Background Essential thrombocythemia (ET) is characterized by thrombocytosis with increased platelet number and persistent activation. The mechanisms of thrombosis and the fate of these platelets are not clear. The aim of the present study is to explore the phagocytosis of platelets of ET patients by endothelial cells (ECs) in vitro and its relevance to the procoagulant activity (PCA). Methods Phosphatidylserine (PS) exposure on platelets was detected by flow cytometry. Phagocytosis of the platelets by ECs was performed using flow cytometry, confocal microscopy, and electron microscopy. The PCA of platelets was evaluated by coagulation time and purified coagulation complex assays. Results The PS exposure on platelets in ET patients is higher than that in healthy controls. The PS-exposed platelets are highly procoagulant and lactadherin reduced 80% of the PCA by blockade of PS. When cocultured, the platelets of ET patients were sequestered by ECs in a time-dependent fashion. Lactadherin enhanced phagocytosis by bridging the PS on activated platelets and the integrin alpha v beta 3 on ECs, and P-selectin played at least a partial role in this process. Furthermore, factor Xa and prothrombinase activity of PS-exposed platelets were decreased after incubation with ECs. Conclusion Our results suggest that phagocytic clearance of platelets by ECs occurs in ET patients, thus representing a novel mechanism to remove activated platelets from the circulation; lactadherin and phagocytosis could cooperatively limit the thrombophilia in ET patients.
Background Disruption of protein folding or inter-subunit interactions in the platelet glycoprotein (GP)Ib-IX complex leads to its abnormally low expression in the plasma membrane, the hallmark of Bernard-Soulier syndrome (BSS). Objective To discover the molecular mechanism by which GPIb alpha in the absence of GPIb beta and GPIX subunits is targeted for rapid degradation. Method The expression of GPIb alpha mutants with deletion or replacement of various domains were measured in transiently transfected Chinese hamster ovary cells. Results We report evidence to suggest that induction of the unfolded protein response by the unaccompanied mechanosensory domain (MSD) is a major factor for intracellular degradation and low expression of GPIb alpha. Removal of the MSD produced the first GPIb alpha variant that, even in the absence of GPIb beta and GPIX, expressed at a level comparable to that of wild-type GPIb alpha in the GPIb-IX complex, while retaining its native ligand-binding activity. Conclusion Our finding has important implications on the molecular pathogenesis of BSS and the function of the GPIb-IX complex.
Essentials Shp2 negatively regulates thrombus stability under pathological shear rate. Shp2 suppresses TXA2 receptor-mediated platelet dense granule secretion. Through alpha IIb beta 3 outside-in signaling, Shp2 targets calmodulin-dependent activation of Akt. Shp2 may serve to prevent the formation of unwanted occlusive thrombi. Background Perpetuation is the final phase of thrombus formation; however, its mechanisms and regulation are poorly understood. Objective To investigate the mechanism of Shp2 in platelet function and thrombosis. Methods and results We demonstrate that the platelet-expressed Src homology region 2 domain-containing protein tyrosine phosphatase Shp2 is a negative regulator of thrombus stability under high shear stress. In a ferric chloride-induced mesenteric arteriole thrombosis model, megakaryocyte/platelet-specific Shp2-deficient mice showed less thrombi shedding than wild-type mice, although their occlusion times were comparable. In accordance with this in vivo phenotype, a microfluidic whole-blood perfusion assay revealed that the thrombi formed on collagen surfaces by Shp2-deficient platelets were more stable under high shear rates than those produced by wild-type platelets. Whereas Shp2 deficiency did not alter platelet responsiveness towards thrombin, ADP and collagen stimulation, Shp2-deficient platelets showed increased dense granule secretion when stimulated by the thromboxane A(2) analog U46619. Shp2 appears to act downstream of integrin alpha(IIb)beta(3) outside-in signaling, inhibiting the phosphorylation of Akt (Ser473 and Thr308) and dense granule secretion. Calmodulin was also shown to bind both Shp2 and Akt, linking Shp2 to Akt activation. Conclusions Platelet Shp2 negatively regulates thrombus perpetuation under high shear stress. This signaling pathway may constitute an important mechanism for the prevention of unwanted occlusive thrombus formation, without dramatically interfering with hemostasis.
