(VEGF) promotes endothelial cell growth and angiogenesis and has been shown to upregulate tissue factor(TF), and elevated VEGF levels have been found to correlate with elevated TF levels in APS. Soluble FMS like tyrosine kinase −1(SLFT-1) and soluble Endoglin(sENG) are antiangiogenic proteins. sFLT1 is a variant of the VEGF receptor released by endothelial cells and monocytes, binds VEGF causing endothelial dysfunction and decreased angiogenesis. sENG is a TGFβ co-receptor which impairs TGF β1 receptor binding and its downstream signalling effects. sFLT-1 and sENG are implicated in the pathogenesis of pre-eclampsia, and are elevated in disorders associated with endothelial dysfunction such as sickle cell disease, chronic kidney disease and coronary artery disease, but have not been studied in patients with aPL. Aims: The aim of this study was to measure sFLT1, sENG, sTF and VEGF in patients with aPL. Materials & methods: Local ethics committee approval was obtained and samples were taken from 182 patients (175 females, 7 males, median age 42 (range 19–73) years) who had PAPS, or had persistent aPL without associated complications. 28 healthy controls (28 females, median age (range 20–58) years) were included. Patients with PAPS included 95 with thrombotic complications, 48 with obstetric complications and 39 with isolated aPL. Patients with intercurrent infection or malignancy were excluded and the control group were not known to have aPL. Blood was drawn into Vacutainers containing EDTA 0.105M and processed within 3 hours of collection. ELISA assays measuring sFLT1, sENG, VEGF and sTF levels were performed according to manufacturer's protocol (Quidel, Pathway diagnostics ltd., Dorking, UK). Intra-assay CV for sFLT1,sENG, VEGF and sTF was 3.0,3.3,4.8 and 6.0 respectively. Inter assay CV for sFLT1,sENG, VEGF and sTF was 6.5,7.6, 7.4 and 5.0 respectively. Statistical analysis of results was performed using the Stata-11 software statistical package. Logarithmic transformation of data was performed and differences between groups were compared using linear regression methods, adjusting for age, ethnicity and medications. Results: Results are shown below in table 1 and are described as means and 95% confidence intervals (adjusted for age, sex, ethnicity and medications). sFLT1 and sENG levels were significantly higher in patients with aPL/PAPS compared to healthy controls(p<0.05). TF levels were significantly lower in patients with aPL/PAPS compared to healthy controls(p<0.05). There were no significant differences in VEGF levels in patients with aPL compared to controls(regardless of complication). When patients were categorised according to aPL related complication (thrombotic APS, obstetric aPS, isolated aPL), sFLT1 and sENG levels were found to be significantly higher in patients with thrombotic APS(p<0.05) compared to controls, sFLT1 levels were also significantly elevated in obstetric APS. TF levels were significantly lower in patients with obstetric APS and isolated aPL compared to controls. There were no differences between patients with aPL and controls when means were adjusted for age, ethnicity or medications. sFLT1 was associated with the presence of aPL/PAPS(area under ROC=0.76), whereas sENG had a weaker association (area under ROC: 0.65). Conclusions: We have demonstrated evidence of increased levels of sENG and sFLT1 in patients with aPL/PAPS.This suggests that there is underlying endothelial dysfunction in patients with APS. The role of sENG and sFLT1 in the pathogenesis of APS requires further exploration. Disclosures: No relevant conflicts of interest to declare.

Serum Asymmetric Dimethylarginine (ADMA) Levels Predict Steroid-Refractory Gvhd before Allogeneic Stem Cell Transplantion

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3908-3908
Author(s):  
Sascha Dietrich ◽  
Aleks Radukjovic ◽  
Michael Hess ◽  
Ute Hegenbart ◽  
Anthony D. Ho ◽  
...  
Keyword(s):  
Stem Cell ◽  
Endothelial Cell ◽  
Risk Score ◽  
Conflicts Of Interest ◽  
Serum Markers ◽  
Steroid Resistance ◽  
Increased Risk ◽  
Steroid Sensitive ◽  
Grade 3 ◽  
Steroid Refractory

