Procoagulant Factors Research Articles

S3612 A Clotting and Bleeding Interplay in Ulcerative Colitis

Introduction: Inflammatory bowel disease (IBD) patients are at increased risk of venous thromboembolic (VTE) events. Here we present a case of a patient with ulcerative colitis (UC) with incidental findings of pulmonary embolism (PE) and, further complications on anticoagulation. Case Description/Methods: A 50-year-old female with a past medical history of anxiety and recently diagnosed UC on steroid taper presented to the hospital with complaints of intractable diarrhea, diffuse abdominal discomfort, and worsening fatigue. Lab findings were consistent with previously diagnosed iron deficiency anemia, downtrending C-reactive protein, ESR, negative C.diff, and gastric pathogen panel. A repeat CT abdomen pelvis with contrast showed improving inflammation of the colon, mesalamine was added to steroid regimen . There was an incidental finding of PE in the bilateral segmental arteries without right heart strain, treated with heparin infusion and transitioned to rivaroxaban.Later, patient had two episodes of hospital admission for rectal bleeding complicating the treatment course. Rivaroxaban was resumed after first episode of bleeding, but since the further episode of rectal bleeding required cardiopulmonary resuscitation, anticoagulation was discontinued. During this time for UC, mesalamine was discontinued and infliximab was initiated. She developed another episode of bilateral deep vein thrombosis off anticoagulation, hence IVC filter was placed to avoid new PE and plan to restart anticoagulation after resolution of bleeding was made. Discussion: Patients with IBD are at higher risk ( 2 to 3 fold) of developing the extraintestinal complication of VTE compared to general population with incidence varying from 1.3% to 6.2% and mortality from 8% to 25%. The exact pathophysiology of thrombosis in IBD is not fully understood and is considered to be a complex interplay of inflammation and coagulation, affecting the procoagulant, anticoagulant and fibrinolytic factors of the coagulation cascade. Patients in an acute flare of IBD are associated with highest risk of VTE compared to patients in remission. Patients admitted for IBD flare should be on pharmacological prophylaxis for VTE and can be safely administered for patients with minor hematochezia. Treatment of VTE in IBD patients is the same as patients without IBD, but duration of therapy is not well established. Risk is lower in patients in remission, another important reason for providers to have IBD patients in endoscopic and clinical remission.

Substrate stiffness modulates endothelial cell function via the YAP-Dll4-Notch1 pathway

Endothelial cells adapt their functions as a consequence of sensing extracellular substrate stiffness; these alterations allow them to maintain their vascular structure and function. Substrate stiffness-mediated yes-associated protein 1 (YAP) activation plays an important role in mechano-transduction and pro-angiogenic phenotype of endothelial cells, and Delta-like ligand 4 (Dll4)-Notch1 signaling is closely related to angiogenesis; however, the impact of substrate stiffness-mediated interrelation of these pathways on endothelial cell functions remains elusive. We confirmed that endothelial cells on softer substrates not only elongate cellular aspects but also attenuate YAP activation compared to cells on stiffer substrates. Endothelial cells on softer substrates also upregulate the vascular endothelial growth factor receptor 1 (VEGFR1) and VEGFR2 mRNA expression that is enhanced by VEGF stimulation. We determined that endothelial cells on softer substrates increased Dll4 expression, but not Notch1 expression, via YAP signaling. Moreover, endothelial cells on soft substrates induced not only VEGFRs upregulation but also suppression of pro-inflammatory interleukin-6 and plasminogen activator inhibitor-1 mRNA expression and the facilitation of anti-coagulant thrombomodulin and pro-coagulant tissue factor mRNA expression. Our results suggest that endothelial cells activate the YAP-Dll4-Notch signaling pathway in response to substrate stiffness and dictate cellular function.

Arterial Thrombosis in Cancer Patients: An Update.

