Thrombotic Activation Research Articles

Targeting KLF2 as a Novel Therapy for Glomerular Endothelial Cell Injury in Diabetic Kidney Disease.

DKD is a microvascular disease, and glomerular endothelial cell injury is a key pathological event in DKD development. Through unbiased screening of glomerular transcriptomes, we previously identified KLF2 as a highly regulated gene in diabetic kidneys. KLF2 exhibits protective effects in endothelial cells by inhibiting inflammation, thrombotic activation, and angiogenesis, all of which are protective for cardiovascular disease. We previously demonstrated that endothelial cell-specific ablation of Klf2 exacerbated diabetes-induced glomerular endothelial cell injury and DKD in mice. Therefore in this study, we sought to assess the therapeutic potential of KLF2 activation in murine models of DKD. We first examined the effects of endothelial cell-specific inducible overexpression of KLF2 (KLF2ov) in streptozotocin-induced diabetic mice. We developed small molecule KLF2 activators and tested whether increased KLF2 activity could impede DKD progression in type 2 diabetic db/db and BTBR ob/ob mice. Diabetic KLF2ov mice had attenuated albuminuria, glomerular endothelial cell injury, and diabetic glomerulopathy compared to control diabetic mice. Novel KLF2 activator, compound 6 (C-6) effectively induced downstream Nos3 expression and suppressed NF-kB activation in glomerular endothelial cells. The administration of C-6 improved albuminuria and glomerulopathy in db/db and BTBR ob/ob mice, which was associated with improved glomerular endothelial cell and podocyte injury. These results validate KLF2 as a potential drug target and KLF2 activators such as C-6 as a novel therapy for DKD.

Krüppel-like factor 2 is an endoprotective transcription factor in diabetic kidney disease.

Diabetic kidney disease (DKD) is a microvascular complication of diabetes, and glomerular endothelial cell (GEC) dysfunction is a key driver of DKD pathogenesis. Krüppel-like factor 2 (KLF2), a shear stress-induced transcription factor, is among the highly regulated genes in early DKD. In the kidney, KLF2 expression is mostly restricted to endothelial cells, but its expression is also found in immune cell subsets. KLF2 expression is upregulated in response to increased shear stress by the activation of mechanosensory receptors but suppressed by inflammatory cytokines, both of which characterize the early diabetic kidney milieu. KLF2 expression is reduced in progressive DKD and hypertensive nephropathy in humans and mice, likely due to high glucose and inflammatory cytokines such as TNF-α. However, KLF2 expression is increased in glomerular hyperfiltration-induced shear stress without metabolic dysregulation, such as in settings of unilateral nephrectomy. Lower KLF2 expression is associated with CKD progression in patients with unilateral nephrectomy, consistent with its endoprotective role. KLF2 confers endoprotection by inhibition of inflammation, thrombotic activation, and angiogenesis, and thus KLF2 is considered a protective factor for cardiovascular disease (CVD). Based on similar mechanisms, KLF2 also exhibits renoprotection, and its reduced expression in endothelial cells worsens glomerular injury and albuminuria in settings of diabetes or unilateral nephrectomy. Thus KLF2 confers endoprotective effects in both CVD and DKD, and its activators could potentially be developed as a novel class of drugs for cardiorenal protection in diabetic patients.

IL-6 and D-dimer Levels at Admission Predict Cardiac Injury and Early Mortality during SARS-CoV-2 Infection

Background: We previously described the mortality associated with cardiac injury in patients with coronavirus disease 2019 (COVID-19). The activation of immune and thrombotic biomarkers at admission, and their ability to predict cardiac injury and mortality patterns in COVID-19, remains unclear. Methods: This retrospective cohort study included 170 patients with COVID-19 with cardiac injury at the time of admission to Tongji Hospital in Wuhan between January 29, 2020, and March 8, 2020. The temporal evolution of inflammatory cytokines, coagulation markers, clinical treatment, and mortality were analyzed. Continuous variables are expressed as median (interquartile range). The Mann-Whitney test was used for two-group comparisons, whereas the Kruskal-Wallis test was used for comparisons among three groups. Categorical variables are expressed as proportions and percentages, and Fisher’s exact test was used to compare differences. A multivariate regression model was used to predict in-hospital death. A simple linear regression analysis was applied to examine the correlation between baseline biomarkers and peak cTnI levels. Results: Of the 170 patients, 60 (35.3%) died early (<21 d), and 61 (35.9%) died after a prolonged stay. The admission laboratory findings correlating with early death were elevated interleukin 6 (IL-6) (P < 0.0001), tumor necrosis factor-α (P = 0.0025), and C-reactive protein (P < 0.0001). We observed the trajectory of biomarker changes in patients after admission hospitalization, and determined that early mortality was associated with a rapidly increasing D-dimer level, and gradually decreasing platelet and lymphocyte counts. Multivariate and simple linear regression models indicated that the risk of death was associated with immune and thrombotic pathway activation. Elevated admission cTnI levels were associated with elevated IL-6 (P = 0.03) and D-dimer (P = 0.0021) levels. Conclusion: In patients with COVID-19 with cardiac injury, IL-6 and D-dimer levels at admission predicted subsequently elevated cTnI levels and early death, thus highlighting the need for early inflammatory cytokine-based risk stratification in patients with cardiac injury.

