Procoagulant phenotype of virus-infected pericytes is associated with portal thrombosis and intrapulmonary vascular dilations in fatal COVID-19.

BACKGROUND & AIMS: Mechanisms and clinical impact of portal microthrombosis featuring severe COVID-19 are unknown. Intrapulmonary vascular dilation (IPVD)-related hypoxia has been described in severe liver diseases. We hypothesized that portal microthrombosis is associated with IPVD and fatal respiratory failure in COVID-19. METHODS: Ninety-three patients who died from COVID-19, were analysed for portal microvascular damage (histology), IPVD (histology and chest-computed tomography, CT), and hypoxemia (arterial blood gas). Seventeen patients who died from COVID-19-unrelated pneumonia served as controls. Vascular lesions and microthrombi were phenotyped for endothelial (vWF) and pericyte (αSMA/PDGFR-ß) markers, tissue factor (TF), viral spike-protein and nucleoprotein (SP, NP), fibrinogen, platelets (CD41a). Viral particles in vascular cells were assessed by transmission electron microscopy (TEM). Cultured pericytes were infected with SARS-CoV-2 to measure TF expression and tubulisation of human pulmonary microvascular endothelial cells (HPMEC) was assessed upon vWF treatment. RESULTS: IPVD was present in 16/66 COVID-19 patients with both liver and lung histology, with a younger age (62 vs 78yo), longer illness (25 vs 14 days), worsening hypoxemia (PaO2/FiO2 from 209 to 89), and more ventilatory support (63 vs 22%) compared to COVID-19/Non-IPVD. IPVD, absent in controls, were confirmed by chest-CT. COVID-19/IPVD liver histology showed portal microthrombosis in >82.5% of portal areas, with a thicker wall of αSMA/PDGFR-ß+/ SP+/NP+ pericytes compared with COVID-19/Non-IPVD. Thrombosed portal venules correlated with αSMA+ area, whereas infected SP+/NP+ pericytes expressed TF. SARS-CoV-2 viral particles were observed in portal pericytes. In-vitro SARS-CoV-2 infection of pericytes up-regulated TF and induced endothelial cells to overexpress vWF, which expanded HPMEC tubules. CONCLUSIONS: SARS-CoV-2 infection of liver pericytes elicits a local procoagulant response associated with extensive portal microthrombosis, IPVD and worsening respiratory failure in fatal COVID-19. IMPACT AND IMPLICATIONS: Vascular involvement of the liver represents a serious complication of COVID-19 infection that must be considered in the work-up of patients with long-lasting and progressively worsening respiratory failure, as it may associate with the development of intrapulmonary vascular dilations. This clinical picture is associated with a pro-coagulant phenotype of portal venule pericytes, which is induced by SARS-CoV-2 infection of pericytes. Both observations provide a model that may apply, at least in part, to other vascular disorders of the liver, featuring obliterative portal venopathy, similarly characterized at the clinical level by development of hypoxemia and at the histological level, by phlebosclerosis and reduced caliber of the portal vein branches in the absence of cirrhosis. Moreover, our findings bring light to an as yet overlooked player of thrombosis pathophysiology, i.e. pericytes, which may provide novel therapeutic tools to halt prothrombotic mechanisms.

Venous Thromboembolism in Children: The Rivaroxaban Experience.