Cancer is associated with an increased incidence of both venous thromboembolism (VTE) and arterial thrombosis (cardiovascular events and ischemic stroke). Cancer-associated arterial thrombotic events are less well studied than VTE, but increasingly recognized, particularly in specific malignancies and in association with specific anticancer therapies. The pathogenesis of arterial thrombotic events in cancer is complex and involves generation of tumor-associated procoagulant factors and a variety of alterations in platelet function as well as in the coagulation and fibrinolytic systems, and endothelial injury and dysfunction, that combine to produce hypercoagulability. The multifactorial interaction between this prothrombotic state, the individual cardiovascular risk, advanced age and presence of comorbidities, and the specific neoplasm characteristics and therapy, may induce the vascular events. Recent studies based on population databases and prospective or retrospective analyses with prolonged follow-up highlight that cancer patients experience an increased (approximately 1.5-2-fold) risk of both cerebrovascular and cardiovascular events compared with noncancer individuals, which peaks in the time period of the diagnosis of cancer but may persist for years. Beyond the type of cancer, the risk reflects the tumor burden, being higher in advanced stages and metastatic cancers. The occurrence of arterial thromboembolic events is also associated with increased overall mortality. We here present an update of the pathophysiology, risk factors, clinical evidence, and treatment considerations on cancer-associated arterial thrombosis, in the light of the need for specific multidisciplinary prevention and surveillance strategies in this setting, in the frame of cardio-oncology approaches.

Rare Defects: Looking at the Dark Face of the Thrombosis.

Venous thromboembolism (VTE) constitutes a serious and potentially fatal disease, often complicated by pulmonary embolism and is associated with inherited or acquired factors risk. A series of risk factors are known to predispose to venous thrombosis, and these include mutations in the genes that encode anticoagulant proteins as antithrombin, protein C and protein S, and variants in genes that encode instead pro-coagulant factors as factor V (FV Leiden) and factor II (FII G20210A). However, the molecular causes responsible for thrombotic events in some individuals with evident inherited thrombosis remain unknown. An improved knowledge of risk factors, as well as a clear understanding of their role in the pathophysiology of VTE, are crucial to achieve a better identification of patients at higher risk. Moreover, the identification of genes with rare variants but a large effect size may pave the way for studies addressing new antithrombotic agents in order to improve the management of VTE patients. Over the past 20 years, qualitative or quantitative genetic risk factors such as inhibitor proteins of the hemostasis and of the fibrinolytic system, including fibrinogen, thrombomodulin, plasminogen activator inhibitor-1, and elevated concentrations of factors II, FV, VIII, IX, XI, have been associated with thrombotic events, often with conflicting results. The aim of this review is to evaluate available data in literature on these genetic variations to give a contribution to our understanding of the complex molecular mechanisms involved in physiologic and pathophysiologic clot formation and their role in clinical practice.

From the Editor’s Desk…

From the Editor’s Desk…

More Pronounced Hypercoagulable State and Hypofibrinolysis in Patients With Cirrhosis With Versus Without HCC.

In patients with cirrhosis, particularly those with hepatocellular carcinoma (HCC), hypercoagulability may be associated with purported increased risks of portal vein thrombosis and cirrhosis progression. In this study, we extensively investigated hemostatic alterations potentially responsible for the thrombotic tendency in HCC, and evaluated whether such alterations were predictive of hepatic decompensation. Patients with cirrhosis at all stages were prospectively recruited and underwent an extensive hemostatic assessment, including all procoagulant factors and inhibitors, thrombin generation with and without thrombomodulin (TG), profibrinolytic and antifibrinolytic factors, and plasmin‐antiplasmin complex. In study part 1 (case control), we compared alterations of coagulation and fibrinolysis in patients with cirrhosis with versus without HCC. In study part 2 (prospective), the subgroup of patients with decompensated cirrhosis was followed for development of further decompensation, and predictors of outcome were assessed by multivariate analysis. One‐hundred patients were recruited (50 each with and without HCC). Severity of cirrhosis was comparable between groups. Median HCC volume was 9 cm3 (range: 5‐16). Compared with controls, patients with HCC demonstrated a significantly more prothrombotic hemostatic profile due to increased TG and reduced activation of fibrinolysis, independent of cirrhosis stage. During a median follow‐up of 175 days, 20 patients with decompensated cirrhosis developed further episodes of decompensation that were predicted by low FVII and high plasminogen activator inhibitor‐1 levels, independent of Model for End‐Stage Liver Disease score. Conclusion: Patients with cirrhosis with HCC have profound hyper‐coagulable changes that can account for their increased thrombotic tendency. In contrast, hypercoagulability in patients with decompensated cirrhosis is more likely a consequence of chronic liver disease rather than a driver for cirrhosis progression.