Obesity-Induced Platelet Mitofusin-1 Expression Causes Vascular Dysfunction

Background: The growing epidemic of obesity is strongly linked to cardiovascular disease (CVD). Obesity leads to endothelial dysfunction, an instigating event that drives vascular injury and subsequent CVD, but the mechanisms by which obesity causes endothelial dysfunction remain unclear. Platelets circulate proximal to the endothelium and are traditionally thought to contribute to obesity-associated vasculopathy through thrombotic activation. However, platelets are metabolically active and release vaso-modulatory molecules into their environment and the role of these functions have not been examined in the context of obesity-induced vasculopathy. We previously showed that alterations in platelet mitochondrial function regulate platelet degranulation. Further, it has been reported that thrombospondin 1 (TSP1), a platelet factor that propagates endothelial dysfunction, is elevated in the plasma of obese humans. We hypothesized that obesity/weight gain modulates platelet mitochondrial function, which stimulates the release of TSP1 from platelets to propagate vascular dysfunction. Methods: Platelets were isolated from lean and obese humans and mitochondrial structure and function measured. To induce diet-induced obesity wildtype (WT) mice were fed with a high fat diet (HFD, 60 kcal% fat) for 10 weeks, and low fat diet (LFD, 10 kcal% fat)- fed WT mice were used as controls. Platelets and plasma from the mice were screened for mitochondrial proteins and plasma TSP-1 was measured by ELISA. A murine model with platelet-specific deletion of mitofusin-1 (MFN1; pltMFN1KO mice), a mitochondrial GTP-ase that increases mitochondrial function was generated and fed either HFD or LFD for 4-10 weeks. Further, endothelial-dependent- and independent- vascular relaxation was measured in both groups by stimulating aortic rings with acetylcholine (Ach) or sodium nitroprusside (SNP) using wire myography. Additionally, platelet-specific TSP1 knockout (plt-TSP1KO) mice were generated and fed with HFD for 10 weeks and measured plasma TSP1 levels. Results: Platelets from obese subjects showed increased levels of MFN1 compared to lean subjects. WT mice fed with HFD had increased platelet MFN1 and plasma TSP1 levels compared to LFD-fed WT mice. Platelet-specific deletion of MFN1 in mice resulted in reduced plasma TSP1 levels compared to control PF4-cre mice after HFD challenge. We found that HFD caused impaired Ach-stimulated vasorelaxation (indicative of endothelial dysfunction) in control PF4-cre mice, and this effect was attenuated in pltMFN1KO mice. Further, we found that HFD-induced increases in plasma TSP1 levels were attenuated in plt-TSP1KO mice, indicating platelets are the major source of TSP1 in HFD induced obesity model. Conclusion: These data demonstrate that obesity/weight gain induces platelet MFN1 expression, which drives TSP1 release from platelets to propagate vascular dysfunction. Our findings elucidate a novel platelet-centric mechanism underlying obesity-associated vasculopathy and have implications for the targeting of non-thrombotic platelet function as a therapeutic strategy for obesity-induced vasculopathy.

Impact of timing and severity of COVID-19 infection in pregnancy on intrauterine fetal growth- a registry-based study from Qatar.

The novel coronavirus disease (COVID-19) pandemic has impacted pregnant women, increasing maternal and neonatal morbidity. The placenta is a potential target for the pathophysiological processes due to the increased thrombotic inflammatory activation and inadequate uteroplacental perfusion and oxygenation, potentially causing intrauterine growth restriction. This study investigates the impact of gestational age at diagnosis of COVID-19 and the presence of symptoms on intrauterine fetal growth in pregnant women. A retrospective review of COVID-19 positive pregnant women in Qatar from March 2020 to March 2021 was conducted. They were divided based on trimester of pregnancy in which they were infected. The outcomes included birthweight, customised fetal birthweight centiles, small for gestational age (SGA) baby and daily growth increments, compared between the trimesters and between symptomatic and asymptomatic women. In our cohort, 218 women (20.5%) were infected in the first trimester, 399 (37.5%) in the second and 446 (42%) in the third. Women in the second trimester were significantly younger and symptomatic. Women infected in the first trimester were least likely to have diabetes. The mean birthweight, risk of SGA (11.5% vs 10% vs 14.6%, p = 0.302), and median customized growth centiles (47.6% vs 45.9% vs 46.1%)were similar between the groups. Symptomatic women had significantly lower mean birthweight (3147 gms vs 3222 gms) and median birthweight centiles (43.9% vs 54.0%)compared to the asymptomatic (p<0.05 for both). In women infected within 20 weeks of gestation, a delay in daily fetal growth increments was noted with symptomatic disease, although not statistically significant. This study shows that women with symptomatic disease had lower birth centiles and birth weights. This was regardless of the gestational age at which they were infected. Early symptomatic disease seems to have an impact on fetal growth velocity; however, larger studies are needed to corroborate these findings.