The incidence of venous thromboembolism (VTE) in the pediatric population has increased more than 10-fold in the last 20 years, as a consequence of the advancement of resuscitation and surgical techniques and the global increase in life expectancy of children suffering from chronic pathologies. Monitoring anticoagulant therapy to achieve outcomes within the target range in childhood VTE, parenteral administration of medications, and frequent blood tests in children are often cumbersome. Availability of safe and effective oral agents with pediatric data to support use would be of clear benefit. A physiologically based pharmacokinetic model was developed to estimate the appropriate dosing schedule for rivaroxaban in children. This incorporated growth/maturation and variability in anthropometrics (e.g., body height, weight, and body mass index), anatomy (e.g., organ weight), physiology (e.g., blood flow rates), metabolism and excretion. Rivaroxaban use in pediatric population underwent a complete investigational program, consisting mainly of one phase I pharmacokinetics/pharmacodynamics trial, three phase II trials, one phase III trial. The phase III trial enrolled 500 patients from birth to <18 years and documented the efficacy and safety of rivaroxaban regimens at dose equivalent to the adult 20 mg dose for the prevention of fatal or symptomatic nonfatal recurrent VTE and major bleeding versus heparin or vitamin K antagonists. Results were similar to those in rivaroxaban studies in adults. The efficacy and safety of rivaroxaban in children reported in the EINSTEIN JUNIOR trial provide further support to previous trials in adults (EINSTEIN Program), which demonstrate a favorable profile for the use of rivaroxaban for the management of VTE in challenging patient populations. Other clinical evidence contributing to the use of rivaroxaban among different risk groups in pediatric VTE population confirms the consistency with principal trial. Our review aims to describe the rationale for using rivaroxaban oral suspension in clinical practice and to summarize its multiple indications in each vascular bed (e.g., cerebral venous thrombosis, symptomatic or asymptomatic central venous catheter-associated thrombosis), etiology, and patients setting.

Partial F8 gene duplication (factor VIII Padua) associated with high factor VIII levels and familial thrombophilia.

High coagulation factor VIII (FVIII) levels comprise a common risk factor for venous thromboembolism (VTE), but the underlying genetic determinants are largely unknown. We investigated the molecular bases of high FVIII levels in 2 Italian families with severe thrombophilia. The proband of the first family had a history of recurrent VTE before age 50 years, with extremely and persistently elevated FVIII antigen and activity levels (>400%) as the only thrombophilic defects. Genetic analysis revealed a 23.4-kb tandem duplication of the proximal portion of the F8 gene (promoter, exon 1, and a large part of intron 1), which cosegregated with high FVIII levels in the family and was absent in 103 normal controls. Targeted screening of 50 unrelated VTE patients with FVIII levels ≥250% identified a second thrombophilic family with the same F8 rearrangement on the same genetic background, suggesting a founder effect. Carriers of the duplication from both families showed a twofold or greater upregulation of F8 messenger RNA, consistent with the presence of open chromatin signatures and enhancer elements within the duplicated region. Testing of these sequences in a luciferase reporter assay pinpointed a 927-bp region of F8 intron 1 associated with >45-fold increased reporter activity in endothelial cells, potentially mediating the F8 transcriptional enhancement observed in carriers of the duplication. In summary, we report the first thrombophilic defect in the F8 gene (designated FVIII Padua) associated with markedly elevated FVIII levels and severe thrombophilia in 2 Italian families.

Use of Glucocorticoids and Risk of Venous Thromboembolism: A Narrative Review.

Glucocorticoids are potent anti-inflammatory agents that are widely used for the treatment of many inflammatory, autoimmune, and neoplastic disorders. However, their beneficial effect is associated with several side effects, including an increased risk of cardiovascular complications, such as myocardial infarction and stroke. Whether their use also contributes to a procoagulant state, and therefore increases the risk of venous thromboembolism (VTE), is still a matter of debate. As an increased risk of venous thrombotic events is described in patients with Cushing's syndrome, which is characterized by endogenous hypercortisolism, it is reasonable to speculate that the chronic administration of glucocorticoids may induce a hypercoagulable state. However, it seems virtually impossible to separate the role of the drug from the underlying condition, which itself predisposes to the development of VTE. Actually, some evidence suggests that the use of exogenous glucocorticoids for the treatment of underlying disease and its exacerbations may further amplify the risk of VTE. Moreover, a procoagulant state has also been reported in healthy participants receiving oral glucocorticoids versus placebo. We have performed a concise narrative review on available data on the influence of exogenous glucocorticoids on hemostasis and their clinical impact on the risk of VTE.

Coagulopathy is not predictive of bleeding in patients with acute decompensation of cirrhosis and acute-on-chronic liver failure.