Abstract Background: Graft-versus-host disease (GvHD) is the major complication of allogeneic stem cell transplantation (alloSCT) and its therapy-resistant form causes significant morbidity and mortality. The pathomechanism of steroid resistance is currently not completely understood, however, endothelial cell dysfunction seems to play an important role. Serum markers indicating endothelial cell distress such as angiopoetin-2 (Ang-2) and nitrates have been demonstrated to predict steroid-refractory GVHD already before alloSCT. In the current study we investigated ADMA, a strong indicator of endothelial dysfunction, as predictor of acute steroid-refractory GVHD. Methods: We performed a retrospective study of 521 patients who were treated by alloSCT between 3/2001-3/2013. Endothelial cell-associated serum markers were correlated with the course of GvHD, non relapse mortality and statin intake. Cut-offs were determined by maximally selected LogRank statistics. Results: During the post transplant period 114 patients developed a steroid-sensitive GvHD grade 1-2, 50 patients a steroid-sensitive GvHD grade 3-4, and 44 patients a steroid-refractory GvHD which had been always grade 3-4 maximum severity. 313 patients had no signs of GvHD. 266 patients were treated with pravastatin since 2010 when statin prophylaxis for cardiovascular long-term toxicity of calcineurin inhibitor immunosuppression was introduced as standard policy in our program. Median follow up of the whole cohort was 47 months. Median age was 53 years (range 17-75). High ADMA levels (>0.6 µM) significantly predicted steroid-refractory GVHD already before alloSCT (p=0.044, HR=3.5, 95% CI 1.1-11.8). In those patients who developed GVHD, high ADMA levels (>0.6 µM) were not only associated with an increased risk of steroid refractoriness, but also translated into increased non-relapse mortality (HR 2.2 CI 1.1-4.8, p=0.049). By multivariate analysis, the association of high ADMA levels with the risk of refractory GVHD remained significant in the whole cohort after adjusting for age, disease specific remission score, donor and conditioning (HR 6.4 CI 1.6-26.2, p=0.009). The ADMA cut-off was used to build a score along with the previously described predictors of GVHD refractoriness pretransplant Ang-2 (cut-off 1µg/ml) and nitrates (cut-off 26.5 µM). Serum levels of above the cut-off were counted as one point and 0-1 points were considered low risk, 2 points intermediate risk and 3 points high risk. The score was capable of separating risk groups of patients with a different likelihood to develop refractory GVHD (p=0.001, Figure 1) already before alloSCT. The highest risk group had also an increased NRM incidence (p= 0.02, Figure 1). Most interestingly, this endothelial risk score completely failed to predict outcome if patients were on statins (p=0.62). Conclusion: High serum ADMA predicts refractory GVHD already prior to alloSCT. Our results support the hypothesis that endothelial damage / vulnerability contribute to refractoriness of acute GVHD. Dept. Medicine V, University of Heidelberg, Germany Figure 1) Endothelial risk score predicts incidence of refractory GVHD before alloSCT. Figure 1). Endothelial risk score predicts incidence of refractory GVHD before alloSCT. Disclosures No relevant conflicts of interest to declare.

Essential Thrombocythemia Patients with CALR Mutations Have Increased Platelet Activation Markers Compared to JAK2V617F Mutated Patients

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3223-3223
Author(s):  
Maya Koren-Michowitz ◽  
Hagit Hauschner ◽  
Yulia Shuly ◽  
Meital Nagar ◽  
Elena Ribakovsky ◽  
...  
Keyword(s):  
Essential Thrombocythemia ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Absolute Number ◽  
Median Number ◽  
Healthy Controls ◽  
Activation Markers ◽  
Increased Risk ◽  
Platelet Aggregates ◽  
Calr Mutations

Abstract Essential thrombocythemia (ET) is associated with an increased risk for thrombo-hemorrhagic complications. The presence of the JAK2V617F mutation, found in approximately 50% of ET patients, has been associated with increased indices of platelet (PLT) activation suggesting its casual role in thrombus formation. Mutations in CALR were recently described in the majority of JAK2V617F negative ET patients, and are associated with a decreased rate of thrombotic events. This has led us to hypothesize that CALR mutations have a different influence on PLT activation compared to JAK2V617F. To evaluate the PLT activation state, surface expression of two PLT activation markers - p-selectin (CD62P) and PAC1 was studied using specific antibodies. MFI was analyzed by flow cytometry at baseline, as well as following ADP addition to PLT rich plasma. Monocyte-platelet aggregates were studied in whole blood samples by gating CD45+/CD14+ cells and calculating the percentage of CD41+ cells in the monocytes population. The immature PLT fraction (IPF) was analyzed with the XE-5000 hematology analyzer (Sysmex UK Ltd., Milton Keynes, UK), and the absolute number of immature PLT (nIP) was calculated from the total PLT count. Low risk ET patients (N-13, M/F-5/8) and healthy controls (N-10, M/F-4/6) are included in this analysis. JAK2V617F and CALR mutations were present in 8 and 5 patients, respectively; low dose aspirin (range 75-100mg) was taken by 85% of patients and 90% of controls. Median PLT count in CALR mutated, JAK2V617F mutated and healthy subjects was 913, 579 and 247 K/uL, respectively (p=0.0002), and it was higher in CALR compared to JAK2V617F positive patients (p=0.09). Both patient subgroups had a lower baseline MFI of p-selectin and PAC1 compared to healthy controls (p-selectin: 2.8, 3 and 4.5 for JAK2V617F [p=0.01], CALR [p=0.05] and controls; PAC1: 3, 3.3 and 5.2 for JAK2V617F [p=0.01], CALR [p=0.02] and controls, respectively) with no difference between CALR and JAK2V617F mutated patients. CALR compared to JAK2V617F mutated patients had higher median number of immature PLT (30 and 10.6 K/uL, p=0.04), and a higher fraction of monocyte- platelet aggregates (90 and 58%, p=0.05). nIP and monocyte- platelet aggregates were also significantly higher in CALR mutated but not in JAK2V617F mutated patients compared to healthy controls. Interestingly, there was no difference in post ADP PLT activation (post/baseline ratio) between ET patients and healthy controls. Finally, there were correlations between the PLT counts and nIP (R=0.8, p<0.0001), monocyte- platelet aggregates (R=0.5, p=0.02), baseline p-selectin MFI (R=-0.5, p=0.02) and PAC1 MFI (R=-0.5, p=0.01). Our preliminary results suggest a correlation between PLT activation markers and the PLT numbers, which can explain why CALR mutated patients in our cohort had higher nIP and monocyte- platelet aggregates fractions. The absence of an increased ADP induced PLT activation between patients and controls in this cohort compared with previous reports could be explained by the use of aspirin in the majority of patients and the high ADP concentration used for PLT activation. These results will be further studied in a lager cohort of patients. Disclosures No relevant conflicts of interest to declare.