Biomimetic polyelectrolyte coating of stem cells suppresses thrombotic activation and enhances its survival and function

Mesenchymal stem cells (MSCs) therapy is a promising approach for treating inflammatory diseases due to their immunosuppressive and tissue repair characteristics. However, allogenic transplantation of MSCs induces thrombotic complications in some patients which limits its potential for clinical translation. To address this challenge, we have exploited the bioactivity of heparin, a well-known anticoagulant and immunosuppressive polysaccharide that is widely used in clinics. We have developed a smart layer-by-layer (LbL) coating strategy using gelatin and heparin polymers exploiting their overall positive and negative charges that enabled efficient complexation with the MSCs' glycocalyx. The stable coating of MSCs suppressed complement attack and mitigated thrombotic activation as demonstrated in human whole blood. Gratifyingly, the MSC coating retained its immunosuppressive properties and differentiation potential when exposed to inflammatory conditions and differentiation factors. We believe the simple coating procedure of MSCs will increase allogenic tolerance and circumvent the major challenge of MSCs transplantation.

An early analysis of Codman® CerebroFlo® Endexo coated ventricular catheters in the setting of intraventricular hemorrhage.

Intraventricular hemorrhages (IVH) are a potentially fatal diagnosis that must be managed properly to avoid devastating complications. While existing catheters have offered relative success, their reactive composition makes them prone to eventual obstruction and thrombotic activation. This problematic outcome has led to emergence of the Codman® CerebroFlo® EVD Catheter (Integra LifeSciences, Princeton, NJ, USA). This catheter is the first of its kind to incorporate the Endexo additive, a slightly reactive, surface modifying molecule that prevents protein adhesion and biomatter accumulation. Despite the promising early outcomes, there are no PubMed articles characterizing this device, with minimal literature highlighting its clinical value. Through a contemporary literature review, the authors will characterize the technological principles, indications, and advantages of this novel device. The authors reported the early findings of this catheter, with studies showing 99% reduction in thrombotic activity with an additional 89% reduction in catheter obstruction compared to its competitors. Areas of uncertainty regarding this device are discussed, with its lack of antibiotic coating being a possible area for clinical concern. In conclusion, the CerebroFlo® catheter is a novel and effective tool in the management of IVH that should be widely considered for the management of IVH.

Thrombotic activation before and after total hip arthroplasty. A prospective cohort study

BackgroundTotal hip arthroplasty (THA) causes acute blood loss. It may lead to a deficiency in coagulation factors, which, in turn, may lead to increased bleeding during the postoperative period.MethodsThirty patients (18 women) with a mean age of 67 years (range: 63–72 years) participated in this prospective diagnostic study. THA was performed without tranexamic acid administration in the perioperative period. Activities of clotting factors II, VIII, X, and fibrinogen concentration were evaluated before surgery, 6 hours after the procedure, 2, 4, and 6 days after the operation. All laboratory tests were performed using ACL TOP 500 CTS analyzer.ResultsNo thromboembolic complications were noted during hospitalization. Mean fibrinogen concentration was 366 mg/dL before surgery, which decreased to 311 mg/dL 6 hours after the operation and peaked at 827 mg/dL on the 4th day after the procedure. Activities of factors II and X decreased on the second and fourth days after surgery. Although the activity of factor VIII decreased after the procedure, it remained within the normal range. Increased baseline fibrinogen concentrations were observed in 6 out of 30 (20%) patients. Mean blood loss was 1332 mL (range, 183–2479 mL) and did not correlate with changes in clotting factor activities.ConclusionsIn patients undergoing THA, fibrinogen acts as an acute-phase protein. Activities of clotting factors II and X normalize within 6 days, and although the activity of factor VIII decreases, it remains within the normal range.Trial registrationThe study was pre-registered May 1st, 2020 on ClinicalTrials.gov

Hemodialysis exacerbates proteolytic imbalance and pro-fibrotic platelet dysfunction