BACKGROUND & AIMS: Understanding factors responsible for the increased bleeding tendency in acute-on-chronic liver failure (ACLF) would improve the management of these complications. We investigated coagulation alterations in ACLF and assessed whether they were predictive of bleeding. METHODS: Cirrhosis patients with ACLF (cases) and acute decompensation (AD, controls) were prospectively recruited and underwent an extensive haemostatic assessment including standard tests, pro and anticoagulant factors, thrombomodulin-modified thrombin generation (TG) and thromboelastometry (ROTEM® ). In study part 1 (case-control), we compared coagulation in ACLF vs AD. In study part 2 (prospective), all patients were followed for bleeding, and predictors of outcome were assessed. RESULTS: Ninety-one patients were included (51 with ACLF, 40 with AD). Infections and ascites/renal dysfunction were the most common precipitating and decompensating events. Platelet count was lower while INR and activated partial thrombin time were longer in ACLF cohort vs AD. Regarding clotting factors, fibrinogen and factor VIII were comparable between groups while protein C and antithrombin were significantly reduced in ACLF. Endogenous thrombin potential by TG was comparable between groups. Clotting formation time and clot stability by ROTEM® were significantly lower in ACLF, indicative of a more hypocoagulable state. No haemostasis alteration could discriminate between patients who had bleeding complications during hospitalization and those who did not. CONCLUSION: We found coagulation changes in ACLF to largely overlap with that of AD and evidence of preserved coagulation capacity in both groups. ROTEM alterations were indicative of a more pronounced hypocoagulable state in ACLF; however, no correlation was found between such alterations and bleeding.

Thrombin generation in patients with COVID-19 with and without thromboprophylaxis.

OBJECTIVES: Thrombin generation (TG) with and without thrombomodulin (TM) was evaluated in COVID-19 patients with different disease severity and thromboprophylaxis regimen, in order to understand the prothrombotic profile. METHODS: We enrolled consecutive patients with confirmed diagnosis of COVID-19 admitted to Medical Departments (MD) or Intensive Care Units (ICU), and 54 healthy controls. RESULTS: Eighty-nine patients were included (mean age 60.4±16.1 years, 68.5% male); 33.7% admitted to ICU. Twenty-four patients (26.9%) were enrolled before thromboprophylaxis administration; 45 patients (50.6%) received standard and 20 (22.5%) intermediate sub-therapeutic dose thromboprophylaxis. Overall, patients with COVID-19 showed a TG profile comparable to that of healthy subjects (i.e. comparable peak height, endogenous thrombin potential [ETP] with and without TM). The only exception was lag time and time to peak, prolonged in COVID-19 patients vs. controls. MD patients showed a similar TG profile to healthy controls, and ICU patients showed significantly decrease ETP (p=0.030) compared to MD. As for thromboprophylaxis, TG profile was significantly increased in COVID-19 patients without thromboprophylaxis vs. controls and vs. those with thromboprophylaxis. In this latter group, ETP inhibition was significantly decreased (p=0.0003) and positively correlated with anti-Xa activity (r=0.49, p=0.0017). However, patients with thromboprophylaxis had similar TG profile vs. controls. Intermediate dose thromboprophylaxis more effectively inhibited TG in severe COVID-19 patients by increasing ETP inhibition via ETP with TM reduction vs. standard dose. CONCLUSIONS: COVID-19 patients showed increased TG at diagnosis. Standard thromboprophylaxis reduced TG to levels of healthy controls. Intermediate sub-therapeutic thromboprophylaxis more effectively inhibited TG by decreasing ETP with TM.

Cardiopulmonary bypass-induced coagulopathy in pediatric patients: The role of platelets in postoperative bleeding. A preliminary study.