Objective Clinical and laboratory studies have demonstrated that platelets become hyperactive and prothrombotic in conditions of inflammation. We have previously shown that the proinflammatory cytokine interleukin (IL)-6 forms a complex with soluble IL-6 receptor alpha (sIL-6R alpha) to prime platelets for activation by subthreshold concentrations of collagen. Upon being stimulated with collagen, the transcription factor signal transducer and activator of transcription (STAT) 3 in platelets is phosphorylated and dimerized to act as a protein scaffold to facilitate the catalytic action between the kinase Syk and the substrate phospholipase C gamma 2 (PLC gamma 2) in collagen-induced signaling. However, it remains unknown how collagen induces phosphorylation and dimerization of STAT3. Methods and results We conducted complementary in vitro experiments to show that the IL-6 receptor subunit glycoprotein 130 (GP130) was in physical proximity to the collagen receptor glycoprotein VI (GPVI in membrane lipid rafts of platelets. This proximity allows collagen to induce STAT3 activation and dimerization, and the IL-6-sIL-6R alpha complex to activate the kinase Syk and the substrate PLC gamma 2 in the GPVI signal pathway, resulting in an enhanced platelet response to collagen. Disrupting lipid rafts or blocking GP130-Janus tyrosine kinase (JAK)-STAT3 signaling abolished the cross-activation and reduced platelet reactivity to collagen. Conclusion These results demonstrate cross-talk between collagen and IL-6 signal pathways. This cross-talk could potentially provide a novel mechanism for inflammation-induced platelet hyperactivity, so the IL-6-GP130-JAK-STAT3 pathway has been identified as a potential target to block this hyperactivity.
Background: Hemostasis, thrombosis and surgical stress-induced immune reactions are important for perioperative morbidity and recovery after major surgical operations. Objectives: To evaluate the effects of combined administration of low-dose epinephrine (LDEPI) and tranexamic acid (TXA) on perioperative blood loss, thromboembolic complications and inflammatory responses in patients undergoing total hip arthroplasty (THA). Patients/Methods: Patients scheduled for THA (n = 195) were randomized into three interventions: intravenous LDEPI plus TXA (group IV); topical diluted epinephrine plus TXA (group TP); and TXA alone as control (group CT). The primary outcome was perioperative blood loss on postoperative day (POD) 1. Secondary outcomes included perioperative blood loss on POD 3, intraoperative blood loss, volume of drainage, transfusion values, coagulation and fibrinolysis parameters, inflammatory cytokine levels, cases of thrombosis, intravenous fluid on the operation day, and length of hospital stay. Results: The mean calculated amounts of total blood loss in groups IV, TP and CT were 631.2 mL, 760.5 mL, and 825.6 mL, respectively, on POD 1; treatment effects (differences) were 194.4 mL (95% confidence interval [CI] 146.7-242.0) and 65.0 mL (95% CI 17.4-112.7). Groups IV and TP had lower levels of proinflammatory cytokines (tumor necrosis factor-a and interleukin [IL]-1b) and higher levels of the anti-inflammatory cytokine IL-10, and showed faster development of coagulation and fibrinolysis (without change in peak levels), than group CT early postoperation. No differences were observed in transfusion, thromboembolic and other outcomes among the groups. Conclusion: The combined administration of LDEPI and TXA was more effective in reducing perioperative blood loss and alleviating the inflammatory response than TXA alone, without increasing the incidence of thromboembolic and other complications.
Background. Despite great progress in the prevention and control of cardiovascular disease, it remains the leading cause of global mortality and morbidity, with new unexpected risk factors emerging and few effective new pharmaceutical treatments. ADAMTS-13 is involved in a clotting disorder, thrombotic thrombocytopenic purpura, for which new treatments are being developed. Observationally, ADAMTS-13 activity is inversely associated with ischemic heart disease (IHD) but positively associated with diabetes. Objectives: To obtain unconfounded estimates of the effect of ADAMTS-13 on IHD, diabetes and lipids. Methods: We applied genetic variants strongly, (P < 5 x 10(-8)), solely and independently associated with ADAMTS-13 to the largest available extensively geno-typed case-control studies of IHD and diabetes and to a large study of lipids to obtain Mendelian randomization inverse variance weighted (IVW) estimates. Sensitivity was evaluated through weighted median and MR-Egger estimates. Results: Genetically predicted ADAMTS-13 activity, based on three genetic variants, was consistently inversely associated with IHD (IVW odds ratio [OR] 0.91 per effect size; 95% confidence interval [CI] 0.86-0.97) but not with diabetes (OR 0.94, 95% CI 0.88-1.01) or high or low-density lipoprotein cholesterol (0.01, 95% CI -0.02 to 0.04; -0.01, 95% CI -0.04 to 0.02, respectively). ADAMTS-13 antigen, based on four genetic variants, was not associated with any outcome. Conclusions: This genetic validation of ADAMTS-13 activity as a target of intervention in IHD raises the possibility of new ways of prevention and treatment being developed by repurposing therapeutics that raise ADAMTS-13 activity, or by other environmental or dietary interventions that raise ADAMTS-13 activity.