Multi-organ fibrosis among end stage renal disease (ESRD) patients cannot be explained by uremia alone. Despite mitigation of thrombosis during hemodialysis (HD), subsequent platelet dysfunction and tissue dysregulation are less understood. We comprehensively profiled plasma and platelets from ESRD patients before and after HD to examine HD-modulation of platelets beyond thrombotic activation. Basal plasma levels of proteolytic regulators and fibrotic factors were elevated in ESRD patients compared to healthy controls, with isoform-specific changes during HD. Platelet lysate (PL) RNA transcripts for growth and coagulative factors were elevated post-HD, with upregulation correlated to HD vintage. Platelet secretome correlations to plasma factors reveal acutely induced pro-fibrotic platelet phenotypes in ESRD patients during HD characterized by preferentially enhanced proteolytic enzyme translation and secretion, platelet contribution to inflammatory response, and increasing platelet dysfunction with blood flow rate (BFR) and Vintage. Compensatory mechanisms of increased platelet growth factor synthesis with acute plasma matrix metalloproteinase (MMP) and tissue inhibitor of MMPs (TIMP) increases show short-term mode-switching between dialysis sessions leading to long-term pro-fibrotic bias. Chronic pro-fibrotic adaptation of platelet synthesis were observed through changes in differential secretory kinetics of heterogenous granule subtypes. We conclude that chronic and acute platelet responses to HD contribute to a pro-fibrotic milieu in ESRD.

HMGB1-Mediated Platelet Activation Is Independent of Platelet Mitochondrial Reactive Oxygen Species Generation

Background: Sickle cell disease (SCD) is a hemolytic disorder that exhibits pathologic platelet activation. Notably, hemolysis is tightly associated with platelet activation and thrombotic complications of SCD such as stroke, leg ulceration, and pulmonary hypertension. To this end, we and others have shown that free hemoglobin (Hb) released into the plasma via hemolysis directly activates healthy platelets ex vivo in a concentration-dependent manner. Treatment with Hb stimulates the production of mitochondrial reactive oxygen species (mtROS) within the platelet, resulting in thrombotic activation. Accordingly, scavenging mtROS prevents Hb-induced platelet activation. While it is established that hemolysis mediates platelet activation, the factors that modulate this response remain unknown. High mobility group box 1 (HMGB1) is an inflammatory mediator, which signals through toll-like receptor-4 (TLR4), to instigate thrombosis. Notably, we previously showed that HMGB1 is elevated in the plasma of SCD patients and augments platelet activation on its own. Importantly, complementary interactions between Hb and other pro-thrombotic plasma components such as HMGB1 have not been investigated. Preliminary data show that HMGB1, like Hb, stimulates platelet mtROS generation. Thus, we hypothesized that elevated levels of HMGB1 and Hb synergize in SCD to stimulate platelet mtROS production and downstream platelet activation. Methods: Whole blood samples were collected from healthy human control subjects or SCD subjects in steady state (n=5 per group). Plasma HMGB1 concentrations were quantified by ELISA. Platelets were isolated and treated with reagents including HMGB1, ferric Hb, or the mtROS scavenger MitoTEMPO. Platelet activation was measured by flow cytometry using PE antibody to GPIIb (CD41) to select for platelets, and two markers of platelet activation: PAC1 to detect the activation-dependent conformational change in integrin αIIbβ3 (GP IIb-IIIa), and APC to detect exposure of surface CD62P (P-selectin). Platelet mtROS were estimated using MitoSOX Red and fluorescence spectroscopy. Data was analyzed using FlowJo software and nonparametric statistical tests. Results: The baseline level of platelet activation in SCD was 7-fold higher than in controls (P=0.009). However, there was no difference in platelet activation levels between isolated SCD and control platelets in response to ex vivo treatment with agonists ADP (5 uM), collagen (50 ug/ml), or thrombin (0.1 U/ml) (P=0.38), suggesting that SCD platelets have the same agonist-induced response as control platelets when they are removed from their native plasma. We found that the concentration of HMGB1 in SCD plasma is 1.5 fold higher than controls (P=0.04), and that plasma HMGB1 concentrations positively correlate with increased platelet activation (P=0.04). Co-incubation of isolated healthy control platelets with HMGB1 (10 ug/ml) and major hemolytic byproduct Hb (10 uM, 30 uM, 50 uM) enhanced platelet activation at all doses of Hb by 3-fold compared to that of Hb alone. Mechanistically, platelets exposed to Hb alone generated mtROS which are necessary for Hb-induced platelet activation. We found that HMGB1 similarly induced platelet mtROS generation, but scavenging with MitoTEMPO did not prevent HMGB1-mediated platelet activation. However the addition of MitoTEMPO to platelets co-treated with HMGB1 + Hb attenuated platelet activation 2-fold, indicating that scavenging mtROS neutralizes the enhanced effect of Hb + HMGB1. Conclusions: We show that HMGB1 synergizes with Hb to enhance platelet activation in SCD, but unlike Hb, HMGB1-dependent platelet activation is independent of mtROS generation. Accordingly, we observed that MitoTEMPO partially attenuates the enhanced activation from co-stimulation by HMGB1 + Hb. These data characterize the synergy between HMGB1 and Hb for the first time and demonstrate differential platelet signaling between the two agonists. Disclosures No relevant conflicts of interest to declare.

Can We Predict the Duration of Treatment Requirement by Complete Blood Count in Chronic Spontaneous Urticaria Patients Receiving Omalizumab?