Platelet Surface Protein Expression and Reactivity upon TRAP Stimulation after BNT162b2 Vaccination.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection induces a coagulopathy characterized by platelet activation and a hypercoagulable state with an increased incidence of cardiovascular events. The viral spike protein S has been reported to enhance thrombosis formation, stimulate platelets to release procoagulant factors, and promote the formation of platelet-leukocyte aggregates even in absence of the virus. Although SARS-CoV-2 vaccines induce spike protein overexpression to trigger SARS-CoV-2-specific immune protection, thrombocyte activity has not been investigated in this context. Here, we provide the first phenotypic platelet characterization of healthy human subjects undergoing BNT162b2 vaccination. Using mass cytometry, we analyzed the expression of constitutive transmembrane receptors, adhesion proteins, and platelet activation markers in 12 healthy donors before and at five different time points within 4 weeks after the first BNT162b2 administration. We measured platelet reactivity by stimulating thrombocyte activation with thrombin receptor-activating peptide. Activation marker expression (P-selectin, LAMP-3, LAMP-1, CD40L, and PAC-1) did not change after vaccination. All investigated constitutive transmembrane proteins showed similar expressions over time. Platelet reactivity was not altered after BNT162b2 administration. Activation marker expression was significantly lower compared with an independent cohort of mild symptomatic COVID-19 patients analyzed with the same platform. This study reveals that BNT162b2 administration does not alter platelet protein expression and reactivity.

The prevalence of Factor V Leiden (Arg506Gln) mutation in King Khalid University Hospital patients, 2017-2019.

ABSTRACTArg506Gln mutation is responsible for one of the procoagulant factors and most common inherited thrombophilia in the Factor V Leiden (FVL) family. The replacement of the missense mutation for Arg506Gln / R506Q is at 1691st position from Guanine to Adenine with the modification of the amino acid from arginine to glutamine. The aim of this study was to investigate the current prevalence of the G1691A mutation in the FVL gene in the capital city’s King Khalid University Hospitals (KKUH). Since 2017–2019 we have recruited 482 patients in these cross-sectional studies to test the G1691A mutation in KKUH’s FVL gene. DNA was extracted using 2mL of the EDTA blood and genotyping was performed with polymerase chain reaction and the data was analyzed using Sanger sequencing. In this study, 4.4% of the G1691A mutation was found to be positive (combined heterozygous-GA and homozygous-AA variants) and 95.6% of them with negative, i.e., homozygous normal-GG genotypes. Our study concludes that with the advances in genetic testing and their recent availability, early mutation detection could approve the genotype risks for many patients and this mutation is not as rare as previously believed in the Saudi region as our study has established with a 4.4 percent prevalence.

Cullin 2-RBX1 E3 ligase and USP2 regulate antithrombin ubiquitination and stability.

Hemophilia A and B are congenital bleeding disorders caused by a deficiency in pro-coagulant factor VIII or IX that is treated by downregulation of antithrombin. However, the molecular mechanisms that regulate antithrombin expression remain poorly understood. Here, we identified Cullin 2 and USP2 (ubiquitin-specific peptidase-2) as novel regulators of antithrombin expression that act by modulating antithrombin ubiquitination. Inhibition of the proteasome caused accumulation of antithrombin and its ubiquitinated forms in HepG2 and SMMC7721 cells. Notably, inhibition of neddylation with MLN4924 suppressed both ubiquitination and degradation of antithrombin, which is recapitulated by silencing of the neddylation enzymes, NAE1, UBA3, and UBE2M, with small interfering RNA (siRNA). We identified Cullin 2 as the interaction partner of antithrombin, and siRNA-mediated Cullin 2 knockdown reduced antithrombin ubiquitination and increased antithrombin protein. We further found that USP2 interacted with antithrombin and regulated antithrombin expression, showing that overexpression of USP2 inhibits the ubiquitination and proteasomal clearance of antithrombin, whereas pharmacological inhibition or siRNA-mediated knockdown of USP2 downregulates antithrombin. Collectively, these results suggest that Cullin 2 E3 ubiquitin ligase and USP2 coordinately regulate antithrombin ubiquitination and degradation. Thus, targeting Cullin 2 and USP2 could be a potential strategy for treatment of hemophilia.

Causes of an elevated international normalized ratio in the intensive care unit and the implications for plasma transfusion.