Background The human genome encodes two paralogs of vitamin-K-epoxide reductase, VKORC1 and VKORL1, that support blood coagulation and other vitamin-K-dependent processes. Warfarin inhibits both enzymes, but VKORL1 is relatively resistant to warfarin. Objectives To understand the difference between VKORL1 and VKORC1, and the cause of warfarin-resistant (WR) mutations in VKORC1. Methods We performed systematic mutagenesis and analyzed warfarin responses with a cell-based activity assay. Mass spectrometry analyses were used to detect cellular redox state. Results VKORC1 and VKORL1 adopt a similar intracellular redox state with four-transmembrane-helix topology. Most WR mutations identified in VKORC1 also confer resistance in VKORL1, indicating that warfarin inhibits these paralogs at a common binding site. A group of WR mutations, distant from the warfarin-binding site, show significantly less resistance in VKORL1 than in VKORC1, implying that their different warfarin responses are determined by peripheral interactions. Remarkably, we identify a critical peripheral region in which single mutations, Glu37Lys or His46Tyr, drastically increase the warfarin sensitivity of VKORL1. In the background of these warfarin-sensitive VKORL1 mutants, WR mutations showing relative less resistance in wild-type VKORL1 become much more resistant, suggesting a structural conversion to resemble VKORC1. At this peripheral region, we also identified a human single nucleotide polymorphism that confers warfarin sensitivity of VKORL1. Conclusions Peripheral regions of VKORC1 and VKORL1 primarily maintain the stability of their common warfarin-binding pocket, and differences of such interactions determine their relative sensitivity to warfarin inhibition. This new model also explains most WR mutations located at the peripheral regions of VKORC1.
Background Tranexamic acid (TXA) can reduce blood loss and the inflammatory response at multiple doses in total knee arthroplasty patients. However, the optimal regimen has not been determined. Objectives To identify the most effective regimen for achieving maximum reductions in blood loss and the inflammatory response. Patients/Methods Two hundred and seventy-five patients were randomized to receive a placebo (group A), a single 2-g oral dose of TXA 2 h preoperatively followed by 1 g of oral TXA 3 h postoperatively (group B), a single dose followed by 1 g of oral TXA 3 h and 7 h postoperatively (group C), a single dose followed by 1 g of oral TXA 3 h, 7 h and 11 h postoperatively (group D), or a single dose followed by 1 g of oral TXA 3 h, 7 h, 11 h and 15 h postoperatively (group E). The primary outcome was total blood loss on postoperative day (POD) 3. Secondary outcomes included a decrease in the hemoglobin level, coagulation parameters, inflammatory marker levels, and thromboembolic complications. Results Groups D and E had significantly lower blood loss and smaller decreases in hemoglobin level than groups A, B, and C, with no significant difference on POD 3 between groups D and E. Significantly enhanced coagulation was identified for the four multiple-dose regimens; however, all thromboelastographic parameters remained within normal ranges. Group E had the lowest inflammatory marker levels and pain, and the greatest range of motion. No thromboembolic complications were identified. Conclusion The four-dose regimen yielded the maximum reductions in blood loss and inflammatory response, improved analgesia, and promoted early rehabilitation. Further studies are required to ensure that these findings are reproducible.