Objectives: The main assay recommended in chronic urticaria is complete blood count (CBC). It is known that changes in the numerical values and rates of blood cells in the CBC are associated with inflammation and thrombotic risk. In addition, the improvement of some patients with short-term Omalizumab treatment, while others need long-term treatment raises the question of whether this difference can be predicted with parameters on the CBC. This study aimes to evaluate the effect of the drug on CBC parameters in patients with chronic spontaneous urticaria (CSU) receiving Omalizumab treatment.&#x0D; Methods: The study included 66 patients who received Omalizumab treatment with CSU and 34 healthy individuals as a control group. The values of 17 parameters from the biomarkers in the CBC were recorded in the patient group (before the treatment/after 12 weeks of treatment) and in the control group. In addition, patients with CSU according to the Omalizumab treatment requirement was divided into two groups as those who recovered in a short time (≤6 months) (group-1) and those who need longer treatment (&gt; 6 months) (group-2). All data were compared statistically.&#x0D; Results: It was observed that many inflammatory and thrombotic activation markers in the CBC increased in the patient group before treatment compared to the control group, and even most of these values approached the values of healthy individuals after treatment. When the pre-treatment and post-treatment data of the patients are compared within themselves; neutrophil and platelet numbers and neutrophil-monocyte ratio decreased after treatment and there was a statistically significant difference. When the pre-treatment values of the two groups determined according to the treatment requirement period were compared, it was determined that the eosinophil-basophil ratio (EBR) value was significantly higher in the group-2 patients. It was observed that the number of platelets in group-1 decreased and eosinophil-lymphocyte ratio and eosinophil-neutrophil ratio increased after treatment compared to pre-treatment; platelet, neutrophil counts and EBR values of patients in group-2 decreased, mean platelet volume (MPV) and platelet distribution width (PDW) values increased.&#x0D; Conclusion: It has been determined that Omalizumab treatment has positive effects on patients with CSU that it reduces many inflammatory and thrombotic activation markers in CBC. It was thought that an increase in EBR may be a biomarker that predicts that patients with CSU will need Omalizumab treatment for a longer period. In addition, it was concluded that MPV and PDW values increased during Omalizumab treatment of patients with long-term treatment needs, and that thrombotic activation markers of these patients should be followed more closely during the treatment.

Differential Augmentation of Thrombin Generation by Andexanet Alfa in Lymphoma Patients

IntroductionThe prevalence of thrombosis in lymphoma patients is reportedly high and ranges from 3–10%. Vascular malfunction and inflammatory processes further contribute to the thrombotic activation processes in these patients. Andexanet alfa is an antidote for factor Xa inhibitors and its usage has been reported with thrombotic complications. This study was designed to compare effect of andexanet alfa (AA) on the thrombin generation potential and its relevance to the generation of thrombin.Materials and MethodCitrated blood samples from 78 patients with confirmed diagnosis of non‐Hodgkin lymphoma (NHL), Hodgkin lymphoma (HL) and Chronic lymphocytic leukemia/Small lymphocytic lymphoma (CLL/SLL) were collected from the Clinic of Hematology Unit, University of Belgrade, Belgrade, Serbia. 50 samples of normal human plasma (NHP) was obtained from George King Biomedical (Overland park, KS). NHP was prepared for referencing purposes. Individual samples were supplemented with andexanet alfa at 100 ug/ml. Thrombin generation studies were carried out using a commercially available a kinetic fluorogenic substrate method (calibrated automated thrombogram; CAT). Thrombin generation parameters such as peak thrombin (PT), lag time (LT) and area under the curve (AUC) were compiled. Results were compiled in terms of mean ±SD.ResultsOn a cumulative basis, lymphoma patients showed an increase in LT (2.9 1.15) in comparison to NHP (2.06 .02) which decreases with AA (1.94 0.1). The PT levels were decreased (120.34 44.3) in comparison to NHP (159.20 4.8) and increases with AA (153.70 37.2). The AUC was also decreased (668.85 284.3) in comparison to NHP (756.46 34.5) which increases with AA (758.32 226.4). When the lymphoma patients were sub‐grouped, peak thrombin levels were similar in HL (128.16), NHL (113.99) and CLL (129.77) were decreased compared to normal human plasma (159.2) which increases with AA, in HL (180.75), NHL (154.16) and CLL (142.46) compared to AA control (153.7). The AUC was increased in the HL (769), decreased in NHL (642.4) and was comparable for CLL (715.4) compared to normal human plasma (756.46). However, with AA the AUC values increases for HL (938.31) and decreases for NHL (744.52) and CLL (725.26) in comparison with AA control (758.32). Variations in lag time were noted in the three groups.ConclusionLymphoma patients represent a heterogenous group of patients in which both the hypercoagulable state and inflammatory responses simultaneously occur. Thrombin is constantly generated in some of these patients leading to the formation of fibrin. The observed decrease in thrombin generation potential in these patients may be due to the consumption of prothrombin and other coagulation factors. Differential increase in thrombin generation in post andexxa supplemented samples suggest that the use of this agent may potentially be associated with thrombotic complications.