Pediatric patients are particularly prone to cardiopulmonary bypass (CPB)-induced coagulopathy mainly due to hemodilution, consumption of coagulation factors and hypothermia. The aim of the present study was to examine the possible role of platelet count and function as it relates to the bleeding risk after CPB in the pediatric population. All consecutive patients (age <13 years) scheduled for elective cardiac surgery between January 2019 and November 2019 were retrospectively considered for the study. We gathered demographic characteristics, perioperative laboratory data (mainly platelet count and function), transfusion requirements, and blood loss for each patient. Patients with a chest tube output during the first 24 hours after surgery >75th percentile were bleeders (cases). Controls were nonbleeders. A total of 31 patients were enrolled [median age 17 (4-57) months]. A significant postoperative reduction in platelet count (P < .001) and function either in ADP-test (P < .001), TRAP-test (P < .001) and ASPI-test (P < .001) was found, with positive correlations between chest tube output within the first 24 hours after surgery and postoperative impairment of platelet count (R = 0.553, P = .001), ADP-test (R = 0.543, P = .001), TRAP-test (R = 0.627, P < .001) and ASPI-test (R = 0.436, P = .014). Eight children (26%) experienced major postoperative bleeding. Bleeders were significantly younger (P = .015) and underwent longer CPB duration (P = .015). Despite no significant differences in postoperative platelet count and function between cases and controls, the postoperative reduction (Δ) in platelet count (P = .002) and function in ADP-test (P = .007), TRAP-test (P = .020) and ASPI-test (P = .042) was significantly greater in bleeders vs. nonbleeders. A ΔPLT >262 500 ×109 /L, a ΔADP-test >29 U, a ΔTRAP-test >44 U and a ΔASPI-test >26 U showed to be predictive of major postoperative bleeding. Postoperative bleeding in children undergoing cardiac surgery with CPB was linked to younger age, longer CPB duration, and significant postoperative reduction in platelet count and function. Larger studies are needed to confirm our results and define strategies to reduce postoperative bleeding in these patients.

Changes in plasma circulating microvesicles in patients with HCV-related cirrhosis after treatment with direct-acting antivirals.

BACKGROUND & AIMS: The eradication of Hepatitis C (HCV) infection by direct-acting antiviral (DAAs) agents has been linked to an amelioration of liver synthesis and regression of fibrosis. Although changes in number and type of circulating microvesicles (MVs) have been reported in cirrhosis, conclusive data on the effect of DAAs treatment on MVs profile in HCV cirrhotic patients remain scarce. METHODS: We measured the levels of endothelial, platelet and hepatocyte MVs, as well as MVs-expressing versican core protein (VCAN+) in patients with HCV-related cirrhosis at baseline, end of treatment (EOT), at 12, 24 and 48 weeks (W) after EOT by new generation flow cytometry. RESULTS: Fifty-eight patients were enrolled (86% Child's A). MVs were increased at EOT vs baseline, though only platelet MVs revealed a statistically significant difference (P < .01). MV levels did not change significantly after EOT notwithstanding a steady downward trend towards baseline levels. Conversely, VCAN + MVs dropped significantly at EOT (P < .001) and remained low throughout the follow-up. Hepatocyte MVs significantly correlated with liver stiffness (r = .40, P = .0021). Eight composite outcomes occurred during the 1-year follow-up: three portal vein thromboses (PVTs), two hepatocellular carcinomas (HCCs) and three liver decompensation. Child's B, the presence of F2 oesophageal varices (OR for interaction 19.2 [95% CI 1.45-253.7], P = .023) and platelet MVs (OR 1.026 [95% CI 1.00-1.05, P = .023) correlated significantly with clinical outcomes. CONCLUSIONS: VCAN + MVs appear to mirror the profibrotic status of the cirrhotic disease; hepatocyte MVs correlate with liver stiffness and increased platelet MV levels could be associated with a worse clinical outcome.

Dengue fever as a rare cause of pulmonary embolism.

Dengue fever is a clinical entity well known for its haemorrhagic complications whose pathophysiology, though not completely understood, may be linked to a systemic inflammatory state caused by the infection itself. Even if rarely described, inflammation may lead as well to thromboembolic manifestations, as in the case we report here.

How haemophilia A impacts severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) treatment: a case report.

The typical symptoms of COVID-19 mimic those of the common season flu. In addition, several changes in the coagulation processes have been observed. To date, it's not fully clear how COVID-19 may affect patients with hereditary bleeding disorders. Anticoagulation in patients with haemophilia is still debated, but in this case could be needed. We are reporting a case of an elderly patient with mild haemophilia A hospitalized for Sars-Cov-2. On the 15th day of hospitalization, we observed an increase of all coagulation parameters. An antithrombotic prophylaxis at low dosage was immediately started, then increased at prophylactic dosage. Even if much more data are needed to ascertain the real thrombotic risk of haemophilia A in COVID-19 patients, it's clear that the FVIII and vWF should be strictly monitored in order to promptly establish an adequate treatment and avoid the onset of thromboembolic events, even fatal, causing many deaths in COVID-19 patients.