The presence of an elevated international normalized ratio (INR) is common in patients in the intensive care unit (ICU), but the cause rarely determined. These patients are at risk to receive prophylactic plasma prior to invasive procedures. Samples from patients with an INR of 1.5 or greater were frozen and subsequently thawed and assayed for procoagulant and anticoagulant clotting factors and anti-Xa to determine the likely cause of the INR. Samples showing a low FVII, FX, PC, and PS were categorized as a vitamin K deficiency pattern. Samples showing a low FV, low or normal fibrinogen, and high FVIII were categorized as a liver disease pattern. Samples showing an anti-Xa >0.01 IU/ml were assayed for anti-Xa DOACs. Samples which could not be categorized were grouped as equivocal. A total of 48 samples were obtained over a 6-month period. Nineteen showed a Vitamin K deficiency pattern, 17 a liver disease pattern, 7 showed an anti-Xa DOAC and 5 were equivocal. High FVIII and D-dimers and reduced levels of the anticoagulant proteins were present in the majority of the samples. FVII levels correlated inversely with the INR (r= -0. 81), as did FX (r= -0.67) but not FV (r= -0.04) nor fibrinogen (r= -0.15). Transfusion of plasma to reverse an elevated INR in the ICU should be discouraged since such a practice is either avoidable by the use of vitamin K or inappropriate in the case of liver disease or an anti-Xa DOAC.

Lifelong dietary omega-3 fatty acid suppresses thrombotic potential through gut microbiota alteration in aged mice

SummaryAging is a major risk factor for cardiovascular diseases, including thrombotic events. The gut microbiota has been implicated in the development of thrombotic risk. Plant-derived omega-3 fatty acid ɑ-linolenic acid (ALA) confers beneficial anti-platelet and anti-inflammatory effects. Hence, antithrombotic activity elicited by ALA may be partly dependent on its interaction with gut microbiota during aging. Here, we demonstrate that lifelong dietary ALA decreases platelet hyperresponsiveness and thrombus formation in aged mice. These phenotypic changes can be partly attributed to alteration of microbial composition and reduction of its metabolite trimethylamine N-oxide and inflammatory mediators including TNF-α, as well as the upregulated production of short-chain fatty acid acetate. ALA-rich diet also dampens secretion of increased procoagulant factors, tissue factor and plasminogen activator inhibitor-1, in aged mice. Our results suggest long-term ALA supplementation as an attractive, accessible, and well-tolerated nutritional strategy against age-associated platelet hyperreactivity and thrombotic potential.

Increased activity of procoagulant factors in patients with small cell lung cancer.

Small cell lung cancer (SCLC) patients have augmented risk of developing venous thromboembolism, but the mechanisms triggering this burden on the coagulation system remain to be understood. Recently, cell-derived microparticles carrying procoagulant phospholipids (PPL) and tissue factor (TF) in their membrane have attracted attention as possible contributors to the thrombogenic processes in cancers. The aims of this study were to assess the coagulation activity of platelet-poor plasma from 38 SCLC patients and to provide a detailed procoagulant profiling of small and large extracellular vesicles (EVs) isolated from these patients at the time of diagnosis, during and after treatment compared to 20 healthy controls. Hypercoagulability testing was performed by thrombin generation (TG), procoagulant phospholipid (PPL), TF activity, Protein C, FVIII activity and cell-free deoxyribonucleic acid (cfDNA), a surrogate measure for neutrophil extracellular traps (NETs). Our results revealed a coagulation activity that is significantly increased in the plasma of SCLC patients when compared to age-related healthy controls, but no substantial changes in coagulation activity during treatment and at follow-up. Although EVs in the patients revealed an increased PPL and TF activity compared with the controls, the TG profiles of EVs added to a standard plasma were similar for patients and controls. Finally, we found no differences in the coagulation profile of patients who developed VTE to those who did not, i.e. the tests could not predict VTE. In conclusion, we found that SCLC patients display an overall increased coagulation activity at time of diagnosis and during the disease, which may contribute to their higher risk of VTE.

Obstructive Sleep Apnea, Hypercoagulability, and the Blood-Brain Barrier.

Obstructive sleep apnea (OSA) is characterized by repeated episodes of intermittent hypoxia (IH) and is recognized as an independent risk factor for vascular diseases that are mediated by a multitude of mechanistic pathophysiological cascades including procoagulant factors. The pro-coagulant state contributes to the development of blood clots and to the increase in the permeability of the blood–brain barrier (BBB). Such alteration of BBB may alter brain function and increase the risk of neurodegenerative diseases. We aim to provide a narrative review of the relationship between the hypercoagulable state, observed in OSA and characterized by increased coagulation factor activity, as well as platelet activation, and the underlying neural dysfunction, as related to disruption of the BBB. We aim to provide a critical overview of the existing evidence about the effect of OSA on the coagulation balance (characterized by increased coagulation factor activity and platelet activation) as on the BBB. Then, we will present the emerging data on the effect of BBB disruption on the risk of underlying neural dysfunction. Finally, we will discuss the potential of OSA therapy on the coagulation balance and the improvement of BBB.