Background Most immune thrombocytopenia (ITP) is sporadic but a positive family history of ITP in some patients suggests that hereditary forms exist. Because of the rarity of familial ITP families available for study and the heterogeneity of sporadic ITP, family linkage analysis or genome-wide association studies are limited. Objectives Based on one ITP pedigree, we try to identify the predisposing gene in familial or sporadic ITP and reveal the way in which it causes thrombocytopenia. Methods Gene expression profiling analysis and whole-exome sequencing were performed on samples from family members with ITP, sporadic ITP cases and healthy individuals. We also evaluated the influence of potential pathogenic mutation on immune function and megakaryocyte apoptosis. Results Whole-exome sequencing identified a potential pathologic p.G76S heterozygous mutation on the TNFRSF13B gene in familial ITP patients. ITP patients harboring the G76S mutation displayed an upregulated cytokine-cytokine receptor interaction' signal, increased serum TNF, IL-17, IFN and BAFF levels, and enhanced binding capacity of APRIL ligand to B cells. Additionally, Epstein-Barr virus (EBV)-transformed B cells with the G76S mutation could induce human megakaryocyte line (Meg-01) apoptosis significantly. Conclusion p.G76S mutation on the TNFRSF13B gene is responsible for ITP, and is a genetic predisposing factor for familial or sporadic ITP.
Background: Gastrointestinal dysfunction and vein thrombosis are well-known acute complications after spinal cord injuries (SCIs). Objective: To determine the frequency and risk factors for deep vein thrombosis (DVT) and small-intestinal bacterial overgrowth (SIBO) in patients with SCI. Methods: A total of 377 consecutive eligible SCI patients tested for SIBO with the glucose hydrogen/methane breath test from January 2011 to December 2015 and who had also undergone venous ultrasound study for suspected DVT were evaluated within 3 months after admission. Results: Seventy-six of the 377 SCI patients were DVT-positive (20.2%; 95% confidence interval [CI] 16.1-24.2%), and 145 were SIBOpositive (38.5%; 95% CI 29.9-59.0%). Among the 76 DVT-positive patients, 60 were SIBO-positive and 16 were SIBO-negative. The difference was statistically significant (41.4% versus 6.9%; odds ratio [OR] 5.99; 95% CI 3.15-9.33). Among the 145 SIBO-positive patients, 60 were DVT-positive and 85 were DVT-negative. The difference was statistically significant (78.9% versus 28.2%; OR 2.88; 95% CI 2.12-4.47). In the stepwise multivariate logistic regression, a family history of venous thrombosis (OR 2.32; 95% CI 1.60-3.79), chronic kidney disease (OR 2.99; 95% CI 1.73-5.08) and the presence of SIBO (OR 3.72; 95% CI 1.97-6.62) remained associated with DVT. Conclusions: These data support an association between SIBO and DVT in SCI patients. Further studies should be carried out with respect to the relationship between SIBO and DVT.
Objective To construct and validate artificial neural networks (ANNs) for predicting the occurrence of portosplenomesenteric venous thrombosis (PSMVT) and compare the predictive ability of the ANNs with that of logistic regression. Methods The ANNs and logistic regression modeling were constructed using simple clinical and laboratory data of 72 acute pancreatitis (AP) patients. The ANNs and logistic modeling were first trained on 48 randomly chosen patients and validated on the remaining 24 patients. The accuracy and the performance characteristics were compared between these two approaches by SPSS17.0 software. Results The training set and validation set did not differ on any of the 11 variables. After training, the back propagation network training error converged to 1 x 10(-20), and it retained excellent pattern recognition ability. When the ANNs model was applied to the validation set, it revealed a sensitivity of 80%, specificity of 85.7%, a positive predictive value of 77.6% and negative predictive value of 90.7%. The accuracy was 83.3%. Differences could be found between ANNs modeling and logistic regression modeling in these parameters (10.0% [95% CI, -14.3 to 34.3%], 14.3% [95% CI, -8.6 to 37.2%], 15.7% [95% CI, -9.9 to 41.3%], 11.8% [95% CI, -8.2 to 31.8%], 22.6% [95% CI, -1.9 to 47.1%], respectively). When ANNs modeling was used to identify PSMVT, the area under receiver operating characteristic curve was 0.849 (95% CI, 0.807-0.901), which demonstrated better overall properties than logistic regression modeling (AUC = 0.716) (95% CI, 0.679-0.761). Conclusions ANNs modeling was a more accurate tool than logistic regression in predicting the occurrence of PSMVT following AP. More clinical factors or biomarkers may be incorporated into ANNs modeling to improve its predictive ability.