Thrombin Generation Profile in Various Lymphoma Sub-Groups and Its Augmentation by Andexanet Alfa.

The prevalence of thrombosis in lymphoma patients is reportedly high and ranges from 3-10%. Vascular malfunction and inflammatory processes further contribute to the thrombotic activation process in these patients. Andexanet alfa (AA) is an antidote for factor Xa inhibitors and its usage has been reported with thrombotic complications. This study was designed to compare the effect of AA on the thrombin generation (TG) potential. Blood samples from 78 patients with confirmed diagnosis of non-Hodgkin lymphoma (NHL), Hodgkin lymphoma (HL) and Chronic lymphocytic leukemia (CLL) were collected from the University of Belgrade Clinic, Serbia. Normal human plasma (NHP) was used for referencing purposes. Individual samples were supplemented with AA at 100 ug/ml. TG studies were carried out using a commercially available fluorogenic substrate method. TG parameters such as peak thrombin (PT), lag time (LT) and area under the curve (AUC) were compiled. Cumulatively, lymphoma patients showed an increase in LT compared to NHP which decreases with AA. The PT and AUC levels were decreased compared to NHP and increases with AA. Upon sub-grouping of lymphoma patients, PT levels for all sub-groups were increased with AA. The AUC values increased for HL and NHL and decreased for CLL with AA. Variations in lag time were noted in all 3 sub-groups. Lymphoma represents a heterogenous group of patients where both the hypercoagulable state and inflammatory responses simultaneously occur. Increased thrombin generation in post AA supplemented samples suggest that the use of this agent may potentially be associated with thrombotic complications.

The Mitochondrion: A Link Between Hemolysis and Platelet Activation

Patients with Sickle Cell Disease (SCD) demonstrate characteristics of chronic hemostatic activation including elevated baseline platelet activation. While it is well established that platelet activation is positively correlated with the magnitude of erythrocytic hemolysis in these patients, the mechanisms linking hemolysis to platelet activation remain unclear. In this study, we investigate the role of the platelet mitochondrion as the molecular link between hemolysis and downstream platelet activation. Using extracellular flux analysis, we demonstrate that platelets isolated from patients with SCD show a distinct alteration in mitochondrial function characterized by a decrease in the activity of the mitochondrial ATP Synthase (electron transport chain Complex V), which leads to enhanced mitochondrial membrane potential and elevated reactive oxygen species (ROS) production by the electron transport chain. Notably, levels of platelet mitochondrial ROS production was significantly correlated with markers of hemolysis (lactate dehydrogenase and plasma free hemoglobin concentration) as well as platelet activation in this cohort. Moreover, the mitochondrial dysfunction observed in platelets from SCD patients was recapitulated by exposing healthy platelets to cell-free hemoglobin ex vivo. Importantly, augmented mitochondrial ROS generation appears to initiate platelet activation, as scavenging of mitochondrial ROS inhibited cell free hemoglobin-induced activation of healthy platelets. Studies in the Berkley (BERK) transgenic murine model of SCD supported ex vivo data demonstrating that hemolysis-induced mitochondrial ROS production stimulates platelet activation. Homozygous BERK mice showed significantly inhibited platelet mitochondrial complex V activity and elevated mitochondrial ROS production, concomitant with enhanced baseline platelet activation compared to hemizygote mice. When subjected to a ferric chloride-induced vascular injury model of thrombosis, BERK homozygous mice showed a significantly shorter time to vascular occlusion due to thrombosis compared to hemizygote mice. Treatment of homozygous BERK mice with the mitochondrial ROS scavenger MitoTEMPO (supplemented in drinking water) significantly increased time to vascular occlusion in the ferric chloride thrombosis model. These ex vivo human platelet data and in vivo studies in murine models demonstrate that hemolysis induces platelet mitochondrial ROS production, which stimulates downstream platelet activation. Ongoing studies are focused on determining the molecular mechanisms by which cell free hemoglobin elicits an inhibition of platelet mitochondrial Complex V and implicate a role for activation of the platelet surface toll like receptor 4 (TLR4). Collectively, these data identify the platelet mitochondrion as an important signaling hub that links hemolysis to platelet activation in SCD patients. The potential of therapeutically modulating platelet mitochondrial ROS production to attenuate hemolysis induced thrombotic activation will be discussed. Disclosures No relevant conflicts of interest to declare.

Thrombotic processes and shear activation of platelets: Pharmacological approaches

Thrombotic processes and shear activation of platelets: Pharmacological approaches

Levels of Matrix Metalloproteinases in Arthroplasty Patients and Their Correlation With Inflammatory and Thrombotic Activation Processes.