Evaluation of coagulation status using clot waveform analysis in general ward patients with COVID-19.

Coronavirus disease (COVID-19)-related systemic cytokine response induces the production of procoagulant factors, which predisposes patients to a prothrombotic state. Viscoelastic testing can identify the degree of hypercoagulability, which is related to outcomes. We aimed to study the changes in clot waveform analysis (CWA) parameters in COVID-19 patients on hospital admission compared to those in a group of healthy individuals. We conducted a retrospective study of COVID-19 patients admitted to general wards and evaluated demographic and clinical parameters as well as laboratory parameters, including coagulation parameters. CWA data from patients (n = 62) with COVID-19 prior to the initiation of anticoagulation therapy were compared with those from healthy controls (n = 67). The measured CWA parameters were min1, min2, max2, and delta change. CWA, fibrinogen, and D-dimer values were higher in COVID-19 patients than in healthy controls (p < 0.001). CWA profiles were consistent with hypercoagulability and characterized by an increase in density, velocity, and acceleration of clot formation. Activated partial thromboplastin time, fibrinogen, D-dimer, and C-reactive protein (CRP) values were higher in patients in whom all CWA parameters were raised than in patients with just a few elevated CWA parameters, while Sequential Organ Failure Assessment scores, prothrombin time, fibrin degradation product levels and platelet counts did not differ between the two groups. CWA variables showed hypercoagulopathy on admission in COVID-19 patients who were hospitalized in the general ward, and this pattern was more pronounced in critically ill patients with elevated fibrinogen, D-dimer, and CRP levels. Our results may help identify patients at high risk of thromboembolism.

Differential effect of statin use on coagulation markers: an active comparative analysis in the NEO study

BackgroundStatins are a potential treatment for venous thromboembolism (VTE) prophylaxis complementary to conventional anticoagulants without associated bleeding complications. This study aimed to compare pro-thrombotic activities of different classes of lipid-lowering drugs in an active comparator design and determine whether there is a relation between statin versus fibrate/niacin use and pro-coagulant factor outcomes.MethodsThis is a cross-sectional analysis of participants from the Netherlands Epidemiology of Obesity study using any class of lipid-lowering drugs, including any types of statins, niacin, and fibrates. We performed linear regression analyses to determine fibrinogen, factor (F) VIII, FIX, and FXI activity in statins versus fibrate/niacin users and adjusted for age, sex, tobacco smoking, body mass index (BMI), hypertension, diabetes, and prevalent cardiovascular disease.ResultsAmong 1043 participants, the mean age was 58.4 ± 5.2 years, 61% were men, and the mean BMI was 31.3 ± 4.5 kg/m2. Clinical characteristics were balanced between statin and fibrate/niacin users. Statin users had lower mean FXI (18.3 IU/dL, 95% confidence interval (CI) 9.4 to 27.3) levels compared to fibrate/niacin users. The level of FVIII (15.8 IU/dL, 95% CI − 0.003 to 31.6), and FIX (11.3 IU/dL, 95% CI − 0.4 to 23.2) were lower in statin users than fibrate/niacin users with marginal statistical significance.ConclusionCurrent statin use was associated with lower plasma levels of FXI than fibrate/niacin use. The effects on coagulation factors may, in part, explain the benefit of statin therapy rendered in primary and secondary prevention of VTE.