ABO blood group and the risk of post-thrombotic syndrome.

Post-thrombotic syndrome (PTS) has been associated to DVT recurrence, increased FVIII, inflammatory biomarker plasma levels, and persistence of vein obstruction. These same features have also been widely reported in non-O blood type subjects. Our aim was to investigate the correlation between the incidence of PTS and ABO blood types. Consecutive patients referred to the Department of Medicine of University of Padua between January 2004 and January 2012 following the diagnosis of a first episode of proximal DVT were enrolled. The presence of PTS was assessed via the Villalta scale at predefined time points (3, 6, 12, 18, 24, 36 months). Hazard ratio (HR) for PTS development was calculated in non-O (exposed) vs O blood (unexposed) type patients. Out of 671 eligible patients, 606 were enrolled. Overall, 192 (31.7%) patients developed PTS: 142 (34.5%) non-O and 50 (25.6%) O blood type patients. Individuals with non-O blood group were associated with a significantly higher risk to develop PTS (HR 1.53, 95% CI, 1.05-2.24; p = 0.028) than O group. Non-O blood type might be a risk factor for the development of PTS.

Reversal of hypercoagulability in patients with HCV-related cirrhosis after treatment with direct-acting antivirals.

BACKGROUND & AIMS: The long-term impact of sustained virological response (SVR) after direct-acting antivirals (DAAs) on the hypercoagulability associated with HCV cirrhosis is unknown. We longitudinally evaluated the effect of DAAs treatment on cirrhotic coagulopathy. METHODS: Pro- and anticoagulant factor levels and thrombin generation were assessed in patients with HCV-related cirrhosis at baseline, end of therapy (EOT), at 12, 24 and 48 weeks (W) after EOT. RESULTS: Fifty-eight patients were enrolled (86% Child's A). SVR was 100%. Median factor VIII activity significantly decreased at EOT, 12 weeks and 24 weeks compared with baseline, whereas protein C significantly increased at 24 weeks and 48 weeks. Cirrhotic patients showed a slight but sustained increase in endogenous thrombin potential (ETP) with a statistically significant difference at EOT, 12 weeks, 24 weeks and 48 weeks compared with baseline. Conversely, thrombomodulin-modified ETP was elevated before treatment and decreased over time to normal levels at 24 weeks and 48 weeks. The ETP ratio decreased slowly at EOT and 12 weeks, and was significantly decreased at 24 weeks and 48 weeks compared with baseline (P < .001 for both comparisons), being not statistically different from ETP ratio measured in healthy controls. Child's B patients showed a significantly higher ETP ratio compared to Child's A at baseline and did not show any significant improvement in ETP ratio through 12 weeks. Two Child's B patients developed PVT with an incidence rate of 1.1% p-yrs (95%CI, 0.18 to 3.58). CONCLUSIONS: DAAs therapy in HCV-related cirrhotic patients is associated with significant changes in thrombin generation suggesting a reversal of hypercoagulability particularly in Child's A patients.

On-treatment platelet reactivity in peripheral and coronary blood in patients undergoing primary PCI for ST-segment elevation myocardial infarction (STEMI).