An imbalance of matrix metalloproteinases (MMPs) and their inhibitors is thought to play a major role in the pathophysiology of joint diseases. The aim of this study is to provide additional insights into the relevance of MMP levels in arthroplasty patients in relation to inflammation and thrombosis. Deidentified plasma samples from 100 patients undergoing total hip arthroplasty or total knee arthroplasty were collected preoperatively, on postoperative day 1, and on postoperative day 3. Tissue inhibitor of MMP 4, tumor necrosis factor α (TNF-α), pro-MMP1, MMP3, MMP9, MMP13, and d-dimer were measured using enzyme-linked immunosorbent assay kits. A biochip array was used to profile interleukin (IL) 2, IL-4, IL-6, IL-8, IL-10, vascular endothelial growth factor (VEGF), interferon gamma, TNF-α, IL-1α, IL-1β, monocyte chemoattractant protein 1, and endothelial growth factor (EGF) levels. The levels of MMP1, MMP9, MMP13, and TNF-α were elevated preoperatively in arthroplasty patients when compared to healthy individuals. The concentrations of MMP1 and MMP9 increased slightly in postsurgical samples. d-Dimer levels were elevated preoperatively, increased postoperatively, and started decreasing on postoperative day 3. Significant correlations between MMP9 with TNF-α, IL-6, IL-8, VEGF, and EGF were identified. Elevated preoperative MMP1, MMP9, and MMP13 concentrations suggest that they may play a role in the pathogenesis of arthritis. There is also evidence of increased coagulation activity and possible upregulation of several MMPs postsurgically. Correlation analysis indicates that MMP9 levels may potentially be related to inflammation and thrombosis in arthroplasty patients.

Inflammation and Hemostatic Activation may Contribute to Postsurgical Thrombosis in Patients With Bladder Cancer.

The alterations of the inflammatory and thrombotic components in patients with cancer are not clearly understood. The purpose of this study was to profile markers of inflammation and thrombotic activation specifically in the patients with bladder cancer undergoing radical cystectomy. For this study, 134 samples were collected from patients undergoing radical cystectomy. Antiphospholipid antibodies (immunoglobulin G subtype), microparticles, and antiglycosaminoglycan antibodies were measured with a commercially available enzyme-linked immunosorbent assay kits. These biomarkers were compared in patients with bladder cancer and normal individuals (n = 20). Patients had an average value of 6.7 ± 11.9 ng/mL (median: 2.8, confidence interval: 4.69-8.75, andPvalue: .0038) of antiphospholipid antibodies versus normal individuals 1.96 ± 0.9 ng/mL (median: 1.8 and confidence interval: 1.5-2.35). Microparticles level in patients was 8.31 ± 6.14 ng/mL, (median: 6.1, confidence interval: 7.26-9.37, andPvalue: <.0001) versus normal individuals 3.57 ± 2.34 ng/mL (median: 2.85 and confidence interval: 2.476-4.664). The antiglycosaminoglycan antibodies in patients had an average value of 0.22 ± 0.1 optical density (OD; median: 0.2, confidence interval: 0.21-0.24, andPvalue: .0213) compared to normal individuals 0.25 ± 0.08 OD (median: 0.25 and confidence interval: 0.22-0.23). The correlation of antiglycosaminoglycan antibodies with antiphospholipid antibodies showed Spearmanrvalue = .2364 (95% confidence interval: 0.05-0.4 andPvalue .009). The correlation of antiglycosaminoglycan antibodies versus microparticles showed Spearmanr= -.195 (95% confidence interval: 0.37-0.01 andPvalue .0321). These data suggest that patients with bladder cancer have subclinical activation of thrombotic and inflammatory processes that may be further exacerbated by surgical procedures and lead to venous thromboembolism-related complications.

JAK2V617F allele burden is associated with thrombotic mechanisms activation in polycythemia vera and essential thrombocythemia patients

The clinical courses of polycythemia vera (PV) and essential thrombocythemia (ET) are characterized by thrombohemorrhagic diathesis. Several groups have suggested an association between JAK2V617F mutation and thrombosis. We hypothesized a relationship between JAK2V617F allele burden, cellular activation parameters, and thrombosis. We evaluated a group of PV and ET patients using flow cytometry: platelet CD62P, CD63, and dense granules, platelet-leukocyte aggregates (PLA), leukocyte CD11b and monocyte tissue factor (TF) expression. All patients had increased baseline platelet CD62P and CD63 expression (p < 0.05); 71 % of PV and 47 % of ET presented with a storage pool disease. Leukocyte CD11b, TF, and PLA were elevated in all patients. TF was higher in PV compared to ET (p < 0.05) and platelet-neutrophil [polymorphonuclear (PMN)] aggregates were increased in ET versus PV (p < 0.05). In ET, PLA were correlated with platelet numbers (p < 0.05). In all patients, JAK2V617F allele burden was directly correlated with monocyte CD11b. Patients with JAK2V617F allele burden >50 % presented higher levels of leukocyte activation. In ET, thrombosis was associated with JAK2V617F mutation (p < 0.05, χ (2) = 5.2), increased monocyte CD11b (p < 0.05) and with platelet-PMN aggregates (p < 0.05). In ET patients, hydroxyurea does not significantly reduce the activation parameters. Our data demonstrate that JAK2V617F allele burden is directly correlated with activation parameters that drive mechanisms that favor thrombosis.