Pregnancy Outcome in Mice with Protein S Complete Deficiency

Introduction:Pregnancy is associated with a shift of the coagulation balance with increased concentration of procoagulant factors, decreased concentration of some anticoagulants and diminished fibrinolytic activity. These changes provoke a state of hypercoagulability that protects pregnant women from fatal hemorrhage during delivery but predispose them to thromboembolism.Protein S (PS) is a natural anticoagulant that acts as a cofactor of activated protein C and tissue factor pathway inhibitor. PS complete deficiency leads to life threatening skin necrosis and disseminated intravascular coagulation (DIC) named purpura fulminans (PF) that leads to death if untreated. Pregnant women with partial PS deficiency are at high risk of late fetal loss. The beneficial role of thromboprophylaxis in ameliorating pregnancy outcome in patients with inherited thrombophilia is debated.Here, we used mice with complete PS deficiency (Pros1-/-) fully rescued from lethality by the lack of factor VIII (F8-/-Pros1-/-). Characterization of F8-/-Pros1-/- mice revealed no signs of PF or DIC. These mice were used to investigate the effect of PS complete deficiency on pregnancy.Methods:Timed matings were set to generate E9-E17.5 embryos from F8-/-Pros1-/-, F8-/-Pros1+/+ and F8+/+Pros1+/+ pregnant mice (n=3-9/genotype). Embryos viability and genotype were evaluated. At the time of sacrifice, blood was drawn from pregnant female to evaluate blood cells count profile and coagulation parameters (fibrinogen, thrombin anti-thrombin complex; TAT) were measured. Anti-fibrin immunostaining was performed in collected embryos and placentas.F8-/-Pros1-/- pregnant females were treated with low-dose aspirin (200mg/kg Lysine acetylsalicylate) or LMWH (Clexane, 100μg/mL delivered by osmotic pump 0.25 μL/hour) from E7 until E20 to assess if thromboprophylaxis could be beneficial for pregnancy.Results:No litters were produced from F8-/-Pros1-/- crosses.F8-/-Pros1-/- females were then mate with F8-/-Pros1+/- male and sacrificed at different gestational stages. A progressively high number of macerated and necrotic embryos were found after E9-E10 in comparison with pregnantcontrols: 0% (E9-10), 57% (E11-E12), 64% (E14-E15) and 67% (E16-E17). In parallel, number of embryos per gestation decreased in F8-/-Pros1-/- compared to F8-/-Pros1+/+ females (4±1 vs 9±1 at E11-E12, 3±1 vs 7±1 at E14-E15, 2±1 vs 7±2 at E16-E17, respectively). Macroscopically, the majority of alive embryos showed the presence of hemorrhages without PF. F8-/-Pros1-/- genotype was underrepresented among alive embryos (33% vs the expected 50%) and it disappeared from those collected after E12.5.The evaluation of DIC parameters in F8-/-Pros1-/- pregnant females during the period of experienced high embryonic mortality (E12-15) showed a significative reduction of platelet counts (486±105 vs 806±134 G/L F8-/-Pros1+/+, P<0.03), fibrinogen (1±0.1 vs 2±0.2 g/L, respectively, P<0.001) and increased TAT (33.2±5.1, 16.6±2.8 ng/L, respectively, P<0.02).Although these results suggest an activated state of blood coagulation, fibrin staining did not reveal fibrin deposition in lungs, liver and placenta from F8-/-Pros1-/- pregnant mice.Treatment of F8-/-Pros1-/- pregnant mice with LMWH or aspirin had a positive pregnancy outcome (n=4/female/treatment). However, litter size was reduced in comparison to F8-/-Pros1+/+ control mice. In particular, the aspirin group showed the lowest litter size while the LMWH group had increased pups number (3±1 vs 6±1, P<0.03). F8-/-Pros1-/- genotype was rescued only among newborns from LMWH treated group (n=1 over 7). Preliminary data from pups monitoring indicated that some pups were smaller, experienced hemorrhages and died after birth.Conclusion:Targeting F8 did not prevent pregnancy loss in F8-/-Pros1-/- mice. Only few F8-/-Pros1+/- embryos survived until E17 indicating that PS expression in the uteroplacental unit is indispensable for embryos development. F8-/-Pros1-/- pregnant mice displayed coagulation activation. However, further investigations are needed if the observed DIC is due to the lack of PS anticoagulant activity or to retained dead embryos. Aspirin or better LMWH partially prevented pregnancy loss indicating that thromboprophylaxis might be beneficial for pregnancy development with very low PS plasma levels. DisclosuresNo relevant conflicts of interest to declare.