Dual antiplatelet therapy is recommended in patients undergoing primary percutaneous coronary intervention (p-PCI) for ST-segment elevation myocardial infarction (STEMI). Pre-analytical variables may influence platelet function analysis results. Our aim was to evaluate the on-treatment platelet reactivity in peripheral artery vs coronary blood in patients with STEMI. We enrolled one hundred and nine patients who consecutively underwent p-PCI at Cardiology Unit of Padua University Hospital between June 2014 and June 2015. Before the procedure, all patients received intravenous aspirin 250 mg and either of the thienopyridines; clopidogrel 600 mg, prasugrel 60 mg or ticagrelor 180 mg. ASPI-test and ADP-test using multiple electrode aggregometry (MEA) were performed in samples collected from both a peripheral artery and the culprit coronary artery. 'Low responders' were patients with an ASPI-test or ADP-test value greater than or equal to a pre-established normal range. No significant differences were observed in ASPI-test values between peripheral (19 (median) [3-49 (10-90 percentiles)] U) vs coronary (12 [1-40] U, p = .06) blood and in ADP-test (40 [14-82] U vs 33 [7-79] U, p =.68) blood. In peripheral blood, fifteen (14%) patients were 'low aspirin' and forty-one (38%) 'low thienopyridines' responders. The prevalence of 'low clopidogrel' responders was higher (45%) than prasugrel (36%) and ticagrelor (33%). Similar results were observed in coronary blood. In patients undergoing p-PCI for STEMI, MEA platelet function observed in coronary arteries was consistent with peripheral artery blood's independently of the antiplatelet drug used. The clinical significance of peripheral and coronary on-aspirin/thienopyridines platelet reactivity needs further clarification.

New Prothrombin Mutation (Arg596Trp, Prothrombin Padua 2) Associated With Venous Thromboembolism.

OBJECTIVE: Two different prothrombin variants, p.Arg596Leu and p.Arg596Gln, conferring antithrombin resistance to patients with venous thromboembolism have been recently reported. Here, we describe a novel substitution affecting Arg596 of prothrombin molecule (Arginine596 to Tryptophan or p.Arg596Trp or Arg221aTrp in the chymotrypsinogen numbering system or prothrombin Padua 2) in 2 Italian families with venous thromboembolism. APPROACH AND RESULTS: Prothrombin Padua 2 has been characterized either in plasma of carriers or using Arg596Trp recombinant prothrombin. Routine coagulation tests, thrombin generation, and antithrombin resistance tests were performed, as well as measurement of the levels of thrombin-antithrombin complexes. All carriers were heterozygotes and presented with a mild reduction of the prothrombin activity. Thrombin generation in carriers showed only a markedly prolonged decay. This finding was confirmed in plasma reconstituted with Arg596Trp recombinant prothrombin mimicking a homozygous condition, which showed longer decay and higher endogenous thrombin potential in thrombin generation than wild-type recombinant prothrombin reconstituted plasma. Patient's plasma as well as Arg596Trp recombinant prothrombin showed a clear thrombin resistance to antithrombin inactivation. These findings were supported by the assessment of thrombin-antithrombin complexes formation, which was strongly reduced for Arg596Trp recombinant prothrombin as compared with wild-type recombinant prothrombin. In a series of 400 unrelated consecutive patients with venous thromboembolism, 2 carriers of prothrombin Padua 2 were found (estimated prevalence of 0.5%). CONCLUSIONS: Our study showed that prothrombin Padua 2 induces antithrombin resistance and is associated with an increased risk of venous thromboembolism. Codon 596 (CGG) of prothrombin is a hot spot for mutations, which constitute a new and relatively frequent cause of inherited thrombophilia.

Modulating thrombotic diathesis in hereditary thrombophilia and antiphospholipid antibody syndrome: a role for circulating microparticles?

Over the past decades, there have been great advances in the understanding of the pathogenesis of venous thromboembolism (VTE) in patients with inherited and acquired thrombophilia [mainly antiphospholipid antibody syndrome (APS)]. However, a number of questions remain unanswered. Prognostic markers capable of estimating the individual VTE risk would be of great use. Microparticles (MPs) are sub-micron membrane vesicles constitutively released from the surface of cells after cellular activation and apoptosis. The effects of MPs on thrombogenesis include the exposure of phopshatidylserine and the expression of tissue factor and MPs have been described in clinical studies as possible diagnostic and prognostic biomarkers for VTE. This review will provide a novel perspective on the current knowledge and research trends on the possible role of MPs in hereditary thrombophilia and APS. Basically, the published data show that circulating MPs may contribute to the development of VTE in thrombophilic carriers, both in mild and severe states. Moreover, the presence of endothelial-MPs and platelet-MPs has been described in antiphospholipid syndrome and seems to be directly linked to antiphospholipid antibodies and not to other underlying autoimmune disorders or the thrombotic event itself. In conclusion, circulating MPs may constitute an epiphenomenon of thrombophilia itself and could be up-regulated in acute particular conditions, promoting a global prothrombotic state up to the threshold of the clinical relevant thrombotic event.