Thrombotic Biomarker Profiling Of Plasma Samples From Patients Undergoing Bypass Surgery Using Protein Chip Array

IntroductionCardiovascular bypass surgical procedures, such as the coronary bypass procedure (CAP), patients are exposed to large doses of heparin and protamine sulfate. There is a high prevalence of anti-heparin platelet factor-4 antibodies in these patients. Although a fraction of these patients develop HIT syndrome, the role of circulating anti-heparin platelet factor-4 antibodies is not clear. Some of these patients may be at a high risk for post-surgical thrombotic complications. Such thrombogenic mediators as the microparticle and tissue factor, may also be up regulated in these patients. Inflammatory processes may also contribute to the overall post-thrombotic complications. The relevance of thrombotic mediators and inflammatory processes remains to be further explored in these patients. Recently protein chip array approach using SELDI/TOF mass spectrometry methods have been employed to identify the unique biomarker in various diseases. The purpose of this study was to determine the protein chip array profile and quantification of various mediators of thrombotic activation in patients who have undergone bypass surgery. Materials & MethodsPlasma samples from 79 patients were collected immediately prior to and two weeks after the CAP. Protein chip array profiling was carried out on a SELDI/TOF mass spectrometric method (PCS4000, BioRad, Richmond, CA) employing a gold chip array in the molecular weight range of 3000-150,000. The intensity of unique peaks was also calculated in terms of relative intensities. Microparticles were measured using a functional method (Hyphen Biomedical, France) and tissue factor antigen levels were measured using an ELISA method. The anti-heparin platelet factor-4 antibodies were also measured using a commercially available ELISA method (Genprobe, Wisconsin). ResultsOf the 79 patients, 20 showed a unique biomarker peak around 11-12kDa in the pre-op samples, which was absent from normal controls. 77 of the patients showed this unique biomarker peak at one week, whereas only 48 patients exhibited at two weeks after surgery. The relative intensity of the 11.6kDa biomarker was much higher at one week (6-fold) and was decreased at two weeks (3-fold). In addition to this unique peak, other biomarker peaks were noted at 15.1 and 15.8kDa. However, these peaks were not changed at different time points. In comparison to the normal, the microparticle levels were higher at the baseline sample (10.1±3.2nM) and increased to 19.3±6.1 and 24.5±8.1nM. Similarly, the tissue factor levels were increased at three weeks' time period. The anti-heparin platelet factor-4 titer rose 28% from the baseline at week one and 33% at week two. ConclusionsThe results on the biomarker profile are consistent to the earlier finding, where the presence of a unique biomarker in the range of 11-12kDa have been reported in patients with high prevalence of anti-heparin platelet factor-4 antibody. The increased level of microparticles and tissue factor at post-surgical periods of one week and two weeks suggest endogenous activation of thrombogenic mechanisms, which appears proportional to the up regulation of the anti-heparin platelet factor-4 antibodies. Thus, this data indicates that the non-functional anti-heparin platelet factor-4 antibodies may result in the activation of cellular processes leading to thrombogenesis. Further characterization of the unique biomarkers identified in these patients may be useful in understanding of the pathogenesis of inflammatory processes and their relevance to thrombogenesis in CABG patients. Clinical ImplicationsThese results indicate that the nonfunctional anti-heparin platelet factor 4 antibodies are capable of mediating inflammatory and thrombotic responses without symptomatic thrombocytopenia. Therefore, the measurement of these antibodies along with inflammatory and thrombogenic mediators may be helpful in the diagnostic and prognostic management of these patients. Disclosures:No relevant conflicts of interest to declare.

The Serpin Solution; Targeting Thrombotic and Thrombolytic Serine Proteases in Inflammation

Serpins in the mammalian body are highly potent serine protease inhibitors which modulate both thrombotic and thrombolytic pathway activation, with direct and indirect crosstalk with immune and inflammatory pathways. In this review, we discuss mammalian and viral serpins as regulators of coagulation and inflammation. We focus first on the thrombotic and thrombolytic serine proteases and known interactions between these protease cascades and elements of the innate immune response. Serpin-mediated regulation of the thrombotic pathway is then discussed, with emphasis on those serpins that have been evaluated as potential new drugs. Finally the potential of viral serpins that target the coagulation and thrombolytic cascades as potential therapeutics for anti-inflammatory properties is discussed from basic molecular activity to studies in animal models. The studies discussed range from thrombosis and hemorrhage to vascular disease and transplant rejection and finally to sepsis and clinical studies in humans. In conclusion, these unique proteins, the serpin family, now have demonstrated therapeutic potential for a wide variety of inflammatory diseases in both animal and human studies and represent a new approach for drug development.