The Role of Platelets in Increased Thrombogenicity Following Splenectomy: Key Platelet-Specific Receptors and Activation Markers

AimThe mechanisms underlying increased thrombogenicity in splenectomy are poorly understood. We assessed a potential role for platelets by analysing surface expression of key platelet-specific receptors glycoprotein (GP)Iba, GPVI and aIIbb3, and a platelet activation marker, P-selectin. GPIba, that binds von Willebrand factor and other ligands, is constitutively shed from aging platelets, and its surface density is crucial for both thrombus formation and platelet clearance through interactions with phagocytic cells. GPIba also forms a non-covalent complex with GPVI, which binds collagen and fibrin. GPIba/GPVI initiate platelet adhesion, leading to platelet activation and aggregation, secretion of procoagulant factors and thrombus formation.MethodsWhole blood from same-day healthy controls (n=15) or splenectomy (n=30) was centrifuged to obtain platelet-rich-plasma, and stained with phycoerythrin (PE)-labelled antibodies against GPIba (PE-AK2), GPVI (PE-1G5), aIIb (PE-CD41a), CD9 (PE-CD9), and P-selectin (PE-CD62P) using standardized protocols, and analysed using a FACSCalibur.ResultsPreliminary analysis suggested that compared to healthy controls, platelets from splenectomy cases showed significantly decreased surface expression of GPIba (p <0.0001) and GPVI (p=0.0035). In contrast, there was no significant difference in relative surface expression of aIIbb3 (p=0.759) and CD9 (p=0.112). Further, consistent with increased platelet activation/secretion, expression of surface P-selectin was more variable and significantly elevated in splenectomy cf . control platelets (p=0.0005). ELISA analysis of stored plasma will be used to confirm whether decreased platelet GPVI is likely due to increased shedding, resulting in increased plasma sGPVI.ConclusionThe results of this pilot study support a role for hyperactivated platelets post-splenectomy, and identify platelet-specific markers related to these changes. Methods and results developed here will enable future larger temporal studies of platelet-related factors, and determine if such measurable changes correlate with clinical haemostatic/thrombotic dysfunction. This can also help explain basic mechanism(s) of thrombotic propensity in this population, and/or indicate potential therapeutic targets for prevention of thrombosis. DisclosuresLuu:Royal College of Pathologists of Australasia: Research Funding. Woolley:Monash Health: Research Funding. Jones:Pfizer: Other: Educational Grant.

Exome Sequencing Studies Identify Mutations in STAB2 As a Genetic Risk for Venous Thromboembolic Disease

Deep vein thrombosis and pulmonary embolism, collectively referred to as venous thromboembolism (VTE), are the third leading cause of cardiovascular death in the United States. Genetic factors account for 50-60% of VTE risk and a recent meta-analysis of genome-wide association studies confirmed that common variants in F5, ABO, F2 and seven other very low effect size loci are associated with VTE. Rare mutations in the anticoagulant genes PROC (protein C), PROS1 (protein S) and SERPINC1 (antithrombin III) have been linked to highly penetrant VTE risk in family studies but no genome-wide study of rare variants has been reported to date.We performed whole exome sequencing (WES) in 373 unrelated individuals of European ancestry with well characterized unprovoked VTE and compared results to a previously genome sequenced control cohort of 5784 unrelated Europeans. We then identified single nucleotide variants, small insertion or deletions that were present at allele frequencies less than 0.005% in ExAC and had equal sequencing coverage between groups. Using a dominant model and a collapsing association analysis, we looked for genes that were more likely to harbor “knockout” (frame shift, nonsense, splice site and damaging missense) mutations in cases versus controls. Strikingly, ranked by p-value, among 17,395 genes tested, the top 4 genes were PROS1 (P= 2.01E-09, odds ratio: 11.8), STAB2 (P=2.70E-7, odds ratio 3.37), PROC (P=3.24E-05, odds ratio 11.0) and SERPINC1 (P=1.10E-04, odds ratio 8.5), Figure. These findings suggest that heterozygous deficiency for the STAB2 encoded protein, stabilin-2, confers VTE susceptibility similar to the previously well described phenotypes of heterozygous protein C, protein S and antithrombin III deficiency.Stabilin-2 is a single transmembrane glycoprotein scavenger receptor expressed on venous endothelium of liver sinusoids and spleen. We detected damaging STAB2 mutations in 29 (7%) VTE cases versus 141 (2%) of controls. Common and uncommon variants in STAB2 have been associated with elevated plasma von Willebrand factor (VWF) and coagulation factor VIII levels, which are known risk factors for VTE. Stabilin-2 is implicated as a clearance receptor for VWF, so that haploinsufficiency of stabilin-2 may increase plasma VWF half-life. To test this hypothesis, we examined 1162 individuals in the Twins UK Study (VTE status unknown) with available whole genome sequencing data and measured their plasma VWF levels by AlphaLISA. VWF levels were adjusted by log transformation and ABO blood type tagging SNPs. We identified 38 individuals (3.2%, all heterozygous) with rare damaging variants in STAB2. VWF levels were significantly higher in individuals (N=30) with rare STAB2 variants compared to those without (N=1016) (Mann-Whitney, P= 0.0046).To further examine stabilin-2 function in vivo, we compared Stab2 deficient mice (Stab2 -/-) to wild type (Stab2 +/+) controls. Stab2 -/- mice had elevated plasma levels of factor VIII:C (118%, p=0.0006), but no differences in VWF (p=0.58). Next, we examined thrombosis in a mouse model where venous blood clotting was induced by inferior vena cava stenosis. Thrombi were weighed and longitudinal sections were analyzed by quantitative immunohistochemistry 24 hours post-stenosis. Stab2 -/- mice developed significantly larger thrombi than Stab2 +/+ mice (194%, n=19, p=0.003) but no difference in DVT incidence (64% vs 61%, P= 0.85). Despite equivalent hematocrit (P= 0.91) and white type thrombus (P> 0.05), thrombi from Stab2 -/- mice were comprised of more red type thrombus (185%, P= 0.004).Through an unbiased analysis of whole exome sequencing in 6,157 individuals, this studyconfirms the role of haploinsufficiency of PROC, PROS1 and SERPINC1 for VTE susceptibility and identifies STAB2 deficiency as a potential new risk factor. Analysis of an independent human cohort of 1162 Europeans demonstrates that STAB2 haploinsufficiency is associated with elevated VWF levels, a known risk factor for VTE. Finally, a mouse model of Stab2 deficiency demonstrates enhanced thrombosis, though without an association with elevated VWF levels. Taken together, these results suggest that rare genetic variants in STAB2 collectively play a role in individuals with unprovoked VTE by prolonging the plasma half-life of procoagulant factors such as VWF, adding an important new gene to the short list of known risk factors for this common disease. [Display omitted] DisclosuresReitsma:Leiden University Medical Center; VarmX B.V.: Equity Ownership, Patents & Royalties: co-inventor on a patent application related to this abstract.