Assembly of Prothrombinase on Endothelial Cells: Receptor-Mediated or Phospholipid-Driven?

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2267-2267
Author(s):  
Mikhail Galkin ◽  
Harlan Bradford ◽  
Sriram Krishnaswamy
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Surface ◽  
Conflicts Of Interest ◽  
Large Body ◽  
Confocal Imaging ◽  
Binding Studies ◽  
Binding Behavior ◽  
Background Levels ◽  
Membrane Bound

Abstract Membrane binding by factors Xa and Va plays an essential role in facilitating their interaction to yield membrane-bound prothrombinase. This concept is backed by a large body of biochemical work using synthetic phospholipid vesicles typically composed of 25% phosphatidylserine (PS) and 75% phosphatidylcholine (PC). However, it remains uncertain whether kinetic and thermodynamic findings with these membranes can be extrapolated to explain the assembly of prothrombinase on cell surfaces relevant to coagulation in the vasculature. We examined the binding of fluorescent derivatives of Xa and Va to endothelial cells cultured in flow chambers using confocal imaging of fluorescence intensity. Cell-bound factor Xa was imaged using Alexa488 or Alexa532 covalently linked to the active site with a peptidyl chloromethyl ketone. Minimal fluorescence was observed on the endothelial surface without intentional activation of the cells by thrombin. Following thrombin activation, total fluorescence of bound Xa in the absence of added Va was marginally different from the unactivated cells but increased 16-fold when excess Va was present. Heterogeneity was evident in the distribution of fluorescence over the surface of the cells that supported Xa binding. Thus, the binding of Xa requires the presence of Va and is distributed in a heterogeneous fashion on the activated endothelial cell surface. Factor Va bound to the endothelial cell was assessed using a derivative singly labeled at Cys539 with Alexa488 or Alexa532. Detection of bound Va also required prior activation by thrombin. The same robust signal for bound but heterogeneously distributed fluorescence was observed following activation both in the presence or absence of added Xa. Together the data indicate that Xa binding to the activated endothelial cell requires bound Va whereas Va binding is unaffected by the presence of Xa, implying a receptor-like role for Va. This is in marked contrast to the behavior on membranes containing 25% PS to which either Xa or Va can bind singly with good affinity. More definitive studies of prothrombinase assembly were conducted using donor fluorescence lifetime imaging on the cells using XaAlexa488 as donor and VaAlexa532 as acceptor. XaAlexa488 bound in the presence of unlabeled Va exhibited an average lifetime of 3.1 ns which was decreased to 2.1 ns in the presence of VaAlexa532. Equivalent lifetimes and energy transfer efficiency were measured using 25% PS containing membranes. Although Xa binding to the cells shows a near-absolute requirement for Va, the resulting cell-bound prothrombinase is comparable to that assembled on synthetic vesicles. An explanation could lie in the binding constraints associated with the exposure of limiting amounts of PS on the activated cell surface expected to disproportionately affect the binding of Xa and Va to membranes. These constraints were replicated using membrane bilayers supported on glass microspheres (lipospheres) containing either 0% or 5% PS balanced by PC and analysis of binding by flow cytometry. Background levels of binding were observed for all approaches in the absence of PS. Va binding was studied using a constant number of lipospheres and increasing concentrations of VaAlexa488 in the presence of different concentrations of unlabeled Xa. Mean fluorescence per 5% PS containing liposphere increased saturably with increasing concentrations of VaAlexa488 and was unaffected by Xa. The binding curves were consistent with a nM dissociation constant. In contrast, background levels of Xa were bound when monitored using varying concentrations of XaAlexa488 in the absence of Va. A family of saturable curves was obtained at different fixed concentrations of unlabeled Va, signifying binding with nM affinity, with amplitude proportional to the concentration of Va. Thus, 5% PS containing lipospheres can replicate the paradoxical receptor-like role for Va in prothrombinase assembly on activated endothelial cells. The binding behavior on lipospheres could be fully accounted for by the markedly decreased affinity of Xa for 5% PS membranes and the major contribution of linked interactions in the stabilization of membrane-bound prothrombinase. Despite appearances to the contrary, a model developed from binding studies with pure phospholipids can be generalized to provide a thermodynamic accounting for the peculiarities of prothrombinase assembly on the endothelial cell. Disclosures No relevant conflicts of interest to declare.

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