Factor VIIa-antithrombin complex: a possible new biomarker for activated coagulation.

The activation of the extrinsic coagulation pathway occurs after endothelial injury when the tissue factor (TF), a transmembrane protein located outside the vasculature, binds factor VII (FVII) or activated FVII (FVIIa). Once formed, the TF-VIIa complex activates both factor IX and X and initiates the coagulation process. The TF-VIIa complex is inhibited by both TF pathway inhibitor (TFPI) and antithrombin (AT). The interaction between TF-VIIa and AT induces FVIIa-AT complex formation, which is released into the plasma. Because AT reacts with FVIIa only when it is bound to TF, the circulating levels of FVIIa-AT reflect the degree of exposure of TF to blood. Preliminary clinical studies have shown higher plasma levels of FVIIa-AT complex both in patients with a prior arterial or venous thrombotic event. Increased plasma levels of FVIIa-AT have also been reported in a number of other prothrombotic conditions - antiphospholipid antibodies, solid and hematological malignancies, pre-eclampsia (PE), obesity and cardiac surgery. However, most of the studies published so far are retrospective and with a limited sample size. Larger prospective clinical studies are needed to confirm these findings and to assess the prognostic role of this possible new biomarker for activated coagulation.

Thromboelastometry guided fibrinogen replacement therapy in cardiac surgery: a retrospective observational study.

This retrospective, observational study compared the impact of a point-of-care rotational thromboelastometry (ROTEM®) method versus conventional bleeding management in terms of postoperative (24-h) blood loss, intraoperative and postoperative (24-h) transfusion requirement and length of stay in the postoperative intensive care unit (ICU) in patients undergoing cardiac surgery. Forty consecutive patients undergoing cardiac surgery under ROTEM®-guided hemostatic management were enrolled; the control population included 40 selected patients undergoing similar interventions without ROTEM® monitoring. Significantly more patients in the thromboelastometry group versus the control group received fibrinogen (45 vs 10 %; p < 0.0001), while fewer received a transfusion (40 vs 72.5 %; p < 0.0033). Compared with control group patients, those in the thromboelastometry group had less postoperative bleeding (285 vs 393 mL; p < 0.0001), a shorter time from cardiopulmonary bypass discontinuation to skin suture (79.3 vs 92.6 min; p = 0.0043) and a shorter stay in the ICU (43.7 vs 52.5 h; p = 0.0002). In our preliminary experience, ROTEM®-guided bleeding management was superior to conventional management of bleeding in patients undergoing complex cardiac surgery with cardiopulmonary bypass in terms of reduced postoperative blood loss, transfusion requirement, and length of ICU stay.

Endocytosis of exogenous factor V by ex-vivo differentiated megakaryocytes from patients with severe parahaemophilia.

Although human megakaryocytes can synthesize factor V (FV), platelet FV derives largely from endocytosis of plasma FV. Recently, it has been shown that plasma transfusions can replenish the platelet FV pool in parahaemophilic patients. Here we corroborate this finding by showing FV endocytosis by ex vivo differentiated megakaryocytes derived from patients with inherited parahaemophilia. Mononuclear stem cells isolated from peripheral blood of healthy subjects and of three patients with severe parahaemophilia were cultured in the presence of thrombopoietin and interleukin-3 and differentiated into CD41-positive polynucleated megakaryocytes. Exogenous purified FV was added to the culture medium to evaluate FV endocytosis. Immunofluorescence staining revealed abundant FV expression in megakaryocytes derived from healthy donors, but no FV expression in those derived from patients with severe parahaemophilia. However, after the addition of purified FV to the culture medium, megakaryocytes from parahaemophilia patients became positive upon FV immunostaining, suggesting endocytosis of exogenous FV. Endocytosed FV retained factor Xa-co-factor activity as assessed by a prothrombin time-based functional test in megakaryocyte lysates. Addition of exogenous FV to culture medium can restore the FV content of megakaryocytes derived from patients with severe FV defects. This rescue mechanism can have important clinical implications in the management of parahaemophilia patients.