Vitamin K2 (Menaquinone-7) supplementation does not affect vitamin K-dependent coagulation factors activity in healthy individuals.

Background:Vitamin K has long been regarded as a procoagulant drug by physicians, and concerns have been raised with regard to its effects on hemostasis. Although many studies have shown that vitamin K supplementation is safe for thrombotic events, the effect of vitamin K supplementation on the activities of vitamin K dependent procoagulation factors in healthy individuals is not available.Objectives:This study aimed to investigate whether vitamin K2 supplementation at recommended doses affects the activity of vitamin K dependent procoagulation factors in healthy individuals without any anticoagulation treatment.Design:Forty healthy volunteers between 25 and 40 years of age were recruited. Menaquinone-7 (MK-7) was administrated at 90 μg for 30 days. Prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), and blood coagulation factors II, VII, IX, and X activities and Protein induced by vitamin K absence or antagonist-II (PIVKA-II) were measured on days 0 and 30 after MK-7 administration.Results:PT, APTT, and TT showed no significant differences on day 30 when compared with baseline. The activities of coagulation factors II, VII, IX, and X on day 30 showed no significant differences with those at baseline. PIVKA-II levels were unchanged after 30 days of MK-7 supplementation.Conclusions:MK-7 supplementation at recommended dosage does not affect vitamin K-dependent coagulation factors’ coagulation activity, and does not enhance the carboxylation of prothrombin in healthy individuals. This indicated that MK-7 administration does not alter hemostatic balance in healthy populations without anticoagulation treatment.

ID: 3526988 PRE-PROCEDURE FRESH FROZEN PLASMA DOES NOT REDUCE BLEEDING RISK IN PATIENTS WITH CIRRHOSIS AND COAGULOPATHY UNDERGOING INVASIVE PROCEDURES

Contrary to old dogma, recent data suggests that in patients with cirrhosis and coagulopathy, prolongation of the INR does not accurately reflect hemostasis or thrombin formation. With chronic liver disease, not only is there a decrease in production of procoagulant factors there is a simultaneous reduction in the production of anticoagulant factors as well, often resulting in normal coagulation. Therefore, because the INR is reflective only of the procoagulant pathway it can be misleading in regards to bleeding risk in cirrhotic patients. The purpose of this study was to investigate whether giving fresh frozen plasma (FFP) prior to invasive procedures for patients with cirrhosis and coagulopathy reduces the incidence of procedure related bleeding.